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rTASSEL: An R interface to TASSEL for analyzing genomic diversity
|
Journal of open source software
| 2,022
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cc-by
| 2,112
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Brandon Monier
1, Terry M. Casstevens
1, Peter J. Bradbury
1,2,
and Edward S. Buckler
1,2 1 Institute for Genomic Diversity, Cornell University, Ithaca, NY 14853 2 United States Department of
Agriculture-Agricultural Research Service, Robert W. Holley Center for Agriculture and Health, Ithaca,
NY 14853 DOI: 10.21105/joss.04530 Monier et al. (2022). rTASSEL: An R interface to TASSEL for analyzing genomic diversity. Journal of Open Source Software, 7(76), 4530.
https://doi.org/10.21105/joss.04530.
1 rTASSEL: An R interface to TASSEL for analyzing
genomic diversity Brandon Monier
1, Terry M. Casstevens
1, Peter J. Bradbury
1,2,
and Edward S. Buckler
1,2 Brandon Monier
1, Terry M. Casstevens
1, Peter J. Bradbury
1,2,
and Edward S. Buckler
1,2 SSEL: An R interface to TASSEL for analyzing genomic diversity. Journal of Open Source Software, 7(76), 4530.
oss 04530
1 Authors of papers retain
copyright and release the work
under a Creative Commons
Attribution 4.0 International
License (CC BY 4.0). Summary The need for efficient tools and applications for analyzing genomic diversity is essential for any
genetics research or breeding program. One commonly used tool, TASSEL (Trait Analysis
by aSSociation, Evolution, and Linkage), provides many core methods for genomic analyses. Despite its efficiency, TASSEL has limited automation potential for reproducible research and to
interact with other analytical tools. Here we present an R package, rTASSEL, that is a front-end
to connect to a variety of highly used TASSEL methods and analytical tools. The goal of
this package is to create a unified scripting workflow that leverages the analytical prowess of
TASSEL, in conjunction with R’s data handling and visualization capabilities, without ever
having the user switch between these two environments. Editor: Frederick Boehm
Reviewers:
• @tkchafin
• @tomsing1
Submitted: 21 June 2022
Published: 10 August 2022
License
Authors of papers retain
copyright and release the work
under a Creative Commons
Attribution 4.0 International
License (CC BY 4.0). Reviewers: Approach Implementation Figure 1: Overview of the rTASSEL workflow. Genotypic and phenotypic data (A) are used to create
an R S4 object (B). From this object, TASSEL functionalities can be called to run various association,
linkage disequilibrium, and relatedness functions (C). Outputs from these TASSEL analyses are returned
to the R environment as data frame objects (D), Manhattan plot visualizations (E), or interactive
visualizations for linkage disequilibrium analysis (F). Figure 1: Overview of the rTASSEL workflow. Genotypic and phenotypic data (A) are used to create
an R S4 object (B). From this object, TASSEL functionalities can be called to run various association,
linkage disequilibrium, and relatedness functions (C). Outputs from these TASSEL analyses are returned
to the R environment as data frame objects (D), Manhattan plot visualizations (E), or interactive
visualizations for linkage disequilibrium analysis (F). rTASSEL combines TASSEL’s abilities to store genotype data as half bytes, bitwise arithmetic
for kinship analyses, genotype filtration, extensive forms of linear modeling, multithreading,
and access to a range of native libraries while providing access to R’s prominent scripting
capabilities and commonly used Bioconductor classes (Gentleman et al., 2004; Lawrence et
al., 2013; Morgan et al., 2021). Since TASSEL is written in Java, a Java to R interface is
implemented via the rJava package (Urbanek, 2021). rTASSEL allows for the rapid import, analysis, visualization, and export of various genomic data
structures. Diverse formats of genotypic information can be used as inputs for rTASSEL. These
include variant call format (.vcf), HapMap (.hmp.txt), and Flapjack (.flpjk.*). Phenotype
data can also be supplied in multiple formats. These include TASSEL formatted data sets or
R data frame objects (Figure 1A). Once data is imported, the function readGenotypePhenotype is used to construct an S4 object,
which is used for all downstream analyses (Figure 1B, Figure 1C). This object contains slots
that exclusively hold references to objects held in the Java virtual machine, which can be
called with downstream functions. Prior to analysis, genotype objects can be quickly imported
and filtered in several ways to help in the reduction of confounding errors. rTASSEL can
filter genotype objects by either variant site properties (filterGenotypeTableSites) or by
individuals (filterGenotypeTableTaxa). License As breakthroughs in genotyping technologies allow for increasing available variant resources,
methods and implementations to analyze complex traits in a diverse array of organisms
are needed. One such resource is TASSEL (Trait Analysis by aSSociation, Evolution, and
Linkage). This software suite contains functionality for analyses in association studies, linkage
disequilibrium (LD), kinship, and dimensionality reduction (e.g., PCA and MDS) (Bradbury et
al., 2007). While initially released in 2001, the fifth version, TASSEL 5, has been optimized
for handling large data sets and has added newer approaches to association analyses for many
thousands of traits (Shabalin, 2012). Despite these improvements, interacting with TASSEL
has been limited to either a graphical user interface with limited workflow reproducibility or
a command-line interface with a higher learning curve that can dissuade novice researchers
and provide unnecessary intermediate files in an analytics workflow (Zhang et al., 2009). To
remediate this issue, we have created an R package, rTASSEL. This package interfaces the
analytical power of TASSEL with R’s data formats and intuitive function handling. Monier et al. (2022). rTASSEL: An R interface to TASSEL for analyzing genomic diversity. Journal of Open Source Software, 7(76), 4530.
https://doi.org/10.21105/joss.04530.
2 Genomic prediction The function genomicPrediction can be used for predicting phenotypes from genotypes. To do
this, genomicPrediction uses genomic best linear unbiased predictors (gBLUPs). It proceeds
by fitting a mixed model that uses kinship to capture covariance between taxa. The mixed
model can calculate BLUPs for taxa that do not have phenotypes based on the phenotypes of
lines with relationship information. Relatedness functions For users to run MLM methods, relatedness estimates need to be calculated. rTASSEL can
efficiently compute this on large data sets by processing blocks of sites at a time using bitwise
operations. This can be accomplished using the function kinshipMatrix, which will generate
a kinship matrix from genotype data. Several methods for calculating kinship in TASSEL are
implemented. By default, a “centered” identity by state (IBS) approach is used (Endelman
& Jannink, 2012). Additionally, normalized IBS (Yang et al., 2011), dominance-centered
IBS (Muñoz et al., 2014), and dominance normalized IBS (Zhu et al., 2015) can be used. rTASSEL can either generate a reference object for association analysis or an R matrix object
via R’s as.matrix function for additional analyses. In addition to kinship generation, principal
components analysis and multidimensional scaling can be used on genotype data using rTASSEL
methods, pca and mds, respectively. Finally, phylogenetic analysis can be performed on genotype
data using the createTree method which will generate phylo objects commonly used by the
ape package (Paradis & Schliep, 2019). The createTree method allows for two clustering
methods: neighbor joining or UPGMA (unweighted pair group method with arithmetic mean). Linear models can be specified following the format used by R’s lm function: Linear models can be specified following the format used by R’s lm function: y ∼A1 + A2 + · · · + An where y is phenotype data, and An is any covariate or factor data. This formula parameter
and several other parameters allow the user to run BLUE, GLM, or MLM modeling. Once
association analysis is completed, TASSEL table reports of association statistics are generated
as an R list which can then be exported as flat files or converted to data frames (Figure 1D). rTASSEL can also visualize association statistics with the function, manhattanPlot, which
utilizes the graphical capabilities of the package, ggplot2 (Wickham, 2016) (Figure 1E). Linkage disequilibrium rTASSEL can also generate linkage disequilibrium (LD) from genotype data via the function
linkageDiseq. LD is estimated by the standardized disequilibrium coefficient, D′, correlation
between alleles at two loci (r2), and subsequent p-values via a two-sided Fisher’s exact test. TASSEL table reports for all pairwise comparisons are generated as data.frame objects, and
heatmap visualizations for each given metric are generated via TASSEL’s legacy LD Java
viewer or ggplot2 (Figure 1F). Monier et al. (2022). rTASSEL: An R interface to TASSEL for analyzing genomic diversity. Journal of Open Source Software, 7(76), 4530.
https://doi.org/10.21105/joss.04530.
3 Association functions One of TASSEL’s most dynamic functionalities is its capability to perform various association
modeling techniques. rTASSEL allows several types of association studies to be conducted
using one primary function, assocModelFitter, with different parameter inputs. This allows
for implementing both least-squares fixed-effects general linear models (GLM) and mixed linear
models (MLM) via the Q + K method (Yu et al., 2006). If no genotypic data is provided
to the GLM model, assocModelFitter can calculate best linear unbiased estimates (BLUEs). Additionally, fast GLM approaches are implemented in rTASSEL, which allow for the rapid
analysis of many phenotypic traits (Shabalin, 2012). Monier et al. (2022). rTASSEL: An R interface to TASSEL for analyzing genomic diversity. Journal of Open Source Software, 7(76), 4530. https://doi.org/10.21105/joss.04530. 2 Acknowledgements This project is supported by the USDA-ARS, the Bill and Melinda Gates Foundation, and NSF
IOS #1822330. We thank Sara J. Miller, Guillaume Ramstein, and Joseph Gage for their
insightful suggestions on this manuscript and pipeline testing. This project is supported by the USDA-ARS, the Bill and Melinda Gates Foundation, and NSF
IOS #1822330. We thank Sara J. Miller, Guillaume Ramstein, and Joseph Gage for their
insightful suggestions on this manuscript and pipeline testing. Additional resources More information about various functionalities and workflows can be found on our project
webpage. Source code can be found on our GitHub repository. An interactive Jupyter notebook
session detailing additional rTASSEL workflows can be found on Binder. Monier et al. (2022). rTASSEL: An R interface to TASSEL for analyzing genomic diversity. Journal of Open Source Software, 7(76), 4530.
https://doi.org/10.21105/joss.04530.
4 References Bradbury, P. J., Zhang, Z., Kroon, D. E., Casstevens, T. M., Ramdoss, Y., & Buckler, E. S. (2007). TASSEL: Software for association mapping of complex traits in diverse samples. Bioinformatics, 23(19), 2633–2635. https://doi.org/10.1093/bioinformatics/btm308 Endelman, J. B., & Jannink, J.-L. (2012). Shrinkage estimation of the realized relationship
matrix. G3: Genes, Genomes, Genetics, 2(11), 1405–1413. https://doi.org/10.1534/g3. 112.004259 Gentleman, R. C., Carey, V. J., Bates, D. M., Bolstad, B., Dettling, M., Dudoit, S., Ellis,
B., Gautier, L., Ge, Y., Gentry, J., Hornik, K., Hothorn, T., Huber, W., Iacus, S., Irizarry,
R., Leisch, F., Li, C., Maechler, M., Rossini, A. J., … Zhang, J. (2004). Bioconductor:
Open software development for computational biology and bioinformatics. Genome Biology,
5(10), R80. https://doi.org/10.1186/gb-2004-5-10-r80 Lawrence, M., Huber, W., Pagès, H., Aboyoun, P., Carlson, M., Gentleman, R., Morgan, M. T., & Carey, V. J. (2013). Software for computing and annotating genomic ranges. PLOS
Computational Biology, 9(8), e1003118. https://doi.org/10.1371/journal.pcbi.1003118 Morgan, M., Obenchain, V., Hester, J., & Pagès, H. (2021). SummarizedExperiment: Summa-
rizedExperiment container. https://bioconductor.org/packages/SummarizedExperiment Muñoz, P. R., Resende, M. F. R., Gezan, S. A., Resende, M. D. V., Campos, G. de los, Kirst,
M., Huber, D., & Peter, G. F. (2014). Unraveling additive from nonadditive effects using
genomic relationship matrices. Genetics, 198(4), 1759–1768. https://doi.org/10.1534/
genetics.114.171322 Paradis, E., & Schliep, K. (2019). Ape 5.0: An environment for modern phylogenetics
and evolutionary analyses in R. Bioinformatics, 35, 526–528. https://doi.org/10.1093/
bioinformatics/bty633 Shabalin, A. A. (2012). Matrix eQTL: Ultra fast eQTL analysis via large matrix operations. Bioinformatics, 28(10), 1353–1358. https://doi.org/10.1093/bioinformatics/bts163 Urbanek, S. (2021). rJava: Low-level R to Java interface. https://CRAN.R-project.org/
package=rJava Wickham, H. (2016). ggplot2: Elegant graphics for data analysis. Springer-Verlag New York. ISBN: 978-3-319-24277-4 Yang, J., Lee, S. H., Goddard, M. E., & Visscher, P. M. (2011). GCTA: A tool for genome-
wide complex trait analysis. The American Journal of Human Genetics, 88(1), 76–82. https://doi.org/10.1016/j.ajhg.2010.11.011 Yu, J., Pressoir, G., Briggs, W. H., Vroh Bi, I., Yamasaki, M., Doebley, J. F., McMullen, M. D., Gaut, B. S., Nielsen, D. M., Holland, J. B., Kresovich, S., & Buckler, E. S. (2006). A
unified mixed-model method for association mapping that accounts for multiple levels of
relatedness. Nature Genetics, 38(2), 203–208. https://doi.org/10.1038/ng1702 Zhang, Z., Buckler, E. S., Casstevens, T. M., & Bradbury, P. J. (2009). Software engineering
the mixed model for genome-wide association studies on large samples. Briefings in
Bioinformatics, 10(6), 664–675. https://doi.org/10.1093/bib/bbp050 Monier et al. (2022). rTASSEL: An R interface to TASSEL for analyzing genomic diversity. Zhu, Z., Bakshi, A., Vinkhuyzen, A. A. E., Hemani, G., Lee, S. H., Nolte, I. M., Vliet-
Ostaptchouk, J. V. van, Snieder, H., Esko, T., Milani, L., Mägi, R., Metspalu, A., Hill,
W. G., Weir, B. S., Goddard, M. E., Visscher, P. M., & Yang, J. (2015). Dominance
genetic variation contributes little to the missing heritability for human complex traits. The
American Journal of Human Genetics, 96(3), 377–385. https://doi.org/10.1016/j.ajhg.
2015.01.001 Monier et al. (2022). rTASSEL: An R interface to TASSEL for analyzing genomic diversity. Journal of Open Source Software, 7(76), 4530.
https://doi.org/10.21105/joss.04530.
5 References Journal of Open Source Software, 7(76), 4530. https://doi.org/10.21105/joss.04530. 4 SSEL: An R interface to TASSEL for analyzing genomic diversity. Journal of Open Source Software, 7(76), 4530.
oss 04530
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https://www.medrxiv.org/content/medrxiv/early/2020/11/10/2020.11.07.20227520.full.pdf
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English
| null |
Execution of intervention matters more than strategy: A lesson from the spatiotemporal assessment of COVID-19 clusters in Nepal
|
medRxiv (Cold Spring Harbor Laboratory)
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cc-by
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is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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this version posted November 10, 2020.
;
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eprint
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doi:
medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. eer review)
The copyright holder for this
this version posted November 10, 2020. ;
10.1101/2020.11.07.20227520 . CC-BY 4.0 International license
It is made available under a . CC-BY 4.0 International license
It is made available under a Execution of intervention matters more than strategy: A
1
lesson from the spatiotemporal assessment of COVID-19
2
clusters in Nepal
3
Bipin Kumar Acharya1#, Laxman Khanal2#, Alnwisi Sameh Mansoor Mahyoub1, Zengliang Ruan1,
4
Yin Yang1, Samir Kumar Adhikari3, Shreejana Pandit4, Basanta Kumar Neupane5 Binod Kumar
5
Paudel6, Hualiang Lin1*
6
1 Department of Epidemiology, School of Public Health, Sun Yat-Sen University,
7
Guangzhou, China
8
2 Central Department of Zoology, Institute of Science and Technology, Tribhuvan
9
University, Kathmandu 44613, Nepal
10
3 Ministry of Health and Population, Government of Nepal, Kathmandu, Nepal
11
4 Kanti Children’s Hospital, Maharajgunj, Kathmandu 44616, Nepal
12
5 University of Chines Academy of Sciences (UCAS), Beijing, China. 13
6 Communication University of China, Beijing, China
14
# Two authors contributed to the manuscript equally. 15
* Correspondence:
16
Huliang Lin, Email: linhualiang@mail.sysu.edu.cn
17 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. Execution of intervention matters more than strategy: A
1
lesson from the spatiotemporal assessment of COVID-19
2
clusters in Nepal
3 Bipin Kumar Acharya1#, Laxman Khanal2#, Alnwisi Sameh Mansoor Mahyoub1, Zengliang Ruan1,
4
Yin Yang1, Samir Kumar Adhikari3, Shreejana Pandit4, Basanta Kumar Neupane5 Binod Kumar
5
Paudel6, Hualiang Lin1*
6 Bipin Kumar Acharya1#, Laxman Khanal2#, Alnwisi Sameh Mansoor Mahyoub1, Zengliang Ruan1,
4
Yin Yang1, Samir Kumar Adhikari3, Shreejana Pandit4, Basanta Kumar Neupane5 Binod Kumar
5
Paudel6, Hualiang Lin1*
6 1 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. . CC-BY 4.0 International license
It is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
eprint
The copyright holder for this
this version posted November 10, 2020. ;
https://doi.org/10.1101/2020.11.07.20227520
doi:
medRxiv preprint . CC-BY 4.0 International license
It is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
preprint
The copyright holder for this
this version posted November 10, 2020. ;
https://doi.org/10.1101/2020.11.07.20227520
doi:
medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. peer review)
The copyright holder for this
this version posted November 10, 2020. ;
rg/10.1101/2020.11.07.20227520 . CC-BY 4.0 International license
It is made available under a Keywords: Disease clusters, Geospatial dynamics, Pandemic, SARS-CoV-2, SaTScan
39 1. Introduction
40 The coronavirus disease 2019 (COVID-19) outbreak has been considered a Public Health
41
Emergency of International Concern on 30 January 2020 and declared a global pandemic on 11
42
March 2020 by the World Health Organization (WHO). This viral disease caused by the severe
43
acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected 39,596,858 people and
44
killed 1107374 lives as of 18 October 2020 (WHO, 2020). COVID-19 is a highly contagious
45
disease with an initial estimated average basic reproductive rate (R0) of 3.28 (Liu et al., 2020) that
46
has been substantially reduced by the multiple intervention approaches (You et al., 2020). The
47
disease has been described to have higher severity to the old-age and immunosuppressed people
48
such as suffering from cardiac and pulmonary disorders (Zheng et al., 2020). Despite the constant
49
efforts of scientists across the globe, we still lack the well-tested and approved vaccines and drugs
50
against the disease. 51 Analogous to many countries in the world, Nepal has hugely suffered from the COVID-19
52
pandemic (Panthee et al., 2020). The first case of the disease was reported on 23 January 2020 in
53
a China-returned 32 years old male (Pun et al., 2020; Shrestha et al., 2020). After the lag period of
54
two months, the second positive case was reported on 23 March 2020 (Piryani et al., 2020). 55
Subsequently the number of the disease victims gradually increased and almost all the cases in the
56
earlier days were among the people returned from Europe, Middle-East countries and others. 57
Despite being neighbored to the China also, the number of SARS-CoV-2 positive cases in Nepal
58
surged up when the Nepalese workers from its southern neighbor India returned home via open
59
border between the two countries. By the date of 18 October 2020, there have been 132246 cases
60
and 739 deaths across the country (MoHP, 2020). The geographical distribution of the COVID-19
61
cases within the Nepal territory is not uniform. Communities associated with the poverty in densely
62
populated growing cities are estimated more vulnerable to the infection (Khanal et al., 2020). 63
Additionally, disease intervention efforts are not equal among the local administrative units of
64
Nepal and so is the pattern of spread of the COVID-19. ABSTRACT
18 (which was not certified by peer review)
preprint
The copyright holder for this
this version posted November 10, 2020. ;
https://doi.org/10.1101/2020.11.07.20227520
doi:
medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. eer review)
The copyright holder for this
this version posted November 10, 2020. ;
10.1101/2020.11.07.20227520 . CC-BY 4.0 International license
It is made available under a ABSTRACT
18 The novel coronavirus disease 2019 (COVID-19) has been the biggest public health problem of
19
the present world. As the number of people suffering from the pandemic is rising, it is likely to
20
claim more life and worsen the global health and economy. Nepal, one of the developing countries
21
in the south Asia has been strongly influenced by the pandemic and struggling to contain it with
22
multiple interventions, however, spatiotemporal dynamics of the epidemic and its linkage with
23
various intervention strategies has not been studied yet. Here, we employed the prospective spatial-
24
temporal analysis with SaTScan assessing dynamics of the COVID-19 cases from 23 January to
25
31 August 2020 at district level in Nepal. The results revealed that COVID-19 dynamics in the
26
early stage of transmission was slower and confined in certain districts. However, from the third
27
week of April, transmission spread rapidly across districts of Province No. 2 and Sudoorpaschim
28
Province, primarily introduced by Nepalese citizens returning from India. Despite nationwide
29
lockdown, nine statistically significant active and emerging clusters were detected between 23
30
January and 21 July 2020, whereas ten emerging clusters were observed for extended period to 31
31
August. The population density and population inflow from India crossing the sealed border had
32
significant effects on the elevated risk of the epidemic. The capital city Kathmandu has become
33
the highest-risk active cluster since August when travel restriction has been suspended. Movement
34
restriction appears to be the most effective non-pharmaceutical intervention against the COVID-
35
19 for resource-scarce countries with limited health care facilities. Our findings could be valuable
36
to the health authorities within Nepal and beyond to better allocate resources and improve
37
interventions on the pandemic for containing it efficiently. 38 Keywords: Disease clusters, Geospatial dynamics, Pandemic, SARS-CoV-2, SaTScan
39 2 . CC-BY 4.0 International license
It is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
eprint
The copyright holder for this
this version posted November 10, 2020. ;
https://doi.org/10.1101/2020.11.07.20227520
doi:
medRxiv preprint . CC-BY 4.0 International license
It is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 1. Introduction
40 65 The magnitude and timing of the interventions matter for the mitigation of the outbreak (Dehning
66
et al., 2020). When the second COVID-19 case was recorded in Nepal and number of cases was
67
also rising in India, the Government of Nepal closed all international flights and borders on 23
68
March 2020 (Sapkota et al., 2020). The very next day, a nation-wide lockdown was further
69 3 . CC-BY 4.0 International license
It is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
eprint
The copyright holder for this
this version posted November 10, 2020. ;
https://doi.org/10.1101/2020.11.07.20227520
doi:
medRxiv preprint . CC-BY 4.0 International license
It is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
preprint
The copyright holder for this
this version posted November 10, 2020. ;
https://doi.org/10.1101/2020.11.07.20227520
doi:
medRxiv preprint . CC-BY 4.0 International license
It is made available under a enforced that continued till 21 July 2020. Besides the diagnosis, isolation and treatment of the
70
COVID-19 patients, Government of Nepal employed multiple public health measures such as
71
border closure, lockdown, social distancing, and personal hygiene which aided Nepal in avoiding
72
the spread of the novel coronavirus during the initial days (Basnet et al., 2020; Dhakal and Karki,
73
2020). Physical distancing measures, such as closure of schools and colleges, retail businesses,
74
and restaurants, cancellation of public events, as well as constraints on individual movements and
75
social interactions, are now in place in many countries with the aim of reducing transmission of
76
SARS-CoV-2 (Cowling et al., 2020; Davies et al., 2020; Yang et al., 2020). Among other
77
measures, travel restrictions, physical distancing, home quarantine, centralized quarantine,
78
compulsion on mask wearing in public places, universal symptom survey, implementation of
79
testing, isolation, and contact tracing probably slowed the transmission dynamics significantly
80
(Davies et al., 2020; Fang et al., 2020; Pan et al., 2020). Despite of continuous lockdown enforced
81
for 120 days, the outbreak of the COVID-19 is reemerging and the efficacy of such interventions
82
have never been assessed in Nepal. 83 Pharmaceutical interventions alone are not enough to contain the COVID-19, hence, countries
84
augmented them with non-pharmaceutical approaches. 1. Introduction
40 However, how different combinations of
85
interventions, timings, and extents have yielded desired outcomes to curb the disease transmission
86
remains unclear (Cowling et al., 2020; Davies et al., 2020; Fang et al., 2020; MacIntyre and Wang
87
2020; Pan et al., 2020; Zhang et al., 2020). The level of vulnerability to the COVID-19 differed
88
among the communities based on the demographic, socioeconomic, accessibility to the health
89
facilities, prevalence of the pulmonary and cardiac disorders, etc. (Khanal et al., 2020). The
90
complex spatial and temporal epidemiology of COVID-19 due to rapid changes in human
91
population dynamics and its demographic and environmental drivers challenges in its efficient
92
control (Alkhamis et al., 2020). One of the important drivers of the spreading of infectious diseases
93
is the human movement, tracking of which is using data sources such as public transportation (bus,
94
train, and flight), social-media data, and mobile-phone data could be critical for the prediction of
95
virus transmission, the identification of risk area, and decisions about control measure (Zhou et
96
al., 2020). Therefore, it is important to analyze the spatiotemporal pattern of the COVID-19
97
outbreak in the light of human dynamics and non-pharmaceutical interventions. 98 4 4 . CC-BY 4.0 International license
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is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
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It is made available under a The use of spatiotemporal analytical tools for rapid risk-based surveillance can offer valuable near-
99
real-time insights into the severity of pandemic spread as well as the effectiveness of intervention
100
measures and can aid decision making, planning and community action (Franch-Pardo et al.,
101
2020). 1. Introduction
40 (which was not certified by peer review)
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It is made available under a 2.1 Data Source
127 This study was conducted covering the entire 77 districts of Nepal (Fig. 1) using three different
128
datasets. COVID-19 cases reported to the Ministry of Health and Population (MoHP), Government
129
of Nepal was the first of its kind. This dataset contains daily COVID-19 positive cases, death and
130
recovery aggregated at district. The COVID-19 were tested using the RT-PCR in various lab
131
distributed across the country. We extracted reported positive case and joined them with district
132
shapefile collected from Department of Survey, Government of Nepal. In addition, we obtained
133
gridded population dataset in 100-meter spatial resolution for the year of 2020 from the worldpop
134
geoportal (https://www.worldpop.org/). We summarized it for each district using the zonal
135
statistics tool of ARC GIS which was used later as a base population to assess underlying risk to
136
COVID-19 in the district. 137 138
Figure 1. Location map of Nepal showing the seven provinces and 77 districts. 139 Figure 1. Location map of Nepal showing the seven provinces and 77 districts. 2.2 Data Analysis
140 1. Introduction
40 Such analyses have been proven to be effective to identify the extent and impact of the
102
pandemic and formulate the intervention strategies in many countries including Bangladesh
103
(Masrur et al., 2020), China (Kang et al., 2020; Xie et al., 2020), Italy (Gatto et al., 2020;
104
Martellucci et al., 2020), Spain (Santamaría and Hortal 2021), USA (Cordes and Castro 2020;
105
Mollalo et al., 2020; Sun et al. 2020), etc. Additionally, findings of such analyses could be decisive
106
for early identification of high-risk groups for disease transmission and efficient deployment of
107
resources for developing economies like Nepal. The spatial scan statistic (SaTScan) is a widely
108
used method for geographical disease surveillance that detects and determines statistical
109
significance of geographical cluster without having to prespecify the cluster size or location. It
110
applies a moving circular window on the map, centered on each of many possible grid points
111
positioned throughout the study region and creates many thousands of distinct geographical circles,
112
each being the possible candidate cluster. The retrospective analysis on the SaTScan can identify
113
all past and current significant clustering events throughout the study period (Kulldorff, 1997). 114 The epidemic crisis management demands estimation of the actual effects of interventions taken
115
not only to make rapid adjustments but also to adapt short-term forecasts (Dehning et al., 2020). 116
Nepal offers an opportunity to assess the impact of non-pharmaceutical interventions on COVID-
117
19 that could be rolled out in resource-limited settings in other countries. The main objective of
118
this study was to assess spatiotemporal dynamics of COVID-19 on the context of various
119
restrictions imposed as preventive measures to contain the disease transmission. We explored
120
active and emerging disease clusters using the prospective space–time scanning (Desjardins et al.,
121
2020; Masrur et al., 2020) for two time periods, 23 January - 21 July, and 23 January - 31 August
122
2020 taking the cutoff date of 21 July. In addition, we investigated biweekly space-time
123
propagation of transmission for locating risk and newly emerged clusters along the timeline
124
accounting two weeks incubation period of the SARS-CoV-2. 125 5 5 . CC-BY 4.0 International license
It is made available under a
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140 We used geospatial analytics to characterize spatiotemporal dynamics of COVID-19 in Nepal. We
141
divided study period in two parts based on the national level lockdown employed by the federal
142 We used geospatial analytics to characterize spatiotemporal dynamics of COVID-19 in Nepal. We
141
divided study period in two parts based on the national level lockdown employed by the federal
142 6 . CC-BY 4.0 International license
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is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
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It is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
eprint
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medRxiv preprint government of Nepal. The country was shut down from 23 March 2020, which was lifted five
143
months later in 21 July 2020. We visualized the temporal dynamics using epidemic curve and
144
several restrictions enforced in different spatiotemporal scales. The spatial distribution of
145
cumulative cases of reported COVID-19 and incidence rate before and after the cutoff date of 21
146
July 2020 is presented through choropleth mapping technique. 147 To quantify spatiotemporal dynamics of the epidemics, we used the SatScan approach (Kulldorff,
148
1997) using the SaTScan version 9.6 (Kulldorff, 2018). The SaTScan statistics has been used
149
widely to identify significant spatial/ temporal and spatiotemporal disease clusters including
150
COVID-19 in different region of the world (Acharya et al., 2016; Desjardins et al., 2020; Masrur
151
et al., 2020). The SaTScan scans across time and/or space using moving window to identify
152
possible clusters by comparing the number of observed cases and expected cases assuming random
153
distribution inside the window at each location. Scanning window is a time interval for purely
154
temporal scan, a circle or ellipse in spatial scan and a cylinder in space-time scan where base of a
155
cylinder represents space dimension and height represents the temporal dimension (Kulldorff,
156
2001; Kulldorff, 2018). 2.2 Data Analysis
140 CC-BY 4.0 International license
It is made available under a where, L(Z)= Log likelihood function for scanning window; L0 is the likelihood function for null
171
hypothesis; nZ is the number of reported cases within the window; µ(Z) is the number of expected
172
cases within the window Z; N is the total number of observed cases for the entire study areas
173
across all time periods; and µ(T) is the total number of expected cases in the study area across all
174
time periods. 175 where, L(Z)= Log likelihood function for scanning window; L0 is the likelihood function for null
171
hypothesis; nZ is the number of reported cases within the window; µ(Z) is the number of expected
172
cases within the window Z; N is the total number of observed cases for the entire study areas
173
across all time periods; and µ(T) is the total number of expected cases in the study area across all
174
time periods. 175 Due to the assumption of uniform relative risk (RR) across a cluster, multiple geographic units can
176
belong to significant space-time clusters. To avoid that assumption, relative risk for each spatial
177
unit that belongs to a cluster is computed. The relative risk is for each location belonging to a
178
cluster is calculated as (Liu et al., 2018; Hohl et al., 2020):
179 Due to the assumption of uniform relative risk (RR) across a cluster, multiple geographic units can
176
belong to significant space-time clusters. To avoid that assumption, relative risk for each spatial
177
unit that belongs to a cluster is computed. The relative risk is for each location belonging to a
178
cluster is calculated as (Liu et al., 2018; Hohl et al., 2020):
179 𝑅𝑅=
𝑐/𝑒
(𝐶−𝑐)/(𝐶−𝑒) … … … … . . (3) (3) where, ‘c’ is the total number reported cases, ‘e’ is the total number of expected cases, and ‘C’ is
181
the total number of observed cases in the entire study area. 182 where, ‘c’ is the total number reported cases, ‘e’ is the total number of expected cases, and ‘C’ is
181
the total number of observed cases in the entire study area. 2.2 Data Analysis
140 The null hypothesis states that there is no difference in risk between inside
157
and outside of the circle or cylinder while alternative hypothesis states the number of observed
158
cases exceeds the number of expected cases derived from null models with elevated risk within
159
the circle/cylinder. The expected number (μ) under the null hypothesis H0 is derived as follows
160
(Desjardins et al., 2020):
161 𝜇= 𝑝∗
𝐶
𝑃 … … … … … .. (1) 𝜇= 𝑝∗
𝐶
𝑃 … … … … … .. (1)
162 𝜇= 𝑝∗
𝐶
𝑃 … … … … … .. (1)
162 𝜇= 𝑝∗
𝐶
𝑃 … … … … … .. (1) where, 𝑝 is the population in a geographic area, C and P are the total number of reported cases and
163
the total estimated population, respectively. 164 where, 𝑝 is the population in a geographic area, C and P are the total number of reported cases and
163
the total estimated population, respectively. 164 The SaTScan also identifies secondary clusters in addition to the most likely cluster for spatial and
166
spatiotemporal scan, and orders them according to their likelihood ratio test. Equation (2) was used
167
for calculating maximum likelihood ratio that identified scanning windows with elevated risk
168
(Kulldorff, 2001). 169 𝐿(𝑍)
𝐿0
=
( 𝑛𝑧
𝜇(𝑧))𝑛𝑍 ( 𝑁−𝑛𝑧
𝑁−𝜇(𝑍)))𝑁−𝑛𝑍
( 𝑁
𝜇(𝑇))𝑁
… … … … … . . (2)
170 170 . (2) . CC-BY 4.0 International license
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medRxiv preprint . 2.2 Data Analysis
140 Results
199
3.1 General overview of the COVID-19 in Nepal
200
A total 39460 cases of COVID-19 were reported in Nepal as of 31 August 2020 out of 693,47
201
tests done by RT-PCR. Among the total infected people, 30,881 (78.25%) were males and 857
202
(21.74%) were females. The fatality rate and cured rate in Nepal due to COVID-19 were 0.6% an
203
54.3%, respectively. 204 2.2 Data Analysis
140 182 In this study, we chose prospective space time analysis to detect emerging or active space-time
183
clusters that are still occurring at the end of the study period based on the discrete Poisson model
184
(Kulldorff, 1997, 2001). We chose discrete Poisson probability model to account heterogeneous
185
distribution of COVID-19 transmission across the space and time (Kim and Castro 2020; Masrur
186
et al., 2020). The space-time scan statistic employs moving cylinders for potential space-time
187
clusters of COVID-19 cases. We performed this analysis on daily reported cases of COVID-19
188
aggregated on 77 districts. As we were interested to locate elevated risk zones to the COVID-19,
189
high rate was chosen for further analysis. We set the upper bounds to have a maximum spatial and
190
temporal scanning window size of 10% of the population at-risk to avoid extremely large clusters;
191
and 50% of the study period, respectively. We utilized Monte Carlo testing with 9999 replications
192
to assess the statistical significance of space-time clusters with default P of 0.05. 193 To understand the space-time propagation of the transmission we computed difference of relative
194
risk between two-study periods and also detected emerging clusters using with shorter temporal
195
scan through biweekly cumulative prospective scanning approach accounting two weeks
196
incubation period (Desjardins et al., 2020) for locating the risk and newly emerged high-risk areas
197
along the timeline. 198 8 8 . CC-BY 4.0 International license
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this version posted November 10, 2020. ;
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It is made available under a . CC-BY 4.0 International license
It is made available under a 3. 3.1 General overview of the COVID-19 in Nepal
200 A total 39460 cases of COVID-19 were reported in Nepal as of 31 August 2020 out of 693,472
201
tests done by RT-PCR. Among the total infected people, 30,881 (78.25%) were males and 8579
202
(21.74%) were females. The fatality rate and cured rate in Nepal due to COVID-19 were 0.6% and
203
54.3%, respectively. 204 205
Figure 2. Epidictic curve of COVID-19 cases in Nepal reported. The primary axis (left) is
206
cumulative count of reported cases and secondary axis (right) is daily reported count
207 205 Figure 2. Epidictic curve of COVID-19 cases in Nepal reported. The primary axis (left) is
206
cumulative count of reported cases and secondary axis (right) is daily reported count
207 The temporal dynamics of the epidemic is presented in the Fig. 2. The epidemic curve started
208
ascend only after third week of April, which was almost four months later the detection of the first
209
case in 23 January 2020. However, sporadically COVID-19 cases were detected from different
210
districts despite nationwide lockdown started on 24 March. From the third week of the May the
211
epidemic curve started to rise abruptly and the trend continued until the June last. In this period,
212
significantly higher number of migrant workers returned home from India. Once the number of
213
returns from India decreased slowly the positive case also shrunk rapidly. However, the number
214
of COVID-19 cases increased substantially again after lifting the nationwide lockdown in 21st July
215 9 . CC-BY 4.0 International license
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is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
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It is made available under a 2020. The rising trend continued in the later part of July and entire August despite the local level
216
restrictions enforced in different districts. 217 2020. 3.2 Emerging district level clusters: 23 January- 21 July 2020
234 Nine statistically significant emerging space-time clusters of COVID-19 were detected at district
235
level between January 23 and July 21, 2020 in Nepal. Table 1 provides the characteristics of these
236
clusters with varying size, relative risk and onset time and duration. The most likely cluster i.e. 237
cluster 1 and other secondary clusters; 3, 4 and 5 emerged from June 12 while cluster 6, 7 and 8
238
lately emerged almost at the end of study period. The relative risk of these clusters also varied
239
significantly. For example, the RR of cluster 1 (most likely cluster) was 16.95 while those of the
240
cluster 2 and 3 were 9.87 and 9.81, respectively. Cluster 5, 8 and 9 were low risk clusters with RR
241
less than 3.00. 242 Table 1. District level emerging space-time clusters of COVID-19 from 23 January to 21 July
243
2020 in Nepal (RR: relative risk). All results are statistically significant at P<0.001. 244 Table 1. District level emerging space-time clusters of COVID-19 from 23 January to 21 July
243
2020 in Nepal (RR: relative risk). All results are statistically significant at P<0.001. 244 Cluster
Radius
Start Date
End Date
# Districts
Observed
Expected
RR
1
82.89
2020/6/12
2020/7/21
11
4574
361.58
16.95
2
145.18
2020/6/14
2020/7/21
19
2884
344.79
9.87
3
53.05
2020/6/12
2020/7/21
5
2843
341.13
9.81
4
0.00
2020/6/12
2020/7/21
1
307
67.52
4.61
5
44.18
2020/6/12
2020/7/21
3
386
222.55
1.75
6
27.29
2020/7/21
2020/7/21
3
36
8.08
4.46
7
51.75
2020/7/16
2020/7/21
2
22
2.97
7.40
8
51.33
2020/7/20
2020/7/21
4
81
36.07
2.25
9
0.00
2020/6/23
2020/7/21
1
50
20.77
2.41 Figure 4a shows the locations and spatial patterns of the nine emerging space-time clusters of
245
COVID-19 at the district level in Nepal between January 23 and July 23, 2020. Cluster 1 contains
246
11 districts of Karnali and Sudoorpaschim province. Cluster 2, the first secondary cluster, is the
247
largest cluster with 145 km radius and covers 19 districts of western Nepal. Cluster 3, 5 and 6 were
248
smaller compared to the first two clusters with radius 53, 44 and 27 km and number of districts
249
inside the clusters were 5, 3 and 3, respectively. Cluster 4 and 9 were single district cluster of
250
Saptari and Sindhupalchok, correspondingly. 3.1 General overview of the COVID-19 in Nepal
200 (which was not certified by peer review)
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It is made available under a 3.1 General overview of the COVID-19 in Nepal
200 The rising trend continued in the later part of July and entire August despite the local level
216
restrictions enforced in different districts. 217 Spatial distribution of cumulative cases and district level incidence rate of COVID-19 reported
218
before and after the cutoff date is presented in the Figures 3a and 3b, respectively. By July 21, the
219
epidemic was more intense in several western districts such as Dailekh, Doti, Achham and Bajura
220
and low lying Tarai districts bordering with India including Rautahat, Kailali, Mahotari and
221
Sarlahi, although it was already spread across the country. Spatial pattern of the incidence rate was
222
slightly different than the patterns of total cumulative cases which determined by the population
223
distribution. Bajura, Doti, Achham, Dailekh were districts with higher incidence. Higher incidence
224
rates were also reported from Palpa, Parbat and Arghaghkanchi districts of Gandaki province. By
225
August 31, the epidemic had become more intense across the country (Figure 3b). The highest
226
number of cases were reported from Kathmandu followed by Parsa, Sarlahi, Rautahat while the
227
least cases were reported from Mustang, Manang and Humla, respectively. In the same period, the
228
highest incidence was observed in Doti, followed by Bajura and Dailekh, where the incidence was
229
above 30/1000. 230 231
Figure 3. Spatial distribution of cumulative number and rate of incidence/10000 of COVID-19
232
cases from a) 23 January – 21 July and b) 23 January – 31 August 2020
233 Figure 3. Spatial distribution of cumulative number and rate of incidence/10000 of COVID-19
232
cases from a) 23 January – 21 July and b) 23 January – 31 August 2020
233 Figure 3. Spatial distribution of cumulative number and rate of incidence/10000 of COVID-19
232
cases from a) 23 January – 21 July and b) 23 January – 31 August 2020
233 10 . CC-BY 4.0 International license
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It is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 3.2 Emerging district level clusters: 23 January- 21 July 2020
234 There were 28 out of 77 districts outside these 9-
251
emerging clusters having RR=0; at the time of this analysis, they were non emerging COVID-19
252
risk districts. 253 Figure 4a shows the locations and spatial patterns of the nine emerging space-time clusters of
245
COVID-19 at the district level in Nepal between January 23 and July 23, 2020. Cluster 1 contains
246
11 districts of Karnali and Sudoorpaschim province. Cluster 2, the first secondary cluster, is the
247
largest cluster with 145 km radius and covers 19 districts of western Nepal. Cluster 3, 5 and 6 were
248
smaller compared to the first two clusters with radius 53, 44 and 27 km and number of districts
249
inside the clusters were 5, 3 and 3, respectively. Cluster 4 and 9 were single district cluster of
250
Saptari and Sindhupalchok, correspondingly. There were 28 out of 77 districts outside these 9-
251
emerging clusters having RR=0; at the time of this analysis, they were non emerging COVID-19
252
risk districts. 253 11 is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
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this version posted November 10, 2020. ;
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It is made available under a Figure 4. Spatial distribution of emerging space-time clusters of COVID-19 at District level. a)
From 23 January – 21 July 2020; and b) 23 January – 31 August 2020. 254
Figure 4. Spatial distribution of emerging space-time clusters of COVID-19
255
From 23 January – 21 July 2020; and b) 23 January – 31 August 2020. 256
3.3 Emerging district level clusters: 23 January- 31 August 2020
257 254 Figure 4. Spatial distribution of emerging space-time clusters of COVID-19 at District level. a)
255
From 23 January – 21 July 2020; and b) 23 January – 31 August 2020. 256 3.3 Emerging district level clusters: 23 January- 31 August 2020
257 267
Cluster
Radius
Start Date
End Date
# Districts
Observed
Expected
RR
1
51.33
2020/7/30
2020/8/31
4
3815
569.21
7.31
2
49.00
2020/8/8
2020/8/31
6
2469
320.65
8.15
3
0.00
2020/8/4
2020/8/31
1
1280
133.03
9.91
4
100.35
2020/7/21
2020/8/31
10
2829
742.70
4.03
5
18.13
2020/8/11
2020/8/31
2
971
94.53
10.51
6
55.39
2020/8/9
2020/8/31
4
1066
232.68
4.68
7
0.00
2020/7/21
2020/8/31
1
430
129.15
3.36
8
77.67
2020/7/21
2020/8/31
8
752
345.89
2.20
9
0.00
2020/8/29
2020/8/31
1
19
2.10
9.07
10
0.00
2020/8/27
2020/8/31
1
20
3.44
5.82 =10.5) had the highest relative risk followed by cluster 3, 2, and 1; while 4, 6, 7, and 9 were the
264
clusters with lower relative risk. 265 =10.5) had the highest relative risk followed by cluster 3, 2, and 1; while 4, 6, 7, and 9 were the
264
clusters with lower relative risk. 265 Table 2. District level emerging space-time clusters of COVID-19 from 23 January to 31 August
266
2020 in Nepal (RR: relative risk). All results are statistically significant at P<0.001. 267 Cluster
Radius
Start Date
End Date
# Districts
Observed
Expected
RR
1
51.33
2020/7/30
2020/8/31
4
3815
569.21
7.31
2
49.00
2020/8/8
2020/8/31
6
2469
320.65
8.15
3
0.00
2020/8/4
2020/8/31
1
1280
133.03
9.91
4
100.35
2020/7/21
2020/8/31
10
2829
742.70
4.03
5
18.13
2020/8/11
2020/8/31
2
971
94.53
10.51
6
55.39
2020/8/9
2020/8/31
4
1066
232.68
4.68
7
0.00
2020/7/21
2020/8/31
1
430
129.15
3.36
8
77.67
2020/7/21
2020/8/31
8
752
345.89
2.20
9
0.00
2020/8/29
2020/8/31
1
19
2.10
9.07
10
0.00
2020/8/27
2020/8/31
1
20
3.44
5.82 Fig. 4b illustrates extent and spatial distribution 10 emerging space-time clusters of COVID-19 at
268
the district level in Nepal between January 23 and August 31, 2020. Cluster 4 is the largest cluster
269
with 100 km radius, which contains 10 districts of Karnali and Sudoorpaschim province. The most
270
likely cluster (Cluster 1) was located in central Nepal covering Parsa, Makawnpur Bara and
271
Chitwan districts. Cluster 3, 7, 9 and 10 were single districts clusters located in the Eastern Nepal. 272
Figure 5 also elucidates the elevated risk of 38 districts lying inside these clusters with varying
273
risk level ranging from 2.2 (Cluster 8) to 10.51 (Cluster 5). Bhaktapur, Lalitpur and Sarlahi were
274
the districts with higher relative risk (RR> 10). 3.3 Emerging district level clusters: 23 January- 31 August 2020
257 The number of the districts with moderate relative
275
risk were 16 (RR= 5-10) while lower risk (RR= 1-5) were observed 19 districts. Other 39 districts
276
exhibited no elevated risk of exposure (RR = 0) to the COVID-19 infection. 277 Fig. 4b illustrates extent and spatial distribution 10 emerging space-time clusters of COVID-19 at
268
the district level in Nepal between January 23 and August 31, 2020. Cluster 4 is the largest cluster
269
with 100 km radius, which contains 10 districts of Karnali and Sudoorpaschim province. The most
270
likely cluster (Cluster 1) was located in central Nepal covering Parsa, Makawnpur Bara and
271
Chitwan districts. Cluster 3, 7, 9 and 10 were single districts clusters located in the Eastern Nepal. 272
Figure 5 also elucidates the elevated risk of 38 districts lying inside these clusters with varying
273
risk level ranging from 2.2 (Cluster 8) to 10.51 (Cluster 5). Bhaktapur, Lalitpur and Sarlahi were
274
the districts with higher relative risk (RR> 10). The number of the districts with moderate relative
275
risk were 16 (RR= 5-10) while lower risk (RR= 1-5) were observed 19 districts. Other 39 districts
276
exhibited no elevated risk of exposure (RR = 0) to the COVID-19 infection. 277 3.3 Emerging district level clusters: 23 January- 31 August 2020
257 Ten statistically significant emerging space-time clusters were detected between 23 January and
258
31 August 2020. Table 2 summarizes the characteristics of these cluster in terms of size, onset
259
time, duration and relative risk level. Clusters 4, 7, 8 emerged from July 21 and persisted till the
260
end of the study period while cluster 9 and 10 were emerged lately and persisted only for few days. 261
The cluster 1, which is the most likely cluster, emerged on June 30 while clusters 2 and 3 arose on
262
first week of August. Relative risk also varied significantly among these clusters. Cluster 5 (RR
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It is made available under a =10.5) had the highest relative risk followed by cluster 3, 2, and 1; while 4, 6, 7, and 9 were the
264
clusters with lower relative risk. 265
Table 2. District level emerging space-time clusters of COVID-19 from 23 January to 31 August
266
2020 in Nepal (RR: relative risk). All results are statistically significant at P<0.001. 3.4 Progression of relative risk of COVID-19 in Nepal
278 The changing patterns of relative risk (RR) over two emerging periods have been shown in the
279
Figure 5. An abrupt reduction of RR was observed in 19 districts of which most of the districts
280
belonged to the cluster 1 and cluster 2 during 23 January- 21 July. A rapid rise of RR (>5) also
281
noticed in 8 districts including Dailekh, Arghakhanchi, Baitadi, Dadeldhura, Rautahat, Parbat,
282
Gulmi and Mahottari while moderate rise and fall in RR was observed in 11 and 12 districts
283
symbolized by light red and light green, respectively. Some districts with RR = 0 over the two
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It is made available under a periods indicated no difference in relative risk which were regarded as “non-emerging” COVID-
285
19 districts. However, it should be noted that these districts had also experienced the outbreak
286
during the study period. Some of them became emerging clusters (with elevated RR) at some point
287
in time when scanned over a shorter temporal window (Figure 6). 288 289
Figure 5. Changes in relative risk (RR) of COVID-19 between two emerging periods 23 January
290
– 21 July and 23 January – 31 August 2020 in Nepal
291 Figure 5. Changes in relative risk (RR) of COVID-19 between two emerging periods 23 January
290
– 21 July and 23 January – 31 August 2020 in Nepal
291 Biweekly spatiotemporal variations of COVID-19 transmission at district level in Nepal from
293
January 23-August 31, 2020 have been shown in the Figure 6. This short temporal window
294
scanning enabled us to assess space-time progression of COVID-19 by locating dispersing risk
295
and newly emerged high-risk areas along the timeline. 296 14 . 3.4 Progression of relative risk of COVID-19 in Nepal
278 (which was not certified by peer review)
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It is made available under a Kalikot and Dailekh districts became hotspots (RR>150) while the risk of Rautahat and Kapilvastu
312
was also significantly high (RR>50). In the later weeks, elevated risk of COVID-19 further
313
expanded but the unexpectedly high RR was more stabilize. From the beginning of July, the
314
districts with higher relative risk further expanded on the proximity of Dailekh and Kailkot and
315
the vicinity of Palpa and Syangja which continue until the first week of August. By the August 31,
316
which is the last date of study period, the elevated RR was expanded to 56 districts. 317 3.4 Progression of relative risk of COVID-19 in Nepal
278 CC-BY 4.0 International license
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Figure 6. Space-time propagation of COVID-19 relative risk in different weeks in Nepal
299 different weeks in Nepal Figure 6. Space-time propagation of COVID-19 relative risk in different weeks in Nepal
299 The elevated risk of COVID-19 transmission was observed in 21 April for the first time in Nepal
300
although sporadic cases were reported from different districts since its first report in 23 January
301
2020. In this time, only six districts had elevated risk with significant variation in RR. The RR of
302
Udayapur was extremely high (RR=297) followed by Jhapa (RR=68). The relative risk of Baglung,
303
Chitawan, Kailali, Parsa and Nawalparasi East were moderately low (RR<20). This was the first
304
cluster level transmission (14 cases) of COVID-19 suspected in Udayapur district of Nepal with
305
migration history of the infected people from India. Two weeks later Banke and Parsa districts
306
bordering India became hotspots while the risk of transmission in Udaypur persisted continuously
307
(RR=86). By the May 21, the elevated risk expanded in 21 districts with significant spatial
308
variation on RR. Dhankuta and Jhapa emerged as new hotspots while elevated risk of Banke
309
remained constantly high (RR= 100). The COVID-19 transmission further spread in the next two
310
weeks, till 07 June and the number of elevated risk (RR>1) districts reached 32. At this time
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318 (which was not certified by peer review)
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It is made available under a people from those areas without testing increased the cases in particular areas of Nepal. 342
Additionally, those districts of elevated incidences are also characterized by higher population
343
density, lower literacy rates, higher poverty, and in turn preeminent vulnerability to the epidemics
344
(Khanal et al., 2020). Population density is one of the important factors in shaping the spatial
345
pattern of the epidemics as the crowded cities worldwide could experience more prolonged
346
epidemics (Rader et al., 2020). Similar results were observed in China where the population inflow
347
from Wuhan, the epicenter of the pandemic and the strength of economic connection were the
348
main factors affecting the epidemic spread (Xie et al., 2020). 349 The spatial analysis and predictive modelling of the evolution of the COVID-19 is important to
350
interpret the epidemic phenomenon (Franch-Pardo et al., 2020). Our prospective space–time
351
scanning analysis revealed nine major emerging clusters for the first phase of the study (23 January
352
- 21 July). The most likely cluster, the C1, emerged on 12 June that included 11 districts from
353
Karnali and Sudoorpaschim provinces. These districts have higher poverty and majority of the
354
households have one or more members of the family working as low-skilled manpower in Indian
355
cities like Mumbai, Delhi, and others (Khanal et al., 2020). The first cluster of COVID-19 observed
356
was the consequence of infected returnees from India. Despite the nationwide lockdown imposed
357
and borders sealed, people from India used resumed Indian railway services after the middle of
358
May and returned back Nepal crossing the open border without taking proper precautions and in
359
many cases violating isolation and quarantine protocols of federal and local governments (Chalise,
360
2020). Clusters 1-5 began on the second week of June and persisted till 21 July that were all
361
associated to the inflow of people from India. 4. Discussion
318 COVID-19 pandemic has greatly affected the south-Asian countries including Nepal where
319
number of cases are still (first week of October) increasing exponentially (MoHP, 2020). This
320
study employed prospective space-time scan statistics for identifying currently active or emerging
321
clusters of COVID-19 at the district level in Nepal, providing results at two distinct time periods
322
of differential intervention attempts. The prospective scanning is valuable surveillance tool in
323
monitoring disease outbreaks and locate active elevated risk of disease in space and time
324
(Desjardins et al., 2020). Our findings can be useful for rapidly monitoring evolving space-time
325
patterns of COVID-19 that will enable government and health officials to take appropriate time-
326
sensitive intervention by considering disease’s space-time diffusion pathways and potentially
327
prepare for future outbreaks of a highly contagious disease (Masrur et al., 2020). 328 After the first recorded positive case of COVID-19 on 21 January, there was a lag period of two
329
months for the very next case. However, the cases increased after the third week of the March and
330
continued to grow exponentially. The first ascend on the epidemic curve was observed after the
331
third week of April when Nepalese migrants working in India returned home after nationwide
332
lockdown there. The open border between the countries and surge of large number of returnees
333
made it impossible to regulate the movement and manage proper test and isolation. Therefore,
334
most of the cases were recorded from the districts of Province No 2 bordering India and that of
335
Sudoorpaschim province, large number of people from those districts are working in India since
336
long (Chalise 2020). Human mobility and control strategy determine the spatial spread of the
337
epidemics (Arimura et al., 2020; Drake et al., 2020; Kraemer et al., 2020; Rader et al., 2020; Zhou
338
et al., 2020). The areas close to the outbreak has a higher risk of contagion, especially in the initial
339
stage of infection (Carteni et al., 2020). Indian cities were severely affected of the COVID-19 since
340
early April (Ray et al., 2020; Tomar and Gupta 2020) and inflow of infected but asymptomatic
341 16 . CC-BY 4.0 International license
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318 It has signified the importance of social distancing
362
and movement restrictions in containing the epidemic. 363 The prospective space–time scanning analyses for wider temporal scale, i.e. from 23 January to 31
364
August revealed 10 emerging clusters. The Cluster 4 in the far-western lowland Nepal was the
365
largest cluster encompassing 10 districts with relative risk of 4.03, which is apparently the
366
continuation of the Cluster 1 of the previous time frame i.e. 23 January- 21 July 2020. The Cluster
367
5 with the highest relative risk of 10.5 included two districts of the capital city Kathmandu valley-
368
Bhaktapur and Lalitpur. The number of COVID-19 cases were much higher in the Kathmandu
369
district of the valley (MoHP, 2020); however, our analysis didn’t account a high relative risk for
370
it due to an enormous base population. When the Government of Nepal lifted the nationwide
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It is made available under a lockdown on 21 July, people from different districts rushed to the capital city and the number of
372
cases raised abruptly that developed a strong cluster (Cluster 5) with very high relative risk. 373
Epidemics in crowded cities disperse rapidly and have larger total attack rates than less populated
374
cities (Rader et al., 2020). To better understand the COVID-19 transmission dynamics, datasets on
375
patient’s travel and contact history need to be incorporated (Masrur et al., 2020), however, there
376
is no proper mechanism of tracking in Nepal. Therefore, the Kathmandu valley with more than
377
four million population within 665 km2 area is under severe risk of COVID-19 outbreak. 378 During nationwide lockdown imposed by the federal government of Nepal, all public places
379
remained shut down and strictly followed government directives. 5. Conclusion
403 The epidemic spread rate in Nepal has an evident spatial variation. Districts of Sudoorpaschim
404
province and Province No 2 bordering India experienced rapid transmission of the COVID-19
405
when the Nepalese migrants returned home in May/June. The unmanaged population inflow from
406
India crossing the sealed border had significant effects on the epidemic spread rate. The capital
407
city Kathmandu and Bharatpur where medical facilities are concentrated have become the highest-
408
risk active clusters since August. It is important to detect emerging clusters that would reveal more
409
updated space-time transmission dynamics of COVID-19 to better allocate resources and improve
410
decision-making as the outbreaks continue to grow. The purposive and time-bound movement
411
restriction appears to be the most important non-pharmaceutical intervention against the COVID-
412
19 for resource-scarce countries with limited health care facilities. 413 4. Discussion
318 (which was not certified by peer review)
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It is made available under a Acknowledgement
414 We thank Ministry of Health and Population, Government of Nepal for providing the valuable data
415
on COVID-19 in Nepal. 416 We thank Ministry of Health and Population, Government of Nepal for providing the valuable data
415
on COVID-19 in Nepal. 416 Author Contributions
417 BKA, LK and HL conceptualized the study. BKA, LK, SKA, SP, BKN and BKP collected and
418
processed data. BKA and LK analyzed the data and prepared the manuscript. ASMM, ZR and YY
419
helped in manuscript improvement. HL supervised the overall study and provided multiple
420
revisions. All authors read and approved the final manuscript. 421 BKA, LK and HL conceptualized the study. BKA, LK, SKA, SP, BKN and BKP collected and
418
processed data. BKA and LK analyzed the data and prepared the manuscript. ASMM, ZR and YY
419
helped in manuscript improvement. HL supervised the overall study and provided multiple
420
revisions. All authors read and approved the final manuscript. 421 Conflicts of Interest
422 The authors declare no conflict of interest. 423 The authors declare no conflict of interest. 423 4. Discussion
318 Many local municipal
380
governments also efficiently implemented the closure, isolation, tracking and quarantine; those
381
which failed to do so experienced initial community outbreaks. Therefore, till June 2020,
382
community level transmission was localized in few districts such as Udaypur, Parsa, and Banke
383
(Figure 6). However, since July, many districts of Lumbini and Sudoorpaschim provinces
384
experienced a high relative risk. Major reason behind such was unpreparedness of the federal
385
government (Thakur et al., 2020) which failed to seal the southern border that imported hundreds
386
of COVID-19 positive people from India, and could not properly test, track and isolate the
387
individuals rescued from the Arabian countries. Another important shortcoming was the use of
388
less reliable and inefficient antibody-based diagnosis (the rapid diagnostic tools) (Bisoffi et al.,
389
2020; Ghaffari et al., 2020) emphasized in place of the antigen-based RT-PCR. By the end of
390
August, densely populated Kathmandu valley that had very few cases of COVID-19 for the first
391
five months of lockdown started having thousands of cases diagnosed every day. Centralization of
392
the health facilities in the capital city Kathmandu caused people to move into it for the diagnosis
393
and treatment of diseases including COVID-19. It is an established fact that people having
394
compromised immunity due to pulmonary and cardiovascular disorders are highly prone to the
395
COVID-19 infection (Fang et al., 2020; Zheng et al., 2020). Large number of old-age people
396
visiting hospital for medical checkup were found positive to the COVID-19 and many were
397
diagnosed positive only after death. Inefficient and inadequate intervention against the epidemic
398
has resulted a strong cluster within the Kathmandu valley and Bharatpur where medical facilities
399
are centralized but becoming short to contain the COVID-19. Therefore, together with medical
400
care, non-pharmaceutical interventions such as travel restrictions, tracking and isolation are
401
inevitable
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medRxiv preprint Table Titles
583 Table Titles
583
Table 3. District level emerging space-time clusters of COVID-19 from 23 January to 21 July
584
2020 in Nepal (RR: relative risk). All results are statistically significant at P<0.001. 585
Table 4. District level emerging space-time clusters of COVID-19 from 23 January to 31 August
586
2020 in Nepal (RR: relative risk). All results are statistically significant at P<0.001. 587
588
589
590 Table 3. District level emerging space-time clusters of COVID-19 from 23 January to 21 July
584
2020 in Nepal (RR: relative risk). All results are statistically significant at P<0.001. 585 Table 3. District level emerging space-time clusters of COVID-19 from 23 January to 21 July
584
2020 in Nepal (RR: relative risk). All results are statistically significant at P<0.001. 585 Table 4. District level emerging space-time clusters of COVID-19 from 23 January to 31 August
86
2020 in Nepal (RR: relative risk). All results are statistically significant at P<0.001. 87 Table 4. District level emerging space-time clusters of COVID-19 from 23 January to 31 August
586
2020 in Nepal (RR: relative risk). All results are statistically significant at P<0.001. 587 590 26 . CC-BY 4.0 International license
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Simple, readily available clinical indices predict early and late mortality among patients with ANCA-associated vasculitis
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BMC nephrology
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© The Author(s). 2017 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: The early identification of patients with ANCA-associated vasculitis (AAV) who are at increased risk for
inferior clinical outcome at the time of diagnosis might help to optimize the immunosuppressive therapy. In this
study we wanted to determine the predictive value of simple clinical characteristics, which may be applicable for
early risk-stratification of patients with AAV. Methods: We retrospectively analyzed the outcome of 101 consecutive patients with AAV receiving a protocolized
immunosuppressive therapy. Baseline Birmingham Vasculitis Activity Score (BVAS) and non-vasculitic comorbidities
were computed, then predictors of early (<90 days) and late (>90 days) mortality, infectious death, relapse and end
stage kidney disease (ESKD) were evaluated. Results: The baseline comorbidity score independently predicted early mortality (HR 1.622, CI 1.006–2.614), and
showed association with infectious mortality (HR 2.056, CI 1.247–3.392). Patients with BVAS at or above median
(=21) had worse early mortality in univariable analysis (HR 3.57, CI 1.039–12.243) (p = 0.031), and had more frequent
relapses (p = 0.01) compared to patients with BVAS below median. Conclusions: Assessing baseline comorbidities, beside clinical indices characterizing the severity and extension of
AAV, might help clinicians in risk-stratification of patients. Future prospective studies are needed to investigate
whether therapies based on risk-stratification could improve both short term and long term survival. Keywords: ANCA, BVAS, Comorbidity, Immunosuppression, Outcome, Vasculitis Keywords: ANCA, BVAS, Comorbidity, Immunosuppression, Outcome, Vasculitis consider when planning the treatment schedule of the indi-
vidual patient at the time of diagnosis. Haris et al. BMC Nephrology (2017) 18:76
DOI 10.1186/s12882-017-0491-z Haris et al. BMC Nephrology (2017) 18:76
DOI 10.1186/s12882-017-0491-z * Correspondence: kokeny.gabor@med.semmelweis-univ.hu
3Institute of Pathophysiology, Semmelweis University, 4 Nagyvárad tér,
Budapest 1089, Hungary
Full list of author information is available at the end of the article Simple, readily available clinical indices
predict early and late mortality among
patients with ANCA-associated vasculitis Ágnes Haris1, Kálmán Polner1, József Arányi1, Henrik Braunitzer1, Ilona Kaszás2, László Rosivall3, Gábor Kökény3*
and István Mucsi4 Background The outcome of ANCA-associated vasculitis (AAV) has
improved significantly since the introduction of im-
munosuppressive therapy. On the other hand, both the
disease and the cytotoxic treatment are associated with
considerable morbidity and mortality [1, 2]. Ideally, pa-
tients should receive a treatment specifically tailored to
the severity of their disease. Other factors, however, such as
age, the extent of organ involvement and also baseline co-
morbidities may influence the outcome [3–7]. Therefore, in
order to optimize the intensity of immunosuppression and
to optimize outcomes, these factors would be important to The Birmingham Vasculitis Activity Score (BVAS) is a
reliable tool to estimate the severity and extent of the
disease [8]. However, studies investigating it’s predictive
value on survival reported conflicting results [9–13]. Be-
side the activity of AAV, patients may have comorbid
conditions, that may also have an impact on their sur-
vival. Comorbidity scores are useful clinical tools for
risk-stratification of patients with chronic disorders, and
the role of comorbid conditions has been emphasized in
the mortality of dialysis patients [14, 15]. Therefore, it
seems reasonable to include comorbidity assessment in
the initial risk-startification at the time of presentation
with AAV. * Correspondence: kokeny.gabor@med.semmelweis-univ.hu
3Institute of Pathophysiology, Semmelweis University, 4 Nagyvárad tér,
Budapest 1089, Hungary
Full list of author information is available at the end of the article Haris et al. BMC Nephrology (2017) 18:76 Page 2 of 7 Page 2 of 7 The aim of our study was to determine if simple clin-
ical and laboratory characteristics, readily available at
the time of diagnosis would predict mortality in patients
with BVAS. We assessed BVAS, and utilized a simplified
score by computing the most important baseline non-
vasculitic comorbidities for risk-stratification of patients
with AAV. the results were comparable to the findings in the whole
cohort. After twelve months azathioprine was introduced,
accompanied by 4 mg methylprednisolone given daily or
every other day as long-term maintenance therapy, at the
discretion of the attending nephrologist. In case of relapse, the induction immunosuppressive
regime was repeated. Remission was defined as disappearance of clinical dis-
ease activity and stabilization or improvement of the kidney
function. Resolution of hematuria was also criteria for re-
mission, but persistent proteinuria was considered as the
consequence of glomerular damage. In patients who
remained dialysis dependent we considered remission if the
extrarenal manifestations and the hematuria completely
ceased. Methods All consecutive patients, diagnosed with AAV at our
nephrology center between January, 1998 and June, 2013
were considered for this study. One patient, who died
within the first month, and 3, who were lost to follow-
up were excluded. Last follow-up was the date of death
or the end of study (December 31, 2013). Patients who
survived longer than the 8 years of follow-up (n = 15)
were censored at that time. The diagnosis of necrotizing small vessel vasculitis was
defined according to the criteria of Chapel Hill consensus
conference [16, 17], by clinical presentation compatible
with AAV, positive ANCA serology and/or kidney biopsy. Histological result confirmed the presence of crescentic/
necrotizing glomerulonephritis in all but 10 subjects, in
whom renal biopsy was not performed either because of
life threatening condition or due to refusal by the patients. All these 10 patients were ANCA positive. Estimated GFR
(eGFR) was computed with CKD-EPI equation [18], and
BVAS (version 3) was calculated by scoring symptoms in
9 organ systems (general, cutaneous, mucus membranes/
eyes, ENT, chest, cardiovascular, abdominal, renal, and
nervous system) at admission [8] (Evaluelogix software by
EPS Research Ltd). Baseline comorbidity score was
assessed by determining conditions that had been present
before the AAV, namely history of myocardial infarction,
congestive heart failure, peripheral vascular disease, cere-
brovascular disease, chronic pulmonary disease, peptic
ulcer disease, liver disease, diabetes or malignancy. Scores
were given 0 if no comorbidity, 1 if a single comorbidity, 2
if two or more comorbidities existed. The main exposure variables were the comorbidity
score (the sum of comorbidities at the time of admis-
sion) and the BVAS score (categorized as below or above
median [median = 21] score). The primary end points were all cause early (<90 days)
and late (>90 days) mortality. Secondary end points con-
sisted of deaths due to infections, rate of relapse and
end stage kidney disease (ESKD). Statistical analyses were performed using SPSS 20.0
(IBM, Chicago, IL) and STATA MP version 12 (Stata
Corporation, College Station, TX). Variables were re-
ported as mean (SD) or median and range, comparison
between
groups
was
analyzed
by
Student’s
t-tests,
Mann–Whitney U tests or χ2 tests, as appropriate. Mor-
tality risk was calculated by Kaplan-Meier method, and
log-rank tests to compare groups. Predictors of death
were evaluated separately for early (<90 days) and late
(>90 days) mortality. Background Relapse was defined as recurrence of presenting
symptoms or appearance of a new organ involvement at-
tributable to AAV. Those, in whom remission could not be
achieved, who died due to active vasculitis, or had low
grade of persistent “grumbling disease” were considered as
treatment resistant patients. Results Baseline data of the 101 individuals are presented in
Table 1. Subjects with BVAS at or above median (median
BVAS = 21) had lower Hgb (p = 0.017), more c-ANCA
positivity (p = 0.024), and needed HD on admission more
often (p = 0.012), compared to the individuals with BVAS
below median. Treatment protocol was strictly followed with only few
exceptions, as excluded iv MP pulses in 1 and 2 patients
and excluded CYC boluses in 3 and 1 patients in the
BVAS at or above and below median groups, respect-
ively; CYC was administered orally in 2 patients in the
BVAS below median group. Subjects with BVAS at or
above median got higher dose of pulse MP (p = 0.002)
compared to the individuals with BVAS below median,
but the dose of CYC and the cumulative dose of MP did
not differ between the groups (Table 1). The median survival in the study sample was 1877
(95%CI 753–2246) days. Mortality during the first year
was 33%. Nineteen patients died within the first 90 days
(“early mortality”), and 41 after the 90th day of follow-up
(“late mortality”). The cumulative probability of survival
was 0.441 (95%CI 0.231–0.633) versus 0.233 (95%CI
0.126–0.359) (p = 0.028) in patients with a BVAS score
below versus at or above median, respectively (Fig. 1). The cumulative probability of early (within 90 days after
diagnosis) survival was also worse in patients with
higher BVAS: 0.921 (95%CI 0.775–0.974) versus 0.746
(95%CI 0.619–0.836) (p = 0.031). Early mortality was also
predicted by baseline comorbidity score, albumin, CRP
and HD requirement on admission in urivariable Cox
regression analysis (Table 2). In a multivariable model
adjusted for BVAS, age, serum albumin, ANCA type and
HD
requirement
on
admission
comorbidity
score
remained a significant predictor for early mortality (HR
1.622, CI 1.006–2.614, p = 0.047) (Table 3). Forty relapses occurred in 24 patients, 10 of them ex-
perienced 2–4 relapses. The proportion of patients with
relapses was 30% in the BVAS at or above median and
13% in the BVAS below median group (p = 0.052). There
was significant difference in the number of relapses
between the subgroups with BVAS at or above and
below median (34 relapses in 63 patients vs. 6 relapses
in 38 patients, p = 0.01). Methods Patients with BVAS below and at or above median
were compared. Although the relatively small number of
events limited multivariable analyses [19], for this pur-
pose those variables were selected, that were considered
important predictors of outcomes of AAV based on clin-
ical experience or the results of the univariable analyses. Patients received protocolized therapy during the entire
observational period: 500–1000 mg intravenous (iv) metly-
prednisolone (MP) for three consecutive days, followed by
1 mg/kg/day per os for one month, then daily 48 mg in the
second, 36 mg in the third, 24 mg in the fourth, 16 then
12 mg in the fifth, and 8 then 4 mg in the sixth months,
continued with the maintenance dose of 4 mg/day, and
10 mg/kg iv bolus cyclophosphamide monthly for six
months, repeated at months 9 and 12. For subjects older
than 65 years the dose of immunosuppressive medications
was decreased by 15%, and for older than 70 years by 20%,
but the CYC dose was not modified by the glomerular fil-
tration rate. In 92 patients five plasmapheresis sessions
were also performed. Eighty-six patients followed the
protocol strictly. When we analyzed their data separately, Multivariable models were sequentially adjusted for
age, serum albumin, HD dependency on admission, and
ANCA type (negative, p- or c-ANCA). Serum CRP was
not used in the multivariable models due to the small
number of events and also because of its strong correl-
ation with serum albumin. Logistic regression models were used to analyze the
association between exposure variables and relapse,
since we considered all relapses for these analyses and
we did not consider the time to events. Page 3 of 7 Haris et al. BMC Nephrology (2017) 18:76 Page 3 of 7 death was unknown in 10 additional cases (1 early, 9 late
mortality), and late malignancy was responsible for one
death. The following types of infections were documented:
bacterial and fungal respiratory tract infections, pulmon-
ary abscess, cerebral abscess, sepsis, disseminated herpes
zoster. Both comorbidity and BVAS predicted infectious
mortality (HR 2.191, 95%CI 1.486–3.231; HR 3.792,
95%CI 1.111–12.949, respectively) in univariable models. The predictive value of comorbidity and BVAS remained
significant after adjustment for age, serum albumin,
ANCA types, and HD dependency (HR 2.056, 95%CI
1.247–3.392; HR 5.079, 95%CI 1.396–18.480, respectively). By
induction
immunosuppression
remission
was
achieved in all but one patient, who survived more than
90 days (81 patients, 80%). Results Although BVAS showed associ-
ation with relapse in univariable logistic regression
model (OR = 1.130 CI 1.028–1.243), after correcting for
the type of ANCA (c-ANCA versus p-ANCA), BVAS
was not a significant predictor of relapse any more. Late (>90 days after diagnosis) mortality was predicted
by age, comorbidity score and HD requirement on ad-
mission in univariable analysis (Table 4). In the most
fully adjusted model adjusted for age, serum albumin,
HD dependency on admission and ANCA type, comor-
bidity was not a significant predictor any more. In this
model, however, BVAS independently predicted all cause
late mortality (HR 2.408, 95%CI 1.081–5.362, Table 5). Methods On the long term, ESKD de-
veloped in 3 patients who had not required dialysis at
diagnosis, but suffered renal failure likely due to low
grade persistent disease activity. Thirty-seven patients
remained dialysis dependent at study end. Serum cre-
atinine and eGFR in patients who were off dialysis at the
end of follow-up (n = 64) were 168 umol/l (83–434) and
33 ml/min (11–88), respectively. In those, who had
BVAS at or above median on admission, serum creatin-
ine at the end of follow-up was significantly higher (191
umol/l (88–418)), compared to patients with BVAS
below median (143 umol/l (83–434), p = 0.041). The
corresponding eGFR values were 26 and 38 ml/min
(11–75 and 12–88, p = 0.092, respectively). Frequency
of long term HD dependency in patients with BVAS at
or above and below median did not differ significantly. Results are expressed as hazard ratios (HRs) with 95%
confidence intervals (CIs) and p values. All tests were
two-tailed, unadjusted for multiple comparisons, and p
values of < 0.05 were considered significant. Discussion The main result of our analysis is that in AAV patients
with predominant renal and pulmonary involvement,
comorbidity score independently predicted short term
survival. It also proved to be a predictor of infectious mor-
tality. On protocolized immunosuppressive therapy, pa-
tients, who had high BVAS at baseline, had significantly
poorer short term survival and more frequent relapses
than subjects with lower than median score. When analyz-
ing early and late mortality separately, BVAS did not pre-
dict outcome in univariable analysis. Seven patients died of infections (37%), 4 of cardiovas-
cular diseases (21%) and 7 of AAV activity (37%) within
the first 90 days. Late mortality occurred from infections
in 13 (32%), cardiovascular diseases in 12 (29%), active
AAV during relapse in 6 patients (15%). Reason for Haris et al. Discussion BMC Nephrology (2017) 18:76 Page 4 of 7 Table 1 Demographics, baseline data and comorbidities at time of diagnosis (mean (SD) or median and range)
Variable
All patients
Patients
with BVAS ≥21
Patients
with BVAS < 21
p value
n of patients
101
63
38
Age (years)
61.4 (13)
60.2 (14)
63.3 (11)
0.237
Male/female
40/61
27/36
13/25
0.389
Time from first symptoms to diagnosis (months)
5.0 (1–36)
5.0 (1–24)
6.0 (1–36)
0.822
Hemoglobin (g%)
8.4 (1.4)
8.1 (1.3)
8.8 (1.5)
0.017
Erythrocyte sedimentation rate (mm/h)
98 (5–138)
98 (5–138)
99 (14–136)
0.762
Serum albumin (g/l)
31.3 (5.4)
30.8 (5.3)
32.0 (5.6)
0.285
CRP (mg/l)
29 (1–221)
40 (2–221)
24 (1–152)
0.054
Urinary protein excretion (mg/day)
1456 (38–8474)
1259 (38–8474)
1843 (184–7344)
0.143
Serum creatinine (umol/l)
554 (84–1904)
573 (146–1904)
428 (84–1722)
0.060
HD requirement on admission (n, %)
56 (55%)
41 (65%)
15 (40%)
0.012
BVAS
21 (11–34)
24 (21–34)
15 (11–20)
<0.001
p-/c-ANCA positivity (n)*
57/36
33/29
24/7
0.024
Anti-MPO level in p-ANCA positive patients (IU/ml)
67 (6–100)
67 (6–100)
70 (11–100)
0.807
Anti-PR3 level in c-ANCA positive patients (IU/ml)
100 (32–100)
100 (50–100)
82 (32–100)
0.354
Dose of iv pulse MP** mg/kg/bw
11.7 (4.1)
12.7 (4.2)
10.1 (3.3)
0.002
Dose of iv bolus CYC*** mg/kg/bw
9.7 (1.6)
9.8 (1.6)
9.5 (1.6)
0.371
Cumulative dose of MP (mg)
11640 (3006–32334)
11238 (3006–32334)
12332 (5076–26364)
0.621
Follow-up time (days)
963 (30–3000)
843 (30–3000)
1393 (51–3000)
0.231
Organ involvement n (%)
Renal
101 (100)
63 (100)
38 (100)
1.000
Respiratory tract
43 (43)
38 (60)
5 (13)
<0.001
Ear-throat-nose
39 (39)
33 (52)
6 (16)
<0.001
Musculoskeletal
55 (55)
33 (52)
22 (58)
0.590
Skin
16 (16)
9 (14)
7 (18)
0.581
Eyes
5 (5)
4 (6)
1 (3)
0.648
Gastrointestinal
9 (9)
9 (14)
0 (0)
0.013
Nervous system
17 (17)
14 (22)
3 (8)
0.062
Cardiovascular
5 (5)
5 (8)
0 (0)
0.154
Baseline comorbidities n (%)
963 (30–3000)
843 (30–3000)
1393 (51–3000)
0.231
History of:
Coronary artery disease
11 (11)
9 (14)
2 (5)
0.201
Congestive heart failure
5 (5)
4 (6)
1 (3)
0.648
Peripheral vascular dis. 1 (1)
1 (2)
0 (0)
1.000
Cerebrovascular disease
11 (11)
7 (11)
4 (11)
1.000
Chronic pulmonary dis. were ANCA negative, all of them had renal biopsy which proved the diagnosis of pauci-immune crescentic glomerulonephriti
rednisolone, administered for 98 patients ***CYC – cyclophosphamide, administered for 95 patients
Comorbidity scores were given 0 if no comorbidity, 1 if a single comorbidity, 2 if two or more comorbidities existed were ANCA negative, all of them had renal biopsy which proved the diagnosis of pauci-immune crescentic glomerulonephriti
rednisolone, administered for 98 patients
hosphamide, administered for 95 patients CYC
cyclophosphamide, administered for 95 patients
morbidity scores were given 0 if no comorbidity, 1 if a single comorbidity, 2 if two or more comorbidities existed *Eight patients were ANCA negative, all of them had renal biopsy which proved the diagnosis of pauci-immune crescentic g
**MP – methylprednisolone, administered for 98 patients hosphamide, administered for 95 patients
ores were given 0 if no comorbidity, 1 if a single comorbidity, 2 if two or more comorbidities existed y p
,
p
CYC – cyclophosphamide, administered for 95 patients nisolone, administered for 98 patients
phamide, administered for 95 patients *Eight patients were ANCA negative, all of them had renal biopsy which proved the diagnosis of pauci-immune crescentic glomerulonephritis
**MP – methylprednisolone, administered for 98 patients
***CYC – cyclophosphamide, administered for 95 patients
Comorbidity scores were given 0 if no comorbidity, 1 if a single comorbidity, 2 if two or more comorbidities existed administered for 98 patients
administered for 95 patients Discussion 16 (16)
9 (14)
7 (18)
0.581
Peptic ulcer disease
8 (8)
6 (10)
2 (5)
0.707
Liver disease
5 (5)
4 (6)
1 (3)
0.648
Diabetes mellitus
8 (8)
3 (5)
5 (13)
0.149
Malignancy
7 (7)
3 (5)
4 (11)
0.421
Number of patients with
0
50
30
20
0.862
1
30
19
11
2 or more comorbidity scores
21
14
7
*Eight patients were ANCA negative, all of them had renal biopsy which proved the diagnosis of pauci-immune crescentic glomerulonephritis
**MP
methylprednisolone administered for 98 patients eline data and comorbidities at time of diagnosis (mean (SD) or median and range) Page 5 of 7 Haris et al. BMC Nephrology (2017) 18:76 Page 5 of 7 Table 3 Comorbidity score predicts “early mortality” in
multivariable Cox regression analysis. Table shows the
parameters of the “comorbidity score” variable in different
models
HR
95% CI
P value
Model 1
1.707
1.176–2.477
0.005
Model 2
1.752
1.225–2.506
0.002
Model 3
1.694
1.072–2.677
0.024
Model 4
1.622
1.006–2.614
0.047
Model 1: comorbidity score
Model 2: Model 1 + BVAS median
Model 3: Model 2 + Age, serum albumin
Model 4: Model 3 + HD dependency on admission and ANCA type
(c-versus p-ANCA) Fig. 1 Kaplan-Meier survival curves of patients with BVAS at or
above and below median in the entire observation period (p = 0.028,
log-rank test) Table 3 Comorbidity score predicts “early mortality” in
multivariable Cox regression analysis. Table shows the
parameters of the “comorbidity score” variable in different
models Fig. 1 Kaplan-Meier survival curves of patients with BVAS at or
above and below median in the entire observation period (p = 0.028,
log-rank test) baseline BVAS of survivors and non-survivors; baseline
BVAS did not, but BVAS at 1 and 3 months predicted
survival [21]. The difference in the association between BVAS and out-
come in these cohorts and ours can be due to a variety of
factors. Event number, therefore statistical power, patient
selection, disease severity and treatment approach were
quite heterogeneous across these studies. It also seems im-
portant to differentiate early and late survival, as the hazard
of mortality is not proportional in these periods. We have
defined the timeframe of early death in 3 months, as risk of
severe complications of AAV, also intensity of immunosup-
pression are the highest during this period. Discussion BVAS, originally designed to standardize disease as-
sessment in AAV, shows good correlation with clinical
activity of the disease [8]. Flossmann et al. documented,
that BVAS was a significant predictor of mortality by
analyzing the data of patients recruited for randomized
controlled trials. Patients in that study were somewhat
different from the ones enrolled in ours, since the me-
dian BVAS was lower, renal function was less severely
compromised, and subjects with life-threatening pul-
monary hemorrhage were excluded [1]. On the contrary,
predictive value of BVAS was not found in several other
investigations. Bakoush and coworkers followed 83 pa-
tients; neither survival nor ESKD was predicted by BVAS
in their cohort, with less severe renal failure compared
to our patients [9]. In Japanese patients with MPO-
ANCA disease, no association was found between BVAS
and mortality during the two years follow-up [20]. In an-
other investigation there was no difference between the To our knowledge, only one study has investigated
the association between comorbidities and risk of all
cause death in AAV patients. Little et al. found, that
the Karnofsky performance score, but not the non-
vasculitic comorbidity showed independent association
with mortality [22]. Although we did not include Karnofsky
performance in our dataset, we found a significant as-
sociation of comorbidity and early mortality, and this
relationship remained independent of other important
clinical characteristics. Discussion Table 2 Predictors of “early mortality” in univariable Cox regression
analysis
HR
95% CI
P value
Age
1.035
0.994–1.078
0.097
Albumin
0.891
0.818–0.970
0.007
ANCA type – ANCA neg
REF
REF
REF
pANCA
0.604
0.215–1.695
0.338
cANCA
0.516
0.067–3.945
0.524
BVAS median
3.57
1.039–12.243
0.043
CRP
1.011
1.003–1.018
0.005
Comorbidity score
1.707
1.176–2.477
0.005
HD on admission
3.404
1.130–20.260
0.030
Table 4 Predictors of “late mortality” in univariable Cox
regression analysis
HR
95% CI
P value
Age
1.059
1.028–1.092
<0.001
Albumin
0.950
0.895–1.009
0.094
ANCA type – ANCA neg
REF
REF
REF
pANCA
0.658
0.341–1.268
0.211
cANCA
0.674
0.159–2.853
0.592
BVAS median
1.483
0.768–2.865
0.241
CRP
0.999
0.993–1.007
0.993
Comorbidity score
1.526
1.106–2.106
0.010
HD on admission
2.157
1.131–4.116
0.020 Table 2 Predictors of “early mortality” in univariable Cox regression
analysis
HR
95% CI
P value
Age
1.035
0.994–1.078
0.097
Albumin
0.891
0.818–0.970
0.007
ANCA type – ANCA neg
REF
REF
REF
pANCA
0.604
0.215–1.695
0.338
cANCA
0.516
0.067–3.945
0.524
BVAS median
3.57
1.039–12.243
0.043
CRP
1.011
1.003–1.018
0.005
Comorbidity score
1.707
1.176–2.477
0.005
HD on admission
3.404
1.130–20.260
0.030 Table 2 Predictors of “early mortality” in univariable Cox regression
analysis Table 4 Predictors of “late mortality” in univariable Cox
regression analysis
HR
95% CI
P value
Age
1.059
1.028–1.092
<0.001
Albumin
0.950
0.895–1.009
0.094
ANCA type – ANCA neg
REF
REF
REF
pANCA
0.658
0.341–1.268
0.211
cANCA
0.674
0.159–2.853
0.592
BVAS median
1.483
0.768–2.865
0.241
CRP
0.999
0.993–1.007
0.993
Comorbidity score
1.526
1.106–2.106
0.010
HD on admission
2.157
1.131–4.116
0.020 Table 4 Predictors of “late mortality” in univariable Cox Table 4 Predictors of “late mortality” in univariable Cox
regression analysis Haris et al. BMC Nephrology (2017) 18:76 Page 6 of 7 Table 5 BVAS predicts “late mortality” in multivariable Cox
regression analysis. The table shows the parameters of the
“BVAS median” variable in the different models
HR
95% CI
P value
Model 1
1.483
0.768–2.865
0.241
Model 2
2.073
1.030–2.435
0.041
Model 3
2.558
1.251–5.231
0.010
Model 4
2.408
1.081–5.362
0.031
Model 1: BVAS median
Model 2: Model 1 + comorbidity score
Model 3: Model 2 + Age, serum albumin
Model 4: Model 3 + HD dependency on admission and ANCA type (c-
versus p-ANCA) Table 5 BVAS predicts “late mortality” in multivariable Cox
regression analysis. Ethics approval and consent to participate The study was approved by the Institutional Ethics Committee of Szent
Margit Hospital, Budapest, Hungary, and conducted in agreement with the
declaration of Helsinki. Since this was a retrospective analysis of patient
records, a waiver for consent has been obtained from the institutional ethics
board. Availability of data and materials y
Anonymised data are available upon request to authors. Discussion The table shows the parameters of the
“BVAS median” variable in the different models tract involvement but no kidney disease have lower BVAS
but more relapses than those with renal AAV [26]. Our study has several limitations. Most importantly,
BVAS was calculated retrospectively. Nevertheless, detailed
source data provided reliable information, and the strictly
followed treatment protocol also assisted our analysis. Mor-
tality rate was fairly high, which likely can be explained by
very late referrals. This resulted in advanced renal failure
and extensive manifestations of AAV in most of our
patients. The extensive comorbidities might also have con-
tributed to the observed high mortality. In conclusion, baseline comorbidities influence both
short and long term outcome of patients with AAV. Risk-stratification would help clinicians to tailor therapy
individually, which might further improve the outcome. Future prospective treatment studies are needed to
assess whether scoring systems based on comorbidities
and BVAS help to individualize therapies in order to im-
prove short and long term survival. We did not find other investigations assessing the as-
sociation between comorbidities and infectious death. Importantly, this reveals the complexity of treatment of
AAV patients: likely those without any comorbidity may
tolerate aggressive immunosuppression and AAV better,
compared to subjects suffering from various chronic
disorders. Remarkably, in another study, accumulation
of adverse events in the first year of treatment - which
influenced survival significantly – was independently as-
sociated with age and renal impairment [2]. Based on
these latter findings we propose, that not only age and
kidney function, but also the presence of non-vasculitic
comorbidities present high risk status for adverse events,
especially for infections, which may provide an explan-
ation for the increased mortality. Authors’ contributions
E
h
h
(ÁH KP JA H Authors contributions
Each author (ÁH, KP, JA, HB, IK, LR, GK and IM) contributed to the design of this
investigation, the analysis of the data and the preparation of the manuscript. All
authors read and approved the final manuscript. Abbreviations AAV: ANCA-associated vasculitis; BVAS: Birmingham Vasculitis Activity Score;
CI: Confidence interval; CYC: Cyclophosphamide; eGFR: Estimated GFR;
ESKD: End stage kidney disease; HR: Hazard ratio; iv: Intravenous;
MP: Methylprednisolone; OR: Odds ratio Acknowledgements
h
d b d Authors are indebted to Dr. Andras Keszei (clinical epidemiologist,
Department of Medical Informatics, RWTH Aachen University) for his
statistical advice in revising the manuscript. Funding
Not applicable. Funding Not applicable. Our findings are in accord with several other reports
showing that the severity of kidney disease at baseline is
an indicator of poor prognosis [1, 6, 9, 10]. Similarly, high
levels of the inflammatory markers (CRP, albumin, etc.)
confer an increased early mortality risk for the individual
patient [5, 7]. The applicability of these predictors is
important, as these are readily available at the first presen-
tation of the patient. Competing interest The frequency of ESKD did not differ in the BVAS
groups in our cohort, similarly to other investigations
[4, 23]. We found more frequent relapses in patients
with higher BVAS; BVAS predicted relapse in Cox regres-
sion analysis, but the association was not significant after
adjustment for ANCA type. The likely explanation for this
observation is, that the higher BVAS in our cohort, com-
prising patients with both respiratory tract and kidney in-
volvement, was associated with c-ANCA disease, which
characteristically confers a higher risk of relapse compared
to p-ANCA positive vasculitis [24]. In comparison, in a
study investigating patients exclusively with c-ANCA posi-
tivity, relapses occurred more often in those who presented
with lower BVAS, compared to more severe cases [25]. A
possible explanation for this difference can be the different
case mix: cohorts with predominantly upper respiratory The authors declare that they have no competing interests. References 2003;63:670–7. 7. Yamagata K, Usui J, Saito C, Yamaguchi N, Hirayama K, Mase K, et al. ANCA-associated systemic vasculitis in Japan: clinical features and
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1 1Nephrology Department, Szent Margit Hospital, 132 Bécsi út, Budapest 1032,
Hungary. 2Pathology Department, Szent Margit Hospital, 132 Bécsi út,
Budapest 1032, Hungary. 3Institute of Pathophysiology, Semmelweis
University, 4 Nagyvárad tér, Budapest 1089, Hungary. 4Department of
Medicine (Nephrology), University of Toronto, Kidney Transplant Program,
Toronto General Hospital, University Health Network, 585 University Avenue,
Toronto M5G 2 N2, ON, Canada. Page 7 of 7 Page 7 of 7 Haris et al. BMC Nephrology (2017) 18:76 Haris et al. BMC Nephrology (2017) 18:76 Received: 2 December 2015 Accepted: 15 February 2017 Received: 2 December 2015 Accepted: 15 February 2017 24. Lionaki S, Blyth ER, Hogan SL, Hu Y, Senior JBA, Jennette CE, et al. Classification
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patient survival in ANCA-associated vasculitis. Ann Rheum Dis. 2011;70:488–94. 1. Flossmann O, Berden A, de Groot K, Hagen C, Harper L, Heijl C, et al. Long-term
patient survival in ANCA-associated vasculitis. Ann Rheum Dis. 2011;70:488–94. 25. Yegin EG, Can M, Yilmaz N, Aydin SZ, Yavuz S, et al. Activity and damage in
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Clin Exp Nephrol. Doi: 10.1007/s10157-013-0926-1. References Jennette JC, Falk RJ, Bacon PA, Basu N, Cid MC, Ferrario F, et al. 2012
Revised International Chapel Hill Consensus Conference Nomenclature of
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and we will help you at every step: Submit your next manuscript to BioMed Central
and we will help you at every step: 19. Vittinghoff E, McCulloch CE. Relaxing the rule of ten events per variable in
logistic and Cox regression. Am J Epidemiol. 2007;165:710–8. • We accept pre-submission inquiries
• Our selector tool helps you to find the most relevant journal
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Submit your manuscript at
www.biomedcentral.com/submit
and we will help you at every step: 20. Koike K, Fukami K, Yonemoto K, Iwatani R, Obata R, Ueda K, et al. A new
vasculitis activity score for predicting death in myeloperoxidase-antineutrophil
cytoplasmic antibody-associated vasculitis patients. Am J Nephrol. 2012;35:1–6. 21. Itabashi M, Takei T, Yabuki Y, Suzuki H, Ando M, Akamatsu M, et al. Clinical
outcome and prognosis of anti-neutrophil cytoplasmic antibody-associated
vasculitis in Japan. Nephron Clin Pract. 2010;115:c21–27. 22. Little MA, Nazar L, Farrington K. Outcome in glomerulonephritis due to
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The heterogeneous effect of forest tenure security on forestry management efficiency of farmers for different forest management types
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Forestry economics review
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The current issue and full text archive of this journal is available on Emerald Insight at:
https://www.emerald.com/insight/2631-3030.htm The current issue and full text archive of this journal is available on Emerald Insight at:
https://www.emerald.com/insight/2631-3030.htm The current issue and full text archive of this journal is available on Emerald Insight at:
https://www.emerald.com/insight/2631-3030.htm 37 Received 14 January 2022
Revised 11 March 2022
Accepted 23 March 2022 Funding: “ Research on Institutional Obstacles and Institutional Innovation of Practicing Xi Jinping’s
theory of ‘Two Mountains’ with Chinese Characteristics in the New Era” (2019BLRD09) supported by
Beijing Forestry University.
C
fli t
f i t
t Th
th
d
l
fli t f i t
t © Yaqin Zou, Xuemei Jiang, Caiyun Wen and Yang Li. Published in Forestry Economics Review.
Published by Emerald Publishing Limited. This article is published under the Creative Commons
Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works
of this article (for both commercial and non-commercial purposes), subject to full attribution to the
original publication and authors. The full terms of this licence may be seen at http://creativecommons.
org/licences/by/4.0/legalcode The heterogeneous effect of forest
tenure security on forestry
management efficiency of farmers
for different forest
management types
Yaqin Zou and Xuemei Jiang
School of Economics and Management, Beijing Forestry University,
Beijing, China, and
Caiyun Wen and Yang Li
Economic Development Research Center,
National Forestry and Grassland Administration, Beijing, China The effect of
forest tenure on
efficiency 37 The authors are indebted to the anonymous reviewers and editors.
di
“
h
i
i
l Ob
l
d
i
i
l Conflicts of interest: The authors declare no conflict of interest. © Yaqin Zou, Xuemei Jiang, Caiyun Wen and Yang Li. Published in Forestry Economics Review.
Published by Emerald Publishing Limited. This article is published under the Creative Commons
Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works
of this article (for both commercial and non-commercial purposes), subject to full attribution to the
original publication and authors. The full terms of this licence may be seen at http://creativecommons.
org/licences/by/4.0/legalcode
The authors are indebted to the anonymous reviewers and editors.
Funding: “ Research on Institutional Obstacles and Institutional Innovation of Practicing Xi Jinping’s
theory of ‘Two Mountains’ with Chinese Characteristics in the New Era” (2019BLRD09) supported by
Beijing Forestry University.
Conflicts of interest: The authors declare no conflict of interest. Forestry Economics Review
Vol. 4 No. 1, 2022
pp. 37-55
Emerald Publishing Limited
2631-3030
DOI 10.1108/FER-01-2022-0001 Abstract The adjustment of management right of forestland elements is of great
significance to improve the internal production relationship of forestry, stimulate the
enthusiasm of farmers in forestry production, alleviate the contradiction between
ecological protection and farmers’ interests and promote the sustainable development of
forestry. In 2018, the National Forestry and Grassland Administration’s opinion on further
enlivening the management right of collective forest pointed out that in accordance with
the idea that “Lucid waters and lush mountains are invaluable assets”, the overall
efficiency and output of forestland can be improved by designing the utilization mode,
approach, intensity and industrial distribution of collective forest resources. The 14th
Five-year Plan for forestry development in 2020 emphasizes in its principles the economical,
intensive and efficient use of resources, and the overall improvement of ecological,
economic and social benefits of forest. However, with the deepening of the CFTR, the
efficiency of forestry management may be weakened by forestland fragmentation and
extensive management of farmers. Due to the long forestry production cycle, low return
rate, scale effect is becoming increasingly important. What is the efficiency of farmers’
forestry management after the decentralization of forest tenure? What are the factors
influencing the differences in efficiency? Is it possible to find the optimal efficient ways to
manage forestland in collective forest regions? These are all questions that need to be
studied if China is to improve forestry management efficiency. 38 A large number of empirical studies have been conducted by academics on the impact of
the CFTR on farmers’ forestry management, and the following views have been broadly
formed. The first group of views believes that the confirmation of forest tenure to households
has strengthened farmers’ perception of forestl tenure security (Yi et al., 2014) and stimulated
the productive activity of farmers (Song et al., 1997). Safe forest tenure promotes farmers to
invest in forestland (Qin and Xu, 2013), thus transforming the advantages of forest resources
into economic advantages, which plays an important role in promoting farmers’ income
growth, rural economic development and sufficient supply of forest products in collective
forest regions (Shi and Wang, 2016). Stable tenure has also accelerated the occurrence of land transfer, which in turn
improves the productivity of forestland (Gao et al., 2021), and the income-generating effect
of the CFTR is sustainable (He et al., 2021). Abstract Purpose – After the Collective Forest Tenure Reform (CFTR) in China, the enthusiasm of farmers for forestry
management is stimulated. However, the forest tenure security varies among farmers, making the research
conclusions of its impact on forestry management efficiency inconsistent. Based on the survey data of 1,627
households from the collective forest regions in 6 provinces of China in 2017, this paper not only discusses the
differences of farmers’ forestry management efficiency after the reform, but also further explores the
heterogeneous impact of forest tenure security on forestry management efficiency in combination with
different forest management types. Design/methodology/approach – This study employed the stochastic frontier production function model to
measure the forestry management efficiency of farmers. Then, Tobit models were used to discuss the
influencing factors of farmers’ forestry management efficiency. Findings – The results demonstrate that the improvement of farmers’ forest tenure security can effectively
improve forestry management efficiency, but the effect is affected by forest management types. For farmers
who manage economic forests and non-timber forests, safe tenure promotes the forestry management
efficiency; while for those who manage ecological public welfare forests, tenure security plays an opposite role. Originality/value – Therefore, satisfying farmers’ differentiated demands for forest tenure according to
forest management types to improve forest tenure security and further refining supporting policies of collective
forestry reform is of great significance to improve the efficiency of farmers’ forestry management in collective
forest regions. Keywords Forest tenure, Tenure security, Forestry management efficiency, SFA Paper type Research paper © Yaqin Zou, Xuemei Jiang, Caiyun Wen and Yang Li. Published in Forestry Economics Review. Published by Emerald Publishing Limited. This article is published under the Creative Commons
Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works
of this article (for both commercial and non-commercial purposes), subject to full attribution to the
original publication and authors. The full terms of this licence may be seen at http://creativecommons. org/licences/by/4.0/legalcode The authors are indebted to the anonymous reviewers and editors. Conflicts of interest: The authors declare no conflict of interest. 1. Introduction
In 2003, the Collective Forest Tenure Refom (CFTR) was piloted in collective forest regions,
which account for 59% of China’s forestland. This is another major change in the
management system of China’s rural forestland after the policy of “Three-fixes” on forest
in 1981. Abstract The second point of view is that tenure
arrangement in collective forest regions emphasizes the fairness of initial distribution of
forestland resources, but fails to achieve the goal of improving efficiency, and it is
highlighted by the fact that the contradiction between family decentralized management
and scale management (Liu and Wang, 2009). The new round of CFTR has intensified the
fragmentation and decentralization of forestland to some extent (Kong et al., 2013; Liu
et al., 2015). After the reform, farmers’ forestland management is extensive and the scale
efficiency is not high, which leads to low comprehensive efficiency of forestland
production (Li et al., 2014). From the perspective of tenure types, independent tenure will
have a significant negative impact on the efficiency of forestry management compared to
joint tenure (Wang et al., 2019). In addition, some scholars believe that although the reform
has accomplished the task of clarifying property rights and realized tenure security, it has
not fully realized tenure stability and the rights owned by farmers in the actual production
and management are not as expected. So tenure security dosen’t have a substantial effect
on the efficiency of forestry management (Chen et al., 2018). y
y
g
(
)
The factors that affect the efficiency of forest management also include the characteristics
of forestland, supporting policies and household characteristics. However, the discussion of collective forest tenure has not been combined with forest management types. It is found that
farmers who manage different forest types have different tenure demands through
investigation, which provides ideas for the adjustment of tenure policies and further detailed
research. The purpose of this study is to verify, through empirical analysis, the differences in
the influence mechanism of forest tenure on forestry management efficiency in combination
with different forest management types, and then to effectively improve the forestry
management efficiency of farmers through precise policies, so that forestry can play a greater
role in rural revitalization and rural ecological construction. 39 Therefore, based on the data from the Economic Research Center of The National Forestry
and Grassland Administration on farmers in China’s collective forest regions in 2017, this
paper not only discusses the differences of farmers’ forestry management efficiency after the
CFTR, but also further explores the heterogeneous impact of different forest management
types on forestry management efficiency. The effect of
forest tenure on
efficiency Abstract The second part of this paper discusses the
influencing mechanism of farmers’ forestry management efficiency, the third part measures
and analyzes farmers’ forestry management efficiency, the fourth part analyzes the
influencing factors of farmers’ forestry management efficiency and the fifth part puts
forward targeted policy recommendations. 2. Study on the influence of farmers’ forest tenure on forestry management
efficiency y
2.1 Literature review on farmers’ forest tenure after the CFTR In the early 1980s, China carried out the policy of “Three-fixes” on forestry in collective forest
regions, which aimed at stabilizing the ownership of mountains and forests. This policy
implemented the responsibility system of forestry production by delineating self-retained
mountainous land for villagers through allocating the right to use forestland and ownership
of trees to peasant households without shifting the ownership of forestland belonging to the
collectives (Liu et al., 2019). Forestland in collective forest regions began to change from
collective management to family decentralized management in the form of “Sub-forest home”,
forming a pattern of fragmented division and scattered use. However, due to the instability of
tenure and the failure to keep up with relevant supporting policies, a large amount of forest
resource was destroyed in a short period of time. In 2003, with the core content of “clarifying tenure, liberalizing management, reducing
taxes and fees, and standardizing circulation”, a new round of CFTR began in Fujian
province. On the premise of ensuring that the ownership of forestland belongs to the
collectives, the right to use forestland and the ownership as well as use of trees are transferred
to families or other economic entities in various ways, such as equalization of mountain and
forests or equalization of shares and profits, and bidding. Since then, the reform rapidly
spread to other provinces. After 2008, the new round of CFTR has been carried out
nationwide, and by the end of 2012, the main task of CFTR in all provinces and autonomous
regions had been basically completed. g
y
p
After the forestland is confirmed to the household, in order to further solve the problem of
unclear property rights of forestland, undone implementation of production subjects,
lagging production mechanism and unfair distribution of benefits, fully mobilize the
enthusiasm of farmers in forestry production, activate the vitality of forestry production, the
government has introduced a series of forestry supporting policies such as “forestry
mortgage loan, forestry insurance, forestry cooperatives”. Studies on forestry supporting
policies mainly focus on forest tenure mortgage loans (Liao et al., 2012), forestland circulation
(Min et al., 2017), forestry subsidies (Wang et al., 2020), forestry insurance (Zhang and Gao,
2011), forestry cooperatives (Han et al., 2018) and forestry technological services (Liao
et al., 2014a). 2. Study on the influence of farmers’ forest tenure on forestry management
efficiency 2.2 Study on the influence of farmers’ forestry management efficiency
2.2.1 Research methods of forestry management efficiency. Most scholars have used
parametric and non-parametric analysis methods in the measurement of forestry
management efficiency. The stochastic frontier analysis (SFA) is widely used in
parametric methods: Kehinde et al. (2010) used SFA to quantify the technical efficiency of
sawmills and ordinary least square (OLS) regression analysis to estimate the determinants;
Xiong et al. (2018) used SFA and panel data model to empirically analyze the factors
influencing forestry production efficiency in northwest China; Chen et al. (2018) used SFA and
Tobit model based on plot survey data to reveal the influencing factors of forestland
management efficiency in collective forest areas; Jia et al. (2019) used SFA and maximum
likelihood estimation (MLE) methods to study the impact of the quality of forestry hired labor
on the technical efficiency of forestry management. Meanwhile, data envelopment analysis
(DEA) is widely used in non-parametric methods: Tong and Wang (2011) conducted an
empirical study on the production efficiency of China Jilin Forest Industry Group based on
DEA and Malmquist index method, emphasizing the importance of technology and intensive
management; Liao et al. (2014b) used the DEA-Tobit two-stage model to examine the factors
influencing the management efficiency of farmers’ economic forests in southern Jiangxi; Li
et al. (2014) classified and evaluated the efficiency of production factors of different types of
commercial forests based on a three-stage DEA model; Chen et al. (2020) conducted a
spatiotemporal empirical study on the coordinated development of forestry management
efficiency and forest ecological security with the help of Super-CCR model, coupling
coordination model and spatial panel models; Ma and Gao (2021) used the DEA-Tobit method
to measure the production efficiency of farmers and used the hierarchical linear model (HLM)
to test the moderating effect of business model on non-agricultural employment and forestry
production efficiency. In addition, a scholar used both DEA and SFA methods to correct the
investment efficiency values of listed forestry companies (Guan et al., 2019). Some scholars
have also applied cost-benefit approach (Clinch, 2000) and case study approach (Li et al., 2007)
to conduct relevant studies. Forestry production is highly influenced by natural and other
random factors, and SFA can better explain the causes of efficiency losses than DEA (Musaba
and Bwacha, 2014), so there is a scientific basis for using SFA to measure the efficiency of
forestry management. The effect of
forest tenure on
efficiency 41 2.3 Influencing mechanism of farmers’ forest tenure on forestry management efficiency
After the CFTR, clear forest tenure improved the enthusiasm of farmers in forestry
production. As a result of the reform, the forestland managed by farmers involves many
types. For example, there are ecological public welfare forests and commercial forests
classified by function; timber forests, charcoal forests, shelter forests, special-use forests
and economic forests classified by concrete purpose. In addition, it can be divided into
timber management and non-timber management according to the management types. Among the various classifications above, different forest management types have
different payback periods and rates of return, and farmers have different input motivation,
resulting in significant differences in management efficiency. In general, forest
management types with short payback periods or high return rates will be more
efficient. For example, compared with the ecological public welfare forests with low
ecological compensation benefits, the return rates of input in commercial forests are more
significant, so farmers are more willing to invest and manage, which will effectively
improve the management efficiency. Compared with timber forests, charcoal forests,
shelter forests and special-use forests with longer operating cycles, economic forests have
shorter investment return cycles and high rates of return, so it will generate higher
management efficiency. Compared with timber production with longer rotation periods,
the management of non-timber forests is more significantly profitable. The operation of
non-timber forests provides a new idea for coordinating the contradiction between
ecological protection and farmers’ income. Farmers are naturally willing to invest more
elements in non-timber forests, which will make it more efficient. These differential impact
mechanisms will be further verified in the later empirical studies. In addition, it
needs further proof whether the effect of tenure security will be more significant for
forestland types that farmers are more positive. And the empirical analysis results will
provide more targeted suggestions for further protection of forest tenure. Therefore, this
study will take into account farmers’ forest management types and explain the mechanism
of forest tenure security on forestry management efficiency from a more subdivided
perspective. 2. Study on the influence of farmers’ forest tenure on forestry management
efficiency FER
4,1
40 40 2.2.2 Factors affecting farmers’ forestry management efficiency. The factors affecting the
efficiency of farmers’ forestry management can be summarized in terms of production
factors, forestland characteristics, management organization forms and householder’s
and family characteristics. (1) Production factors. Forestry capital input can effectively
improve forestry output, but with a certain lag (Zhan et al., 2016). Meanwhile, forestry is a
typical labor-intensive industry, and the effectiveness of labor supply directly affects
farmers’ forestland decision-making behavior, which in turn affects forestland output
efficiency (Liao et al., 2018). (2) Forestland characteristics. After the CFTR, family
management became the main mode of collective forestland management in China. The
characteristics of decentralized and fragmented management make many scholars begin
to pay attention to the relationship between forestland scale and input–output efficiency. And the research mainly focused on the number of forest blocks (Liao et al., 2014b),
forestland stand conditions (Xu et al., 2014) and forestland scale (Tian and Shi, 2017), but
the research conclusions are not consistent. (3) Management organization forms. Cooperative production through family forestry farms and cooperatives can improve
the organization of forestry production and management, and can also overcome
the shortcomings of family operation in terms of scale economy and inefficient
factor utilization (Ke et al., 2014), thus improving the efficiency of forestry management. (4) Householder’s and family characteristics. Age (Tian and Jia, 2004), gender (Zhu et al.,
2018), education (Wang et al., 2011) of the householder, whether the householder is a village The effect of
forest tenure on
efficiency cadre (Ke and Chen, 2016) and family size (Huang et al., 2019) will have impacts on
forestry production and management behavior and even output efficiency; The
degree of part-time farming (Zhai et al., 2013) and the share of forestry income in
household income (Xu et al., 2015a) also have a significant impact on forestry management
efficiency. 41 3.2 Efficiency measurement methods and indicators ff
y
3.2.1 Basic connotation. Efficiency refers to the ratio of input and output of management
subject in operational activities, mainly including technical efficiency and allocation
efficiency. The former refers to the ability to make optimal use of resources, in other
words, to minimize input under given conditions of output, or to maximize output under
given conditions of input factors; the latter is to achieve the optimum of inputs under
certain conditions of factor prices (Xu et al., 2015b). Forestry management efficiency
indicates whether all kinds of production factors have reached the optimal allocation in
the process of forestry production. It reflects the realization degree of forest resource
value (Shi and Zhang, 2012). When the efficiency value equals 1, it means that the forestry
management efficiency has achieved the optimal state. g
y
p
3.2.2 Measurement method and index selection. The SFA is a typical representative of
parametric estimation method to efficiency evaluation, which needs to determine the
specific form of production frontier. Compared with non-parametric estimation, its
advantages are shown by considering the influence of random factors on output, more
suitable for large sample calculation, and more stable results without the influence of
outliers (Li and Fan, 2009). For cross-sectional data, SFA can be expressed as follows: Yi ¼ fðxi; βiÞ expðvi uiÞ
(1) (1) Yi ¼ fðxi; βiÞ expðvi uiÞ
(1)
TEi ¼ expðuiÞ
(2) (1)
(2) TEi ¼ expðuiÞ
(2) (2) In the above equation, Yi and xi represent the output and input of the ith decision-making unit
(DMU) respectively, β is the model parameter, and the compound disturbance term is a
composite structure. vi is the random factor affecting output, which is assumed to obey
independent normal distribution, namely vi ∼Nð0; σ2
vÞ; ui represents the error caused by
technical inefficiency, which is assumed to be a half-normal random variable with
independent identical distribution, namely ui ∼N þð0; σ2
uÞ. TEi is technical efficiency,
which ranges from 0 to 1, and it measures the relative difference between the output of the ith
DMU and the output of a fully efficient DMU with the same inputs, that is, the ratio of actual
output expectation to production frontier expectation. p
p
p
p
In the process of efficiency measurement, scholars are basically consistent in selecting
input and output indicators for forestry production and operation. 3. Measurement and analysis of farmers’ forestry management efficiency
3.1 Data collection This paper uses research data from China’s monitoring project of the CFTR in 2017 to
conduct an empirical analysis. In order to comprehensively understand the progress of
CFTR, the National Forestry and Grass Administration conducted a tracking survey in 7
provinces including Yunnan, Jiangxi, Hunan, Gansu, Fujian, Liaoning and Shaanxi, with 10
counties randomly selected in each sample province, and 5 sample villages were selected
separately in each sample county, and about 10 farm households were randomly selected in
each village for a one-on-one questionnaire survey. The sample data included a total of 70
sample counties, 350 villages and 3,509 households in 2017. The percentage of timber forests
with long production cycles in the full sample was only 3.676%. To ensure the completeness
of information and to meet the data requirements for efficiency measurement, only the sample
of farmers with both input and output data is retained, which can explain the scientific nature of efficiency measurement to a certain extent. At this time, only 26 farmers were left in Gansu
province, which was not included in the final data because of weak representativeness. Finally, the data of 1,627 households in 60 counties in Yunnan, Jiangxi, Hunan, Fujian,
Liaoning and Shaanxi provinces were used. The questionnaires were conducted face-to-face with farmers. The main contents of the
survey include the process of family participation in the CFTR, structure of forest tenure, the
status of forest resources, the input–output situation of forestland, forestry related
supporting policies and the basic household characteristics of farmers and so on. 42 3.2 Efficiency measurement methods and indicators Capital, labor and
forestland area are usually selected as input indicators, while forestland output value is
selected as an output indicator (Xu et al., 2015a). Combined with the actual situation and data
availability, this paper finally selected capital input, labor input and forestland area as input
indicators and selected forestry economic benefit as an output indicator. The descriptions of
forestry input and output indicators are shown in Table 1. The C-D production function is selected as the model of the stochastic frontier production
function and it is constructed as follows: (3) ln Yi ¼ β0 þ β1 ln Ki þ β2 ln Li þ β3 ln Ai þ vi þ ui TEi ¼ expð−uiÞ
(4)
f TEi ¼ expð−uiÞ (4) TEi ¼ expð−uiÞ In the equation above, Yi is the total income of forestry production (yuan), Ki, Li and Ai
denote the capital input (yuan), labor input (day) and forestland area (mu) of the ith decision
unit, respectively; β0, β1, β2 and β3 are parameters to be estimated; TEi is the efficiency of
forestry management. The main advantages of choosing C-D function are its simple form and
direct economic meaning of parameters, β1, β2 and β3 denote the output elasticity of capital,
labor and land, respectively. In the equation above, Yi is the total income of forestry production (yuan), Ki, Li and Ai
denote the capital input (yuan), labor input (day) and forestland area (mu) of the ith decision
unit, respectively; β0, β1, β2 and β3 are parameters to be estimated; TEi is the efficiency of
forestry management. The main advantages of choosing C-D function are its simple form and
direct economic meaning of parameters, β1, β2 and β3 denote the output elasticity of capital,
labor and land, respectively. 43 3.2.3 Parameter estimation results. Frontier 4.1 was used to estimate the parameters
(Table 2), where the variation rate γ was used to determine whether SFA passed the test. The
expression is as follows: γ ¼
σ2
u
σ2
v þ σ2
u
(5) (5) In formula (5), σ2
u and σ2
v are the variance of inefficient terms u and v; The variation rate γ
ranges from 0 to 1, and the approximation to 1 means that the stochastic frontier production
function is reasonable. (4)
d Ai
ision
The effect of
forest tenure on
efficiency 3.2 Efficiency measurement methods and indicators The result shows that the value of γ is 0.838 and is significant at 1% level, which indicates
that there is indeed inefficiency in farmers’ forestry management. The compound error
term of 83.8% comes from technical inefficiency and 16.2% from the disturbance of
random factors, which indicates that the stochastic production function used in the model
is valid. 3.3 Descriptive statistics of farmers’ forestry management efficiency Jiangxi province, second only to Fujian in terms of forest
coverage, has seen an increase in forestry inputs and improved output efficiency in the
background of the CFTR; Yunnan province has a vast forested area, sufficient water and
heat, good quality forest stands and high forestry management efficiency among farmers;
Liaoning and Shaanxi have low forestry management efficiency mainly due to their faint
advantages in resource endowment and secondly due to diseconomies of scale. The low level
of forestry management efficiency in Hunan province is related to the type of main source of
income of farmers. More than 60% of sample farmers in Hunan province go out to work for a
living, while less than 5% of farmers take forestry income as their main source of income
(According to farmer’s answer to the question: “whether the main source of income of the
family is forestry production” in the questionnaire.) and they are not very enthusiastic about
forestry production. In empirical research, “whether forestry is the main source of income”
will be added for control to test the impact of the importance of forestry income on forestry
management efficiency. vitality of forestry development and significantly improved the efficiency of farmers’ forestry
production and management. Jiangxi province, second only to Fujian in terms of forest
coverage, has seen an increase in forestry inputs and improved output efficiency in the
background of the CFTR; Yunnan province has a vast forested area, sufficient water and
heat, good quality forest stands and high forestry management efficiency among farmers;
Liaoning and Shaanxi have low forestry management efficiency mainly due to their faint
advantages in resource endowment and secondly due to diseconomies of scale. The low level
of forestry management efficiency in Hunan province is related to the type of main source of
income of farmers. More than 60% of sample farmers in Hunan province go out to work for a
living, while less than 5% of farmers take forestry income as their main source of income
(According to farmer’s answer to the question: “whether the main source of income of the
family is forestry production” in the questionnaire.) and they are not very enthusiastic about
forestry production. In empirical research, “whether forestry is the main source of income”
will be added for control to test the impact of the importance of forestry income on forestry
management efficiency. 3.3 Descriptive statistics of farmers’ forestry management efficiency The low level
of forestry management efficiency in Hunan province is related to the type of main source of
income of farmers. More than 60% of sample farmers in Hunan province go out to work for a
living, while less than 5% of farmers take forestry income as their main source of income
(According to farmer’s answer to the question: “whether the main source of income of the
family is forestry production” in the questionnaire.) and they are not very enthusiastic about
forestry production. In empirical research, “whether forestry is the main source of income”
will be added for control to test the impact of the importance of forestry income on forestry
management efficiency. 3.3.3 There are significant differences in efficiency of forest management types. In general,
the efficiency of farmers’ forestry management is closely related to the types of forest
Parameter to be estimated
Estimate of parameter
Standard error
T statistic
Constant term
2.585***
0.382
6.767
lnðCapitalÞ
0.661***
0.065
10.123
lnðLaborÞ
0.104
0.067
1.551
ln (Forestland)
0.285***
0.033
8.570
σ2
7.142***
0.477
14.961
γ
0.838***
0.031
26.764
Likelihood function value
3250.282
Likelihood ratio tests one side value
44.427
Sample size
1,627
Note(s): ***, ** and * are significant at 1%, 5% and 10% levels respectively
Efficiency interval
Mean
Median
Sample size
Percentage (%)
0:001 ≤te < 0:108
0.051
0.049
406
24.954
0:108 ≤te < 0:278
0.191
0.188
407
25.015
0:278 ≤te < 0:443
0.366
0.366
407
25.015
0:443 ≤te ≤1
0.748
0.540
407
25.015
Total
0.287
0.278
1,627
100
Province
Mean
Median
Min
Max
Sample size
Yunnan
0.309
0.309
0.012
0.748
208
Jiangxi
0.350
0.366
0.019
0.705
224
Hunan
0.183
0.117
0.006
0.694
283
Fujian
0.359
0.415
0.001
0.739
331
Liaoning
0.277
0.267
0.002
0.730
253
Shaanxi
0.255
0.241
0.004
0.639
328
Total
0.287
0.278
0.001
0.748
1,627
Table 2. Parameter estimation
results of C-D
stochastic frontier
production function
Table 3. Forestry management
efficiency of farmers
Table 4. Efficiency of farmers’
forestry management
in sample provinces
FER
4,1
44 vitality of forestry development and significantly improved the efficiency of farmers’ forestry
production and management. 3.3 Descriptive statistics of farmers’ forestry management efficiency 3.3.1 The overall forestry management efficiency of farmers in collective forest regions is at a
low level. By measuring the forestry management efficiency of 1,627 households, the overall
efficiency values were divided into 4 groups according to the 25th percentile, as shown in
Table 3. Overall, most of the farmers’ forestry management are inefficient. The average value of
farmers’ forestry management efficiency is only 0.287 which has a large gap with the effective
efficiency value of 1 and still has much room for improvement. 3.3.2 Significant differences exist in the efficiency of farmers’ forestry management across
regions. The average level of forestry management efficiency of farmers in Fujian province is
the highest, followed by Jiangxi, Yunnan, Liaoning and Shaanxi, and Hunan is the lowest (see
Table 4). Fujian province has the highest forest coverage rate and has taken the lead in
implementing the CFTR and comprehensive supporting reforms, which has enhanced the The index
type
Level
indicators
The secondary indicators (unit)
Mean
Median
Standard
error
Output
indicator
Economic
benefit
Total forestry output value
(yuan)
18200.085
2297.860
84689.480
Input
indicators
Capital
Forestry operational
expenditure (yuan)
17948.851
4,800
85000.980
Labor
Self-employment and
employment (day)
132.299
36
620.651
Forestland
Forestland area (mu)
131.216
41
413.881
Source(s): Monitoring Project of China’s Collective Forest Tenure Reform in 2017, Economic Research Center,
the National Forestry and Grassland Administration, the same below
Table 1. Input–output
indicators of household
forestry production vitality of forestry development and significantly improved the efficiency of farmers’ forestry
production and management. Jiangxi province, second only to Fujian in terms of forest
coverage, has seen an increase in forestry inputs and improved output efficiency in the
background of the CFTR; Yunnan province has a vast forested area, sufficient water and
heat, good quality forest stands and high forestry management efficiency among farmers;
Liaoning and Shaanxi have low forestry management efficiency mainly due to their faint
advantages in resource endowment and secondly due to diseconomies of scale. The low level
of forestry management efficiency in Hunan province is related to the type of main source of
income of farmers. 3.3 Descriptive statistics of farmers’ forestry management efficiency More than 60% of sample farmers in Hunan province go out to work for a
living, while less than 5% of farmers take forestry income as their main source of income
(According to farmer’s answer to the question: “whether the main source of income of the
family is forestry production” in the questionnaire.) and they are not very enthusiastic about
forestry production. In empirical research, “whether forestry is the main source of income”
will be added for control to test the impact of the importance of forestry income on forestry
management efficiency. 3.3.3 There are significant differences in efficiency of forest management types. In general,
the efficiency of farmers’ forestry management is closely related to the types of forest
Parameter to be estimated
Estimate of parameter
Standard error
T statistic
Constant term
2.585***
0.382
6.767
lnðCapitalÞ
0.661***
0.065
10.123
lnðLaborÞ
0.104
0.067
1.551
ln (Forestland)
0.285***
0.033
8.570
σ2
7.142***
0.477
14.961
γ
0.838***
0.031
26.764
Likelihood function value
3250.282
Likelihood ratio tests one side value
44.427
Sample size
1,627
Note(s): ***, ** and * are significant at 1%, 5% and 10% levels respectively
Efficiency interval
Mean
Median
Sample size
Percentage (%)
0:001 ≤te < 0:108
0.051
0.049
406
24.954
0:108 ≤te < 0:278
0.191
0.188
407
25.015
0:278 ≤te < 0:443
0.366
0.366
407
25.015
0:443 ≤te ≤1
0.748
0.540
407
25.015
Total
0.287
0.278
1,627
100
Province
Mean
Median
Min
Max
Sample size
Yunnan
0.309
0.309
0.012
0.748
208
Jiangxi
0.350
0.366
0.019
0.705
224
Hunan
0.183
0.117
0.006
0.694
283
Fujian
0.359
0.415
0.001
0.739
331
Liaoning
0.277
0.267
0.002
0.730
253
Shaanxi
0.255
0.241
0.004
0.639
328
Table 2. Parameter estimation
results of C-D
stochastic frontier
production function
Table 3. Forestry management
efficiency of farmers
Table 4. Efficiency of farmers’
forestry management
FER
4,1
44 vitality of forestry development and significantly improved the efficiency of farmers’ forestry
production and management. Jiangxi province, second only to Fujian in terms of forest
coverage, has seen an increase in forestry inputs and improved output efficiency in the
background of the CFTR; Yunnan province has a vast forested area, sufficient water and
heat, good quality forest stands and high forestry management efficiency among farmers;
Liaoning and Shaanxi have low forestry management efficiency mainly due to their faint
advantages in resource endowment and secondly due to diseconomies of scale. The effect of
forest tenure on
efficiency 3.3 Descriptive statistics of farmers’ forestry management efficiency 44 g
y
3.3.3 There are significant differences in efficiency of forest management types. In general,
the efficiency of farmers’ forestry management is closely related to the types of forest Parameter to be estimated
Estimate of parameter
Standard error
T statistic
Constant term
2.585***
0.382
6.767
lnðCapitalÞ
0.661***
0.065
10.123
lnðLaborÞ
0.104
0.067
1.551
ln (Forestland)
0.285***
0.033
8.570
σ2
7.142***
0.477
14.961
γ
0.838***
0.031
26.764
Likelihood function value
3250.282
Likelihood ratio tests one side value
44.427
Sample size
1,627
Note(s): ***, ** and * are significant at 1%, 5% and 10% levels respectively
Efficiency interval
Mean
Median
Sample size
Percentage (%)
0:001 ≤te < 0:108
0.051
0.049
406
24.954
0:108 ≤te < 0:278
0.191
0.188
407
25.015
0:278 ≤te < 0:443
0.366
0.366
407
25.015
0:443 ≤te ≤1
0.748
0.540
407
25.015
Total
0.287
0.278
1,627
100
Province
Mean
Median
Min
Max
Sample size
Yunnan
0.309
0.309
0.012
0.748
208
Jiangxi
0.350
0.366
0.019
0.705
224
Hunan
0.183
0.117
0.006
0.694
283
Fujian
0.359
0.415
0.001
0.739
331
Liaoning
0.277
0.267
0.002
0.730
253
Shaanxi
0.255
0.241
0.004
0.639
328
Total
0.287
0.278
0.001
0.748
1,627
Table 2. Parameter estimation
results of C-D
stochastic frontier
production function
Table 3. Forestry management
efficiency of farmers
Table 4. Efficiency of farmers’
forestry management
in sample provinces meter to be estimated
Estimate of parameter
Standard error
T statistic (see Table 5). The highest average efficiency of forests are economic forests, which have
short return on investment cycles, more flexible operation forms and more stable outputs. The efficiency of timber forests is lower than that of economic forests, which may be
related to the longer return on investment periods. The average efficiency of non-timber
forests is higher than the average of population. The development of non-timber forests
can make full use of land resources and shady space, combine with breeding, collection
and other activities. It can realize the transformation from simple utilization of forest
resources to combined utilization of forest resources and forestland resources and finally
reach the goal of “profits in the near term and forests in the long term”. By shortening
management cycles and bringing farmers higher economic returns, farmers’ enthusiasm
for non-timber forests production is stimulated and forestry management efficiency is
improved. The efficiency of farmers who manage the ecological public welfare forests is
the lowest. 3.3 Descriptive statistics of farmers’ forestry management efficiency The main forestry income of these farmers is the ecological public welfare
forest compensation. Due to the implementation of protective policies, logging is strictly
prohibited, and farmers cannot obtain forestry income through market behaviors, which
highlight the contradiction between forestry inputs and outputs, resulting in low efficiency
of forestry management. According to the frequency distribution characteristics of forest
management types, “Management in economic forests”, “Management in non-timber
forests” and “Management in ecological public welfare forests” are finally selected as
dummy variables and crossed with the indicator of tenure security to further analyze the
influence of forest tenure on forestry management efficiency. 45 4. Influencing factors of farmers’ forestry management efficiency
4.1 Empirical model and variable selection Since the efficiency values determined by SFA are restricted to between 0 and 1, using OLS
for regression will lead to the situation that parameters exceed the range. Tobit model was
used for the analysis based on existing studies. The specific model setting is as follows: Tei ¼ β0 þ β1Tenurei þ β2Tenurei * FMTi þ
X
n
1
λiCVi þ εi
(6) (6) In the formula, Tei represents the efficiency of farmers’ forestry management. The key
variable Tenurei is an indicator of forest tenure security, considering four indicators:
“whether there is a forest certificate,” “whether there is forest tenure mortgage loan,”
“whether there is forestland circulation” and “whether there is logging.” Specifically, forest
certificate is an important indicator of tenure security, and only when forestland is registered
and legally protected can the certainty of forest tenure be guaranteed (Brasselle et al., 2002),
and it means formal, legal security of tenure; forest tenure mortgage loan represents the Provinces
Timber forests
Economic forests
Non-timber forests
Ecological public welfare forests
Yunnan
0.494 (19)
0.373 (87)
0.369 (42)
0.255 (98)
Jiangxi
0.457 (11)
0.350 (24)
0.422 (30)
0.335 (109)
Hunan
0.405 (8)
0.427 (8)
0.276 (13)
0.168 (241)
Fujian
0.490 (13)
0.492 (118)
0.413 (34)
0.292 (177)
Liaoning
0.346 (71)
0.403 (65)
0.343 (41)
0.248 (176)
Shaanxi
0.639 (1)
0.335 (58)
0.359 (21)
0.253 (251)
Total
0.400 (123)
0.411 (360)
0.372 (181)
0.248 (1,052)
Note(s): Some farmers are engaged in part-time employment
Table 5. Average efficiency and
frequency of different
forest management
types (household) vinces
Timber forests
Economic forests
Non-timber forests
Ecological public welfare forests mortgage and transaction rights in the land property bundle (Sun and Xu, 2011); forestland
circulation reflects the autonomy of farmers’ forestland management after the CFTR (Cao
et al., 2014); timber harvesting right is a key institutional variable for forest tenure security
(He et al., 2014). These indicators reflect the realization of farmers’ control power of forestland
at the ground-truth level, including right of use, right of disposal and right of profit, as the
main body of forestry production and management. 4. Influencing factors of farmers’ forestry management efficiency
4.1 Empirical model and variable selection Then Tenurei is obtained by assigning
and arithmetic averaging the plot area, and it is expressed as follows: 46 Tenurei ¼
X12
1 ðploti * ifceiÞ þ
X12
1 ðploti * ifmliÞ þ
X12
1 ðploti * ifciiÞ þ
X12
1 ðploti * ifloiÞ
4 *
X12
1 ploti
(7) (7) In equation (7), ploti is the area of the ith forestland managed by peasant household; ifcei
represents whether the ith forestland has a certificate; ifmli represents whether the ith
forestland has forest tenure mortgage loan; iffci represents whether there is forestland
circulation for the ith forestland; ifloi represents whether there is logging in the ith forestland. The value range of tenure security indicator is between 0 and 1, and when Tenurei becomes
larger, it means that the degree of security of forest tenure becomes higher. FMTi is the forest management types of farmers, including “Management in economic
forests,” “Management in non-timber forests,” and “Management in ecological public welfare
forests”; CVi (i ¼ 1; 2; . . . ; n) are control variables. β0 is the constant term, β1, β2 are the
parameters to be estimated and εi is the random disturbance term. The control variables include forestry supporting policies, production factors and family
characteristics. Detailed description and data description are as follows (Table 6): (1)
Forestry supporting policies. i) Forestry subsidies: expressed by forestry subsidies
per mu, can promote farmers’ enthusiasm in forestry production; ii) Participation in
forestry cooperatives: cooperative operation can optimize the allocation of factors,
improve the level of specialization and promote forestry management efficiency; iii)
Adoption of forestry technological services: forestry technology training will improve
farmers’ forestry management skills and methods and ultimately improve the
efficiency of forestry management. (2)
Production factors. i) Capital refers to total household forestry expenditure, which
includes seedlings, fertilizer, machinery, animal power and so on. It is an
indispensable mean of production for developing forestry management activities. ii) Labor refers to the number of household labor that plays a decisive role in forestry
production. Forestry production has a weak quality and a long production cycle. The
availability of sufficient capital and labor will work better with the land factor to
produce higher efficiency. (3)
Family characteristics. i) Gender of householder: in forestry production, the male has
labor efficiency superiority compared with the female in terms of physical ability. 4. Influencing factors of farmers’ forestry management efficiency
4.1 Empirical model and variable selection ii)
Age of householder: The older the householder is, the more experienced in forestry
production. But the ability of acquiring new technology and information is weak,
which is not conducive to the improvement of forestry production efficiency. iii)
Education level of householder: householder with high education can grasp advanced
ideas and management methods, then promote the efficiency. iv) Householder is a
village cadre: village cadre is often a capable person in the village and can quickly
accumulate social capital and policy information, which is conducive to forestry
production and management. v) Family size: sufficient family labor force is a
guarantee to improve the efficiency of forestry management. vi) Forestry is the main
source of income: the main income source represents the characteristics of (3)
Family characteristics. i) Gender of householder: in forestry production, the male has
labor efficiency superiority compared with the female in terms of physical ability. ii)
Age of householder: The older the householder is, the more experienced in forestry
production. But the ability of acquiring new technology and information is weak,
which is not conducive to the improvement of forestry production efficiency. iii)
Education level of householder: householder with high education can grasp advanced
ideas and management methods, then promote the efficiency. iv) Householder is a
village cadre: village cadre is often a capable person in the village and can quickly
accumulate social capital and policy information, which is conducive to forestry
production and management. v) Family size: sufficient family labor force is a
guarantee to improve the efficiency of forestry management. 4. Influencing factors of farmers’ forestry management efficiency
4.1 Empirical model and variable selection vi) Forestry is the main
source of income: the main income source represents the characteristics of Variable type
Variable name
Variable
interpretation
Mean
Standard
deviation
Min
Max
Influence
direction
Explanatory variable
Forestry
management
efficiency
Te
By calculation
0.287
0.191
0.001
0.748
Key explanatory variable
Characteristics
of forestland
Tenure
Calculated by
weighting
0.277
0.084
0
0.759
þ
Forest management types
Management in
economic
forests
1 5 yes,
0 5 otherwise
0.221
0.415
0
1
þ
Management in
non-timber
forests
1 5 yes,
0 5 otherwise
0.111
0.315
0
1
þ
Management in
ecological
public welfare
forests
1 5 yes,
0 5 otherwise
0.647
0.478
0
1
Forestry supporting policies
Forestry
subsidies
Ten- thousand-
yuan per mu
0.003
0.015
0
0.323
þ
Participation in
forestry
cooperatives
1 5 yes,
0 5 otherwise
0.086
0.281
0
1
þ
Adoption of
forestry
technological
services
1 5 yes,
0 5 otherwise
0.368
0.482
0
1
þ
Production factors
Total
household
forestry
expenditure
Ten- thousand-
yuan
1.795
8.500
0.010
277.500
þ
Number of
household
labor
Person
2.778
1.330
0
9
þ
Family characteristics
Gender of
householder
1 5 male,
0 5 female
0.933
0.250
0
1
þ
Age of
householder
year
55.853
10.492
22
88
Education level
of householder
1 5 primary and
below, 2 5 middle
school, 3 5 high
school,
4 5 university or
above
1.821
0.769
1
4
þ
Householder is
a village cadre
1 5 yes,
0 5 otherwise
0.281
0.450
0
1
þ
Family size
Person
4.724
1.903
1
14
þ
Forestry is the
main source of
income
1 5 yes,
0 5 otherwise
0.147
0.354
0
1
þ
Table 6. Definitions and
descriptive statistics of
variables
The effect of
forest tenure on
efficiency
47 household’s livelihood, and farmer who takes forestry as the main income source will
tilt production factors to improve forestry yield. 4.2 Results
Farmers’ forestry management efficiency is the explanatory variable, and forest tenure
security is the key explanatory variable. The indicator “forest tenure security” is multiplied
by different forest management types to test the previous hypothesis (see Table 7). 48 4.2.1 Impact of tenure security on forestry management efficiency. Forest tenure security
has a significant positive impact on the efficiency of forestry management. The issuance of
certificates gives farmers clear and legally guaranteed bundle of rights. 4. Influencing factors of farmers’ forestry management efficiency
4.1 Empirical model and variable selection The forest tenure
mortgage loan, forestland circulation and the timber logging reflect farmers’ disposal and
profit right of forestland resources from the fact level. The full realization of the forest tenure
security leads to more forestland rights actually owned by farmers. The degree of forest
tenure security becomes higher, and farmers are motivated to manage forestland, which
eventually leads to the improvement of forestry management efficiency. For farmers who manage economic forests, the positive effect of tenure security on
forestry management efficiency has been verified for many times. With the improvement of
tenure security, the efficiency of forestry management is also increasing. The initial stage of
managing economic forests requires a large amount of capital, and secure tenure will increase
the intrinsic enthusiasm of farmers in forest production, increase forestry inputs and realize
the increase of income, thus driving the improvement of efficiency. For farmers who manage non-timber forests, the higher the forest tenure security is, the
better the efficiency of forestry management is. In multiple regression models, the positive
effect of tenure security on forestry management efficiency is significant at the level of 1%. Secure tenure contributes significantly to efficiency growth and facilitate farmers to increase
their income from engaging in forestry production and operation. For farmers who manage ecological public welfare forests, the improvement of tenure
security will restrain forestry management efficiency, which has been verified in models. At
present, ecological public welfare forests are strictly prohibited from logging, but the subsidy
standard is not perfect and flexible. So there is a far gap between the benefits of timber
harvesting and subsidy income. The strict restriction of revenue right leads to low efficiency
in forestry management. 4.2.2 Influence of control variables on forestry management efficiency. The positive effect of
forestry subsidies on forestry management efficiency has been repeatedly verified, and it is
significant at the level of 1% in models. In the process of forestry production, policy subsidies
are beneficial to increase farmers’ motivation in forestry production and improve the efficiency. Participation in forestry cooperatives is an effective mean for farmers to improve forestry
management efficiency. It makes it possible to improve market competitiveness and promote
the improvement of farmers’ forestry management efficiency by integrating resources and
optimizing forestry production factors. The age of householder has a significant negative effect on the efficiency of forestry
production and management. 5.1 Conclusions 5.1.1 The efficiency of farmers’ forest management in collective forest regions is at low lev
Although there are some differences in the efficiency of farmers’ forestry management amo 5.1.1 The efficiency of farmers’ forest management in collective forest regions is at low level. Although there are some differences in the efficiency of farmers’ forestry management among
the sample provinces, in general, they have not achieved efficient production, with an average
efficiency value of 0.287, which is still a long way from achieving the relative efficiency value
of “1”. It indicates that most of the farmers in collective forest regions use relatively crude
ways in forestry production and management, and the utilization of various resources does
not reach the optimal allocation, and the input and output do not achieve the best effect. p
,
p
p
5.1.2 Forest tenure security has a significant effect on forestry management efficiency. Secure forest tenure has a significant positive effect on the improvement of forestry
management efficiency. During the implementation of the new round of CFTR, farmers have
been given more property rights, and their rights to occupy, use, dominate and dispose
forestland have been further guaranteed. The definition of forest tenure ultimately affects the
efficiency level of forestry production by stimulating investment and rational allocation of
factors. Safe forest tenure has increased the enthusiasm of farmers who engage in forestry
production, and it highlights the contribution to the efficiency of forestry management. After
controlling the difference of provinces, the positive effect of tenure security on the efficiency
of forestry management is not significant. It is probably due to the differences in the
understanding and implementation of policies in different provinces and the existence of
problems such as property rights disputes, which make the bundle of forest tenure obtained
by farmers not as expected. At the same time, traditional ways of forestry production and
management have not changed substantially, offsetting the positive incentive effect of tenure
security. 5.1.3 The influence of forest tenure security on efficiency varies according to forest
management types. Foresttypesmanaged byfarmers influencedthedegreeand direction ofthe
effect of tenure security on management efficiency. Forest tenure security has a significant
positive effect on forestry management efficiency for farmers who manage economic forests
and non-timber forests. Both of these forms are flexible and can achieve economic benefits in
short periods. 4. Influencing factors of farmers’ forestry management efficiency
4.1 Empirical model and variable selection Among other control variables, the adoption of forestry technological services, total
household forestry expenditure, the number of household labor and householder’s personal
characteristics such as gender and education level as well as family size all show no
significant correlation with forestry management efficiency. 5.2 Suggestions
5.2.1 Fully guarantee forest tenure, reduce the restriction and make clear tenure promote the
efficiency of forestry management. After forest tenure is clearly defined, it will be necessary to
further ensure its long-term security and stability in order to effectively play an incentive role 5. Conclusions and recommendations
C
l
i 50 4. Influencing factors of farmers’ forestry management efficiency
4.1 Empirical model and variable selection It indicates that the limitation of physical and learning ability
will affect the efficiency of forestry management as the age of householder increases. The householder serving as the village cadre has certain positive influence on forestry
management efficiency. There will be some advantages for a village cadre to have more social
capital and channels to obtain forest management rights. When forestry income is the main source of family income, the efficiency of forestry
management of farmers is significantly higher than that of farmers who regard other income
as the main source of livelihood. This means that the greater the importance of forestry in
families’ livelihood, the greater the incentive for farmers’ forestry production. Variable type
Variable name
Te
Te
Te
Te
Te
Characteristics of
forestland tenure
Tenure
0.2750*** (0.0626)
0.2780*** (0.0620)
0.2780*** (0.0621)
0.2330*** (0.0617)
0.0777 (0.0676)
Tenure * Management in
economic forests
0.4700*** (0.0469)
0.4570*** (0.0472)
0.4570*** (0.0473)
0.4140*** (0.0472)
0.4010*** (0.0465)
Tenure * Management in
non-timber forests
0.3710*** (0.0467)
0.3610*** (0.0475)
0.3630*** (0.0477)
0.3530*** (0.0481)
0.3340*** (0.0475)
Tenure * Management in
ecological public welfare
forests
0.1980*** (0.0347)
0.1990*** (0.0346)
0.2000*** (0.0346)
0.1860*** (0.0345)
0.1460*** (0.0339)
Forestry supporting
policies
Forestry subsidies
1.1660*** (0.2780)
1.1660*** (0.2760)
1.2270*** (0.2790)
1.1920*** (0.3200)
Participation in forestry
cooperatives
0.0359** (0.0148)
0.0362** (0.0148)
0.0270* (0.0148)
0.0210 (0.0150)
Adoption of forestry
technological services
0.0131 (0.0091)
0.0132 (0.0091)
0.0074 (0.0090)
0.0082 (0.0095)
Production factors
Total household forestry
expenditure
0.0006 (0.0007)
0.0008 (0.0008)
0.0010 (0.0008)
Number of household
labor
0.0035 (0.0034)
0.0060 (0.0043)
0.0025 (0.0043)
Family
characteristics
Gender of householder
0.0243 (0.0173)
0.0177 (0.0172)
Age of householder
0.0011** (0.0004)
0.0009** (0.0004)
Education level of
householder
0.0027 (0.0060)
0.0049 (0.0060)
Householder is a village
cadre
0.0168* (0.0102)
0.0033 (0.0101)
Family size
0.0035 (0.0031)
0.0016 (0.0031)
Forestry is the main
source of income
0.0636*** (0.0130)
0.0550*** (0.0127)
Region dummy
variables
No
No
No
No
Yes
Constant
0.2060*** (0.0195)
0.1950*** (0.0194)
0.1870*** (0.0213)
0.2420*** (0.0382)
0.2730*** (0.0414)
Observations
1,627
1,627
1,627
1,627
1,627
Note(s): ***, ** and * are significant at the level of 1%, 5% and 10% respectively. Standard error is indicated in brackets The effect of
forest tenure on
efficiency 49 Among other control variables, the adoption of forestry technological services, total
household forestry expenditure, the number of household labor and householder’s personal
characteristics such as gender and education level as well as family size all show no
significant correlation with forestry management efficiency. 5.2 Suggestions 5.1 Conclusions Compared with farmers engaged in non-timber forests production, those who
manage economic forests is more sensitive to tenure security. As for farmers operating
ecological public welfare forests, China implements ecological public welfare forest
compensation policy to alleviate the contradiction between ecological public welfare forest
protection and farmers’ livelihoods, which restricts farmers’ independent management
activities and adversely affects farmers’ forestry production activities. Although ecological
public welfare forest compensation can compensate farmers’ losses to a certain extent, it is far
from the opportunity cost of managing forestland. In fact, farmers are not highly satisfied with
the compensation policy, which finally has a negative impact on forestry management
efficiency. Therefore, it shows that the effect of forest tenure security on forestry management
efficiency may be influenced by forest management types, investment return cycles and so on. gg
5.2.1 Fully guarantee forest tenure, reduce the restriction and make clear tenure promote the
efficiency of forestry management. After forest tenure is clearly defined, it will be necessary to
further ensure its long-term security and stability in order to effectively play an incentive role and give farmers a “pill of confidence”. It is necessary to ensure that certificates are issued to
farmers to improve forest tenure in legal and factual dimensions. Through the
implementation of management and use rights such as mortgage, circulation and logging,
farmers’ rights to benefit and disposal of forest resources will be guaranteed, and they will
have enough room for management. Only in this way can the enthusiasm of farmers be fully
mobilized and the efficiency of forestry management will be improved. 5.2.2 Develop differentiated policies according to forest management types to enhance the
role of forest tenure in promoting management efficiency. According to forest management
types, targeted differentiated policies should be discussed to meet the needs of farmers on
forest tenure security. Economic forests and non-timber forests have short return on
investment cycles, and farmers are more responsive to forest tenure security. The
government should guide and encourage the circulation of forestland to improve the scale
effect on the basis of stabling forest tenure, while giving appropriate financial and technical
assistance to further promote the efficiency of forestry management. The main source of
income for farmers who manage ecological public welfare forests is the ecological public
welfare forest compensation, but it is a drop in the bucket. 5.1 Conclusions The compensation mechanism of
ecological public welfare forests should be further improved, taking into account the level of
economic development and the status forest resources of each region. Instead of adopting a
“one-size-fits-all” policy, reasonable mechanism should be explored for the use of
undergrowth resources in ecological public welfare forests. Establishing the adjustment
and withdrawal mechanism of ecological public welfare forests and respecting the will of
contracted management subjects are of great significance to improve the efficiency of
forestry management. 51 5.2.3 Improve forestry supporting policies, strengthen the effect of the CFTR to assist in
promoting forestry management efficiency. Supporting policies related to the CFTR play an
important role in guiding farmers’ forestry production and management, so further
perfecting supporting policies can stimulate farmers’ forestry production and consolidate
the effect of increasing forestry income. In terms of forest tenure mortgage loans, the
threshold for mortgage application should be appropriately lowered, the approval
procedures should be simplified, the loan periods and interest rates should be rationalized
to ease the financial tension of farmers in forestry production. In terms of forestry subsidies,
there are some problems such as inconsistent standards, delayed payment or even
unpopularity, so financial support needs to be increased. Especially in terms of ecological
public welfare forest compensation, it is necessary to focus on social benefits while
safeguarding ecological benefits. In terms of forestry insurance system, the government
needs to play the role of service-oriented department, carry out policies tilt to forestry
insurance companies and promote the establishment of forestry insurance market. At the
same time, the government is supposed to guide farmers to improve their rational cognition
of forest insurance products and reasonably avoid forestry management risks. In terms of
forestry professional cooperatives, they can provide farmers with technological support and
market docking services. They can also enhance the ability to absorb capital and improve
farmers’ risk resistance and market competitiveness by standardizing management and
actively integrating resources. In terms of forestry technological services, it is necessary to
vigorously promote the popularization of forestry technologies, carry out multi-type forestry
science and technology services according to the types and characteristics of local forestry
management, and finally meet the needs of farmers’ forestry production and finally
effectively improve the utilization efficiency of input factors in forestry. 5.2.4 Promote factors circulation and moderate scale management through government
and market-led means to improve forestry management efficiency. The effect of
forest tenure on
efficiency 5.1 Conclusions Under the policy of “Three rights separated” on the land tenure
structure, moderate scale operation could be carried out, such as the promotion of family
forestry. In addition to increasing government financial input, market-based financing
channels should also be explored, and external social funds should be pulled in to realize the
exchange of forest tenure and capital elements and solve the problem of insufficient funds and
financing difficulties. Finally, the forestry labor force employment service market should be
improved, and new types of farmers should be cultivated to improve the level of human capital
in forestry. Besides, the flow of labor to deep processing and high value-added forestry
industries should be facilitated. At last, in order to overcome the shortage of forestry labor
caused by aging of rural labor force and labor migration, cooperatives may lead its members
to engage in labour in the form of joint or mutual assistance. 52 5.1 Conclusions In order to drive the
transformation of forestry operation from traditional extensive management to intensive and
efficient management, it is vital to promote the establishment of standardized forestland circulation market and rational allocation of resources through scale management. As the
most important management subjects in collective forest regions, farmers’ forest management
ways are not only related to the efficiency of forestry management, but also related to the
sustainable development of forestry management in collective forest regions. The
combination of a competent government and an effective market is the key to reverse the
decentralization of forestland management and promoting moderate scale management of
forestry. In order to reduce the production cost caused by fragmentation, improve the
management efficiency and increase forestry income, the circulation and re-incorporation of
forestland should be sped up. Under the policy of “Three rights separated” on the land tenure
structure, moderate scale operation could be carried out, such as the promotion of family
forestry. In addition to increasing government financial input, market-based financing
channels should also be explored, and external social funds should be pulled in to realize the
exchange of forest tenure and capital elements and solve the problem of insufficient funds and
financing difficulties. Finally, the forestry labor force employment service market should be
improved, and new types of farmers should be cultivated to improve the level of human capital
in forestry. Besides, the flow of labor to deep processing and high value-added forestry
industries should be facilitated. At last, in order to overcome the shortage of forestry labor
caused by aging of rural labor force and labor migration, cooperatives may lead its members
to engage in labour in the form of joint or mutual assistance. circulation market and rational allocation of resources through scale management. As the
most important management subjects in collective forest regions, farmers’ forest management
ways are not only related to the efficiency of forestry management, but also related to the
sustainable development of forestry management in collective forest regions. The
combination of a competent government and an effective market is the key to reverse the
decentralization of forestland management and promoting moderate scale management of
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(in Chinese). 55 Zhan, L.H., Su, S.P., Chen, S.F. and Li, H.D. (2016), “Forest fragmentation’s and forest land circulation
behavior’s impact on efficiency of forest resource allocation”, Resource Development and
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Agrotechnical Economics, No. 5, pp. 104-111, (in Chinese). The effect of
forest tenure on
efficiency Corresponding author Xuemei Jiang can be contacted at: jiangxm@bjfu.edu.cn Xuemei Jiang can be contacted at: jiangxm@bjfu.edu.cn For instructions on how to order reprints of this article, please visit our website:
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PI-Rads Scores and MRI-Targeted Prostate Biopsy
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JOJ urology & nephrology
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Introduction 27% of patients avoid a primary biopsy and allow to detect 5% fewer
clinically insignificant cancers. Compared with the standard
TRUS biopsy 18% percent more cases of clinically significant
cancer might be detected. Moreover, MRI as a triage test before
first prostate biopsy could reduce unnecessary biopsies by
a quarter [3]. Multiparametric MRI and MRI-guided biopsy
techniques thoughbear the risk to overlook or misscancers,
especially cancers with small volumes (<0.5ml, and low grade,
Gleason score ≤6) [4,5,9]. In a study of De Visschere the negative
predictive value for the detection rate of high-grade cancers
(tumor volume >0.5ml, Gleason score ≥7) was 95, 4% [3-5,9]. When stratifying multiparametric MRI and in-bore MRI-guided
prostate biopsies according to PI-RADS 2 the system could
identify 95% of prostate cancer foci ≥0.5ml, but this was limited
to the assessment of tumors with a Gleason score, GS≥4+3=7
[1,5,10]. One study with a critical look at PI-RADS System,
version2 summarized that a wide range of questions remains
to be answered regarding how to apply the system in directing
patient management [8]. For instance, which overall PI-RADS
assessment categories are targeted by a reasonable biopsy? In
lesions with PI-RADS 3, which are declared as intermediate “the
presence of clinically significant cancer is equivocal” [7] the
system recommends clinical follow-up. However, should we not
in contrast to that debate applying a targeted biopsy for patients
with rising PSA and a PI-RADS 3 lesion? Prostate cancer is the second most prevalent cause of cancer
death of men in Western Europe and in the United States [1]. For patients with high clinical suspicion for prostate cancer
(abnormal digital rectal examination and/or elevated prostate-
specific antigen (PSA) level, the standard diagnostic tool for the
diagnosis is the systematic trans rectal ultrasonography (US)-
guided 10 to 12 core biopsy [1-4]. Multiparametric prostate
magnetic resonance (MR) imaging by applying T2-weighted
imaging, diffusion-weighted imaging (DWI), and dynamic
contrast-enhanced imaging (DCI) has been proven to improve
the early detection, and localization of prostate cancer [1-5]. Over the past 10 years, the use of multiparametric MRI as
a tool for biopsy targeting of suspicious lesions for prostate
cancer revealed significantly higher cancer detection rates. Opinion
Volume 4 Issue 2 - November 2017
DOI: 10.19080/JOJUN.2017.04.555633
JOJ uro & nephron
Copyright © All rights are reserved by Karl Engelhard Opinion
Volume 4 Issue 2 - November 2017
DOI: 10.19080/JOJUN.2017.04.555633
JOJ uro & nephron
Copyright © All rights are reserved by Karl Engelhard Opinion
Volume 4 Issue 2 - November 2017
DOI: 10.19080/JOJUN.2017.04.555633 Copyright © All rights are reserved by Karl Engelhard Karl Engelhard* and Florian Schneider Karl Engelhard* and Florian Schneider University Erlangen-Nuremberg, Hospital Martha-Maria, Germany
Submission: October 27, 2017; Published: November 07, 2017 *Corresponding author: Karl Engelhard, University Erlangen-Nuremberg, Hospital Martha-Maria, Nuremberg, Germany, Tel:
;
Fax: +499119591174; Email: Abbrevations: PSA: Prostate Specific Antigen; US: Ultra Sonography; ACC: American College of Radiology; ESUR: European Society of
Urogenital Radiology; PI-RADS: Prostate Imaging Reporting and Data System Introduction Overall, there exist a variety of methods, such as cognitive
fusion, MRI-Ultrasound (US) fusion and direct in-bore MR-
guided biopsy which can be compared with standard template-
based the Prostate Imaging Reporting and Data System (PI-
RADS), an expert consensus document has been introduced by
the European Society of Urogenital Radiology (ESUR) and the
American College of Radiology (ACC) as a standardized lexicon
and diagnostic instrument for interpretation of multiparametric
prostate MR images [6]. This stratified the malignant capability
of individual lesions that could be detected on MR images. PI-
RADS version 2, the improved system of the first version was
updated and published in 2104. With PI-RADS version 2, the
evaluation of DCE imaging was simplified and the criteria for
category 3 lesions were elucidated [1,7,8]. In a multicenter,
paired-cohort, confirmatory study [3]. In our institute we evaluated the impact of PI-RADS 3 score
in differentiating these equivocal lesions as benign or malignant
for 54 men with elevated PSA levels (PSA>4ng/ml) and abnormal
multiparametric MRI. For patients with PI-RADS score 4 and 5
we ruled out the sensitivity and the positive predictive value of
MRI-guided prostate biopsy in determining positive histological
tumor results. 34 men were biopsy naive, 20 patients had prior Hashim U Ahmed could show, that the use of multi-parametric
MRI for patients with a suspicion of prostate cancer might allow JOJ uro & nephron 4(2): JOJUN.MS.ID.5555633 (2017) 001 JOJ Urology & Nephrology tothe Gleason Score with Postprostatectomy Histopathologic control-a
Targeted Biopsy-Only Strategy with Limited Number of Cores. Acad
Radiol 22(11): 1409-1418. tumor negative biopsies. The in-bore MRI-guided prostate
biopsies were performed in a standard 1.5 Tesla scanner. A mean
of 2.2 cores was taken from each tumor suspected lesion. The PI-
RADS scores of the tumor suspected areas were compared with
the histological findings of the biopsy. By applying the PI-RADS
system 54% of cancers were detected by MRI-guided biopsy with
a rate of 90% of significant tumors. In 25 cases of PI-RADS score
3 no cancerous tissue was found in the histology report. In one
man a high grade prostatic intraepithelial neoplasia (PIN) was
detected. The sensitivity of PI-RADS scores 4 and 5 was 90%, by
a positive predictive value of 70%. 3. Ahmed HU, Bosaily AS, Brown LC, Gabe R, Kaplan R, et al. (2017)
Diagnostic accuracy of multi-parametric MRI and TRUS biopsy in
prostate cancer (PROMIS): a paired validating confirmatory study. Introduction Lancet 389(10071): 815-822. 4. Felker ER, Lee-Felker SA, Feller J, Margolis DJ, Lu DS, et al. (2016) In-
bore magnetic resonance-guided transrectal biopsy forthe detection
of clinically significant prostate cancer. Abdom Radiol 41(5): 954-962. 5. Wang R, Wang H, Zhao C, Hu J, Jiang Y, et al. (2015) Evaluation of
Multiparametric Magnetic Resonance Imaging in Detection and
Prediction of Prostate Cancer. PLoS One 10(6): e0130207. Conclusion 6. Arsov C, Rabenalt R, Blondin D, Quentin M, Hiester A, et al. (2015)
Prospective Randomized Trial Comparing Magnetic Resonance
Imaging (MRI)-guided In-bore Biopsy to MRI-ultrasound Fusion and
Transrectal Ultrasound-guided Prostate Biopsy in Patients with Prior
Negative Biopsies. Eur Urol 68(4): 713-720. Based on our population we could assess a highly sensitivity
and a fair positive predictive value in detecting significant
prostate cancers by stratifying the MRI-guided biopsy with the
PI-RADS system. For patients with PI-RADS 3 lesions a patient
management is necessary in order to differentiate candidates
fitting for a clinical follow up or suitable for a targeting biopsy. PI-RADS 3 score could not be confirmed as an absolute marker in
patient clinical management care. In our study PI-RADS 3 lesions
revealed only benign conditions. 7. Turkbey B, Choyke PL (2015) PIRADS 2.0: what is new? Diagn Interv
Radiol 21(5): 283-285. 8. Rosenkrantz AB, Aytekin O, Turkbey B, Westphalen AC (2016) Prostate
Imaging Reporting and Data System (PI-RADS), Version 2: A Critical
Look. AJR Am J Roentgenol 206(6): 1179-1183. 9. De Vischere PJL, Naesens L, libbrecht L, Van Praet C, Limen N, et al. (2016) What kind of prostate cancers do we miss on multiparametric
magnetic resonance imaging? Eur Radiol 26(4):1098-1107. 2. Garmer M, Busch M, Mateiescu S, Fahlbusch DE, Wegener B, et al.
(2015) Accuracy of MRI-Targeted in-Bore Prostate Biopsy According This work is licensed under Creative
Commons Attribution 4.0 License
DOI: 10.19080/JOJUN.2017.04.555633 References 1. Tan N, Lin WC, Khosnoodi P, Asvadi NH, Yoshida J, et al. (2016) In-Bore
3-T MR-guided Transrectal Targeted Prostate Biopsy: Prostate Imaging
Reporting and Data System Version 2-based Diagnostic Performance
for Detection of Prostate Cancer. Radiology 283(1) 130-139. 1. Tan N, Lin WC, Khosnoodi P, Asvadi NH, Yoshida J, et al. (2016) In-Bore
3-T MR-guided Transrectal Targeted Prostate Biopsy: Prostate Imaging
Reporting and Data System Version 2-based Diagnostic Performance
for Detection of Prostate Cancer. Radiology 283(1) 130-139. 10. Vargas HA, Hötker AM, Goldman DA, Moskowitz CS, GondoT,et al. (2016) Updated prostate imaging reporting and data system (PIRADS
v2) recommendations for the detection of clinically siginificant
prostate cancer using multiparametric MRI: critical evaluation using
whole.mount pathology as standard of reference. Eur Radiol 26(6):
1606-1612. 2. Garmer M, Busch M, Mateiescu S, Fahlbusch DE, Wegener B, et al. (2015) Accuracy of MRI-Targeted in-Bore Prostate Biopsy According Your next submission with Juniper Publishers
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https://juniperpublishers.com/online-submission.php article: Karl Engelhard, Florian Schneider. PI-Rads Scores and MRI-Targeted Prostate Biopsy. JOJ uro & nephron. 2017; 4(2):
19080/JOJUN.2017.04.555633. 002
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Distinct TP53 Mutation Types Exhibit Increased Sensitivity to Ferroptosis Independently of Changes in Iron Regulatory Protein Activity
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International journal of molecular sciences
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Received: 13 August 2020; Accepted: 12 September 2020; Published: 15 September 2020 Received: 13 August 2020; Accepted: 12 September 2020; Published: 15 September 2020 Abstract: The tumor suppressor gene TP53 is the most commonly mutated gene in human cancer. In addition to loss of tumor suppressor functions, mutations in TP53 promote cancer progression by
altering cellular iron acquisition and metabolism. A newly identified role for TP53 in the coordination
of iron homeostasis and cancer cell survival lies in the ability for TP53 to protect against ferroptosis,
a form of iron-mediated cell death. The purpose of this study was to determine the extent to
which TP53 mutation status affects the cellular response to ferroptosis induction. Using H1299 cells,
which are null for TP53, we generated cell lines expressing either a tetracycline inducible wild-type
(WT) TP53 gene, or a representative mutated TP53 gene from six exemplary “hotspot” mutations
in the DNA binding domain (R273H, R248Q, R282W, R175H, G245S, and R249S). TP53 mutants
(R273H, R248Q, R175H, G245S, and R249S) exhibited increased sensitivity ferroptosis compared to
cells expressing WT TP53. As iron-mediated lipid peroxidation is critical for ferroptosis induction,
we hypothesized that iron acquisition pathways would be upregulated in mutant TP53-expressing
cells. However, only cells expressing the R248Q, R175H, and G245S TP53 mutation types exhibited
statistically significant increases in spontaneous iron regulatory protein (IRP) RNA binding activity
following ferroptosis activation. Moreover, changes in the expression of downstream IRP targets
were inconsistent with the observed differences in sensitivity to ferroptosis. These findings reveal
that canonical iron regulatory pathways are bypassed during ferroptotic cell death. These results
also indicate that induction of ferroptosis may be an effective therapeutic approach for tumor cells
expressing distinct TP53 mutation types. Keywords: mutant TP53; ferroptosis; iron metabolism; iron regulatory proteins; cancer Laurie R. Thompson, Thais G. Oliveira
, Evan R. Hermann, Winyoo Chowanadisai
,
Stephen L. Clarke and McKale R. Montgomery * Laurie R. Thompson, Thais G. Oliveira
, Evan R. Hermann, Winyoo Chowanadisai
,
Stephen L. Clarke and McKale R. Montgomery * Department of Nutritional Sciences, Oklahoma State University, Stillwater, OK 74074, USA;
laurie.thompson@okstate.edu (L.R.T.); thais.g.oliveira@okstate.edu (T.G.O.);
evan.hermann@okstate.edu (E.R.H.); winyoo.chowanadisai@okstate.edu (W.C.);
stephen.clarke@okstate.edu (S.L.C.) Department of Nutritional Sciences, Oklahoma State University, Stillwater, OK 74074, USA
laurie.thompson@okstate.edu (L.R.T.); thais.g.oliveira@okstate.edu (T.G.O.);
evan.hermann@okstate.edu (E.R.H.); winyoo.chowanadisai@okstate.edu (W.C.);
stephen.clarke@okstate.edu (S.L.C.) * Correspondence: mckale.montgomery@okstate.edu; Tel.: +1-405-744-4437 * Correspondence: mckale.montgomery@okstate.edu; Tel.: +1-405-744-4437 International Journal of
Molecular Sciences International Journal of
Molecular Sciences 1. Introduction Iron is an essential, yet potentially toxic nutrient that can contribute to both the initiation
and progression of cancer [1–4]. The tumor suppressor TP53 can protect against carcinogenesis by
contributing to the regulation of cellular iron homeostasis [5–7]. Unfortunately, TP53 is mutated
in nearly half of all human cancers. Mutations in TP53 can lead to both loss of tumor suppressive
functions and the acquisition of oncogenic traits, but the influence on cellular iron homeostasis has yet
to be fully described. The cytosolic mRNA binding proteins, iron regulatory proteins (IRP1 and IRP2), function as
key regulators of cellular iron homeostasis by coordinating iron uptake, storage, and utilization in Int. J. Mol. Sci. 2020, 21, 6751; doi:10.3390/ijms21186751 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms 2 of 18 Int. J. Mol. Sci. 2020, 21, 6751 accordance with cellular iron availability. When iron availability is limited, the binding of IRPs to
iron responsive elements (IREs) within the 5′ untranslated region of mRNA such as ferritin heavy
chain 1 (FTH1) results in translational inhibition [8]. Conversely, binding to IRE in the 3′ untranslated
regions of mRNA, such transferrin receptor 1 (TFRC) promotes transcript stabilization. The net effect
is to subsequently decrease iron storage and increase iron uptake, respectively. Under iron sufficient
conditions, IRP2 is subject to proteasomal degradation, while IRP1 is regulated via the insertion of a
Fe-S cluster, which prevents IRE binding [9]. In cancer, IRP signaling can be corrupted in an effort to acquire sufficient iron to support rapid cell
proliferation. For example, IRP2 overexpression in breast cancer results in increased TFRC expression,
decreased ferritin expression, and subsequently an increased labile iron pool [10]. Increased expression
of TFRC also worsens clinical prognosis in patients with renal cell carcinoma [11]. As mentioned
above, increased expression of TFRC is typically mediated by increased IRP mRNA binding activity,
but overexpression of IRP1 was actually found to decrease tumor growth in vivo [12]. Thus, despite
their similar roles in the maintenance of iron homeostasis, IRP1 and IRP2 exhibit opposing phenotypes
in the reduction and promotion of tumor growth, respectively. Therefore, continued investigation into
the roles IRPs play in cancer progression is warranted. Ferroptosis is an iron-dependent form of programmed cell death with broad chemotherapeutic
potential [13,14]. Driven by the iron-dependent accumulation of lipid reactive oxygen species (ROS),
the import of iron by TFRC-mediated iron uptake is an essential component of ferroptotic cell death [15]. 2. Results 2.1. TP53 Mutation Status Influences Sensitivity to Erastin-Induced Ferroptotic Cell Death Mutation Status Influences Sensitivity to Erastin-Induced Ferroptotic Cell Death To assess the influence of TP53 mutation status on ferroptosis sensitivity, H1299 cells expressing
tetracycline-inducible wild-type (WT) or mutant TP53 plasmids were generated, and their protein
expression was validated via Western blot (Figure 1A). These mutations were selected because they
represent the most commonly observed TP53 mutation types in human cancers [21]. TP53 expression
levels were variable between WT and mutant TP53-expressing subtypes, but we have previously
demonstrated that even a low level induction of WT TP53 protein is sufficient to induce expression
of the WT TP53 target, CDKN1A [22]. We also showed that CDKN1A expression was not increased
by the induction of any of the mutant TP53 subtypes examined [22]. This is important because
upregulation of CDKN1A expression appears to be critical for suppression of ferroptosis [18]. Thus,
we hypothesized that mutant TP53-expressing cells would be more sensitive to ferroptosis induction. TP53-dependent differences in ferroptosis sensitivity were determined by comparing differences in cell
viability following treatment with erastin, a potent inducer of ferroptosis [13]. A two-factor ANOVA
was conducted to compare the effects of TP53 mutation type on cell viability following treatment
with erastin or erastin + ferrostatin confirmed a significant erastin effect, indicating that all cells
examined were susceptible to at least some degree of ferroptotic cell death (Figure 1B). Additional
post hoc analyses revealed that induction of R273H, R248Q, R175H, G245S, and R249S TP53 mutations
significantly increased sensitivity to ferroptosis induction, as evidenced by reduced cell viability
compared to the WT TP53-expressing cells following erastin treatment (Figure 1B). The TP53 null
(H1299) cells and cells expressing the R282W mutation type did not exhibit differences in sensitivity to
erastin treatment compared to the cells expressing WT TP53 (Figure 1B). Importantly, co-treatment
with ferrostatin, a potent ferroptosis inhibitor [23], was sufficient to rescue all cell types, demonstrating
that the reduction in cell viability was indeed due to ferroptosis (Figure 1B). These results indicate that
induction of distinct TP53 mutation type is sufficient to increase sensitivity of cells to iron-mediated
ferroptotic cell death. 2.2. Ferroptosis Induction Differentially Affects IRP mRNA Binding Activity in Cells Expressing Distinct
TP53 Mutation Types We have previously established that induction of distinct TP53 mutation types differentially
influences IRP RNA binding activity and diminishes IRP responsiveness to changes in cellular iron
availability [22]. 1. Introduction The increased expression of IRP2 and degradation of FTH1 have also been hypothesized to serve as
critical contributors to ferroptosis induction, presumably as a means of increasing the redox-active
labile iron pool [13,16]. Yet, the contribution of the IRE-IRP system to iron accumulation during
ferroptotic cell death remains unclear. Intriguingly, wild-type (WT) TP53 can contribute to both IRP regulation [5,7] and ferroptosis
sensitivity in human cancer cells, though contradictory roles have been reported [17–19]. These mixed
findings are likely attributable to the context-dependent TP53-mediated upregulation of Cyclin
Dependent Kinase Inhibitor 1A (CDKN1A) expression that appears to be critical for suppression of
ferroptosis [18]. Posttranslational modifications and/or genetic mutations within TP53 can render it
unable to induce CDKN1A in some cell types resulting in increased ferroptosis sensitivity [17,20]. However, the mutants in these studies were acetylation defective mutants and not representative of
the more common TP53 mutations within the DNA binding domain most often observed in human
cancers. Moreover, the capacity for distinct TP53 subtypes to protect against ferroptosis by modulating
IRP has yet to be fully investigated. The purpose of this study was to determine the extent to which ferroptosis induction influences
iron metabolism and ferroptotic cell death in cells with distinct TP53 mutation types. We hypothesized
that impaired IRP-mediated signaling pathways would render cells harboring TP53 mutations more
susceptible to ferroptotic cell death than cells expressing wild-type TP53. To test this hypothesis we
assessed cell viability, IRP mRNA binding activity, and iron-mediated lipid peroxidation following
erastin induction in isogenic cell lines expressing inducible versions of the six most commonly observed
TP53 mutations in human cancers. We established that induction of distinct TP53 mutations alone
significantly increased sensitivity to ferroptotic cell death. Contrary to our hypothesis, however,
we show that mutant TP53-dependent differences in ferroptotic sensitivity are independent of IRP
regulation, but rather, are likely multifactorial and dependent upon TP53 mutation type. These findings
provide further evidence of the many phenotypic differences that can be observed between distinct TP53
mutation types, and illustrate the importance of distinguishing between subtypes when investigating
mutant TP53-dependent outcomes. Int. J. Mol. Sci. 2020, 21, 6751 3 of 18 2. Results 2. Results As such, we hypothesized that mutant TP53-dependent differences in ferroptosis
sensitivity might be influenced by mutant TP53-dependent differences in IRP RNA binding activity
in response to erastin treatment. To examine the impact of ferroptosis induction on IRP function
and expression, we quantitatively assessed spontaneous mRNA binding activity and total IRP
mRNA binding capacity through an electrophoretic mobility assay (EMSA). Spontaneous IRP binding
measures the amount of IRP1 and IRP2 in their active RNA binding forms under the specified control
or treatment conditions. As iron-mediated lipid peroxidation is critical for ferroptosis induction [13],
we hypothesized that spontaneous IRP mRNA binding activity would be increased in response to
erastin treatment. However, despite upward trends in the TP53 null, WT TP53, and R273H TP53
mutants, only cells expressing the R248Q (p = 0.019), R175H (p = 0.015), and R245S (p = 0.027) TP53
mutation types exhibited statistically significant increases in spontaneous IRP RNA binding activity
following erastin treatment (Figure 2A,B). Thus, erastin-mediated increases in IRP RNA binding activity
cannot fully explain the increased sensitivity to ferroptotic cell death in all mutant TP53-expressing
cell types. 4 of 18
ivity of Int. J. Mol. Sci. 2020, 21, 6751
results indicate that ind
cells to iron-mediated fe Figure 1. TP53 mutation status influences ferroptosis sensitivity. H1299 (TP53 null) cells were
transfected with either a tetracycline inducible wild-type (WT) TP53 or a representative contact (273H,
248Q, 282W) or conformational (175H, 245S, 249S) mutant TP53. (A) Tetracycline-inducible
expression of WT and mutant TP53 expression was confirmed by Western blot. Glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Cell viability measured
following 24 h of treatment with DMSO (Control), 10 µM erastin, or 10 µM each of erastin and
ferrostatin-1 (n = 4 per cell type and treatment group). Superscripts (a,b) denote statistical significance,
p < 0.05. Treatments that share the same superscripts are not significantly different. Error bars
Figure 1. TP53 mutation status influences ferroptosis sensitivity. H1299 (TP53 null) cells were
transfected with either a tetracycline inducible wild-type (WT) TP53 or a representative contact (273H,
248Q, 282W) or conformational (175H, 245S, 249S) mutant TP53. (A) Tetracycline-inducible expression
of WT and mutant TP53 expression was confirmed by Western blot. Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) was used as a loading control. 2. Results (B) Cell viability measured following 24 h of
treatment with DMSO (Control), 10 µM erastin, or 10 µM each of erastin and ferrostatin-1 (n = 4 per
cell type and treatment group). Superscripts (a,b) denote statistical significance, p < 0.05. Treatments
that share the same superscripts are not significantly different. Error bars indicate SEM. Figure 1. TP53 mutation status influences ferroptosis sensitivity. H1299 (TP53 null) cells were
transfected with either a tetracycline inducible wild-type (WT) TP53 or a representative contact (273H,
248Q, 282W) or conformational (175H, 245S, 249S) mutant TP53. (A) Tetracycline-inducible
expression of WT and mutant TP53 expression was confirmed by Western blot. Glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Cell viability measured
following 24 h of treatment with DMSO (Control), 10 µM erastin, or 10 µM each of erastin and
ferrostatin-1 (n = 4 per cell type and treatment group). Superscripts (a,b) denote statistical significance,
p < 0.05. Treatments that share the same superscripts are not significantly different. Error bars
Figure 1. TP53 mutation status influences ferroptosis sensitivity. H1299 (TP53 null) cells were
transfected with either a tetracycline inducible wild-type (WT) TP53 or a representative contact (273H,
248Q, 282W) or conformational (175H, 245S, 249S) mutant TP53. (A) Tetracycline-inducible expression
of WT and mutant TP53 expression was confirmed by Western blot. Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) was used as a loading control. (B) Cell viability measured following 24 h of
treatment with DMSO (Control), 10 µM erastin, or 10 µM each of erastin and ferrostatin-1 (n = 4 per
cell type and treatment group). Superscripts (a,b) denote statistical significance, p < 0.05. Treatments
that share the same superscripts are not significantly different. Error bars indicate SEM. Figure 1. TP53 mutation status influences ferroptosis sensitivity. H1299 (TP53 null) cells were
transfected with either a tetracycline inducible wild-type (WT) TP53 or a representative contact (273H,
248Q, 282W) or conformational (175H, 245S, 249S) mutant TP53. (A) Tetracycline-inducible
expression of WT and mutant TP53 expression was confirmed by Western blot. Glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Cell viability measured
following 24 h of treatment with DMSO (Control), 10 µM erastin, or 10 µM each of erastin and
ferrostatin-1 (n = 4 per cell type and treatment group). Superscripts (a,b) denote statistical significance,
p < 0.05. Treatments that share the same superscripts are not significantly different. Error bars
Figure 1. 2. Results TP53 mutation status influences ferroptosis sensitivity. H1299 (TP53 null) cells were
transfected with either a tetracycline inducible wild-type (WT) TP53 or a representative contact (273H,
248Q, 282W) or conformational (175H, 245S, 249S) mutant TP53. (A) Tetracycline-inducible expression
of WT and mutant TP53 expression was confirmed by Western blot. Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) was used as a loading control. (B) Cell viability measured following 24 h of
treatment with DMSO (Control), 10 µM erastin, or 10 µM each of erastin and ferrostatin-1 (n = 4 per
cell type and treatment group). Superscripts (a,b) denote statistical significance, p < 0.05. Treatments
that share the same superscripts are not significantly different. Error bars indicate SEM. indicate SEM. 2.2. Ferroptosis Induction Differentially Affects IRP mRNA Binding Activity in Cells Expressing Distinct
TP53 Mutation Types
The addition of 2-mercaptaethanol transiently prevents Fe-S assembly which allows for
the measurement of total IRP1 protein levels, and thus reflects total IRP1 mRNA binding capacity [24]. This assay also informs us about the relative distribution of IRP1 in its mRNA binding form or its Fe-S
cluster containing enzymatic form [25]. Total IRP1 mRNA binding activity was not affected by erastin
treatment (Figure 2A,C). These results indicate that changes in spontaneous IRP binding activity were
not the result of changes in the total pool of available IRP1 protein. Because human IRP1 and IRP2 do
not separate during standard gel-shift analyses [26], the relative contributions of IRP1 versus IRP2 to
IRE binding cannot be determined based on our current data. Therefore, the increased spontaneous
IRP mRNA binding observed reflects either a removal of the Fe-S cluster from IRP1 RNA binding site,
or an increased stability of IRP2 protein in response to erastin treatment. 2.3. Ferroptosis Induction and TP53 Status do not Influence Iron-Related mRNA Abundance The import of iron by TFRC and degradation of ferritin via ferritinophagy are critical components
of ferroptotic cell death, with both IRP-dependent and -independent modes of regulation [15,16]. Therefore, we also investigated the influence of TP53 mutation types and ferroptosis induction on
the expression of TFRC and the ferritinophagy-related genes, nuclear receptor coactivator 4 (NCOA4),
autophagy related 5 (ATG5), and CDGSG iron sulfur domain 1 (CISD1). First, we assessed the influence
of induction of TP53 expression alone on iron-related mRNAs by normalizing changes in the relative
abundance of the indicated mRNAs to that expressed in the TP53 null H1299 cell line (Figure 3A–E). A one-way between-subjects ANOVA comparing the effects of TP53 mutation type TFRC mRNA
expression identified a significant effect at the p < 0.05 level for the eight cell types (F(7,16) = 5.302,
p = 0.003). However, further post hoc analyses using Tukey HSD only revealed a significant difference
between the TP53 null H1299 cells and the G245S-expressing mutants (Figure 3A). No other differences
were observed following the induction of WT or mutant TP53 expression (Figure 3B–E). TFRC mRNA 5 of 18 Int. J. Mol. Sci. 2020, 21, 6751 abundance was not affected by erastin treatment in any of the cell lines examined (Figure 3F). Thus,
even though TFRC plays an essential role in ferroptosis [15], our data indicate that upregulation of
TFRC is not necessary for ferroptosis to occur. NCOA4 mRNA expression significantly increased
following erastin treatment in H1299 cells expressing the R248Q TP53 mutant (Figure 3G). Conversely,
CISD1 mRNA abundance significantly decreased following erastin treatment in H1299 cells expressing
the R273H TP53 mutant (Figure 3I). We also examined the mRNA abundance of Solute Carrier family 7
member 11 (SLC7A11), which is required for cystine import and has been shown to be upregulated
following erastin treatment [18,23]. Importantly, there was a significant increase in SLC7A11 mRNA
abundance following erastin in each TP53-expressing subtype (Figure 3J). Thus, the observed increase
in SLC7A11 mRNA expression provides supporting evidence that ferroptosis was induced despite
the lack of change in iron-related gene expression. . Mol. Sci. 2020, 21, x FOR PEER REVIEW
5 o gure 2. Erastin treatment increases iron regulatory protein (IRP)1/2 RNA binding activity in distin
utant TP53-expressing subtypes. 2.3. Ferroptosis Induction and TP53 Status do not Influence Iron-Related mRNA Abundance Relative mRNA expression of (A) transferrin receptor 1 (TFRC) (B) nuclear
receptor coactivator 4 (NCOA4), (C) autophagy related 5 (ATG5), (D) CDGSG iron sulfur domain 1
(CISD1), and (E) solute carrier family 7 member 11 (SLC7A11) in TP53 null H1299 cells (H1299) or
H1299 cells transfected with wild-type (WT) TP53, or the indicated TP53 mutant (n = 3 per cell type
and treatment group). Relative mRNA expression of (F) TFRC, (G) NCOA4, (H) ATG5, (I) CISD1, and
(J) SLC7A11 in TP53 null H1299 cells (H1299) or H1299 cells transfected with wild-type (WT) TP53, or
the indicated mutant TP53 following treatment with DMSO (control) or 10 µM erastin for 24 h. a
Denotes statistical difference from H1299. * Denotes statistical differences from respective control, p
< 0 05 Error bars indicate SEM
Figure 3. Iron-uptake and ferritinophagy-related gene expression are not affected by TP53 mutation
status or erastin treatment. Relative mRNA expression of (A) transferrin receptor 1 (TFRC) (B) nuclear
receptor coactivator 4 (NCOA4), (C) autophagy related 5 (ATG5), (D) CDGSG iron sulfur domain 1
(CISD1), and (E) solute carrier family 7 member 11 (SLC7A11) in TP53 null H1299 cells (H1299) or
H1299 cells transfected with wild-type (WT) TP53, or the indicated TP53 mutant (n = 3 per cell type
and treatment group). Relative mRNA expression of (F) TFRC, (G) NCOA4, (H) ATG5, (I) CISD1,
and (J) SLC7A11 in TP53 null H1299 cells (H1299) or H1299 cells transfected with wild-type (WT) TP53,
or the indicated mutant TP53 following treatment with DMSO (control) or 10 µM erastin for 24 h. a Denotes statistical difference from H1299. * Denotes statistical differences from respective control,
p < 0.05. Error bars indicate SEM. J
,
, Figure 3 Iron uptake and ferritinophagy related gene expression are not affected by TP53 mutation
Figure 3 Iron-uptake and ferritinophagy-related gene expression are not affected by TP53 mutation Figure 3. Iron-uptake and ferritinophagy-related gene expression are not affected by TP53 mutation
Figure 3. Iron-uptake and ferritinophagy-related gene expression are not affected by TP53 mutation Figure 3. Iron-uptake and ferritinophagy-related gene expression are not affected by TP53 mutation
status or erastin treatment. 2.3. Ferroptosis Induction and TP53 Status do not Influence Iron-Related mRNA Abundance (A,B) Spontaneous IRP RNA binding was in tetracycline-induce
T and mutant TP53 (n = 3–4 per cell type and treatment) expressing subtypes following treatmen
th DMSO (control) or 10 µM erastin (Erastin) for 24 h. * Denotes statistical differences from
pective control, p < 0.05. (A,C) Total IRP RNA binding capacity was measured by assaying the cel
der the same conditions in the presences of 2-mercaptoethanol. Error bars indicate SEM. Figure 2. Erastin treatment increases iron regulatory protein (IRP)1/2 RNA binding activity in distinct
mutant TP53-expressing subtypes. (A,B) Spontaneous IRP RNA binding was in tetracycline-induced
WT and mutant TP53 (n = 3–4 per cell type and treatment) expressing subtypes following treatment
with DMSO (control) or 10 µM erastin (Erastin) for 24 h. * Denotes statistical differences from respective
control, p < 0.05. (A,C) Total IRP RNA binding capacity was measured by assaying the cells under
the same conditions in the presences of 2-mercaptoethanol. Error bars indicate SEM. ure 2. Erastin treatment increases iron regulatory protein (IRP)1/2 RNA binding activity in distin
tant TP53-expressing subtypes. (A,B) Spontaneous IRP RNA binding was in tetracycline-induc
and mutant TP53 (n = 3–4 per cell type and treatment) expressing subtypes following treatme
h DMSO (control) or 10 µM erastin (Erastin) for 24 h. * Denotes statistical differences fro
pective control, p < 0.05. (A,C) Total IRP RNA binding capacity was measured by assaying the ce
der the same conditions in the presences of 2 mercaptoethanol Error bars indicate SEM
Figure 2. Erastin treatment increases iron regulatory protein (IRP)1/2 RNA binding activity in distinct
mutant TP53-expressing subtypes. (A,B) Spontaneous IRP RNA binding was in tetracycline-induced
WT and mutant TP53 (n = 3–4 per cell type and treatment) expressing subtypes following treatment
with DMSO (control) or 10 µM erastin (Erastin) for 24 h. * Denotes statistical differences from respective
control, p < 0.05. (A,C) Total IRP RNA binding capacity was measured by assaying the cells under
the same conditions in the presences of 2-mercaptoethanol. Error bars indicate SEM. 6 of 18
7 of 19 Int. J. Mol. Sci. 2020, 21, 6751
Int J Mol Sci 2020 21 x FOR J M
S
0 0,
, 6 5
6 o
nt. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW
7 of
Figure 3. Iron-uptake and ferritinophagy-related gene expression are not affected by TP53 mutation
status or erastin treatment. 2.3. Ferroptosis Induction and TP53 Status do not Influence Iron-Related mRNA Abundance Relative mRNA expression of (A) transferrin receptor 1 (TFRC) (B) nuclear
receptor coactivator 4 (NCOA4), (C) autophagy related 5 (ATG5), (D) CDGSG iron sulfur domain 1
(CISD1), and (E) solute carrier family 7 member 11 (SLC7A11) in TP53 null H1299 cells (H1299) or
H1299 cells transfected with wild-type (WT) TP53, or the indicated TP53 mutant (n = 3 per cell type
and treatment group). Relative mRNA expression of (F) TFRC, (G) NCOA4, (H) ATG5, (I) CISD1, and
(J) SLC7A11 in TP53 null H1299 cells (H1299) or H1299 cells transfected with wild-type (WT) TP53, or
the indicated mutant TP53 following treatment with DMSO (control) or 10 µM erastin for 24 h. a
Denotes statistical difference from H1299. * Denotes statistical differences from respective control, p
< 0.05. Error bars indicate SEM. Figure 3. Iron-uptake and ferritinophagy-related gene expression are not affected by TP53 mutation
status or erastin treatment. Relative mRNA expression of (A) transferrin receptor 1 (TFRC) (B) nuclear
receptor coactivator 4 (NCOA4), (C) autophagy related 5 (ATG5), (D) CDGSG iron sulfur domain 1
(CISD1), and (E) solute carrier family 7 member 11 (SLC7A11) in TP53 null H1299 cells (H1299) or
H1299 cells transfected with wild-type (WT) TP53, or the indicated TP53 mutant (n = 3 per cell type
and treatment group). Relative mRNA expression of (F) TFRC, (G) NCOA4, (H) ATG5, (I) CISD1,
and (J) SLC7A11 in TP53 null H1299 cells (H1299) or H1299 cells transfected with wild-type (WT) TP53,
or the indicated mutant TP53 following treatment with DMSO (control) or 10 µM erastin for 24 h. a Denotes statistical difference from H1299. * Denotes statistical differences from respective control,
p < 0.05. Error bars indicate SEM. Int. J. Mol. Sci. 2020, 21, 6751 7 of 18 2.4. FTH1, but not TFRC Protein Expression, Is Altered by Erastin Treatment in Cells Expressing Distinct tp53
Mutation Types 2.4. FTH1, but not TFRC Protein Expression, Is Altered by Erastin Treatment in Cells Expressing Distinct tp53
Mutation Types 2.4. FTH1, but not TFRC Protein Expression, Is Altered by Erastin Treatment in Cells Expressing Distinct tp53
Mutation Types While the IRP-mediated regulation of TFRC is modulated at the level of mRNA transcript stability,
IRPs regulate FTH1 expression via translational inhibition [8]. Therefore, we used Western blots
analyses to examine the effects of erastin treatment on the expression FTH1 in each of the cell lines
described above. 2.3. Ferroptosis Induction and TP53 Status do not Influence Iron-Related mRNA Abundance Unexpectedly, there was a significant increase in FTH1 protein expression following
ferroptosis induction in R282W (p = 0.004), and R249S (p = 0.013) mutant TP53-expressing cells, but a
significant decrease in FTH1 expression in the R273H-expressing mutants (p = 0.020) (Figure 4A). It is worth noting however, that IRP RNA binding activity was not significantly altered in any of
the cell types in which FTH1 expression was changed. Independently of iron and IRPs, FTH1 can also
be transcriptionally activated in response to oxidative stress [27,28]. As increased lipid peroxidation
is a hallmark of ferroptosis, the increase in FTH1 expression in the R282W and R249S TP53 mutants
could reflect an oxidative stress response. Given the importance of TFRC-mediated iron uptake to
ferroptosis induction [15], and previous reports of post-translational regulation of TFRC expression
independently of IRP [29–31], we also examined the protein expression of TFRC following erastin
treatment. Consistent with our mRNA data however, there were no significant changes in TFRC
protein expression following ferroptosis induction in any of the cell lines examined (Figure 4B). Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW
8 of 19 Figure 4. Erastin treatment differentially affects TFRC and ferritin heavy chain 1 (FTH1) protein
expression in cells expressing distinct TP53 mutation types. Representative images and quantification
of Western blots examining the expression of (A,B) transferrin receptor 1 (TFRC) and (C,D) ferritin
heavy chain 1 (FTH1) in TP53 null H1299 cells (H1299) or H1299 cells transfected with wild-type (WT)
TP53, or the indicated TP53 mutant following treatment with DMSO (control) or 10 µM erastin for 24
h (n = 3-4 per cell type and treatment). Relative expression was normalized using glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) as a loading control. * Denotes statistical differences from
respective control, p < 0.05. Error bars indicate SEM. 2.5. TP53 Mutation Status Influences Basal Levels of Lipid Peroxidation and Subsequent Responsiveness to
Figure 4. Erastin treatment differentially affects TFRC and ferritin heavy chain 1 (FTH1) protein
expression in cells expressing distinct TP53 mutation types. Representative images and quantification
of Western blots examining the expression of (A,B) transferrin receptor 1 (TFRC) and (C,D) ferritin
heavy chain 1 (FTH1) in TP53 null H1299 cells (H1299) or H1299 cells transfected with wild-type (WT)
TP53, or the indicated TP53 mutant following treatment with DMSO (control) or 10 µM erastin for
24 h (n = 3–4 per cell type and treatment). 2.3. Ferroptosis Induction and TP53 Status do not Influence Iron-Related mRNA Abundance Relative expression was normalized using glyceraldehyde
3-phosphate dehydrogenase (GAPDH) as a loading control. * Denotes statistical differences from
respective control, p < 0.05. Error bars indicate SEM. Figure 4. Erastin treatment differentially affects TFRC and ferritin heavy chain 1 (FTH1) protein
expression in cells expressing distinct TP53 mutation types. Representative images and quantification
of Western blots examining the expression of (A,B) transferrin receptor 1 (TFRC) and (C,D) ferritin
heavy chain 1 (FTH1) in TP53 null H1299 cells (H1299) or H1299 cells transfected with wild-type (WT)
TP53, or the indicated TP53 mutant following treatment with DMSO (control) or 10 µM erastin for 24
h (n = 3-4 per cell type and treatment). Relative expression was normalized using glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) as a loading control. * Denotes statistical differences from
respective control, p < 0.05. Error bars indicate SEM. Figure 4. Erastin treatment differentially affects TFRC and ferritin heavy chain 1 (FTH1) protein
expression in cells expressing distinct TP53 mutation types. Representative images and quantification
of Western blots examining the expression of (A,B) transferrin receptor 1 (TFRC) and (C,D) ferritin
heavy chain 1 (FTH1) in TP53 null H1299 cells (H1299) or H1299 cells transfected with wild-type (WT)
TP53, or the indicated TP53 mutant following treatment with DMSO (control) or 10 µM erastin for
24 h (n = 3–4 per cell type and treatment). Relative expression was normalized using glyceraldehyde
3-phosphate dehydrogenase (GAPDH) as a loading control. * Denotes statistical differences from
respective control, p < 0.05. Error bars indicate SEM. Int. J. Mol. Sci. 2020, 21, 6751 8 of 18 2.5. TP53 Mutation Status Influences Basal Levels of Lipid Peroxidation and Subsequent Responsiveness to
Erastin Treatment 2.5. TP53 Mutation Status Influences Basal Levels of Lipid Peroxidation and Subsequent Responsiveness to
Erastin Treatment 2.5. TP53 Mutation Status Influences Basal Levels of Lipid Peroxidation and Subsequent Responsiveness to
Erastin Treatment Reduced expression of functional WT TP53 has previously been demonstrated to amplify
erastin-mediated lipid peroxidation [32]. Thus, we next investigated whether mutant TP53-dependent
differences in erastin sensitivity were driven by variations in levels of lipid peroxidation. To do so,
cells were incubated with a fluorescent lipid peroxidation sensor and differences in the level of oxidized
probe in cells following tetracycline induction of WT and mutant TP53 expression. Intriguingly, R273H
mutant TP53-expressing cells displayed higher levels of basal lipid peroxidation than any of there TP53
subtypes examined (Figure 5A,B). 2.6. Human Cancer Cells Expressing Distinct Endogenous TP53 Mutation Types Exhibit Increased
Ferroptotic Sensitivity 2.6. Human Cancer Cells Expressing Distinct Endogenous TP53 Mutation Types Exhibit Increased
Ferroptotic Sensitivity We next sought to determine whether cells derived from tumors expressing endogenous TP53
mutations also displayed differences in sensitivity to ferroptosis induction. To assess the influence of
distinct endogenous TP53 mutation types, we examined a panel of cell lines expressing endogenous
WT TP53 (SW48), or similar representative hotspot TP53 mutations that were used in the experiments
described above: R273H (MDA-MB-468), R248Q (HCC70), R282W (NCI-H510), R175H (AU565), G245S
(SU.86.86), and R249S (BT549). One exception was made as NCI-H510 cells express an R282G instead of
an R282W mutation, but an endogenously expressing R282W mutant was not commercially available. These cell lines were, in general, more resistant to ferroptosis than the H1299 cell line, and thus
differences in ferroptotic sensitivity were assessed by treating cells with 20 µM of erastin for 24 h
(instead of 10 µM). However, in agreement with our findings in the exogenously expressing TP53
mutant cell lines, cells expressing endogenous R248Q, R175H, G245S, and R249S TP53 mutation types
exhibited increased sensitivity to ferroptosis induction compared to the cells expressing endogenous
WT TP53 (Figure 6A). Cells endogenously expressing the R282G mutation type exhibited a similar
erastin response to the WT TP53-expressing cells, as did the exogenously expressing R282W mutants. In contrast to the findings in the exogenously expressing R273H mutants, the endogenously expressing
R273H mutants were no more sensitive to erastin treatment than their WT TP53-expressing controls. Given the similarities in erastin-mediated changes cell in viability compared to the cells expressing
exogenous TP53, we also investigated whether endogenous mutant TP53-dependent differences in
erastin sensitivity might drive discrepancies in levels of lipid peroxidation. Following erastin treatment,
the endogenous WT and mutant TP53-expressing cell lines were incubated with the same fluorescent
lipid peroxidation sensor used above and differences in the levels of oxidized probe were measured
using a plate reader with fluorescent capabilities. In line with the cells expressing exogenous TP53,
cells expressing endogenous WT TP53, as well as R248Q, G245S, and R249S TP53 mutants displayed a
significant increase in lipid peroxidation in response to erastin treatment (Figure 6B). The increase in
lipid peroxidation following erastin treatment in the cells expressing the endogenous R175H mutant
did not reach statistical significance. 2.3. Ferroptosis Induction and TP53 Status do not Influence Iron-Related mRNA Abundance We then assessed the influence of ferroptosis on lipid peroxidation
by measuring changes in the amount of oxidize probe following 24 h of treatment with erastin or
DMSO (control). To account for differences in basal levels of lipid peroxidation, the changes in relative
fluorescence ratio in the erastin-treated cells were normalized to that of their respective controls. Erastin treatment significantly increased lipid peroxidation in all cells tested, except for those cells
expressing the R282W and R273H TP53 mutation types (Figure 5A,C). The lack of erastin-mediated lipid
peroxidation in the R273H-expressing TP53 mutants may be due to high basal levels lipid peroxidation,
which perhaps represent a maximal threshold. 2.6. Human Cancer Cells Expressing Distinct Endogenous TP53 Mutation Types Exhibit Increased
Ferroptotic Sensitivity 2.6. Human Cancer Cells Expressing Distinct Endogenous TP53 Mutation Types Exhibit Increased
Ferroptotic Sensitivity However, also in agreement with the H1299 cells expressing
the exogenous R273H and R282W TP53 mutations, the endogenously expressing R273H and R282G
mutant cell lines did not exhibit an increase in lipid peroxidation in response to erastin treatment. Moreover, as with the exogenously expressing R273H mutants, the cells expressing an endogenous
R273H mutant also displayed remarkably high levels of basal lipid peroxidation compared to the other
cell lines tested (Figure 6C). 9 of 18
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I t J M l S i 2020 21
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9 of 1
Figure 5. Mutant TP53 expression influences basal and erastin-induced levels of lipid peroxidation. (A) Unoxidized and oxidized C11-BODIPY in tetracycline-induced WT and mutant TP53-expressing
following treatment with DMSO (control) or 10 µM erastin (Erastin) for 24 (n = 3–4 per cell type and
treatment). (B,C) Changes in relative amounts of oxidized probe were quantified using ImageJ
software following normalization to Hoechst nuclear staining to account for differences in cell
number. (B) Student’s t-tests were used to identify statistically significant erastin responses relative
to their controls within a given cell type. Relative fluorescence ratios for erastin-treated cells are
shown normalized to the relative fluorescence ratios of their respective controls. * Denotes significant
difference from control. (C) A one-way between-subjects ANOVA identified a significant effect of
TP53 mutation type on basal oxidized probe at the p <0.05 level for the 8 cell types (F (7,16) = 13.563,
p < 0.001). * Post hoc comparisons using the Tukey LSD test indicated that basal levels of lipid
peroxidation in the R273H-expressing mutants was significantly different from the WT TP53-
expressing cells (p < 0.001). All images were acquired using a 100X objective under immersion oil. Error bars indicate SEM
Figure 5. Mutant TP53 expression influences basal and erastin-induced levels of lipid peroxidation. (A) Unoxidized and oxidized C11-BODIPY in tetracycline-induced WT and mutant TP53-expressing
following treatment with DMSO (control) or 10 µM erastin (Erastin) for 24 (n = 3–4 per cell type
and treatment). (B,C) Changes in relative amounts of oxidized probe were quantified using ImageJ
software following normalization to Hoechst nuclear staining to account for differences in cell number. (B) Student’s t-tests were used to identify statistically significant erastin responses relative to their
controls within a given cell type. 2.6. Human Cancer Cells Expressing Distinct Endogenous TP53 Mutation Types Exhibit Increased
Ferroptotic Sensitivity Relative fluorescence ratios for erastin-treated cells are shown
normalized to the relative fluorescence ratios of their respective controls. * Denotes significant
difference from control. (C) A one-way between-subjects ANOVA identified a significant effect of TP53
mutation type on basal oxidized probe at the p < 0.05 level for the 8 cell types (F (7,16) = 13.563, p < 0.001). * Post hoc comparisons using the Tukey LSD test indicated that basal levels of lipid peroxidation in
the R273H-expressing mutants was significantly different from the WT TP53-expressing cells (p < 0.001). All images were acquired using a 100X objective under immersion oil. Error bars indicate SEM. Figure 5. Mutant TP53 expression influences basal and erastin-induced levels of lipid peroxidation. (A) Unoxidized and oxidized C11-BODIPY in tetracycline-induced WT and mutant TP53-expressing
following treatment with DMSO (control) or 10 µM erastin (Erastin) for 24 (n = 3–4 per cell type and
treatment). (B,C) Changes in relative amounts of oxidized probe were quantified using ImageJ
software following normalization to Hoechst nuclear staining to account for differences in cell
number. (B) Student’s t-tests were used to identify statistically significant erastin responses relative
to their controls within a given cell type. Relative fluorescence ratios for erastin-treated cells are
shown normalized to the relative fluorescence ratios of their respective controls. * Denotes significant
difference from control. (C) A one-way between-subjects ANOVA identified a significant effect of
TP53 mutation type on basal oxidized probe at the p <0.05 level for the 8 cell types (F (7,16) = 13.563,
p < 0.001). * Post hoc comparisons using the Tukey LSD test indicated that basal levels of lipid
peroxidation in the R273H-expressing mutants was significantly different from the WT TP53-
expressing cells (p < 0.001). All images were acquired using a 100X objective under immersion oil. Figure 5. Mutant TP53 expression influences basal and erastin-induced levels of lipid peroxidation. (A) Unoxidized and oxidized C11-BODIPY in tetracycline-induced WT and mutant TP53-expressing
following treatment with DMSO (control) or 10 µM erastin (Erastin) for 24 (n = 3–4 per cell type
and treatment). (B,C) Changes in relative amounts of oxidized probe were quantified using ImageJ
software following normalization to Hoechst nuclear staining to account for differences in cell number. (B) Student’s t-tests were used to identify statistically significant erastin responses relative to their
controls within a given cell type. 2.6. Human Cancer Cells Expressing Distinct Endogenous TP53 Mutation Types Exhibit Increased
Ferroptotic Sensitivity Error bars indicate SEM. Figure 6. Cells expressing distinct TP53 mutation types exhibit increased ferroptotic sensitivity. (A)
Cell viability measured following 24 h of treatment with 20 µM erastin in cells expressing endogenous
WT TP53, or one of the TP53 mutation types indicated. * Denotes difference from WT TP53, p < 0.05. (B,C) Lipid peroxidation was assessed by measuring relative changes in fluorescence of unoxidized
versus oxidized C11-BODIPY probe following treatment with DMSO (control) or 20 µM erastin
(Erastin) for 24 h (n = 3–4 per cell type and treatment). (B,C) Changes in relative amounts of oxidized
probe were quantified according to cell viability. (B) Student’s t-tests were used to identify statistically
significant erastin responses relative to their controls within a given cell type. * Denotes significant
difference from control. (C) A one-way between-subjects ANOVA identified a significant effect of
TP53 mutation type on basal oxidized probe at the p < 0.05 level for the 8 cell types (F (6,14) = 24.954,
p = 0.000). * Post hoc comparisons using the Tukey LSD test indicated that basal levels of lipid
peroxidation in the R273H- (p = 0.000) and R175H- (p = 0.041) expressing mutants were significantly
different from the WT TP53-expressing cells. Error bars indicate SEM. Figure 6. Cells expressing distinct TP53 mutation types exhibit increased ferroptotic sensitivity. (A) Cell
viability measured following 24 h of treatment with 20 µM erastin in cells expressing endogenous
WT TP53, or one of the TP53 mutation types indicated. * Denotes difference from WT TP53, p < 0.05. (B,C) Lipid peroxidation was assessed by measuring relative changes in fluorescence of unoxidized
versus oxidized C11-BODIPY probe following treatment with DMSO (control) or 20 µM erastin (Erastin)
for 24 h (n = 3–4 per cell type and treatment). (B,C) Changes in relative amounts of oxidized probe were
quantified according to cell viability. (B) Student’s t-tests were used to identify statistically significant
erastin responses relative to their controls within a given cell type. * Denotes significant difference
from control. (C) A one-way between-subjects ANOVA identified a significant effect of TP53 mutation
type on basal oxidized probe at the p < 0.05 level for the 8 cell types (F (6,14) = 24.954, p = 0.000). 2.6. Human Cancer Cells Expressing Distinct Endogenous TP53 Mutation Types Exhibit Increased
Ferroptotic Sensitivity Relative fluorescence ratios for erastin-treated cells are shown
normalized to the relative fluorescence ratios of their respective controls. * Denotes significant
difference from control. (C) A one-way between-subjects ANOVA identified a significant effect of TP53
mutation type on basal oxidized probe at the p < 0.05 level for the 8 cell types (F (7,16) = 13.563, p < 0.001). * Post hoc comparisons using the Tukey LSD test indicated that basal levels of lipid peroxidation in
the R273H-expressing mutants was significantly different from the WT TP53-expressing cells (p < 0.001). All images were acquired using a 100X objective under immersion oil. Error bars indicate SEM. 10 of 18
1 of 19 Int. J. Mol. Sci. 2020, 21, 6751
Int J Mol Sci 2020 21 x FO Figure 6. Cells expressing distinct TP53 mutation types exhibit increased ferroptotic sensitivity
Cell viability measured following 24 h of treatment with 20 µM erastin in cells expressing endoge
WT TP53, or one of the TP53 mutation types indicated. * Denotes difference from WT TP53, p <
(B,C) Lipid peroxidation was assessed by measuring relative changes in fluorescence of unoxi
versus oxidized C11-BODIPY probe following treatment with DMSO (control) or 20 µM e
(Erastin) for 24 h (n = 3–4 per cell type and treatment). (B,C) Changes in relative amounts of oxi
probe were quantified according to cell viability. (B) Student’s t-tests were used to identify statist
significant erastin responses relative to their controls within a given cell type. * Denotes signi
difference from control. (C) A one-way between-subjects ANOVA identified a significant eff
Figure 6. Cells expressing distinct TP53 mutation types exhibit increased ferroptotic sensitivity. viability measured following 24 h of treatment with 20 µM erastin in cells expressing endo
WT TP53, or one of the TP53 mutation types indicated. * Denotes difference from WT TP53, p
(B,C) Lipid peroxidation was assessed by measuring relative changes in fluorescence of unox
versus oxidized C11-BODIPY probe following treatment with DMSO (control) or 20 µM erastin (
for 24 h (n = 3–4 per cell type and treatment). (B,C) Changes in relative amounts of oxidized prob
quantified according to cell viability. (B) Student’s t-tests were used to identify statistically sig
erastin responses relative to their controls within a given cell type. * Denotes significant diff
from control. (C) A one-way between-subjects ANOVA identified a significant effect of TP53 m Figure 6. 2.6. Human Cancer Cells Expressing Distinct Endogenous TP53 Mutation Types Exhibit Increased
Ferroptotic Sensitivity Cells expressing distinct TP53 mutation types exhibit increased ferroptotic sensitivity. (A)
Cell viability measured following 24 h of treatment with 20 µM erastin in cells expressing endogenous
WT TP53, or one of the TP53 mutation types indicated. * Denotes difference from WT TP53, p < 0.05. (B,C) Lipid peroxidation was assessed by measuring relative changes in fluorescence of unoxidized
versus oxidized C11-BODIPY probe following treatment with DMSO (control) or 20 µM erastin
(Erastin) for 24 h (n = 3–4 per cell type and treatment). (B,C) Changes in relative amounts of oxidized
probe were quantified according to cell viability. (B) Student’s t-tests were used to identify statistically
significant erastin responses relative to their controls within a given cell type. * Denotes significant
difference from control. (C) A one-way between-subjects ANOVA identified a significant effect of
TP53 mutation type on basal oxidized probe at the p < 0.05 level for the 8 cell types (F (6,14) = 24.954,
p = 0.000). * Post hoc comparisons using the Tukey LSD test indicated that basal levels of lipid
peroxidation in the R273H- (p = 0.000) and R175H- (p = 0.041) expressing mutants were significantly
different from the WT TP53-expressing cells. Error bars indicate SEM. Figure 6. Cells expressing distinct TP53 mutation types exhibit increased ferroptotic sensitivity. (A) Cell
viability measured following 24 h of treatment with 20 µM erastin in cells expressing endogenous
WT TP53, or one of the TP53 mutation types indicated. * Denotes difference from WT TP53, p < 0.05. (B,C) Lipid peroxidation was assessed by measuring relative changes in fluorescence of unoxidized
versus oxidized C11-BODIPY probe following treatment with DMSO (control) or 20 µM erastin (Erastin)
for 24 h (n = 3–4 per cell type and treatment). (B,C) Changes in relative amounts of oxidized probe were
quantified according to cell viability. (B) Student’s t-tests were used to identify statistically significant
erastin responses relative to their controls within a given cell type. * Denotes significant difference
from control. (C) A one-way between-subjects ANOVA identified a significant effect of TP53 mutation
type on basal oxidized probe at the p < 0.05 level for the 8 cell types (F (6,14) = 24.954, p = 0.000). * Post hoc comparisons using the Tukey LSD test indicated that basal levels of lipid peroxidation in
the R273H- (p = 0.000) and R175H- (p = 0.041) expressing mutants were significantly different from
the WT TP53-expressing cells. 3. Discussion Cancer cells are extravagant users of iron, and as such, much effort has been devoted to taking
advantage of cancers cells’ “iron addiction” by restricting iron availability (reviewed in [33–35]). However, these approaches are confounded by the essential nature of iron for noncancerous cells
as well. Ferroptosis has been described as a novel approach to exploiting the toxic nature of iron to
promote programmed cell death, but the toxic potential of iron for all cell types must be considered. Thus, we sought to investigate the potential to exploit the frequency of TP53 mutations in human
cancer to more favorably induced iron-mediated cell death in tumors harboring these mutation types. y
g
yp
Though the TP53 mutation spectrum is vast, the majority of mutations occur within the DNA
binding domain. TP53 mutation types can generally be classified as either DNA contact mutants or
conformational-type mutants depending on whether the mutation affects contact with target DNA
or disrupts the structure of the TP53 protein, respectively. Such distinctions are important because
they can dramatically influence phenotypic effects. In this study, we utilized cell lines expressing
the most common examples of TP53 contact (R248Q, R273H and R282W) and conformational (R175H,
G245S, and R249S) mutants [21]. Of the three contact mutation types investigated in the current
study, only the R273H and R248Q TP53 exogenous mutant-expressing cell lines were more sensitive
to ferroptotic cell death than WT TP53-expressing cells. However, the R282W mutants responded
similarly to the WT TP53-expressing cells. On the other hand, the three conformation mutation types
examined in this study (175H, G245S, and R249S) were all consistently more sensitive to ferroptosis
induction than WT TP53-expressing cells. Importantly, we were then able to validate these findings in
cells expressing endogenous WT and mutant TP53 as well. In the cells expressing endogenous TP53
mutation types, only one of the contact mutants (R248Q) was more sensitive to ferroptotic cell death,
while all three conformational mutants examined exhibited increased ferroptotic sensitivity. Further
investigation is needed to determine whether conformational-type TP53 mutants are uniformly more
susceptible to iron-mediated cell death. To investigate the extent to which the increase in IRP RNA binding activity was indeed responsible
for the increase in ferroptotic sensitivity in the R248Q-, R175H-, and G245S-expressing mutants,
we examined the expression of IRP targets, TFRC and FTH1. 2.6. Human Cancer Cells Expressing Distinct Endogenous TP53 Mutation Types Exhibit Increased
Ferroptotic Sensitivity * Post hoc comparisons using the Tukey LSD test indicated that basal levels of lipid peroxidation in
the R273H- (p = 0.000) and R175H- (p = 0.041) expressing mutants were significantly different from
the WT TP53-expressing cells. Error bars indicate SEM. Int. J. Mol. Sci. 2020, 21, 6751 11 of 18 11 of 18 3. Discussion p
g
Independently of how iron becomes available, it is the peroxidation of lipids by free iron within
the cell that ultimately leads to ferroptotic cell death [13,39]. Therefore, we examined whether mutant
TP53-dependent changes in oxidized lipid accumulation in response to ferroptosis induction could
explain the observed differences ferroptosis sensitivity. Intriguingly, though all cells succumbed to
some level of ferroptotic cell death, erastin treatment did not significantly increase lipid peroxidation in
the R273H and R282W TP53-expressing mutants. This was also true in the cells expressing endogenous
R273H and R282W TP53 mutation types. To further investigate why lipid peroxidation did not increase in the R273H and R282W TP53
mutant-expressing cell lines in response to erastin treatment, we analyzed basal levels of lipid
peroxidation following induction of TP53 expression alone. Both the exogenous and endogenous
R273H TP53-expressing mutants displayed significantly higher levels of basal lipid peroxidation
compared to the other TP53 subtypes tested. These results are in agreement with previous work
demonstrating that H1299 cells expressing an R273H mutation have impaired glutamate release
and diminished baseline glutathione levels [40]. Future work should investigate why the endogenously
expressing R273H TP53 mutants were more resistant to ferroptosis. On the contrary, despite a lack of
erastin responsiveness, in either the exogenous R282W or endogenous R282G TP53-expressing mutants,
basal levels of lipid peroxidation were similar to WT in both groups. These findings indicate that
R282W and R282G TP53 mutations may have more capacity to combat lipid peroxidation than other
TP53 mutation types through a yet undefined mechanism. Future studies should examine differences
in antioxidative capacity between cells expressing distinct mutant TP53 types. A recognized limitation of the current study was the primary use of a single isogenic cell line
(H1299). However, by removing the variability associated with differences in genetic backgrounds
and tumor sites, the use of a single cell line is a significant strength as well. Thus, the results can be
attributed solely to differences in TP53 mutation type. Our findings also highlight the importance of
distinguishing between mutant TP53 subtypes when investigating phenotypic effects. Previous work
has shown that substitution of a different amino acid, even at the same position, can substantially
alter phenotypic outcomes [41]. Herein, we show that induction of five of the six most common
TP53 mutations observed in human cancers are increases in sensitivity to ferroptosis in the human
lung adenocarcinoma H1299 cell line. 3. Discussion As transferrin-bound iron uptake via
TFRC is essential for ferroptosis [15], we hypothesized that increased IRP RNA binding activity in these
cells would result in increased TFRC expression, thereby increasing free iron availability and promoting
ferroptotic cell death. However, we did not observe any changes in TFRC mRNA or protein expression
following erastin treatment in any of the cell lines examined. Previous research investigating
the regulation of TFRC upon ferroptosis induction has produced conflicting results. Wang et al. reported reduced TFRC expression following erastin treatment [36]. Such results are consistent with an
appropriate cellular response, wherein IRPs sense a relative cellular iron overload and decrease mRNA
binding to reduce TFRC expression and subsequently cellular iron uptake. Conversely, Alvarez et al. reported an increase in TFRC expression following erastin treatment [37]. The authors speculated
that the increase in TFRC expression resulted from a decrease in Fe-S biogenesis/stability and a
subsequent increase in IRP1 RNA binding activity. Nonetheless, neither IRP1 nor IRP2 expression
or activity were assessed in either of these studies. The inconsistency between observations of TFRC
responsiveness between these studies, and ours, is likely due to the differences in experimental models. Nonetheless, these findings demonstrate that iron availability during ferroptosis can be mediated via
IRP-independent mechanisms. Similarly, ferritin expression was not changed in any of the cell lines for which an increase in IRP
RNA binding activity was observed. Further confounding our initial hypotheses, ferritin expression
was decreased in the R273H-expressing mutants, but increased in the R249S and R282W mutants. The degradation of ferritin via ferritinophagy is an IRP-independent mode of ferritin regulation
with an established role in ferroptotic cell death [16,38]. Therefore, we investigated the potential for
ferritinophagy to contribute to ferritin regulation and ferroptosis sensitivity in mutant TP53-expressing
cells. We did not detect differences in the expression of any of the ferritinophagy-related genes Int. J. Mol. Sci. 2020, 21, 6751 12 of 18 following erastin treatment in any of the cell lines examined. Thus, the mechanisms contributing to
increased ferroptotic sensitivity in mutant TP53-expressing cells are complex and may not be consistent
between distinct TP53 mutation types. Further investigation is warranted because increased ferritin
expression in the R282W mutant TP53-expressing cells, which were less sensitive to erastin treatment,
may be indicative of a unique protective mechanism against ferroptosis in this particular TP53 mutation
type. Future studies should investigate the IRP-independent modes of iron regulation in mutant
TP53-expressing cells. 3. Discussion We further support these findings by validating our results
in cell lines derived from a diverse set of malignancies. Future studies should seek to determine
whether ferroptosis induction will be a viable approach for targeting tumors expressing distinct TP53
mutation types. 4.2. Ferroptosis Induction and Cell Viability Assessment H1299 cells expressing exogenous WT or mutant TP53 were seeded at 4 × 103 cells per well in a
96-well plate and allowed to grow for 24 h before treatment with 5 µM erastin, or a vehicle control
(DMSO) along with 10 µg tetracycline for 24 h. Co-treatment with 10 µM ferrostatin-1, a ferroptosis
inhibitor, was used as a negative control. Cell viability was assessed using PrestoBlue reagent
(Thermo Fisher Scientific, Waltham, MA, USA) by adding 10 µL of PrestoBlue reagent to each followed
by incubation at 37 ◦C for 20 min. Differences in fluorescence absorbance were measured using a Biotek
Synergy HT (Biotek, Winooski, VT, USA) plate reader. Each assay included four technical replicates
and was repeated at least three times. Differences in cell viability were determined by normalizing
reductions in fluorescent absorbance relative to the vehicle control group for each cell line. To adjust for
differences in size in the cells expressing endogenous WT or mutant TP53, cells were plated such that
they were ~70% confluent on the day of treatment. Cells were treated with a vehicle control (DMSO)
or 20 µM erastin for 24 h, and cell viability was assessed using PrestoBlue reagent as described above. 4. Materials and Methods 4.1. Cell Culture and Creation of Stable Mutant TP53-Expressing Cell Lines Cells containing endogenous WT TP53 (SW48) or an endogenous R273H (MDA-MB-468), R248Q
(HCC70), R282G (NCI-H510), R175H (AU565), G245S (SU.86.86), or R249S (BT549) TP53 mutation
were also obtained from the American Type Culture collection (ATCC). All cell lines were maintained
according to the instructions provided on the provider’s website. TP53 null, H1299 cells, were also
obtained from ATCC and maintained in RPMI-40 (Corning) containing 10% tetracycline-free FBS
(Atlanta Biologicals, Norcrsoss, GA, USA) and 100 IU/mL penicillin and 100 (µg/mL) streptomycin
in a temperature and humidity-controlled incubator. Tetracycline-inducible expression plasmids Int. J. Mol. Sci. 2020, 21, 6751 13 of 18 13 of 18 (pcDNA5/TO; ThermoFisher; Waltham, MA, USA) containing either wild-type TP53, or a representative
“hotspot” TP53 mutant (R273H, R248Q, R282W, R175H, G245S, R245G, or R249S) were generated
and validated by Sanger sequencing (See Supplementary Files 1–13) using a custom cloning service
GenScript (Piscatawy, MJ, USA). To obtain stable, tetracycline-inducible TP53-expressing cell lines,
H1299 cells were co-transfected with a Tet-repressor plasmid (pcDNA/TR; ThermoFisher; Waltham, MA,
USA) and one of the tetracycline-inducible plasmids mentioned above using lipofectamine 3000 reagent
(ThermoFisher; Waltham, MA, USA) followed by selection with 6 µg/mL blasticidin and 600 µg/mL
hygromycin. In total, eight cell lines were generated. H1299 cells expressing only the pcDNA6/TR
and empty pcDNA5/TO vectors (H1299) were used as TP53 null control cells. The seven other cell lines
expressed both pcDNA6/TR and either a wild-type TP53 gene (WT), or one of the representative TP53
hotspot mutations mention above. These mutations were selected because they represent the most
common examples of p53 contact (R248Q, R273H and R282W) and conformational (R175H, G245S,
and R249S) mutation types. TP53 expression was induced by supplementing the media with 10 µg/mL
tetracycline for 24 h. The efficacy of tetracycline-dependent induction of TP53 was validated by
Western blot. 4.3. Electrophoretic Mobility Shift Assays Spontaneous and total IRP RNA binding activity was determined by EMSA as previously
described [42]. Briefly, cells were treated with 10 µM erastin and 10 µg tetracycline for 24 h and harvested
from 25-cm plates at 90% confluency. Cytosolic protein was collected by lysing cells in two-volumes
cytosol buffer (1 M HEPES 10 mM, 10 mM KCl, 1%, 0.1 mM EGTA, 0.1 mM EDTA) supplemented with
1 mM citrate, 1 mM DTT, 0.1 M PMSF, and 100X Halt Protease and Phosphatase inhibitor cocktail
(ThermoFisher; Waltham, MA, USA). After 15 min of incubation on ice, NP40 was added to a final
volume of 1%, and the samples were vortexed for 10 s. Following centrifugation at 12,000× g for 10 min
at 4 ◦C, the supernatant (cytosol) was collected, concentration was determined by bicinchoninic acid
(BCA) assay (Pierce, ThermoFisher; Waltham, MA, USA), and samples were store in liquid nitrogen
until use. Spontaneous IRP RNA binding activities were assessed by incubating 10 µg cytosolic protein
with saturating levels (1 nM) of [32P]-labeled RNA from the rat L-ferritin IRE. Total IRP1 RNA binding
activity was measured by adding 10 µg of cytosolic protein to saturating levels of [32P]-labeled RNA
in the presence of 4% 2-mercaptoethanol. RNA binding activity was quantified using Optiquant
Acquisition and Analysis software (Packard Bioscience, Meriden, CT, USA) (Supplementary Figure S1). 4.4. Western Blots Following their respective treatments, cells were collected into 1.5-mL microcentrifuge tubes
and washed with 1X PBS. Total protein was isolated by lysing cells in radioimmunoprecipitation
(RIPA) buffer (50 mM Tris-HCL, pH 8.0, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 2 mM EDTA, Int. J. Mol. Sci. 2020, 21, 6751 14 of 18 150 mM NaCl) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (ThermoFisher;
Waltham, MA, USA), 1 mM DTT, 1 mM citrate, 0.1 mM phenylmethylsulfonyl fluoride, and 10 µM
MG-132. Following centrifugation at 14,000× g for 20 min, the supernatant was collected, and protein
concentration was determined by BCA assay. For Western blot analysis, 30 µg of total protein was solubilized in 2× Laemmli sample buffer (4%
SDS, 20% glycerol. 0.0004% bromophenol blue, 0.125 M Tris-HCl, pH 6.8, 10% 2-mercaptoethanol) then
boiled at 95 ◦C for 4 min. Proteins were then separated using a (Bio-Rad Mini-PROTEAN® TGX™,
Hercules, CA, USA) 4–20% gradient SDS gel at 150 volts for 60 min before being transferred to a PVDF
membrane. Equal transfer was confirmed with Ponceau-S staining before blocking of the membrane
for 60 min in 5% non-fat dried milk in 1× TBS and 0.01% Tween-2) (5% NDFM-T) at room temperature. The membranes were incubated with primary antibodies diluted in the 5% NFDM-T at the following
concentrations: CD71 (sc-9099) (H-300) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) at 1:100
dilution, CD71 (D7S5Z) #13208 (Cell Signaling Technology, Danvers, MA, USA) at 1:1000 dilution,
FTH1 (D1D4) #4393S (Cell Signaling Technology, MA, USA) at 1:1000 dilution and loading control
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-47724) (0411) (Santa Cruz Biotechnologies,
Santa Cruz, CA, USA) at 1:1000 dilution. The membranes were then washed three times in 5% NFDM-T to remove any residual primary
antibody. The membranes were then incubated in the following secondary antibodies (diluted in
the 5% non-fat dry milk—TBS Blotto): Anti-Rabbit IgG, HRP-linked Antibody #7074P2 (Cell Signaling
Technology, MA, USA) and Anti-Mouse IgG, HRP-linked Antibody #7076P2 (Cell Signaling Technology,
MA, USA) both at 1: 10,000 for one hour at 4 ◦C. Membranes were then washed three times using
the 5% NFDM-T for five minutes each time, then washed two times with 1X TBS. Protein bands were
then visualized using (SuperSignal™West Pico PLUS Chemiluminescent Substrate kit; Pierce, WA,
USA) following the manufacturer’s protocol (Supplementary Figure S2). 4.4. Western Blots Chemiluminescence signal
was then viewed using a FluorChem R ProteinSimple fluorescence imaging system (R&D Systems;
Minneapolis, MN, USA) and analyzed using ImageJ software [43]. 4.5. RNA Isolation and Real-Time qPCR To assess changes in mRNA abundance following induction of ferroptosis, cells were seeded
in a 6-well plate at 1 × 105 cells/well and incubated for 24 h before co-treatment with 10 µM erastin
and 10 µg tetracycline for 24 h. Total RNA was isolated using TRIzol reagent (ThermoFisher; Waltham,
MA, USA). RNA purity and integrity were confirmed by Nanodrop (ThermoFisher) and agarose gel
electrophoresis, respectively, before reverse-transcription using SuperScript II (Invitrogen, Carlsbad,
CA, USA). The relative abundance of TFRC, NCOA4, ATG5, CISD1, SLC7A11, and ferroportin
(Supplementary Figure S3) were determined by real-time qPCR using SYBR green chemistry on an
ABI 7900HT Real-Time PCR system (ThermoFisher; Waltham, MA, USA) and normalized relative to
peptidylprolyl isomerase B (PPIB) abundance using the 2−∆∆Ct method (User Bulletin no. 2, Applied
Biosystems). Primer sequences for each mRNA of interest were obtained from previously published
sources: PPIB [22], TFRC [44], NCOA4 [45], ATG5 [46], CISD1 [47], and SLC7A11 [48]. 4.7. Statistical Analysis One-way ANOVA was used to assess differences in treatment responses between cell lines
following induction of TP53 expression. A two-factor mixed design ANOVA was performed to
assess differences in responsiveness to treatment between cells. When statistically significant effects
were identified by ANOVA, post hoc analyses were performed to make pairwise comparisons using
the Tukey HSD method. Student’s t-tests were used to identify statistically significant treatment
responses relative to their controls within a given cell type. Differences were considered statistically
significant at the 95% confidence level (alpha = 0.05). Descriptive statistics were calculated for all
variables and include mean ± SEM. All experiments were repeated 3 times, with n = 3 per group. All tests were performed using SPSS v23.0 software (IBM-SPSS; Chicago, IL, USA). 4.6. Lipid Peroxidation Assays To assay TP53-dependent differences in lipid peroxidation following erastin treatment in
the tetracycline-inducible cells, all cell lines were plated in an 8-well chamber slide (Ibidi, Martinsried,
Germany) at 10,000 cells/well. Cells were treated with 10 µM erastin and 10 µg tetracycline for
24 h. The cells were then washed with 1X Hank’s balanced salt solution (HBSS) before incubation
with 5 µM BODIPY 581/591 C11 and Hoechst stain (1:1000) (ThermoFisher; Waltham, MA, USA)
for 10 min at 37 ◦C. Then, the mixture of 1X HBSS and reagent was aspirated and 1× HBSS was
added to the cells. Cells were imaged using the BZ-X700 Life Science Microscope (Keyence, Osaka,
Japan) with a 100× objective lens under immersion oil. Low photobleach settings and exposure times
were held consistent through the imaging process. Increased lipid peroxidation was shown with Int. J. Mol. Sci. 2020, 21, 6751 15 of 18 15 of 18 oxidation of the polyunsaturated butadienyl portion of the dye resulting in a change from 590 to 510 nm
excitation. ImageJ software was used to measure differences in the ratio of red and green fluorescence
intensities, which were then normalized to cell number by counting the number of Hoechst stained
nuclei [43]. To measure lipid peroxidation in the cell lines expressing endogenous WT or mutant TP53,
cells were plated into black-wall, clear bottom 96-well plates such that they were 70% confluent on
the day of treatment. Cells were treated with DMSO as a vehicle control or with 20 µM erastin for
24 h. On the day of measurement, cells were washed once with HBSS, incubated with 5 µM BODIPY
581/591 C11 (ThermoFisher; Waltham, MA, USA) at 37 ◦C for 20 min, and then washed once more
with HBSS. Fluorescence was read at 488 nmexcitation/510 nmemission and 581 nmexcitation/591 nmemission
wavelengths using Biotek Synergy H1 (Biotek, Winooski, VT, USA) plate reader. The fluorescence ratio,
591 nm[reduction]/510 nm[oxidation], was calculated and normalized to cell viability, which was assessed
in parallel plates using PrestoBlue reagent (Thermo Fisher Scientific, Waltham, MA, USA). Conflicts of Interest: The authors have no conflict of interest to declare. Author Contributions: Conceptualization, M.R.M.; methodology, M.R.M. and L.R.T.; validation, M.R.M., L.R.T.,
and T.G.O.; formal analysis, M.R.M., and L.R.T.; investigation, M.R.M., L.R.T., T.G.O., E.R.H.; data curation, M.R.M.
and L.R.T.; writing—original draft preparation, M.R.M.; writing—review and editing, L.R.T., T.G.O., E.R.H.,
W.C., and S.L.C.; visualization, M.R.M. and L.R.T.; supervision, M.R.M., W.C., and S.L.C.; project administration,
M.R.M.; funding acquisition, M.R.M. All authors have read and agreed to the published version of the manuscript. Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/18/6751/s1.
Supplementary Files 1–13; Figure S1. Raw Gel Shift Images for Manuscript; Figure S2. Raw Western Blot Images
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149, 1060–1072. 5. Conclusions To date, the targeting of mutant TP53 has primarily focused on restoring its wild-type activity,
or promoting its degradation, while iron chelation has been a primary emphasis for the development of
iron-based chemotherapy. In this study, we have established that induction of distinct TP53 mutation
types increases sensitivity to ferroptotic cell death. These findings are novel because they describe an
approach that would allow for the exploitation of mutant TP53 expression to more favorably induce
iron-mediated cell death via the activation of ferroptosis. We have also demonstrated that the IRP
response to erastin treatment is dependent upon TP53 mutation type and is not essential for ferroptosis
induction. Our findings are strengthened by examining the most prevalent TP53 mutations that
represent exemplary models of both contact and conformational mutants. As these mutation types
represent some of the most prevalent TP53 mutations in human tumors, these findings are relevant to
a variety of clinically important cancers. Supplementary Materials: The following are available online at http://www.mdpi.com/1422-0067/21/18/6751/s1. Supplementary Files 1–13; Figure S1. Raw Gel Shift Images for Manuscript; Figure S2. Raw Western Blot Images
for Manuscript; Figure S3. Ferroportin (SLC40A1) mRNA expression. Author Contributions: Conceptualization, M.R.M.; methodology, M.R.M. and L.R.T.; validation, M.R.M., L.R.T.,
and T.G.O.; formal analysis, M.R.M., and L.R.T.; investigation, M.R.M., L.R.T., T.G.O., E.R.H.; data curation, M.R.M. and L.R.T.; writing—original draft preparation, M.R.M.; writing—review and editing, L.R.T., T.G.O., E.R.H.,
W.C., and S.L.C.; visualization, M.R.M. and L.R.T.; supervision, M.R.M., W.C., and S.L.C.; project administration,
M.R.M.; funding acquisition, M.R.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by start-up funding from the Vice President of Research’s office at Oklahoma
State University. Conflicts of Interest: The authors have no conflict of interest to declare. Int. J. Mol. Sci. 2020, 21, 6751 16 of 18 16 of 18 22.
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article distributed under the terms and conditions of the Creative Commons Attribution
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Number of vaginal lactobacilli in postmenopausal women with vaginal atrophy before and after treatment with erbium–YAG laser: a randomized sham-controlled trial
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Abstract Primary objective To evaluate the effect of erbium–YAG laser on the number of vaginal lactobacilli in
postmenopausal women. Secondary objectives To evaluate the effect of erbium–YAG laser on vaginal atrophy symptoms and vaginal pH in
postmenopausal women. Materials and methods A total of 44 postmenopausal women who met the inclusion criteria were randomized in
the laser group (n = 22) and sham group (n = 22). Vaginal lactobacilli grading, vaginal pH, vaginal atrophy score, and
vaginal atrophy symptoms were assessed before and after treatment with erbium–YAG laser for two consecutive
times, with a four-week interval; the results were compared with the effects of the sham procedure. Any adverse
events after the treatment were recorded. Results A total of 44 women were included, and five were lost to follow-up. Compared with sham procedure, vaginal
lactobacilli grading improved in the laser group (5/20 in the laser group and 1/19 in the sham group). However, the
improvement did not reach statistical significance (adjusted odds ratio = 5.32, 95% CI = 0.5–56.21). Vaginal atrophy
symptoms measured by the visual analog scale (VAS) and vaginal pH were improved in both groups without a
statistically significant difference between the two groups. Vaginal “dryness” VAS and vaginal atrophy score after
treatment were significantly lowered in the laser group compared with the sham group. Conclusions This study showed an improvement in vaginal lactobacilli grading after vaginal laser treatment. However, the difference in vaginal lactobacilli grading after treatment in both groups was not statistically significant. Keywords Erbium–YAG laser, Vaginal lactobacilli, Randomized sham-controlled trial, Menopause, Vaginal atrophy,
Vaginal pH *Correspondence:
Krasean Panyakhamlerd
Krasean.P@chula.ac.th
1Department of Obstetrics and Gynecology, Faculty of Medicine,
Chulalongkorn University, Bangkok, Thailand *Correspondence:
Krasean Panyakhamlerd
Krasean.P@chula.ac.th
1Department of Obstetrics and Gynecology, Faculty of Medicine,
Chulalongkorn University, Bangkok, Thailand © 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. RESEARCH Open Access BMC Women's Health BMC Women's Health (2023) 23:513 Panyawongudom et al. BMC Women's Health (2023) 23:5
https://doi.org/10.1186/s12905-023-02590-y Abstract If material is not included
in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data. Number of vaginal lactobacilli
in postmenopausal women with vaginal
atrophy before and after treatment
with erbium–YAG laser: a randomized sham-
controlled trial Nuttanun Panyawongudom1, Krasean Panyakhamlerd1* and Ammarin Suwan1 © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,
sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included
in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data. Materials and methodsh This randomized sham-controlled study was conducted
at King Chulalongkorn Memorial Hospital, Bangkok,
Thailand, between May 2019 and September 2020. The
target population was postmenopausal women attend
ing gynecology clinic or gender health clinic with vaginal
atrophy symptoms (dryness, dyspareunia, irritation, and
pain). The inclusion criteria were as follows: postmeno
pausal women with at least one symptom of moderate-
to-severe vaginal atrophy and age more than 40 years. (Moderate-to-severe vaginal atrophy was defined by the
most bothersome symptom score at least “2” in at least
one vaginal atrophy symptom [0 = no symptom, 1 = mild,
2 = moderate, 3 = severe]). As the inclusion criteria of our
study were women older than 40 years, the postmeno
pausal women in this study included natural menopause,
surgical menopause, primary ovarian insufficiency, and
participants with history of chemoradiation therapy for
cancers. Both sexually active and sexually inactive women
were included in this study. The exclusion criteria were as
follows: history of hormonal therapy, vaginal lubricants,
vaginal moisturizers, or spermicide in previous three
months; active or recent genitourinary tract infection in
previous one month; abnormal vaginal bleeding; genital
organ prolapse stage II; history of vaginal surgery in pre
vious three months; and previous vaginal laser treatment. Vaginal laser is a novel treatment for vaginal atrophy. Two types of laser can be used for intravaginal therapy,
namely, CO2 and erbium–YAG laser [5]. Erbium–YAG
laser has 15 times more water absorption than CO2 laser,
thereby causing less penetration, faster tissue healing
phase, fewer side effects, and lesser pain compared to the
CO2 laser [4].f Vaginal laser has a thermal effect on vaginal epithe
lium, resulting in the expression of heat-shock proteins,
which stimulate growth factor activities, neovasculariza
tion, neocollagenesis, and extracellular matrix formation
as well as increased vaginal thickness and elasticity. How
ever, the thermal effect of erbium–YAG laser is less than
that of CO2 laser, thereby causing no tissue damage [4]. Many prospective studies reported that vaginal
erbium–YAG laser can improve vaginal atrophy symp
toms, and its effect is comparable to that of vaginal
estrogen therapy [6–8]. A systematic review and meta-
analysis of laser therapy for GSM published in 2017,
included 14 studies of CO2 and erbium–YAG laser,
showed that vaginal laser improved GSM symptoms,
female sexual function index, vaginal health index score,
and vaginal maturation value. Introduction A prospective study of vaginal CO2 laser and vaginal
flora reported a significant increase of vaginal lactoba
cilli and a significant decrease of vaginal pH after three
applications of CO2 laser [9] . owever, no study has been
conducted on the effect of vaginal erbium–YAG laser and
number of vaginal lactobacilli to date. Vaginal atrophy or genitourinary syndrome of meno
pause (GSM) is commonly found in postmenopausal
women. About 50% of postmenopausal women suffer
from vaginal atrophy symptoms [1]. This condition is due
to low estrogen levels after menopause [2]. The diagno
sis of vaginal atrophy is clinical, which is manifested by
vaginal dryness, irritation, pain, dyspareunia, and urinary
symptoms [3]. This study aimed to measure the effect of vaginal
erbium–YAG laser on a number of vaginal lactobacilli. We also evaluate vaginal pH, vaginal atrophy symptoms,
and adverse effects after vaginal erbium–YAG laser
treatment. Vaginal atrophy can be treated by either local hor
monal treatment or non-hormonal treatment. At present,
vaginal estrogen therapy is a standard treatment for vag
inal atrophy. However, caution must be observed in hor
mone-sensitive cancer survivors [4]. Moreover, a number
of patients denied hormonal treatment. Thus, non-hor
monal treatments, including vaginal erbium–YAG laser,
have been considered. Panyawongudom et al. BMC Women's Health (2023) 23:513 Panyawongudom et al. BMC Women's Health (2023) 23:513 Page 2 of 7 Panyawongudom et al. BMC Women's Health Results A total of 44 participants were recruited and random
ized to the laser group and sham group, with 22 partic
ipants in each group. Of the participants, five were lost
to follow-up, and a total of 39 participants completed
the study (the detail was shown by a consort diagram in
Fig. 1). Table 2 shows similar baseline characteristics of
participants in the laser group and sham group, but the
participants in the laser group were younger than those
in the sham group (the mean age in the sham group was
60.4, and the mean age in the laser group was 55.5). No
significant difference in parity, menopausal age, year
since menopause, baseline vaginal atrophy symptoms
measured by VAS, baseline vaginal pH, and baseline vagi
nal atrophy score was observed among participants in
both groups. In addition, no significant difference in BMI
was found between the two groups. Similar baseline vagi
nal lactobacilli were observed between the laser group
and sham group. The most prominent symptom of the
participants was dyspareunia (54.5% in both groups). For sample size calculation, two independent propor
tion formulas were used. The sample size was calculated
using data obtained from our pilot study. The propor
tion in the study group and control group was 0.4 and
0.9, respectively. (The result of sample size calculation
was the proportion of participants with grade 1 vaginal
lactobacilli after vaginal erbium laser treatment and after
sham operation). Using 5% type I error and 20% type II
error, the calculated sample size was 17 per group. Add
ing 20% drop out, a total of 42 participants were needed. The randomization sequence was generated by com
puter generated block of 4 randomization and the allo
cation concealment was done by sequentially numbered
sealed envelopes. The first author (N.P.) kept the ran
domization schedule. All participants were blinded dur
ing the study period. All vaginal swab slides were sent
to a microbiologist with identification numbers that
did not mention about the study group. Therefore, a Table 3 shows vaginal lactobacilli grading in each visit
of both groups. Visit 1 is pre-treatment. Visits 2 and 3
are after the first and second treatments, respectively. In the laser group, the grade of vaginal lactobacilli was
significantly higher after the second treatment (p = 0.02). However, no statistically significant change in vaginal
lactobacilli was observed after two sham procedures. Materials and methodsh Vaginal atrophy score (Table 1) was assessed
in every visit. Vaginal swabs were sent to a microbiologist
to evaluate the vaginal lactobacilli grading. Vaginal lacto
bacilli were reported into four grades in accordance with
the number of lactobacilli per high power field (grade 1:
< 6/HPF, grade 2: 6–20/HPF, grade 3: 21–50/HPF, and
grade 4: > 50/HPF). All participants were not received
reimbursement to be enrolled but the authors provided
travelling expenses for each follow-up visit. microbiologist who evaluated vaginal lactobacilli grading
was also blinded. However, all the vaginal laser and sham
applications were performed by the first author (N.P.) and
those procedures cannot be blinded to the provider. This study was funded by Ratchadapisek Sompot and
approved by the Institutional Review Board of Faculty
of Medicine, Chulalongkorn University. This study was
registered in Thai Clinical Trial Registry on 30/10/2019
(TCTR20191030001; https://www.thaiclinicaltrials.org/
export/pdf/TCTR20191030001). Statistical analysis was performed by SPSS version 22.0. For descriptive statistics, the number, percentage, and
mean ± standard deviation (SD) were used for continuous
data, while the number and percentage were used for cat
egorical data. For analytical statistics, repeated measures
ANOVA and Friedman test were used for continuous and
categorial data, respectively. A p value of < 0.05 was con
sidered statistically significant. Materials and methodsh However, the quality of
evidence was “low” or “very low” because no randomized
sham-controlled study can decrease the bias and placebo
effect [5].l This study was conducted at King Chulalongkorn
Memorial
Hospital
(KCMH),
Bangkok,
Thailand,
between May 2019 and September 2020. The study was
not advertised. The primary outcome of this study was to
evaluate the effect of erbium YAG laser on the number
of vaginal lactobacilli. The secondary outcomes were to
evaluate the effect of erbium YAG laser on vaginal atro
phy symptoms and vaginal pH. f
Vaginal lactobacilli are normal vaginal flora that can
produce antimicrobial compounds, such as hydrogen
peroxide and lactic acid, and compete with pathogens for
vaginal adherence [9]. A number of vaginal lactobacilli
increase after vaginal epithelial maturation because of
an increase of glycogen storage in superficial vaginal epi
thelium [10, 11]. Therefore, it can be used as an objective
measurement of vaginal atrophy. As vaginal lactobacilli
decrease during postmenopausal period, the postmeno
pausal women are at increased risk of vaginal pathogenic
bacteria and mycotic infection. After obtaining written informed consent, all partici
pants were interviewed to collect demographic data and
randomized into the laser group and sham group using
block-of-four randomization with a sealed envelope. Par
ticipants in the laser group were treated with erbium in
yttrium aluminum–garnet crystal (Er:YAG) laser (Fotona
Smooth™ XS, Fotona, Ljubljana, Slovenia) at a wave
length of 2940 nm, with a spot size of 7 mm, frequency of Panyawongudom et al. BMC Women's Health (2023) 23:513 Page 3 of 7 Panyawongudom et al. BMC Women's Health 1.6 Hz, and laser energy of 6.0 J/cm [2], two times every
four weeks. Participants in the sham group were treated
with two sham operations every four weeks. Sham opera
tion was conducted while the patient was in the lithot
omy position, and the same equipment as vaginal erbium
laser was used, but no laser energy was emitted from the
equipment. The duration of sham operation was equal
to that of vaginal erbium laser treatment or about 5 min. All participants were appointed for follow-up visit four
weeks after the second laser or sham application. Vagi
nal atrophy symptoms were measured by using the visual
analog scale (VAS) in every visit. Vaginal swabs were
collected from the posterior fornix, and vaginal pH was
measured using a pH indicator strip applied to the lateral
vaginal wall. Results In comparison between the two groups, using logistic
regression for calculation of odds ratio (OR) of improve
ment after two laser treatments, the adjusted OR for age
was 5.32 (95% CI = 0.5–56.21, p = 0.17). Therefore, com
pared with the sham group, the improvement of vaginal
lactobacilli grading of participants in the laser group did
not reach statistical significance. Table 1 Assessment of vaginal atrophy score
Not present (0)
Mild (1)
Moderate
(2)
Severe
(3)
Dryness
Normal
lubrication
Slightly
decreased
Minimal
lubrication
Dry
Rugae
Normal number
and depth
Reduced
rugae
Rare rugae
Smooth
vagina
Pallor
Normal pink
Light pink
Very pale
White/
deep red
Petechiae
None
Bleeds on
scraping
Bleeds on
contact
Clearly
seen
Mucosal
elasticity
Normal
decreased
None
Stenosis Table 1 Assessment of vaginal atrophy score
Not present (0)
Mild (1)
Moderate
(2)
Severe
(3) Page 4 of 7 Panyawongudom et al. BMC Women's Health (2023) 23:513 Fig. 1 Consort diagram of the recruitment and drop out of the participants in the laser group and sham group Fig. Results 1 Consort diagram of the recruitment and drop out of the participants in the laser group and sham group Table 2 Baseline characteristics of the subjects in the laser
group and sham group
Laser group
(mean ± SD)
Sham group
(mean ± SD)
p
value
Age (year)
55.5 ± 7.6
60.4 ± 6.6
0.03
Parity
1.5 ± 1.0
1.8 ± 1.1
0.47
Menopausal age (year)
47.3 ± 4.3
49.7 ± 5.3
0.10
Year since menopause (year)
9.0 ± 6.4
10.7 ± 7.1
0.41
Vaginal atrophy symptoms (VAS) 17.5 ± 8.6
16.3 ± 7.5
0.62
Vaginal pH
6.3 ± 1.4
6.6 ± 1.2
0.35
Vaginal atrophy score
6.5 ± 2.9
7.9 ± 2.9
0.13
Laser group
N (%)
Sham group
N (%)
p
value
BMI
1.00
< 18.5
1(4.5)
0(0)
18.5–24.99
12(54.6)
13(59.1)
25–29.99
7(31.8)
7(31.8)
≥ 30
2(9.1)
2(9.1)
Lactobacilli grade
0.55
1
17(77.3)
21(91)
2
2(9.1)
1(4.5)
3
1(4.5)
0(0)
4
2(9.1)
1(4.5)
Most symptoms
0.27
Dryness
8(36.5)
4(18.3)
Dyspareunia
12(54.5)
12(54.5)
Irritation
1(4.5)
5(22.7)
Pain
1(4.5)
1(4.5) ble 2 Baseline characteristics of the subjects in the laser Table 2 Baseline characteristics of the subjects in the laser
group and sham group Table 2 Baseline characteristics of the subjects in the laser In determining secondary outcomes, mixed-effect
multiple linear regression was used, and it showed a sig
nificant change in vaginal dryness (mean change = − 2.28,
p = 0.01) and vaginal atrophy score (mean change = − 1.49,
p = 0.03) before and after treatment in the laser group as
compared with the sham group. The changes in vaginal
pH, total vaginal atrophy symptoms, pain, irritation, and
dyspareunia were not statistically significant. The sec
ondary outcome results are shown in Table 4. For the
adverse event, only one participant in the sham group
complained about pain during operation, but it was
improved after the operation. Discussion
l
b Vaginal erbium–YAG laser is a novel treatment for vagi
nal atrophy, which is currently known as GSM. Literature
showed the effectiveness and safety of intravaginal laser
therapy, while erbium–YAG laser is considered as a safer
option as compared with fractional CO2 laser because of
its non-ablative effect [4–8]. f
The mechanism of action of erbium–YAG laser is its
photothermal effect on the vaginal epithelium, which
causes superficial tissue shrinkage, neovascularization,
and neocollagenesis, resulting in vaginal tissue thicken
ing, and increases vaginal elasticity [4]. Based on the literature, few studies have reported an
improvement of vaginal atrophy symptoms after vagi
nal erbium–YAG laser treatment [6–8]. However, no
study has been conducted on vaginal erbium–YAG laser
and vaginal lactobacilli, which can be used as an objec
tive measurement. Vaginal lactobacilli are vaginal normal
flora that dynamically changed with vaginal maturation,
and they play a role in maintaining vaginal acidity and
compete with pathogens [9, 10, 12]. Vaginal atrophy symptoms, which were measured by
VAS, were significantly decreased after treatment in the
laser group. However, no statistical difference in vaginal
atrophy symptoms was observed after sham operations. Table 3 also demonstrates that the vaginal atrophy score
after treatment was significantly lowered in the laser
group and sham group (p < 0.001). In addition, no statisti
cally significant difference in vaginal pH before and after
treatment was observed in both groups (p = 0.20 and 0.15
in the laser group and sham group, respectively). To our knowledge, this study is the first randomized
sham-controlled study to assess the effect of vaginal
erbium–YAG laser on vaginal lactobacilli, vaginal pH, and
vaginal atrophy symptoms. This study reports the objec
tive and subjective measurements of vaginal atrophy after Panyawongudom et al. Discussion
l
b BMC Women's Health (2023) 23:513 Page 5 of 7 Table 3 Vaginal lactobacilli grading, vaginal atrophy symptoms (VAS), vaginal atrophy score, and vaginal pH in each visit of
participants in the laser group and sham group
Laser group
Sham group
Lactobacilli grade
Grade 1
Grade 2
Grade 3
Grade 4
Grade 1
Grade 2
Grade 3
Grade 4
Visit 1
15
2
1
2
17
1
0
1
Visit 2
16
0
1
3
16
2
0
1
Visit 3
13
1
1
5
17
1
1
0
p value
0.02
0.72
Vaginal atrophy symptoms (VAS)
Laser group (mean ± SD)
Sham group (mean ± SD)
Visit 1
17.5 ± 8.6
16.3 ± 7.5
Visit 2
10.5 ± 8.7
13.8 ± 8.9
Visit 3
6.8 ± 5.5
10.0 ± 8.2
p value
< 0.001
0.08
Vaginal atrophy score
Visit 1
6.5 ± 2.9
7.9 ± 2.9
Visit 2
4.3 ± 2.1
6.6 ± 2.3
Visit 3
3.9 ± 2.4
5.6 ± 1.9
p value
< 0.001
< 0.001
Vaginal pH
Visit 1
6.3 ± 1.4
6.6 ± 1.2
Visit 2
6.0 ± 1.3
6.6 ± 1.2
Visit 3
6.1 ± 1.2
6.2 ± 1.1
p value
0.20
0.15 Table 3 Vaginal lactobacilli grading, vaginal atrophy symptoms (VAS), vaginal atrophy score, and vaginal pH in each visit of
participants in the laser group and sham group nal lactobacilli grading, vaginal atrophy symptoms (VAS), vaginal atrophy score, and vaginal pH in each visit of
n the laser group and sham group Their vaginal epithelium contains only few lactobacilli
that cannot produce enough lactic acid to lower vaginal
pH. Their vaginal epithelium contains only few lactobacilli
that cannot produce enough lactic acid to lower vaginal
pH. Table 4 Secondary outcome adjusted with time and age
because of imperfect randomization Table 4 Secondary outcome adjusted with time and age
because of imperfect randomization
Outcome
Mean
change
p value
95% CI
VAS
−2.73
0.15
−6.44 to 0.98
Dryness
−2.28
0.01
−4.11 to
− 0.46
Irritation
−0.36
0.57
−1.64 to 0.91
Soreness
−1.18
0.07
−2.44 to 0.08
Dyspareunia
−1.50
0.09
−3.22 to 0.22
Vaginal atrophy score
−1.49
0.03
−2.82 to
− 0.16
pH
−0.0
0.86
−0.61 to 0.51 A recent large prospective observational study of frac
tional CO2 laser conducted in Italy has shown a signifi
cant improvement of vaginal lactobacillus species at four
weeks after CO2 laser treatment [17]. Author contributions lactobacilli, vaginal pH, and vaginal atrophy score) out
comes of vaginal atrophy after vaginal laser treatment. N.P. wrote the main manuscript text, prepared all figures and tablesK.P and A.S. reviewed the manuscript. However, this study also has a limitation, which is the
different baseline characteristics between the laser group
and sham group (participants in the laser group were
younger that those in the sham group), which results
from imperfect randomization. Moreover, the age dif
ference between groups can have an impact on vaginal
lactobacilli grading. Therefore, the main findings of this
study were the improvement of vaginal atrophy symp
toms and vaginal atrophy score after the vaginal laser
treatments. References 1. Berek J, Novak D, Berek. & Novak’s gynecology. 16th ed. Philadelphia: Wolters
Kluwer; 2020. Chapter 18, Menopause;435. 2. Portman DJ, Gass MLS. Genitourinary syndrome of menopause: new
terminology for vulvovaginal atrophy from the international society for the
study of women’s sexual health and the north american menopause society. Climacteric. 2014;17(5):557–63. For further research, increasing the sample size or vagi
nal laser applications might show a statistically significant
improvement of subjective and objective measurements
of vaginal atrophy after vaginal erbium laser treatment. Moreover, further research may focus on the possible
benefits of the vaginal erbium laser in other study pop
ulations such as menopause women after ovarian tissue
cryopreservation and transplantation or patients with
refractory lower urinary tract dysfunction [19, 20]. 3. Stephanie S, Richa S, Ekta K. Genitourinary syndrome of menopause: man
agement strategies for the clinician. Mayo Clin Proc. 2017;92(12):1842–9. 3. Stephanie S, Richa S, Ekta K. Genitourinary syndrome of menopause: man
agement strategies for the clinician. Mayo Clin Proc. 2017;92(12):1842–9. 4. Walter JE, Larochelle A. No. 358-Intravaginal laser for genitourinary syndrome
of menopause and stress urinary incontinence. J Obstet Gynaecol Can. 2018;40(4):503–11. 4. Walter JE, Larochelle A. No. 358-Intravaginal laser for genitourinary syndrome
of menopause and stress urinary incontinence. J Obstet Gynaecol Can. 2018;40(4):503–11. 5. Pitsouni E, Grigoriadis T, Falagas ME, Salvatore S, Athanasiou S. Laser therapy
for the genitourinary syndrome of menopause. A systematic review and
meta-analysis. Maturitas. 2017;103(9):78–88. 6. Gaspar A, Brandy H, Gomez V, Luque D. Efficacy of Erbium:YAG laser treat
ment compared to topical estriol treatment for symptoms of genitourinary
syndrome of menopause. Laser Surg Med. 2017;49(2):160–8. 7. Gambacciani M, Levacini M, Cervigni M. Vaginal erbium laser: the second-
generation thermotherapy for the genitourinary syndrome of menopause. Climacteric. 2015;18(5):757–63. Data Availability Data Availability
All data and materials are described in the manuscript and tables. Conclusion No statistically significant difference in vaginal lacto
bacilli grading was observed before and after treatment
with erbium–YAG laser as compared with the sham
procedure. However, the grade of lactobacilli tended
to increase after two applications of vaginal laser. Vagi
nal “dryness” was improved significantly after treatment
with erbium–YAG laser. Furthermore, no serious adverse
events were reported. 8. Gambacciani M, Levacini M, Russo E, Vacca L, Simoncini T, Cervigni M. Long-term effects of vaginal erbium laser in the treatment of genitourinary
syndrome of menopause. Climacteric. 2018;21(2):148–52. 9. Athanasiou S, Pitsouni E, Antonopoulou S, Zacharakis D, Salvatore S, Falagas
ME. The effect of microablative fractional CO2 laser on vaginal flora of post
menopausal women. Climacteric. 2016;19(5):512–8. 10. Amabebe E, Anumba DOC. The vaginal microenvironment: the physiologic
role of lactobacilli. Front Med. 2018;13(5):181. 11. Gambacciani M, Palacios S. Laser therapy for the restoration of vaginal func
tion. Maturitas. 2017;99(5):10–5. 12. Redondo-Lopez V, Cook RL, Sobel JD. Emerging role of lactobacilli in the
control and maintenance of the vaginal bacterial microflora. Rev Infect Dis. 1990;12(5):856–72. Discussion
l
b This study also
demonstrates the improvement of vaginal atrophy symp
toms indicated by the most bothersome symptoms. How
ever, compared with the result of our study, a significant
improvement in vaginal pH was observed after the treat
ment. This result can be explained by the differences in
laser energies and percentage of participants with lacto
bacillus-predominated vaginal wet smear.h vaginal erbium–YAG laser treatment. The results show
an increase of vaginal lactobacilli grading after two laser
applications. The increase of vaginal lactobacilli grading
can be described by the effect of vaginal laser on vaginal
epithelial maturation, that is, superficial cells of the vagi
nal epithelium are increased after treatment with vaginal
laser. The superficial cells are glycogen-rich cells, and
lactobacilli use glycogen as their nutrients and produce
lactic acid. Moreover, vaginal atrophy symptoms assessed
by VAS and vaginal atrophy scores were improved after
laser treatments. The reduction in VAS score in this study
is similar to the results of previous studies [5–8, 13–15]. However, compared with several reports,[9, 16] no sta
tistically significant difference in vaginal pH before and
after vaginal laser applications is observed in this study. This result can be due to the majority of participants with
grade 1 vaginal lactobacilli after vaginal laser treatment. The safety of intravaginal laser therapy is a major con
cern. The result from previous studies showed only few
minor adverse events after vaginal laser application
[5, 8, 16, 18] In our study, only one patient in the sham
group complained about pain after treatment, which
was improved during the follow-up period. In addition,
no side effects were reported after treatment in the laser
group. Therefore, the safety result from our study is con
cordant with previous studies,[5, 8] confirming the short-
term safety of vaginal erbium–YAG laser treatment. However, long-term follow-up is necessary to evaluate
the long-term safety of this treatment. This study has several strengths. First, this study has a
randomized sham-controlled design, which can dimin
ish the bias and placebo effect. Second, our study evalu
ates the subjective (VAS score) and objective (vaginal Panyawongudom et al. BMC Women's Health (2023) 23:513 Panyawongudom et al. BMC Women's Health Page 6 of 7 Competing interests the authors declare that they have no competing interests the authors declare that they have no competing interests Received: 3 October 2022 / Accepted: 6 August 2023 Received: 3 October 2022 / Accepted: 6 August 2023 Received: 3 October 2022 / Accepted: 6 August 2023 A significant number of participants were lost to fol
low-up during the study period because of the COVID-
19 situation and personal illness that is not related to the
procedure. However, the number of participants who
were lost to follow-up was similar in the laser and sham
groups (2/22 and 3/22, respectively). Therefore, the effect
of loss to follow-up might not interfere with the outcome
of this study. Funding The study was funded by the Ratchadapisek Sompoch Endowment Fund
Chulalongkorn University Fund. Ethics approval and consent to participate Ethics approval and consent to participate
This study had been performed in accordance with the Declaration of Helsinki
and was approved by Institutional Review Board of Faculty of Medicine,
Chulalongkorn University. All participants were completed written informed
consent before data collection and all procedures. Another limitation of our study was lacking an active
control group (such as vaginal estrogen, vaginal lactoba
cilli, or other non-hormonal treatments). Thus, for clini
cal implication, clinicians should be acknowledged this
limitation. However, as this study showed the improve
ments of vaginal atrophy after the vaginal erbium laser
procedure, it might lead to further clinical trials with an
active control group. Declarations Ethics approval and consent to participate Acknowledgements We would like to thank the Gynecologic Outpatient Clinic and Gender
Health Clinic at King Chulalongkorn Memorial Hospital for their support in
collecting the data. Special thanks to Dr.Tanittha Chatsuwan, Department
of Microbiology, Faculty of Medicine, Chulalongkorn University and her
colleagues who evaluated the grade of vaginal lactobacilli. Of course, we are
grateful to all subjects in this study. 13. Hutchinson-Colas J, Segal S. Genitourinary syndrome of menopause and the
use of laser therapy. Maturitas. 2015;82(4):342–5. 14. Rabley A, O’Shea T, Terry R, Byun S, Moy ML. Laser therapy for genitourinary
syndrome of menopause. Curr Urol Rep. 2018;19(10):83. Panyawongudom et al. BMC Women's Health (2023) 23:513 Page 7 of 7 Page 7 of 7 Page 7 of 7 (2023) 23:513 20. Culmone S, Speciale P, Guastella E, Puglisi T, Cucinella G, Paizza F, et al. Sacral
neuromodulation for refractory lower urinary tract dysfunctions: a single-
center retrospective cohort study. Ital J Gynaecol Obstet. 2022;34(4):317–23. 17. Di Donato V, D’Oria O, Giannini A, Scudo M, Sher C, Fischetti M, et al. The
efficacy of fractional CO2 laser in the treatment of Genitourinary Syndrome
of Menopause: a large prospective observational study. Clin Exp Obstet
Gynecol. 2022;49(9):212. 18. Gambacciani M, Elia D, Berreni N, Bohbot JM, Druckmann R, Geoffrion H, et al
Genitourinary syndrome of menopause (GSM) and laser VEL: a review. Horm
Mol Biol Clin Investig. 2020;41(1):307–11. 16. Mothes AR, Runnebaum M, Runnebaum IB. Ablative dual-phase Erbium:YAG
laser treatment of atrophy-related vaginal symptoms in post-menopausal
breast cancer survivors omitting hormonal treatment. J Cancer Res Clin
Onco. 2018;144(5):955–60. 19. Gullo G, Etrusco A, Cucinella G, Basile G, Fabio M, Perino A, et al. Ovarian tis
sue cryopreservation and transplantation in menopause: new perspective of
therapy in postmenopausal women and the importance of ethical and legal
frameworks. Eur Rev Med Pharmacol Sci. 2022;26:9107–16. 15. Jha S, Wyld L, Krishnaswamy PH. The impact of vaginal laser treatment for
genitourinary syndrome of menopause in breast cancer survivors: a system
atic review and meta-analysis. Clin Breast Cancer. 2019;19(4):556–62.
16. Mothes AR, Runnebaum M, Runnebaum IB. Ablative dual-phase Erbium:YAG
laser treatment of atrophy-related vaginal symptoms in post-menopausal
breast cancer survivors omitting hormonal treatment. J Cancer Res Clin
Onco. 2018;144(5):955–60.
17. Di Donato V, D’Oria O, Giannini A, Scudo M, Sher C, Fischetti M, et al. The
efficacy of fractional CO2 laser in the treatment of Genitourinary Syndrome
of Menopause: a large prospective observational study. Clin Exp Obstet
Gynecol. 2022;49(9):212.
18. Gambacciani M, Elia D, Berreni N, Bohbot JM, Druckmann R, Geoffrion H, et al.
Genitourinary syndrome of menopause (GSM) and laser VEL: a review. Horm
Mol Biol Clin Investig. 2020;41(1):307–11. 15. Jha S, Wyld L, Krishnaswamy PH. The impact of vaginal laser treatment for
genitourinary syndrome of menopause in breast cancer survivors: a system
atic review and meta-analysis. Clin Breast Cancer. 2019;19(4):556–62. 19. Gullo G, Etrusco A, Cucinella G, Basile G, Fabio M, Perino A, et al. Ovarian tis
sue cryopreservation and transplantation in menopause: new perspective of
therapy in postmenopausal women and the importance of ethical and legal
frameworks. Eur Rev Med Pharmacol Sci. 2022;26:9107–16.
20. Culmone S, Speciale P, Guastella E, Puglisi T, Cucinella G, Paizza F, et al. Sacral
neuromodulation for refractory lower urinary tract dysfunctions: a single-
center retrospective cohort study. Ital J Gynaecol Obstet. 2022;34(4):317–23. 15. Jha S, Wyld L, Krishnaswamy PH. The impact of vaginal laser treatment for
genitourinary syndrome of menopause in breast cancer survivors: a system
atic review and meta-analysis. Clin Breast Cancer. 2019;19(4):556–62.
16. Mothes AR, Runnebaum M, Runnebaum IB. Ablative dual-phase Erbium:YAG
laser treatment of atrophy-related vaginal symptoms in post-menopausal
breast cancer survivors omitting hormonal treatment. J Cancer Res Clin
Onco. 2018;144(5):955–60. Publisher’s Note
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Supplementary Figure 6 from Ang-2-VEGF-A CrossMab, a Novel Bispecific Human IgG1 Antibody Blocking VEGF-A and Ang-2 Functions Simultaneously, Mediates Potent Antitumor, Antiangiogenic, and Antimetastatic Efficacy
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Suppl. Figure 1
A
B
Supplemental Figure 1. (A) Schematic representation of Ang2-VEGF-A
CrossMab (RO5520985) blocking VEGF A and Ang 2 function simultaneously
CrossMab (RO5520985) blocking VEGF-A and Ang-2 function simultaneously.
Blue color represents the Ang-2 crossed (CH1-Cκ crossover) Fab domain.
Yellow color represents the original bevacizumab (Avastin) light chain. Heavy
chain heterodimerization is enforced using KiH (“knobs into holes”) technology
by introducing a “knob” in the Fc part of bevacizumab and a “hole” in the Fc part Kienast et al. Suppl. Figure 1
A
B
Supplemental Figure 1. (A) Schematic representation of Ang2-VEGF-A
CrossMab (RO5520985) blocking VEGF A and Ang 2 function simultaneously
CrossMab (RO5520985) blocking VEGF-A and Ang-2 function simultaneously. Blue color represents the Ang-2 crossed (CH1-Cκ crossover) Fab domain. Yellow color represents the original bevacizumab (Avastin) light chain. Heavy
chain heterodimerization is enforced using KiH (“knobs into holes”) technology
by introducing a “knob” in the Fc part of bevacizumab and a “hole” in the Fc part A Kienast et al.
Suppl. Figure 1 A A B
Supplemental Figure 1. (A) Schematic representation of Ang2-VEGF-A
CrossMab (RO5520985) blocking VEGF A and Ang 2 function simultaneously
CrossMab (RO5520985) blocking VEGF-A and Ang-2 function simultaneously. Blue color represents the Ang-2 crossed (CH1-Cκ crossover) Fab domain. Yellow color represents the original bevacizumab (Avastin) light chain. Heavy
chain heterodimerization is enforced using KiH (“knobs into holes”) technology
by introducing a “knob” in the Fc part of bevacizumab and a “hole” in the Fc part
of the Ang-2 targeting antibody fragment. (B) Simultaneous binding of A2V B B B Supplemental Figure 1. (A) Schematic representation of Ang2-VEGF-A
CrossMab (RO5520985) blocking VEGF A and Ang 2 function simultaneously
CrossMab (RO5520985) blocking VEGF-A and Ang-2 function simultaneously. Blue color represents the Ang-2 crossed (CH1-Cκ crossover) Fab domain. Yellow color represents the original bevacizumab (Avastin) light chain. Heavy
chain heterodimerization is enforced using KiH (“knobs into holes”) technology
by introducing a “knob” in the Fc part of bevacizumab and a “hole” in the Fc part
of the Ang-2 targeting antibody fragment. (B) Simultaneous binding of A2V
CrossMab to VEGF-A and Ang-2 as shown by SPR.
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Association of central obesity with sex hormonebinding globulin: a cross-sectional study of 1166 Chinese men
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Open Medicine
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1 Introduction Sex hormone-binding globulin (SHBG) is a kind of gly-
coprotein that binds to androgen or estrogen hormones
in blood circulation and is mainly produced by human
hepatocytes. Low serum level of SHBG has been found a
risk factor of metabolic syndrome (MS) in both cross-sec-
tional and longitudinal studies [1-3]. Also, a reverse rela-
tion was found between SHBG and cardiovascular disease
(CVD) [4]. *Corresponding author: Yuanzhong Zhou, School of Public Health,
Zunyi Medical University, No.6 of Xuefuxi Road, Honghuagang
District, Zunyi of Guizhou Province, PR of China, E-mail: zhouyuanz-
hong@163.com
Fangwei Liu, Xubo Shen, Shimin Xiong, School of Public Health,
Zunyi Medical University, Zunyi, China
Ruifeng Wang, Department of Chronic Noncommunicable Diseases,
Huichuan District Center for Disease Control and Prevention, Zunyi,
China
Na Yu, School of Public Health, Guangdong Medical University, Dong
Guan, China
Yongjun Shi, Department of Neonatology, Guiyang Maternal and
Child Healthcare Hospital, Guiyang, China
Chengliang Xiong, Institute of Family Planning, Huazhong University
of Science and TechnologyTongji Medical College, Wuhan, China
Open Access. © 2018 Fangwei Liu et al., published by De Gruyter. *Corresponding author: Yuanzhong Zhou, School of Public Health,
Zunyi Medical University, No.6 of Xuefuxi Road, Honghuagang
District, Zunyi of Guizhou Province, PR of China, E-mail: zhouyuanz-
hong@163.com
Fangwei Liu, Xubo Shen, Shimin Xiong, School of Public Health,
Zunyi Medical University, Zunyi, China
Ruifeng Wang, Department of Chronic Noncommunicable Diseases,
Huichuan District Center for Disease Control and Prevention, Zunyi,
China
Na Yu, School of Public Health, Guangdong Medical University, Dong
Guan, China
Yongjun Shi, Department of Neonatology, Guiyang Maternal and
Child Healthcare Hospital, Guiyang, China
Chengliang Xiong, Institute of Family Planning, Huazhong University
of Science and TechnologyTongji Medical College, Wuhan, China *Corresponding author: Yuanzhong Zhou, School of Public Health,
Zunyi Medical University, No.6 of Xuefuxi Road, Honghuagang
District, Zunyi of Guizhou Province, PR of China, E-mail: zhouyuanz-
hong@163.com Central obesity, also known as abdominal obesity, is
one of the National Cholesterol Education Program Adult
Treatment Panel (ATP-III) criteria [5], the most widely
used criteria in diagnosing MSat present. Moreover, CVD
risk is associated more strongly with central obesity than
general obesity [6-8]. Open Med. 2018; 13: 196-202 Fangwei Liu, Xubo Shen, Ruifeng Wang, Na Yu, Yongjun Shi, Shimin Xiong, Chengliang Xiong,
Yuanzhong Zhou*
Association of central obesity with sex hormone-
binding globulin: a cross-sectional study of 1166
Chinese men https://doi.org/10.1515/med-2018-0030
received August 7, 2017; accepted March 26, 2018 https://doi.org/10.1515/med-2018-0030
received August 7, 2017; accepted March 26, 2018 nmol/L), farm occupation (43.58±20.60nmol/L), non-al-
cohol or former user (44.78±20.94 nmol/L) and long-term
medication history (44.79±21.50 nmol/L). Factors inde-
pendently associated with sex hormone binding globulin
level on multiple regression were waist-to-height ratio (β=-
11.84 [95% confidence interval -13.96,-9.72]), age(β=12.40
[9.63,15.17]) and diastolic blood pressure (β=-5.07 [-7.44,-
2.71]). Abstract: Background Both sex hormone-binding globu-
lin and central obesity have been found to be associated
with metabolic and cardiovascular diseases. However,
the direct relation between sex hormone-binding globulin
and central obesity has not been demonstrated. Methodology We performed a cross-sectional study of 1166
male participants from Zunyi, Guizhou, western China, in
2013. Each participant completed a questionnaire and had
a brief clinical exam with a fasting blood sample taken. All blood samples underwent standard laboratory testing
for sex hormone-binding globulin. Level of serum sex hor-
mone-binding globulin was compared by demographic
characteristics, and multiple linear regression was used
to evaluate the independent association of variables and
sex hormone-binding globulin level. Conclusions Central obesity has an independent inverse
relation with serum level of sex hormone binding globulin
among western Chinese men Keywords: Cardiovascular risk; Metabolism; Cross-sec-
tional study Results The mean serum level of sex hormone-binding
globulin was increased in old-aged men (older than 40
years; mean 44.68±20.58 nmol/L), low diastolic blood
pressure (<90mmHg; 43.76±20.50 nmol/L), waist-to-height
ratio <0.5 (48.73±20.59 nmol/L), no education (52.36±22.91 Open Access. © 2018 Fangwei Liu et al., published by De Gruyter.
This work is licensed under the Creative Commons Attribution-NonCom-
mercial-NoDerivatives 4.0 License. al., published by De Gruyter.
This work is licensed under the Creative Commons Attribution-NonCom- Central obesity related to SHBG
197 often defined by waist circumference (WC) only, even in
the ATP-III criteria. Waist-to-height ratio (WHtR) is more
appropriate in defining central obesity than WC and waist-
to-hip (WHR) ratio because WC and WHR do not include
height, which can influence the distribution of abdominal
fat and differs by age and race. Also, WHtR shows better
predictive ability than WC and BMI for diabetes, hyperten-
sion, and CVD [6, 9, 10]. The most commonly use index,
body mass index (BMI), is used for substance adipose
tissue not visceral adipose tissue, and it cannotdistin-
guish muscle type obesity or adipose type obesity. The questionnaire mainly collected the basic informa-
tion of participants, including age, marital status, educa-
tion status, smoking, drinking, occupation and previous
history, including vasectomy and long-term medication
status. Trained study staff measured body weight, height,
waist circumference, systolic blood pressure, diastolic
blood pressure, and WHtR [waist circumference (cm)/
height (cm)] for participants. Fasting venous blood
samples were collected by trained nurses and were centri-
fuged for 15 min at 4°C to obtain serum and stored at -80°C
until analysis. The data for this cross-sectional study were from the
Chinese Middle-aged and Elderly Men of Reproduction
Health Project. In this study, we used WHtR rather than
WC or WHR to define central obesity to investigate the
direct association of SHBG level with central obesity after
adjustment for confounding factors (demographic char-
acteristics and lifestyle), to support existing evidence of
both biochemical and epidemiological research. Ethical approval: The research related to human use
has been complied with all the relevant national regula-
tions, institutional policies and in accordance the tenets
of the Helsinki Declaration. 2.3 Laboratory assessments We used chemiluminescent immunoassays to measure
SHBG on a Beckman Access Immunoassay System
(Beckman Coulter, Brea, CA, USA). 2.4 Statistical analysis We performed this cross-sectional study from August 20
to September 20, 2013, in Zunyi, Guizhou, located in the
southwest of China, with a population of 1.2 million. We
used a stratified cluster design. Among 80 communities in
this city, 50 km away from the downtown, 7 communities
were targeted (2 urban communities, 2 suburban commu-
nities and 3 rural communities). Males older than 20 years
from the 7 communities were qualified to participate in a
questionnaire and a brief clinical exam voluntarily. We
included 1213 participants initially, and 1166 participants
were finally included. See in the flow chart below. Data were analyzed by using analyzed by SPSS 18.0 (SPSS
Inc., Chicago, IL, USA). We re-coded the independent
variables in the multiple models as binary variables. The
cutoff of WHtR was set as 0.5 as per the literature and as
a suitable predictor of diabetes, CVD and MS(11-15). The
other independent variables were defined by common
clinical standards. Quantitative data are presented as
mean±SD and categorical data as frequency (%). SHBG
level in groups was compared by one-way ANOVA. P<0.05
was considered statistically significant. We used multiple
linear regression to evaluate the association of independ-
ent variables with SHBG level and the results are shown as
beta values and 95% confidence intervals (95%CIs). 2.2 Study design
Every participant was asked to sign consent before the test
and each was anonymized for research and confidential-
ity purposes. 2.2 Study design 1 Introduction Ruifeng Wang, Department of Chronic Noncommunicable Diseases,
Huichuan District Center for Disease Control and Prevention, Zunyi,
China Na Yu, School of Public Health, Guangdong Medical University, Dong
Guan, China Yongjun Shi, Department of Neonatology, Guiyang Maternal and
Child H
l h
H
i
l G i
Chi Some diseases seem to be part of both SHBG and
central obesity. However, studies that focused on their
direct relation are few. Furthermore, central obesity is Chengliang Xiong, Institute of Family Planning, Huazhong University
of Science and TechnologyTongji Medical College, Wuhan, China Chengliang Xiong, Institute of Family Planning, Huazhong University
of Science and TechnologyTongji Medical College, Wuhan, China 197 3 Results 2.2 Study design 198
Fangwei Liu et al. Table 1: Demographic characteristics by WHtR
Characteristics
WHtR
Total
≥0.5
n=631
<0.5
n=535
Education, N(%)
Uneducated
63(5.4)
47(4.0)
110(9.4)
Educated
538(48.7)
488(41.9)
1056(90.6)
Occupation, N(%)
Farmer
503(43.1)
453(38.9)
956(82.0)
Other
128(11.0)
82(7.0)
210(18.0)
Marital status, N(%)
Married (including co-habitating)
596(51.1)
494(42.4)
1090(93.5)
Single, divorced or widowed
35(3.0)
41(3.5)
76(6.5)
Vasectomy, N(%)
Yes
52(4.5)
45(3.9)
97(8.3)
No
579(49.7)
490(42.0)
1069(91.7)
Smoking, N(%)
Current
491(42.1)
428(36.7)
919(78.8)
Never or former
140(12.0)
107(9.2)
247(21.2)
Alcohol use, N(%)
Current
373(32.0)
295(25.3)
668(57.3)
Never or former
258(22.1)
240(20.6)
498(42.7)
Long-term medication, N(%)
Yes
128(11.0)
88(7.5)
216(18.5)
No
503(43.1)
447(38.3)
950(81.5)
Uneducated=Never went to school for formal education; Educated=At least received a primary education; WHtR=waist-to-height ratio;
SBP=systolic blood pressure; DBP=diastolic blood pressure; FBG=fasting blood glucose Uneducated=Never went to school for formal education; Educated=At least received a primary education; WHtR=waist-to-height ratio;
SBP=systolic blood pressure; DBP=diastolic blood pressure; FBG=fasting blood glucose. 3.3 Factors associated with SHBG level on
multiple regression mmHg and 5.56±1.56nmol/L respectively. Other details are
shown in Table 1. Table 3 shows factors associated with SHBG level on
adjusted multiple linear regression. Except for SBP
because of its colinearity with DBP, factors significantly
associated with SHBG level were WHtR(β=-11.84), age(β=-
12.04), DBP(β=-5.07), education(β=-8.70) and occupa-
tion(β=-4.03). 3.1 Demographic characteristics Every participant was asked to sign consent before the test
and each was anonymized for research and confidential-
ity purposes. For 631 participants, WHtR was ≥ 0.5 and for 535 it was <
0.5. The mean age, systolic blood pressure (SBP), diastolic
blood pressure (DBP) and fasting blood glucose (FBG) was
51.56±12.82 years old, 128.81±19.04 mmHg, 83.79±12.03 Table 2: Mean SHBG level by demographic characteristics
Characteristics
SHBG, nmol/L
P value
Age
0.000
≥40 years
44.68±20.57
<40 years
31.67±14.59
DBP(mmHg)
0.000
≥90
38.11±18.91
<90
43.76±20.50
WHtR
0.000
≥0.5
36.56±18.11
<0.5
48.73±20.59
Education
0.000
Uneducated
52.36±22.91
Educated
41.08±19.62
Occupation
0.000
Farmer
43.58±20.60
Other occupation
35.63±16.93
Alcohol use
0.000
Current
40.18±19.44
Never or former
44.78±20.94
Long-term medication
0.033
Yes
44.79±21.50
No
41.55±19.87
Data are mean±SD. SHBG=Sex hormone-binding globulin; DBP=di-
astolic blood pressure; WHtR=waist-to-height ratio; Uneducat-
ed=Never went to school for formal education; Educated=At least got
a primary education. Table 3: Multiple linear regression of factors associated with SHBG
level. Variables
β
95%CI
WHtR
-11.84**
(-13.96,-9.72)
Age
12.40**
(9.63,15.17)
DBP
-5.07**
(-7.44,-2.71)
FBG
-0.86
(-2.96,1.25)
Education
-8.70**
(-12.34,-5.06)
Occupation
-4.03*
(-6.79,-1.26)
Marital status
-2.16
(-6.42,2.09)
Vasectomy
-0.17
(-3.96,3.62)
Smoking
0.97
(-1.62,3.56)
Alcohol
-2.04
(-4.19,0.12)
Long-term medication
1.67
(-1.10,4.43) Table 2: Mean SHBG level by demographic characteristics
Characteristics
SHBG, nmol/L
P value
Age
0.000
≥40 years
44.68±20.57
<40 years
31.67±14.59
DBP(mmHg)
0.000
≥90
38.11±18.91
<90
43.76±20.50
WHtR
0.000
≥0.5
36.56±18.11
<0.5
48.73±20.59
Education
0.000
Uneducated
52.36±22.91
Educated
41.08±19.62
Occupation
0.000
Farmer
43.58±20.60
Other occupation
35.63±16.93
Alcohol use
0.000
Current
40.18±19.44
Never or former
44.78±20.94
Long-term medication
0.033
Yes
44.79±21.50
No
41.55±19.87 3.4 Relation between WHtR and SHBG level The following scatter grams show the relation between
WHtR and SHBG level by factors presented in Table 2. The range of the X-axis was 0.35-0.72(16). Despite central
obesity, SHBG level was higher for male solder than
younger than 40 years and was higher with DBP <90 than
> 90 mmHg (Figure 1). SHBG level was higher for uned-
ucated than educated males. It was higher for farmers
than other occupations in the non-central-obesity group,
but with increasing WHtR in the central-obesity group,
the SHBG level between groups approached concord-
ance (Figure 2). SHBG level was lower for men who were
alcohol users than never or former users. It was higher for
men with than without long-term medication history in
the non-central-obesity group, but in the central-obesity
group, SHBG level between groups approached concord-
ance with increasing WHtR (Figure 3). 3.2 Serum level of SHBG by demographic
characteristics. Table 2 shows only significant results of the association
of characteristics with SHBG level. Mean serum level of
SHBG significantly differed by age, DBP, WHtR, educa-
tion, occupation, alcohol use and long-term medication. Central obesity related to SHBG 199 Figure 2: Relation between WHtR and SHBG level by sociological
characteristics (A) education and (B) occupation. In our findings, central obesity defined by WHtR was
inversely related to SHBG level. The result is similar to
research from the Endocrinology Unit at the University
Hospital of Los Andes Merida and Venezuela, finding in 70
men aged 20 to 62 years old, that SHBG level was inversely
correlated with WC (r=-0.322, P<0.01) (17). A cross-sectional
study from Korea also found SHBG level inversely related
to WC and BMI [18]. However, those two indices cannot
define central obesity better than WHtR. Our result might
show a more precise relation between central obesity and
SHBG level. A transgenic animal experiment from Selva et
al [19] showed the mechanism of SHBG decreasing with
increased lipid levels in hepatocytes: increasing monosac-
charide-induced lipogenesis caused a down-regulation
of hepatocyte nuclear factor 4and reduced expression of
SHBG gene in hepatocytes, thus decreased SHBG level. This research provides a biological explanation for our
study results showing decreased SHBG level with obesity. Our results show that SHBG level was in dependently
and positively associated with age. A large cross-sectional
study offered more precise results to support this rela-
tion(20): the study of 58,162 men among 110,712 partici-
pant saged from 10 to 90 years old tested blood testoster-
one, SHBG and calculated free testosterone levels together
with sex and age and created smoothed age-specific cen-
tiles (2.5%, 5%, 25%, 50%, 75%, 95%, 97.5%) for males
and females. After early childhood, serum SHBG level
declined to a nadir in males at age 20 years and remained
stable until the sixth decade of age, with a gradual, pro-
gressive increase thereafter. Our study did not reveal a
decline in SHBG level in young males because we exam-
ined male solder than 20 years, so the results of SHBG
level only show the increasing trend with age. Longitudi-
nal study from two Australian [21], geographically widely
separated regions, of 610 men at baseline and adding 370
and 200 men on the second and third occasion from 1989
to 2004, revealed that SHBG level increased annually and
the increase was steeper in middle-aged and older men
versus young men(P<0.001). The result of SHBG level
increasing with age in our study is consistent with both
cross-sectional and longitudinal studies. However, we
lack the molecular biology evidence of this phenomenon. 4 Discussion This was a cross-sectional study of1166 male participants
from the Chinese Middle-aged and Elderly Men of Repro-
duction Health Project. Each participant took a ques-
tionnaire, a brief clinical exam and a blood sample; the
one-way ANOVA was used to compare the differences Data are mean±SD. SHBG=Sex hormone-binding globulin; DBP=di-
astolic blood pressure; WHtR=waist-to-height ratio; Uneducat-
ed=Never went to school for formal education; Educated=At least got
a primary education. Data are mean±SD. SHBG=Sex hormone-binding globulin; DBP=di-
astolic blood pressure; WHtR=waist-to-height ratio; Uneducat-
ed=Never went to school for formal education; Educated=At least got
a primary education. Figure 1: Relation between WHtR and SHBG level by physiologic
characteristics (A) age and (B) DBP. The 0.50 cutoff of WHtR is shown
by the upright dotted line. Table 3: Multiple linear regression of factors associated with SHBG
level. Variables
β
95%CI
WHtR
-11.84**
(-13.96,-9.72)
Age
12.40**
(9.63,15.17)
DBP
-5.07**
(-7.44,-2.71)
FBG
-0.86
(-2.96,1.25)
Education
-8.70**
(-12.34,-5.06)
Occupation
-4.03*
(-6.79,-1.26)
Marital status
-2.16
(-6.42,2.09)
Vasectomy
-0.17
(-3.96,3.62)
Smoking
0.97
(-1.62,3.56)
Alcohol
-2.04
(-4.19,0.12)
Long-term medication
1.67
(-1.10,4.43)
WHtR=Waist-to-height ratio; DBP=diastolic blood pressure; FBG=-
fasting blood glucose. **p<0.001, *p<0.05
Figure 1: Relation between WHtR and SHBG level by physiologic
characteristics (A) age and (B) DBP. The 0.50 cutoff of WHtR is shown
by the upright dotted line. Table 3: Multiple linear regression of factors associated with SHBG
level. Table 3: Multiple linear regression of factors associated with SHBG
level. Variables
β
95%CI
WHtR
-11.84**
(-13.96,-9.72)
Age
12.40**
(9.63,15.17)
DBP
-5.07**
(-7.44,-2.71)
FBG
-0.86
(-2.96,1.25)
Education
-8.70**
(-12.34,-5.06)
Occupation
-4.03*
(-6.79,-1.26)
Marital status
-2.16
(-6.42,2.09)
Vasectomy
-0.17
(-3.96,3.62)
Smoking
0.97
(-1.62,3.56)
Alcohol
-2.04
(-4.19,0.12)
Long-term medication
1.67
(-1.10,4.43)
WHtR=Waist-to-height ratio; DBP=diastolic blood pressure; FBG=- Figure 1: Relation between WHtR and SHBG level by physiologic
characteristics (A) age and (B) DBP. The 0.50 cutoff of WHtR is shown
by the upright dotted line. 200
Fangwei Liu et al. a progressive impairment in left-ventricle relaxation
with increasing number of MS criteria, which indicates
impaired diastolic function with increasing burden of MS. Another more than4-year longitudinal study used similar
techniques to measure ventricular-arterial function under
general and central adiposity. Weight gain was associated
with significant increases in LV diastolic stiffness in both
men and women, whereas central obesity and insulin
resistance were associated with large increases in end-sys-
tolic elastance in women but not men, which indicates sex
difference in the biology of age-related ventricular systolic
stiffening [24]. Our finding of central obesity inversely
associated with SHBG level can explain why SHBG level is
negatively related with only DBP. a progressive impairment in left-ventricle relaxation
with increasing number of MS criteria, which indicates
impaired diastolic function with increasing burden of MS. Another more than4-year longitudinal study used similar
techniques to measure ventricular-arterial function under
general and central adiposity. Weight gain was associated
with significant increases in LV diastolic stiffness in both
men and women, whereas central obesity and insulin
resistance were associated with large increases in end-sys-
tolic elastance in women but not men, which indicates sex
difference in the biology of age-related ventricular systolic
stiffening [24]. Our finding of central obesity inversely
associated with SHBG level can explain why SHBG level is
negatively related with only DBP. We cannot declare whether the relation of SHBG
level with central obesity was causal because this was a
cross-sectional study. The data in this study came from
the Chinese Middle-aged and Elderly Men Reproduction
Health Project, and it did not survey all risk factors for
metabolic and CVD. Also, we did not exclude the subjects
with some special diseases that may affect SHBG level,
such as hepatic or thyroid disease [27]. Further studies
could focus on the mechanism of how SHBG influences
diastolic function and blood glucose. Funding: This work was supported by the Ministry of
Science and Technology of the People’s Republic of China
(grant no.2012BAI32B03) SHBG level is inversely related with insulin resist-
ance (IR) among men [17], so SHBG should have the same
relation with blood glucose because IR would lead to
high blood glucose status. However, in our study, SHBG
showed no relation with FBG. The reasons may be that 1)
we did not collect the biochemical criteria for IR in high
FBG participants, so we can not declare whether is the
IR or the pancreas beta cell dysfunction leading to high
fasting blood glucose condition; 2)even if some partici-
pants had IR, the resistant status might be weak so that
FBG did not increase enough to be defined by a medical
test; 3) a cohort of participants who were normoglycemic
at baseline but hyperglycemic at 3years(glycemia≥6.1m-
mol/L or Type2 diabetes) and matched for sex, age, and
BMI with control participants who remained normogly-
cemicin 3 years found serum SHBG level significantly
associated with risk of developing hyperglycemia among
women but not men [25]. This suggests that the interaction
between SHBG level and blood glucose may be bridged by
estrogens in part. Conflicts of interest: The authors have no conflicts of
interest to declare. Figure 2: Relation between WHtR and SHBG level by sociological
characteristics (A) education and (B) occupation. Figure 3: Relation between WHtR and SHBG level by life style charac-
teristics (A) alcohol use and (B) long-term medication. Figure 3: Relation between WHtR and SHBG level by life style charac-
teristics (A) alcohol use and (B) long-term medication. We observed SHBG level inversely related to DBP, but
with no significant difference in SBP (Tables 2-3). In MS,
lower serum SHBG concentration is found related to DBP
and SBP [1, 22]. We have no direct evidence to prove this
phenomenon in our study. Across-sectional study [23]
used echocardiography, pulse-wave Doppler and tissue
Doppler imaging to measure the structure and function
of the left ventriclein participantsgrouped according to
number of MS criteria (ATP-III) met; the results suggested between groups, and significant results were also showed
in figures (Figure1-3). The main results were revealed in
the multiple linear regression (Table 3). Serum level of
SHBG was inversely related with central obesity (WHtR). In addition, age, DBP, education and occupation were
independently related to serum SHBG level. 201 Central obesity related to SHBG
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Palliative care for persons with Parkinson’s disease: a qualitative study on the experiences of health care professionals
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Download date: 2024-10-24 Palliative care for persons with Parkinson's disease: a qualitative study on the experiences of
health care professionals
Lennaerts, H.H.; Steppe, M.A.; Munneke, M.; Meinders, M.J.; Steen, J.T. van der; Brand, M van den;
Vissers, K.; Bloem, B.R.; Groot, M. 2019, Article / Letter to editor (BMC Palliative Care, 18, (2019), article 53)
Doi link to publisher: https://doi.org/10.1186/s12904-019-0441-6 Note: Note:
To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). Lennaerts et al. BMC Palliative Care (2019) 18:53
https://doi.org/10.1186/s12904-019-0441-6 Open Access © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Parkinson’s disease (PD) is a chronic and neurodegenerative disease associated with a wide variety of
symptoms. The risk of complications increases with progression of the disease. These complications have a
tremendous impact on the quality of life of people with PD. The aim of this study was to examine health
care professionals’ experiences of potential barriers and facilitators in providing palliative care for people with
PD in the Netherlands. Methods: This was a qualitative descriptive study. The data were collected from 10 individual in-depth interviews and
three focus groups (n = 29) with health care professionals. Health care professionals were selected based on a positive
answer to the question: “In the past 2 years, did you treat or support a person with PD who subsequently died?” The
data were analyzed by thematic text analysis. Results: Health care professionals supported the development of a palliative care system for PD but needed to better
understand the essence of palliative care. In daily practice, they struggled to identify persons’ needs due to interfering
PD-specific symptoms such as cognitive decline and communication deficits. Timely addressing the personal
preferences for providing palliative care was identified as an important facilitator. Health care professionals
acknowledged being aware of their lack of knowledge and of their little competence in managing complex
PD. Findings indicate a perceived lack of care continuity, fragmentation of services, time pressure and
information discontinuity. Conclusions: Health care professionals experienced several facilitators and barriers to the provision of palliative care
to people with PD. There is a need to improve the knowledge on complex PD and the continuity of information, as
well as optimize coordination and deliver care based on a persons’ preferences. Additional training can help
to become more knowledgeable and confident. Keywords: Parkinson’s disease, Palliative care, Barriers, Facilitators, Focus groups, Qualitative approaches disciplines are involved in the care for persons with PD
and their family caregivers. Because PD progresses
slowly, in the early phase it is generally not considered
as a terminal illness. But PD does increase mortality, al-
though PD is usually not listed as the immediate cause
of death [4, 5]. Most persons with PD die from compli-
cations such as dementia, pneumonia, infections or fall-
related injuries [5–8]. Furthermore, late-stage PD is
associated with considerable suffering, comparable with
end-stage cancer [9]. Palliative care for persons with Parkinson’s
disease: a qualitative study on the
experiences of health care professionals Herma Lennaerts1*, Maxime Steppe1, Marten Munneke1, Marjan J. Meinders6, Jenny T. van der Steen3,4,
Marieke Van den Brand2, Dorian van Amelsvoort5, Kris Vissers2, Bastiaan R. Bloem1 and Marieke Groot2 Background Parkinson’s disease (PD) is a chronic, progressive disease
for which there is no cure. Affected persons may expe-
rience a wide range of symptoms, such as immobility,
pain, fatigue, sleeping problems, cognitive deficits or
dementia [1–3]. Therefore, professionals from various * Correspondence: Herma.lennaerts@radboudumc.nl
1Department of Neurology, Donders Institute for Brain, Cognition and
Behaviour, Radboud university medical center, Reinier Postlaan 4, 6500
Nijmegen, AB, The Netherlands
Full list of author information is available at the end of the article * Correspondence: Herma.lennaerts@radboudumc.nl
1Department of Neurology, Donders Institute for Brain, Cognition and
Behaviour, Radboud university medical center, Reinier Postlaan 4, 6500
Nijmegen, AB, The Netherlands
Full list of author information is available at the end of the article * Correspondence: Herma.lennaerts@radboudumc.nl
1Department of Neurology, Donders Institute for Brain, Cognition and
Behaviour, Radboud university medical center, Reinier Postlaan 4, 6500
Nijmegen, AB, The Netherlands
Full list of author information is available at the end of the article Lennaerts et al. BMC Palliative Care (2019) 18:53 Page 2 of 9 Lennaerts et al. BMC Palliative Care (2019) 18:53 The World Health Organization (WHO) has defined
palliative care as an approach that improves quality of
life of patients and families facing the problems asso-
ciated with life-threatening illnesses [10]. Many health-
care professionals from a wide range of professional
disciplines can provide “generalist palliative care”. In
addition, specialist palliative care (specialised or specialty
palliative care) can be considered when the patient’s or
family’s needs exceed the competence and confidence of
healthcare professionals or when access to certain ser-
vices is required. The need to further develop and imple-
ment specialised palliative care for persons with PD has
received more attention over the last decades [11–13]. However, despite these efforts, it is often not a common
component of current PD care programs. Furthermore,
it is unclear how “generalist palliative care” in PD should
be provided. Yet, it is important to initiate palliative care
at the appropriate disease stage, preferably in addition to
a more disease-oriented approach [12–14]. to seek professional care from members of the Parkinson-
Net, but this is not compulsory, and patients are free to
also select more generically active professionals. Import-
antly, recent work showed that patients treated by Parkin-
sonNet professionals are very comparable to patients
treated by generically trained therapists, e.g. in regards to
age, gender or education level [25]. The coverage of Par-
kinsonNet depends on the professional discipline, and var-
ies between around 60% of patients for physiotherapy to
around 90% for speech-language therapy (Darweesh et al.,
unpublished observations). p
All health care professionals from ParkinsonNet re-
ceived an email and those who confirmed they had
treated or supported a person with PD in the past 2
years, who subsequently died, were invited to fill in a
questionnaire (unpublished data). The final question
concerned willingness to elaborate further on the topic
in a semi-structured interview or a focus group discus-
sion. Purposive sampling was employed to ensure the
sample represented a diverse range of health care profes-
sionals and practice characteristics such as age, sex, ex-
perience and professional background. Next, we divided
health care professionals in two groups, individual inter-
view or focus group discussion. Standard procedures
were employed for obtaining informed consent from the
professionals who eventually participated in the individ-
ual interviews or focus group discussions. Selection of participants Health care professionals were recruited from Parkinson-
Net. This concept is reviewed in detail elsewhere [20, 21]. Briefly, ParkinsonNet is a Dutch professional healthcare
network with nationwide coverage, consisting of 70
regional networks which encompass health care profes-
sionals specialized in PD (n = 3,171) [20, 22, 23]. The
central idea is that persons with PD should preferentially
be treated by a small group of selected professionals with
a high degree of expertise in PD. This expertise is gained
initially during a 3-day training course, and is maintained
at a high level because patients are being referred specific-
ally to these ParkinsonNet therapists, resulting in high
case loads and opportunities to maintain or even increase
expertise [24]. Patients in the Netherlands are motivated Study design This qualitative study is part of the ‘ParkinsonSupport–
project’ that aims to improve the palliative care for per-
sons with PD and their families in the Netherlands [19]. We conducted individual semi-structured interviews with
10 health care professionals and held three focus group
discussions with 29 health care professionals. Recruitment
and data collection took place from September 2016 till
March 2017. To develop the interview guide, we first conducted
a literature review in PubMed in 2016 using search
terms like ‘palliative care’, ‘Parkinson’s disease’ and
‘end of life care’. Our aim was to find relevant studies
on persons with PD and (defining) palliative care in-
cluding expert opinions, case studies and empirical
studies [9, 12, 15–18, 26–37]. Furthermore, as indi-
cated earlier, we sent out a questionnaire to gain
more information on professionals’ knowledge and
competence in palliative care. An interview guide was
developed starting from the research gap and ques-
tionnaire results (unpublished data). The guide was
discussed and amended if needed by HL, MS and
MG. An expert panel consisting of three health care
professionals and two researchers with experience in
either PD, palliative care or both, reviewed this ver-
sion and, where thought appropriate, adjusted it. It
consisted of four topics, each comprising multiple
open questions (Additional file 1). Most non-
health care professionals declined because of time issues. A detailed description of health care professionals in the
interviews and focus group discussions is presented in
Table 1 “Characteristics of participants”. Health care professionals’ experiences with and percep-
tions of palliative care in PD are known from a few studies
[15–18]. It appears that many professionals lack the neces-
sary competences for palliative care, feel inexperienced
and want more education in this field [15–17]. We con-
ducted a large qualitative study based on previous re-
search to gain a more in-depth view on these aspects and
add new ones that can help to improve palliative care. Data analysis All semi-structured interviews and focus group discus-
sions were audio-taped and transcribed. We used an in-
ductive
analysis,
involving
the
conceptualization
of
themes from the transcripts. Two researchers (HL & MS)
read and reread the transcripts and coded the first four
transcripts using open coding. To increase coding reliabi-
lity, all transcripts were initially coded by HL and MS
separately. Open codes were compared and discussed
until an initial code tree was established. All interviews
and focus group transcripts were further analysed indep-
ently. Codes were discussed, added, modified or merged if
necessary. After coding three interviews and two focus
group discussions with the code tree no new codes
emerged and data saturation was reached. Afterwards a process of sorting and classifying codes
into subthemes and themes started. When differences in
interpretation between researchers remained, a third
senior researcher (MG) was consulted. The reliability of
the findings was enhanced further by scrutiny from the
project group, which included researchers and practicing
clinicians from different fields (experiences in palliative
care or parkinson care). Once consensus was reached, a
final set of themes and subthemes was decided upon
[38–40]. The software package Atlas Ti-8 supported the
qualitative data analysis [41]. Data collection Two of the authors (HL or MS) held individual semi-
structured interviews with 10 health care professionals. Lennaerts et al. BMC Palliative Care (2019) 18:53 Lennaerts et al. BMC Palliative Care Page 3 of 9 Page 3 of 9 Each interview took between 60 and 90 min. All interviews
took place at professional’s place of work or preferred loca-
tion, so that the interviewers could get a good feeling with
Table 1 Characteristics of participants
Individual interview (n = 10)
Focus group
discussion
(n = 29)
Gender
Women
7
26
Professional background
Neurologist
1
1
Elderly care physician
1
2
Psychologist
–
1
PD nurse practitioner
1
6
Community nurse
1
1
Physiotherapist
1
10
Speech therapist
1
3
Occupational therapist
1
3
Dietician
1
2
Othera
2
–
Age
25–34
1
2
35–44
4
4
45–54
1
11
55–64
4
11
65+
–
1
Highest educational level
Secondary education
–
2
Higher education
5
20
University
5
7
Setting
Academic hospital
3
3
Community hospital
1
3
Nursing home
1
12
Private clinic
2
8
Home care
2
2
Hospice
–
–
Rehabilitation centre
–
2
Primary healthcare centre
1
4
aOther: general practitioner & psychiatrist Table 1 Characteristics of participants
Individual interview (n = 10)
Focus group
discussion
(n = 29) prior relationship between the researchers and the partici-
pants and before the start of the interview, only the inter-
viewers’ names and occupations were mentioned. See
Table 2 “Researchers’ characteristics” for more information
regarding researchers’ characteristics. The number of health
care professionals in the focus groups ranged from eight to
ten. The focus group discussions served to engage the parti-
cipating professionals in considering findings from the
individual interviews. Specifically, we used the individual in-
terviews to solicit individual views and experiences. In con-
trast, the focus group discussions served to engage the
professionals in further discussions about similar topics. Addressing needs of persons with PD and family
caregivers cannot clarify needs or make decisions, health care pro-
fessionals intensified collaboration with family caregivers
so as to be able to offer care in accordance with the per-
son’s needs. However, a few health care professionals
recognized that people with PD and caregivers may have
different needs, in which case it is difficult to determine
whose needs must be prioritized (Table 4, Q3). Health
care professionals also noted that emotionally burdened
family caregivers can hinder persons’ adjustment to their
disease (Table 4, Q3). Care for family caregivers was
seen as an important, but also as a complicating factor. Furthermore, many professionals experienced a lack of
time for interaction with family caregivers and/or be-
reavement support. One of the key issues in late-stage PD is that the per-
son’s needs and wishes can be difficult to elicit due to
communication
problems
and/or
cognitive
decline
(Table 4, Q1). A number of respondents cited that not
speaking timely about needs might complicate treat-
ment. In some cases, when the person with PD could
not understand the purpose of a proposed treatment, it
was hard to tell whether the treatment might be too bur-
densome. Health care professionals mentioned that they
were not certain about what persons with PD themselves
want. Early discussions about wishes was seen as a facili-
tator for improving palliative care (Table 4, Q2). A
barrier that was mentioned by a few HCP was that a PD
trajectory is less predictable than for example for cancer. Persons with PD might be focusing on stabilizing instead
of their general decline (Table 4, Q2). HCP emphasized
the urgency of timely speaking about wishes and needs
(due PD specific symptoms) as it might enable them to
provide future care that is based on a person’s needs. However, ‘timely’ was not well defined. On the other
hand, HCP argued that in some cases persons were not
‘open’ for having these conversations. Results The analysis resulted in four themes and 13 subthemes
(see Table 3 “Perceived facilitators & barriers for pallia-
tive care for persons with PD”) relating to perceived bar-
riers and facilitators for palliative care for persons with
PD and their families. The themes are described in detail
below with supporting data that are presented in Table 4
“Quotes taken from the interview and focus group dis-
cussions”. In the discussions we departed from the defin-
ition on palliative care (see Additional file 1; interview
guide theme 1 ‘defining palliative care in PD’) from this
current analysis. Each interview took between 60 and 90 min. All interviews
took place at professional’s place of work or preferred loca-
tion, so that the interviewers could get a good feeling with
the matters discussed. We conducted three focus group
discussions which, on average, took 90 min each. Focus
group discussions were organized at a central place in the
Netherlands. The sessions were chaired by either MG or
MM, assisted by either HL or MS. One served as a mode-
rator who fostered an active and open discussion, and the
other served as an assistant who took notes. There was no Lennaerts et al. BMC Palliative Care (2019) 18:53 Lennaerts et al. BMC Palliative Care Page 4 of 9 Table 2 Researchers’ characteristics
Code
Initials
Gender
Age
Occupation and experience
I1
H.L. Female
35
PhD candidate, master degrees in Social Sciences. 5 years experience on multiple projects for PD,
nursing care and 14 years experience in PD-patient care. I2
M.S. Female
29
Master degrees in Psychology. Two years experience on multiple projects for PD and 5 years
employed as coordinator at ParkinsonNet, a Dutch nationwide PD network
I3
M.G. Female
51
Senior researcher, assistant professor, PhD in Palliative Care, over 18 years of experience on research
projects in palliative care, senior lecturer Qualitative Research, Nurse (NP)
I4
M.M. Male
50
Associate professor in healthcare innovation, director of strategy Movement Disorders Centre of Expertise, Managing
Director ParkinsonNet, 17 years of experience on multiple multidisciplinary research and innovation projects for PD I4
M.M. Male
50
Associate professor in healthcare innovation, director of strategy Movement Disorders Centre of Expertise, Managing
Director ParkinsonNet, 17 years of experience on multiple multidisciplinary research and innovation projects for PD Addressing needs of persons with PD and family
caregivers Addressing needs of persons with PD and family
caregivers Disease management We do not know
what she wants and how she think of her end of life…. Now, she has several cognitive
problems… shouldn’t these conversations have been introduced earlier in the trajectory when this
women was cognitively at a better level? (individual interview, nurse practitioner)
Many people with PD at advanced stage can’t communicate anymore. They can live their life but
we can’t communicate on that moment about what they want and what is important for them. (focus group discussion) People will stay focused on something like hope. Maybe there are still opportunities that will help
them. They try, have new hopes, but also are often disappointed. (focus group interview,
occupational therapist) Speaking about prognosis should be done earlier. You see that if a person goes to a nursing home
(...) they often expect that the condition will stabilize. But PD will continue and you need to
discuss scenarios before things get worse. (focus group discussion, elderly care physician) Q3
Theme: Addressing needs of persons with PD
and their family caregivers What I find really difficult is when a family caregiver is too overburdened and puts a patient in a
wheelchair for the whole day. As a caregiver said: “At least he doesn’t stand in the way then and
cannot fall. The more immobile my husband is the better, that’s easier for me. But it is very
emotionally taxing. (focus group discussion, occupational therapist)
When a patient became ill, his partner pushed for a hospital admission. I don’t think that it was what
he wanted, or what maybe was best for him. But this partner, she wanted to do something… (focus
group discussion, physiotherapist) What I find really difficult is when a family caregiver is too overburdened and puts a patient in a
wheelchair for the whole day. As a caregiver said: “At least he doesn’t stand in the way then and
cannot fall. The more immobile my husband is the better, that’s easier for me. But it is very
emotionally taxing (focus group discussion occupational therapist) y
g
g
When a patient became ill, his partner pushed for a hospital admission. I don’t think that it was what
he wanted, or what maybe was best for him. But this partner, she wanted to do something… (focus
group discussion, physiotherapist) Q4
Theme: Disease management When you get diagnosed with PD, you will in fact fight against the disease. Disease management People do not always know what is agreed upon (focus group discussion) these become too frail to attend an outpatient clinic. An-
Table 4 Quotes taken from the interviews and focus group discus
Quote number
Q1
Theme: Addressing needs of persons with PD
and their family caregivers
This female with PD is in a
what she wants and how s
problems… shouldn’t these
women was cognitively at
Many people with PD at ad
we can’t communicate on
(focus group discussion)
Q2
Theme: Addressing needs of persons with PD
and their family caregivers
People will stay focused on
them. They try, have new h
occupational therapist)
Speaking about prognosis s
(...) they often expect that t
discuss scenarios before thi
Q3
Theme: Addressing needs of persons with PD
and their family caregivers
What I find really difficult is
wheelchair for the whole d
cannot fall. The more immo
emotionally taxing. (focus g
When a patient became ill,
he wanted, or what maybe
group discussion, physiothe
Q4
Theme: Disease management
When you get diagnosed w
many years with your spec
for a specialist to say, I cann
patient is tough. (individua
Saying that a patient is in h
more on comfort than on t
explicitly say it. (focus grou
Q5
Theme: Disease management
You can have all kind of sid
therapy carries a high risk o
very difficult. (focus group d
Parkinson medication is tro
we give more medication o
Or should we quit trying to
elderly care physician)
Q6
Theme: Professionals need for training
I would never mention the
occupational therapist)
You have to get used to sp
this and it will eventually g
Q7
Theme: Professionals need for training
I think doctors won’t speak
course, our core business a
pays attention to this. (indiv
Q8
Theme: Professionals need for training
Well, I won’t speak about d
with a patient, it is different
thought of when the situat
A personal relation and con
physiotherapist, or a cleane
occupational therapist)
Q9
Theme: Connection between services
I am also a member of a pa
PD is depressingly low. I th
they not consult us? (indivi
Q10
Theme: Connection between services
What I often hear, is that th
Especially when a patient c
and home. There are chang
People do not always know This female with PD is in a phase that we need to make choices for the future. Disease management You form an alliance for
many years with your specialist too fight for a better quality of life or to remain stable. It is very hard
for a specialist to say, I cannot do anything more for you, I have to quit this fight. Telling this to a
patient is tough. (individual interview, elderly care physician) p
g
y
p y
Saying that a patient is in his last month of his life, I think you get different interventions, focusing
more on comfort than on treatment. I think we (professionals) feel and think about it, but don’t
explicitly say it. (focus group discussion) Q5
Theme: Disease management You can have all kind of side effects through increasing the medication doses. For example, clozapine
therapy carries a high risk of dizziness and balance problems. Balancing between pros and cons is
very difficult. (focus group discussion, elderly care physician) You can have all kind of side effects through increasing the medication doses. For example, clozapine
therapy carries a high risk of dizziness and balance problems. Balancing between pros and cons is
very difficult. (focus group discussion, elderly care physician)
Parkinson medication is troublesome. It’s hard to judge whether medication is effective or not. Should
we give more medication or should we give less medication instead to reduce side effects? py
g
p
very difficult. (focus group discussion, elderly care physician) y
g
p
y
p y
Parkinson medication is troublesome. It’s hard to judge whether medication is effective or not. Should
we give more medication or should we give less medication instead to reduce side effects? Or should we quit trying to improve balance in medication completely?” (focus group discussion,
elderly care physician) Q6
Theme: Professionals need for training
Q7
Theme: Professionals need for training
Q8
Theme: Professionals need for training I would never mention the word palliative to a patient. No, that’s not my thing. (individual interview,
occupational therapist) You have to get used to speak about difficult issues. However, you need to persuade yourself to do
this and it will eventually get easier. (individual interview, nurse practitioner) I think doctors won’t speak in depth about spirituality with their patients. Medical treatment is, of
course, our core business and this (spirituality) often is not a subject. I hope also that our PD-nurse
pays attention to this. Disease management The theme disease management occurred on a meso-
and a micro level. On the meso level, health care profes-
sionals were unsure of the timing and introduction of
palliative care into the care pathway and by whom. Many healthcare professionals noticed that palliative
care should be the role and responsibility of a physician. There was a lack of role descriptions in providing pallia-
tive care and a need to clarify the roles and responsibil-
ities of different healthcare professionals. According to health care professionals, involvement of
family caregivers in the care for PD was seen as a facili-
tator, but also as a barrier. In situations where a person Health care professionals mentioned that in case of
neurologists, they often lose track of their patients when Table 3 Perceived facilitators & barriers for palliative care for persons with PD
Themes
Barriers
Facilitators
Addressing needs of persons with PD
and family caregivers
A persons cognitive deficits and
communication problems
Tension between needs from a person and his/her
family caregivers
A lack of time in interaction with family caregivers
Early speaking about wishes and needs with person,
family and health care professionals
Disease management
Lack of clear responsibilities and roles in
(introducing) palliative care
Limited resources; lack of time, high workloads
and financials
More evidence and guidance in offering adequate
disease management
Professionals need for training
A lack of competences and specifically for the
spiritual domain
Training helps in feeling more confident
Communication skills; an open and sensitive attitude
Connection between services
Limited communication between health care
professionals
Availability of specialized palliative care services
Care coordination; need for a central figure to
coordinate palliative care Table 3 Perceived facilitators & barriers for palliative care for persons with PD
Themes
Barriers Page 5 of 9 Lennaerts et al. BMC Palliative Care (2019) 18:53 Lennaerts et al. BMC Palliative Care these become too frail to attend an outpatient clinic. An-
other probable reason for not discussing palliative care
for a person with PD to accept that drug treatment is
less effective and for doctors to verbalize ‘bad news’
Table 4 Quotes taken from the interviews and focus group discussions
Quote number
Q1
Theme: Addressing needs of persons with PD
and their family caregivers
This female with PD is in a phase that we need to make choices for the future. Disease management We do not know
what she wants and how she think of her end of life…. Now, she has several cognitive
problems… shouldn’t these conversations have been introduced earlier in the trajectory when this
women was cognitively at a better level? (individual interview, nurse practitioner)
Many people with PD at advanced stage can’t communicate anymore. They can live their life but
we can’t communicate on that moment about what they want and what is important for them. (focus group discussion)
Q2
Theme: Addressing needs of persons with PD
and their family caregivers
People will stay focused on something like hope. Maybe there are still opportunities that will help
them. They try, have new hopes, but also are often disappointed. (focus group interview,
occupational therapist)
Speaking about prognosis should be done earlier. You see that if a person goes to a nursing home
(...) they often expect that the condition will stabilize. But PD will continue and you need to
discuss scenarios before things get worse. (focus group discussion, elderly care physician)
Q3
Theme: Addressing needs of persons with PD
and their family caregivers
What I find really difficult is when a family caregiver is too overburdened and puts a patient in a
wheelchair for the whole day. As a caregiver said: “At least he doesn’t stand in the way then and
cannot fall. The more immobile my husband is the better, that’s easier for me. But it is very
emotionally taxing. (focus group discussion, occupational therapist)
When a patient became ill, his partner pushed for a hospital admission. I don’t think that it was what
he wanted, or what maybe was best for him. But this partner, she wanted to do something… (focus
group discussion, physiotherapist)
Q4
Theme: Disease management
When you get diagnosed with PD, you will in fact fight against the disease. You form an alliance for
many years with your specialist too fight for a better quality of life or to remain stable. It is very hard
for a specialist to say, I cannot do anything more for you, I have to quit this fight. Telling this to a
patient is tough. (individual interview, elderly care physician)
Saying that a patient is in his last month of his life, I think you get different interventions, focusing
more on comfort than on treatment. I think we (professionals) feel and think about it, but don’t
explicitly say it. Disease management (focus group discussion)
Q5
Theme: Disease management
You can have all kind of side effects through increasing the medication doses. For example, clozapine
therapy carries a high risk of dizziness and balance problems. Balancing between pros and cons is
very difficult. (focus group discussion, elderly care physician)
Parkinson medication is troublesome. It’s hard to judge whether medication is effective or not. Should
we give more medication or should we give less medication instead to reduce side effects? Or should we quit trying to improve balance in medication completely?” (focus group discussion,
elderly care physician)
Q6
Theme: Professionals need for training
I would never mention the word palliative to a patient. No, that’s not my thing. (individual interview,
occupational therapist)
You have to get used to speak about difficult issues. However, you need to persuade yourself to do
this and it will eventually get easier. (individual interview, nurse practitioner)
Q7
Theme: Professionals need for training
I think doctors won’t speak in depth about spirituality with their patients. Medical treatment is, of
course, our core business and this (spirituality) often is not a subject. I hope also that our PD-nurse
pays attention to this. (individual interview, neurologist)
Q8
Theme: Professionals need for training
Well, I won’t speak about dying if I see a patient for only a short time. But when I build a relationship
with a patient, it is different. Even when a patient is physically stable, I probe whether they had ever
thought of when the situation gets worse. (individual interview, physiotherapist)
A personal relation and connection with a patient is important; sometimes it is a nurse, a
physiotherapist, or a cleaner who has known a person for a couple of years. (individual interview,
occupational therapist)
Q9
Theme: Connection between services
I am also a member of a palliative care team in our region. The number of referrals for persons with
PD is depressingly low. I think that should be different… I know there are many cases and why do
they not consult us? (individual interview, elderly care physician)
Q10
Theme: Connection between services
What I often hear, is that there is a need for better coordination by for example a case manager. Especially when a patient comes from a situation of transfers between hospital, rehabilitation centre
and home. There are changes in medication regimen, feeding or therapy from a physiotherapist. Q3
Theme: Addressing needs of persons with PD
and their family caregivers Disease management BMC Palliative Care (2019) 18:53 Lennaerts et al. BMC Palliative Care (2019) 18:53 Page 6 of 9 Lennaerts et al. BMC Palliative Care Page 6 of 9 brought forward that adjusting medication becomes
extremely difficult when PD progresses. Few medication
treatment goals remain and often the therapeutic effect
aimed for does not weigh against the occurring side-
effects.(Table 4, Q5) Furthermore, occupational and
physical therapists specifically mentioned the need for
more knowledge on how to support persons with PD in
day-time tasks, comfortable sitting and lying, or preven-
tion from pressure ulcers. Another topic, mentioned by
physicians, dieticians and speech therapists, was the place-
ment of a feeding tube as a life-prolonging intervention. If
a feeding tube was already in place, deciding whether to
leave it in place or to remove it was perceived as difficult. Ethical issues concerning boundaries between curative
care and palliative care as well as possible legal implica-
tions of end-of-life decisions are not clear. Although
health care professionals realized that to provide patient
centered
care
achievable,
tailored
interventions
are
needed. However, more general guidance and evidence are
wished for disease management in PD. brought forward that adjusting medication becomes
extremely difficult when PD progresses. Few medication
treatment goals remain and often the therapeutic effect
aimed for does not weigh against the occurring side-
effects.(Table 4, Q5) Furthermore, occupational and
physical therapists specifically mentioned the need for
more knowledge on how to support persons with PD in
day-time tasks, comfortable sitting and lying, or preven-
tion from pressure ulcers. Another topic, mentioned by
physicians, dieticians and speech therapists, was the place-
ment of a feeding tube as a life-prolonging intervention. If
a feeding tube was already in place, deciding whether to
leave it in place or to remove it was perceived as difficult. Ethical issues concerning boundaries between curative
care and palliative care as well as possible legal implica-
tions of end-of-life decisions are not clear. Although
health care professionals realized that to provide patient
centered
care
achievable,
tailored
interventions
are
needed. However, more general guidance and evidence are
wished for disease management in PD. also connected with personal attitude. An open and sen-
sitive attitude to persons with PD might help them to
speak freely and honestly about expectations and fears. Talking about difficult issues was easier if trust had been
established between a health care professionals and a
person with PD. Connection between services The availability of specialist palliative care (SPC) services
in a hospital was seen as a facilitator to improve PD
care. However, some professionals had not heard from
SPC services before and other health care professionals
did not know how SPC could contribute in late-stage
PD. Only a small group had often contact with a SPC
services. An elderly care physician who was also working
in an SPC service, said he hardly saw persons with PD
and his expertise could be used more often. (Table 4,
Q9) A further integration of SPC services could help the
organization of palliative care for people with PD. Fur-
thermore, a number of health care professionals identi-
fied a lack of communication and information continuity
in situations where multiple health care professionals
were involved. More multidisciplinary communication
could also help in using each specific knowledge in pro-
viding palliative care. Furthermore, health care profes-
sionals highlighted the need for a central figure in
palliative care for PD. Although many participant agreed
that the general practitioner could be this person, there
was some questioning of the appropriateness and in-
volvement of GP’s in PD. Health care professionals cited
barriers, such as their lack of PD knowledge, lack of time
for care coordination and limited accessibility for other
professionals. Other health care professionals pointed
out that a case manager liaising with other professionals
might be a facilitator to improve and ensure continuity
of palliative care. (Table 4, Q10). A few professionals
mentioned that a casemanager could be ‘a new person’,
while others saw a casemanager more as an approach
supporting teamwork, common goals, and a willingness
to involve whoever had the appropriate skills. Overall, many health care professionals reported barriers
to the provision of palliative care such as high workloads,
a lack of time and restrained financial resources. Disease management A longstanding or intensive patient-
professional relation was another facilitator mentioned. (Table 4, Q8). Professionals need for training A range of experiences from clinical practice in relation
to limited competences and skills in working with per-
sons with PD were described. Health care professionals
found themselves at the stage of ‘conscious incompe-
tence’ [42]. They recognized the deficit of not knowing
how to offer palliative care or not knowing what skills
are needed. It was suggested that additional training can
help overcome these deficits. Training could also help in
feeling more confident in using the knowledge and skills. But also in helping health care professionals to address
issues related to death and dying. Some professionals felt
reluctant because of their own personal issues, such as
taboo, uncertainty or personal life experiences and
beliefs. (Table 4, Q6). Spiritual care was much less actively discussed among
health care professionals than the other domains of pallia-
tive care (physical, social, psychological). A few health care
professionals remarked that spirituality was addressed
only if the person with PD explicitly raised spiritual issues
and was not always a standard subject in care. (Table 4,
Q7) When professionals were confronted with spiritual
care, they frequently referred to or involved others. Health
care professionals underpinned spiritual care as an im-
portant part of palliative care. However, the awareness for
spiritual needs and how to respond or act as a professional
remains unclear. Some suggested that it was not enough
to develop training on this topic. Disease management (individual interview, neurologist) Well, I won’t speak about dying if I see a patient for only a short time. But when I build a relationship
with a patient, it is different. Even when a patient is physically stable, I probe whether they had ever
thought of when the situation gets worse. (individual interview, physiotherapist)
A personal relation and connection with a patient is important; sometimes it is a nurse, a
physiotherapist, or a cleaner who has known a person for a couple of years. (individual interview,
occupational therapist) Well, I won’t speak about dying if I see a patient for only a short time. But when I build a relationship
with a patient, it is different. Even when a patient is physically stable, I probe whether they had ever
thought of when the situation gets worse. (individual interview, physiotherapist) A personal relation and connection with a patient is important; sometimes it is a nurse, a
physiotherapist, or a cleaner who has known a person for a couple of years. (individual interview,
occupational therapist) physiotherapist, or a cleaner who has known a person for a couple of years. (individual interview,
occupational therapist) I am also a member of a palliative care team in our region. The number of referrals for persons with
PD is depressingly low. I think that should be different… I know there are many cases and why do
they not consult us? (individual interview, elderly care physician) What I often hear, is that there is a need for better coordination by for example a case manager. Especially when a patient comes from a situation of transfers between hospital, rehabilitation centre
and home. There are changes in medication regimen, feeding or therapy from a physiotherapist. People do not always know what is agreed upon (focus group discussion) for a person with PD to accept that drug treatment is
less effective and for doctors to verbalize ‘bad news’
(Table 4, Q4). these become too frail to attend an outpatient clinic. An-
other probable reason for not discussing palliative care
is the longstanding relationship between a neurologist
and a person with PD with its focus on optimizing med-
ical treatment to suppress symptoms. If suppressing
symptoms is not feasible anymore, it might be painful On micro level, many health care professionals men-
tioned a lack of evidence about interventions as a barrier
in offering adequate symptom management. Physicians’ Lennaerts et al. Strengths and limitations partnership with family caregivers becomes more crucial
in late-stage PD, although this may not be easy. The
needs of persons with PD and caregivers may not always
be congruent and family dynamics may hinder the
provision of optimal support [43, 44]. Persons with PD,
family caregivers and health care professionals need to
work together to plan care based on a person’s wishes
and needs. From studies on the perspectives of persons
with PD and caregivers, it becomes clear that prefer-
ences on when to discuss end-of-life issues vary and that
only half of the persons are ready to discuss advanced
care documents in an early stage of the disease [45]. One of the reasons for this hesitant attitude is the lack
of information and support from health care services
[31, 35]. From the health care professionals’ perspective,
however, early planning, before a person with PD loses
communication capacity, is preferable for providing opti-
mal palliative care [16, 17, 46]. partnership with family caregivers becomes more crucial
in late-stage PD, although this may not be easy. The
needs of persons with PD and caregivers may not always
be congruent and family dynamics may hinder the
provision of optimal support [43, 44]. Persons with PD,
family caregivers and health care professionals need to
work together to plan care based on a person’s wishes
and needs. From studies on the perspectives of persons
with PD and caregivers, it becomes clear that prefer-
ences on when to discuss end-of-life issues vary and that
only half of the persons are ready to discuss advanced
care documents in an early stage of the disease [45]. One of the reasons for this hesitant attitude is the lack
of information and support from health care services
[31, 35]. From the health care professionals’ perspective,
however, early planning, before a person with PD loses
communication capacity, is preferable for providing opti-
mal palliative care [16, 17, 46]. g
The use of multiple qualitative methods (individual in-
terviews and focus groups discussions) and investigator
triangulation (data coding by more than one person) en-
sured the validity of our approach. A particular strength
of this study is that it inventoried the experiences of a
wide range of health care professionals from several set-
tings. This allowed us to gather a broad view of palliative
care for PD. Discussion This study provides insight into how a variety of health
care professionals experienced palliative care provision
for persons with PD. Health care professionals find it
hard to identify needs of persons with late-stage PD due
to impaired communication and declining cognitive
functioning. Therefore, speaking timely on a persons’
wishes and needs for future care can help them to pre-
pare for what might come. As a result, health care pro-
fessionals are more able at the end of life to provide
patient centered care. It also appeared that working in Many health care professionals noted that ‘good com-
munication skills’ is a facilitator. Communication was Lennaerts et al. BMC Palliative Care (2019) 18:53 Lennaerts et al. BMC Palliative Care (2019) 18:53 Page 7 of 9 Future perspectives / clinical relevance p
p
Early identifying needs and discussing the preferences of a
person with PD should become a part of PD care. Further-
more, educational strategies are needed to increase health
care professionals’ knowledge of palliative care. Appropri-
ate strategies include workshops, written material and on-
line learning modules. Based on the outcomes of this
study, we developed an information film for persons with
PD and their families (only available in Dutch: https://
www.youtube.com/watch?v=8W02j6fzd3g). Collaboration
with an SPC service can help to learn on a case-by-case
basis. Competence gaps can have implications for care as
persons with PD may experience significant delay in
symptom control, until they are referred to appropriate
specialist services. Measures to increase health care pro-
fessionals’ competence should be implemented to improve
quality of care. Obviously, it would be relevant to gain
more insight into the experiences of persons with PD
themselves at advanced disease stages [54]. Exploring this
is notoriously difficult due to communication and cogni-
tive deficits that increase with advancing PD. The interviewed health care professionals were posi-
tive about collaboration with SPC services, although
such collaborations were still rare in daily practice. Con-
crete examples of how this collaboration could be real-
ized are needed. Earlier studies [16, 17] also found that
PD care should include a collaboration with an SPC ser-
vice so as to be able to meet the palliative care needs of
persons with PD. Patients who received care from an
SPC service were more satisfied about care [50]. Health
care professionals overall were not well informed about
whom to refer to and when, and they were uncertain
about when to initiate palliative care [16, 17, 51]. This
can lead to inappropriate or no referral at all, and per-
sons with PD missing the benefits of a palliative care
approach. In the literature, different models of impro-
ving palliative care have been proposed; this includes
consultative palliative care teams, integrated palliative
care programs and complementary models (including
primary palliative care, mobile consultation team, an
acute palliative care unit, and an outpatient supportive
care clinic) [11–13, 52, 53]. Although different models
can help to optimize palliative care in PD, further re-
search is needed on outcomes of these various models in
daily practice. Strengths and limitations As a limitation, we did not included health
care professionals with no or short experience in pallia-
tive care. The participating health care professionals
might have been keener to have a good palliative care
system because they have a recent experience with a per-
son with PD who died in the past 2 years. Consequently
these findings do not reflect views from more inexperi-
enced health care professionals. A further limitation is
that our data present health care professionals’ reported
practice, we did not quantitatively measure the health
care professionals’ knowledge and experiences. Lastly,
our findings represent the situation in the Netherlands
and may not generalize to a wider international context. Despite these limitations, the findings of this study do
point to ways of improving the quality of palliative care
for persons with PD. We found a need for further education about “general
palliative care” for health care professionals. Additional
training was seen as a facilitator for health care pro-
fessionals to become more competent and confident. Two studies have reported similar findings regarding
competences and knowledge [15–17]. Improving one’s
knowledge about palliative care can help health care
professionals overcome barriers in palliative care [47, 48]
Special attention may be paid to spirituality as also many
other health care professionals struggle with the concept
of spirituality in relationships with patients, caregivers
and themselves [49]. Conclusion Our results has identified several barriers and facilitators
in providing palliative care for people with PD. Due to
specific PD-symptoms there is a need of an early pro-
active approach to identify palliative care needs. Further-
more, health care professionals experienced a need to
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d This study is funded by The Netherlands Organization for Health Research
and Development (ZonMw). (Grant reference number 80–84400–98-086). Prof. Bastiaan R. Bloem was supported by a research grant of the Parkinson’s
Foundation. The funding party had no role in the design of the study, or in
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The authors declare that they have no competing interests. 20. Bloem BR, Munneke M. Revolutionising management of chronic disease: the
ParkinsonNet approach. BMJ. 2014;348:g1838. Consent for publication
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JTvdS contributed to analysis and interpretation of the data. HL, MS, MG
were main contributors in writing the manuscript. KV, BB, MM, MB and DA
contributed to the study design, and provided feedback on the analysis and
results. KV and BB also supervised the process. All other authors contributed
to critically revising the manuscript. All authors read, reread and approved
the final manuscript. All authors read and approved the final manuscript. 10. WHO Definition of palliative care. 2015. http://www.who.int/cancer/
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approach from The Netherlands. Health Aff. 2017;36(11):1987. 1Department of Neurology, Donders Institute for Brain, Cognition and
Behaviour, Radboud university medical center, Reinier Postlaan 4, 6500
Nijmegen, AB, The Netherlands. 2Department of Anesthesiology, Pain and
Palliative Medicine, Radboud university medical center, Nijmegen, The
Netherlands. 3Department of Primary and Community Care, Radboud
university medical center, Nijmegen, The Netherlands. 4Department of Public
Health and Primary Care, Leiden University Medical Center, Leiden, The
Netherlands. 5Dutch Parkinson’s Disease Association, Bunnik, The
Netherlands. 6Scientific Center for Quality of Healthcare, Radboud Institute
for Health Sciences, Radboud university medical center, Nijmegen, The
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care in people with idiopathic Parkinson’s disease who die in hospital. BMJ
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311(7000):299–302. 39. Powell RA, Single HM. Focus groups. Int J Qual Health Care. 1996;8(5):499–504. 39. Powell RA, Single HM. Focus groups. Int J Qual Health Care. 1996;8(5):499–504. 40. Sofaer S. Qualitative research methods. Int J Qual Health Care. 2002;
14(4):329–36. 40. Sofaer S. Qualitative research methods. Int J Qual Health Care. 2002;
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research (COREQ): a 32-item checklist for interviews and focus groups. Int J
Qual Health Care. 2007;19(6):349–57. 42. Burch N. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
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Case report: septic shock after endometrial polypectomy with tissue removal system
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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. Case report: septic shock after endometrial
polypectomy with tissue removal system Danna Su1, Jiajie She1, Yuting Xu2, Ying Li1, Yan Guo1, Yajie Yang3, Qiao Tan1, Liping Wang1 and Ruiying Diao1* Keywords
l transformation of untreated Eps is unknow [2]. How-
ever, hysteroscopic surgery is recommended for large and
symptomatic polyps [3]. BMC Women’s Health BMC Women’s Health Su et al. BMC Women’s Health (2023) 23:546
https://doi.org/10.1186/s12905-023-02690-9 Open Access Introduction Endometrial polyps (Eps) represent a frequent benign
focal overgrowth of endometrial mucosa. The forma-
tion of Eps is related to the high level of estrogen [1]. Other risk factor including age, hypertension, obesity,
and tamoxifen use have been associated with the devel-
opment of Eps [2]. Eps can lead to abnormal uterine
bleeding and infertility, while the potential for malignant Hysteroscopic electrosurgical techniques are the com-
monest method to remove Eps [4]. The complication rate
of hysteroscopic polypectomy is low, only about 0.22%
[5]. As a new surgical device emerging in recent years,
the mechanical hysteroscopic tissue removal systems
have significantly shortened operation time and improves
success rate of surgery while not increasing complication
rates [4, 6, 7]. Herein, we reported a patient with a his-
tory of recurrent vaginitis who suffered septic shock after
endometrial polypectomy with the tissue removal sys-
tem. This is the first case report of serious complication
after hysteroscopic morcellation. *Correspondence:
Ruiying Diao
15889753127@163.com
1 Reproductive Medicine Centre, The First Affiliated Hospital of Shenzhen
University, Shenzhen Second People’s Hospital, Shenzhen 518035, China
2 Shantou University Medical College, Shantou, Guangdong, China
3 Department of Pathology, The First Affiliated Hospital of Shenzhen
University, Shenzhen Second People’s Hospital, Shenzhen, China *Correspondence:
Ruiying Diao
15889753127@163.com
1 Reproductive Medicine Centre, The First Affiliated Hospital of Shenzhen
University, Shenzhen Second People’s Hospital, Shenzhen 518035, China
2 Shantou University Medical College, Shantou, Guangdong, China
3 Department of Pathology, The First Affiliated Hospital of Shenzhen
University, Shenzhen Second People’s Hospital, Shenzhen, China © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract As an emerging surgical technology, tissue removal systems have been widely used in the treatment of endometrial
polyps due to its characteristics of less endometrial damage, shorter learning curve and clearer vision of the opera-
tive field. There are few cases in the literature reporting serious complications after endometrial polypectomy using
tissue removal systems. As known, septic shock is a rare complication following hysteroscopic polypectomy. Now, we
present the case of a 23-year-old woman who developed septic shock after polypectomy with tissue removal system. The patient had a history of recurrent vaginitis for more than half a year. Due to endometrial polyps, she was admitted
to our hospital and scheduled to undergo hysteroscopic endometrial polypectomy. Three hours after the endometrial
polypectomy using the tissue removal system, the patient had shock symptoms such as increased body tempera-
ture, decreased blood pressure and increased heart rate. Then, the patient was successfully treated and discharged
after anti-infection and anti-shock treatments. The purpose of this case report is to remind clinicians to consider
the possibility of serious infection and comprehensively evaluate the risk of infection before choosing hysteroscopic
devices for endometrial polyps, especially for patients who choose the mechanical hysteroscopic tissue removal
systems. Furthermore, the mechanical hysteroscopic tissue removal systems should be used with caution in patients
with previous recurrent vaginitis. Keywords Hysteroscopy, Hysteroscopic tissue removal system, Endometrial polypectomy, Postoperative
complications, Shock, Septic *Correspondence:
Ruiying Diao
15889753127@163.com
1 Reproductive Medicine Centre, The First Affiliated Hospital of Shenzhen
University, Shenzhen Second People’s Hospital, Shenzhen 518035, China
2 Shantou University Medical College, Shantou, Guangdong, China
3 Department of Pathology, The First Affiliated Hospital of Shenzhen
University, Shenzhen Second People’s Hospital, Shenzhen, China Case presentation were cut to the base by the rotating movements of the
inner blade, and the abraded fragments were aspirated
and sent for pathological examination. The operation was
successfully completed in about 8 min and the patient
was sent to the ward for observation after surgery. On June 29, 2022, a 23-year-old young woman with a
BMI of 18.2 kg/m2 was admitted to our hospital because
of Eps found more than half a year ago. The patient lacked
exercise at ordinary times and denied previous history of
surgery as well as chronic diseases in the past. The exami-
nation of ovarian reserve function in another hospital
found that AMH was low (less than 1 ng/ml), indicating
primary ovarian insufficiency. She had regular menstrua-
tion and had never been pregnant. No special family his-
tory of disease was identified in this patient. g
y
About 3 h after surgery, the patient developed mild
lower abdominal pain with elevated body temperature
(up to 38.7 °C)、heart rate (up to 92–120 bpm) and
hypotension (about 72–83/35-54 mmHg) (shown in
Fig. 1). Laboratory examination showed that white blood
cells decreased to 3.18 × 109/L (up to 22.39 × 109/L later),
the proportion of neutrophil increased (up to 98.5%), and
the index of infection increased significantly, including
hypersensitive C-reactive protein (up to 45.13 mg/l), pro-
calcitonin (up to 42 ng/ml), interleukin (up to 3120 pg/
ml) (shown in Fig. 2). Combined with the clinical mani-
festations and laboratory examination, the patient was
considered to have postoperative septic shock. Then she
was transferred to Intensive Care Unit (ICU) for further
treatment.h i
More than half a year ago, the uterine ultrasound
examination of this patient revealed the presence of
hyperechoic lesions with smooth and regular contours
within the uterine lumen. These lesions were encircled by
thin hyperechoic haloes. These findings are indicative of
endometrial polyps. Meanwhile, vaginal discharge were
positive for mycoplasma, bacteria and fungi. The vagi-
nal discharge was performed again after treatment with
"nifuratel nystatin vaginal soft capsules and doxycycline
hyclate tablets", and was negative for mycoplasma and
bacteria but still positive for fungi. The patient was then
treated with "clotrimazole tablet" for 4 times. However,
her subsequent vaginal discharge still showed positive for
fungus. Luckily, the fungus finally turned negative after
receiving the treatment of "fluconazole tablets". There-
fore, the patient was admitted to the hospital for hystero-
scopic surgery. Open Access T
d
d Su et al. BMC Women’s Health (2023) 23:546 Page 2 of 6 Case presentation The ICU physicians administered a combination of
piperacillin tazobactam and tinidazole for antimicrobial
treatment, while epinephrine was given for vasoconstric-
tive purposes. Additionally, fluid replacement and albu-
min supplementation were provided. After treatment,
the patient’s condition was gradually relieved, which was
characterized by stable recovery of vital signs, gradual
decline of white blood cells and infection indicators. At
this moment, the blood culture result was negative. So,
she was returned to Gynecology ward on the 3rd day
after operation. After antibiotic therapy for 10 days,
abdominal ultrasound was performed and showed a
small amount of effusion. The results of white blood cells
and various infection indicators were normal before dis-
charged. Ultrasound imaging and hysteroscopic view
were shown in Fig. 3. Histopathologic examination of Eps
was shown in Fig. 4. Following admission, a series of testing projects were
conducted including blood routine, coagulation func-
tion, liver and kidney function, preoperative assessment
for infectious diseases, electrocardiogram, and chest
X-ray. No significant abnormalities were found. Given
the patient had fertility requirements, she met the inclu-
sion criteria of a clinical study being conducted in our
hospital (clinical trial numbers: ChiCTR2200058712). After signing informed consent, the patient was
recruited into the study (ethical review approval num-
ber: 20210620213357026-FS01). In addition, the patient
required tubal hydrotubation during the operation
because she failed in trying to conceive for more than
half a year. Discussion Our study was the first case to report a septic shock, after
the hysteroscopic polypectomy with the tissue removal
system. Different kinds of tissue removal systems share
the same structural design and operating principle with
others. All tissue removal devices use mechanical energy
to simultaneously cut and aspirate tissue and the shaver
blades consist of an outer sheath and an inner hollow
tube with windows for simultaneous suction and cut-
ting. Tissue removal system has advantages compared
with conventional electroresection, being able to control
the cutting depth by wrapping around the inner rotating
blade with outer sheath, which plays an important role
in the protection for endometrium. This technique has
very promising features [8], making the operation faster, The vital signs of the patient were stable before the
operation (shown in Fig. 1). The intervention was accom-
plished under general anesthesia. During the operation, a
catheter was inserted into the uterine cavity, and 20 mL
of physiological saline was injected without obvious
resistance, indicating that the fallopian tube was unob-
structed. The cervix was dilated to 8.5 mm and a 8 mm-
sheathed hysteroscopic morcellator was inserted into the
uterus. Hysteroscopic inspection showed five endome-
trial polyps. The largest one was about 0.8*0.5 cm, while
the smallest one was about 0.3*0.3 cm. All the polyps Page 3 of
Su et al. BMC Women’s Health (2023) 23:546
Fig. 1 Line Chart Illustrating Patient’s Vital Signs During Hospitalization. a Temperature during hospitalization. b Daily pulse during hospitalization. c Perioperative changes in systolic blood pressure (SBP) and diastolic blood pressure (DBP). * PRD: preoperative day; DO: postoperative day; D:
postoperative day; H1-H20: the first 20 postoperative hours Page 3 of 6 Su et al. BMC Women’s Health (2023) 23:546 Fig. 1 Line Chart Illustrating Patient’s Vital Signs During Hospitalization. a Temperature during hospitalization. b Daily pulse during hospitalization. c Perioperative changes in systolic blood pressure (SBP) and diastolic blood pressure (DBP). * PRD: preoperative day; DO: postoperative day; D:
postoperative day; H1-H20: the first 20 postoperative hours Fig. 1 Line Chart Illustrating Patient’s Vital Signs During Hospitalization. a Temperature during hospitalization. b Daily pulse during hospitalization. c Perioperative changes in systolic blood pressure (SBP) and diastolic blood pressure (DBP). * PRD: preoperative day; DO: postoperative day; D:
postoperative day; H1-H20: the first 20 postoperative hours easier and with low potential complication rates [9, 10]. Discussion Therefore, it is meaningful to improve the clinicians’
understanding of complications when using this system
through this case.l of the postoperative specimens revealed no positive
staining for CD38 or CD138 (shown in Fig. 4), which
might not be supposed chronic endometritis. Therefore,
the available evidence was inadequate to propose that the
patient was afflicted with chronic endometritis. Although
no signs of vaginitis were identified in the preoperative
leucorrhea routine detection, but this patient was prone
to recurrent vaginitis probably due to the impaired
immune response. So, the dysbiosis in vaginal microbiota
or imbalance in vaginal microbiota microenvironment
might not be excluded. Before the surgery, six courses of anti-inflammatory
treatment were performed on the patient’s vaginitis. After
the routine vaginal examination indicated the absence of
vaginitis, she was prepared for endometrial polypectomy. The reusable surgical instrument blade was sterilized. Aseptic procedures were also strictly followed during
the whole operation. Therefore, postoperative complica-
tions caused by contamination of surgical instruments
were not considered. Endometritis might not be excluded
in this patient who failed in trying to conceive for more
than half a year. However, immunohistochemical staining Using mechanical energy to cut tissue is the most sig-
nificant feature of tissue removal system, indicating a
potential disadvantage is the lack of high-frequency elec-
trocoagulation possibilities. In other words, it is unable Page 4 of 6 Su et al. BMC Women’s Health (2023) 23:546 Page 4 of 6
Su et al. BMC Womens Health (2023) 23:546
Fig. 2 Changes of infection indicators during hospitalization. a Changes in the white blood cell count. b Changes in the proportion of neutrophils. c The development of hypersensitive C-reactive protein(CRP) values after Surgery. d The development of procalcitonin(PCT) values after Surgery. *
PRD: preoperative day; DO: operation day; D: postoperative day Fig. 2 Changes of infection indicators during hospitalization. a Changes in the white blood cell count. b Changes in the proportion of neutrophils. c The development of hypersensitive C-reactive protein(CRP) values after Surgery. d The development of procalcitonin(PCT) values after Surgery. *
PRD: preoperative day; DO: operation day; D: postoperative day Fig. 3 Ultrasound images of the uterus (a) and hysteroscopic view of the uterine cavity (b) before and after hysteroscopic polypectomy. * The
red dotted circle represents the outline of the uterine cavity and endometrial polyps before polypectomy. The white dashed circle represents
the outline of the uterine cavity after hysteroscopic polypectomy Fig. Availability of data and materials Availability of data and materials
All data for the case reports are available in this manuscript. All data for the case reports are available in this manuscript. References 1. Kossaï M, Penault-Llorca F. Role of hormones in common benign uterine
lesions: endometrial polyps, leiomyomas, and adenomyosis. Adv Exp Med
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People’s Hospital(ethical review approval number: 2023–019-01PJ). Written
informed consent was obtained from the patient before publishing. Received: 27 June 2023 Accepted: 5 October 2023 Received: 27 June 2023 Accepted: 5 October 2023 Acknowledgements
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management of endometrial polyps: a critical review of the literature. J Minim Invasive Gynecol. 2011;18(5):569–81. https://doi.org/10.1016/j.
jmig.2011.05.018. This study was funded by the Shenzhen Science and Technology Innovation
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guide. Eur J Obstet Gynecol Reprod Biol. 2021;260:70–7. https://doi.org/
10.1016/j.ejogrb.2021.03.017. Discussion In this case report, the possibility of
Gram-positive bacterial (in vivo) infection was consid-
ered according to the laboratory test indicators and suc-
cessful empirical antibiotic treatment although the blood
culture was negative. Therefore, for patients with a his-
tory of repeated vaginitis or poor body resistance, hyst-
eroscope with tissue removal system should be carefully
or not chosen for avoiding the risk of serious infection. Competing interests Competing interests
The authors declare no competing interests. The authors declare no competing interests. In conclusion, the purpose of this case report is to
remind clinicians to pay attention to the selection and use
of hysteroscope with tissue removal system, especially for
patients with a history of repeated vaginitis. In addition,
early identification of septic shock and timely application
of antibiotic treatment are crucial, which can improve the
prognosis of patients and avoid more serious outcomes. Consent for publication Written informed consent was obtained from the individual for the publica-
tion of any identifiable images or data included in this article. Competing interests
The authors declare no competing interests. Discussion 3 Ultrasound images of the uterus (a) and hysteroscopic view of the uterine cavity (b) before and after hysteroscopic polypectomy. * The
red dotted circle represents the outline of the uterine cavity and endometrial polyps before polypectomy. The white dashed circle represents
the outline of the uterine cavity after hysteroscopic polypectomy Page 5 of 6 Su et al. BMC Women’s Health (2023) 23:546 Fig. 4 Pathological figures in the reported patient with endometrial polyps. a-b Endometrial polyps with hematoxylin and eosin(H&E),
magnification(10 ×). The area indicated in a dark box is enlarged in the high power view on the right(40 ×). c-d Immunohistochemical staining
for CD38(lower left) and CD138(lower right) reveales no plasma cell infiltration Fig. 4 Pathological figures in the reported patient with endometrial polyps. a-b Endometrial polyps with hematoxylin and eosin(H&E),
magnification(10 ×). The area indicated in a dark box is enlarged in the high power view on the right(40 ×). c-d Immunohistochemical staining
for CD38(lower left) and CD138(lower right) reveales no plasma cell infiltration created figures and tables. Y.X. and R.D. reviewed and revised the manuscript. All authors have read and approved the manuscript. to cauterize blood vessels through thermal energy during
the removal of lesions. It remains to be explored whether
the slow closure of blood vessels provides an opportunity
for pathogens to enter vascular system, thus leading to
an increased risk of surgical infection. During the cutting
process, the pathogens may spread through the unclosed
blood vessels, thus having the possibility of inducing
infectious shock. In this case report, the possibility of
Gram-positive bacterial (in vivo) infection was consid-
ered according to the laboratory test indicators and suc-
cessful empirical antibiotic treatment although the blood
culture was negative. Therefore, for patients with a his-
tory of repeated vaginitis or poor body resistance, hyst-
eroscope with tissue removal system should be carefully
or not chosen for avoiding the risk of serious infection. to cauterize blood vessels through thermal energy during
the removal of lesions. It remains to be explored whether
the slow closure of blood vessels provides an opportunity
for pathogens to enter vascular system, thus leading to
an increased risk of surgical infection. During the cutting
process, the pathogens may spread through the unclosed
blood vessels, thus having the possibility of inducing
infectious shock. D.S. and R.D. designed the case report. D.S. and J.S. wrote the first draft of the
manuscript. Y.L., Y.G. and T.Q. systematically edited the manuscript. Y.Y. and L.W. Authors’ contributions D.S. and R.D. designed the case report. D.S. and J.S. wrote the first draft of the
manuscript. Y.L., Y.G. and T.Q. systematically edited the manuscript. Y.Y. and L.W. Page 6 of 6 Su et al. BMC Women’s Health (2023) 23:546 Su et al. BMC Women’s Health (2023) 23:546 4. Raz N, Feinmesser L, Moore O, Haimovich S. Endometrial polyps: diag-
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cia E, Buron Pust A, Mateu Pruñonosa JC, et al. Outpatient Hysteroscopic
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8. Smith PP, Middleton LJ, Connor M, Clark TJ. Hysteroscopic morcellation
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1097/aog.0000000000000187. 9. Hamerlynck TW, Schoot BC, van Vliet HA, Weyers S. Removal of endome-
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doi.org/10.1016/j.jmig.2015.07.006. 10. Arnold A, Ketheeswaran A, Bhatti M, Nesbitt-Hawes E, Abbott J. A
prospective analysis of hysteroscopic morcellation in the management
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Obinutuzumab-atezolizumab-lenalidomide for the treatment of patients with relapsed/refractory follicular lymphoma: final analysis of a Phase Ib/II trial
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Blood cancer journal
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Obinutuzumab-atezolizumab-lenalidomide for the
treatment of patients with relapsed/refractory follicular
lymphoma: final analysis of a Phase Ib/II trial
Franck Morschhauser, Nilanjan Ghosh, Izidore S. Lossos, M. Lia Palomba,
Amitkumar Mehta, Olivier Casasnovas, Don Stevens, Sudhakar Katakam,
Andrea Knapp, Tina Nielsen, et al. To cite this version: Franck Morschhauser, Nilanjan Ghosh, Izidore S. Lossos, M. Lia Palomba, Amitkumar Mehta, et
al.. Obinutuzumab-atezolizumab-lenalidomide for the treatment of patients with relapsed/refractory
follicular lymphoma: final analysis of a Phase Ib/II trial. Blood Cancer Journal, 2021, Blood Cancer
Journal, 11, pp.147. 10.1038/s41408-021-00539-8. hal-04227342 Distributed under a Creative Commons Attribution 4.0 International License 1University of Lille, CHU Lille, ULR 7365 - GRITA - Groupe de Recherche sur les formes Injectables et les Technologies Associées, Lille, France. 2Hematologic Oncology and Blood
Disorders, Levine Cancer Institute/Atrium Health, Charlotte, NC, USA. 3Division of Hematology, Department of Medicine, Sylvester Comprehensive Cancer Center, University of
Miami, Miami, FL, USA. 4Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA. 5Division of Hematology and Oncology, University of Alabama
School of Medicine, Birmingham, AL, USA. 6Service d’Hématologie Clinique, CHU Dijon Bourgogne – Hôpital François Mitterrand, Dijon, France. 7Norton Cancer Institute, Norton
Healthcare, Louisville, KY, USA. 8Product Development Oncology, F. Hoffmann-La Roche Ltd, Basel, Switzerland. 9Genentech Inc., South San Francisco, CA, USA. 10Haematology
Department, Université Claude Bernard de Lyon, Lyon University Hospital, Pierre Benite, France. ✉email: franck.morschhauser@chru-lille.fr ARTICLE
OPEN
Obinutuzumab-atezolizumab-lenalidomide for the treatment of
patients with relapsed/refractory follicular lymphoma: final
analysis of a Phase Ib/II trial analysis of a Phase Ib/II trial
Franck Morschhauser
1✉, Nilanjan Ghosh2, Izidore S. Lossos
3, M. Lia Palomba
4, Amitkumar Mehta5, Olivier Casasnovas6,
Don Stevens7, Sudhakar Katakam8, Andrea Knapp8, Tina Nielsen8, Ron McCord9 and Gilles Salles
10 Franck Morschhauser
1✉, Nilanjan Ghosh2, Izidore S. Lossos
3, M. Lia Palomba
4, Amitkumar Mehta5, Olivier Casasnovas6,
Don Stevens7, Sudhakar Katakam8, Andrea Knapp8, Tina Nielsen8, Ron McCord9 and Gilles Salles
10 © The Author(s) 2021 We evaluated the triplet regimen obinutuzumab-atezolizumab-lenalidomide (G-atezo-len) for patients with relapsed/refractory
(R/R) follicular lymphoma (FL) in an open-label, multicenter phase Ib/II study (BO29562; NCT02631577). An initial 3 + 3 dose‐
escalation phase to define the recommended phase II dose of lenalidomide was followed by an expansion phase with G-atezo-len
induction and maintenance. At final analysis, 38 patients (lenalidomide 15 mg, n = 4; 20 mg, n = 34) had completed the trial. Complete response rate for the efficacy population (lenalidomide 20 mg, n = 32) at end-of-induction was 71.9% (66.7% in double‐
refractory patients [refractory to rituximab and alkylator] [n = 12]; 50.0% in patients with progressive disease within 24 months of
first-line therapy [n = 12]). The 36-month progression-free survival rate was 68.4%. All treated patients had ≥1 adverse event (AE;
grade 3–5 AE, 32 patients [84%]; serious AE, 18 patients [47%]; AEs leading to discontinuation of any study drug, 11 patients [29%]). There were 2 fatal AEs (1 merkel carcinoma, 1 sarcomatoid carcinoma; both unrelated to any study drug). The G‐atezo-len regimen
is effective and tolerable in patients with R/R FL. AEs were consistent with the known safety profile of the individual drugs. Blood Cancer Journal (2021) 11:147 ; https://doi.org/10.1038/s41408-021-00539-8 HAL Id: hal-04227342
https://hal.univ-lille.fr/hal-04227342v1
Submitted on 3 Oct 2023 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
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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 Blood Cancer Journal www.nature.com/bcj Received: 5 May 2021 Revised: 4 August 2021 Accepted: 6 August 2021 INTRODUCTION induction and maintenance treatment with the novel glycoengi-
neered humanized type II anti-CD20 antibody obinutuzumab plus
lenalidomide also showed favorable activity and tolerable safety in
patients with R/R FL in the phase II GALEN study [17, 18]. Follicular lymphoma (FL) is the most common indolent non-
Hodgkin’s lymphoma (NHL) in the Western world [1]. Although the
vast majority of patients treated for FL usually respond to initial
chemoimmunotherapy regimens [2], most will ultimately relapse,
and experience increasing refractoriness to subsequent lines of
therapy [3]. This has led to research into novel treatment regimens
such as phosphoinositide 3-kinase inhibitors (PI3K); [4, 5] or those
combining an anti-CD20 monoclonal antibody (mAb) and an
immunomodulatory agent. p
p
y
Since FL has been considered as particularly immune respon-
sive, further targeting of the immune microenvironment may be
beneficial [19, 20]. In patients with FL, programmed death-ligand 1
(PD-L1) is expressed on tumor-infiltrating lymphocytes, macro-
phages, peripheral blood T cells, and monocytes, but not on tumor
cells [21]. Although the impact of lenalidomide on programmed
death 1 (PD-1)/PD-L1 expression has not been specifically
reported in patients with FL, it has been reported to downregulate
PD-L1 expression on plasma cells and to downregulate PD-1
expression on T cells in multiple myeloma [22]. Furthermore, it has
been observed that activated NK cells express PD-1 and that
PD-L1 engagement could suppress NK-cell mediated anti-tumor
immunity [23]. Of note, lenalidomide triggers NK-cell activation
and increases antibody-dependent cell cytotoxicity in patients
with FL [11], suggesting that combination of lenalidomide with a
PD-1/PD-L1 inhibitor could have synergistic effects on NK cell anti-
tumor activity in these patients. Lenalidomide is an orally active immunomodulatory agent with
direct anti-tumor activity as well as indirect effects mediated
through T-cell and natural killer (NK) cell function [6]. Specifically,
lenalidomide promotes degradation of the hematopoietic tran-
scription factors Ikaros and Aiolos, leading to apoptosis of
neoplastic B cells [7–9]. Adding lenalidomide to rituximab has
been reported to enhance anti-tumor activity by reversing or
reducing the impairment in tumor‐infiltrating T-cell immunologic
synapse formation present in patients with FL [10, 11]. In phase II/
III studies in patients with NHL, including those with relapsed/
refractory (R/R) FL, lenalidomide in combination with rituximab (R2
regimen) demonstrated manageable safety and superior efficacy
over rituximab alone [12–16]. Furthermore, chemotherapy-free y
p
Atezolizumab is a humanized immunoglobulin G1 mAb that
targets PD-L1, inhibiting interaction with its receptors, PD-1 and F. Study design
Thi
h This was a phase Ib/II, open-label, multicenter, non-randomized study. The
study comprised an initial 3 + 3 dose-escalation phase to determine the
recommended phase II dose (RP2D) for lenalidomide when combined with
fixed doses of obinutuzumab and atezolizumab in the G-atezo-len triplet
regimen for induction treatment. The dose-escalation phase was followed
by an expansion phase. Minimal residual disease (MRD) was evaluated at EOI (at 10−5 sensitivity)
using the Adaptive ClonoSEQ® with next-generation sequencing platform
(v2), with assessment of immunoglobulin heavy (IGH) and light chain (IGK),
and BCL2-IGH alterations in DNA from peripheral blood mononuclear cells. During the dose-escalation phase, patients received induction with 6,
28-day cycles of obinutuzumab 1000 mg intravenously (IV; Days 1, 8, 15 of
Cycle 1; Day 1, Cycles 2–6), atezolizumab 840 mg IV (Days 1, 15, Cycles 2–6),
and lenalidomide 15 or 20 mg orally (Days 1–21, Cycles 1–6). Patients
enrolled in the subsequent expansion phase received the same G-atezo-
len induction regimen as used in the dose-escalation phase, but with
administration of lenalidomide at the established RP2D (20 mg) (Supple-
mentary Fig. 1). Assessments All patients were closely monitored for AEs (criteria provided in the
Supplementary section), with nature, frequency, severity, and timing of AEs
reported throughout the study and for at least 35 days after the last dose
of study treatment. Changes in vital signs, electrocardiograms, and clinical
laboratory results during and following study treatment administration
were recorded. Statistical analyses
h
d
l The estimated sample size for the study was determined by the dose-
escalation rules for a 3 + 3 algorithm. It was anticipated that enrolment
of two dosing groups of 3–6 patients each, for a total of 6-12 patients
with R/R FL, was required to establish the RP2D of lenalidomide during
the dose-escalation phase. Approximately 40 patients were planned to
be enrolled during the expansion phase. It was assumed that the PET-
CT-defined CR rate with obinutuzumab-lenalidomide was ~40% in the
R/R setting, as assessed by Cheson 2007 criteria [29]. A sample size of 40
patients was deemed sufficient to provide adequate precision for the
point estimate and for the lower bound of the two-sided 90%
confidence interval (CI) to rule out a clinically uninteresting probability
of response of <46%, assuming an observed PET-CT-defined CR rate
with G-atezo-len of 60%. Patients who achieved a complete response (CR), partial response (PR), or
stable disease (SD) at the end-of-induction (EOI) during the dose-escalation
and expansion phases were eligible to receive extended dosing with G-
atezo-len as maintenance treatment for up to 24 months or until disease
progression or unacceptable toxicity. Maintenance treatment comprised
obinutuzumab 1000 mg IV Day 1 every 2 months and atezolizumab 840 mg
IV Day 1 and 2 every month with lenalidomide 10 mg orally (Days 1–21,
months 1–12) started 8 weeks (±1 week) after Day 1 of Cycle 6. The study was reviewed and approved by the ethics review boards of
the relevant institutions and was conducted in accordance with the
Declaration of Helsinki and Good Clinical Practice guidelines. All patients
provided written informed consent. EOI response and safety analyses were performed on the primary
population (cutoff date 23 October 2018), which included patients who
received at least one dose of each study drug in the triplet combination. PFS was evaluated in the efficacy-evaluable population (patients in the
lenalidomide 20 mg cohort who received all three drug components), at
the time all patients had completed the 36-month visit or discontinued
treatment (cutoff date 7 October 2020). Patients who received lenalido-
mide at the RP2D during the dose-escalation phase were pooled with
patients in the expansion phase for the efficacy and safety analyses. INTRODUCTION Morschhauser et al. 2 B7-1 (also known as CD80) [24, 25]. Antibody-mediated PD-1
blockade has already been successfully exploited as a therapeutic
strategy in several solid tumors and is currently being evaluated in
hematologic malignancies [26]. The combination of obinutuzu-
mab and atezolizumab has previously been shown to be well
tolerated, with no new or unexpected safety signals, and with
evidence of clinical activity in R/R FL in a phase Ib study [27]. CT scan had to be met; if bone marrow involvement was present at baseline,
CR had to be confirmed with a negative bone marrow result at EOI. CT scan had to be met; if bone marrow involvement was present at baseline,
CR h d t
b
fi
d
ith
ti
b
lt t EOI CT scan had to be met; if bone marrow involvement was present at baseline,
CR had to be confirmed with a negative bone marrow result at EOI. ;
p
,
CR had to be confirmed with a negative bone marrow result at EOI. Safety endpoints included evaluating the safety and tolerability of the G-
atezo-len triplet regimen through the incidence of adverse events (AEs). Secondary efficacy endpoints included: CR rate at EOI assessed by the
investigator (INV; PET-CT) and by the IRC and INV (CT scans alone; standard
Lugano 2014 criteria; [28] and objective response rate (ORR; defined as a
CR or PR) at EOI assessed by the IRC and INV (PET-CT/CT scans alone). We hypothesized that combining obinutuzumab, atezolizumab,
and lenalidomide (G-atezo-len) in a triplet regimen could have the
potential to enhance the anti-lymphoma immune response of the
individual drugs. To explore this, we conducted a phase Ib/II study
(BO29562; NCT02631577) to assess the safety and efficacy of the
novel triplet combination of G-atezo-len as induction and
maintenance therapy in patients with R/R FL. Data from the final
analysis of this study are reported. y
Exploratory endpoints included: duration of response (DOR, all patients),
progression-free survival (PFS), overall survival (OS), and ORR and CR rate at
EOI among patients with and without progression of disease within
24 months (POD24) of first-line therapy. METHODS Response was determined by examination of PET and CT scans by the
IRC and the INV using modified Lugano Response Criteria for Malignant
Lymphoma. CT scans were performed at screening, at the EOI Cycle 2
(within 7 days prior to Day 1, Cycle 3), at 12, 18, and 24 months after
initiation of induction treatment, and every 3 months post-treatment. Patients with radiographic signs of progression at the EOI Cycle 2 could
continue to receive study treatment if the findings were considered to be
due to pseudoprogression/tumor flare, but they were required to have a
CT scan repeated 4–8 weeks later. PET-CT scans were performed at
screening (within 35 days prior to Day 1, Cycle 1), at EOI in patients who
had received ≥2 doses of induction treatment, and at 12 months after
initiation of induction treatment if the PET-CT scan was positive at EOI. Bone marrow examinations were required at screening (within approxi-
mately 3 months prior to Day 1, Cycle 1) for staging purposes in all
patients. If bone marrow infiltration was present at screening, a bone
marrow biopsy was required at the EOI response assessment for all
patients who may have achieved a CR, as defined per imaging methods. In
patients with less than a CR at EOI, a bone marrow examination was also
required to confirm a CR that was achieved after the EOI response
assessment. 5 Patients aged ≥18 years were eligible for inclusion if they had histologically
documented CD20-positive R/R FL (grade 1–3a), an Eastern Cooperative
Oncology Group performance status of 0–2, at least 1 bi-dimensionally
measurable lesion (>1.5 cm in its largest dimension by computed
tomography [CT] scan or magnetic resonance imaging), and had received
at least one prior anti-CD20 mAb-containing immunochemotherapy. Patients with grade 3b FL or a history of transformation of indolent
disease to diffuse large B-cell lymphoma were excluded. To rule out the
possible transformation, a core-needle biopsy was strongly recommended,
but not mandatory, for patients with a biopsy taken more than 12 months
prior to Day 1, Cycle 1 of treatment, or for patients who received anti-
lymphoma treatment between the time of the most recent available
biopsy and Day 1, Cycle 1. An overview of the full inclusion/exclusion
criteria is provided in the Supplementary section. Blood Cancer Journal (2021) 11:147 Study endpoints The primary endpoint of phase Ib was to determine the RP2D for
lenalidomide in combination with obinutuzumab and atezolizumab based
on the incidence of dose-limiting toxicities (DLT; criteria provided in the
Supplementary section) during Cycle 2 of study treatment. For all efficacy endpoints, point estimates are presented, along with the
corresponding two-sided 90% Clopper-Pearson exact CIs. Patients without
an EOI tumor assessment were considered to be non-responders. PFS and
DOR were summarized descriptively using the Kaplan–Meier method. For
the PFS analysis, data for patients without an event of interest were
censored at the date of the last tumor assessment. In phase II, the primary endpoint was efficacy, defined as CR by positron
emission tomography-computed tomography (PET-CT) and assessed by
independent review committee (IRC; modified Lugano 2014 criteria) at EOI
in the RP2D expansion cohort. Modifications to the standard Lugano criteria
were as follows: for the designation of a PR on PET, criteria for CR or PR on Blood Cancer Journal (2021) 11:147 F. Morschhauser et al. 3 Patients enrolled
N = 38
Completed induction
N = 3
Started maintenance
N = 3
Completed maintenance
N = 2
Started induction
N = 4
G-atezo-len 15mg
N = 4
Completed induction
N = 28
Started maintenance
N = 27
Discontinued
N = 1
PD
Discontinued
N = 1
Death
Discontinued
N = 6
Death (n = 2), AE (n = 2), PD
(n = 1), withdrew consent (n = 1)
Did not start maintenance
N = 1
Discontinued
N = 10
Death (n = 7), withrew
consent (n = 3)
In maintenance
N = 25
Started induction
N = 34
G-atezo-len 20mg
N = 34
Fig. 1
Patient disposition (final analysis). AE, adverse event; atezo, atezolizumab; G, obinutuzumab; L, lenalidomide; PD, progressive disease. Discontinued
N = 1
Death Fig. 1
Patient disposition (final analysis). AE, adverse event; atezo, atezolizumab; G, obinutuzumab; L, lenalidomide; PD, progressive disease. nalysis). AE, adverse event; atezo, atezolizumab; G, obinutuzumab; L, lenalidomide; PD, progressive disease. Fig. 1
Patient disposition (final analysis). AE, adverse event; atezo, atezolizumab; G, obinutuzumab; L, Fig. 1
Patient disposition (final analysis). AE, adverse event; atezo, at An interim analysis was conducted during the expansion phase of the
study and data from the first 20 patients treated at the RP2D of
lenalidomide were analyzed for PET-CT-defined CR at EOI. Enrolment was
stopped early based on sponsor decision. Table 1. Baseline characteristics (safety population). RESULTS
Patients Following sponsor assessment, and unrelated to safety findings,
enrolment was stopped after 38 patients; this was deemed a
sufficient
sample
size to perform
the
planned
benefit-risk
assessment. At the time of the final analysis (cutoff date
7 October 2020), 38 patients (lenalidomide 15 mg, n = 4; 20 mg,
n = 34) had completed the trial; 7 patients discontinued treatment
during induction (progressive disease, n = 4 [death due to
progressive disease in Cycle 1, n = 2]; AEs, n = 2; withdrawal of
consent, n = 1) and 31 patients completed induction therapy. At
the final cutoff date, 27 patients had completed maintenance
treatment (Fig. 1). Patient
baseline
characteristics
for
the
safety
population
(N = 38) are summarized in Table 1. Median age was 61.5 (range
38–79) years; 79% of patients had Ann Arbor stage III/IV disease at
diagnosis, and 26% had a high-risk Follicular Lymphoma Interna-
tional Prognostic Index (≥3); 47% had received ≥2 prior lines of
therapy. Forty-five percent of patients were refractory to (did not
respond to or progressed within 6 months of) their last line of
treatment (29% to last line of anti-CD20 mAb) and 37% of patients
had POD24 on first-line treatment. 71.9% and 85.7%; and lenalidomide, 76.5% and 85.7%; the
respective proportions of patients receiving ≥75% dose intensity
during induction and maintenance were obinutuzumab, 100%
and 100%; atezolizumab, 90.6% and 85.7%; and lenalidomide,
88.2% and 92.9% (Supplementary Table 1). Study endpoints Characteristic, [n (%), unless stated]
Safety population
(N = 38)
Median age, years (range)
61.5 (38–79)
Male
19 (50)
ECOG PS 0–1
38 (100)
Ann Arbor stage III/IV at diagnosis
30 (79)
FLIPI risk group [low (0–1);
intermediate (2); high (≥3)]
6 (16); 22 (58); 10 (26)
Elevated LDH >1 × ULN
9 (24)
Prior lines of therapy [1; ≥2]
20 (53); 18 (47)
Prior treatment
Bendamustine
12 (32)
CHOP
24 (63)
Obinutuzumab
1 (3)
Rituximab
35 (92)
Refractory to last line of treatment
17 (45)
Refractory to last line of anti-CD20
antibody
11 (29)
POD24 on first-line treatment
14 (37)
Bulky disease (≥7 cm)
6 (16)
Bone marrow infiltration
13 (35)*
Extranodal involvement
20 (53)
*N = 37. CHOP cyclophosphamide, doxorubicin, vincristine, prednisone, ECOG PS
Eastern Cooperative Oncology Group performance status; FLIPI Follicular
Lymphoma International Prognostic Index, LDH lactate dehydrogenase,
POD24 progression of disease within 24 months, ULN upper limit of normal. Table 1. Baseline characteristics (safety population). Treatment exposure and follow-up No DLTs were reported with either lenalidomide 15 mg or 20 mg
during Cycle 2 of the dose-escalation phase; therefore, lenalido-
mide 20 mg was selected as the RP2D for expansion. At the time
of the primary analysis, median follow-up was 30.0 months (range
2.7–32.1) in the lenalidomide 15 mg cohort and 14.2 months
(range 0.6–24.8) in the lenalidomide 20 mg cohort. Data sharing statement Qualified researchers may request access to individual patient level data
through the clinical study data request platform (https://vivli.org/). Further
details on Roche’s criteria for eligible studies are available here (https://
vivli.org/members/ourmembers/). For further details on Roche’s Global
Policy on the Sharing of Clinical Information and how to request access to
related clinical study documents, see here (https://www.roche.com/
research_and_development/who_we_are_how_we_work/clinical_trials/
our_commitment_to_data_sharing.htm). Efficacy
A
l Efficacy
A total of 32 patients were evaluated for efficacy in the
lenalidomide 20 mg cohort (Table 2; primary analysis). The IRC-
assessed CR rate based on modified Lugano 2014 PET-CT criteria
at EOI for the lenalidomide 20 mg cohort (primary efficacy
endpoint) was 71.9%. The corresponding INV-assessed CR rate
was
75.0%. Among
patients
who
were
double
refractory
(refractory to rituximab and alkylator), the CR rate (IRC-assessed;
modified Lugano 2014 PET-CT criteria) was 66.7% (95% CI, At the time of final analysis, in the lenalidomide 20 mg cohort,
the overall median treatment duration was 26.4 (range 0.4–29.5)
months. The proportions of patients receiving >90% dose
intensity during induction (n = 34) and maintenance (n = 28),
respectively, were: obinutuzumab, 91.2% and 100%; atezolizumab, Blood Cancer Journal (2021) 11:147 G-atezo-len 20 mg (N = 32)
Censored
0
3
6
9
12
15
18
Time (months)
21
24
27
30
33
48
No. of patients at risk
Probability of PFS
0.0
0.2
0.4
0.6
0.8
1.0
36
39
42
45
32
30
28
PFS time point
36 months
Failed
At risk
8
PFS estimate
68.4
95% CI
(47.7–82.3)
28
25
25
23
23
20
20
15
14
8
5
2
1
12
Fig. 2
Kaplan–Meier estimate of INV-assessed progression-free
survival amongst patients with relapsed/refractory follicular
lymphoma (efficacy-evaluable population; 36-month cutoff: 7
October 2020). The median observation time was 35.9 months
[range 3–47 months]. F. Morschhauser et al. 4 G-atezo-len 20 mg (N = 32)
Censored
0
3
6
9
12
15
18
Time (months)
21
24
27
30
33
48
No. of patients at risk
Probability of PFS
0.0
0.2
0.4
0.6
0.8
1.0
36
39
42
45
32
30
28
PFS time point
36 months
Failed
At risk
8
PFS estimate
68.4
95% CI
(47.7–82.3)
28
25
25
23
23
20
20
15
14
8
5
2
1
12 Table 2. IRC- and INV-assessed response rates at EOI (lenalidomide
20 mg cohort, n = 32 evaluable patients; primary analysis). Efficacy
A
l The
most common non-hematologic grade ≥3 AEs were increased
lipase (3 patients [8%]) and increased alanine aminotransferase
(ALT, 2 patients [5%]). The most common hematologic AEs (any grade) were neutro-
penia (17 patients [45%]), thrombocytopenia (10 patients [26%])
and anemia (7 patients [18%]) (Supplementary Table 3). Grade ≥3
hematologic AEs occurred in 27 patients (71%); the most common
of these were neutropenia (16 patients [42%]), thrombocytopenia (7
[18%]) and anemia (3 [8%]). The most common non-hematologic
AEs (any grade) were diarrhea (58%), constipation (40%), asthenia
(37%), cough (37%) and infusion-related reactions (34%); these AEs
were predominantly grade 1 or 2 (Supplementary Table 3). The
most common non-hematologic grade ≥3 AEs were increased
lipase (3 patients [8%]) and increased alanine aminotransferase
(ALT, 2 patients [5%]). 36-month efficacy data. In the efficacy-evaluable population, the
36-month
PFS
rate
(data
cutoff
7
October
2020;
median
observation time 35.9 months [range 3–47 months]) was 68.4%
(95% CI, 48–82) (Fig. 2). There were 14 INV-assessed progression
events. A total of 24 patients (75.0%) who received G-atezo-len as
maintenance treatment had durable clinical responses (>1 year)
and 18 patients had clinical response lasting >36 months (Fig. 3). The median DOR was 38 months (95% CI, 35-not estimable). The
36-month OS rate was 90.0% (95% CI, 72–97) (Supplementary Fig. 3). Two patient deaths in treatment Cycle 1 were considered by
the investigators to be related to disease progression, although
there was no radiologic or biopsy confirmation. These deaths were
unrelated to atezolizumab treatment, which did not commence
until Cycle 2. p
A total of 28 serious AEs (SAEs) were reported in 18 (47.4%)
patients (lung disorder was reported in two patients). Study
drug withdrawal (permanent discontinuation of any treatment)
due to an AE was reported in 11 (28.9%) patients who
experienced a total of 13 AEs; these included colitis, diarrhea,
increased lipase, arthralgia, myalgia, acute myeloid leukemia,
malignant lung neoplasm, ischemic stroke, lung disorder,
pneumonitis, maculopapular rash, and urticaria. The severity Efficacy
A
l PET-CT scan
(modified Lugano 2014)
CT-MRI scan
(Lugano 2014)
IRC-
assessed
Patients,
n (%)
90% CI
Patients,
n (%)
90% CI
ORR
25 (78.1)
62.8–89.3
26 (81.3)
66.3–91.5
CR
23 (71.9)
56.1–84.5
10 (31.3)
18.0–47.2
PR
2 (6.3)
16 (50.0)
SD
2 (6.3)
1 (3.1)
PD
3 (9.4)
4 (12.5)
INV-assessed
ORR
27 (84.4)
69.9–93.6
28 (87.5)
73.6–95.6
CR
24 (75.0)
59.4–86.9
16 (50.0)
34.4–65.6
PR
3 (9.4)
12 (37.5)
SD
1 (3.1)
1 (3.1)
PD
1 (3.1)
1 (3.1)
CR complete response, CT computed tomography, EOI end of induction,
INV investigator, IRC independent review committee, MRI magnetic
resonance imaging, ORR objective response rate, PD progressive disease,
PET positron emission tomography, PR partial response, SD stable disease. Table 2. IRC- and INV-assessed response rates at EOI (lenalidomide
20 mg cohort, n = 32 evaluable patients; primary analysis). Fig. 2
Kaplan–Meier estimate of INV-assessed progression-free
survival amongst patients with relapsed/refractory follicular
lymphoma (efficacy-evaluable population; 36-month cutoff: 7
October 2020). The median observation time was 35.9 months
[range 3–47 months]. Fig. 3
Duration of response* in 32 patients receiving G-atezo-len
(INV-assessed, efficacy-evaluable population; 36-month cutoff: 7
October 2020). *Duration of response was defined as time from the
first occurrence of a documented objective response to the time of
disease progression or relapse, as determined by the investigator on
the basis of CT scans alone or death from any cause, whichever
occurred first. CR complete response, CT computed tomography, EOI end of induction,
INV investigator, IRC independent review committee, MRI magnetic
resonance imaging, ORR objective response rate, PD progressive disease,
PET positron emission tomography, PR partial response, SD stable disease. CR complete response, CT computed tomography, EOI end of induction,
INV investigator, IRC independent review committee, MRI magnetic
resonance imaging, ORR objective response rate, PD progressive disease,
PET positron emission tomography, PR partial response, SD stable disease. 39.1–87.7; ORR 66.7%) compared with 75.0% (95% CI, 54.4–89.6;
ORR 85.0%) among non-refractory patients (P = 0.6960; Supple-
mentary Fig. 2A). Among POD24 patients, the CR rate (IRC-
assessed; modified Lugano 2014 PET-CT criteria) was 50.0% (95%
CI, 24.5–75.5; ORR 58.3%) compared with 85.0% (95% CI, 65.6–95.8;
ORR 90.0%) among non-POD24 patients (P = 0.0493; Supplemen-
tary Fig. 2B). 39.1–87.7; ORR 66.7%) compared with 75.0% (95% CI, 54.4–89.6;
ORR 85.0%) among non-refractory patients (P = 0.6960; Supple-
mentary Fig. 2A). Efficacy
A
l Among POD24 patients, the CR rate (IRC-
assessed; modified Lugano 2014 PET-CT criteria) was 50.0% (95%
CI, 24.5–75.5; ORR 58.3%) compared with 85.0% (95% CI, 65.6–95.8;
ORR 90.0%) among non-POD24 patients (P = 0.0493; Supplemen-
tary Fig. 2B). Fig. 3
Duration of response* in 32 patients receiving G-atezo-len
(INV-assessed, efficacy-evaluable population; 36-month cutoff: 7
October 2020). *Duration of response was defined as time from the
first occurrence of a documented objective response to the time of
disease progression or relapse, as determined by the investigator on
the basis of CT scans alone or death from any cause, whichever
occurred first. Response according to MRD status. A total of 22/28 MRD-
evaluable patients had a circulating clone detected at baseline;
6 MRD-evaluable patients had no circulating clone at baseline. Of
the 22 patients with a circulating clone at baseline, 21 were MRD
evaluable at EOI (1 patient sample was not evaluable due to
inadequate preparation of the sample). Among the 21 MRD-evaluable patients at EOI, 16 (76.2%) were
MRD negative; of these patients, 15 achieved a CR (93.8%) and
1 achieved a PR (6.3%), as determined by the IRC. Among the
MRD-positive patients at EOI (n = 5), 1 patient (20.0%) had SD,
3 patients (60.0%) had disease progression, and 1 patient was not
evaluable for response as determined by the IRC. treatment period is summarized in Supplementary Table 2. All
treated patients experienced at least 1 AE during induction,
31/38 patients (81.6%) who started maintenance experienced
AEs during maintenance and 11/38 patients (28.9%) who
started follow-up experienced AEs during the follow-up period. treatment period is summarized in Supplementary Table 2. All
treated patients experienced at least 1 AE during induction,
31/38 patients (81.6%) who started maintenance experienced
AEs during maintenance and 11/38 patients (28.9%) who
started follow-up experienced AEs during the follow-up period. The most common hematologic AEs (any grade) were neutro-
penia (17 patients [45%]), thrombocytopenia (10 patients [26%])
and anemia (7 patients [18%]) (Supplementary Table 3). Grade ≥3
hematologic AEs occurred in 27 patients (71%); the most common
of these were neutropenia (16 patients [42%]), thrombocytopenia (7
[18%]) and anemia (3 [8%]). The most common non-hematologic
AEs (any grade) were diarrhea (58%), constipation (40%), asthenia
(37%), cough (37%) and infusion-related reactions (34%); these AEs
were predominantly grade 1 or 2 (Supplementary Table 3). DISCUSSION
I
hi
h bColitis, diarrhea, increased lipase, arthralgia, myalgia, acute myeloid
leukemia, myelodysplastic syndrome, malignant lung neoplasm, ischemic
stroke, lung disorder, pneumonitis, maculopapular rash, urticaria. cThe primary reason for discontinuation was death and the primary cause
of death was a fatal AE (1 merkel carcinoma, 1 sarcomatoid carcinoma). d≥5%, in either group (len 15 mg or 20 mg); all AESIs were grade ≤2 and
resolved without any drug discontinuations. bColitis, diarrhea, increased lipase, arthralgia, myalgia, acute myeloid
leukemia, myelodysplastic syndrome, malignant lung neoplasm, ischemic
stroke, lung disorder, pneumonitis, maculopapular rash, urticaria. cThe primary reason for discontinuation was death and the primary cause
of death was a fatal AE (1 merkel carcinoma, 1 sarcomatoid carcinoma). d≥5%, in either group (len 15 mg or 20 mg); all AESIs were grade ≤2 and cThe primary reason for discontinuation was death and the primary cause
of death was a fatal AE (1 merkel carcinoma, 1 sarcomatoid carcinoma). d≥5%, in either group (len 15 mg or 20 mg); all AESIs were grade ≤2 and
resolved without any drug discontinuations. Our results with the G-atezo-len regimen showing a 3-year PFS
rate of 68.4% look encouraging. Prior series evaluating anti-CD20
mAb + len reported a 65% 2-year PFS rate for the G-len regimen
[18] and 53% for the R2 regimen [12]. Additionally, pivotal phase II
studies in R/R FL, have reported PFS rates for the PI3K inhibitors,
idelalisib and duvelisib, of 47% (IRC-assessed PFS rate at 48 weeks)
[5] and 62% (IRC-assessed PFS rate at 6 months) [4], respectively. However, patients in the PI3K inhibitor clinical trials were high-risk,
having received a median of ≥3 lines of prior therapy and in some
cases, were double refractory to rituximab and/or chemotherapy/
radioimmunotherapy. Additionally, any cross-trial comparison is
challenging due to differences between studies in dosing
regimen, patient population/inclusion criteria, endpoints, and
response criteria (PET based in our series). of death was a fatal AE (1 merkel carcinoma, 1 sarcomatoid carcinoma). d≥5%, in either group (len 15 mg or 20 mg); all AESIs were grade ≤2 and
resolved without any drug discontinuations. and timing of each of these permanent study drug discontinua-
tions due to an AE (atezolizumab, 6 patients; lenalidomide,
3
patients;
obinutuzumab,
3
patients)
is
summarized
in
Supplementary Table 4. Safety
ll y
All treated patients (lenalidomide 15 mg and 20 mg cohorts)
experienced ≥1 AE and 32 patients (84.2%) had a grade 3–5 AE
(Table 3; final analysis). The incidence of AEs according to Blood Cancer Journal (2021) 11:147 F. Morschhauser et al. 5 Table 3. Summary of adverse events (final analysis). Patient, n (%)
G-atezo-len
15 mg (n = 4)
G-atezo-
len 20 mg
(n = 34)
All patients
(N = 38)
Any AE
4 (100.0)
34 (100.0) 38 (100.0)
Grade 3–5 AE
4 (100.0)
28 (82.4)
32 (84.2)
Grade 5 (fatal) AEa
0
2 (5.9)
2 (5.3)
Serious AE
2 (50.0)
16 (47.1)
18 (47.4)
AE leading to
discontinuation of any study
drugb
1 (25.0)
10 (29.4)
11 (28.9)
AE leading to study
discontinuationc
0
2 (5.9)
2 (5.3)
AE leading to dose
interruption of any
treatment
4 (100.0)
30 (88.2)
34 (89.5)
Atezolizumab-related AESI (≥5%)d
Hyperthyroidism
0
5 (14.7)
5 (13.2)
Hypothyroidism
0
4 (11.8)
4 (10.5)
ALT increased
1 (25.0)
2 (5.9)
3 (7.9)
AST increased
1 (25.0)
2 (5.9)
3 (7.9)
Lipase increased
0
3 (8.8)
3 (7.9)
Hepatocellular injury
0
2 (5.9)
2 (5.3)
Rash
0
2 (5.9)
2 (5.3)
Rash maculopapular
0
2 (5.9)
2 (5.3)
Squamous cell carcinoma
0
2 (5.9)
2 (5.3)
Pneumonitis
1 (25.0)
0
1 (2.6)
Bronchiolitis
1 (25.0)
0
1 (2.6) 2 atypical fibroxanthoma, and grade 2 and grade 3 squamous cell
carcinoma) which resolved following treatment. There were no
cases of tumor lysis syndrome. The most common atezolizumab
AESIs
included
hyperthyroidism
(13%;
based
on
laboratory
abnormalities detected through frequent testing of thyroid
hormones), hypothyroidism (11%), increased ALT and aspartate
aminotransferase (both 8%), increased lipase (8%), hepatocellular
injury, rash, maculopapular rash and squamous cell carcinoma (5%
each). Two patients received hormone-replacement treatment for
hypothyroidism. Table 3. Summary of adverse events (final analysis). Blood Cancer Journal (2021) 11:147 DISCUSSION
I
hi
h In this phase Ib/II study, the chemotherapy-free triplet regimen
G-atezo-len (lenalidomide 20 mg) demonstrated marked efficacy
and an acceptable and manageable toxicity profile when used as
induction and maintenance therapy in patients with R/R FL who
had received at least one prior anti-CD20 mAb-containing
immunochemotherapy regimen. The primary endpoint was met:
G-atezo-len resulted in a CR rate at EOI of 71.9% and a 36-month
PFS rate of 68.4%. Most responses were durable, with 18 patients
experiencing clinical responses lasting longer than 36 months. The
efficacy of the G-atezo-len triplet regimen was also reflected in
high molecular response rates. Of 21 MRD-evaluable patients at
EOI, 76% (16/21) were MRD negative, which was strongly
associated with achievement of a CR (15/16 MRD-negative
patients; 93.8%). treatment
Atezolizumab-related AESI (≥5%)d
Hyperthyroidism
0
5 (14.7)
5 (13.2)
Hypothyroidism
0
4 (11.8)
4 (10.5)
ALT increased
1 (25.0)
2 (5.9)
3 (7.9)
AST increased
1 (25.0)
2 (5.9)
3 (7.9)
Lipase increased
0
3 (8.8)
3 (7.9)
Hepatocellular injury
0
2 (5.9)
2 (5.3)
Rash
0
2 (5.9)
2 (5.3)
Rash maculopapular
0
2 (5.9)
2 (5.3)
Squamous cell carcinoma
0
2 (5.9)
2 (5.3)
Pneumonitis
1 (25.0)
0
1 (2.6)
Bronchiolitis
1 (25.0)
0
1 (2.6) A promising CR rate of 67% was reported for the sub-group of
patients with double-refractory disease. Among the 12 patients
with POD24, the CR rate was 50% compared with 85% for patients
without POD24 (N = 20). Of note, two patients died early in Cycle
1 of treatment, which is quite uncommon for true FL patients. These deaths were unrelated to atezolizumab because it was not
administered until the start of Cycle 2. The authors consider that
these deaths may be a consequence of misdiagnosed histological
transformation in these patients at study entry, as there was no
mandatory histological biopsy confirmation at baseline. It has
been recently shown that the negative prognostic impact of
POD24 is strongly related to histological transformation [30] and
that anti-CD20 len-based combinations are not an adequate
treatment option in this setting [31]. Given the small sample size,
this may have affected response and PFS findings in our series. AE adverse event, AESI adverse event of special interest, ALT alanine
aminotransferase, AST aspartate aminotransferase, atezo atezolizumab, G
obinutuzumab, len lenalidomide. aThe 2 fatal AEs were merkel carcinoma and sarcomatoid carcinoma; both
unrelated to any study drug. unrelated to any study drug. DISCUSSION
I
hi
h The incidence and severity of
known AEs associated with obinutuzumab (i.e., infusion-related
reactions, hypersensitivity reactions, neutropenia, thrombocyto-
penia) was consistent with the known safety profile of obinutu-
zumab [6, 34]. AESI to obinutuzumab were uncommon; there were
2 reports of grade 2-second malignancy, which subsequently
resolved following treatment, and there were no events of tumor
lysis syndrome. Among the AESIs associated with atezolizumab
were hyperthyroidism, hypothyroidism, elevated hepatic transa-
minases and lipase, hepatocellular injury, and maculopapular rash. The incidence of thyroid-related AEs, particularly hyperthyroidism,
which was often transient but, in some cases, clinically relevant in
this study, was slightly higher than expected. However, it should
be noted that the study protocol mandated a high frequency of
laboratory testing that may have contributed to this increased
incidence and that thyroid dysfunction is a well-documented side-
effect of checkpoint inhibitors, including atezolizumab [37]. study drug due to AEs was 29%. The incidence and severity of
known AEs associated with obinutuzumab (i.e., infusion-related
reactions, hypersensitivity reactions, neutropenia, thrombocyto-
penia) was consistent with the known safety profile of obinutu-
zumab [6, 34]. AESI to obinutuzumab were uncommon; there were
2 reports of grade 2-second malignancy, which subsequently
resolved following treatment, and there were no events of tumor
lysis syndrome. Among the AESIs associated with atezolizumab
were hyperthyroidism, hypothyroidism, elevated hepatic transa-
minases and lipase, hepatocellular injury, and maculopapular rash. The incidence of thyroid-related AEs, particularly hyperthyroidism,
which was often transient but, in some cases, clinically relevant in
this study, was slightly higher than expected. However, it should
be noted that the study protocol mandated a high frequency of
laboratory testing that may have contributed to this increased
incidence and that thyroid dysfunction is a well-documented side-
effect of checkpoint inhibitors, including atezolizumab [37]. repaired with lenalidomide: implications for the tumor microenvironment and
immunotherapy. Blood. 2009;114:4713–20. 11. Chiu H, Trisal P, Bjorklund C, Carrancio S, Toraño EG. Combination lenalidomide-
rituximab immunotherapy activates anti-tumour immunity and induces tumour
cell death by complementary mechanisms of action in follicular lymphoma. Br J
Haematol. 2019;185:240–53. 12. Leonard JP, Trneny M, Izutsu K, Fowler NH, Hong X, Zhu J, et al. AUGMENT: A
phase III study of lenalidomide plus rituximab versus placebo plus rituximab in
relapsed or refractory indolent Lymphoma. J Clin Oncol. 2019;37:1188–99. 13. Tuscano JM, Dutia M, Chee K. Lenalidomide plus rituximab can produce durable
clinical responses in patients with relapsed or refractory, indolent non-Hodgkin
lymphoma. REFERENCES 1. National Comprehensive Cancer Network: NCCN Clinical Practice Guidelines:
Non-Hodgkin’s lymphoma, version 3; 2016. 1. National Comprehensive Cancer Network: NCCN Clinical Practice Guidelines:
Non-Hodgkin’s lymphoma, version 3; 2016. 25. Seetharamu N, Preeshagul IR, Sullivan KM. New PD-L1 inhibitors in non-small cell
lung cancer - impact of atezolizumab. Lung Cancer 2017;8:67–78. 2. Freedman A. Follicular lymphoma: 2018 update on diagnosis and management. Am J Hematol. 2018;93:296–305. 2. Freedman A. Follicular lymphoma: 2018 update on diagnosis and management. Am J Hematol. 2018;93:296–305. 26. Gong J, Chehrazi-Raffle A, Reddi S, Salgia R. Development of PD-1 and PD-L1
inhibitors as a form of cancer immunotherapy: a comprehensive review of
registration trials and future considerations. J Immunother Cancer. 2018;6:8. 3. Cheah CY, Chihara D, Ahmed M, Davis RE, Nastoupil LJ, Phansalkar K, et al. Factors
influencing outcome in advanced stage, low-grade follicular lymphoma treated
at MD Anderson Cancer Center in the rituximab era. Ann Oncol. 2016;27:895–901. 27. Palomba ML, Till BG, Park SI. A phase Ib study evaluating the safety and clinical
activity of atezolizumab combined with obinutuzumab in patients with relapsed
or refractory non‐Hodgkin lymphoma (NHL). Hematol Oncol. 2017;35:137–8. 4. Flinn IW, Miller CB, Ardeshna KM, Tetreault S, Assouline SE, Mayer J, et al. DYNAMO: a phase II study of duvelisib (IPI-145) in patients with refractory
indolent non-Hodgkin lymphoma. J Clin Oncol. 2019;37:912–22. 4. Flinn IW, Miller CB, Ardeshna KM, Tetreault S, Assouline SE, Mayer J, et al. DYNAMO: a phase II study of duvelisib (IPI-145) in patients with refractory
indolent non-Hodgkin lymphoma. J Clin Oncol. 2019;37:912–22. 28. Cheson BD, Fisher RI, Barrington SF, Cavalli F, Schwartz LH, Zucca E, et al. Recommendations for initial evaluation, staging, and response assessment of
Hodgkin and non-Hodgkin lymphoma: the Lugano classification. J Clin Oncol. 2014;32:3059–68. 5. Gopal AK, Kahl BS, de Vos S, Wagner-Johnston ND, Schuster SJ, Jurczak WJ, et al. PI3Kδ inhibition by idelalisib in patients with relapsed indolent lymphoma. N. Engl J Med. 2014;370:1008–18. 29. Cheson BD, Pfistner B, Juweid ME, Gascoyne RD, Specht L, Horning SJ, et al. Revised response criteria for malignant lymphoma. J Clin Oncol. 2007;25:579–86. g
6. Gribben JG, Fowler N, Morschhauser F. Mechanisms of action of lenalidomide in
B-cell non-Hodgkin lymphoma. J Clin Oncol. 2015;33:2803–11. 6. Gribben JG, Fowler N, Morschhauser F. Mechanisms of action of lenalidomide in
B-cell non-Hodgkin lymphoma. J Clin Oncol. 2015;33:2803–11. 30. Freeman CL, Kridel R, Moccia AA, Savage KJ, Villa DR, Scott DW, et al. CONCLUSIONS In conclusion, this phase Ib/II study provides evidence of activity
for the immunomodulatory triplet combination of G-atezo-len in
R/R FL. The high rate of MRD negativity observed in our study is
encouraging given the previously reported prognostic value of
MRD status at EOI for long-term PFS in patients with R/R FL
(GADOLIN trial) [39]. Although the sample size is too limited to
draw definitive conclusions, data from this final analysis suggest
that the addition of atezo to G-len may contribute to the durability
of response. 20. Ysebaert L, Morschhauser F. Immunomodulatory agents in follicular lymphoma. Hematol Oncol Clin North Am. 2020;34:715–26. 21. Myklebust JH, Irish JM, Brody J, Czerwinski DK, Houot R, Kohrt HE, et al. High PD-1
expression and suppressed cytokine signaling distinguish T cells infiltrating fol-
licular lymphoma tumors from peripheral T cells. Blood. 2013;121:1367–76. 22. Jelinek T, Paiva B, Hajek R. Update on PD-1/PD-L1 inhibitors in multiple myeloma. Front Immunol. 2018;9:2431. 23. Hsu J, Hodgins JJ, Marathe M, Nicolai CJ, Bourgeois-Daigneault MC, Trevino TN,
et al. Contribution of NK cells to immunotherapy mediated by PD-1/PD-L1
blockade. J Clin Invest. 2018;128:4654–68. 24. Lee HT, Lee JY, Lim H, Lee SH, Moon YJ, Pyo HJ, et al. Molecular mechanism of PD-
1/PD-L1 blockade via anti-PD-L1 antibodies atezolizumab and durvalumab. Sci
Rep. 2017;7:5532. DISCUSSION
I
hi
h Br J Haematol. 2014;165:375–81. 14. Leonard JP, Jung SH, Johnson J, Pitcher BN, Bartlett NL. Randomized trial of
lenalidomide alone versus lenalidomide plus rituximab in patients with recur-
rent
follicular
lymphoma:
CALGB
50401
(Alliance). J
Clin
Oncol. 2015;33:3635–40. 15. Sacchi S, Marcheselli R, Bari A, Buda G, Molinari AL, Baldini L, et al. Safety and
efficacy of lenalidomide in combination with rituximab in recurrent indolent non-
follicular lymphoma: final results of a phase II study conducted by the Fonda-
zione Italiana Linfomi. Haematologica. 2016;101:e196–199. Of note, despite some safety concerns of lenalidomide in
combination with PD-1/PD-L1 inhibitors in two phase III trials in
patients with multiple myeloma, which reported excessive and
unpredictable toxicity necessitating trial discontinuation, and an
increased death rate, following treatment with an anti-PD-1 mAb
and immunomodulatory agent (KEYNOTE-185: pembrolizumab/
lenalidomide/dexamethasone;
KEYNOTE-183:
pembrolizumab/
pomalidomide/dexamethasone) [36, 38], the addition of atezolizu-
mab to G-len did not lead to an unacceptable increase in the
incidence of immune-based toxicities in R/R FL patients in our study. 16. Andorsky D, Coleman M, Yacoub A. MAGNIFY Phase IIIb interim analysis of
induction R2 followed by maintenance in relapsed/refractory NHL. J Clin Oncol. 2020;38:8046. 17. Morschhauser FA, Cartron G, Thieblemont C, Solal-Céligny P, Haioun C, Bouab-
dallah R, et al. Obinutuzumab (GA101) monotherapy in relapsed/refractory dif-
fuse large b-cell lymphoma or mantle-cell lymphoma: results from the phase II
GAUGUIN study. J Clin Oncol. 2013;31:2912–9. 18. Morschhauser F, Le Gouill S, Feugier P, Bailly S, Nicolas-Virelizier E, Bijou F, et al. Obinutuzumab combined with lenalidomide for relapsed or refractory follicular
B-cell lymphoma (GALEN): a multicentre, single-arm, phase 2 study. Lancet
Haematol. 2019;6:e429–37. 19. Amé-Thomas P, Tarte K. The yin and the yang of follicular lymphoma cell niches:
role of microenvironment heterogeneity and plasticity. Semin Cancer Biol. 2014;24:23–32. DISCUSSION
I
hi
h Three of these AEs, acute myeloid
leukemia, ischemic stroke, and increased lipase (2 incidences in
the same patient), were categorized as grade 4 AEs; and four of
these AEs (acute myeloid leukemia, ischemic stroke, lung
disorder, and malignant lung neoplasm) were classed as SAEs. Three of these AEs resulted in permanent discontinuation of all
three study drugs (acute myeloid leukemia, ischemic stroke,
and malignant lung neoplasm), but not discontinuation from
the study itself. and timing of each of these permanent study drug discontinua-
tions due to an AE (atezolizumab, 6 patients; lenalidomide,
3
patients;
obinutuzumab,
3
patients)
is
summarized
in
Supplementary Table 4. Three of these AEs, acute myeloid
leukemia, ischemic stroke, and increased lipase (2 incidences in
the same patient), were categorized as grade 4 AEs; and four of
these AEs (acute myeloid leukemia, ischemic stroke, lung
disorder, and malignant lung neoplasm) were classed as SAEs. Three of these AEs resulted in permanent discontinuation of all
three study drugs (acute myeloid leukemia, ischemic stroke,
and malignant lung neoplasm), but not discontinuation from
the study itself. The acceptable and manageable toxicity profile of the G-atezo-
len regimen in the current study was substantiated by the high
proportion of patients (>85%) receiving >75% dose intensity
during induction and maintenance. Furthermore, the safety and
tolerability profile of G-atezo-len was generally consistent with the
known profiles for the individual drugs and the double combina-
tion regimens, obinutuzumab plus lenalidomide, and obinutuzu-
mab plus atezolizumab, with no new safety signals identified
[18, 27, 32–36]. Most AEs in the study were manageable with
appropriate medical care or dose modifications. The majority of
AEs were grade 1–2, and most grade 3–5 AEs and all SAEs were
isolated events. The rate of permanent discontinuation of any y
Overall, AEs led to dose modification/interruption of any
study drug in 34 patients (89.5%). The most common events
(≥10%)
leading
to
dose
modification/interruption
were
hyperthyroidism (18.4%) and hematologic toxicities including
neutropenia (13.2%), and thrombocytopenia (10.5%). Two fatal
AEs were reported; both were unrelated to any study drug
(1 merkel carcinoma, 1 sarcomatoid carcinoma). Reported
adverse
events
of
special
interest
(AESI)
with
obinutuzumab included 3 cases of second malignancies (grade F. Morschhauser et al. 6 study drug due to AEs was 29%. REFERENCES Early
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lymphoma cells induce T-cell immunologic synapse dysfunction that can be Blood Cancer Journal (2021) 11:147 Blood Cancer Journal (2021) 11:147 ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41408-021-00539-8. Reprints and permission information is available at http://www.nature.com/
reprints Reprints and permission information is available at http://www.nature.com/
reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims
in published maps and institutional affiliations. AUTHOR CONTRIBUTIONS FM and GS designed the study; AM, DS, FM, ISL, GS, MLP, and NG conducted the
study; AM, DS, FM, GS, ISL, MLP, OC, and NG recruited and followed-up with patients;
AM, DS, GS, ISL, MLP, OC and NG were responsible for data collection; AK, RM, SK, OC,
and TN were responsible for data analysis; AM, FM, GS, ISL, MLP, NG, RM, OC and TN
were involved in the interpretation of data. All authors contributed to the drafting of
the manuscript and provided final approval. Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made. The images or other third party
material in this article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. ACKNOWLEDGEMENTS Correspondence and requests for materials should be addressed to F.M. This study was sponsored by F. Hoffmann-La Roche Ltd. Medical writing support for
the development of this manuscript, under the direction of the lead author, was
provided by Louise Profit, PhD, and Aisling Lynch, PhD, of Ashfield MedComms, an
Ashfield Health company, and was funded by F. Hoffmann-La Roche Ltd. F. Morschhauser et al. 7 34. FDA. GAZYVA: Highlights of Prescribing Information. Available at: https://www. accessdata.fda.gov/drugsatfda_docs/label/2017/125486s017s018lbl.pdf. Vol. 2020; 2020. Roche Ltd, Forty Seven Inc./Gilead, Genentech Inc., Incyte, Innate Pharma, Juno
Therapeutics/Bristol Myers Squibb, Kite Pharma/Gilead, Miragen Therapeutics Inc.,
OncoTartis Inc., Seattle Genetics, Takeda, TG Therapeutics Inc., Merck and Rhizen
Pharmaceuticals and personal fees from AstraZeneca, Carevive Systems Inc,
Gilead, Pharmacyclics, Incyte, Kyowa Kirin, MorphoSys/Incyte, Seattle Genetics,
TG
Therapeutics
and
Rigel
Pharmaceuticals
Inc.;
OC
reports
grants
from
F. Hoffmann-La Roche Ltd and Gilead; personal fees and non-financial support from
F. Hoffmann-La Roche
Ltd,
Janssen,
Takeda,
Bristol
Myers
Squibb,
Amgen,
AbbVie; personal fees from Gilead and Merck. DS has been an investigator for
AbbVie-sponsored clinical trials. SK is an employee of IQVIA; AK and TN are
employees of F. Hoffmann-La Roche Ltd. RM is an employee of Genentech Inc. and
has equity ownership in F. Hoffmann-La Roche Ltd. GS reports personal fees from
F. Hoffmann-La Roche Ltd, Gilead, Celgene, Janssen Pharmaceuticals, Novartis,
Amgen, AbbVie, Autolus, Epizyme, MorphoSys, Takeda, Genmab, Allogene, VelosBio
Inc., and BeiGene. 35. TECENTRIQ Summary of Product Characteristics. Available at: https://www.ema. europa.eu/en/documents/product-information/tecentriq-epar-product-
information_en.pdf Last updated October 2019 [Last accessed 11 Feb 2020]. 36. Mateos MV, Blacklock H, Schjesvold F, Oriol A, Simpson D, George A, et al. Pembrolizumab plus pomalidomide and dexamethasone for patients with
relapsed or refractory multiple myeloma (KEYNOTE-183): a randomised, open-
label, phase 3 trial. Lancet Haematol. 2019;6:e459–69. label, phase 3 trial. Lancet Haematol. 2019;6:e459–69. 37. Ferrari SM, Fallahi P, Galetta F, Citi E, Benvenga S, Antonelli A. Thyroid disorders
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brolizumab plus lenalidomide and dexamethasone for patients with treatment-
naive multiple myeloma (KEYNOTE-185): a randomised, open-label, phase 3 trial. Lancet Haematol. 2019;6:e448–58. 39. Pott C, Sehn LH, Belada D, Gribben J, Hoster E, Kahl B, et al. MRD response in
relapsed/refractory FL after obinutuzumab plus bendamustine or bendamustine
alone in the GADOLIN trial. Leukemia. 2020;34:522–32. ADDITIONAL INFORMATION COMPETING INTERESTS FM reports personal fees from AbbVie, Celgene, Epizyme, F. Hoffmann-La Roche Ltd/
Genentech Inc., Gilead, Janssen and Servier. NG reports grants and personal fees from
AstraZeneca, Bristol Myers Squibb, TG Therapeutics, and Pharmacyclics, personal fees
from Genmab, Seattle Genetics, Janssen, Karyopharm Therapeutics, AbbVie and
Gilead and grants from Genentech Inc., and Forty Seven Inc. ISL has served on
advisory boards from Seattle Genetics, Janssen Scientific and Verastem Inc.; MLP has
served on advisory boards for Novartis, Kite Pharma and Merck. AM reports grants
from ADC Therapeutics, Affimed, Celgene/Bristol-Myers Squibb, F. Hoffmann-La © The Author(s) 2021 Blood Cancer Journal (2021) 11:147 Blood Cancer Journal (2021) 11:147
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Exploring Spatial UI Transition Mechanisms with Head-Worn Augmented Reality
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ABSTRACT everyday scenarios [29, 35, 41]. For example, recent work by Lu
& Bowman suggested that AR head-worn displays (HWDs) could
support easier and less distracting everyday information acqui
sitions as compared to mobile phones [41]. However, in existing
state-of-the-art AR operating systems (OS) (e.g., the Magic Leap
One and the HoloLens 2), AR content defaults to staying at a fxed
location until users manually move or re-instantiate it. This kind
of mechanism assumes that the main use cases for AR are confned
in one space, limiting the mobility and accessibility of the digital
content when users move around. Imagine in the future people comfortably wear augmented reality
(AR) displays all day, how do we design interfaces that adapt to the
contextual changes as people move around? In current operating
systems, the majority of AR content defaults to staying at a fxed
location until being manually moved by the users. However, this
approach puts the burden of user interface (UI) transition solely on
users. In this paper, we frst ran a bodystorming design workshop to
capture the limitations of existing manual UI transition approaches
in spatially diverse tasks. Then we addressed these limitations by
designing and evaluating three UI transition mechanisms with dif
ferent levels of automation and controllability (low-efort manual,
semi-automated, fully-automated). Furthermore, we simulated im
perfect contextual awareness by introducing prediction errors with
diferent costs to correct them. Our results provide valuable lessons
about the trade-ofs between UI automation levels, controllability,
user agency, and the impact of prediction errors. With mobile computing (e.g., smartphones, smartwatches), peo
ple can access diferent applications and information on-the-go. However, most of the time, these systems still rely very much on
the users’ efort to fnd and open the application that is needed
at that time. This poses challenges to the users who need to fo
cus on real-world tasks with their attention and hands occupied. How could we enable easier access to the digital content as users
move across diferent environments while needing access to some
information? Exploring Spatial UI Transition Mechanisms with Head-Worn
Augmented Reality Yan Xu
yanx@fb.com
Reality Labs Research
Meta
Redmond, Washington, USA Feiyu Lu
feiyulu@vt.edu
Center for Human-Computer Interaction
Virginia Tech
Blacksburg, Virginia, USA ACM Reference Format: ACM Reference Format: Feiyu Lu and Yan Xu. 2022. Exploring Spatial UI Transition Mechanisms
with Head-Worn Augmented Reality. In CHI Conference on Human Factors
in Computing Systems (CHI ’22), April 29-May 5, 2022, New Orleans, LA, USA. ACM, New York, NY, USA, 16 pages. https://doi.org/10.1145/3491102.3517723 We see the great opportunity to leverage prediction and automa
tion with AR systems. AR devices have the potential to understand
users’ intent accurately and just-in-time, due to the wearability,
world-facing sensors (e.g. egocentric videos, depth cameras), and
user-facing sensors (e.g. eye-tracking cameras). Combined with
the increased AI capability, AR can help ofoad the users’ efort of
fnding the digital content to the system. CCS CONCEPTS One direction is to predict what the user tries to do and surface
the corresponding functions. With the advancements in Artifcial
Intelligence (AI) and computational power, recent user interfaces
have become more capable of predicting user intent and suggest
ing potential interactions to be performed by the users [34, 64]. Recently, more and more of this kind of prediction is applied to
mobile systems, where the mobile applications make interaction
suggestions based on the time of the day, the history of interactions,
and location [8, 55, 68, 72]. For example, Google Maps occasionally
pops up a suggestion to navigate to a certain destination based on
past uses. • Human-centered computing → Mixed / augmented reality;
Virtual reality; Interaction techniques. KEYWORDS agency, automation, controllability, adaptive interfaces agency, automation, controllability, adaptive interfaces 1 INTRODUCTION Compared to Virtual Reality (VR) headsets where users are im
mersed in a virtual environment, Augmented Reality (AR) glasses
enable people to interact with their everyday physical world with
the digital augmentation [3]. In a typical everyday-life activity, peo
ple will need to move around to carry out diferent tasks, changing
their information needs on-the-go. Recent research has shed light
on the potential of AR glasses to support such needs in common To explore the intersection between AI and AR, a lot of questions
need to be answered. First, how would people respond to user
interfaces (UI) that try to predict and adapt to their needs? What
do they like or dislike about it? Second, how is such automated
experience compared to manually controlling the UIs, the latter
of which is more familiar to users? Third, how would efciency,
usability, and agency be afected when the interfaces automatically
adapt to user needs with diferent levels of user control? Finally,
given that it is virtually impossible for any prediction to reach 100% This work is licensed under a Creative Commons Attribution International
4.0 License. CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA
© 2022 Copyright held by the owner/author(s). ACM ISBN 978-1-4503-9157-3/22/04. https://doi.org/10.1145/3491102.3517723 This work is licensed under a Creative Commons Attribution International
4.0 License. CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA
© 2022 Copyright held by the owner/author(s). ACM ISBN 978-1-4503-9157-3/22/04. https://doi.org/10.1145/3491102.3517723 This work is licensed under a Creative Commons Attribution International
4.0 License. CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA
© 2022 Copyright held by the owner/author(s). ACM ISBN 978-1-4503-9157-3/22/04. https://doi.org/10.1145/3491102.3517723 CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA Feiyu Lu and Yan Xu accuracy, how would the user experience be afected when the
system predicts user intent incorrectly, and how to mediate the
consequences when an error happens? information. Widget UIs have been the common form of displaying
information on current mobile phone interfaces [62, 67]. Similarly,
in this research, we focused on everyday information access in AR
systems with widget UIs. In this research, we answer these questions by designing, de
veloping, and evaluating several mechanisms to spatially transit
AR UIs when people move in space. 2.2 Mobility of UIs in AR UIs in AR are usually rendered at a fxed location in the real world. However, in everyday situations. information could be needed on
the-go in a less-controlled manner [15, 57]. As such, recent research
has explored the possibility of carrying AR content with the users
while moving. Lages & Bowman explored an adaptive walking
interface in which AR windows become body-referenced and follow
the users around [37]. Lu et al. explored display-referenced and
body-referenced layouts for carrying the AR content with the users
[42]. The major limitation of these approaches is scalability. Because
the system has no knowledge of what the users might need, it has
to bring all the AR content that the user will possibly need, while
increased pieces of information could cause information overload
and distract the users. An early study by Sohn et al. found that 72%
of the information needs were prompted by contextual factors such
as location changes and activities to be done [57]. In this research,
we explored the possibility of automated UI placements based on
location changes and activities, and compared them with existing
solutions such as display-referenced follow behaviors and manual
drag and drops of the AR UIs. Through the design workshop and the user study, We learned
valuable lessons about users’ needs for on-the-go AR UIs. We also
learned users’ performance and preferences with diferent UI tran
sition mechanisms, which reveals the relation among automation,
controllability, and user agency. Furthermore, we found that predic
tion errors were perceived diferently with diferent controllability
and diferent error-recovery cost. 1 INTRODUCTION To inform our design direc
tions, we frst conducted a body-storming design workshop with
expert user experience (UX) designers, in which we learned about
the major problems participants encountered when they use AR
glasses for acquiring information on-the-go. We then designed and
implemented three UI transition mechanisms as outcomes of the
workshop. These interfaces have diferent levels of automation
and controllability, which required diferent levels of user efort
to access AR content on-the-go. Moreover, we simulated the inac
curacy/error of prediction about what UI widgets users may need
at diferent locations. With this simulation of errors, we looked
into how users perceive and handle the error in the context of
spatial tasks while given diferent levels of user control over the
automation results. Finally, we ran a within-subject study with 40
users to compare the three UI transition mechanisms, plus the base
line of the manual UI manipulations which is available on existing
commercial AR glasses. 2.3 Levels of System Automation and User
Control The main contributions of this work include: (1) explorations
of the challenges users encounter when trying to access AR con
tent on-the-go; (2) designing and implementing three interface
solutions with diferent levels of automation controllability; (3) em
pirical fndings about users’ performance and preference among the
diferent interfaces and how prediction error afects the experience. (4) design implications for future implementations of automated UI
transition mechanisms for AR. Roy et al. defned automation as the programming of complex tasks
to be automatically executed by a machine with the goal of reduc
ing tedious manual efort, workload and improving productivity of
everyday human users [51]. Automated systems may encompass
a wide range of low-to-high automation levels and user controls. A higher level of system automation could lead to a lower level of
user control because the system would take over, performing more
decision-making and task-executions with less user interference. In 2004, Findlater and McGrenere proposed three levels of automa
tion: (1) adaptive: the system controls all the interface changes with
no user control; (2) adaptable: the users control all the interface
changes with no system control; and (3)mixed-initiative: the control
is shared between the user and the system [23]. A more widely
adopted automation level standard could be found in the feld of au
tonomous driving. The standard J3016_202104 (SAE 2021) defned
six levels of automation in automated driving: no driving automation
(level 0); driver assistance (level 1); partial driving automation (level
2); conditional driving automation (level 3); high driving automation
(level 4); and full driving automation (level 5) [52]. A lower level of
system automation allows customization for certain needs, while
a higher level of automation reduces complexity and friction to
interact with the UIs [71]. In our work, we are interested in under
standing how diferent levels of automation impact users’ efciency
and agency. 2.1 Everyday Information Acquisition with AR
HWDs People encounter a variety of information needs in their every
day lives [14, 21]. AR HWDs have the potential to address such
needs by displaying relevant information directly in the real-world
environment in front of the users. ARWin is an early attempt of
displaying everyday information such as calendar, weather, and
clock in AR on a tabletop [22]. In recent work, Colley et al. explored
displaying virtual information on top of relevant objects at home
to augment user memory [18]. Ventä-Olkkonen et al. explored dis
playing everyday information on home windows [61]. Knierim et
al explored the use of AR for displaying information in home envi
ronments [35]. Lu et al. explored displaying everyday information
as glanceable UIs at the periphery of the user’s view [42]. These
work shed light on the potential of AR displays to fulfll people’s
everyday information needs. Most of these work involved the idea
of “widgets”, which are compact glanceable UIs for quick access to Automation is powered by the advancement in Machine Learning
and AI. The algorithm generates an output (“prediction”) based on
the past training data, the input, and the model. Some autonomous CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA Exploring Spatial UI Transition Mechanisms with Head-Worn Augmented Reality systems may be capable of predicting user intent and making deci
sions on the users’ behalf [19, 20]. However, the predictions may
be inaccurate. In addition to trying to improve the accuracy of pre
diction, it’s proven to be critical to design for controllability, which
“refects to what extent the users can control the automation or
alter its result to reach their goal, and how easily and rapidly can
this control be carried out [51].” There have been long-time debates
on how much the system should be involved in the automation
of UI components, as well as how much controllability should the
user hold over the automation. Findlater and McGrenere found
that adaptable was more preferred and signifcantly more efcient
than adaptive in 2D menus [23]. Gajos et al. found that adaptive
interfaces were not necessarily advantageous purely because of
their theoretical benefts [26]. Zhang et al. found that combining
adaptive with adaptable could lead to higher usability [71]. Roy et al. found that manual approaches were more preferred as compared to
automated systems [51]. 3.3 Bodystorm Activities With the AR prototype application, participants were asked to go
through a sequence of physical and digital tasks while moving
around in their own home environments, including the kitchen, the
living room, and the home ofce room. The tasks in the workshop
were designed to represent common at-home user scenarios where
digital information may be needed. Participants started in their
home ofce. First, they were asked to place all eight widgets in their
ofce environments. Second, participants were asked to monitor
the stock widget while going to the kitchen to make a cofee. They
were asked to set up a timer with the timer widget above the cofee
machine. Third, participants were asked to open their fridge and
check what ingredients they do not have according to the list in
the recipe widget. Fourth, participants were asked to go back to the
home ofce, record the current stock price and the missing recipe
ingredients in a notepad application on their laptop. Last, partici
pants were asked to go to the living room and check out the current
weather in the weather widget. During the activities, whenever
participants encountered any pain-points or challenges, they were
instructed to write down notes on post-its and take screenshots
with their HoloLens 2 device. In total, we allocated 25 minutes
during the workshop for the bodystorming activities. Participants
were also encouraged to complete more custom activities if they
had time left. 3.1 Research Goals We frst conducted a design workshop to identify user needs for
accessing AR content on-the-go. The workshop was conducted
online with video-conferencing software. Specifcally, we aimed to:
(1) identify the gaps between user needs and the existing manual
UI transition mechanisms available on commercial AR systems; (2)
brainstorm with experienced designers about potential solutions to
address these gaps. 2.1 Everyday Information Acquisition with AR
HWDs However, little research has been explored
for AR displays about how automation and controllability would
impact the multi-faceted user experience. In this research, we aim
to explore the trade-of between automation and user agency, and
the roles that controllability and prediction errors play in this kind
of trade-ofs. 2.4 Automated UI Placements in AR Interfaces 2.4 Automated UI Placements in AR Interfaces
Research in automated UI placements in AR mostly lies in label
placements and view management [43, 50]. Little research has been
conducted to explore automated placements of everyday AR UIs. In
2019, Lages and Bowman explored an adaptive walking UI, in which
AR windows were placed adaptively around the user’s body or on
the wall based on manual input [37]. Lindlbauer et al. explored au
tomated placements of AR content based on task and eye-tracking
data [40]. Cheng et al. explored automatic adaptation of UI’s spatial
layouts based on environmental changes when users move to dif
ferent locations [12]. Their results shed light on the potential of AR
systems to predict user needs and assist the placements of AR UIs. In this research, we explore the idea of automated UI placements
with diferent levels of automation and controllability when users
move across diferent locations. 3.2 Prototype for the Workshop For the design workshop, we developed a prototype on the HoloLens
2 device. In the prototype, eight AR widgets were integrated in
the system, including calendar, weather, timer, email, recipe, so
cial, stock, and news. The widgets contain pre-defned information
that we programmed in the system. All the widgets were world-
referenced by default. We implemented the three common solutions
for transitioning AR user interfaces. The frst one was drag&drop
(see Figure 1 (a)). Users performed a pinch gesture to grab the wid
gets, then they could walk to a new location and drop the widgets. The second one was tag-along (see Figure 1 (b-c)), in which users
could touch a button to trigger the widgets to follow them around. While following, the widgets became loosely display-referenced
and stayed within the feld of view (FoV) of the users. By either
dragging the widgets or pressing the “follow” button again, users
could unfollow the widgets and make them world-fxed. Similar to
how tag-along is implemented on HoloLens 2, at most one widget
could be triggered to follow the users at a time. The last one was re-
instantiate (see Figure 1 (d-e)). Users could bring out a home menu
by showing their left-hand palm to the front camera of the headset. Then they could tap on the icon of a widget to re-instantiate the
widget on the right side of their hands. 3.4 Participants & Procedures Third, a brief overview was given
to all participants about the workshop background and schedule (10
min). Fourth, participants were asked to go through the bodystorm
activities (25 min). Fifth, after participants fnished all the activities,
they were instructed to go back to their home ofce and import the
comments and screenshots in a shared online whiteboard2 (15 min). Sixth, in the same online whiteboard board, participants were asked
to brainstorm about how these pain-points could be resolved if the
AR system has diferent levels of knowledge on their contextual
changes while moving around. Each participant was encouraged
to brainstorm three to fve interface solutions (25 min). In the end,
participants shared their solutions with each other and voted for
their favourite ones. The entire workshop took around 90 minutes
to complete. P2. Awareness & Recall. The second pain-point, mentioned by
three participants, was the difculty of memorizing where and why
a widget was placed beforehand in the previous environment (see
Figure 2 (b)). One participant commented that “I felt confused about
why the timer was opened in the ofce”; and the other one mentioned
that “I forgot where I placed a widget, so I had to scan the whole area
around my screen”. When being asked about improvements, one
designer mentioned that “I wish there were some guidance about
where I placed what widget within my feld of view”, and the other
one mentioned that “(I want to be more aware) of the locations of the
widgets, what’s opened, and how I’m bringing a widget from place to
place”. P3. High efort of widget acquisition. The last pain-point, which
was mentioned by three participants, was the high-level user efort
required to access the widgets. Participants mentioned that they
did not want to fnd the widget they placed in the previous location. They wish they could easily bring multiple widgets with them and
have access to certain information without the need to relocate or
reopen a widget. For example, one commented that “I wish I didn’t
have to reload the weather app just to check the weather” (see Figure 2
(c)); and the other one commented that “Only one widget following is
too little. I would like to put ‘quick’ widgets such as weather and timer
on to my forearms”. 3.4 Participants & Procedures To design for the embodied nature of AR interactions in the
space, we conducted a bodystorming workshop [54] with fve UX
experts. They needed to walk through their house for a sequence
of physical and digital tasks. During the tasks, they experienced
and refected on a Hololens prototype we developed to represent
the current UI transition mechanisms available on commercial AR
devices. We recruited participants with rich experience in designing AR, VR,
or MR user experiences in the industry, including fve participants
(three designers, one design technologist, and one UX researcher). All participants came from the AR/VR industry. They all had ac
cess to a HoloLens 2 hardware and were very experienced with
AR/VR platforms. The workshop was conducted remotely via a CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA Feiyu Lu and Yan Xu Figure 1: An illustration of the AR prototype application: (a) drag & drop: users approach a widget with their hands and drag
them around; (b-c) tag-along: users press a “follow” button beside a widget and the widget will loosely stay in the FoV of the
display; (d-e) re-instantiate: users press the icon of a widget in the hand menu to instantiate the widget in front of them; (f)
users show their palms to see a hand menu and instruction tasks to be completed in the bodystorming session. Figure 1: An illustration of the AR prototype application: (a) drag & drop: users approach a widget with their hands and drag
them around; (b-c) tag-along: users press a “follow” button beside a widget and the widget will loosely stay in the FoV of the
display; (d-e) re-instantiate: users press the icon of a widget in the hand menu to instantiate the widget in front of them; (f)
users show their palms to see a hand menu and instruction tasks to be completed in the bodystorming session. they talked about the system automation to help organize the wid
gets. (e.g., “I wish the system can help me organize the widgets so
they don’t take up too much space around me”). video-conferencing application called BlueJeans1. The procedure
for the workshop included seven phases. First, before the workshop,
the prototype application was sent to participants together with
instructions about how to sideload it on their own HoloLens 2. Sec
ond, participants joined the virtual conference room and introduced
themselves to each other (5 min). 3.4 Participants & Procedures Lastly, participants talked about leveraging the
contextual change for the UI transition, for example, one asked
“Could the weather widget appear as I am walking to the door? Or as
I am about to head out?” 1https://www.bluejeans.com/
2https://start.mural.co/ 3.5 Results In this solution, designers suggested that all
widgets were shown in low level-of-detail (LoD) icons and attached
to the user’s wrist and stays with the users by default similar to
smartwatches (“I would like to put ’quick’ widgets such as weather
and timer on to my forearms.”) Users could easily glance at the icons,
or open the widget in full size if they need high LoD information. S2. Snap to planes or objects. The second interface solution,
voted by three of the participants (60%), aimed to solve P.1 and P.2. It required some level of knowledge of the environments. In this
solution, the widgets automatically snap to physical surfaces and
planes, or near relevant objects after being opened. Participants
suggested that “widgets should align/snap to my physical surround
ing or other already placed widgets”; and “widgets understand what
is around it and adapt to the environment”. S1. Wrist-based glanceable UIs. The frst interface solution, voted
by four of the participants (80%), aimed to solve P.3 and required a
low level of contextual understanding with some level of input re
quired from the users. In this solution, designers suggested that all
widgets were shown in low level-of-detail (LoD) icons and attached
to the user’s wrist and stays with the users by default similar to
smartwatches (“I would like to put ’quick’ widgets such as weather
and timer on to my forearms.”) Users could easily glance at the icons,
or open the widget in full size if they need high LoD information. which incorporated diferent levels of automation and controlla
bility. We chose these three because they represent the three au
tomation levels proposed by Findlater and McGrenere: adaptable,
mixed-initiative, and adaptive, with an increased level of system au
tomation and decreased level of user control. [23]. We also simulated
error/inaccuracy that is unavoidable in any of the prediction-based
automation algorithms. To evaluate and understand how these interfaces are used while
people move in spaces, we conducted a user study. Due to chal
lenges of running in-person studies during COVID-19 and to avoid
technological limitations of current AR devices, the study was con
ducted in a VR-simulated AR environment. 4.2 Interface Conditions In general, through the workshop, we learned about the user
needs and challenges when trying to use AR interfaces while car
rying out real-world tasks. New interface solutions need to be
explored for solving the pain-points mentioned above. The design
workshop highlighted some of the potential directions, such as re
ducing the efort to remember, carrying AR content, and leveraging
the system’s contextual awareness to trigger certain widgets. We
designed multiple UI transition mechanisms in sketch based on the
workshop learnings. 4.2.1 Wristpack. The frst interface solution took inspirations from
S.1. When users leave the current room and head towards the other
one, all the widgets become automatically attached to user’s wrist
and forearms, displaying as icons and names. In previous work,
Harrison et al. explored projecting UIs on the wrist for interacting
with menus [30]. Grubert et al. explored extending wrist-worn dis
plays with widget UIs for convenient access to mobile applications
[28]. Similarly, in our Wristpack solution, when users need access
to a widget later, they can pull the widget of their arms and place
them around in the new location (see Figure 3 (a-b)). This interface
represents the adaptable metaphor from Findlater and McGrenere’s
work [23], in which users take most of the control about when and
where the widgets are placed in the real world; the system only 4.1 Research Questions In this study, our goal was to evaluate and compare four conditions
(Wristpack, Semi-Auto, Fully-Auto, Baseline) for transitioning AR
widget UIs. Specifcally we aim to answer the following questions
through the user study: S3. Everything in the right place. The third interface solution,
voted by four of the participants (80%), aimed to solve all the three
pain-points. It required a high level of contextual understanding of
the environment. In this solution, the system automatically opened
the widget and placed it right when and where the users needed
it. Participants suggested that “the UI populates in the right place
where it is most relevant, where the user’s attention is, what the user’s
intention might be”; and “the UI should be displayed around the
system’s best guess of the object or activity it’s related to, the user can
move this.” S3. Everything in the right place. The third interface solution,
voted by four of the participants (80%), aimed to solve all the three
pain-points. It required a high level of contextual understanding of
the environment. In this solution, the system automatically opened
the widget and placed it right when and where the users needed
it. Participants suggested that “the UI populates in the right place
where it is most relevant, where the user’s attention is, what the user’s
intention might be”; and “the UI should be displayed around the
system’s best guess of the object or activity it’s related to, the user can
move this.” • How do these interface transition mechanisms perform in
terms of efciency, usability, workload, and agency? • How do diferent levels of automation and controllability
afect agency and users’ preference? • How do people perceive and handle system inaccuracy/error
when it occurs? How is the overall experience afected by
the errors? 3.5 Results 3.5.1 Most common pain-points. In this section, we listed the most
frequently appearing pain-points mentioned by the workshop par
ticipants in the bodystorm session. P1. Placements of the widget UIs. The frst pain-points, which
was mentioned by all participants, was the high level of efort
required to manually place the widgets (see Figure 2 (a)). One par
ticipant commented that “I need to spend a lot of time arranging. The widgets look messy and topsy turvy”, and another participant
commented, “manually laying out the widgets felt tedious”. When
being asked how they wish the AR interface could be improved, 3.5.2 Most common interface solutions. In this section, we high
light the most frequently appearing solutions mentioned by the
workshop participants in the brainstorming session. CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA Exploring Spatial UI Transition Mechanisms with Head-Worn Augmented Reality Figure 2: Three examples from the screenshots taken by the workshop participants, each highlights one of the pain-points: (a)
P1. placements of the widget UIs, in which participants wish that the AR system could help them arrange the UIs around the
physical monitor; (b) P2. awareness & recall, in which one participant forgot why the timer widget was placed in the kitchen
after returning there from the ofce; (c) P3. high efort of widget acquisition, in which a participant wish that did not have to
reopen the weather widget and manually place it in the living room just to access the weather information. Figure 2: Three examples from the screenshots taken by the workshop participants, each highlights one of the pain-points: (a)
P1. placements of the widget UIs, in which participants wish that the AR system could help them arrange the UIs around the
physical monitor; (b) P2. awareness & recall, in which one participant forgot why the timer widget was placed in the kitchen
after returning there from the ofce; (c) P3. high efort of widget acquisition, in which a participant wish that did not have to
reopen the weather widget and manually place it in the living room just to access the weather information. S1. Wrist-based glanceable UIs. The frst interface solution, voted
by four of the participants (80%), aimed to solve P.3 and required a
low level of contextual understanding with some level of input re
quired from the users. 4 EVALUATION: USER STUDY Inspired by the solutions we generated from the design workshop,
we implemented three interfaces (Wristpack, Semi-Auto, Fully-Auto), CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA Feiyu Lu and Yan Xu Figure 3: An illustration of the three interfaces: (a-b) Wristpack, in which all widgets UIs are attached to participants’ wrist
when spatial changes are detected and can be “pulled out” if needed; (c-d) Semi-Auto, in which the system would suggest three
widgets on participants’ wrist, and participants hold the decision of when and which one to place; (e) Fully-Auto, in which the
system would place the most recommended widget automatically for the users without any input needed (a purple dot was
displayed at the corner of the display as a visual indicator when automation happened in the system). Figure 3: An illustration of the three interfaces: (a-b) Wristpack, in which all widgets UIs are attached to participants’ wrist
when spatial changes are detected and can be “pulled out” if needed; (c-d) Semi-Auto, in which the system would suggest three
widgets on participants’ wrist, and participants hold the decision of when and which one to place; (e) Fully-Auto, in which the
system would place the most recommended widget automatically for the users without any input needed (a purple dot was
displayed at the corner of the display as a visual indicator when automation happened in the system). provided a small amount of automation when a spatial change was
detected. predictions, the user needed to fnd the previous location of the
widget to access it. The Fully-Auto condition represents the adaptive
metaphor [23], in which the system takes full control over which,
when and where the widgets are placed. The users could not change
the predictions made by the system even when it is incorrect. 4.2.2 Semi-Automated Placements (Semi-Auto). The second inter
face solution took inspiration from S.1, S.2 and S.3. The system
would predict user needs and suggest three widgets on the user’s
wrist to be placed in these spaces, so it has a higher level of automa
tion as compared to the Wristpack interface. The three widgets will
be spawned on users’ wrist, the widget with the largest probability
of being needed had the biggest size and was the most visible, while
the widgets that were less possibly needed was smaller and less
visible (see Figure 3 (c)). 4 EVALUATION: USER STUDY The user has high controllability by making
the fnal decision of which widget to open and when to place it
(see Figure 3 (d)). If a prediction error happened, meaning that the
top-recommended widget was not a match to the task, the user can
look through the rest of the two less prominent recommendations
and fnd the correct widget. This interface represents the mixed-
initiative metaphor [23], in which the user and the system take
shared control over which and when the widgets are placed in the
real world. 4.2.4 Baseline. We also included a Baseline interface, which was
the drag & drop and tag-along behaviors of the widgets. Similar to
the AR prototype, participants could either grab a widget with their
controller and drop it at a new room, or trigger tag-along mode so
the widget would follow them around automatically. Note that we
removed the re-instantiation function in the VR study because: (1)
we wanted to keep computational resource allocation consistent for
all interface conditions; (2) our research focus was about interface
transition rather than interface initiation. 4.3 Study Design Users could use a raycasting technique to point the
right controller at the objects to use them. 4.3.4 Simulation of predictability & accuracy. Predictability and
accuracy are two important aspects of adaptive UIs. According to
Gajos et al., accuracy refers to “the percentage of time that the
necessary UI elements are contained in the adaptive area”, and pre
dictability refers to “if the adaptation follows a strategy the users
could easily model in their heads [27].” Our tasks setup simulated
high predictability because users know that the system’s recom
mended widget(s) will appear after they interact with an object
during Semi-Auto and Fully-Auto conditions. Since it is extremely
challenging for adaptive interfaces to reach 100% accuracy on pre
dicting user intent, we simulated imperfect accuracy in both the
Semi-Auto and Fully-Auto interface conditions. For the Semi-Auto
condition, the system would suggest the widget needed to answer
the question in the second or third slots 25% of the time (3 out of 12
trials), which posed a low cost on the users to retrieve the correct
widget when imperfect prediction happened. For the Fully-Auto
condition, the system would place the incorrect widget in front of
the users 25% of the time. If that happens, since users could not
interfere with system automation results, they needed to manu
ally fnd the widget and access the information in it similar to the
Baseline condition, which posed a high cost on the users to correct
the prediction errors. While we understand that 25% error rate is
relatively high for regular well-trained machine learning classi
fers, the value is close to the accuracy levels of state-of-the-art
predictive systems that predict user’s interaction intent in order
to provide the relevant apps, tools, and information at the right
time [11, 32, 46, 59, 66], which is the use case we are targeting by 4.3.3 Tasks. A within-subject design was used for the study, in
which interface condition was the only independent variable. Latin-
square counterbalancing was applied to the order of conditions. In
the task, participants were instructed to imagine that they were the
owner of the virtual home. They wanted to interact with various
objects in the three diferent rooms, for example, use the stove
in the kitchen, use the laptop in the home ofce, or turn on the
TV in the living room. 4.3 Study Design We conducted a within-subject user study to experience the above
four conditions of UI transition mechanisms in VR. Due to the
limitation of not being able to run in-person studies during the
COVID-19 pandemic, we simulated the AR interfaces in a VR envi
ronment so that we could recruit from a larger pool of VR headset
owners who have access to consumer VR hardware. The study was
conducted remotely and unsupervised. 4.2.3 Fully-Automated Placements (Fully-Auto). The third interface
solution was similar to the Semi-Auto condition, but with a higher
automation level and lower controllability level, in the sense that
the user could not interfere with the system automation results. As such, a higher cost was introduced while the prediction was
wrong because users were not allowed to make any change to
system predictions. After predicting the widget that the user may
need for a new task, the system would automatically place the
top-recommended widget in front of the user without any input
from the users (see Figure 3 (e)). When an error happened in the 4.3.1 System. The experiment used a simulated AR setting im
plemented in a VR system, to avoid the limitations of current AR
devices (e.g., limited FoV, unstable wide-area multiple-room track
ing), to allow us to systematically control key features of the en
vironment and task, and to potentially recruit from a larger base
of participant pool online. This approach, known as Mixed Reality CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA Exploring Spatial UI Transition Mechanisms with Head-Worn Augmented Reality Figure 4: An illustration of the virtual home environment with three rooms: (a) the kitchen; (b) the home ofce; and (c) the
living room. Participants could touch the buttons with the blue outlines on the door to travel between the three rooms. Each
room has four usable objects, yielding a total of 12 objects in the virtual home (kitchen: stove, microwave, blender, fridge;
ofce: laptop, lamp, bookshelf, smartphone; living room: TV, plant, remote control, trash bin). Figure 4: An illustration of the virtual home environment with three rooms: (a) the kitchen; (b) the home ofce; and (c) the
living room. Participants could touch the buttons with the blue outlines on the door to travel between the three rooms. 4.3 Study Design Each
room has four usable objects, yielding a total of 12 objects in the virtual home (kitchen: stove, microwave, blender, fridge;
ofce: laptop, lamp, bookshelf, smartphone; living room: TV, plant, remote control, trash bin). Simulation, has been used in a variety of prior AR experiments
and was proven to be efective [6, 25, 38, 39]. The Oculus Quest 2
device was used for the implementation of the VR experience. The
device has 1832 × 1920 resolution per eye with 90 Hz refresh rate. The Oculus Touch controllers were used for interactions with the
widgets in the VR environment. The experimental software was
developed via Unity 2020.3.16f1 with the SDK provided by Oculus. answer the questions. As such, in a single trial, participants were
asked to go to a diferent room (see Figure 5 (a)), interact with a
virtual object by pointing the ray at the object and press the trigger
button (see Figure 5 (b)), and answer the questions prompted on
the object about information in a widget (see Figure 5 (c)). For the
Semi-Auto and the Fully-Auto conditions, the automation results
were dependent on the questions asked (i.e., which widget was
needed by the users in order to answer a question). A total of 12
trials were included for each interface. Participants were asked to
answer the questions as fast as possible while prioritizing accuracy. As such, our setup simulates a scenario in which users are in a
hurry and want to obtain the information they need quickly and
efciently. 4.3.2 Virtual Environment. In the task, participants were placed in
a virtual home environment with three rooms, the kitchen, the liv
ing room, and the home ofce (see Figure 4). Each room has a 2 by 2
meters walking area for participants to freely move around. In case
that participants do not have access to a large enough walking area,
we implemented a teleportation technique so participants could
teleport in the same room. To move between the three rooms, partic
ipants could move to the virtual door and touch the corresponding
button with the controllers (see Figure 4). The virtual scene would
then be switched to the new room. As such, we were able to reuse
the same walking space in the real world for interactions in difer
ent virtual rooms. In each room, there were four “usable” objects
(see Figure 4). 4.3 Study Design A total of twelve objects were scattered
in the home environment, four in each of the three rooms (see
Figure 4). They were asked to move between the three virtual rooms
in order to use these objects. After they interact with an object, they
suddenly wanted to check some information. For example, they
wanted to know the ingredients needed in the recipe after opening
the fridge, or know the next calendar event after turning on the
laptop. Similar to the prototype application in the design workshop,
eight widgets were integrated in the system. A multiple-choice
question popped up near the object simulating their thoughts of
mind, and they needed to check the information in the widgets to CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA Feiyu Lu and Yan Xu Figure 5: An illustration of a single task trial: (a) participants followed an instruction board and went to a diferent room to
use an object; (b) participants used the object by pointing the ray at the object and press the trigger button; and (c) a multiple-
choice question popped up on the object about a widget, and participant accessed information in that widget and answered
the question by selecting an option. Figure 5: An illustration of a single task trial: (a) participants followed an instruction board and went to a diferent room to
use an object; (b) participants used the object by pointing the ray at the object and press the trigger button; and (c) a multiple-
choice question popped up on the object about a widget, and participant accessed information in that widget and answered
the question by selecting an option. transitioning the right widget to the right place right when the
users need them. For example, Huang et al. proposed a system that
predicts which app the user will open on mobile phones based
on contextual information. The system prompted three apps, the
hit rate of which by the users fell between 67% to 79% maximum
[32]. Qu et al. compared diferent machine learning algorithms for
predicting user intent in information seeking with conversation
assistants, the accuracy levels of which lie between 63% to 69% [46]. Xia et al. proposed IntentCapsuleNet-ZSL, a zero-shot deep neural
network classifer for predicting everyday interaction intent such as
play music or get weather information, the accuracy levels of which
fell at 75.87% the lowest [66]. Chen et al. 4.4 Measures 4.4.1 Performance measures. For evaluating user performance on
the tasks, we calculated (1) the time of completion (how long did
it take for participants to fnish a task); (2) the distance travelled
(including distance teleported and distance walked); as well as (3)
the accuracy of the answers for each interface condition. 4.3.5 Procedure. The study was completely remote and unsuper
vised on the dscout platform3. The study includes six phases. In
the frst phase, a screener questionnaire was sent out to the dscout
platform. Participants were required to have access to the Oculus
Quest 2 hardware, internet connection, and at least a 5 by 5 feet
area to move around safely. Second, qualifed participants were
invited to the project on dscout, which granted them access to the
test software and the questionnaire. Participants were instructed
to complete a background questionnaire, and install the test soft
ware on their own Quest device. Third, participants opened the
test application. The application started with a tutorial about the
environment, controls, and tasks. Fourth, participants experienced
the four interface conditions one by one. Before the formal testing
session of each interface condition, a training session was provided
to participants in VR to teach them how to use the interface. After
they fnish the 12 trials for each interface, they were instructed to
take of the VR headset, go to their laptop and complete one page
of the questionnaire on dscout. The questionnaire asked about the
usability, workload, agency, as well as what they like and dislike 4.4.2 Subjective Measure. We used the Single Easement Question
naire (SEQ) [53], System Usability Scale (SUS) [5] and NASA TLX
workload questionnaire [31] to gauge the usability, efectiveness
and workload of each interface condition. We also asked partic
ipants to rate the level of agency on each interface using three
questions adapted from the work by Tapal et al. (see Figure 9 (b))
[58], and rank the interface based on their own preferences. 4.3 Study Design proposed a reinforcement
learning system for predicting user’s query intent in automated
customer service, in which the hit rate of the top three instance
in the recommended list reached 75.95% [11]. As such, 25% could
be an ideal simulated error rate value in order to make our results
relevant to the current technological contexts in predicting user’s
intent specifcally in everyday interactions such as information
seeking or opening an app. about the interface in the condition they just experienced. Fifth,
after participants fnished all the four interfaces, they clicked on
an upload button to upload the logged data to a cloud server. Last,
participants were instructed to rank the four interfaces based on
their own experience. They were also asked about how they felt
when the system suggested the wrong widgets in the tasks. The
study took about 80 minutes in total. Participants are compensated
with $70 US dollars for their time. To encourage participants to
achieve as good performance as they can, we rewarded half of the
participants who performed more accurately and faster than the
other half another $10 dollars. 4.3.6 Participants. Participants were recruited from the dscout
platform. The dataset includes 40 participants (23 M, 17 F) between
18 to 55 years old (M = 34.53, SD = 9.33). All participants had prior
experience with VR and were regular users of the Oculus Quest 2
device. 3https://dscout.com/ 4.5.1 Performance measures. In this subsection, results about the
performance measures are reported in detail. Figure 6 (b) shows the average time it took for participants to
answer the questions under four scenarios: when the system pre
diction was correct or wrong for Semi-Auto or Fully-Auto inter
faces. A Box-Cox transformation with λ = −0.6 was applied. RM
ANOVA yielded signifcant main efect of scenario on the average
time (F(3, 117) = 440.201, p < .001, η2 p = .919). Post-hoc pairwise
comparisons indicated that when a prediction error happened dur-
ing the Fully-Auto condition, participants spent signifcantly longer
time to answer the questions as compared to when error happened
in the Semi-Auto condition (p < .001), as well as when the predic
tions were correct in Semi-Auto (p < .001) and Fully-Auto (p < .001). No diference was found for Semi-Auto between when the system
suggested the correct and when the system suggested the wrong
AR content (
086). This result shows that in the Fully-Auto
p = . condition, users spent more time handling the prediction error, as
compared to the
condition. Semi-Auto Agency. Figure 9 (b) shows participants’ responses to the three
questions regarding agency (A.1: To what extent did you feel
the decision of where and when to place a widget was within
your hands; A.2: To what extent did you feel the widgets
were placed with your intent; A.3: I felt that I am responsible
for the speed and accuracy of completing the task). Friedman
test yielded signifcant main efects of interface on the ratings for
all the three questions (all p < .002). Participants found that the
decision of where and when to place the widgets was signifcantly
less in their hand for the Fully-Auto condition as compared to Base
line (Z = -4.800, p < .001, r = .537), Wristpack (Z = -4.214, p < .001, r
= .471), and Semi-Auto (Z = -3.846, p < .001, r = .429). When being
asked to what extent did they feel that the widgets were placed with
their intent, Fully-Auto was rated signifcantly lower as compared
to Baseline (Z = -3.153, p = .012, r = .352) and Semi-Auto (Z = -3.707,
p < .001, r = .414). 4.5.1 Performance measures. In this subsection, results about the
performance measures are reported in detail. SEQ. Figure 8 (b) shows participants’ response to the SEQ. Fried
man test yielded signifcant main efect of interface on the ratings
(χ2(3) = 22.610, p < .001). Wilcoxon signed-rank tests showed that
the Semi-Auto condition was rated signifcantly higher than the
Baseline condition (Z = -3.408, p = .003, r = .381). No signifcant
diferences were identifed between other pairs. Time. A Box-Cox transformation with λ = 0.3 was applied to
correct non-normal residuals. RM-ANOVA indicated signifcant
main efect on interface on the average time it took for participant
to answer each question (F (2.017, 78.680) = 44.556,p < .001, η2
.533). Post-hoc pairwise comparisons with Bonferroni adjustments
p =
indicated that Baseline yielded signifcantly more time to answer
the question as compared to the Wristpack (p < .001), Semi-Auto (p
< .001), and Fully-Auto (p < .001) conditions. Semi-Auto also took
signifcantly less time as compared to Wristpack (p < .001) and
Fully-Auto (p < .001) (see Figure 6 (a)). -3.484, p = .002, r = .390). Similarly, for U2, Semi-Auto was rated
signifcantly higher than Baseline (Z = -4.401, p < .001, r = .492) and
Usability. Figure 9 (a) shows participants’ responses towards
three questions in the SUS questionnaire. Friedman tests found
signifcant main efect of interface on all the three statements (U1:
I thought the interface was easy to use; U2: I would imag
ine that most people would learn to use the interface very
quickly; and U3: I felt very confdent using the interface) (all
p < .001). For U1, Semi-Auto was rated signifcantly higher than
Baseline (Z = -4.979, p < .001, r = .556) and Wristpack (Z = -4.861,
p < .001, r = .543). Fully-Auto was also rated signifcantly higher
than Baseline (Z = -3.953, p < .001, r = .442) and Wristpack (Z =
Wristpack (Z = -3.714, p < .001, r = .415). Fully-Auto was also rated
signifcantly higher than Baseline (Z = -3.399, p = .004, r = .496) and
Wristpack (Z = -2.855, p = .042, r = .319). For U3, Semi-Auto was
rated signifcantly higher than Baseline (Z = -4.679, p < .001, r =
.523), Wristpack (Z = -4.438, p = .001, r = .496), and Fully-Auto (Z =
-3.714, p = .024, r = .415). 4.5 Results We conducted a series of analyses to our results. Shapiro-Wilk
test indicated that both the time and distance were not normally
distributed for Wristpack, Semi-Auto and Fully-Auto. As such, we
applied Box-Cox transformations to correct non-normal residuals
[4, 7, 60, 69], followed by Repeated-Measure One-way ANOVA
(RM-ANOVA) tests to reveal the main efect of independent vari
ables. A Greenhouse-Geisser correction was applied for violations CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA Exploring Spatial UI Transition Mechanisms with Head-Worn Augmented Reality Baseline condition resulted in signifcantly lower accuracy as com
pared to Wristpack (p < .001), Semi-Auto (p = .001), and Fully-Auto
(p = .002) conditions. of sphericity. For Likert measures, Friedman tests were applied with
Wilcoxon signed-rank test as post-hoc pairwise analysis. The Pear
son’s correlation coefcient r was reported as a measure of efect
size [9, 49]. According to Cohen’s measure [16, 17], 0.1 ≤ r < 0.3,
0.3 ≤ r < 0.5, and r ≥ 0.5 would be considered as small, medium
and large efects respectively. Bonferroni correction was applied to
all pairwise comparisons. We used an α level of 0.05 in all signif
cance tests. In the result fgures, pairs that are signifcantly diferent
are marked with * when p ≤ .05, ** when p ≤ .01 and *** when
p ≤ .001. 4.5.2 Subjective measures. In this subsection, results about the
subjective measures are shown in detail. User preference. Figure 8 (a) shows the distributions of the sub
jective rankings. 27 our of 40 (72.5%) participants ranked Baseline as
the least favored interface. 31 participants (77.5%) ranked Semi-Auto
as the most favored interface. 21 participants (52.5%) ranked the
Fully-Auto condition as the second most favored interface. 4.5.1 Performance measures. In this subsection, results about the
performance measures are reported in detail. 4.5.1 Performance measures. In this subsection, results about the
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answers for each of the interface conditions. We processed the
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CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA
Feiyu Lu and Yan Xu
Figure 6: (a) The average time it took for participants to answer each question (in seconds); (b) the average time took for
participants to answer the questions when failure happened / did not happen in the prediction for Semi-Auto and Fully-Auto
interfaces (in seconds) (±S.E.). CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA Feiyu Lu and Yan Xu .... "'
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Figure 8: (a) The ranking distributions for each interface; (b) the average ratings of the SEQ questionnaire (±S.E.). • e ranking distributions for each interface; (b) the average ratings of the SEQ questionnaire (±S.E.). Workload. Figure 10 shows the NASA TLX ratings for fve cate-
gories. Pairwise comparisons showed that the Baseline condition
yielded a signifcantly higher level of mental workload and efort
than Wristpack. It also resulted in a signifcantly higher level of
mental, physical, efort, and frustration as compared to Semi-Auto
and Fully-Auto. The Semi-Auto condition resulted in a signifcantly
lower level of mental, physical, efort and frustration as compared
to Wristpack. Meanwhile, it also yielded a lower level of efort and
frustration than the Fully-Auto condition. Workload. Figure 10 shows the NASA TLX ratings for fve cate-
gories. Pairwise comparisons showed that the Baseline condition
yielded a signifcantly higher level of mental workload and efort
than Wristpack. It also resulted in a signifcantly higher level of
mental, physical, efort, and frustration as compared to Semi-Auto
and Fully-Auto. The Semi-Auto condition resulted in a signifcantly
lower level of mental, physical, efort and frustration as compared
to Wristpack. Meanwhile, it also yielded a lower level of efort and
frustration than the Fully-Auto condition. 4.5.3 Qalitative feedback. To understand why participants liked
or disliked the interfaces, we collected qualitative feedback by ask-
ing participants to comment on what they like and dislike about
each interface. Below we highlight most commonly appeared com-
ments by participants. Baseline. Participants liked the “intuitiveness” and “the sense of
being in control;” they disliked that it was “sometimes cumbersome
and slow,” as shown in the quotes like, “I always have to travel to Ill
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tasks that require users to move in spaces. These mechanisms in
clude: Wristpack (users carry the widgets on the wrist when moving
from one space to another), Semi-Auto (the system predicts three
widgets that may be needed for the task, and the user makes the
fnal choice, in which the cost of automation error is low), Fully-
Auto (the system predicts what widgets may be needed for the task
and presents the top one directly to the user, in which the cost of
automation error is high) and the Baseline conditions (the widgets
need to be manually moved or tethered by the user). We found: • The Semi-Auto condition performed the best both objectively
(time of completion and traveled distance) and subjectively
(user preference, workload, usability, and agency) among all
four conditions; Figure 10: The average workload ratings from NASA-TLX
subcategories (±S.E.). get the widget I need,” and “I need to recall where I placed the widgets
beforehand to fnd them.” • The Baseline condition performed the worst among all con
ditions; • The participants felt signifcantly less agency during Fully-
Auto condition than the more manual conditions (i.e., Base
line and Wristpack). In contrast, participants felt an equal
or even higher level of agency on Semi-Auto condition as
compared to the manual conditions. Wristpack. Participants liked the “convenience,” “similarity to
smart watch,” and “no need to think about carrying the widgets with
them;” they disliked that they “had to search for the correct widget
on their wrist to open.” Semi-Auto. Participants liked the “ease of use, accuracy.” More
over, they liked “the sense of being in control,” as witnessed in quotes
like “I really liked that I was completely in control of the widgets that
I wanted to see up here,” and “it still gives you the convenience of
having things pop up, but then you can control and make sure you’re
getting the correct one.” They also liked they were able “to fnd the
right widget in the recommended list, even when it was not the top
one.” They disliked that they “can’t easily select another widget if
they accidentally picked the wrong widget.” • When a prediction error happened, users spent a lot shorter
time in handling the error in the Semi-Auto condition than
in the Fully-Auto condition. 4.5.1 Performance measures. In this subsection, results about the
performance measures are reported in detail. 2.00
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Exploring Spatial UI Transition Mechanisms with Head-Worn Augmented Reality
CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA
Figure 9: (a) The average ratings for the three questions from the SUS questionnaire; (b) the average ratings to three questions
about agency for each interface condition (±S.E.). Exploring Spatial UI Transition Mechanisms with Head-Worn Augmented Reality CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA ■
Baseline DWristpack
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Figure 10: The average workload ratings from NASA-TLX
subcategories (±S.E.). 4.6 Summary of Findings • From the qualitative feedback, we found that users consid
ered the sense of control and ease of recovering from error
(could be from user error or system’s prediction error) as the
key factors when deciding their preference. 4.7 Discussion & Design Implications Fully-Auto. Participants liked the “the prediction was correct most
of the time” and “it was absolutely awesome when it worked;” they
disliked “not being able to correct the widget when failure happens”
and “high efort to correct the error when it happens by manually
fnding the widget they need.” 4.7.1 The need for ultra-low-friction interfaces on-the-go. Our re
sults provided strong evidence that the current mechanism (i.e.,
manual movement of the AR widgets) was not optimal for tran
sitioning widget UIs spatially. It was the least preferred interface
for most participants, resulted in a lower level of accuracy and CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA Feiyu Lu and Yan Xu the users, but could happen falsely and change the typed words to
undesired phrases (i.e., auto-correction), the structure of which is
similar to our Fully-Auto condition in terms of system-level control. Previous work has found that word-prediction could reduce the
required keystrokes by giving users both automation and control
over world selections [24]. However, it might also introduce extra
interaction and perception costs by requiring users to pay continu
ous attention to the list of suggested words [47, 48]. In our work,
we successfully indicated the importance of both controllability
and automation by demonstrating the advantages of the Semi-Auto
condition. However, the fndings of our work were restrained to
interface placements in AR in a hurried scenario with 25% errors. A higher level of controllability may introduce extra interaction
and perception costs, which could outweigh the benefts brought
by the automation. In diferent task contexts or systems, the degree
level of user control and system automation needs to be carefully
considered and balanced to achieve the optimal user experience. efciency, and posed high workload on the users. The major reason
was that in Baseline, users had to remember which information
was needed, recall where the widgets were located in the previous
environment, and manually acquire them in order to answer each
question. The heavy mental and physical workload made it chal
lenging for the users to obtain the correct answers. The Wristpack
interface ofoaded part of the workload by carrying the widgets
automatically on user’s wrist. However, it was still not optimal in
that the users need to manually locate the widget on their wrist,
open it, and place it in the new location. 4.7 Discussion & Design Implications Controllability also plays a critical role in user agency. In our study, the Fully-Auto condition had lower agency ratings
than the more manual conditions (i.e., Baseline and Wristpack). But
we also found that participants felt an equal or even higher level of
agency on Semi-Auto condition as compared to the more manual
conditions. This result, combined with users’ positive comments
around controllability, indicates that giving users the control for
decision-making can keep the agency high while leveraging system
automations. 4.7.3 The cost of correcting prediction errors. Error has always
been one of the biggest concerns for intelligent systems [45], which
motivated us to study the user experience outcomes when an error
occurs. In both Semi-Auto and Fully-Auto conditions, the top rec
ommendation from the system was occasionally wrong. However,
Semi-Auto condition is diferent from Fully-Auto condition in two
ways, one is that the correct widget can be found among the other
recommended items, just in a lower order and a smaller size; and
the other is that users need to choose which widget to use from
the recommended list. As a result of the diference, users spent
signifcantly more time handling the prediction error during the
Fully-Auto condition than the Semi-Auto condition. y
While being asked about their feelings when the system sug
gested the wrong widget, participants gave very diferent responses
for the Semi-Auto and Fully-Auto conditions. For the Fully-Auto con
dition, most participants mentioned “Annoying” (65%) and “Frus
trated” (50%), as shown in comments like the following: “The un
certainty of knowing if it would be right or not was very annoying
and made me anxious”; “I was slightly annoyed because I have to
fnd the widget I truly need, which adds lots of unnecessary work”;
and “I would feel less frustrated if I could’ve grabbed the correct wid
get from a UI after failure happens.” In summary, a big source of
frustration came from the efort of correcting the error. On the
contrary, for Semi-Auto condition, the majority of the participants
did not fnd it bothering when the top-recommended widget was
not correct. They can easily fnd the correct widget from the rest
of recommended list. 4.7 Discussion & Design Implications For scenarios that the
users move around in diferent spaces to carry out diferent tasks,
users are already multitasking - they navigate the space, look for
diferent physical objects, and sometimes handle social encounters. When users need digital content in such scenarios, they have less
cognitive bandwidth to maneuver UI widgets, therefore needing
the ultra-low-friction interface mechanisms. In both our design
workshop and user study, we confrmed that this user need does
exist, calling for more solution explorations from the HCI research
and design community. Overall, our study confrms that automation is a promising de
sign direction that can greatly reduce users’ efort and attention
cost on-the-go. Controllability is especially critical to ensure higher
user agency when the system provides automation functions. More
over, we call out for further explorations about how to combine
automation with controllability. The tasks in our study were easy
to combine both because the system can wait on the user to make
the choices. What if the user choices are more time-sensitive (e.g. decisions when driving), how do we balance the automation and
controllability in such tasks? 4.7.2 Automation, controllability and agency. One of the motiva
tions of our work is to explore how automation and controllability
levels can make a diference in addressing the dynamic UI needs
on-the-go. We designed the Wristpack, Semi-Auto and Fully-Auto in
terfaces to integrate diferent levels of automation and user control. In Wristpack condition, the previously opened widgets automati
cally minimize and attach to the wrist when major spatial difer
ences are detected. In Semi-Auto condition the system automatically
fnds a list of matching widgets for the task. In Fully-Auto condi
tion the system automatically places the best matching widgets
for the task. All three interfaces were able to reduce the workload
and increase the accessibility of the widgets. However, Semi-Auto
condition, where the system suggests a few widgets for the task
and the user makes the choice, did the best objectively and sub
jectively. Clearly, under situations where errors are inevitable and
happen 25% of the time, more automation does not necessarily lead
to better user experiences. Along the same line with the previous
work, we confrm the importance of controllability, which is how
much the user is in control of an automated task [51, 56]. More
over, our results confrmed that a combination of automation and
controllability creates the best user experience outcomes for tasks
on-the-go. 4.7 Discussion & Design Implications They commented “sometimes the widget (I
need) was not at the top, which is totally fne cause I could still fnd
it in the list;” “even though I had to click something other than the We would also like to highlight the relevance of our fndings
with previous work in predicting typing intent during text entry. While entering texts, keyboards with predictive features could (1)
recommend a list of words (usually 2-3 words on mobile interfaces)
based on what is already typed (i.e., word-prediction), the structure
of which is similar to our Semi-Auto interface; and (2) automatically
correct the typed word to another word without any input from CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA Exploring Spatial UI Transition Mechanisms with Head-Worn Augmented Reality fully automated one, I could always check and get the right widget.”
Needing to select the widget actually gave users a good opportunity
for double-checking and recognizing the recommendation error. Interestingly, if the user accidentally selected the wrong widget
(i.e. user slip), they also complained about the efort they had to
take to correct it, not too dissimilar to the comments about the
efort required for correcting the system error during Fully-Auto
condition. research community about the importance of studying error from
prediction algorithms with more depth and nuance. To conclude, for the design space of AR UI transitions on-to
go, our results show that user experiences could beneft from in
troducing automation, such as detecting contextual changes and
predicting the user intent. At the same time, we need to creatively
combine automation with controllability to ensure high agency and
overall satisfaction. Moreover, we should always provide an easy
way for the users and the system to recognize and recover from the
always-gonna-be-there prediction errors. Our fndings were established on an accuracy level of 75% in
a demanding scenario when accuracy was prioritized. While it
is true that the users might be more tolerant of having a higher
error-recovery cost when errors happen less frequently or when
users are in a lightweight scenario, our results demonstrated that
in worst-case scenarios where the errors happen inevitably and
users are in a hurry during AR UI transitions, the cost of correcting
them could play a crucial role in performance and user experience. In recent work, Lafreniere et al. 5 LIMITATION & FUTURE WORK There are several limitations of our work. First of all, our study was
conducted in VR due to COVID-19 restrictions and the limitations
of current AR devices. Future work could evaluate the interface
conditions in AR with real-world environments and tasks. Second,
to ensure a safe walking environment and overcome space limita
tions, we implemented teleportation for locomotion in the virtual
environment in the remote study. Based on recent research, tele
portation may hinder spatial cognition performances as compared
to real walking [13, 33]. Future research could consider involving
real walking of the participants to compare these interfaces. Third,
our task setup simulated a scenario that encouraged efciency. The
users were incentivized to access the widgets they needed as fast
and accurately as possible. However, in everyday AR scenarios,
users may access AR content at their own pace. Perhaps there will
be more need for UIs that suggest non-utilitarian widgets. Future
work could capture and design these AR use cases, and situate the
UI mechanisms with more diverse scenarios. Fourth, we adopted
a 75% accuracy level for the Semi-Auto and Fully-Auto conditions. Future research could explore how diferent accuracy levels could
afect the user behavior and user experience of using these inter
faces, and the design choices to be made. Fifth, our fndings indicate
that a Semi-Auto interface with high controllability and low error-
recovery cost would likely be more favored in a hurried scenario. In future work, we plan to explore the benefts and drawbacks of
having such interfaces. Last, we are interested in designing and
researching lightweight/low-efort methods to recover from predic
tion errors, without compromising usability and agency. The error recognition and recovery could be achieved through a
human-system team efort. For example, on the user side, the users
could learn from the prediction errors about the limitations of the
systems, thus becoming more prepared to correct the automation
errors quickly. One limitation of this approach, as indicated by
previous work in text-entry, would be that the users’ abilities of
identifying and adapting to errors vary among individuals and are
largely afected by how long they have been using the system and
how frequently the errors occur [2, 10]. A more reliable way would
be from the system side, in which the system could incorporate
functions to involve users in the loop to help it identify and learn
from the prediction errors [65, 70]. 4.7 Discussion & Design Implications proved that the temporal cost of
recovering automation errors could signifcantly afect user frus
tration and experience [36]. Similarly, in previous work about the
auto-correction feature in text-entry, it was found that when users
have to manually correct the system’s faulty auto-corrections, the
cost of it could outweigh the reduced efort when the corrections
were desired [1]. How much efort is needed for error recovery (in
cluding both system-generated prediction error and user error) is
crucial to the user experience. The usability heuristics about “help
ing users recognize, diagnose, and recover from errors [44]” needed
to be expanded and emphasized for today’s automated/intelligence
systems. Errors are not edge cases anymore, it always happens with
the probabilistic output of AI systems. We need to “always enable
an easy path to recognize and recover from error.” 5 LIMITATION & FUTURE WORK Moreover, the system could
even auto-detect its error based on the confdence level and users’
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Interaction 24, 6 (2008), 574–594. 6 CONCLUSIONS In this research, we aimed to support the UI transition needs when
people use AR interfaces on-the-go. We conducted an AR design
workshop to reveal the existing challenges when accessing AR
content across multiple spaces. We designed three interfaces to
address these challenges with diferent levels of automation and
user control. In a VR-simulated AR user study, we found that the
semi-automated condition stood out as the best performing and
most favored one. Thanks to the balance between automation and
controllability in the semi-automated condition, user agency was
not compromised when the automation level increased as com
pared to the manual conditions. Moreover, our study indicated the
importance of error recovery cost when an error happens in pre
dicting the exact AR content that users may need. We would like
to further explore ways to fail gracefully with backup plans for 4.7.4 The stakes of error occurrences. We would also like to point
out that what is at stake when errors happen could largely afect
user behaviors of using automated interfaces. Although the defni
tion of automation level in our use case is similar to the automated
driving use case, there are two major diferences: (1) the conse
quence of an error is much less severe (lower cost of error); and (2)
it is much easier to recover from the error, as the user can always
fnd the widgets manually when the system’s prediction was wrong. Our application scenario is more general-purpose and focuses on
the use of AR widgets on-the-go. We do not consider our learnings
here to be directly applicable to a scenario that has much higher
stakes for prediction errors. Through this work, we call out to the CHI ’22, April 29-May 5, 2022, New Orleans, LA, USA Feiyu Lu and Yan Xu Feiyu Lu and Yan Xu automated interfaces that rely on predictions. The fndings and de
sign insights from this work can provide valuable lessons to design
ultra-low-friction AR interfaces with automation, controllability,
and low error-recovery cost, especially for scenarios where users
have limited attention bandwidth. 21, 2, Article 10 (feb 2014), 46 pages. https://doi.org/10.1145/2552 [15] Karen Church and Barry Smyth. 2008. Understanding Mobile Information Needs. In Proceedings of the 10th International Conference on Human Computer Interaction
with Mobile Devices and Services (Amsterdam, The Netherlands) (MobileHCI ’08). Association for Computing Machinery, New York, NY, USA, 493–494. https:
//doi.org/10.1145/1409240.1409325 [16] Jacob Cohen. 1988. ACKNOWLEDGMENTS g (
)
p
g
[17] Jacob Cohen. 1992. A power primer. Psychological bulletin 112, 1 (1992), 155. https://doi.org/10.1037/0033-2909.112.1.155 We would like to thank Sophie Kim, Hiroshi Horii, Mark Parent,
Peiqi Tang, Missie Smith, Michael Shvartsman, Nicci Yin, Ryan
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Molecular cloning and functional characterisation of an H+-pyrophosphatase from Iris lactea
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Scientific reports
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cc-by
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Molecular cloning and functional
characterisation of an H+-
pyrophosphatase from Iris lactea
Lin Meng, Shanshan Li, Jingya Guo, Qiang Guo, Peichun Mao & Xiaoxia Tian Received: 16 November 2016
Accepted: 5 December 2017
Published: xx xx xxxx Tonoplast H+-pyrophosphatases (VPs) mediate vacuolar Na+ sequestration, a process important
for salt tolerance of plants. The function of VP in the highly drought- and salt-tolerant perennial Iris
lactea under salt stress is unclear. Here, we isolated IlVP from I. lactea and investigated its function in
transgenic tobacco. IlVP was found to comprise 771 amino acid residues and showed 88% similarity
with Arabidopsis AtVP1. IlVP was mainly expressed in shoots and was up-regulated by salt stress. Overexpression of IlVP enhanced growth of transgenic tobacco plants compared with wild-type (WT)
plants exposed to salt stress. Transgenic plants accumulated higher quantities of Na+ and K+ in leaves,
stems, and roots under salt stress, which caused higher leaf relative water content and decreased cell
membrane damage compared with WT plants. Overall, IlVP encoding a tonoplast H+-pyrophosphatase
can reduce Na+ toxicity in plant cells through increased sequestration of ions into vacuoles by enhanced
H+-pyrophosphatase activity. Plant physiological drought leads to ionic imbalance in cells, depressed functioning of cell membranes and met-
abolic activity, and even cell death owing to excessive soil Na+ concentrations1. To cope with salinity stress, strat-
egies adopted by plants cells to Na+ compartmentalisation into vacuoles alleviated the cellular Na+ toxicity to
maintain osmotic balance using Na+ as a osmoregulation substance, thus to improve salt tolerance of plant2. Previous studies suggested that tonoplast Na+/H+ antiporters (NHXs) could mediate Na+ compartmentation
into vacuolar3. The process is driven by electrochemical gradient of protons across tonoplast generated by the
H+- ATPase and H+-pyrophosphatase (H+-PPase) in tonoplast4,5. It has been suggested that H+-PPase plays an
important role in salt tolerance via the establishment of a transmembrane electrochemical gradient6,7. First cloned
from Arabidopsis thaliana, H+-PPase genes have subsequently been cloned from other plants, such as Hordeum
vulgare8, Beta valgaris9, Pyrus serotina10, Triticum aestivum11, Thellungiella halophila12, and Haloxylon ammo-
dendron13. H+-PPase activity and transcript levels can vary among different plant species, organ types, growth
stages, and Na+ concentrations in nutrient solutions14. In Daucus carota15, Helianthus annuus16, Suaeda salsa17,
and Thellungiella halophila18 subjected to different NaCl concentrations, tonoplast H+-PPase activity was higher
in treated plants than in the control, further demonstrating that NaCl may induce an increase in H+-PPase
activity. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Beijing Research and Development Center for Grass and Environment, Beijing Academy of Agriculture and Forestry
Sciences, Beijing, 100097, P. R. China. Correspondence and requests for materials should be addressed to L.M.
(email: menglin9599@sina.com) Received: 16 November 2016
Accepted: 5 December 2017
Published: xx xx xxxx Results
l
i These findings suggest that IlVP expression is induced by salt. Compartmentalisation of cytoplasmic Na+ into the vacuole would help reduce salt-induced cell damage. Production and molecular characterisation of tobacco plants over-expressing IlVP. To inves-
tigate the potential benefit of transferring IlVP into other plant species, we identified eight independent IlVP
transgenic tobacco lines by PCR amplification (data not shown). To further examine IlVP transcript levels in
transgenic tobacco, we used Northern hybridisation to analyse IlVP expression in young leaves of all PCR-positive
lines and WT plants. We observed that the relatively lower expression levels in L4 and the highest expression lev-
els in L18, but had not detected in the WT (Supplementary Figure 2a). We therefore used lines 4 and 18 in the
following assay. Subsequently, we randomly selected four transgenic tobacco lines and determined whether IlVP was intro-
duced into the tobacco genome using Southern hybridisation analysis. As shown in Supplementary Figure 2b,
only one band was visible in transgenic tobacco lines 4, 6, 14, and 18 after the hybridisation, whereas line 10
yielded two copies (Supplementary Figure 2b). The Southern blot analysis thus confirmed the integration and
expression of IlVP in tobacco. In addition, membrane proteins of isolation increased markedly the IlVP protein in T4 and T18 by Western
blot analysis compared to WT, and the protein level in T18 was higher than in T4 (Supplementary Figure 2c),
expecting that heterologous expression of IlVP could enhance salt tolerance in transgenic tobacco. Effect of NaCl stress on transgenic tobacco growth. The dry weights of both WT and transgenic
tobacco plants decreased gradually with increasing NaCl concentration. The weights of the transgenic tobacco
plants declined more slowly, and the dry weights were higher in the transgenic tobacco plants than the WT
plants (Fig. 3a). In particular, the dry weights of the roots, stems, and leaves were 2.0-, 1.1-, and 2.6-fold higher
in T4 and 2.7-, 1.9-, and 3.1-fold higher in T18, respectively, under 200 mM NaCl compared with the WT plants
(Fig. 3b,c,d). The growth of the transgenic plants was thus significantly better under salt stress compared with the
WT plants, and this growth increased as the relative expression of IlVP increased. Effect of NaCl stress on leaf relative water content and plasmalemma permeability. The rela-
tive water content is an important physiological indicator of plant growth status. Results
l
i Isolation and characterisation of IlVP. An 893-bp fragment was first obtained by RT-PCR using degen-
erate primers P1 and P2 (Supplementary Figure 1). A nucleotide BLAST search revealed that the isolated cDNA
fragment shared high sequence homology with many known VPs from other plants (e.g. Oryza sativa), indicat-
ing that a partial potential VP had been isolated from I. lactea. Sequences of the 5′ and 3′ ends were obtained by
rapid amplification of cDNA ends (RACE), which yielded products of 1,117 bp and 881 bp, respectively. The open
reading frame (ORF) of IlVP was 2,316 bp long and encoded a polypeptide protein consisting of 771 amino acid
residues (Supplementary Figure 1). The predicted protein had an isoelectric point of 5.16 and a molecular weight
of 80.7 kDa. The cDNA sequence of IlVP was submitted to GenBank under accession number KY406740. h
q
Analysis using the TMpred tool indicated that IlVP contained 14 transmembrane regions (Fig. 1a). Both
of N- and C-terminus were located in vacuole. Multiple sequence alignment showed that the PPi binding site
sequences were GGG, DVGADLVGK, and DNVGDNVGD, all located in the loop sequence connecting domains
TM5 and TM6 in the cytoplasm. The core VP sequence, essential for implementation of proton transfer functions,
was highly conserved and corresponded to that of PdVP, OsVP1, and AtVP. Alignment of H+-PPase amino acid
sequences of I. lactea and other plant species showed that IlVP was 96%, 93%, and 88% similar to PdVP, OsVP,
and AtVP, respectively (Fig. 1b). Phylogenetic analysis indicated that IlVP was most closely related to PdVP and
MaVP, and only distantly related to GmVP (Fig. 1b). Consequently, IlVP may have the same function as other
plant vacuolar membrane H+-PPases such as AtVP and may play an important role in drought resistance and salt
tolerance. Expression pattern analysis of IlVP. To investigate the tissue-specific expression of IlVP, plants were
subjected to 200 mM NaCl for 24 h. In the absence of NaCl, IlVP was constitutively expressed in roots and
shoots (Fig. 2a). In the presence of 200 mM NaCl, IlVP transcripts were detected in both organs, with level 7.6
times higher in shoots than in roots (Fig. 2a). Subsequently, I. lactea plants were treated with 0, 25, 50, 100, or
200 mM NaCl for 0, 6, 12, 24, and 48 h. IlVP expression levels in shoots increased significantly as salt concentra-
tion and stress duration were increased (Fig. 2b). Molecular cloning and functional
characterisation of an H+-
pyrophosphatase from Iris lactea
Lin Meng, Shanshan Li, Jingya Guo, Qiang Guo, Peichun Mao & Xiaoxia Tian In contrast, however, Matsumoto and Chung19 reported that H+-PPase activity in Hordeum vulgare
roots treated with 200 mM NaCl was half that of the control, and similar results were obtained in a study of
Mesembryanthemum crystallinum treated with 400 mM NaCl20. These suggest that overexpressing the H+-PPase
resulted in enhanced resistance to salt in various transgenic plants linked with the increased Na+ compartmen-
tation into the vacuoles. Iris lactea Pall. var. chinensis (Fisch.) Koidz., a wild perennial monocotyledonous halophyte, is widely dis-
tributed in desert steppe and saline lowland meadows in northern China, Siberian regions, eastern Russia, and
Mongolia21. Moreover, this species has attractive leaves and flowers, a wide range abundant seeds, stronger salt
and drought tolerance, higher pest and disease resistance, and easy cultivation, which has become a popular
groundcover plant for landscape design and park greenspace construction in northern China because of its orna-
mental foliage and flower22,23. Our previous research showed that the salt sensitive BJCY-ML035 in meadow
grassland (37°31′12′′ N, 112°19′00′′ E; altitude 760 m) and the salt tolerant BJCY-ML007 in saline lowland
meadow (43°45′15′′ N, 83°10′30′′ E; altitude 1,071 m) were screeed out from the sixteen accessions of I. lactea in
northern China by the comprehensive assessment of salinity soils24. Further research suggested that the specific Beijing Research and Development Center for Grass and Environment, Beijing Academy of Agriculture and Forestry
Sciences, Beijing, 100097, P. R. China. Correspondence and requests for materials should be addressed to L.M. (email: menglin9599@sina.com) SCiEntiFiC REPOrTS | (2017) 7:17779 | DOI:10.1038/s41598-017-18032-3 1 www.nature.com/scientificreports/ locus ISSR841-220 associated with the VP gene was found in the BJCY-ML007 compared with BJCY-ML03525. However, the role of IlVP in the salt tolerance of I. lactea is still unclear. To test whether the overexpression of IlVP confers improved salt tolerance in plant, we introduced the gene
into tobacco to measure and analyse the growth performance and Na+, K+ concentrations in the transgenic
tobacco plants and in wild-type (WT) plants subjected to salinity stress. The results indicate that IlVP-mediated
compartmentalisation of Na+ into vacuoles may play a key role in salt tolerance of plant. This would provide a
potential benefits for generating engineered plants to increased tolerance to salinity conditions. Results
l
i The plant water content can be
divided into free and bound water, with the majority present as free water. The free water ratio can reflect plant
salt resistance in response to the metabolic situation. With increasing salt concentration, the leaf relative water
content distinctly decreased in both the WT and transgenic tobacco plants compared with the control, but that
of the transgenic tobacco plants declined more slowly and remained higher than that of the WT plants (Fig. 4). In response to treatment with 200 mM NaCl, the leaf relative water content of the transgenic tobacco plants was
1.05 times higher in T4 and 1.08 times higher in T18 compared with the WT (Fig. 4a). IlVP was therefore found
to enhance the salt resistance of the tobacco plants. The water-retention capacity of the transgenic tobacco plants
increased significantly under salt stress. SCiEntiFiC REPOrTS | (2017) 7:17779 | DOI:10.1038/s41598-017-18032-3 2 www.nature.com/scientificreports/ Figure 1. (a) Alignment of amino acid sequences of H+-PPase genes from Iris lactea
from Phoenix dactylifera (PdVP), Oryza sativa (OsVP), and Arabidopsis thaliana (AtV
enclosed in red frames represent the PPi binding sites and activity domains of H+-PP
H+-PPase genes from Iris lactea and other plant species. Genes and GenBank accessi
AtVP (Arabidopsis thaliana, NM_101437), BdVP (Brachypodium distachyon, XM_00
rubrum, AF533336), CsVP (Citrus sinensis, XM_006474322), EgVP (Eucalyptus gran
(Glycine max, XM_003528254), HbVP (Hevea brasiliensis, AY514019), HvVP (Horde
IlVP (Iris lactea, KY406740), MaVP (Musa acuminata, XM_009386846), NtVP (Nico
XM_009630002), NnVP (Nelumbo nucifera, XM_010246610), OsVP (Oryza sativa, D
dactylifera, XM_008790581), PpVP (Prunus persica, AF367446), PtVP (Populus trich
(Ricinus communis, XM_002530709), SbVP (Sorghum bicolor, HM143921), SiVP (Se Figure 1. (a) Alignment of amino acid sequences of H+-PPase genes from Iris lactea var. chinensis (IlVP) with those
from Phoenix dactylifera (PdVP), Oryza sativa (OsVP), and Arabidopsis thaliana (AtVP). Amino acid sequences
enclosed in red frames represent the PPi binding sites and activity domains of H+-PPase. (b) Phylogenetic tree of
H+-PPase genes from Iris lactea and other plant species. Results
l
i Genes and GenBank accession numbers are as follows:
AtVP (Arabidopsis thaliana, NM_101437), BdVP (Brachypodium distachyon, XM_003564169), CrVP (Chenopodium
rubrum, AF533336), CsVP (Citrus sinensis, XM_006474322), EgVP (Eucalyptus grandis, XM_010035677), GmVP
(Glycine max, XM_003528254), HbVP (Hevea brasiliensis, AY514019), HvVP (Hordeum vulgare, AK360389),
IlVP (Iris lactea, KY406740), MaVP (Musa acuminata, XM_009386846), NtVP (Nicotiana tomentosiformis,
XM_009630002), NnVP (Nelumbo nucifera, XM_010246610), OsVP (Oryza sativa, D45383), PdVP (Phoenix
dactylifera, XM_008790581), PpVP (Prunus persica, AF367446), PtVP (Populus trichocarpa, XM_006381029), RcVP
(Ricinus communis, XM_002530709), SbVP (Sorghum bicolor, HM143921), SiVP (Setaria italic, XM_004964638),
SlVP (Solanum lycopersicum, NM_001278976), TcVP (Theobroma cacao, XM_007023235), VvVP (Vitis vinifera,
XM_002273171), and ZmVP (Zea mays, BT086232). Figure 1. (a) Alignment of amino acid sequences of H+-PPase genes from Iris lactea var. chinensis (IlVP) with those
from Phoenix dactylifera (PdVP), Oryza sativa (OsVP), and Arabidopsis thaliana (AtVP). Amino acid sequences
enclosed in red frames represent the PPi binding sites and activity domains of H+-PPase. (b) Phylogenetic tree of
H+-PPase genes from Iris lactea and other plant species. Genes and GenBank accession numbers are as follows:
AtVP (Arabidopsis thaliana, NM_101437), BdVP (Brachypodium distachyon, XM_003564169), CrVP (Chenopodium
rubrum, AF533336), CsVP (Citrus sinensis, XM_006474322), EgVP (Eucalyptus grandis, XM_010035677), GmVP
(Glycine max, XM_003528254), HbVP (Hevea brasiliensis, AY514019), HvVP (Hordeum vulgare, AK360389),
IlVP (Iris lactea, KY406740), MaVP (Musa acuminata, XM_009386846), NtVP (Nicotiana tomentosiformis,
XM_009630002), NnVP (Nelumbo nucifera, XM_010246610), OsVP (Oryza sativa, D45383), PdVP (Phoenix
dactylifera, XM_008790581), PpVP (Prunus persica, AF367446), PtVP (Populus trichocarpa, XM_006381029), RcVP
(Ricinus communis, XM_002530709), SbVP (Sorghum bicolor, HM143921), SiVP (Setaria italic, XM_004964638),
SlVP (Solanum lycopersicum, NM_001278976), TcVP (Theobroma cacao, XM_007023235), VvVP (Vitis vinifera,
XM_002273171), and ZmVP (Zea mays, BT086232). SCiEntiFiC REPOrTS | (2017) 7:17779 | DOI:10.1038/s41598-017-18032-3 3 www.nature.com/scientificreports/ Figure 2. Expression analysis of IlVP in shoots and roots of Iris lactea var. chinensis under different NaCl
treatments. The expression levels of IlVP in shoot and root under control (0 mM) and 200 mM were normalized
with that in shoot in control. (a) IlVP expression in roots and shoots under control and 200 mM NaCl treatment
for 24 h as indicated by quantitative real-time PCR (qRT-PCR); (b) IlVP expression in shoots after treatment
with different concentrations of NaCl (0, 25, 50, 100, and 200 mM) for 0, 6, 12, 24, and 48 h as indicated by qRT-
PCR. Each bar represents the mean (n = 3), and bars indicate the standard deviation (SD). Figure 2. Expression analysis of IlVP in shoots and roots of Iris lactea var. Results
l
i chinensis under different NaCl
treatments. The expression levels of IlVP in shoot and root under control (0 mM) and 200 mM were normalized
with that in shoot in control. (a) IlVP expression in roots and shoots under control and 200 mM NaCl treatment
for 24 h as indicated by quantitative real-time PCR (qRT-PCR); (b) IlVP expression in shoots after treatment
with different concentrations of NaCl (0, 25, 50, 100, and 200 mM) for 0, 6, 12, 24, and 48 h as indicated by qRT-
PCR. Each bar represents the mean (n = 3), and bars indicate the standard deviation (SD). Figure 3. (a) Growth of wild-type and IlVP-transgenic tobacco plants in response to 200 mM NaCl treatment
for 7 days. WT: wild type; T4, T18: transgenic tobacco. (b–d) Root, stem and leaf dry weight of wild-type and
IlVP-transgenic tobacco plants in response to salt stress, respectively. Each bar represents the mean (n = 7), and
error bars indicate the standard deviation (SD). Columns with different letters indicate a significant difference at
P < 0.05 (Duncan’s multiple range test). Figure 3. (a) Growth of wild-type and IlVP-transgenic tobacco plants in response to 200 mM NaCl treatment
for 7 days. WT: wild type; T4, T18: transgenic tobacco. (b–d) Root, stem and leaf dry weight of wild-type and
IlVP-transgenic tobacco plants in response to salt stress, respectively. Each bar represents the mean (n = 7), and
error bars indicate the standard deviation (SD). Columns with different letters indicate a significant difference at
P < 0.05 (Duncan’s multiple range test). Maintenance of the cell microenvironment and normal metabolism relies on plant cell membranes. The rel-
ative plasma membrane permeability of the WT and transgenic tobacco plants increased with NaCl treatment,
with a lower increase observed in the transgenic than the WT plants (Fig. 4b). For instance, the relative plasma SCiEntiFiC REPOrTS | (2017) 7:17779 | DOI:10.1038/s41598-017-18032-3 4 www.nature.com/scientificreports/ tificreports/
Figure 4. Leaf relative water content (a) and relative membrane permeability (b) of wild-type and IlVP-
transgenic tobacco plants in response to salt stress for 7 days. Each bar represents the mean (n = 7), and error
bars indicate the standard deviation (SD). Columns with different letters indicate a significant difference at
P < 0.05 (Duncan’s multiple range test). Figure 4. Results
l
i Leaf relative water content (a) and relative membrane permeability (b) of wild-type and IlVP-
transgenic tobacco plants in response to salt stress for 7 days. Each bar represents the mean (n = 7), and error
bars indicate the standard deviation (SD). Columns with different letters indicate a significant difference at
P < 0.05 (Duncan’s multiple range test). membrane permeability was 28.3% (T4) and 43.2% (T18) lower than that of the WT plants subjected to 200 mM
NaCl treatment for 7 days. Damage to the cell membranes of the transgenic plants under salt stress was thus less
severe than that of the WT plants, while the salt resistance of the transgenic plants was higher than that of the WT. membrane permeability was 28.3% (T4) and 43.2% (T18) lower than that of the WT plants subjected to 200 mM
NaCl treatment for 7 days. Damage to the cell membranes of the transgenic plants under salt stress was thus less
severe than that of the WT plants, while the salt resistance of the transgenic plants was higher than that of the WT. Effect of NaCl stress on Na+ and K+ concentrations. Concentrations of Na+ in the tissues (roots,
stems, and leaves) of the WT and transgenic plants (T4 and T18) increased with increasing NaCl concentrations;
considerably higher increases were observed in the transgenic compared with the WT plants. Under 200 mM
NaCl treatment for 7 days, the Na+ concentrations in the roots, stems, and leaves of T4 were 38.7%, 15.7% and
12.2% higher, and those of T18 were 188.0%, 29.5% and 33.5% higher, respectively, compared with the WT
(Fig. 5a,b,c). The K+ concentration in the tissues of transgenic plants was significantly higher than that in the WT
plants in the presence of 200 mM NaCl. Although the accumulation of K+ in T4, T18 and WT decreased with
external NaCl treatment, the tissues of transgenic plants retained more K+ (Fig. 5d,e,f). Under NaCl treatment,
the transgenic lines showed significantly higher concentrations of Na+ and K+ than the WT plants. SCiEntiFiC REPOrTS | (2017) 7:17779 | DOI:10.1038/s41598-017-18032-3 Discussion In a previous study, we determined that I. lactea has strong salt resistance24, and a rapid tissue culture propagation
system was subsequently established26. It is well known that tonoplast H+-PPase is involved in the sequestration
of Na+ into vacuoles, which contributes to salt tolerance of plants27.h In a previous study, we determined that I. lactea has strong salt resistance24, and a rapid tissue culture propagation
system was subsequently established26. It is well known that tonoplast H+-PPase is involved in the sequestration
of Na+ into vacuoles, which contributes to salt tolerance of plants27.h p
The tonoplast H+-PPase is encoded by a highly hydrophobic, single-subunit protein with a calculated molec-
ular mass of 80 kDa28. In higher plants, H+-PPase cDNA commonly contains a 2,283–2,319 bp ORF encoding
761–773 amino acid residues with a deduced calculated molecular mass of 79–81 kDa29–31. Our results showed
that IlVP consists of 2,316 bp, encodes a protein of 771 amino acids with a calculated molecular mass of 80.7 kDa,
and contains 14 trans-membrane domains. Two contrasting responses in tonoplast H+-PPase activity under salt
stress have been reported. Some researchers have reported that H+-PPase activity declines in response to salt
treatment19, whereas other studies have shown that NaCl may enhance H+-PPase activity15,16,18. H+-PPase hydro-
lytic activity in barley roots and leaves has been found to increase under different salt concentrations17. IlVP was
mainly expressed in shoots under the NaCl concentrations in our study, and transcript abundance of IlVP in
shoots increased gradually with increasing NaCl concentrations (50 and 200 mM). This response is conducive
for compartmentalisation of Na+ in the vacuole of the leaf cytoplasm, with a consequent reduction in the salt
damage caused to plants32. Overexpression of the H+-PPase gene may enhance trans-membrane electrochemical
gradients and improve secondary transport carrier efficiency across the vacuole membrane under salt stress33. A SCiEntiFiC REPOrTS | (2017) 7:17779 | DOI:10.1038/s41598-017-18032-3 5 www.nature.com/scientificreports/ Figure 5. Cation concentration in tissues of wild-type and IlVP-transgenic tobacco plants in response to salt
stress. Na+ (a–c) and K+ (d–f) concentrations were measured after treatment for 7 days with different NaCl
concentrations (0, 50, 100, and 200 mM). Each bar represents the mean (n = 7), and error bars indicate the
standard deviation (SD). Columns with different letters indicate a significant difference at P < 0.05 (Duncan’s
multiple range test). Figure 5. Cation concentration in tissues of wild-type and IlVP-transgenic tobacco plants in response to salt
stress. Discussion Na+ (a–c) and K+ (d–f) concentrations were measured after treatment for 7 days with different NaCl
concentrations (0, 50, 100, and 200 mM). Each bar represents the mean (n = 7), and error bars indicate the
standard deviation (SD). Columns with different letters indicate a significant difference at P < 0.05 (Duncan’s
multiple range test). variety of inorganic ions accumulate in the vacuoles to maintain the balance between ionic equilibrium, osmotic
equilibrium, and cell turgor-pressure stability; in this way, damage to cells by inorganic ions is reduced, and salt
or osmotic stress tolerance of cells is enhanced14. Thus, H+-PPase plays an important role as a proton pump in the
process of salt or osmotic stress resistance and adaptation. Plant salt resistance is enhanced by excessive expression of tonoplast H+-PPase. Overexpression of SaVP1 in
Arabidopsis enhances tolerance to drought and salt stresses; this overexpression also results in the up-regulation
of several K+ and Ca2+ channel/transporter genes that show a function similar to that of vacuolar H+-PPase
from other plants33. Overexpression of KfVP1 increases salt and drought tolerance of Arabidopsis31. The AVP1
protein content in AVP1-transgenic Arabidopsis seedlings was significantly higher than that of WT plants34. Recovery of salt resistance can be achieved by overexpression of Arabidopsis AVP1 in yeast salt-sensitive enal
mutants35. In one study, inorganic ion accumulation was higher in the roots and leaves of AVP1 genetically
modified alfalfa (Medicago sativa) than in WT plants, and the leaf osmotic potential of transgenic plants was
reduced. In addition, the salt and drought resistance of the transgenic plants was significantly enhanced36. PvVP1 has been transferred to non-halophytic grass, thus providing a feasible basis to improve the salt resist-
ance of Paspalum vaginatum37. Overexpression of the H+-PPase gene provides a stimulus for ion compartmen-
talisation, thereby maintaining the balance between ionic balance and osmotic equilibrium within the cell and
enhancing plant salt resistance10. In the present study, using the anti-AtVP found that exogenous IlVP protein
was largely expressed in transgenic plants by Western blots analysis compared to WT. This suggested the salt
tolerance of the IlVP transgenic tobacco plants was higher than that of the WT plants. Materials and Methods Material culture and main experimental reagents. Seeds of I. lactea Pall. var. chinensis (Fisch.) Koidz. were collected from the National Experiment Station of Precision Agriculture, Xiao Tang Shan, China, located
approximately 55 km from Beijing (39°34′ N, 116°28′ E). Plump seeds were sterilised with sodium hypochlorite
solution (5%) for 5 min, rinsed thoroughly with distilled water, incubated in 40 °C water for 56 h, and then germi-
nated on moistened filter paper for 10 days at 25 °C in the dark. After plumule emergence, uniform seedlings were
transferred to plastic containers (19 cm long, 13.5 cm wide and 7.5 cm high) containing modified Hoagland’s solu-
tion (2 mM KNO3, 1 mM NH4H2PO4, 0.5 mM Ca(NO3)2·4H2O, 0.5 mM MgSO4·7H2O, 60 µM Fe-citrate, 92 µM
H3BO3, 18 µM MnCl2·4H2O, 1.6 µM ZnSO4·7H2O, 0.6 µM CuSO4·5H2O, and 0.7 µM (NH4)6-Mo7O24·4H2O) for 5
weeks. The nutrient solution was renewed every 3 days. All seedlings were grown in the same chamber under a
day/night cycle of 16 h/8 h at 25 °C/18 °C, a relative humidity of 50%–60%, and 600 µmol m−2 s−1 photosyntheti-
cally active radiation. Cloning of IlVP. A pair of degenerate primers, P1 and P2, were designed based on VP gene sequences in
GenBank. Six-week-old seedlings of I. lactea were treated with 100 mM NaCl for 24 h. After treatment, fresh
roots (100 mg) were washed in sterile water and then ground in liquid nitrogen. Total RNA was extracted using a
Takara RNA extraction kit. After synthesis of cDNA using a First-Strand PrimeScript RTase cDNA synthesis kit,
reverse transcription and PCR amplification were conducted. The PCR protocol was as follows: 94 °C for 2 min,
followed by 30 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min, and a final step of 72 °C for 10 min. The
amplified fragment was sequenced and analysed using the BLAST tool (http://www.ncbi.nlm.nih.gov/BLAST). IlVP 5′- and 3′-ends were obtained using Clontech SMARTer RACE and Takara 3′-Full RACE kits in accord-
ance with the manufacturers’ instructions and the gene-specific primers P3 and P4, respectively (Supplementary
Table 1). The ORF of IlVP was amplified using a Tks Gflex DNA Polymerase PCR kit with primers P5 and P6
(Supplementary Table 1). DNA sequence and phylogenetic analyses. The IlVP sequence was analysed, and the coding regions
were predicted using DNAMAN 6.0 software. Conclusions
l
l
d IlVP was cloned from I. lactea. IlVP expression was observed mainly in shoots, and its transcript abundance
changed with increasing salt concentration and duration of exposure. IlVP was cloned from I. lactea. IlVP expression was observed mainly in shoots, and its transcript abundance
changed with increasing salt concentration and duration of exposure. Phenotypes, the leaf relative water content, relative plasma membrane permeability, and concentrations of
Na+ and K+ in roots, stems, and leaves were measured in response to treatment with different NaCl concentra-
tions for 7 days. The transgenic tobacco plants displayed enhanced tolerance to NaCl stress compared with the
WT plants. These results suggest that overexpression of IlVP in tobacco plants enhances sequestration of Na+ into
vacuoles to alleviate Na+ toxicity in the cytoplasm, further maintaining cellular K+ and Na+ homeostasis and cell
membrane stability, thereby enhancing tobacco salt tolerance. Discussion Compared with the WT
plants, the physiological indices of the transgenic tobacco plants showed greater stability and slower changes, SCiEntiFiC REPOrTS | (2017) 7:17779 | DOI:10.1038/s41598-017-18032-3 6 www.nature.com/scientificreports/ which indicated that the physiological system of the transgenic tobacco plants had not been severely damaged
under NaCl stress. In addition, overexpression of IlVP enhanced the accumulation of Na+ in tissues. On the one
hand, these differences might be ascribed to enhanced sequestration of Na+ into the vacuole and maintained
the balance between K+ and Na+, and increased the osmotic regulation ability, because of overexpressing of
H+-PPase38–40. On the other hand, potassium is required for plant growth, tropisms, cell expansion, enzyme
activity, ion homeostasis and stomatal movements41. Our study showed that the K+ concentrations in trans-
genic tobacco were higher than in the WT plants under salt stress. The increased accumulation of potassium
is likely to be overexpression of the H+-PPase resulted in the enhanced K+ uptake and the release of organic
acids, which contribute to an increased rhizosphere acidification and to enhanced phosphorus uptake, and thus
improved salt tolerance in transgenic plants42. Genetic transformation and identification of transgenic tobacco.
Chemically competent cells o
b
f
d
ft
f
h
l After prehybridisation for 2 h, a radiolabeled probe was added and hybridised overnight.it Probe Synthesis kit (Beijing Mylab Corporation). DNA samples (30 µg) were cut with DraI. The enzyme-digested
product was purified and electrophoresed on a 1% agarose gel at 20 V. A capillary siphon was used for transfer of
the purified product. After prehybridisation for 2 h, a radiolabeled probe was added and hybridised overnight. DIG-PCR amplification was used to label probes for the Northern blot assay. After carrying out 1.1% for-
maldehyde denaturing agarose gel electrophoresis for 3 h at 50 V, a capillary siphon was used for transfer of the
purified product. After prehybridisation for 2 h, the radiolabeled probe was added and hybridised overnight. Membrane washing and signal detection were conducted using a DIGD-210 Hybridization Detection II kit in
accordance with the manufacturer’s instructions. pi
pt
p
y
p
y
g
DIG-PCR amplification was used to label probes for the Northern blot assay. After carrying out 1.1% for-
maldehyde denaturing agarose gel electrophoresis for 3 h at 50 V, a capillary siphon was used for transfer of the
purified product. After prehybridisation for 2 h, the radiolabeled probe was added and hybridised overnight. Membrane washing and signal detection were conducted using a DIGD-210 Hybridization Detection II kit in
accordance with the manufacturer’s instructions. Assessment of salt tolerance of transgenic tobacco. Transgenic tobacco (T4 and T18) and WT
plants grown under identical growth conditions were irrigated for 7 days using Hoagland’s solution contain-
ing 0, 50, 100, or 200 mM NaCl. Seven biological replicates were conducted, with three seedlings of each strain
used per replicate. Root, stem, and leaf fresh weights, leaf relative water content, relative electrical conductivity,
and Na+ and K+ concentrations were measured. After fresh weight measurements, roots, stems, and leaves were
oven-dried at 80 °C to a constant weight, and the dry weight of each organ was recorded. The leaf relative water
content and relative electrical conductivity were measured using Gao’s method43. Na+ and K+ concentrations in
roots, stems, and leaves were measured using a flame emission spectrophotometer. Tonoplast vesicles isolation and Western blot. According to the method of Wang et al. (2000)44 with
minor modifications, tonoplast enriched membrane vesicles were isolated. Genetic transformation and identification of transgenic tobacco.
Chemically competent cells o
b
f
d
ft
f
h
l Genetic transformation and identification of transgenic tobacco. Chemically competent cells of
Agrobacterium tumefaciens strain EHA105 were prepared. After fusing the plant expression vector into EHA105
cells using the freeze–thaw method, tobacco strain ‘W38’ was transformed using the leaf-disc method. Tobacco
leaf strips that showed expansion after placement on Murashige and Skoog (MS) culture medium lacking kan-
amycin were transfected by Agrobacterium for 7 min, blotted with sterile filter paper to remove excess liquid,
and co-cultured for 2–3 days. The leaf strips were placed on kanamycin-containing differentiation medium (MS
medium supplemented with 1 mg L−1 6-benzylaminopurine, 0.1 mg L−1 naphthaleneacetic acid, 50 mg L−1 kana-
mycin, and 500 mg L−1 carbenicillin). Callus was visible after 4 weeks of culturing on this medium. When gener-
ated shoots were 1–3 cm tall, they were transplanted onto MS medium supplemented with 50 mg L−1 kanamycin
and 500 mg L−1 carbenicillin. Disinfected leaf strips were used as a control and were cultured on MS medium. When space was insufficient for growth, the generated shoots were transplanted into plastic culture pots con-
taining a mixture of vermiculite and perlite (v/v, 3:1) and grown under a 16 h/8 h (light/dark) photoperiod and
a light intensity of 600 µmol m−2 s−1 at 25 °C and 60% relative humidity. The plants were watered every 2 days
with Hoagland’s solution. Total genomic DNA was extracted from the leaves of regenerated and WT plants in
accordance with the Takara MiniBEST Plant Genomic DNA Extraction kit manual. PCR amplification was car-
ried out with a TksGflex DNA Polymerase kit following the manufacturer’s instructions using plasmid DNA as a
positive control, WT plant DNA as a negative control, and the following pair of specifically designed primers: F1
(5′-CATTGCTGGGATGGGTTC-3′) and R1 (5′-TCGTGGCTGCTCCTGTTC-3′). The PCR protocol consisted
of pre-denaturation at 94 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 30 s, annealing at 55 °C
for 15 s, and elongation at 68 °C for 1 min, followed by storage at 4 °C. Southern Northern blot assays. Preparation of the Southern blot probe was carried out using a PCR DIG
Probe Synthesis kit (Beijing Mylab Corporation). DNA samples (30 µg) were cut with DraI. The enzyme-digested
product was purified and electrophoresed on a 1% agarose gel at 20 V. A capillary siphon was used for transfer of
the purified product. Genetic transformation and identification of transgenic tobacco.
Chemically competent cells o
b
f
d
ft
f
h
l Briefly, about 100 mg leaves of WT and
transgenic tobacco (T4 and T18) were selected under 200 mM NaCl treatment for 10 days, which were homog-
enized in extraction medium (pH 7.8) containing 250 mM mannitol, 1 mM DTT, 3 mM EGTA, 1% (w/v) PVP,
0.25 mM PMSF, 100 mM Tricine, 3 mM MgSO4. The homogenate was filtered using four layers of cheesecloth that
was centrifuged at 12,000 × g for 15 min at 4 °C. Subsequently, these supernatant were centrifuged at 300,000 × g
for 45 min and was suspended in suspension buffer (pH 7.5) with 250 mM mannitol, 2 mM DTT, 3 mM EGTA,
10 mM Hepes. The microsomal membrane vesicle suspension was loaded on a 1%/18% (w/w) Dextran T70 gra-
dient in suspension buffer and then centrifuged at 100,000 × g for 2 h. The tonoplast-enriched membrane vesicle
fraction located at the 1%/8% (w/w) Dextran T70 interface was carefully collected, diluted 4–5 fold with dilution
buffer (pH 7.0) with 1 mM DTT, 3 mM MgSO4, 50 mM Hepes, 0.2 mM PMSF and then centrifuged at 300,000 × g
for 45 min. For western blot, 100 μg tonoplast proteins were separated using 12% SDS-PAGE (sodium dodecyl
sulfate polyacrylamide gel electrophoresis), and was immunoblotted with antibody against H+-PPiase (Agrisera,
Vännäs, SWEDEN). Blots were performed according to describe the methods of Sarafian et al. (1999)45 and
Venancio et al. (2014)46. Statistical analysis. Excel 2010 was used for data processing. SAS11.0 was used for variance and clustering
analyses. fl
(
),
(
)
3. Blumwald, E. & Poole, R. J. Na+/H+ antiporter in isolated tonoplast vesicles from storage tissue of Beta vulgaris. Plant Physiol. 78,
163–167 (1985). Materials and Methods IlVP sequence homology analysis and phylogenetic tree construc-
tion was carried out using MEGA 6.0 software. The isoelectric point and molecular mass were predicted using the
online Compute pI/Mw tool (http://web.expasy.org/compute_pi/). Expression analysis of IlVP. Six-week-old seedlings of I. lactea var. chinensis were treated with 200 mM
NaCl for 24 h. After washing the roots in sterile water, 200 mg of fresh roots and shoots were ground sepa-
rately in liquid nitrogen. Total RNA was extracted using a Takara RNA extraction kit, after which cDNA was
synthesised using a PrimeScript RT Reagent Kit with gDNA Eraser. In addition, 6-week-old seedlings of I. lactea were treated with 0, 25, 50, 100, and 200 mM NaCl for 0, 6, 12, 24, and 48 h; total RNA was extracted,
cDNA was synthesised as described above, and the expression of IlVP in shoots was analysed. Quantitative
real-time RT-PCR (qRT-PCR) was performed using SYBR Premix Ex Taq II (Perfect Real Time) (Takara) on a
StepOnePlus Real-Time PCR system (ABI) to monitor the amplification of each cDNA fragment. The qRT-PCR
amplification was performed using the IlVP-specific primer pair P7 and P8. Actin amplified with the primer pair
A1 and A2 was used as an internal reference in the qRT-PCR analysis. All experiments were carried out with
three biological replicates.hi g
p
The qRT-PCR protocol consisted of three steps: predenaturation at 95 °C for 30 s; PCR amplification for 40
cycles of 95 °C for 5 s and 60 °C for 1 min; and finally, dissociation, consisting of 95 °C for 10 s, 65 °C for 5 s, and
95 °C for 5 s. Construction of plant expression vectors. The plasmid containing the IlVP gene and the plasmid
pBI121 were cut with QuickCut SmaI and QuickCut ScaI, respectively, and incubated for 15 min at 30 °C. The two fragments were ligated in accordance with the DNA Ligation Kit Version 2.1 manual. A plant expres-
sion vector that included the CaMV 35S promoter, a NOS terminator and the IlVP gene was constructed. The SCiEntiFiC REPOrTS | (2017) 7:17779 | DOI:10.1038/s41598-017-18032-3 7 www.nature.com/scientificreports/ recombinant plasmid was transformed into Escherichia coli strain DH5α. The cells were screened for kanamycin
and rifampicin resistance. After purification of cells harbouring the recombinant plasmid, restriction enzyme
digestion was performed. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 4. Apse, M. P., Aharon, G. S., Snedden, W. A. & Blumwald, E. Salt tolerance conferred by overexpression of a vacuolar Na+/H+
antiporter in Arabidopsis. Science 285, 1256–1258 (1999).hf p
p
5. Wei, A., He, C., Li, B., Li, N. & Zhang, J. The pyramid of transgenes TsVP and BetA effectively enhances the drought tolerance o
maize plants. Plant Biotechnol. J. 9(2), 26–29 (2011). p
6. Britten, C. J., Zhen, R. C., Kim, E. J. & Rea, P. A. Reconstitution of transport function of vacuolar H+-translocating inorganic
pyrophosphatase. J. Biol. Chem. 267, 21850–21855 (1992). 6. Britten, C. J., Zhen, R. C., Kim, E. J. & Rea, P. A. Reconstitution of transport function of vacuolar H+-
pyrophosphatase. J. Biol. Chem. 267, 21850–21855 (1992). py
p
p
. Maeshima, M. Vacuolar H+-pyrophosphatase. Biochimica et Biop py
p
p
p y
8. Tanaka, Y., Chiba, K., Maeda, M. & Maeshima, M. Molecular cloning of cDNA for vacuolar membrane proton-translocating
inorganic pyrophosphatase in Hordeum vulgare. Biochem. Bioph. Res. Co. 190, 1110–1114 (1993). + g
py
p
p
g
p
9. Kim, Y., Kim, E. J. & Rea, P. A. Isolation and characterization of cDNAs encoding the vacuolar H+-pyrophosphatase of Beta vulgaris
Plant Physiol. 106, 375–352 (1994). y
0. Suzuki, Y. & Maeshima, M. Molecular cloning of vacuolar H+-pyrophosphatase and its expression during the development of pear
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xanthoxylum improves alfalfa plant growth under salinity, drought and field conditions. Plant Biotechnol. J. 14, 9 40. Bao, A. K. et al. Co-expression of tonoplast Cation/H+ antiporter and H+-pyrophosphatase from xerophyte Zygophyllum
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This research was financially supported by the Beijing Natural Science Foundation (grant no. 6142007) and
the Scientific Innovation Ability Construction Project (grant no. KJCX20140103) of the Beijing Academy of
Agriculture and Forestry Sciences. Author Contributions L.M. conceived and designed the experiments. S.L., J.G., Q.G., P.M. and X.T. prepared for sample and performed
the experiments. L.M. analysed the data and wrote the manuscript. www.nature.com/scientificreports/ Co expression of tonoplast Cation/H antiporter and H
pyrophosphatase from xerophyte Zygophyllum
xanthoxylum improves alfalfa plant growth under salinity, drought and field conditions. Plant Biotechnol. J. 14, 964–975 (2016). i
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pyrophosphatase. Plant Biotechnol. J. 5, 735–745 (2007). pyrophosphatase. Plant Biotechnol. J. 5, 735–745 (2007). p
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45. Retamal, C. A., Thiebaut, P. & Alves, E. W. Protein purification from polyacrylamide gels by sonication extraction. Anal. Biochem. 268, 15–20 (1999).f 46. Venancio, J. B. et al. A vacuolar H+-pyrophosphatase differential activation and energy coupling integrate the responses of weeds
and crops to drought stress. Biochim. Biophys. Acta 1840, 1987–1992 (2014). SCiEntiFiC REPOrTS | (2017) 7:17779 | DOI:10.1038/s41598-017-18032-3 9 www.nature.com/scientificreports/ SCiEntiFiC REPOrTS | (2017) 7:17779 | DOI:10.1038/s41598-017-18032-3 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-18032-3 Competing Interests: The authors declare that they have no competing interests. ublisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
nstitutional affiliations. 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
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Pengelolaan Informasi Kesehatan secara Terintegrasi untuk Memaksimalkan Layanan Kesehatan kepada Pasien di Rumah Sakit
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IJPST (Indonesian Journal Pharmaceutival Science and Technology)/IJPST (Indonesian Journal of Pharmaceutical Science and Technology)
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Abstrak Banyaknya masyarakat yang memperoleh perawatan pada beberapa instansi layanan kesehatan
menjadikan adanya alasan mengenai pentingnya kegiatan pengelolaan informasi kesehatan pasien
yang terintegrasi. Hal ini berfungsi untuk menyediakan informasi kesehatan secara komprehensif
kepada pasien dan staf medis dalam membantu pelayanan kesehatan yang lebih baik. Artikel ini
mengkaji elemen-elemen yang diperlukan dan model dalam kegiatan Health Information
Management yang terintegrasi. Kajian artikel ini dilakukan secara deskriptif dengan mengkaji 23
literatur yang terbit dari tahun 2007-2017, yang diperoleh dari jurnal-jurnal yang dilanggan
perpustakaan UGM, Google Scholar dan PubMed Health, mengenai pengelolaan informasi kesehatan
pasien. Hasil penelitian ini menjelaskan bahwa elemen yang diperlukan dalam penerapan Health
Information Management yang terintegrasi dapat dibagi ke dalam lima elemen, yaitu: 1. Sumber Daya
Manusia, 2. Kebijakan dan kelembagaan, 3. Penerapan sistem, 4. Data dan pengelolaannya, 5. Akses
dan integrasi data. Pada model pengelolaan informasi kesehatan pasien yang terintegrasi, harus
menyediakan akses terhadap informasi kesehatan pasien dan memungkinkan pertukaran data yang
dikelola beberapa petugas medis, pengelola data pasien dan rumah sakit lain. Hal ini didukung oleh
sebuah sistem informasi kesehatan yang dilengkapi dengan sistem keamanan data dan adanya
dukungan dari kepala rumah sakit. Kata kunci: Pengelolaan informasi kesehatan; layanan kesehatan di rumah sakit; integrasi informasi
kesehatan pasien Pengelolaan Informasi Kesehatan Secara Terintegrasi untuk
Memaksimalkan Layanan Kesehatan kepada Pasien di Rumah Sakit Nuzlil Laily Nur Azizah1, M. Very Setiawan2
1Fakultas Farmasi Universitas Padjadjaran, Sumedang Jawa Barat, Indonesia
2Fakultas Sekolah Pascasarjana, Universitas Gadjah Mada, Sleman, Yogyakarta, Indonesia IJPST IJPST IJPST Volume 4, Nomor 3, Oktober 2017 Abstract The large number of people who receive care in some healthcare establishments make sense
of the importance of integrated patient health information management activities. This serves
to provide comprehensive health information to patients and medical staff to help better
health care. This article examines the necessary elements and models in integrated Health
Information Management activities. The review of this article was conducted descriptively by
reviewing 23 literatures published from 2007-2017, obtained from journals subscribed to
UGM libraries, Google Scholar and PubMed Health, on the management of patient health
information. The results of this study explain that the elements needed in the application of
integrated Health Information Management can be divided into five elements, namely: 1. Human Resources, 2. Policies and institutions, 3. Implementation of the system, 4. Data and
its management, 5. Access And data integration. In an integrated patient health information
management model, it must provide access to patient health information and enable the
exchange of data managed by some medical officers, managers of patient data and other
hospitals. This is supported by a health information system equipped with a data security
system and support from the head of the hospital. Key words: Health information management; Health services in hospitals; Integration of patient health
information Key words: Health information management; Health services in hospitals; Integration of patient health
information Korespondensi: Nuzlil Laily Nur Azizah
Nuzlillaily@gmail.com 79 Volume 4, Nomor 3, Oktober 2017 IJPST IJPST teknologi Atas dasar ini penulis ingin mengkaji
elemen-elemen yang diperlukan dan model
dalam
kegiatan
Health
Information
Management yang terintegrasi. Pendahuluan informasi kesehatan pasien tersedia dan
mudah diakses saat dibutuhkan. Sihingga
HIM
dapat
membantu para petugas
pelayanan kesehatan dalam memberikan
pelayanan kesehatan kepada pasien secara
lebih baik.1 Atas dasar ini adanya
pengelolaan informasi kesehatan pasien
yang terintegrasi merupakan hal yang
penting, agar dapat memfasilitasi beberapa
penyedia layanan medis dalam kegiatan
pertukaran
dan
berbagi
informasi
kesehatan pasien.5 Banyaknya masyarakat yang saat ini
memperoleh perawatan pada beberapa
instansi layanan kesehatan, menjadikan
adanya alasan pentingnya kegiatan Health
Information Management (HIM). Hal ini
karena seseorang mungkin melakukan
pemeriksaan kesehatan pada rumah sakit
di kota lain atau melakukan pengobatan ke
luar negeri.1 Gagasan
mengenai
pengelolaan
informasi kesehatan pasien sebenarnya
bukan merupakan hal baru. Di sekitar kita
banyak instansi pelayanan kesehatan yang
telah melakukan pengelolaan informasi
kesehatan pasien dengan menyimpan data
hasil lab, arsip-arsip tentang pengobatan
dan lain sebagainya.2 Namun data tersebut
hanya
dikelola
pada
masing-masing
instansi dalam bentuk record konvensional
berbasis kertas.3 Seperti yang disampaikan oleh WHO
bahwa sistem kesehatan elektronik harus
dibangun untuk memfasilitasi pertukaran
data.1 Selain itu informasi kesehatan
pasien
yang
tersedia
harus
dapat
dilayankan
dengan
sebuah
sistem
informasi, agar orang yang membutuhkan
tidak perlu datang secara langsung ke
tempat instansi pengelola data.3 Dengan
adanya sistem informasi kesehatan yang
menyediakan akses data pasien dalam
format digital, diharapkan memungkinkan
petugas medis melacak data pasien dari
waktu ke waktu, membantu mengetahui
bagaimana
keadaan
pasien
dan
meningkatkan
kualitas
pelayanan
kesehatan.6 Sehingga dengan pengelolaan
informasi kesehatan pasien yang dilakukan
secara elektronik dan terintegrasi, dapat
membantu
setiap
petugas
pelayanan
kesehatan dalam memberikan kegiatan
pelayanan kesehatan kepada pasien secara
lebih baik.2 Perkembangan
teknologi
memberikan banyak kontribusi untuk
meningkatkan pelayanan kesehatan yang
diberikan kepada pasien secara lebih
efektif.4
Termasuk
dalam
kegiatan
pengelolaan informasi kesehatan, beberapa
instansi
layanan
kesehatan
mulai
menerapkan sistem informasi kesehatan
secara elektronik agar pengelolaan data
kesehatan pasien dapat dilakukan secara
lebih efektif. Beberapa rumah sakit juga
telah melakukan alih media data kesehatan
pasien dari bentuk kertas ke format
digital.2 Mereka juga mulai membuat
catatan Electronic Health Record (EHR)
yang berisi data demografi pasien, riwayat
medis
dan
obat-obatan,
informasi
diagnostik, tanda vital, riwayat kesehatan,
data laboratorium, dan laporan radiologi.1
Yang
disayangkan
yaitu
informasi-
informasi tersebut belum dapat terbuka
untuk dapat diakses lembaga pelayanan
kesehatan lain, karena sistem pengelolaan
informasi
kesehatan
pasien
hanya
dilakukan secara mandiri, pada masing-
masing instansi layanan kesehatan.5 Hasil 1. Elemen
dalam
kegiatan
Helath
Information
Management
(HIM)
yang terintegrasi Metode Kajian artikel ini dilakukan secara
deskriptif
dengan
mengkaji
beberapa
literatur terdahulu mengenai pengelolaan
data dan informasi kesehatan pasien. Pengumpulan literatur dilakukan melalui
pencarian
dari
jurnal-jurnal
yang
dilanggan perpustakaan UGM, Google
Scholar dan PubMed Health. Jurnal yang
dipilih hanya jurnal berbasa inggris yang Health
Information
Management
sebenarnya
bertujuan
untuk
mengumpulkan, menyimpan dan membuat Health
Information
Management
sebenarnya
bertujuan
untuk
mengumpulkan, menyimpan dan membuat 80 Volume 4, Nomor 3, Oktober 2017 IJPST IJPST diterbitkan pada 10 tahun terakhir, yaitu
tahun
2007-2017. Proses
pencarian
literatur dilakukan menggunakan metode
“boolean logic” dengan menambahkan
kata “and” untuk menggabungkan masing-
masing kata kunci. Kata kunci yang
digunakan dalam pencarian yaitu “health
information management” and “electronic”
and “system” and “patient” and “hospital”
and
“integrated”. Secara
keseluruhan
literatur
yang
muncul
pada
proses
pencarian berjumlah 5.185, namun yang
menurut penulis relevan dengan topik
bahasan artikel ini hanya berjumlah 38. sakit.7 Cludia dkk, menjelaskan elemen
penting dalam kegiatan EHR yaitu: 1. Privasi dan keamanan, 2. Pemilihan model
sistem informasi yang dapat mewakili
sistem yang digunakan di setiap rumah
sakit, 3. Staf dan organisasi untuk semua
rumah sakit.8 Pada penelitian Ashish dkk
yang
mengkaji
pemanfaatan
Health
Information Technologi dalam membantu
pelaksanaan
EHR
dan
pertukaran
informasi
kesehatan
pasien,
harus
memperhatikan: 1. Bagaimana penerapan
EHR di rumah sakit dan 2. Bagaimana
EHR memfasilitasi pertukaran informasi
kesehatan pasien.9 Dari 38 literatur yang terkumpul,
penulis
mengkaji
23
literatur
yang
membahas mengenai elemen-elemen yang
diperlukan dan model dalam pengelolaan
informasi
kesehatan
pasien
yang
terintegrasi. Menurut Stephen and Jeffrey hal
yang perlu diperhatikan untuk dapat
menciptakan inovasi sistem informasi
kesehatan elektronik yaitu: 1. Aplikasi IT
untuk
menunjang kegiatan EHR,
2. Kebijakan, seperti siapa saja yang dapat
melakukan
proses
input
data
atau
kebijakan dalam penggunaan data.10 Esther
Suter dkk yang menjelaskan 10 elemen
kunci untuk menyediakan sistem informasi
kesehatan kepada pasien. Hal yang harus
diperhatikan yaitu: 1. Layanan yang
komprehensif
yang
terintegrasi,
2. Layanan yang berorientasi pada pasien dan
dapat memenuhi kebutuhan informasi
pasien, 3. Cakupan geografis pasien untuk
dapat melakukan akses terhadap informasi,
4. Standar pengiriman data antar lembaga
kesehatan, 5. Adanya standar format data
yang
digunakan,
5. Kemampuan
pengelolaan sistem, 6. Sistem informasi, 7. Dukungan pimpinan, 8. Petugas medis
yang terintegrasi, 9. Integrasi dari instansi
pelayanan kesehatan, 10. Pengelolaan
anggaran.11 1. Elemen
dalam
kegiatan
Helath
Information
Management
(HIM)
yang terintegrasi Artikel ini mengkaji 23 literatur yang
membahas mengenai Health Information
Management,
Medical
Record
Management, Elektronik Health Record
atau
tema
lain
berkaitan
dengan
pengelolaan data atau informasi kesehatan
pasien. Penjelasan dari hasil penelitian-
penelitian tersebut yaitu: Penelitian
Carroll
dkk
yang
membahas mengenai penerapan Elektronik
Helath Record (EHR) untuk membantu
memberikan perawatan kesehatan kepada
pasien secara lebih baik. EHR diharapkan
dapat memfasilitasi pertukaran informasi
kesehatan pasien di beberapa penyedia
layanan kesehatan. Menurut Carroll dkk
elemen yang harus ada pada penerapan
EHR yaitu: 1. Adanya portal untuk
menyediakan akses
informasi
kepada
pengguna, 2. Adanya pimpinan instansi
yang mendukung implementasi EHR, 3. Adanya keterlibatan staf medis dalam
Implementasi EHR, 4. Adanya pelatihan
untuk staf pengelola EHR, 5. Adanya
penggabungan data dari setiap rumah Jennifer dkk menjelaskan dalam
pelaksanaan Personal Health Records
(PHR)
yang
terintegrasi,
hal
yang
diperlukan yaitu: 1. Data medis elektronik,
2. Akses untuk pasien 3. Adanya proses
berbagi
data.12
Penelitian
Hussain
mengenai penerapan Health Information
Exchance
(HIE)
untuk
membantu
meningkatkan
kualitas
layanan
yang
diberikan
rumah
sakit,
menjelaskan 81 Volume 4, Nomor 3, Oktober 2017 IJPST IJPST elemen yang diperlukan yaitu: 1. Staff
medis (farmasi, lab, radiologi) untuk
membantu mengumpulkan data, 2. Sistem
pengelolaan
informasi,
3. Adanya
pertukaran informasi.13 Penelitian Oliver
dkk yang mengkaji implementasi dari
Regional Health Information Network
(RHIN) menyatakan komponen yang harus
dipenuhi yaitu: 1. Sistem informasi pada
layanan kesehatan yang terintegrasi, 2. Elektronik health record, 3. Akses data
yang
disediakan
untuk
pasien,
4. Kebijakan privasi data pasien.14 Sedangkan
untuk memberika akses terhadap data
secara efektif menurut Dean dkk rumah
sakit dapat menyediakan: 1. Akses data
menyeluruh kepada pasien, 2. Informasi
dalam bentuk ringkasan yang mudah
dipahami.15 Menurut James hal ini dapat
didukung dengan adanya: 1. Hardware
sebagai pendukung sistem informasi, 2. Software untuk kegiatan, 3. Perubahan
pengelolaan dari pengelolaan konvensional
beralih ke pengelolaan secara elektronik.16 perlu memperhatikan beberapa hal seperti:
1. Bagaimana validitas data pasien, karena
beberapa data yang dikelola mungkin
hilang atau rusak, 2. Bagaimana kebijakan
yang diberikan untuk akses ke data pasien
lain, 3. Adanya personalisasi data seperti
pengelompokkan data menurut keluarga
dan sebagainya, 4. 1. Elemen
dalam
kegiatan
Helath
Information
Management
(HIM)
yang terintegrasi Bagaimana kebijakan
jika data dikelola oleh jasa pengelola
informasi dari luar rumah sakit.20 Terkait
data yang disediakan untuk pengguna data,
menurut Severin dkk rumah sakit dapat
memutuskan apakah data yang dapat
diakses hanya data hasil diagnosis pasien
atau data lengkap dari proses awal pasien
melakukan pendaftaran hingga proses
perawatan
selesai.21
Pada
penelitian
Tsung-Chih dan Zhen-Yu menyatakan
bahwa, untuk mengembangkan kegiatan
pelayanan kesehatan, sistem informasi
medis harus memenuhi elemen: 1. Adanya
integrasi data antar departemen di dalam
rumah sakit, 2. Adanya integrasi data antar
rumah sakit, 3. Pengelolaan data medis
dalam bentuk elektronik.22 p
g
Penelitian Muhammad dan Moazzam
mengenai sistem informasi kesehatan
rumah sakit di Pakistan memerlukan
komponen-komponen: 1. Kebijakan dan
perencanaan
penerapan
sistem,
2. Pengelolaan informasi, 3. Adanya tranmisi
perubahan format data yang sesuai sistem,
3. Sistem informasi kesehatan yang
terintegrasi.17 Vincent dkk menambahkan,
selain sistem informasi hal yang perlu
dipersiapkan
dalam
kegiatan
Health
Information Management Sistem yaitu: 1. Adanya pelatihan untuk staf pengelola
sistem dan 2. Adanya proses perpindahan
data untuk memperoleh format yang sama
dari
tiap
rumah
sakit.18
Angelo
menambahkan, selain melakukan pelatihan
kepada staf, dalam pengelolaan sistem
informasi kesehatan diperlukan panduan,
seperti pedoman yang dapat berbentuk
booklet, untuk membantu para pengelola
sistem secara lebih mudah memahami
sistem yang digunakan.19 Ross yang menjelaskan mengenai
penerapan teknologi
informasi
dalam
pelayanan kesehatan dapat membantu
mempercepat pengumpulan data pasien
antar instansi medis.23 Hal ini dapat
dilakukan dengan berbagai cara seperti
yang disampaikan oleh Ann dan John,
melalui pengembangan aplikasi kesehatan
berbasis mobile phone dapat membantu
menyediakan akses terhadap data pasien
secara lebih efektif.24 Penelitian Kenneth
dkk yang mengkaji bagaimana aplikasi
“invido” memungkinkan beragam sumber
data medis dari berbagai rumah sakit dapat
integrasi. Kenneth
dkk
menjelaskan
elemen-elemen yang harus dipenuhi yaitu:
1. Rumah sakit sebagai penyedia layanan
informasi kepada pasien, 2. Adanya
pertukaran data pasien dari penyedia
layanan kesehatan, 3. Pasien, peneliti, dan
provider informasi sebagai pengguna, 4. Sistem aplikasi (invido), 5. Server aplikasi
invido yang menjaga keamanan data.25 Corine yang mengkaji kegiatan HIM
pada sebuah rumah sakit yang ditinjau dari
segi etik menyatakan bahwa, rumah sakit Penelitian Andrew mengenai dampak
dari
era
digital
terhadap
kegiatan 82 Volume 4, Nomor 3, Oktober 2017 IJPST pelayanan kesehatan. 1. Elemen
dalam
kegiatan
Helath
Information
Management
(HIM)
yang terintegrasi Hal ini menuntut
adanya pengelolaan data kesehatan pasien
secara
elektronik
agar
lebih
mudah
dikelola
dan
perlunya
sistem
untuk
menjaga
keamanan
data
pasien.26
Penelitian Joan dkk menjelaskan bahwa
selain menyediakan sistem keamanan data
pasien, menjaga privasi data pasien juga
merupakan
hal
yang
diperlukan.27
Penelitian Francois dan Ebere
yang
menyatakan, hal penting yang harus
diperhatikan
dalam
pengelolaan
data
eletronik
pasien
yaitu:
1. Adanya
pemanfaatan
teknologi,
2. Adanya
kebijakan penerapan EHR, 3. Adanya
privasi data.28 Selanjutna penelitian Martha
yang menjelaskan, pentingnya pertukaran
data pasien yang dikelola secara elektronik
akan
dapat
membantu
memberikan
pelayanan kesehatan pasien secara lebih
baik.29 kesehatan pasien. Empat dari literatur
tersebut menjelaskan bahwa pentingnya
peran
staf
medis
dalam
membantu
pengumpulan data pasien yang akan
dikelola.7,25,8 Empat penelitian diantaranya
menjelaskan bahwa rumah sakit perlu
memperhatikan kemampuan staf pengelola
sistem
informasi
dalam
pemanfaatan
sistem. Hal ini dapat dibantu dengan
mengadakan pelatihan penggunaan sistem
informasi
kesehatan yang baik
atau
memberikan buku panduan penggunaan
sistem.7,11,18,19 Berdasarkan 23 literatur yang dikaji
39% diantaranya membahas mengenai hal-
hal yang berkaitan dengan kebijakan,
organisasi
dan
kelembagaan
sebagai
elemen pendukung kegiatan HIM. Lima
dari
penelitian
tersebut
menjelaskan
bahwa, dalam pelaksanaan pengelolaan
informasi kesehatan pasien, diperlukan
adanya kebijakan mengenai privasi dan
sistem keamanan data pasien.8,14,26,27,28
Tiga dari literatur tersebut membahas
mengenai pentingnya kebijakan dalam
pengelolaan data terkait dengan kebijakan
pengumpulan, pengelolaan, akses terhadap
data, hingga kebijakan pengelolaan data
berbasis elektronik.10,28,17 Dua dari literatur
tersebut
juga
membahas
mengenai
pentingnya dukungan dari kepala instansi
dalam pembuatan kebijakan mengenai
pelaksanaan
pengelolaan
informasi
kesehatan
pasien.7,11
Satu
literatur
menambahkan adanya kebijakan mengenai
anggaran yang harus dipersiapkan untuk
mendukung
kegiatan
pengelolaan
informasi kesehatan pasien dan perlu
adanya struktur organisasi pada masing-
masing instansi layanan kesehatan, yang
berfungsi
sebagai
pengawas
dalam
pelaksanaan
pengelolaan
informasi
kesehatan pasien yang terintegrasi.11
D i
23
lit
t
t
b t
48% Pada 23 penelitian tersebut memiliki
berapa
fokus
kajian
yang
berbeda. Beberapa penelitian mengkaji kegiatan
HIM berkaitan dengan privasi data pasien
dan akses yang disediakan.8,14 Penelitian
lain mengkaji penerapan HIM berkaitan
dengan pemanfaatan aplikasi berbasis web
dengan nama “invido”25 dan pemanfaatan
aplikasi berbasis mobile phone untuk
menyediakan
akses
terhadap
data
kesehatan pasien secara lebih mudah.10
penelitian lain juga mengkaji pelaksanaan
HIM dari segi etik dalam pengelolaan dan
akses data pasien yang disediakan.20 Secara
garis
besar
artikel
ini
membagi elemen-elemen yang diperlukan
dalam pelaksaan HIM dari hasil kajian 23
penelitian tersebut ke dalam lima bagian. Yaitu: 1. HIM berkaitan dengan Sumber
Daya Manusia (SDM), 2. 1. Elemen
dalam
kegiatan
Helath
Information
Management
(HIM)
yang terintegrasi HIM berkaitan
dengan kebijakan dan kelembagaan, 3. HIM berkaitan dengan penerapan sistem,
4. HIM berkaitan dengan data dan
pengelolaannya, 5. HIM berkaitan dengan
akses dan integrasi data. Dari 23 literatur tersebut 48%
literatur membahas mengenai penerapan
sistem dalam pelaksanaan HIM yang
terintegrasi. Mengani
pentingnya
pemanfaatan sistem, empat dari literatur
tersebut membahas mengenai pentingnya
penerapan
sistem
dan
pemanfaatan Dari 23 penelitian yang dikaji 31%
dari
penelitian
tersebut
membahas
mengenai SDM sebagai faktor pendukung
pelaksanaan
pengelolaan
informasi 83 IJPST Volume 4, Nomor 3, Oktober 2017 teknologi informasi untuk menunjang
kegiatan pengelolaan informasi kesehatan
pasien.10,18,28,13
Empat
literatur
juga
membahas pentingnya penerapan sistem
yang dapat mewakili semua instansi
kesehatan yang berbeda, agar dapat
menjadi sarana pendukung kegiatan HIM
yang terintegrasi. Hal ini karena pada
beberapa instansi
kesehatan mungkin
menggunakan
sistem
operasi
pada
komputer
yang
berbeda.8,11,16,22
Pada
bidang ini dua literatur lainnya membahas
mengenai
model
sistem
yang
dapat
digunakan. Salah satu penelitian tersebut
membahas
mengenai
model
sistem
berbasis web dengan nama “invido”25, satu
lainnya
membahas
model
penerapan
sistem berbasis aplikasi mobile phone.24
Penelitian lain menjelaskan mengenai
adanya satu sistem pusat dari HIM yang
terintegrasi
pada
antar
rumah
sakit,
sehingga
sistem
sentral
ini
dapat
digunakan pada setiap anggota instansi
kesehatan
dan
memudahkan
proses
berbagi data.17 Dari 23 literatur tersebut 61%
literatur membahas mengenai akses dan
integrasi data dalam pelaksanaan HIM
yang terintegrasi. 10 dari literatur tersebut
mengkaji mengenai pentingnya integrasi
data untuk dapat menyediakan akses
terhadap data secara komprehensif kepada
pasien, dari berbagai sumber informasi
dari rumah sakit dimana mereka pernah
dirawat. Selain itu adanya data yang
terintegrasi juga dapat membantu para
provider informasi (staf medis pada
instansi
layanan
kesehatan)
untuk
memperoleh
data
pasien
yang
dibutuhkan.7,9,14,15,17,22,23,29,13,12
Dua
literatur lainnya membahas mengenai
akses yang disediakan dapat dilakukan
melalui sebuah portal pada suatu aplikasi
berbasis website atau aplikasi yang dapat
diakses melalui mobile phone.7,25 Dua
literatur lainnya membahas mengenai data
apa saja yang dapat diakses oleh pasien,
apakah
data
keseluruhan
atau
data
berkaitan dengan laporan diagnosis.15,21
Satu literatur lainnya membahas mengenai
akses yang dapat dilakukan oleh pengguna
lainnya, dalam hal ini yaitu pasien lain.20 48% dari 23 literatur yang dikaji
membahas
mengenai
data
dan
pengelolaannya
dalam
mendukung
pelaksanaan
pengelolaan
informasi
kesehatan pasien yang terintegrasi. 1. Elemen
dalam
kegiatan
Helath
Information
Management
(HIM)
yang terintegrasi Enam
dari
literatur
tersebut
menjelaskan
mengenai
pentingnya
perubahan
pengelolaan data pasien yang awalnya
dilakukan
secara
konvensional
untuk
berpindah menuju pengelolaan secara
elektronik.9,12,14,16,22,26 Tiga dari literatur
tersebut menjelaskan bahwa perlu adanya
format standar dari data-data yang dikelola
pada masing-masing instansi kesehatan,
sehingga dapat memastika data yang ada
dapat digunakan oleh instansi lain yang
membutuhkan.11,17,18 Dua literatur lainnya
menjelaskan
mengenai
adanya
personalisasi data, seperti data mungkin
dapat dikelompokkan dalam beberapa
bagian
seperti
keluarga,
atau
dari
banyaknya
informasi
yang
tersedia,
informasi dapat ditampilkan dalam bentuk
ringkasan.20,15 48% dari 23 literatur yang dikaji
membahas
mengenai
data
dan
pengelolaannya
dalam
mendukung
pelaksanaan
pengelolaan
informasi
kesehatan pasien yang terintegrasi. Enam
dari
literatur
tersebut
menjelaskan
mengenai
pentingnya
perubahan
pengelolaan data pasien yang awalnya
dilakukan
secara
konvensional
untuk
berpindah menuju pengelolaan secara
elektronik.9,12,14,16,22,26 Tiga dari literatur
tersebut menjelaskan bahwa perlu adanya
format standar dari data-data yang dikelola
pada masing-masing instansi kesehatan,
sehingga dapat memastika data yang ada
dapat digunakan oleh instansi lain yang
membutuhkan.11,17,18 Dua literatur lainnya
menjelaskan
mengenai
adanya
personalisasi data, seperti data mungkin
dapat dikelompokkan dalam beberapa
bagian
seperti
keluarga,
atau
dari
banyaknya
informasi
yang
tersedia,
informasi dapat ditampilkan dalam bentuk
ringkasan.20,15 2. Model kegiatan Health Information
Management
(HIM)
yang
terintegrasi g
Banyak penelitian yang memberikan
gambaran model yang dapat diterapkan
dalam pengelolaan data dan informasi
kesehatan pasien. Diantaranya yaitu model
yang
dijelaskan
oleh
Kenneth
dkk
mengenai bagaimana aplikasi berbasis
website “invido” dapat membantu pasien
untuk melakukan akses kepada data
mereka dan membantu terciptanya proses
pertukaran
data
dari
masing-msiang
penyedia data kesehatan pasien. Dari
gambar
pertama
menjelaskan
pasien,
peneliti dan provider (tenaga medis rumah
sakit) yang menjadi pengguna data. Pada
gambar
kedua
menjelaskan
proses
pertukaran data dari provider data yang
terdiri dari beberapa rumah sakit dan
apoteker sebagai penyedia obat. Selain
pengguna data, pada gambar 3 juga
menjelaskan bahwa rumah sakit sebagai 84 IJPST Volume 4, Nomor 3, Oktober 2017 Aplikasi berbasis website ini dikelola oleh
server
yang
akan
membantu
proses
enkripsi data untuk menjaga keamanan
data. penyedia layanan kesehatan juga dapat
melakukan akses terhadap data kesehatan
pasien untuk membantu menyediakan
layanan kesehatan secara lebih baik. Gambar 1 Alur Integrasi Data Menggunakan Aplikasi Invido25 Gambar 1 Alur Integrasi Data Menggunakan Aplikasi Invido25 Model
lain
dari
Hussain
yang
menggambarkan sebuah sistem dapat
memungkinkan terjadinya pertukaran data
kesehatan pasien. Pertukaran ini berasal
dari beberapa petugas medis yang berasal
dari staf radiologi, staf lab dan apoteker di
bagian atas, dan para pengelola data pasien
atau instansi layanan kesehatan lain di
bagian
kiri. Dalam
kegiatan
ini membutuhkan
adanya
kontrol
dari
pengelola sistem dan pengelola informasi
kesehatan pasien di bagian bawah. Data
yang tersedia ini dapat digunakan oleh
para rumah sakit atau pengguna lain yang
membutuhkan. Penggunaan data tersebut
tentunya sesuai dengan kebijakan dari
masing-masing instansi kesehatan. Gambar 2 Rangkaian Alur Pertukaran Informasi Kesehatan9 Gambar 2 Rangkaian Alur Pertukaran Informasi Kesehatan9 Kedua
model
tersebut
menjelaskan
mengenai
pentingnya
sebuah
sistem
aplikasi
berbasi
untuk
memfasilitasi
pertukaran data. Sistem tersebut dilengkapi
dengan adanya server sebagai pengelola sistem,
untuk
melakukan
enkripsi
keamanan data pasien. Dalam pertukaran
data pasien dilakukan oleh staf medis dari
instansi kesehatan, pengelola data dari
instansi kesehatan dan klinik lain. Data ini 85 Volume 4, Nomor 3, Oktober 2017 IJPST IJPST rumah sakit atau pengguna lain yang
membutuhkan. rumah sakit atau pengguna lain yang
membutuhkan. c. SDM sebagai pengguna informasi c. SDM sebagai pengguna informasi c. SDM sebagai pengguna informasi
User dalam hal ini yaitu: pasien dan
provider (staff medis). Bagi pasien,
mereka dapat mengetahui informasi yang
dibutuhkan
untuk
kesehatan
mereka. Sedangkan bagi provider dapat membantu
dalam
pengambilan
keputusan
dalam
pelayanan kesehatan.25 Sumber
Daya
Manusia
(SDM)
merupakan
faktor
penting
dalam
implementasi
pengelolaan
informasi
kesehatan pasien.7 SDM tidak hanya
berkaitan dengan penyedia informasi,
dalam hal ini SDM juga berkaitan dengan
pengguna
informasi,
staf
pengelola
informasi dan staf pengelola sistem.13,25 2. Model kegiatan Health Information
Management
(HIM)
yang
terintegrasi selanjutnya akan terkumpul pada server
pengelola sistem, yang nantinya akan
dapat diakses oleh pasien, staf medis dan Oleh karena itu, adanya pelatihan untuk
staff pengelola sistem merupakan hal yang
penting.7,18,19 Tidak hanya menyediakan
pelatihan kepada staf pengelola sistem
informasi,
rumah
sakit
juga
dapat
menyediakan pedoman seperti booklet
tentang cara pengelolan sistem informasi
yang baik.11 Adanya hal ini rumah sakit
akan memperoleh staf pengelola sistem
yang terampil.13 Pembahasan Berdasarkan literatur yang dikaji
dalam artikel ini, secara garis besar elemen
yang diperlukan dalam penerapan HIM
yang terintegrasi dapat dibagi ke dalam
lima kompenen, yaitu: 1. Sumber Daya
Manusia
(SDM),
2. Kebijakan
dan
kelembagaan, 3. Penerapan sistem, 4. Data
dan pengelolaannya, 5. Akses dan integrasi
data. 2. Kebijakan dan kelembagaan Dalam
kegiatan
pengelolaan
informasi
kesehatan
pasien
yang
terintegrasi, diperlukan adanya kebijkan
mengenai pengelolan informasi.17 Adanya
kebijakan dalam pengelolaan data akan
dapat membantu untuk menentukan siapa
saja yang dapat melakukan input data atau
bagaimana standar pengelolaan data yang
dilakukan.10
Selain
menyediakan
kebijakan mengenai pengelolaan data,
rumah sakit juga harus memiliki kebijakan
berkaitan
dengan
akses
data
yang
disediakan.20
Rumah
sakit
dapat
memutuskan apakah data yang dapat
diakses hanya data hasil diagnosis pasien
atau data lengkap dari proses awal pasien
melakukan pendaftaran hingga proses
perawatan selesai.21 a. SDM sebagai staf pelayanan kesehatan
dan provider informasi SDM sebagai pelayanan kesehatan berasal
dari staf medis pada sebuah rumah sakit. Selain melakukan pelayanan kesehatan,7
para staf medis ini juga berperan sebagai
staf yang membantu untuk menentukan
data mana yang penting untuk dikelola.25
Staf medis tersebut dapat berasal dari
beberapa bidang pelayanan kesehatan
seperti, staf farmasi, staf lab dan staf
radiologi.13 Para staf medis ini dapat juga
berperan sebagai provider data perawatan
pasien, data hasil uji kesehatan, hingga
catatan mengenai obat yang telah atau
sedang dikonsumsi pasien.25 b. SDM sebagai pengelola data dan sistem
Dalam
pengelolaan
sistem
informasi
kesehatan pasien, memerlukan adanya
staff IT yang bertanggung jawab untuk
mengelola sistem informasi kesehatan dan
database pasien.13 Staf pengelola sistem
informasi juga bertugas merawat sistem
informasi.25
Sehingga
kemampuan
pengelola sistem informasi untuk dapat
memfaatkan sisitem informasi dengan
baik, merupakan hal sangat dibutuhkan.11 Hal penting lainnya yaitu kebijkan
mengenai privasi dan keamanan data.27
Hal ini penting karena seluruh informasi
pribadi dan medis pasien banyak dianggap
sebagai informasi yang sensitif, rahasia
dan pribadi.30 Banyak pasien khawatir
mengenai
bagaimana
keamanan
dan
privasi data peronal mereka terutama hal
yang berkaitan dengan gangguan mental 86 Volume 4, Nomor 3, Oktober 2017 IJPST dan seks.8 Sehingga diperlukan kebijakan
privasi dan keamanan untuk menjaga data
pasien.28 Untuk mengatasi hal ini pihak
instansi dapat melakukan beberapa hal
seperti melakukan sistem keamanan data
dengan password yang mungkin hanya
dapat dibuka oleh pasien atau petugas
tertentu.30 mobile phone untuk lebih memudahkan
dalam
menyediakan
akses
informasi
kesehatan.24 4. Data dan pengelolaannya Data
kesehatan
pasien
dapat
berbentuk kertas ataupun berbentuk digital
(elektronik).34 EHR dapat juga berupa
catatan medis elektronik yang berisi
demografi pasien, riwayat medis, obat-
obatan, dan informasi diagnostik dan lain
sebagainya.28
Bordoloi
dan
Nazrul
menjelaskan bahwa pada EMR catatan
medis pasien harus dapat disimpan dan
diambil dalam format digital/ elektronik.35
Hal ini dapat dilakukan dengan cara
beralih dari pengelolaan konvensional ke
pengelolaan secara elektronik.16 Semua kegiatan tersebut tidak dapat
terlaksana tanpa adanya dukungan kepala
instansi
sebagai
pembuat
kebijakan. Sehingga dukungan dari kepala rumah
sakit
merupakan
hal
penting
untuk
menciptakan
pelaksanaan
pengelolaan
informasi kesehatan yang terintegrasi.11
Dukungan
ini
dapat
dengan
cara
menyediakan anggaran untuk kegiatan
sistem kesehatan yang terintegrasi.11 Pada pengelolaan data kesehatan
pasien
rumah
sakit
juga
harus
memperhatikan validitas data pasien. Hal
ini karena, terkadang informasi yang
dikelola mungkin kurang lengkap karena
hilang atau lain sebagainya.20 Beberapa hal
lain
juga
harus
diperhatikan
dalam
kegiatan pengelolaan informasi kesehatan
pasien seperti bagaimana jenis data yang
akan dikelola, apakah data-data tersebut
hanya hasil alihmedia berbasis kertas ke
bentuk elektronik atau data dalam bentuk
gambar digital dan data yang telah diolah
dalam bentuk video, dan lain sebagainya.30 3. Penerapan sistem Teknologi
informasi
yang
terus
berkembang turut membantu kegiatan
pelayanan kesehatan.10 Diantaranya dapat
digunakan untuk membantu memperoleh
informasi kesehatan pasien terkini dengan
waktu
yang
cepat
dan
membantu
meningkatkan
efisiensi
pelayanan
kesehatan31 Oleh karena itu, banyak rumah
sakit mulai menggunakan sistem informasi
kesehatan yang digunakan untuk berbagai
keperluan seperti, pengelolaan data pasien
dan sebagainya.32 Adanya sistem informasi
kesehatan, juga dapat membantu beberapa
instansi layanan kesehatan untuk dapat
menggabungkan
semua
data
yang
dibutuhkan
oleh
pembuat
kebijakan,
dokter dan pengguna layanan kesehatan.33
Sehingga pemilihan sistem informasi yang
sesuai yang dapat mewakili semua instansi
merupakan hal yang penting.16 Hal ini
karena beberapa rumah sakit mungkin
menggunakan sistem yang berbeda,8 atau
beberapa pengguna informasi mungkin
menggunakan beberapa operating sistem
(OS) pada PC yang berbeda. Sehingga
sistem yang dapat digunakan pada seluruh
rumah sakit merupakan hal yang penting.16
Sistem yang diterapkan dapat bermacam-
macam. Kita dapat menerapkan sistem
apliaksi
berbasis
website25
atau
memanfaatkan sistem aplikasi berbasis Pada pengelolaan data kesehatan
pasien, rumah sakit juga dapat mengelola
data pasien dalam bentuk ringkasan15 atau
data dikelompokkan pada hal tertentu,
semisal data kesehatan sebuah keluarga. Sehingga hal ini dapat lebih memudahkan
pasien atau staf medis untuk memahami
informasi yang tersedia.20 Hal lain yang
perlu diperhatikan yaitu adanya standar
format data yang memastikan data dapat
digunakan pada seluruh rumah sakit.11
Sehingga akan lebih baik jika data yang
tersedia dapat ditransmisikan ke dalam
format tertentu, semisal dalam bentuk
microsoft exel.14,18 5. Akses dan integrasi data Adanya sistem informasi kesehatan
di rumah sakit dapat membantu penyedia
layanan kesehatan untuk memperoleh 87 Volume 4, Nomor 3, Oktober 2017 IJPST informasi kesehatan pasien secara real
time.33 Pada layanan informasi kesehatan
pasien, rumah sakit dapat menetapkan
apakah
informasi
yang
disediakan
merupakan data pasien secara keseluruhan
semisal data keseluruhan dari awal pasien
melakukan proses pendaftaran hingga
pemeriksaan kesehatan selesai, atau hanya
beberapa data seperti hasil diagnosis
pasien.15,21 Keuntungan lain dari adanya integrasi data
yaitu, rumah sakit dapat meningkatkan
kegiatan berbagi data kepada pasien.12 Unutk mengintegrasikan informasi
kesehatan pasien, dapat dilakukan dengan
bermacam-macam
cara,
seperti
mengintegrasikan sistem informasi pada
beberapa rumah sakit, melalui sistem
pengelolaan
informasi
kesehatan
tersentral.17 Dalam pelaksanaannya kita
dapat memanfaatkan aplikasi atau software
tertentu.28
Untuk
menyediakan
akses
informasi, sistem ini dapat menyediakan
sebuah portal, sehingga hanya pengguna
tertentu yang dapat melakukan akses
terhadap informasi kesehatan pasien.7 Hal yang penting yaitu bagaimana
akses terhadap data dapat disediakan
secara komprehensif, pada seluruh instansi
kesehatan. Atas dasar ini perlu adanya
integrasi data kesehatan pasien pada
beberapa
rumah
sakit.11
Dengan
melakukan
pengelolaan
informasi
kesehatan pasien yang terintegrasi, pasien
dan staf medis akan dapat melakukan
akses ke record pasien, dimanapun dan
kapanpun dibutuhkan secara cepat.28,23 Atas dasar ini penulis memiliki
usulan
model
pengelolaan
informasi
kesehatan pasien yang terintegrasi sebagai
berikut: Gambar 3 Alur Pengelolaan dan Pertukaran data dalam HIM yang Terintegrasi Gambar 3 Alur Pengelolaan dan Pertukaran data dalam HIM yang Terintegrasi Pada gambar 3 menjelaskan
mengenai alur penerapan HIM pasien
yang terintegrasi. Pada alur pertama
menjelaskan mengenai asal informasi
kesehatan pasien yang dapat diperoleh
dari staf medis, rumah sakit lain dan data
konvensional yang telah dialih media. Semua data ini akan terkumpul pada
sistem HIM yang dikelola oleh server,
yang menjadi admin pada masing-
masing rumah sakit. Sebagai pengelola
sistem, server akan mengatur akses dan
keamanan informasi data pasien. Terkait privasi informasi, data setiap pasien ini
hanya akan diperbolehkan diakses untuk
membantu
pelaksanaan
pelayanan
kesehatan dari pasien terkait, sehingga
informasi seorang pasien yang akan
digunakan untuk pasien lain, harus
memperoleh ijin dari pemilik informasi. Staf medis sebagai petugas layanan
kesehatan dapat mengakses informasi
pasien
untuk
membantu
dalam
memberian layanan kesehatan kepada
pasien. Rumah sakit lain juga dapat
melakukan akses terhadap data pasien 88 Volume 4, Nomor 3, Oktober 2017 IJPST IJPST atau menjadi elemen dalam pertukaran
informasi kesehatan pasien. Hal ini tentu
tidak
akan
berjalan
tanpa
adanya
kebijakan dari masing-masing kepala
rumah sakit. terintegrasi. 5. Akses dan integrasi data Hal ini dapat dilakukan
dengan mengintegrasikan data pada
sebuah sistem pengelolaan informasi
kesehatan. Adapun model dalam pengelolaan
informasi
kesehatna
pasien
yang
terintegrasi
harus
memungkinkan
terciptanya pertukaran data kesehatan
pasien yang dikelola beberapa staf
medis, para pengelola data pasien dan
instansi
rumah
sakit
lain. Dalam
kegiatan
ini
membutuhkan
adanya
kontrol dari server sebagai pengelola
sistem
dan
pengelola
informasi
kesehatan
pasien,
yang
menjaga
keamanan informasi. Server juga akan
menentukan data mana dan siapa yang
dapat melakukan akses. Sistem yang
disediakan dapat berupa aplikasi berbasi
website. Simpulan p
Berdasarkan
hasil
dan
pembahasan, dapat disimpulkan bahwa
elemen
yang
diperlukan
dalam
penerapan HIM yang terintegrasi dapat
dibagi ke dalam lima kompenen yaitu: 1. Sumber Daya Manusia (SDM), yang
berperan
sebagai
staf
pelayanan
kesehatan
dan
provider
informasi,
pengelola data dan sistem dan pengguna
informasi. 2. Kebijakan
dan
kelembagaan. Kebijakan
ini
terkait
dengan pengelolan informasi, akses data,
privasi
serta
keamanan
data
dan
kebijakan
kepala
instansi
untuk
mendukung pelaksanaan HIM yang
terintegrasi,
diantaranya
dengan
memberikan anggaran. 3. Penerapan
sistem. Sistem ini harus disesuaikan dan
dapat digunakan pada seluruh instansi
yang
menjadi
anggota
HIM
yang
terintegrasi dan memudahakan pasien
untuk melakukan akses informasi. 4. Data
dan
pengelolaannya. Pada
pelaksaan HIM yang terintegrasi, semua
data pasien akan dikelola dalam format
elektronik. Data tersebut dapat juga
dikelola
dalam
bentuk
ringkasan
sehingga
memudahkan
pasien
dan
tenaga medis untuk menggunakannya. Hal yang penting dalam pengelolaan
data pasien yaitu adanya format data
general yang dapat digunakan seluruh
rumah sakit. 5. Akses dan integrasi data,
diperlukan untuk memudahakn para
pasien
dan
petugas
medis
dalam
memperoleh data pasien secara real
time. Data
yang
disediakan
dapat
merupakan
data
pasien
secara
keseluruhan
atau
data
tertentu,
tergantung dari kebijakan rumah sakit
dan ijin dari pasien. Hal penting yaitu
bagaimana
data
kesehatan
seorang
pasien dari berbagai rumah sakit dapat Daftar Pustaka 1. World Health Organization, Global
Observatory
for
eHealth. Management of Patient Information:
Trends and Challenges in Member
States: Based on The Findings of
The Second Global Survey on
eHealth Seri ke-6. Geneva: Global
Observatory for eHealth Series;
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and
International
Departement. Personal
Health
Records
and
Information Management Helping
Patients, Clients and Their Parents/
Carers to Make The Most of Health
Information. London: Royal College
of Nursing; 2014. 2. Royal College of Nursing, Policy
and
International
Departement. Personal
Health
Records
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Patients, Clients and Their Parents/
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of Nursing; 2014. 3. Smith E, Macdonald R. Managing
Health
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During
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EE. Accelerating
the
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DC,
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Heinze,
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Birkle,
Lennart Köster and Björn Bergh. Architecture
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Elise
Russo,
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Hardeep Singh. Safe and Effective
Care for Adults and Children. Health Inf. 2016;4: 21-38. 7. Sharon Silow-Carroll, Jennifer N. EdwardS, dan Diana Rodin. Using
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Suleman
Qazi,
Moazzam Ali. Health Management
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the way forward. BioSci. 2011;
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Matovu, Fredrick Makumbi, Carol
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Eddie
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Rhoda K Wanyenze. Daftar Pustaka Strengthening
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through
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Daniel Grandt, Tim Scott dan David
W. Bates. The use of health
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English
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Ethanol contamination of cerebrospinal fluid during standardized sampling and its effect on 1H-NMR metabolomics
|
Analytical and bioanalytical chemistry/Analytical & bioanalytical chemistry
| 2,015
|
cc-by
| 3,443
|
Ethanol contamination of cerebrospinal fluid during standardized
sampling and its effect on 1H-NMR metabolomics Sonia A. van der Sar1 & Ronald Zielman2 & Gisela M. Terwindt2 &
Arn M. J. M. van den Maagdenberg2,3 & André M. Deelder1 &
Oleg A. Mayboroda1 & Axel Meissner1 & Michel D. Ferrari2 Received: 22 January 2015 /Accepted: 25 March 2015 /Published online: 3 May 2015
# The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Standardization of body fluid sampling, processing
and storage procedures is pivotal to ensure data quality in
metabolomics studies. Yet, despite strict adherence to standard
sampling guidelines, we detected variable levels of ethanol in
the 1H-NMR spectra of human cerebrospinal fluid (CSF) sam-
ples (range 9.2×10−3–10.0 mM). The presence of ethanol in
all samples and the wide range of concentrations clearly indi-
cated contamination of the samples of some sort, which af-
fected the 1H-NMR spectra quality and the interpretation. To
determine where in the sampling protocol the ethanol contam-
ination occurs, we performed a CSF sampling protocol simu-
lation with 0.9 % NaCl (saline) instead of CSF and detected
ethanol in all simulation samples. Ethanol diffusion through
air during sampling and preparation stages appeared the only
logical explanation. With a bench study, we showed that eth-
anol easily diffuses into ex vivo CSF samples via air transmis-
sion. Ethanol originated from routinely used skin disinfectants
containing ethanol and from laboratory procedures. Ethanol
affected the CSF sample matrix at concentrations above ~9.4 mM and obscured a significant part of the 1H-NMR
spectrum. CSF sample preparation for 1H-NMR-based meta-
bolomics analyses should therefore be carried out in a well-
ventilated atmosphere with laminar flow, and use of ethanol
should be avoided. ~9.4 mM and obscured a significant part of the 1H-NMR
spectrum. CSF sample preparation for 1H-NMR-based meta-
bolomics analyses should therefore be carried out in a well-
ventilated atmosphere with laminar flow, and use of ethanol
should be avoided. Keywords Metabolomics . Cerebrospinal fluid . Ethanol . NMR . Biobank Anal Bioanal Chem (2015) 407:4835–4839
DOI 10.1007/s00216-015-8663-9 Anal Bioanal Chem (2015) 407:4835–4839
DOI 10.1007/s00216-015-8663-9 NOTE NOTE Introduction Cerebrospinal fluid (CSF) is generally believed to reflect brain
physiology and is therefore frequently used to assess bio-
markers for brain disorders [1–3]. CSF sampling, processing,
and storage procedures are pivotal to prevent biochemical
ex vivo changes which may invalidate the observed profiles. Recently, a consensus protocol was published [4] to standard-
ize CSF sampling and storage for CSF biobanks. However,
despite rigorous adherence to this and other protocols, we
detected substantial amounts of ethanol disturbing the 1H-
NMR spectra of human CSF samples. Other studies reporting
the presence of ethanol in CSF have interpreted this as either a
contaminant [5, 6] or due to the disease process [7, 8]. Sonia A. van der Sar and Ronald Zielman contributed equally to this
work. Electronic supplementary material The online version of this article
(doi:10.1007/s00216-015-8663-9) contains supplementary material,
which is available to authorized users. In the present study, we set out to identify the origin of
ethanol contamination and to assess the effect it has on the
sample matrix. * Michel D. Ferrari
M.D.Ferrari@lumc.nl * Michel D. Ferrari
M.D.Ferrari@lumc.nl * Michel D. Ferrari
M.D.Ferrari@lumc.nl 1
Center for Proteomics and Metabolomics, Leiden University Medical
Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands 2
Department of Neurology, Leiden University Medical Center,
P.O. Box 9600, 2300 WB Leiden, The Netherlands 3
Department of Human Genetics, Leiden University Medical Center,
P.O. Box 9600, 2300 RC Leiden, The Netherlands 1
Center for Proteomics and Metabolomics, Leiden University Medical
Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
2
Department of Neurology, Leiden University Medical Center,
P.O. Box 9600, 2300 WB Leiden, The Netherlands
3
Department of Human Genetics, Leiden University Medical Center,
P.O. Box 9600, 2300 RC Leiden, The Netherlands Human CSF samples Human CSF samples Human CSF samples The cohort in which we initially discovered ethanol contami-
nation consisted of human CSF samples that were collected 4836 S.A. van der Sar et al. for research purposes, namely to investigate the patho-
physiology of migraine. The study protocol was approved
by the Medical Ethical Committee of Leiden University
Medical Center (LUMC), and all subjects gave written
informed consent prior to collection. For disinfection of
the skin, chlorhexidine (5 g/L)/denatured ethanol 70 %
(Pharmacy LUMC, art. no.: 909602) was used. CSF sam-
ples were taken via lumbar puncture. Three different sam-
ple handling protocols were used simultaneously for the
preparation of samples for mass spectrometry (MS)-based
metabolomics (methods 1 and 2) and for 1H-NMR-based
metabolomics (method 3) (see Fig. 1). Method 3 was used
for the initial 1H-NMR measurements in which we detect-
ed ethanol contamination. For sampling of method 3,
4.8 mL of CSF dripped through air into a 15-mL polypro-
pylene falcon tube. Directly after sampling, the CSF was
centrifuged at 4 °C for 5 min (2000 rpm, 747 g). Follow-
ing centrifugation, the supernatant was transferred to a
15-mL polypropylene falcon tube and divided in 0.5-mL
aliquots, placed on dry ice within 30 min of sampling and
transferred to −80 °C for storage within 60 min of sam-
pling. In sampling methods 1 and 2, cold ethanol was
added to the CSF during the sample handling to denature
proteins and to be able to thaw the samples before mea-
surements at lower temperatures. Because this was a like-
ly source of ethanol contamination, we also analysed three
CSF samples obtained for clinical diagnostic purposes to
check whether ethanol is also present in these samples. Disinfection of the skin with chlorhexidine (5 g/L)/dena-
tured ethanol 70 %, sample collection and sample pro-
cessing of these clinical samples were similar to the re-
search samples with the exception that ethanol was not
used anywhere in the handling of these samples. For for research purposes, namely to investigate the patho-
physiology of migraine. The study protocol was approved
by the Medical Ethical Committee of Leiden University
Medical Center (LUMC), and all subjects gave written
informed consent prior to collection. For disinfection of
the skin, chlorhexidine (5 g/L)/denatured ethanol 70 %
(Pharmacy LUMC, art. no.: 909602) was used. CSF sam-
ples were taken via lumbar puncture. Simulation of CSF sampling protocol with saline To determine the origin of ethanol during our sample process-
ing procedure, we performed a CSF sampling protocol simu-
lation. A 0.9 % NaCl solution (saline) was used instead of
CSF in an exact simulation of the CSF sampling protocol
(see Fig. 1). Different pipetting methods were used for mass
spectrometry (MS)-based metabolomics (methods 1 and 2)
and for 1H-NMR-based metabolomics (method 3). In methods
1 and 2, one pipette was used throughout the entire sampling
protocol, including pipetting of ethanol. In method 3, a differ-
ent pipette was used which had never been exposed to ethanol;
only these samples were measured with 1H-NMR as described
below. Human CSF samples Three different sam-
ple handling protocols were used simultaneously for the
preparation of samples for mass spectrometry (MS)-based
metabolomics (methods 1 and 2) and for 1H-NMR-based
metabolomics (method 3) (see Fig. 1). Method 3 was used
for the initial 1H-NMR measurements in which we detect-
ed ethanol contamination. For sampling of method 3,
4.8 mL of CSF dripped through air into a 15-mL polypro-
pylene falcon tube. Directly after sampling, the CSF was
centrifuged at 4 °C for 5 min (2000 rpm, 747 g). Follow-
ing centrifugation, the supernatant was transferred to a
15-mL polypropylene falcon tube and divided in 0.5-mL
aliquots, placed on dry ice within 30 min of sampling and
transferred to −80 °C for storage within 60 min of sam-
pling. In sampling methods 1 and 2, cold ethanol was
added to the CSF during the sample handling to denature
proteins and to be able to thaw the samples before mea-
surements at lower temperatures. Because this was a like-
ly source of ethanol contamination, we also analysed three
CSF samples obtained for clinical diagnostic purposes to
check whether ethanol is also present in these samples. Disinfection of the skin with chlorhexidine (5 g/L)/dena-
tured ethanol 70 %, sample collection and sample pro-
cessing of these clinical samples were similar to the re-
search samples with the exception that ethanol was not
used anywhere in the handling of these samples. For additional details on sampling and processing of the re-
search and clinical CSF samples, see the Electronic Sup-
plementary Material (ESM). 1H-NMR data acquisition and processing 1H-NMR data were obtained using a Bruker 600 MHz AVAN
CE II spectrometer equipped with a 5-mm TCI cryoprobe and
a z-gradient system; a Bruker SampleJet sample changer sys-
tem was used for sample transfer; samples were kept at 6 °C
while queued for acquisition. One-dimensional (1D) 1H-NMR
spectra were recorded at 300.0 K using the first increment of a
NOESY pulse sequence [9] with presaturation (γB1=50 Hz)
during a relaxation delay of 4 s and a mixing time of 10 ms for
efficient water suppression [10]. The duration of 90° pulses
was automatically calibrated for each individual sample using
a homonuclear gated nutation experiment [11] on the locked
and shimmed samples after automatic tuning and matching of
the probe head. Sixteen scans of 200,704 points covering 18,
028 Hz were recorded and zero filled to 262,144 complex
points prior to Fourier transformation; an exponential window
function was applied with a line-broadening factor of 1.0 Hz. The spectra were manually phased and baseline corrected and
automatically referenced to the internal standard (TSP=
0.0 ppm). Phase offset artifacts of the residual water resonance
were manually corrected using a polynomial of degree 5 least
square fit filtering of the free induction decay (FID) [12]. Fig. 2 Cross-contamination of CSF by diffusion of ethanol through air. a
Schematic representation of experimental set-up to test for air diffusion of
ethanol. b 1H-NMR overlay spectrum showing the methyl signal of eth-
anol; here, it is used to visualize the relative amount of ethanol found in
test samples from the air diffusion experiment. c Table comparing the
amount of ethanol quantified in the air to sample diffusion test with the
simulation of CSF sampling protocol with saline, clinical CSF samples
and research CSF samples Air to sample diffusion of ethanol To test how fast ethanol can diffuse into CSF via air, we
created a work area simulation. Ethanol (1 mL, Biosolve,
96 % analytical grade) was placed into 2 × 1.8-mL
cryotubes (Nunc, art. no. 368632). An up-turned 500-mL
glass beaker was placed over two open cryotubes contain-
ing 1 mL of saline and one open cryotube containing etha-
nol (see Fig. 2a). After 5 min, one saline cryotube was
removed and capped firmly until 1H-NMR analysis. The
second cryotube was removed after 30 min. This process
was repeated without the beaker in the same well-ventilated
work space. All samples were then prepared for and Fig. 1 In-house sample processing protocol used for preparation of
samples for mass spectrometry (MS)-based metabolomics (methods 1
and 2) and for 1H-NMR-based metabolomics (method 3). All aliquots
were immediately placed on dry ice within 30 min of sampling and then
transferred to −80 °C for storage within 60 min were immediately placed on dry ice within 30 min of sampling and then
transferred to −80 °C for storage within 60 min Fig. 1 In-house sample processing protocol used for preparation of
samples for mass spectrometry (MS)-based metabolomics (methods 1
and 2) and for 1H-NMR-based metabolomics (method 3). All aliquots 4837 Ethanol contamination and its effect on 1H-NMR metabolomics 1H-NMR sample preparation Fig. 2 Cross-contamination of CSF by diffusion of ethanol through air. a
Schematic representation of experimental set-up to test for air diffusion of
ethanol. b 1H-NMR overlay spectrum showing the methyl signal of eth-
anol; here, it is used to visualize the relative amount of ethanol found in
test samples from the air diffusion experiment. c Table comparing the
amount of ethanol quantified in the air to sample diffusion test with the
simulation of CSF sampling protocol with saline, clinical CSF samples
and research CSF samples In 1.5-mL eppendorf tubes that were placed on ice, 225 μL of
CSF, 0.9 % NaCl, or CSF/ethanol was added to 25 μL of pH
7.0 phosphate buffer (50 mM) in D2O containing 4 mM of
sodium 3-trimethylsilyltetradeuteriopropionate (TSP) and
2.0 mM NaN3. Following thorough mixing by repeated inver-
sion, 190 μL of the sample was transferred into 3-mm NMR
tubes in a cooled rack at 6 °C. Effect of ethanol on the sample matrix A serial dilution was made to test at which level of ethanol
contamination the CSF matrix and thereby the whole 1H-
NMR spectrum is affected. Two hundred microlitres of CSF
was aliquoted into 10×1.5-mL eppendorf tubes containing
ethanol (Biosolve, 96 % analytical grade), decreasing in con-
centration in regular stepped increments so that the final eth-
anol concentrations were 9.1×10−3–500.0 mM. Samples were
prepared for 1H-NMR analysis as described below. analysed by quantitative 1H-NMR spectroscopy as de-
scribed below. Fig. 2 Cross-contamination of CSF by diffusion of ethanol through air. a
Schematic representation of experimental set-up to test for air diffusion of
ethanol. b 1H-NMR overlay spectrum showing the methyl signal of eth-
anol; here, it is used to visualize the relative amount of ethanol found in
test samples from the air diffusion experiment. c Table comparing the
amount of ethanol quantified in the air to sample diffusion test with the
simulation of CSF sampling protocol with saline, clinical CSF samples
and research CSF samples Discussion We showed that CSF samples can be contaminated with
ethanol during routine sampling and processing steps via
diffusion through air. Most likely sources of ethanol are (i)
disinfection of the skin with chlorhexidine/70 % ethanol
prior to lumbar puncture and (ii) use of ethanol in the same
room as where the CSF samples were prepared for 1H-NMR
measurements. Ethanol affected the CSF sample matrix at
concentrations above ~9.4 mM and obscured a significant
part of the 1H-NMR spectrum. Ethanol in human CSF samples An effect of high ethanol concentrations on the CSF matrix
was demonstrated in a spiking experiment. A distinctive up-
field shift of small metabolite signals is observed at ethanol
concentrations of 9.38 mM and higher (Fig. 3). Ethanol was detected in 1H-NMR spectra of not only all re-
search CSF samples but also in three clinical CSF samples
where ethanol is only used during disinfection and not during
sample processing (see Fig. 2c). In the research samples, the
ethanol levels varied between 0.08 and 10.0 mM and, in the
three clinical samples, the ethanol levels were 9.2×10−3,
0.023 and 0.063 mM. Simulation of CSF sampling protocol with saline In the simulation experiment, ethanol was detected in all sam-
ples, even in method 3 (Fig. 1) in which no ethanol was used. Furthermore, we observed a trend of increasing ethanol con-
centration from 0.070 to 0.203 mM (mean) in the aliquots of
method 3, which was related to the time the cryotubes were
uncapped during aliquoting. These findings suggested that the
ethanol arose from cross-contamination by air diffusion dur-
ing sampling and processing procedures. The most important effect of ethanol contamination is on
the interpretation of the 1H-NMR spectra. The two signal
groups of ethanol at 1.18 and 3.66 ppm can mask metabo-
lites that lie in the same chemical shift region of 1H-NMR
spectra. The methylene quartet at 3.66 ppm will itself be
masked, as it lies in the heavily crowded glucose region of
the spectrum. The methyl signal of the small metabolite 3-
hydroxybutyric acid, however, normally occurs in the same
region of the CSF spectrum as the ethanol methyl triplet
(1.18 ppm). In the presence of ethanol, the detection and
subsequent quantification of this metabolite are heavily
compromised. 1H-NMR data analysis Quantification of ethanol peaks was carried out by iterative
line fitting in the frequency domain (Topspin version 2.1,
Bruker Biospin; MDCON command). analysed by quantitative 1H-NMR spectroscopy as de-
scribed below. 4838 S.A. van der Sar et al. References References spectrum alignment might be required prior to analysis in cases
of severe matrix distortion; 3) processing of CSF samples for
1H-NMR analysis in an ethanol-free zone or well-ventilated
area such as a laminar flow cabinet; 4) use of non-ethanol-
containing disinfectant instead of chlorhexidine/ethanol. 1. Mandal R, Guo AC, Chaudhary KK et al (2012) Multi-platform
characterization of the human cerebrospinal fluid metabolome: a
comprehensive and quantitative update. Genomics Med 4(4):38 2. Tumani H, Teunissen C, Süssmuth S et al (2008) Cerebrospinal
fluid biomarkers of neurodegeneration in chronic neurological dis-
eases. Expert Rev Mol Diagn 8(4):479–494 We believe that the recommendations resulting from this
study should be considered when collecting CSF for biomark-
er research in brain disorders. 3. Zhang AH, Sun H, Wang XJ (2013) Recent advances in metabolo-
mics in neurological disease, and future perspectives. Anal Bioanal
Chem 405:8143–8150 Acknowledgments
This study was supported by the Netherlands Or-
ganisation for Scientific Research (VICI grant 918.56.601), the Nether-
lands Organisation for Health Research and Development (Clinical Fel-
lowship 90700217), the Center of Medical System Biology (CMSB)
established by the Netherlands Genomics Initiative/Netherlands Organi-
sation for Scientific Research (NGI/NWO) and by funding from the Eu-
ropean Union’s Seventh Framework Programme (2007–2013) under
grant agreement no. 602633. 4. Teunissen CE, Petzold A, Bennett JL et al (2009) A consensus
protocol for the standardization of cerebrospinal fluid collection
and biobanking. Neurology 73(22):1914–1922 5. Holmes E, Tsang TM, Huang JT et al (2006) Metabolic pro-
filing of CSF: evidence that early intervention may impact on
disease progression and outcome in schizophrenia. PLoS Med
3(8):1420–1428 6. Wijeyesekera A, Selman C, Barton RH et al (2012) Metabotyping
of long-lived mice using (1)H NMR spectroscopy. J Proteome Res
11(4):2224–2235 Conflict of interest
S.A. van der Sar reports no disclosures. R. Zielman
has received support for conference visits from Menarini and Allergan. G.M. Terwindt has received grants and consultancy/industry support
from Merck, Janssen-Cilag, Almirall, Allergan and Menarini, and inde-
pendent support from the Netherlands Organisation for Scientific Re-
search (NWO). A.M.J.M. van den Maagdenberg reports no disclosures. A.M. Deelder reports no disclosures. O.A. Mayboroda reports no disclo-
sures. A. Meissner reports no disclosures. M.D. Ferrari has, in the past
3 years, received grants and consultancy/industry support from
Medtronic, Menarini and Merck, and independent support from the Neth-
erlands Organisation for Scientific Research (NWO). 7. Air to sample diffusion of ethanol To test the hypothesis of ethanol diffusion through air, we
created a work area simulation. Ethanol readily diffused into
an open vessel containing saline. After 5 min under a beaker,
ethanol was detected at 0.82 mM compared to 7.2×10−3 mM
after 5 min open on the lab bench. After 30 min, ethanol had
increased to 2.5 and 0.055 mM, respectively (see Fig. 2b, c). These levels are in the same range as the CSF and saline
samples prepared using sampling handling method 3 (see
Fig. 2c). There are several solutions to minimize the risk of ethanol
contamination and to mitigate its effects on metabolomics anal-
ysis, as follows: 1) ethanol regions can sometimes be removed
from 1H-NMR spectra prior to multivariate analysis; 2) Fig. 3
1H-NMR spectra overlay (a) and graph (b) showing the effect of ethanol on the chemical shift of the α-anomeric proton of D-glucose as an
example Fig. 3
1H-NMR spectra overlay (a) and graph (b) showing the effect of ethanol on the chemical shift of the α-anomeric proton of D-glucose as an
example Ethanol contamination and its effect on 1H-NMR metabolomics 4839 References Meshitsuka S, Morio Y, Nagashima H, Teshima R (2001) 1H-NMR
studies of cerebrospinal fluid: endogenous ethanol in patients with
cervical myelopathy. Clin Chim Acta 312(1–2):25–30 8. Agapejev S, Vassilieff I, Curi PR (1992) Alcohol levels in cerebro-
spinal fluid and blood samples from patients under pathological
conditions. Acta Neurol Scand 86(5):496–500 9. Kumar A, Ernst RR, Wuethrich K (1980) A two-dimensional nu-
clear overhauser enhancement (2D NOE) experiment for the eluci-
dation of complete proton-proton cross-relaxation networks in bio-
logical macromolecules. Biochem Biophys Res Commun 95(1):1–
6 10. Price WS (1999) Water signal suppression in NMR spectroscopy. Ann Rep NMR Spectrosc 38(1):289–354 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. 11. Wu PSC, Otting G (2005) Rapid pulse length determination in high-
resolution NMR. J Magn Reson 176(1):115–119 12. Coron A, Vanhamme L, Antoine JP, Van Hecke P, Van Huffel S
(2001) The filtering approach to solvent peak suppression in MRS:
a critical review. J Magn Reson 152(1):26–40
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Interplay of beam polarisation and systematic uncertainties at future $e^+e^-$ colliders
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∗Talk presented at
*** The European Physical Society Conference on High Energy Physics (EPS-HEP2021), ***
*** 26-30 July 2021 ***
*** Online conference, jointly organized by Universität Hamburg and the research center DESY *** Interplay of beam polarisation and systematic
uncertainties at future e+e- colliders Jakob Beyer∗1,2 and Jenny List1 1Deutsches Elektronen-Synchrotron DESY, Notkestr. 85, 22607 Hamburg, Germany
2Universität Hamburg, Hamburg, Germany iv:2111.06687v1 [hep-ex] 12 Nov 2021 arXiv:2111.06687v1 [hep-ex] 12 Nov 2021 November 15, 2021 1
Introduction The latest update of the European strategy of particle physics identifies “[a]n electron-positron
Higgs factory [as] the highest-priority next collider” [1]. Proposed future e+e−Higgs factories
have either no beam polarisation, e−beam polarisation or polarisation of both beams. This
situation raises the question which expected advantages beam polarisation can bring. Among
the frequently studied advantages are the increase in effective luminosity, the suppression of
backgrounds and the analysis of chiral behaviour. In the context of dark matter searches, it
has also been shown that the combination of datasets with different beam polarisations reduces
the impact of global scaling systematic uncertainties drastically, if they are at least partially
correlated between the data sets [2]. The study presented here investigates to which extent
beam polarisation could help to control both global and shape-dependent systematic effects in the
context of electroweak precision measurements. For this purpose a framework to simultaneously
fit differential distributions from multiple physics processes and various data sets with different
polarisations has been developed [3]. The fit performs a Poissonian log-likelihood maximisation
using the Minuit2 framework of ROOT. Proposed future colliders motivate the polarisation and luminosity configurations used in this
study [4]. The polarisation scenarios are an unpolarised scenario (Pe−= Pe+ = 0%) with only
one unpolarised dataset, a e−-only polarised scenario with Pe−= ±80% and Pe+ = 0% and
a luminosity sharing of 50 : 50 between the two datasets, and a fully polarised scenario with
Pe−= ±80% and Pe+ = ±30% and an optimized luminosity sharing of 45 : 45 : 5 : 5 where
the first two are the opposite-sign and the second two the same-sign datasets. Two luminosity
scenarios with a factor 5 difference are tested: L = 2 ab−1 and L = 10 ab−1. All polarisations and the luminosity can vary freely and can be given a Gaussian constraint
to represent external measurements, e.g. here [5, 6]: ∆L/L = 3 × 10−3 and ∆P/P = 2.5 × 10−3
(∆P = 2.5×10−3) for polarised (unpolarised) beams. The helicity flip between polarised datasets
is not assumed to be perfect, so that each signed polarisation is a separate parameter. As a prototype for shape-depending systematic effects, a simplified model of the muon ac-
ceptance is introduced as a parametrised detector effect as described in [3]. 1
Introduction The model assumes
that muons are perfectly reconstructed in the central region down to a specific limiting angle
below which they can not be reconstructed. The limiting angles can be varied independently for
the forward and backward direction; their nominal values are chosen to be 7◦here, inspired by
the ILD detector [7]. Abstract arXiv:2111.06687v1 [hep-ex] 12 Nov Future high-energy e+e−colliders will provide some of the most precise tests of the
Standard Model. Statistical uncertainties on electroweak precision observables and triple
gauge couplings are expected to improve by orders of magnitude over current measurements. This provides a new challenge in accurately assessing and minimising the impact of
systematic uncertainties. Beam polarisation may hold a unique potential to isolate and
determine the size of systematic effects. So far, studies have mainly focused on the statistical
improvements from beam polarisation. This study aims to assess, for the first time, its
impact on systematic uncertainties. arXiv:2111.06687v1 [hep-ex] A combined fit of precision observables, such as chiral fermion couplings and anoma-
lous triple gauge couplings, together with experimental systematic effects is performed on
generator-level differential distribution of 2-fermion and 4-fermion final-states. Different
configurations of available beam polarisations and luminosities are tested with and without
systematic effects, and will be discussed in the context of the existing projections on fermion
and gauge boson couplings from detailed experimental simulations. 1 2 2
Physical parameters and systematic effects A measurement of all chiral parameters at an unpolarised
collider would require the reconstruction of final state polarisations, i.e. from τ-leptons. 2
Physical parameters and systematic effects This contribution focuses on di-muon production at 250 GeV, separated into events returning
to the Z pole and high-
√
s′ events, using generator-level events created for the 250 GeV ILD
production [8], produced with WHIZARD2.8 [9, 10]. Previous work used a set of processes with
two and four fermions in the final state [11, 3], but did not yet include the angular acceptance. The di-muon distributions are one-dimensional distributions of the µ−polar angle in the
di-muon rest frame, separated into a high-energy part with
√
s′ ∈[180, 275] GeV (“high-Q2”) and
radiative-return part with
√
s′ ∈[81, 101] GeV (“return-to-Z”). The di-muon distributions are one-dimensional distributions of the µ−polar angle in th
di-muon rest frame, separated into a high-energy part with
√
s′ ∈[180, 275] GeV (“high-Q2”) and
radiative-return part with
√
s′ ∈[81, 101] GeV (“return-to-Z”). √ These distributions are described by six fit parameters for each of the two
√
s′ ranges. The
first three are the unpolarised total cross-section σ0 as well as the initial- and final-state fermion
asymmetries Ae and Aµ, as known from classic Z-pole physics, e.g. at LEP and SLC [12]. At
√s above the Z-pole, a fourth parameter ϵµ is needed to model the effect of Z/γ interference. Finally, two correction parameters kL and kR account for radiative corrections in the shape of
the initial state radiation included in the Monte-Carlo samples. 3 3 0/
SM
0
Ae
A
AFB, 0
kL
kR
k0
e + e
parameters at return-to-Z
0
5
10
15
20
25
30
35
Uncertainty [1E-4]
(Pe , Pe+), L
(80%, 30%), 2ab
1
(80%, 0%), 2ab
1
(0%, 0%), 2ab
1
all P fixed
Figure 1: Precision on the return-to-Z muon pair production parameters for varius collider
setups. Crosses represent result with fixed polarisation amplitudes. Figure 1: Precision on the return-to-Z muon pair production parameters for varius collider
setups. Crosses represent result with fixed polarisation amplitudes. In the case of an unpolarised collider, the Ae, Aµ and ϵµ parameters combine to AF B, and kL
and kR merge into the unpolarised k0. A measurement of all chiral parameters at an unpolarised
collider would require the reconstruction of final state polarisations, i.e. from τ-leptons. In the case of an unpolarised collider, the Ae, Aµ and ϵµ parameters combine to AF B, and kL
and kR merge into the unpolarised k0. 3
Accessing chiral behavior with beam polarisation Figure 1 shows the resulting precisions on the six (or three in the unpolarised case) fit parameters
describing the Z-return samples for various assumptions on the availability of beam polarisation,
all for an integrated luminosity of 2 ab−1, and with fixed angular acceptance parameters. The total unpolarised cross-section is determined in all cases at the level of 3×10−3, i.e. is – in
this setup – only limited by the assumed precision of the luminosity measurement. In the case that
both beams are polarised (blue), the two asymmetry parameters Ae and Aµ can be determined
at the level of 7×10−4, which is about two times more precise than AF B in the unpolarised case
(grey). This illustrate the significant additional information provided by the polarised data sets. It can also be noted that if both beams are polarised, there is hardly any additional uncertainty
from the finite knowledge of the beam polarisation, which can be seen from the fact that there
is no (Aµ) or just a tiny (Ae) improvement when the polarisation parameters are fixed in the
fit (crosses). The situation changes drastically in the absence of positron polarisation (orange):
in this case, Ae cannot be disentangled from the positron polarisation parameter and is limited
directly by the assumed polarimeter precision — in other words, a deviation from P(e+) = 0
cannot be distinguished from a non-SM value of Ae. This can be seen more explicitly in Fig. 2, which shows the degree of correlation between all
the parameters in the fit, in Fig. 2a for the case of only the electron beam polarised, and in Fig. 2b
for the case of both beams polarised. The orange boxes highlight the correlation between the
polarisation parameters and Ae in the Z-return sample, clearly showing how a non-zero positron
polarisation reduces the correlations. The same effect can also be seen for Ae in the high-Q2
sample. On a more subtle level, also correlations between the radiative correction parameters 4 (a)
(b)
Figure 2: Correlation matrices of all parameters included in the fit for L = 2 ab−1 and
scenarios of: (a) only e−polarisation, (b) both beams polarised. The correlations between
the return-to-Z Ae parameter and the polarisation amplitudes are emphasized. (b) (a) (a) (b) Figure 2: Correlation matrices of all parameters included in the fit for L = 2 ab−1 and
scenarios of: (a) only e−polarisation, (b) both beams polarised. 3
Accessing chiral behavior with beam polarisation The correlations between
the return-to-Z Ae parameter and the polarisation amplitudes are emphasized. on one hand and the total cross section and the Ae parameters on the other hand are reduced i
both beams are polarised. Once the 4-fermion processes will be included, there will be further contraints on the po-
larisation amplitudes, so that the polarisation will be known better than just the polarimeter
precision [13]. Still, the full correlation between Ae and Pe+ remains, propagating any potential
bias of Pe+ directly to Ae. For the same reason, a contribution of the di-fermion final states to
the polarisation measurement is only possible if both beams are polarised. 4
Isolating detector effects with beam polarisation The notion of separating effects by their chiral behavior can be translated to the interplay of
physical and systematic effects. Detector effects are uniquely chirality-independent, compared
to their generally chirality-independent physical counterparts. A combined fit to all polarised
datasets of a polarised collider could use the different chiral behaviour to reduce correlation
between physical and systematic effects. This may reduce the impact of systematic uncertainties
(e.g from detector effects) which can be assumed to be correlated between data sets with different
polarisation signs. This is usually the case when the beam helicities can be flipped sufficiently
fast, e.g. at the level of a few Hz, and in a randomised way. This has been shown for global
scaling uncertainties, like the luminosity measurement [2], and will be investigated in this study
with a shape-dependent effect, taking as example the angular acceptance for muon reconstruction
introduced above. In this very simplified case of just two parameters describing a perfect “box-like” acceptance,
it turns out that the only change to the results presented in the previous section occurs for the
radiative-correction parameters k. Figure 3 compares the precisions on these parameters for the
case of fixed muon acceptance (stars) with the case of two additional free acceptance parameters. The additional systematic uncertainty due to the muon acceptance makes up 14% of the total 5 kL
kR
k0
e + e
parameters at return-to-Z
0
5
10
15
20
25
30
35
Uncertainty [1E-4]
(Pe , Pe+), L
(80%, 30%), 2ab
1
(80%, 0%), 2ab
1
(0%, 0%), 2ab
1
(0%, 0%), 10ab
1
acc. fixed
Figure 3: Precision on the radiative correction parameters for return-to-Z muon pair produc-
tion for varius collider setups. Stars represent result with fixed muon acceptance parameters. Figure 3: Precision on the radiative correction parameters for return-to-Z muon pair produc-
tion for varius collider setups. Stars represent result with fixed muon acceptance parameters. uncertainty on the k parameters in the unpolarised case. Adding e−beam polarisation leads to
a relative impact of 7(5)% on individual kL(kR) parameters. 1Subsequent studies have shown that this a consequence of the choice of parameters which lead to correlations
between kL and kR. The same 14% impact is observed in the polarised cases when choosing a more appropriate
linear combination of kL and kR. 4
Isolating detector effects with beam polarisation An additional polarisation of the
e+ does not lead to a further reduction of the relativ systematic uncertainty.1 In case of the di-muon events, small variations the perfect box-acceptance around the default
7◦in the detector system thus lead to very limited changes in the angular distributions in
the restframe on the di-muon, which have very different shapes that the the effects of the other
parameters and thus can be easily determined from the data themselves. Even if so far only tested
in this very simplified Ansatz, this robustness of the physics parameters w.r.t. the acceptance is
in principle very good news. In remains to be studied in the future, however, whether this statement holds up also for the
four-fermion case, with more subtle angular effects e.g. from anomlous triple gauge couplings,
and for more realistic parametrisations of the acceptance. 5
Conclusion Beam polarisation separates effects by their chiral behaviour. This is especially relevant for those
effects with the same or a similar differential shape. This chiral sensitivity is essential to gain
direct access to all chiral parameters in muon pair production - and difermion production in
general. A single polarised beam gives access to the full set of chiral parameters. Polarising
both beams also removes remaining full correlations between the chiral parameters and the
polarisation amplitudes. In general, every additional beam polarisation decreases correlations
between parameters. A first, very simplified Ansatz to include a systematic uncertainty on the
muon acceptance profed the fit results to be very robust against the exact knowledge of the onset
of the acceptance in the forward (and backward) region of the detector. In the future it needs
to be investigated in how far this result holds also in the case of more realistic parametrisations. 6 Acknowledgments This work was funded by the Deutsche Forschungsgemeinschaft under Germany’s Excellence
Strategy – EXC 2121 “Quantum Universe” – 390833306. It has benefited from the computing
services provided by the German National Analysis Facility (NAF)[14]. This work was funded by the Deutsche Forschungsgemeinschaft under Germany’s Excellence
Strategy – EXC 2121 “Quantum Universe” – 390833306. It has benefited from the computing
services provided by the German National Analysis Facility (NAF)[14]. References [1] European Strategy Group, “2020 Update of the European Strategy for Particle Physics,”
CERN-ESU-013, doi:10.17181/ESU2020. [2] M. Habermehl, M. Berggren and J. List, Phys. Rev. D 101 (2020) no.7, 075053. [3] J. Beyer and J. List, “Isolating systematic effects with beam polarisation at e+e−colliders,”
arXiv:2105.09691 [hep-ex]. [4] R. K. Ellis et al. “Physics Briefing Book: Input for the European Strategy for Particle
Physics Update 2020,” arXiv:1910.11775 [hep-ex]. [5] B. Vormwald, J. List and A. Vauth, JINST 11 (2016) no.01, P01014. [6] I. Božović Jelisavčić et al., JINST 8 (2013), P08012. [6] I. Božović Jelisavčić et al., JINST 8 (2013), P08012. [7] H. Abramowicz et al. [ILD Concept Group], “International Large Detector: Interim Design
Report,” arXiv:2003.01116 [physics.ins-det]. [8] M. Berggren, “Generating the full SM at linear colliders,” PoS ICHEP2020 (2021), 903. [9] W. Kilian, T. Ohl and J. Reuter, Eur. Phys. J. C 71 (2011), 1742. [10] M. Moretti, T. Ohl and J. Reuter, “O’Mega: An Optimizing matrix element generator,”
arXiv:hep-ph/0102195 [hep-ph]. [11] J. Beyer, R. Karl and J. List, “Precision measurements of Triple Gauge Couplings at future
electron-positron colliders,” arXiv:2002.02777 [hep-ex]]. [12] M. E. Peskin, Conf. Proc. C 8708101 (1987) 1-45, SLAC-PUB-4601. [13] R. Karl, PhD Thesis, Hamburg University (2019), doi:10.3204/PUBDB-2019-03013. [14] A. Haupt et al. [NAF], “The NAF: National Analysis Facility at DESY,” J. Phys. Conf. Ser. 219 (2010), 052007.
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The influence of maternal singing on well-being, postpartum depression and bonding – a randomised, controlled trial
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BMC pregnancy and childbirth
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Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501
https://doi.org/10.1186/s12884-021-03933-z Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501
https://doi.org/10.1186/s12884-021-03933-z (2021) 21:501 Open Access The influence of maternal singing on
well-being, postpartum depression and
bonding – a randomised, controlled trial Verena Wulff1*
, Philip Hepp2,3, Oliver T. Wolf4, Tanja Fehm5 and Nora K. Schaal1* Verena Wulff1*
, Philip Hepp2,3, Oliver T. Wolf4, Tanja Fehm5 and Nora K. Schaal1* * Correspondence: verena.wulff@hhu.de; nora.schaal@hhu.de * Correspondence: verena.wulff@hhu.de; nora.schaal@hhu.de
1Department of Experimental Psychology, Heinrich-Heine-University
Düsseldor, Universitätsstraße 1, 40225 Düsseldorf, Germany
Full list of author information is available at the end of the article * Correspondence: verena.wulff@hhu.de; nora.schaal@hhu.de
1Department of Experimental Psychology, Heinrich-Heine-University
Düsseldor, Universitätsstraße 1, 40225 Düsseldorf, Germany
Full list of author information is available at the end of the article Abstract Background: Postpartum depression is fairly common in new mothers and moreover associated with impaired
bonding and poor maternal well-being. The aim of the present study was to investigate the impact of a mother-
infant singing intervention within the first three months after birth on maternal well-being, depressive symptoms
and bonding. Methods: 120 women who were recruited at the maternity ward at the University Clinic in Düsseldorf took part in
this prospective, randomised-controlled study. Beside the baseline measurement 1–3 days after childbirth,
depressive symptoms, maternal well-being and mother-infant bonding were evaluated with questionnaires before
(two weeks after birth) and after (twelve weeks after birth) the intervention took place. The experimental group
(n = 59) participated in several singing intervention sessions while the control group (n = 61) did not. In the
intervention group, salivary cortisol as well as attachment and mood were assessed immediately before and after
the singing sessions. Results: The participants of the intervention group showed a significant reduction of cortisol (p = .023) and an
improvement of attachment and mood from start to end of the intervention session (all p ≤.008). However, no
prolonged effects were revealed beyond the intervention sessions as the two groups did not differ regarding the
alterations of the primary outcomes postpartum depression (interaction effect p = .187) and postpartum bonding
(interaction effect p = .188) in the 10-week period from two up to twelve weeks after childbirth (all p > .05). Additional analyses of singing habits at home in both groups, revealed that only in the singing group more
frequent singing was associated with less anxiety and more well-being of the mother. Conclusion: Singing towards the infant seems to have positive immediate effects on the well-being of new
mothers (on subjective variables as well as physiological measurements). However, the intervention did not lead to
more long lasting positive effects although several limitations should be considered. Trial registration: DRKS00015178 (registered at the German Clinical Trial Registry), date of registration: 09.11.2018. Trial registration: DRKS00015178 (registered at the German Clinical Trial Registry), date of registration: 09.11.2018. Keywords: Maternal health Mother-infant bonding Music Singing Postpartum depression Keywords: Maternal health, Mother-infant bonding, Music, Singing, Postpartum depression Background Women who have developed postpartum depression
suffer from impaired mental and psychological health (like
lower self-esteem, anxiety or emotional problems) as well
as lower quality of life, less social relationships and an in-
creased risk to develop addictive behaviors [6]. However,
postpartum depression is not only a problem for the af-
fected woman but can also have short- and long-term im-
pact on the child. As a consequence of depression, the
mother shows less interaction with the infant, less positive
responsiveness to the infant’s affect and overall less posi-
tive emotions [7, 8]. This lack of response and interaction
can have a substantial impact on the baby. Studies have
shown that postpartum depression of the mother can
cause eating and sleeping difficulties of the baby [7], influ-
ence the temperament of the infant [9] and the emotional
tie between mother and baby [10] which is known as
mother-infant bonding or attachment. Disturbances in
parts of the attachment system (such as disturbed respon-
siveness, less interaction or flattened maternal emotions
due to stress, anxiety or depression) can have a wide range
of negative consequences such as impaired cognitive or
emotional development that may last over years [11, 12]. Thus it is desirable to develop suitable interventions to
improve postnatal maternal well-being in order to prevent
negative consequences such as depressive symptoms and
impaired mother-infant bonding. In recent years, an increasing body of research showed For the time period after birth, only a few studies ex-
amined the effects of music on the emotional state of
the mother. A review and meta-analysis of Yang et al. [21], summerised positive effects on postpartum depres-
sion and anxiety. Of the analysed seven studies that con-
ducted daily music therapy sessions with a duration of a
minimum of 30 min, the majority found positive effects
on postpartum depressive symptoms and anxiety while
one study reported additional positive effects on pain,
sleep satisfaction and attachment. Although positive ef-
fects were found, the authors highlighted the heterogen-
eity of study designs and sample sizes and concluded
that more research is needed to confirm the promising
effects of music interventions after childbirth. As a special form of making music, singing is used by
mothers around the world as a tool to relax and calm
the baby [22–24]. Background Hinterberger [16] demonstrated in their review of 28
studies. In sum, they found a positive impact of different
kinds of music interventions like passive music listening,
active singing and playing music or improvising on de-
pression score improvements. Beyond that, positive ef-
fects of music have been reported in the context of
childbirth [17]. A review of Lin et al. [18] summarises
positive effects such as significant decreases of anxiety
scores or improvements in physiological parameters like
heart rate and blood pressure of the mother during
labour. Furthermore, Nwebube, Glover and Stewart [19]
showed that pregnant women who listened to special
composed songs for pregnant women report lower anx-
iety and depression scores compared to a control group
that conducted daily relaxation. In a study by Wulff
et al. [20] the immediate and more prolonged effects of
a music and a singing intervention were explored in
pregnant women during the last trimester of pregnancy. Immediate improvements of salivary cortisol, oxytocin
and maternal mood were found while the expect-
ant mother listened to music or sang lullabies for the
unborn baby. In addition to that, more prolonged effects
of the interventions were reported for the perceived
closeness to the baby and self-efficacy when compared
to a control group. g
After birth, about half of new mothers suffer from tem-
porary mood disturbances such as tearfulness, emotional
lability, feelings of inability to cope with the baby as well
as worries about the baby’s well-being, that are known as
the "Baby Blues", which usually occur within the first week
after birth and resolve after a few hours or several days [1,
2]. One reason for the mood disturbances after birth is the
withdrawal of reproductive hormones that impact further
systems in the brain like the hypothalamic-pituitary-
adrenal axis and the limbic system that are associated with
depression [3]. Normally, the biological systems regulate
and stabilise over time but sometimes the disturbances re-
main and result in postnatal depression [3, 4]. In the case
that the depressive symptoms last longer than two weeks,
it is possible that a postpartum depression manifests [5]. Postpartum depression is not uncommon and about 10–
15% of new mothers are affected by it within the first
twelve months after childbirth. The highest prevalence for
developing postnatal depression is up to the third month
after childbirth and thereafter the prevalence decreases
[4]. © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
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licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
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data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 15 Page 2 of 15 Page 2 of 15 Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 Background There is evidence that music and sing-
ing can affect bonding and interactions in social con-
texts, as well as possessing evolutionary aspects of infant
care and parental attention [25–27]. It has been shown
that directly after birth, infants have the ability to per-
ceive and process complex musical stimuli [28, 29]. Add-
itionally, maternal singing has the power to modulate
the infant’s arousal and even its physiological state after
birth [30–32]. The mother is able to adjust the state of
the child for example through the singing style and can
induce decreasing arousal as a response to soothing
singing as well as to particular musical aspects of lulla-
bies [33, 34]. In the course of this, studies showed that In recent years, an increasing body of research showed
beneficial effects of music interventions and music
assisted relaxation techniques on arousal, stress and anx-
iety that are visible in psychological and physiological
parameters [13–15]. Furthermore, beneficial effects of
music were reported in clinical contexts, for example re-
garding the treatment of depression as Leubner and Page 3 of 15 Page 3 of 15 Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 features of songs can indicate context and intention. For
example lullabies can be identified as calming songs in the
context of infant care independent of culture, familiarity
or language [22, 34, 35]. They also seem to have an uni-
versal influence on infants´ states which indicates that
there is a predisposed ability to respond to music [34]. features of songs can indicate context and intention. For
example lullabies can be identified as calming songs in the
context of infant care independent of culture, familiarity
or language [22, 34, 35]. They also seem to have an uni-
versal influence on infants´ states which indicates that
there is a predisposed ability to respond to music [34]. infant bonding [41], the aim of the present study was to
investigate the effect of a music intervention (group
singing with the infants) in comparison to a control
group (no intervention) on some of the most relevant
factors that are associated with maternal and infant
health after birth in particular on maternal depressive
symptoms, well-being and mother-infant bonding. Background In
contrast to previous studies, the impact of singing was
investigated in a randomised controlled trial design and
for the first time, the effects on the subjective factors
mother-infant bonding, depressive symptoms and mater-
nal well-being as well as on salivary cortisol as a physio-
logical marker for stress were explored within one study. In a non-obstretical setting, a positive impact of sing-
ing on affect and arousal was found in a sample of choir
singers that was visible in a significant reduction of
negative affect and a trend of salivary cortisol reduction
in the singing compared to a non-singing group [36]. The release of cortisol is caused by an activation of the
hypothalamus-pituitary-adrenal axis through the limbic
system during stress and therefore it is often used as a
physiological stress-marker that can be easily collected
with saliva samples [37, 38] and shows decreases in the
context of relaxing music and singing interventions [15,
36]. Overall, singing seems to promote health and well-
being [39]. g
p
y
While a baseline measurement was conducted 48 h
after childbirth (baseline) in order to control for group
differences at the time of recruitment, a factorial design
with two parallel arms was used to investigate the im-
pact of a singing intervention in a randomised controlled
study. Therefore, measurements took place before (two
weeks postpartum (T1)) and after (12 weeks after child-
birth (T2)) the intervention group participated in the
intervention sessions, while the control group received
no further care in this period. The effects on mother-
infant bonding, depressive symptoms and maternal well-
being were investigated with questionnaires. Addition-
ally, in the intervention group saliva samples for cortisol
determination were taken to investigate the immediate
effect of the intervention session on a physiological
stress parameter. In relation to previous findings regard-
ing direct effects of music interventions [36, 41], an im-
mediate positive effect was expected during the 45 min
long sessions (from pre to post session) i.e. an increase
of positive affect, a decrease of negative affect and a
stress reduction visible through a cortisol reduction dur-
ing the intervention. Furthermore, in line with previous
studies [41, 42], it was expected that the singing inter-
vention will have a positive effect on all variables and
therefore lower despressive symptoms, higher mother-
infant bonding as well as higher well-being at T2 were
expected. Background With regard to the time of pregnancy, Persico et al. [40] revealed positive effects of a prenatal singing inter-
vention only on postnatal mother-infant bonding and
neonatal crying episodes but no effects during pregnancy
in comparison to a control group. Only a few studies ex-
plored the effect of maternal singing towards the baby in
the postpartum period. Fancourt and Perkins [41] inves-
tigated the effect of mother-infant singing compared to
playing with the baby on the emotional closeness to the
infant and the affect of the mother. With a sample of 43
mother-infant-dyads with three - up to 14-month-olds,
the study revealed in a within-subject design that singing
in a 35 min musical workshop leads to a significant
greater increase of emotional attachment to the baby
and to a greater increase in positive maternal affect as
well as to a larger decrease in cortisol when compared
to a 35 min workshop without singing components. An-
other study, that was conducted with a sample of de-
pressive mothers with their up to ten months old babies,
compared a music group to a play group and a control
group and investigated the impact on depressive symp-
toms
with
a
ten-week-intervention
program
[42]. Women that participated in the music group sang to-
gether and learned new songs whereas the play group in-
cluded creative and sensory play with the babies and the
control group did not receive any kind of intervention. In case of higher depressive symptoms at baseline,
women in the music group showed the fastest improve-
ment after six weeks of workshop participation com-
pared to the play and control group. No significant
impact of group allocation was found for women with a
medium score of depression at baseline. Methods
Sample Therefore, the study was not
blinded as the participants as well as the team members
were aware of the group allocation. The study was ap-
proved by the ethics committee of the Medical depart-
ment of the Heinrich-Heine-University in Düsseldorf
(Germany) and was registered in the “Deutsches Register
Klinischer Studien” (DRKS00015178). The study adheres
to CONSORT guidelines. No harms or unintended
effects were revealed in this study. participants. Due to a larger drop-out through missing
questionnaires (n = 63) or refused attendance to the
intervention sessions (n = 55), the final sample contains
120 women (see Fig. 1). A post hoc power analysis with
G*Power with the given sample of 120 participants, a
low effect size of f = 0.10 and a correlation between mea-
sures of r = .60 revealed a power of 68%. The partici-
pants were aged between 19 and 44 years (M = 33.73,
SD = 4.74) and had a gestational age between 35 + 1 and
42 + 1 weeks (M = 38.81, SD = 1.62) at the time of child-
birth. As criteria for inclusion, women had to be aged
over 18 years, have sufficient knowledge of the German
language and no serious comorbidities or pregnancy and
birth complications. All pregnant women, who met our
inclusion criteria, were offered participation. We did not
screen patients regarding their level of depression or dis-
tress as the aim was to include the whole potential
population and not only women with moderate or high
levels of depression and distress. All participants re-
ceived detailed information for study participation from
a member of the study team on the maternity unit and
gave informed written consent. Following that, they
filled in the first questionnaire and the study team mem-
ber allocated them to the control or intervention group
via a computer-assisted permuted block randomisation
(1:1 allocation ratio). Therefore, the study was not
blinded as the participants as well as the team members
were aware of the group allocation. The study was ap-
proved by the ethics committee of the Medical depart-
ment of the Heinrich-Heine-University in Düsseldorf
(Germany) and was registered in the “Deutsches Register
Klinischer Studien” (DRKS00015178). The study adheres
to CONSORT guidelines. No harms or unintended
effects were revealed in this study. subjective feeling of anxiety and the subscore Trait was
used to measure the general tendency toward anxiety. Methods
Sample Between November 2018 and October 2019, study par-
ticipation was offered to 616 new mothers within 48 h
after childbirth at the Clinic for Gynecology and Obstet-
rics at the University Hospital Duesseldorf and 238
women agreed to take part in the study. A sample size
calculation was conducted with the programm G*Power
[43]. Based on an expected low to medium effect size
(f = 0.15), a power of 80% and an alpha-error of 0.05 in
the present study design, the required sample size was
176 participants (88 per group). We expected several
drop-outs and therefore reached a sample of 238 Based on first promising findings that music and sing-
ing can have a positive effect on postnatal depressive
symptoms of the mother [21, 42] as well as on mother- Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 Page 4 of 15 Page 4 of 15 participants. Due to a larger drop-out through missing
questionnaires (n = 63) or refused attendance to the
intervention sessions (n = 55), the final sample contains
120 women (see Fig. 1). A post hoc power analysis with
G*Power with the given sample of 120 participants, a
low effect size of f = 0.10 and a correlation between mea-
sures of r = .60 revealed a power of 68%. The partici-
pants were aged between 19 and 44 years (M = 33.73,
SD = 4.74) and had a gestational age between 35 + 1 and
42 + 1 weeks (M = 38.81, SD = 1.62) at the time of child-
birth. As criteria for inclusion, women had to be aged
over 18 years, have sufficient knowledge of the German
language and no serious comorbidities or pregnancy and
birth complications. All pregnant women, who met our
inclusion criteria, were offered participation. We did not
screen patients regarding their level of depression or dis-
tress as the aim was to include the whole potential
population and not only women with moderate or high
levels of depression and distress. All participants re-
ceived detailed information for study participation from
a member of the study team on the maternity unit and
gave informed written consent. Following that, they
filled in the first questionnaire and the study team mem-
ber allocated them to the control or intervention group
via a computer-assisted permuted block randomisation
(1:1 allocation ratio). Methods
Sample Each scale consists of 20 statements with response op-
tions on a 4-point likert scale from “almost never” to “al-
most always”. For analysis, an overall sum score was
calculated for each subscale with a possible range from
20 to 80 for which a higher score indicates higher anx-
iety levels. For the measurement of postpartum depression, the
German version of the Edinburgh Postnatal Depression
Scale [45] was used. The Scale consists of 10 statements
and participants are asked to rate the appropriate feeling
within the last 7 days on a 4-point likert scale from 0 to
3. A sum score was calculated which could range from 0
to 30. A higher score reflects more symptoms of depres-
sion and a higher probability to suffer from depression. Mother-infant attachment was measured with the
German version of the Postpartum Bonding Question-
naire [1] that consists of 16 items. Participants had to
state the frequency of attachment-related emotions on a
5-point likert scale from “always” to “never”. The calcu-
lated overall sum score indicates the impairment of
bonding with a higher score indicating greater impair-
ment (possible range 0–80). In addition to the Postpartum Bonding Questionnaire,
mother-infant bonding was measured with a visual
analogue scale where participants had to rate their per-
ceived closeness to the baby with a cross on a 10 cm
line. According to the actual subjective feeling, they were
asked “How close do you feel to your baby?” (visual
analogue scale closeness to the baby) and the response
was given between the anchors “No closeness to the
baby” on the left end and “Maximum closeness to the
baby” on the right end. The score was measured in cm
from the left end of the scale with a possible score be-
tween 0 and 10 cm. Another visual analogue scale was
used to measure the perceived comfort with the mater-
nal role (visual analogue scale comfort with maternal
role). Participants had to rate their answer to the Procedure They were
asked to take part at least once between the third and
the 10th week of the baby’s life so that the starting point
of the intervention period varied slightly between partici-
pants. Women of the intervention group had the possi-
bility to take part in the sessions with their babies up to
three times with the latest possibility of participation in
the 12th week after birth. The control group only re-
ceived standard care, which does not include any mental
health care or screening from hospital staff. At T2 (12
weeks postpartum), all participants received an e-mail
with the link to the second online questionnaire that
contained the same questionnaires as T1 and with a re-
quest for completion within three days. Reminders were
also sent in case of missing completion. As soon as a
woman completed T2, a baby rattle was sent to them as
a thank you for study participation. In the case that par-
ticipants reached cut-off scores for clinical relevance in Additionally, several questions about the use of
music and singing were asked at each time of meas-
urement. Participants were asked “How often did you
sing for your baby since birth?” (frequency singing
(baby)), “How often did you sing for yourself?” (fre-
quency singing (oneself)), “How often did you play
music for your baby since birth?” (frequency playing
music (baby)) and “How often did you listen to music
for yourself?” (frequency listening music (oneself))
and had to rate their answer on a 5-point likert-scale
(“never”, “once per week”, “several times per week”,
“once per day”, “several times per day”). Furthermore, additional items were presented in the
intervention group at T2 to inquire the satisfaction with
the intervention. A visual analogue scale was used where
participants had to rate their answer to the question
“How satisfied were you with the intervention?” (visual
analogue scale satisfaction with intervention) on a 10 cm
line between “Not at all” and “Very satisfied”. They were
also asked whether they would participate again with
“Yes” and “No” as possible answers. In order to assess the emotional state at the beginning
and at the end of the first intervention session, partici-
pants filled in the Self-Assessment Manikin (SAM) [46]. The questionnaire has three items where participants
rate their actual feeling regarding the dimension valence,
arousal and dominance via visual figures. Procedure At T2 (12
weeks postpartum), all participants received an e-mail
with the link to the second online questionnaire that
contained the same questionnaires as T1 and with a re-
quest for completion within three days. Reminders were
also sent in case of missing completion. As soon as a
woman completed T2, a baby rattle was sent to them as
a thank you for study participation. In the case that par-
ticipants reached cut-off scores for clinical relevance in
the questionnaires, they were informed by phone and in-
formation about contact points and possibilities for Eligible women were visited on the maternity unit within
48 h after childbirth and participation was offered to
them. An information sheet was given to the participants
containing information that prior studies showed posi-
tive effects of singing on several aspects of well-being
and therefore the intervention was designed to explore
effects of singing in the postpartum context. In this
course, it was not explicitly mentioned on which out-
comes effects were expected. After informed written
consent was obtained, they were randomised into the
control or intervention group and received the baseline
questionnaire that comprised the State-Trait-Anxiety In-
ventory State and Trait, Postpartum Bonding Question-
naire,
Edinburgh
Postnatal
Depression
Scale,
visual
analogue scales about well-being and closeness to the
baby as well as questions about the use of music and
singing in paper-pencil format. Some general informa-
tion such as age and gestational age was taken from the
medical record. A member of the study team returned a
few hours later to collect the completed questionnaire
and to give a pair of baby socks away as a thank you for
participation. Two weeks after the baseline measure-
ment, participants received an invitation with a link to
fill in the first questionnaire (T1) that contained the
State-Trait-Anxiety Inventory Stubscore State, Postpar-
tum Bonding Questionnaire, Edinburgh Postnatal De-
pression Scale, visual analogue scales about well-being
and closeness to the baby as well as questions about the
use of music and singing via the online-platform SoSci-
Survey [47] with the request to complete the survey
within the next three days. In case of a missing comple-
tion, up to two reminders were sent. Participants of the
intervention group made appointments for the interven-
tion sessions during the following weeks. Questionnaires To measure anxiety, the State-Trait-Anxiety Inventory
[44] was used. The subscale State of the State-Trait-
Anxiety Inventory was used to measure the temporary Fig. 1 Flow chart of the sample. The final sample participated in all measurement time points Fig. 1 Flow chart of the sample. The final sample participated in all measurement time points Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 Page 5 of 15 Page 5 of 15 question “How comfortable do you feel with the mater-
nal role?” between the anchors “Not comfortable” on the
left end and “Maximum comfortable” on the right end. question “How comfortable do you feel with the mater-
nal role?” between the anchors “Not comfortable” on the
left end and “Maximum comfortable” on the right end. Procedure Procedure
Eligible women were visited on the maternity unit within
48 h after childbirth and participation was offered to
them. An information sheet was given to the participants
containing information that prior studies showed posi-
tive effects of singing on several aspects of well-being
and therefore the intervention was designed to explore
effects of singing in the postpartum context. In this
course, it was not explicitly mentioned on which out-
comes effects were expected. After informed written
consent was obtained, they were randomised into the
control or intervention group and received the baseline
questionnaire that comprised the State-Trait-Anxiety In-
ventory State and Trait, Postpartum Bonding Question-
naire,
Edinburgh
Postnatal
Depression
Scale,
visual
analogue scales about well-being and closeness to the
baby as well as questions about the use of music and
singing in paper-pencil format. Some general informa-
tion such as age and gestational age was taken from the
medical record. A member of the study team returned a
few hours later to collect the completed questionnaire
and to give a pair of baby socks away as a thank you for
participation. Two weeks after the baseline measure-
ment, participants received an invitation with a link to
fill in the first questionnaire (T1) that contained the
State-Trait-Anxiety Inventory Stubscore State, Postpar-
tum Bonding Questionnaire, Edinburgh Postnatal De-
pression Scale, visual analogue scales about well-being
and closeness to the baby as well as questions about the
use of music and singing via the online-platform SoSci-
Survey [47] with the request to complete the survey
within the next three days. In case of a missing comple-
tion, up to two reminders were sent. Participants of the
intervention group made appointments for the interven-
tion sessions during the following weeks. They were
asked to take part at least once between the third and
the 10th week of the baby’s life so that the starting point
of the intervention period varied slightly between partici-
pants. Women of the intervention group had the possi-
bility to take part in the sessions with their babies up to
three times with the latest possibility of participation in
the 12th week after birth. The control group only re-
ceived standard care, which does not include any mental
health care or screening from hospital staff. Procedure g
g
The intervention session took place every second week
in a gymnastic room of the Clinic for Gynecology and
Obstetrics at the University Hospital Duesseldorf and
the women that were randomised into the intervention
group participated between one and three times. Five to
ten women participated simultaneously in one interven-
tion session. When the participants arrived with their
babies, they were welcomed by a member of the study
team and one music therapist that moderated the inter-
vention class. All participants filled in the pre interven-
tion session questionnaire that comprised the Self-
Assessment Manikin and the visual analogue scales
about well-being and closeness to the baby insalivated a
saliva sample. After a short welcome that always con-
tained a “welcome song” where all participants and in-
fants were addressed with names, the concept of the
intervention was explained. Due to the explanations
given, the participants were aware of the goal of the
intervention, which was to implement a singing- and
music-based interaction between the new mother and
the baby at home. Therefore, elements of finger games,
lullabies and movements to music were used and all par-
ticipants practiced them together with their infants dur-
ing
the
intervention
session
which
lasted
45 min. Although a standard repertoire of songs and games
existed, the music therapist incorporated the partici-
pants´ requests and wishes if present. The participants
were asked to implement the intervention daily at home. At the end of the intervention session, all participants
filled in the questionnaire again and insalivated a second
saliva sample. To investigate 10-week effects over the two times of
measurement (T1 and T2), 2 × 2 mixed factorial ANO-
VAs with the independent variable group (control vs. intervention group) and the repeated-measure variable
timepoint (T1 and T2) were applied with the dependent
variables Trait-Anxiety Inventory Stubscore State, Edin-
burgh Postnatal Depression Scale, visual analogue scale
comfort with the maternal role, visual analogue scale
perceived closeness to the baby and Postpartum Bonding
Questionnaire respectively. In order to check for normality, Shapiro-Wilk tests
were calculated for all dependent variables. The Shapiro-
Wilk tests revealed that most variables were not nor-
mally distributed (p > .05). Even though the normality as-
sumption was violated, all analyses were conducted as
intended because of the proved robustness of ANOVAs
[49, 50] and the absence of appropriate non-parametric
alternatives for repeated-measures ANOVAs. Procedure The figures
depicture a range of each dimension on a 9-point likert-
scale and participants are asked to mark the appropriate
figure. There is a range of 5 figures with 4 possible interim
points from “pleasant” to “unpleasant” for the valence
dimension, from “excited” to “calm” regarding the di-
mension of arousal and from “dependent” to “inde-
pendent”
for
the
dominance
dimension
and
each
score has a range from 0 to 5. Higher scores indicate
less pleasure, less arousal and a greater feeling of
independence. Maternal salivary cortisol was measured in order to
evaluate a physiological marker for stress of the partici-
pants when they participated in the intervention session
for the first time whereas no samples were taken from
the infants. Cortisol is widely used as a biomarker in
stress research due to its reflection of the activity of the
sympathetic nervous system [37]. Saliva samples were
taken with Salivettes (Sarstedt, Germany) at the begin-
ning and at the end of the intervention session. Partici-
pants insalivated cotton swabs for at least 30 s. After the
samples were taken, they were stored at −18 °C until
further analysis. Cortisol levels were determined in the
laboratory of the DresdenLAB (Dresden, Germany) by
using immunoassay (IBL, Hamburg, Germany). Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 Page 6 of 15 Page 6 of 15 check ups (gynaecologist, hospital staff, and midwives)
were given. An overview of the procedure as well as in-
formation about when which questionnaire was admi-
nistred is given in Fig. 1. baseline were checked with t-tests for independent sam-
ples regarding maternal age, gestational age and State-
Trait-Anxiety Inventory Trait. In order to explore the immediate effects during the
intervention session, dependent-sample t-tests were used
to compare pre-post differences for the dependent vari-
ables Self-Assessment Manikin valence, arousal and
dominance as well as for saliva cortisol. For the anaylsis
of the immediate effects of the intervention on the three
dimensions of the Self-Assessment Manakin (valence,
arousal and dominance) as well as on salivary cortisol,
only the data of the first intervention session for each
women were used. All women in the intervention group
had immediate effects data. Statistical analysis For the statistical analysis, the statistical software pack-
age SPSS 24 (IBM Inc., Armonk, NY) was used. When
sphericity was violated, Greenhouse-Geisser corrected
values were reported. Only the data of participants that
performed by protocol were used for calculations (i.e. participants who were randomized into the intervention
group but did not attend the intervention, were excluded
from the analysis). Outliers above two standard devia-
tions from the mean, were excluded separately for each
calculation. The maximal number of excluded outliers
was six per measure. This outlier correction did not
affect the results in a substantive way. In case of up to
two missing values, replacements with the mean scores
of the norm sample were conducted for the State-Trait-
Anxiety Inventory as suggested by Laux et al. [44] and
for the other questionnaires with the sample mean for
each
item
[48]. Furthermore,
group
differences
at Procedure As explorative analysis, Mann-Whitney U tests were
conducted in order to check for group differences be-
tween the intervention and the control group regarding
the ordinal scaled frequencies of the use of music and
singing at T2. Additionally, the relations between the
frequencies of the use of music and singing and the
variables at T2 (State-Trait-Anxiety Inventory State,
Edinburgh Postnatal Depression Scale, visual analogue
scale comfort with the maternal role, visual analogue
scale closeness to the baby and Postpartum Bonding
Questionnaire) were explored with Spearman correla-
tions. Furthermore, Fisher’s z tests were conducted to
examine group differences for the correlations. No cor-
rections for multiple comparisons were applied due to
the explorative character of the calculations [51]. 10-week effects A mixed-factorial ANOVA with the dependent variable
State-Trait-Anxiety Inventory State showed a significant
main effect of the factor time of measurement [F (1,
111) = 30.61, p < .001, d = 0.49 (CI 95% 0.22, 0.75)] with
both groups showing a reduction of State-Trait-Anxiety
Inventory State scores from T1 to T2. The main effect
of the factor group was not significant [F (1, 111) = 0.33,
p = .566, d = 0.11 (CI 95% -0.26, 0.48)] as well as the
interaction effect [F (1, 111) = 0.99, p = .32, d = 0.19 (CI
95% -0.19, 0.56)]. See Table 2 for the descriptive
statistics. With a mixed-factorial ANOVA with the dependent
variable Edinburgh Postnatal Depression Scale, a signifi-
cant main effect for the factor time of measurement was
revealed [F (1, 108) = 14.81, p < .001, d = 0.34 (CI 95%
0.07, 0.60)] and a reduction of depressive symptoms was
visible from T1 to T2. No significant main effect of the
factor group [F (1, 108) = 0.03, p = .859, d = 0.03 (CI 95%
-0.34, 0.41)] and no significant interaction effect [F (1,
108) = 1.76, p = .188, d = 0.25 (CI 95% -0.12, 0.63)] were
revealed. Furthermore a significant difference between pre and
post measurement was revealed for the score of visual
analogue scale perceived closeness to the baby [t (51) = −
3.40, p = .001, d = 0.43 (CI 95% 0.04, 0.82)]. After the
intervention session, women reported higher feelings of A mixed-factorial ANOVA with the dependent vari-
able visual analogue scale comfort with the maternal role
showed a significant main effect of the factor time of Table 1 Sample characteristics. Descriptive statistics (means (standard deviations)) and results of calculations of group differences (p-
values) at baseline Descriptive statistics (means (standard deviations)) and results of calculations of group differences (p- Table 1 Sample characteristics. Sample characteristics From the final sample (N = 120), 59 women were in the
experimental group and 61 were in the control group. No group differences were revealed regarding maternal
age, gestational age at the time of childbirth and State- Page 7 of 15 Page 7 of 15 Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 Trait-Anxiety Inventory Trait (p-values ≥.394) at the
start of measurements (baseline). In regard to the rele-
vant dependent variables (State-Trait-Anxiety Inventory
State,
Edinburgh
Postnatal
Depression
Scale,
visual
analogue scale comfort with the maternal role, visual
analogue scale closeness to the baby and Postpartum
Bonding
Questionnaire)
no
group
differences
were
found at baseline (p-values ≥.277). See Table 1 for the
descriptive statistics
and
the
p-values
of the
test-
statistics regarding group differences at baseline. closeness and attachment. The score of visual analogue
scale comfort with the role of the mother also differed
from pre to post measurement [t (48) = −5.59, p < .001,
d = 0.74 (CI 95% 0.33, 1.15)]. At the end of the inter-
vention session, women reported more feelings of
well-being and confidence with the maternal role (see
Fig. 2b). For salivary cortisol, a t-test for dependent samples
showed a significant difference between the pre and post
measurement [t (49) = 2.35, p = .023, d = 0.32 (CI 95%
0.07, 0.72)]. The cortisol level decreased significantly
during the intervention session (see Table 3). Immediate effects during the intervention In the context of the intervention session, dependent-
sample t-tests showed significant improvements of the
emotional state from pre to post measurement (see
Table 2 for the descriptive statistics and the number of
participants included in each analysis). Regarding the
Self-Assessment Manikin valence score, a significant dif-
ference from pre to post was revealed [t(50) = 3.33, p =
.002, d = 0.43 (CI 95% 0.03, 0.82)] with lower scores at
the end of the intervention indicating more happiness at
the end of the session compared to the start. A signifi-
cant difference was also revealed for the Self-Assessment
Manikin arousal score [t(49) = −5.11, p < .001, d = 0.65
(CI 95% 0.25, 1.05)] that increased during the interven-
tion session which indicates less arousal at the end of the
intervention session. The difference between the pre and
post measurement regarding Self-Assessment Manikin
dominance score was also significant [t (52) = −2.76, p =
.008, d = 0.37 (CI 95% -0.01, 0.76)]. The participating
women reported a higher feeling of independence and
self-confidence at the end of the intervention session (see
Fig. 2a). e. A = in years at the time of childbirth, B = in weeks at the time of childbirth, C = result of an independent-samples t-test. STAI: State-T
DS: Edinburgh Postnatal Depression Scale; VAS: visual analogue scale; PDQ: Postpartum Bonding Questionnaire; n = participants include me of childbirth, B = in weeks at the time of childbirth, C = result of an independent-samples t-test. STAI: State-Trait-Anxiety Inventory;
l Depression Scale; VAS: visual analogue scale; PDQ: Postpartum Bonding Questionnaire; n = participants included in the analysis 10-week effects Descriptive statistics (means (standard deviations)) and results of calculations of group differences (p-
values) at baseline
Control group
Intervention group
M
SD
n
M
SD
n
p-value
AgeA
34.31
(4.19)
61
33.67
(3.94)
58
p = .394C
Gestational AgeB
38.76
(3.67)
61
39.05
(1.51)
57
p = .713C
STAI Trait
47.00
(5.25)
60
46.53
(4.85)
53
p = .627C
STAI State
36.06
(9.89)
58
36.63
(9.10)
54
p = .753 C
EPDS
7.13
(4.34)
56
6.36
(4.04)
54
p = .341 C
VAS comfort with the maternal role
8.59
(1.25)
57
8.58
(1.15)
53
p = .970 C
VAS closeness to the baby
9.49
(0.65)
55
9.34
(0.82)
53
p = .277 C
PBQ
5.51
(4.12)
54
5.14
(3.98)
54
p = .630 C
Note. A = in years at the time of childbirth, B = in weeks at the time of childbirth, C = result of an independent-samples t-test. STAI: State-Trait-Anxiety Inventory;
EPDS: Edinburgh Postnatal Depression Scale; VAS: visual analogue scale; PDQ: Postpartum Bonding Questionnaire; n = participants included in the analysis Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 Page 8 of 15 Page 8 of 15 Table 2 Descriptive statistics (mean values and in parentheses standard deviations) of the variables that were measured during the
music intervention session (pre and post)
Pre intervention
Post intervention
p – valueA
n
SAM valence
1.85 (0.58)
1.61 (0.50)
.002
51
SAM arousal
4.38 (0.77)
4.90 (0.55)
< .001
50
SAM dominance
4.30 (0.75)
4.49 (0.68)
.008
53
VAS perceived closeness to the baby
8.85 (1.08)
9.26 (0.73)
.001
52
VAS comfort with maternal role
7.73 (1.27)
8.71 (1.07)
< .001
49
Saliva Cortisol (nmol/l)
3.55 (1.71)
3.01 (1.54)
.023
50
Note. A = t-tests for dependent samples for the comparison between pre and post intervention. SAM: Self-Assessment Manikin; VAS: visual analogue scale; n =
participants included in each analysis Table 2 Descriptive statistics (mean values and in parentheses standard deviations) of the variables that were measured during the
music intervention session (pre and post) Fig. 2 Results of the immediate effects during the 45-min intervention session with a comparison between pre and post intervention
measurement; * = p < .05; error bars represent standard deviations. A: Results of the SAM scores valence, arousal and dominance. A significant
improvement was found from pre to post intervention for all dimensions of SAM. 10-week effects Comparison between
the control group and the intervention group measurement [F (1, 110) = 4.82, p = .030, d = 0.19 (CI
95% -0.07, 0.46)] but no significant main effect or the
factor group [F (1, 110) = 0.06, p = .805, d = 0.05 (CI 95%
-0.32, 0.42)] or an interaction effect [F (1, 110) = 0.65,
p = .422, d = 0.16 (CI 95% -0.21, 0.53)] were revealed. Both groups showed an increase of comfort from T1 to
T2. group but overall, the majority of mothers reported the
use of singing and music in everyday life independent of
group allocation. See Table 4 for an overview of the de-
scriptive statistics (mean rank scores). In order to explore the relation between the frequen-
cies of listening to music or singing and the dependent
variables at T2, correlations were calculated. Due to the
group differences regarding the mentioned frequencies,
Spearman correlations were conducted separately for
each group. The results of the correlations are displayed
in Table 5 as well as the between group comparison with
Fisher’s z tests. It should be highlighted that in the inter-
vention group, a higher frequency of singing for the baby
was significantly associated with less maternal anxiety
(State-Trait-Anxiety Inventory State) and with greater
comfort with the maternal role (visual analogue scale). Additionally in the intervention group, the frequency of
singing for oneself is significantly correlated with the Ed-
inburgh Postnatal Depression Scale, visual analogue
scale comfort with maternal role and Postpartum Bond-
ing Questionnaire. The correlations indicate that a
higher frequency of singing for oneself is associated with
less depressive symptoms, greater comfort and closer
bonding. The analysis of visual analogue scale closeness to the
baby showed no significant main effect of the factor time
of measurement [F (1, 106) = 1.91, p = .170, d = 0.13 (CI
95% -0.13, 0.40)]. The main effect of the factor group
was also non significant [F (1, 106) = 0.60, p = .442, d =
0.15 (CI 95% -0.23, 0.53)] but a significant interaction ef-
fect was revealed [F (1, 106) = 4.19, p = .043, d = 0.97 (CI
95% 0.57, 1.37)]. However, post-hoc analyses showed no
significant group difference at T1 [t (91.18) = 1.54, p =
.127, d = 0.30 (CI 95% -0.08, 0.68)] or at T2 [t (107) = −
0.39, p = .695, d = 0.06 (CI 95% -0.32, 0.44)]. 10-week effects B: Results of the VAS scores closeness to baby and comfort
with maternal role. Both scores increased significantly from pre to post intervention, indicating an improvement of bonding and comfort. C: Comparison of salivary cortisol levels between pre and post intervention session. Saliva cortisol decreases significantly during the
intervention session Fig. 2 Results of the immediate effects during the 45-min intervention session with a comparison between pre and post intervention
measurement; * = p < .05; error bars represent standard deviations. A: Results of the SAM scores valence, arousal and dominance. A significant
improvement was found from pre to post intervention for all dimensions of SAM. B: Results of the VAS scores closeness to baby and comfort
with maternal role. Both scores increased significantly from pre to post intervention, indicating an improvement of bonding and comfort. C: Comparison of salivary cortisol levels between pre and post intervention session. Saliva cortisol decreases significantly during the
intervention session Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 Page 9 of 15 Page 9 of 15 Table 3 Descriptive statistics (means (standard deviations)) of the variables that were measured at T1 and T2. Comparison between
the control group and the intervention group
T1
T2
CG
n
IG
n
CG
n
IG
n
STAI State
37.30
(7.01)
57
36.04
(8.04)
56
33.44
(5.92)
57
33.36
(6.71)
56
EPDS
5.05
(4.09)
56
5.37
(3.55)
54
4.25
(3.38)
56
3.72
(2.95)
54
VAS comfort with the maternal role
8.01
(1.38)
56
7.98
(1.31)
56
8.17
(1.35)
56
8.32
(1.12)
56
VAS closeness to the baby
9.30
(1.02)
56
9.55
(0.61)
52
9.55
(0.67)
56
9.50
(0.66)
52
PBQ
6.82
(5.02)
57
6.63
(5.16)
56
6.04
(4.40)
57
6.77
(4.48)
56
Note. CG = control group, IG = intervention group. STAI: State-Trait-Anxiety Inventory; EPDS: Edinburgh Postnatal Depression Scale; VAS: visual analogue scale;
PDQ: Postpartum Bonding Questionnaire. N = participants included in each analysis Table 3 Descriptive statistics (means (standard deviations)) of the variables that were measured at T1 and T2. Comparison between
the control group and the intervention group Table 3 Descriptive statistics (means (standard deviations)) of the variables that were measured at T1 and T2. 10-week effects For the Postpartum Bonding Questionnaire no signifi-
cant main effect of the factor time of measurement was
revealed [F (1, 111) = 0.85, p = .359, d = 0.08 (CI 95%
-0.18, 0.35)]. Furthermore, no significant main effect for
the factor group [F (1, 111) = 0.104, p = .748, d = 0.06 (CI
95% -0.31, 0.43)] nor a significant interaction effect [F
(1, 111) = 1.76, p = .187, d = 0.25 (CI 95% -0.12, 0.62)]
was revealed. Additional analyses of the variables regarding the satis-
faction with the intervention revealed that the majority
of mothers would participate in the intervention lessons
again (88.33%). Moreover the participants of the inter-
vention group were very pleased with the intervention
showing high scores of visual analogue scale pleasure
with intervention (M = 8.47, SD = 1.98). The majority of
the intervention group participated twice in the lessons
(62.71%) whereas some women even participated three
times (10.17%) and 27.12% of the intervention group
participated only once. Further analysis In order to investigate whether the group allocation is
related to the frequencies of singing and listening to
music at the second time of measurement (T2), Mann-
Whitney U tests were performed. For the frequency sing-
ing (baby) no significant relation was found [U =
1625.50, z = −.99, p = .324]. However the group alloca-
tion was significantly related to the frequency of singing
(oneself) [U = 1434.00, z = −2.00, p = .045], the frequency
of playing music (baby) [U = 1232.50, z = −3.07, p = .002]
and to the frequency of playing music (oneself) [U =
1390.50, z = −2.20, p = .028]. Regarding these dependent
variables, the intervention group reported higher fre-
quencies of singing and music compared to the control Discussion In the present study, the effects of a singing intervention
for mothers with their infants in the first weeks after
birth were explored. Immediate effects of the singing
intervention were found from pre to post intervention Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 Page 10 of 15 Table 4 Descriptive statistics (absolute values of the frequencies of singing and playing music for onself and for the baby) at the
second time of measurement (T2), separately listed for the intervention and the control group. Mean rank scores are additionally
listed for interpretion of the Mann-Whitney U tests Table 4 Descriptive statistics (absolute values of the frequencies of singing and playing music for onself and for the baby) at the
second time of measurement (T2), separately listed for the intervention and the control group. Mean rank scores are additionally
listed for interpretion of the Mann-Whitney U tests Table 4 Descriptive statistics (absolute values of the frequencies of singing and playing music for onself and for the baby) at the
second time of measurement (T2), separately listed for the intervention and the control group. Mean rank scores are additionally
listed for interpretion of the Mann Whitney U tests p
y
Frequency singing
(baby)
Frequency singing
(oneself)
Frequency playing music
(baby)
Frequency listening music
(oneself)
IG
never
1
17
1
8
once per week
1
15
5
11
several times per week
13
15
17
19
once per day
13
6
20
12
several times per day
31
6
16
9
Mrank
63.45
66.69
70.11
67.43
CG
never
1
29
9
19
once per week
3
12
12
15
several times per week
17
13
16
11
once per day
12
2
15
7
several times per day
28
5
9
9
Mrank
57.65
54.51
51.20
53.80
Note. IG = intervention group (n = 59), CG = control group (n = 61) Note. IG = intervention group (n = 59), CG = control group (n = 61) second and the 12th week after birth. Regarding this
time window, only time-effects but no group or inter-
action effects were revealed for the variables measuring
maternal well-being. session. The data revealed a significant immediate reduc-
tion of cortisol levels as well as an immediate improve-
ment
of
maternal
scores
on
valance,
arousal
and
dominance and mother-infant attachment (see Fig. Note. IG = intervention group, CG = control group, * p < .05, ** p < .01. STAI: State-Trait-Anxiety Inventory; EPDS: Edinburgh Postnatal Depression Scale; VAS: visual
analogue scale; PDQ: Postpartum Bonding Questionnaire = intervention group, CG = control group, * p < .05, ** p < .01. STAI: State-Trait-Anxiety Inventory; EPDS: Edinburgh Postnatal Depressio
e scale; PDQ: Postpartum Bonding Questionnaire oup, CG = control group, * p < .05, ** p < .01. STAI: State-Trait-Anxiety Inventory; EPDS: Edinburgh Postnatal Depression Scale; VAS: visua
partum Bonding Questionnaire Discussion One limitation is that the variables
that were captured during the intervention (Self-Assess-
ment Manikin, visual analogue scale closeness to the
baby, visual analogue scale comfort with the maternal
role, salivary cortisol) were not measured in the control
group. Thus the immediate effects from pre to post
intervention should be carefully interpreted because a
comparison with the control group is not possible. In
this respect, it would also be desirable to include an p
g
p
In contrast to our hypotheses that the singing interven-
tion would also show positive 10-week effects from T1 to
T2, both groups showed similar reductions in State-Trait-
Anxiety Inventory State scores and Edinburgh Postnatal
Depression Scale scores. The alternation over time is in
line with the fact that in the postpartum period, symptoms
of anxiety and depression are common but decrease over
time [56, 57]. Overall, the sample of the present study
showed Edinburgh Postnatal Depression Scale scores (see
Table 2) that are much lower than the cut-off score (EPDS
≥13) for a high probability of depression [45] while the
State-Trait-Anxiety Inventory State comply with the
scores of the German norm sample [44]. This indicates
that the women of the present sample were in a good
mental state and therefore little room for improvement
was evident. As Fancourt and Perkins [42] revealed, the
positive effects of the singing intervention were only vis-
ible in a subgroup of women with Edinburgh Postnatal
Depression Scale scores ≥13, it may be hypothesised that
women in a good mental health might not further im-
prove their mood with an additional intervention. Beyond
that, the additional exploratory analysis regarding the fre-
quencies of the use of singing and music revealed no rela-
tion between group allocation and the frequency of
singing for the baby showing that both groups sang for
their babies in a similar amount which could explain the
missing 10-week impact of the singing intervention during
the time window from T1 to T2. Furthermore, a signifi-
cant time effect but no group or interaction effects
occured for the visual analogue scale comfort with the ma-
ternal role where high scores at all times of measurement
(see Table 2) also indicate a ceiling effect. No influence of
the intervention was found, which is also in contrast to
our main hypotheses. Discussion 2),
highlighting a significant positive immediate effect of the
intervention on maternal stress levels, emotional state
and bonding. However, no impact of the intervention
could be revealed for the 10-week period between the Significant immediate positive effects were found from
pre to post intervention session on affect, well-being and
attachment in accordance with the hypotheses and pre-
vious studies [36, 41]. Regarding the Self-Assessment Table 5 Results of the correlations (Spearman correlation coefficients r) between the frequencies of listening to music and singing
with the dependent subjective variables at T2. Results of the comparison between the intervention group and the control group are with the dependent subjective variables at T2. Results of the comparison between the intervention group and the control group are
listed with Fisher’s z
n
Frequency singing
(baby)
Frequency singing
(oneself)
Frequency playing music
(baby)
Frequency listening music
(oneself)
STAI State
IG
58
- .26
*
- .24
- .23
- .10
CG
58
.05
- .39
**
- .16
- .20
z
−1.65
*
0.88
−0.38
0.53
EPDS
IG
56
- .08
- .28
*
- .08
- .22
CG
57
.12
- .23
- .14
- .15
z
0.99
−0.28
0.33
−0.33
VAS comfort maternal role
IG
57
.35
**
.41
**
.37
**
.24
CG
58
.02
.31
*
.11
.05
z
1.81
*
0.62
1.42
0.99
VAS closeness to the baby
IG
54
.14
.21
- .07
.14
CG
60
.20
.13
.15
.04
z
−0.34
0.42
−1.12
0.52
PBQ
IG
57
- .19
- .29
*
- .23
- .33
*
CG
59
.01
- .08
- .06
- .07
z
−1.04
−1.16
−0.91
−1.40
Note. IG = intervention group, CG = control group, * p < .05, ** p < .01. STAI: State-Trait-Anxiety Inventory; EPDS: Edinburgh Postnatal Depression Scale; VAS: visual
analogue scale; PDQ: Postpartum Bonding Questionnaire Page 11 of 15 Page 11 of 15 Page 11 of 15 Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 active control group (i.e. a playgroup without music ele-
ments) in future studies, as it may be that the positive
immediate effects of the music group were influenced by
the social experience of a group intervention. Discussion Manikin scores, significant improvements were found
for valence, arousal and dominance showing that women
were happier, more relaxed and more self-confident at
the end of the intervention session compared to the be-
ginning. The stress-reducing effect was also evident in
the physiological stress marker salivary cortisol that de-
creased significantly during the intervention session. The
improvement
of
well-being,
mood
and
relaxation
through music and singing is in line with other studies
that showed similar improvements in other contexts [15,
36, 39]. The results are also in accordance with results
of Fancourt and Perkins [41], who showed beneficial
positive effects of a singing workshop for mothers and
babies on maternal positive affect, cortisol and addition-
ally on perceived closeness to the baby. Likewise, posi-
tive
effects
on
attachment
(visual
analogue
scale
perceived closeness) and maternal well-being (visual
analogue scale comfort with maternal role) were found
in the present study with significant improvements. In
accordance with our hypotheses, the maternal well-being
and the attachment towards the baby seems to be posi-
tively affected by the music assisted interaction during
the intervention session. Like Fancourt and Perkins [41]
showed in their study, a music and singing intervention
seems to encourage mother-infant bonding. Besides the
mood improving and relaxing effects that were reported
for singing in general [36, 39], singing also impacts social
mechanisms. While feelings of social connectedness, so-
cial flow and bonding can be enhanced in particular by
group singing [52, 53], similar social mechanisms seem
to work during the interventions of the present study
through the interaction with the newborn. In the initial
period after birth, the variety of interaction is very re-
stricted and during the first weeks the interaction is
dominated by caring for the substantial infant’s needs
and in particular (breast-) feeding [54]. Beyond that,
singing can be an additional way to interact with the
baby and leads to a stronger bonding between mother
and child [20, 31]. Furthermore it is possible to induce
infant feedback through singing like attention towards
the mother or relaxation [31, 55]. This hypothesis is re-
inforced by the individual feedback of the participants
who reported for example that they were “more sensitive
in regard to interaction”, “interacted more with the
baby” and “experienced a way to entertain the child” due
to the intervention. Discussion A missing or limited feedback of the baby dur-
ing the intervention or at home could have led to a per-
ception of unsatisfied interactions. Larger effects might
be visible when the intervention takes place later as done
in other studies [41, 42] where the infants´ age was a
few months up to almost one year. In spite of these limi-
tations and the missing significant effects, the majority
of participants reported that they would like to partici-
pate again (88.33%) and the mean visual analogue scale
satisfaction with intervention score was very high (M =
8.47, SD = 1.98). Another limitation is that the post-hoc
power analysis revealed a power of 68% in the current
sample of 120 participants, which is lower than the
power of 80% that was presumed in advance. This
should be kept in mind when interpreting the results. Additionally, it would be desirable if future studies
would replicate the present findings with an increased
sample size and power. p
p
The exploratory analyses that were performed beyond
the main hypotheses revealed that the intervention
group used music and singing for oneself more fre-
quently in every day life even though the group differ-
ences
regarding
singing
for
the
baby
were
not
significant. Although the main goal of increasing singing
to the baby can therefore not be supported, the women
who participated in the intervention sang significantly
more frequently for themselves and additionally, used
music more often as well as they played music for their
babies more regularly in comparison to the control
group. This highlights that the overall musical activity
was increased by the intervention. The difference regard-
ing the frequency of singing for the baby was not signifi-
cantly related to the group allocation which can be
explained by the common use of singing lullabies to
calm a baby that is widespread almost all over the world
[22]. This could also explain the absent impact of the
intervention in the 10-week period. Unfortunately, it is
not possible to restrict singing or playing music in the
control group which in turn restrains the interpretation
of the results of the comparison between groups and
should be taken into account as a limitation. Discussion y
With regard to mother-infant bonding, a significant
time effect was only revealed for the Postpartum Bonding
Questionnaire, but, against our hypotheses, no group or
interaction effect was found showing that the intervention
did not influence mother-infant bonding measured with
the Postpartum Bonding Questionnaire in the time frame
from T1 to T2. The Postpartum Bonding Questionnaire
scores decreased over time indicating an improvement of
bonding, whereas the visual analogue scale perceived close-
ness scores remained stable in a high range. Although the
interaction effect turned out significant for the visual
analogue scale, the post-hoc t-tests showed no significant
group effect at both time points. The significant inter-
action can be traced back to a slight descriptive group dif-
ference at T1, although it was not significant. The result
of an increase in mother-infant bonding over time, which Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 Page 12 of 15 Page 12 of 15 Page 12 of 15 was visible in the Postpartum Bonding Questionnaire
scores, is in line with results of O’Higgins, Roberts, Glover
and Taylor [58] who observed stronger bonding along
with time after childbirth. The lack of an observable im-
pact of the intervention on attachment can also be ex-
plained with a ceiling-effect because all participating
women showed strong mother-infant attachment at both
times of measurements (see Table 2). In particular the
Postpartum Bonding Questionnaire scores are even lower
than the scores reported by Reck and colleagues [1] for
non-depressed (M = 7.33, SD = 6.14) postpartum women. It has to be noted that in the current study participation
was offered to the whole potential population of pregnant
women without a pre-screening for depression or risk for
bonding impairment in order to include a sample which is
representative of the general (pregnant) population. We
would hypothesise that related to the results regarding a
greater improvement of depression in clinical samples
[42], the impact of the intervention on bonding could be
also greater in a high-risk sample with mothers who have
an impaired attachment towards the infant. This hypoth-
esis should be explored in future studies in which the ef-
fects of singing interventions should be investigated
particular in mothers who report a very low and impaired
mother-infant attachment. processes due to a reciprocal dependency of reactions
[59, 60]. Discussion It is pos-
sible that the intervention did not specifically influence
the frequency of singing for the baby, but led to an in-
creased sensitivity to include music and singing in every-
day life as well as an enriched musical environment at
home. It would be interesting to investigate musical ac-
tivities in everyday life in depth as part of further
studies. Besides the potential ceiling effects, it is possible that
the intervention period was too short because the
women participated in the intervention only once to a
maximum of three times. In contrast to the study of
Fancourt and Perkins [42], where the mothers partici-
pated over 10 weeks in the intervention lessons, the
intervention of the current study was less frequent. Overall, 55 women were excluded from the analysis be-
cause they refused to participate in the intervention. Un-
fortunately, we did not evaluate why participants refused
to take part in the intervention sessions. However, it
would be interesting to try to evaluate this in future
studies, as it would provide valuable information for
planning and improving study protocols in the future, in
order to make participation in the early postpartal phase
more convenient. In this respect, another limitation of
the study is that the time point where the women took
part for the first time varied between the second and the
ninth week of the baby’s life. As a result, a later partici-
pation reduced the intervention period. Although, we
made sure that the number of attended sessions were
comparable (one to three attended sessions), the inter-
vention period until the post-intervention measurement
varied. Future studies should try to optimize the stand-
ardisation of the time window. Furthermore, we hy-
pothesise that the intervention period may also have
been too early. In the first weeks of life the infant’s feed-
back and interaction repertoire is very restricted which
can lead to maternal dissatisfaction during interaction A correlation analysis revealed that the frequency of
singing and playing music is significantly associated with
relevant variables measuring well-being and attachment
at the second time of measurement. The significant
group difference regarding the relation between singing Page 13 of 15 Page 13 of 15 Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 Wulff et al. BMC Pregnancy and Childbirth (2021) 21:501 Wulff et al. Received: 26 August 2020 Accepted: 7 June 2021 Received: 26 August 2020 Accepted: 7 June 2021 Received: 26 August 2020 Accepted: 7 June 2021 Author details
1 Author details
1Department of Experimental Psychology, Heinrich-Heine-University
Düsseldor, Universitätsstraße 1, 40225 Düsseldorf, Germany. 2Clinic for
Gynecology and Obstetrics, University Clinic, Augsburg, Germany. 3University
Witten/Herdecke, Landesfrauenklinik, Wuppertal, Germany. 4Department of
Cognitive Psychology, Institute of Cognitive Neuroscience, Faculty of
Psychology, Ruhr-University Bochum, Bochum, Germany. 5Clinic for
Gynecology and Obstetrics, Heinrich-Heine-University Düsseldorf, Düsseldorf,
Germany. Consent for publication
Not applicable. Consent for publication
Not applicable. Consent for publication
Not applicable. Discussion BMC Pregnancy and Childbirth (2021) 21:501 for the baby and anxiety (State-Trait-Anxiety Inventory
State) and maternal well-being (visual analogue scale
comfort with maternal role) highlighted that only in the
intervention group more frequent singing for the baby
was associated with less anxiety and greater well-being. We assume that the intervention group was more sensi-
tive and aware to the use of singing and music as they
received instructions to do so in the intervention ses-
sions. The music therapist gave several examples for the
practical use at home and underlined the aspects of re-
laxation, consciousness and pleasure that are relevant
for the effects of music [61, 62]. This could have made
the use of music and singing “more effective” and led to
greater associations in the intervention group even
though no 10-week effects of the intervention were
revealed. References
1
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Erratum to: Is bottomonium suppression in proton-nucleus and nucleus-nucleus collisions at LHC energies due to the same effects?
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Published for SISSA by
Springer
Received: January 25, 2019
Accepted: March 4, 2019
Published: March 12, 2019 Published for SISSA by
Springer Published for SISSA by
Springer Received: January 25, 2019
Accepted: March 4, 2019
Published: March 12, 2019 Received: January 25, 2019
Accepted: March 4, 2019
Published: March 12, 2019 JHEP03(2019)063 Open Access, c⃝The Authors.
Article funded by SCOAP3. Erratum: Is bottomonium suppression in
proton-nucleus and nucleus-nucleus collisions at LHC
energies due to the same effects? 03(2019)063
energies due to the same effects? E.G. Ferreiroa,b and J.P. Lansbergc
aLaboratoire Leprince-Ringuet, Ecole polytechnique, CNRS/IN2P3, Universit´e Paris-Saclay,
Palaiseau, France
bDepartamento de F´ısica de Part´ıculas and IGFAE, Universidade de Santiago de Compostela,
15782 Santiago de Compostela, Spain
cIPNO, Universit´e Paris-Saclay, Univ. Paris-Sud, CNRS/IN2P3,
F-91406, Orsay, France
E-mail: elena@fpaxp1.usc.es, Jean-Philippe.Lansberg@in2p3.fr
Erratum to: JHEP10(2018)094
ArXiv ePrint: 1804.04474
Open Access, c⃝The Authors. Article funded by SCOAP3. https://doi.org/10.1007/JHEP03(2019)063 E.G. Ferreiroa,b and J.P. Lansbergc E.G. Ferreiroa,b and J.P. Lansbergc
aLaboratoire Leprince-Ringuet, Ecole polytechnique, CNRS/IN2P3, Universit´e Paris-Saclay,
Palaiseau, France
bDepartamento de F´ısica de Part´ıculas and IGFAE, Universidade de Santiago de Compostela,
15782 Santiago de Compostela, Spain
cIPNO, Universit´e Paris-Saclay, Univ. Paris-Sud, CNRS/IN2P3,
F-91406, Orsay, France E-mail: elena@fpaxp1.usc.es, Jean-Philippe.Lansberg@in2p3.fr Erratum to: JHEP10(2018)094 Erratum to: JHEP10(2018)094 ArXiv ePrint: 1804.04474 Open Access, c⃝The Authors. Article funded by SCOAP3. https://doi.org/10.1007/JHEP03(2019)063 https://doi.org/10.1007/JHEP03(2019)063 In the presence of a relative suppression of the different quarkonium states and due to
the feed downs, the statement on pg. 1 & 2 “one is entitled [...] to square the measured
suppression factor [12] in pPb collisions to extrapolate to PbPb collisions.” is unfortunately
incorrect. As such, the extrapolated values in eq. (1.1) are not correct and should be
disregarded. Such an extrapolation is in fact not possible without knowing all the involved
yields and feed downs. In the presence of a relative suppression of the different quarkonium states and due to
the feed downs, the statement on pg. 1 & 2 “one is entitled [...] to square the measured
suppression factor [12] in pPb collisions to extrapolate to PbPb collisions.” is unfortunately
incorrect. As such, the extrapolated values in eq. (1.1) are not correct and should be
disregarded. Such an extrapolation is in fact not possible without knowing all the involved
yields and feed downs. We however stress that this mistake only applies to this introductory statement and
does not affect our results, which follow from a complete computation of the yield suppres-
sions and of their effect on the lower lying states via feed downs. JHEP03(2019)063 Acknowledgments We would like to thank Michael Strickland who drew our attention, during a discussion at
the Munich Institute for Astro- and Particle Physics (MIAPP) programme “Probing the
quark-gluon plasma with collective phenomena and heavy quarks”, to this issue. Open Access. This article is distributed under the terms of the Creative Commons
Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in
any medium, provided the original author(s) and source are credited. – 2 –
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https://openalex.org/W4308720012
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https://www.mdpi.com/1424-8220/22/21/8305/pdf?version=1667439347
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English
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xRatSLAM: An Extensible RatSLAM Computational Framework
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Sensors
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Citation: de Souza Muñoz, M.E.;
Chaves Menezes, M.; Pignaton de
Freitas, E.; Cheng, S.; de Almeida
Ribeiro, P.R.; de Almeida Neto, A.;
Muniz de Oliveira, A.C. xRatSLAM:
An Extensible RatSLAM
Computational Framework. Sensors
2022, 22, 8305. https://doi.org/
10.3390/s22218305
Academic Editors: Chee Kiat Seow,
Henrik Hesse, Yanliang Zhang, Torr
Polakow and Kai Wen
Received: 27 August 2022
Accepted: 27 October 2022
Published: 29 October 2022 Citation: de Souza Muñoz, M.E.;
Chaves Menezes, M.; Pignaton de
Freitas, E.; Cheng, S.; de Almeida
Ribeiro, P.R.; de Almeida Neto, A.;
Muniz de Oliveira, A.C. xRatSLAM:
An Extensible RatSLAM
Computational Framework. Sensors
2022, 22, 8305. https://doi.org/
10.3390/s22218305 Keywords: robotics; simultaneous localization and mapping; RatSLAM; image SLAM Mauro Enrique de Souza Muñoz 1
, Matheus Chaves Menezes 1
, Edison Pignaton de Freitas 2
, Sen Cheng 3
,
Paulo Rogério de Almeida Ribeiro 4
, Areolino de Almeida Neto 5
and Alexandre César Muniz de Oliveira 5,* Mauro Enrique de Souza Muñoz 1
, Matheus Chaves Menezes 1
, Edison Pignaton de Freitas 2
, Sen Cheng 3
,
Paulo Rogério de Almeida Ribeiro 4
, Areolino de Almeida Neto 5
and Alexandre César Muniz de Oliveira 5,* 1
LACMOR, Federal University of Maranhão, Av. dos Portugueses, 1966, São Luís 65080-805, MA, Brazil
2
INF, Federal University of Rio Grande do Sul, Av. Bento Gonçalves, 9500, 2
INF, Federal University of Rio Grande do Sul, Av. Bento Gonçalves, 9500,
Porto Alegre 91501-970, RS, Brazil g
3
INI, Ruhr University Bochum, 44801 Bochum, Germany y
y
4
ECP, Federal University of Maranhão, Av. dos Portugueses, 1966, São Luís 65080-805, MA, Brazil
5
DEINF, Federal University of Maranhão, Av. dos Portugueses, 1966, São Luís 65080-805, MA, Brazil y
y
4
ECP, Federal University of Maranhão, Av. dos Portugueses, 1966, São Luís 65080-805, MA, Brazil 5
DEINF, Federal University of Maranhão, Av. dos Portugueses, 1966, São Luís 65080-805, MA, Brazil
*
Correspondence: alexandre.cesar@ufma.br 5
DEINF, Federal University of Maranhão, Av. dos Portugueses, 1966, São Luís 65080-805, MA, Brazil
*
Correspondence: alexandre.cesar@ufma.br *
Correspondence: alexandre.cesar@ufma.br Abstract: Simultaneous localization and mapping (SLAM) refers to techniques for autonomously
constructing a map of an unknown environment while, at the same time, locating the robot in this
map. RatSLAM, a prevalent method, is based on the navigation system found in rodent brains. It
has served as a base algorithm for other bioinspired approaches, and its implementation has been
extended to incorporate new features. This work proposes xRatSLAM: an extensible, parallel, open-
source framework applicable for developing and testing new RatSLAM variations. Tests were carried
out to evaluate and validate the proposed framework, allowing the comparison of xRatSLAM with
OpenRatSLAM and assessing the impact of replacing framework components. The results provide
evidence that the maps produced by xRatSLAM are similar to those produced by OpenRatSLAM
when they are fed with the same input parameters, which is a positive result, and that implemented
modules can be easily changed without impacting other parts of the framework. sensors sensors sensors 1. Introduction Simultaneous localization and mapping (SLAM) deals with the robotic problem of
autonomously building the map of an initially unknown environment at the same time
as it locates the robot on this map. The map defines the robot’s localization by a pose
(position and orientation). The map can be understood as an abstract representation of a set
of resources describing the environment, such as walls, obstacles, landmarks, and so on [1]. Therefore, a robot running SLAM incrementally builds a representation of the environment
through pose estimations obtained from the data collected by its sensors [2]. Academic Editors: Chee Kiat Seow,
Henrik Hesse, Yanliang Zhang, Torr
Polakow and Kai Wen Besides these more general SLAM approaches, some bioinspired algorithms have also
been proposed for some specific autonomous robotic applications, such as exploration of
hazardous areas [3,4], robotic area surveillance [5], and robotic search and rescue [6–8]. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. One such bioinspired SLAM algorithm is RatSLAM [9], which was inspired by the
navigation system found in rodent brains, where the hippocampus and entorhinal cortex
play an important role in spatial navigation [10–12]. RatSLAM works both in indoor and
outdoor environments and requires only a monocular vision sensor [9,13–16]. Several
RatSLAM-based algorithms have been proposed recently, demonstrating continued interest
in bioinspired approaches for both robotics and neuroscience applications [17–19]. Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). The RatSLAM algorithm has three main computational modules: LocalView, Pose-
Cell and ExperienceMap. The LocalView module processes the robot’s camera inputs. The PoseCell module keeps track of the robot’s pose over time, combining odometry (path
integration) and sensor information from the LocalView module. The ExperienceMap
module collects experiences and embeds them into a topological graph representing the
environment’s topology. https://www.mdpi.com/journal/sensors Sensors 2022, 22, 8305. https://doi.org/10.3390/s22218305 2 of 15 Sensors 2022, 22, 8305 The work reported in [15] proposed a MatLab implementation of RatSLAM and
demonstrated how a rat animat robot could learn a spatial layout, including the ability to
close physical loops based only on visual input (image sequences) and robot odometry
(dead reckoning). 1.1. Main Objective In this work, we propose xRatSLAM, an extensible, parallel, open-source framework
(code available at [22]) intended to facilitate the development and testing of new RatSLAM
algorithm variations, allowing researchers and developers to share RatSLAM modules code
and compare their experiment results easily. The xRatSLAM framework was conceived so
that each RatSLAM module could be recoded without needing to change any other module,
i.e., applying good practices to achieve high cohesion and low coupling. Furthermore,
by mixing the features of a framework and a library, xRatSLAM allows us to dynamically
choose different module versions or biological behaviours. g
Researchers have proposed libraries and open-source implementations to reduce
unnecessary self-research (or knowledge recycling). A recent example is an augmented
reality system design that can be used in mobile devices [23] based on the previously
proposed OpenVSLAM [24]. The development of open-source libraries and frameworks is
justified whenever there is a growing interest by the academic community in responding to
scientific and technological demands, such as new propositions of methods for bioinspired
mapping or even immersion in indoor augmented reality [23]. 1. Introduction However, this implementation was not designed to be used by different
programmable robot interfaces since it was too slow to be applied in real-time for large
environments [16]. OpenRatSLAM, proposed in [16], is an open-source RatSLAM implementation based
on the Robot Operating System (ROS) [20], widely used in robotics. OpenRatSLAM
implements each module as an ROS node inheriting the ROS intrinsic node parallelisation
and the ability to integrate more than one robot architecture. However, OpenRatSLAM
cannot be used as a library since it cannot be easily integrated as a module by other
computational applications. p
pp
Therefore, the C++ library libratslam was developed [21], which employed a couple
of threads to run RatSLAM modules in parallel and could be easily integrated with other
computational systems. A drawback of that library was that it was not easy to modify its
modules’ code to implement another biological concept, as the classes that implemented
the RatSLAM modules were not designed as classes expected to be specialised. 1.2. Contributions and Organisation The main contribution of this work is an open-source, modular implementation of
RatSLAM mainly featured by: •
Processing: suitable to produce maps from batch and real-time image streams; •
Flexibility: developers can focus on implementing all or some specific RatSLAM
components, allowing them to use third part modules; •
Library: an efficient, easy-to-use, and integrated C++ library; •
Compatibility: compatible with well-settled RatSLAM implementations by using the
same input and output; •
Debugging: easy access to specific RatSLAM internal records for logging, tracing,
and monitoring tasks. This work also contributes to the literature on RatSLAM both in the conceptual and
practical fields. It is essential to mention that the technical specification of the proposed
piece of software is also a relevant result as it allows other developers to design new
frameworks and tools for bioinspired mapping and localization. This paper is divided into sections as follows: Section 2 presents an overview of the
main RatSLAM concepts and algorithms. In Sections 3 and 4, the proposal is detailed. Section 5 highlights the main experiments designed to evaluate and validate the pro- Sensors 2022, 22, 8305 3 of 15 posed framework. Finally, in Section 6, conclusions and new directions are summarised
and discussed. 2. The RatSLAM Dynamics RatSLAM is a SLAM system inspired by computational models of the neural pro-
cesses underlying navigation in the hippocampus and the entorhinal cortex of rodents [9]. Over time, RatSLAM has been enhanced to work with general real-world examples of
localization and mapping using a video camera as its main input sensor [13,25,26]. Since
RatSLAM has been discussed extensively in the literature (e.g., [9,14,16]), we only briefly
describe the system in the following. The RatSLAM architecture is composed of three main modules: PoseCell, LocalView,
and ExperienceMap. The system uses two external modules to capture input information:
the RobotVision module, which captures camera images of the environment, and the
RobotOdometry module, which captures robot self-motion cues. Figure 1 shows RatSLAM’s
internal and external modules and their interactions. An overview of the three RatSLAM original modules’ structures and behaviours is
presented in the next section. A more detailed algorithm description of the RatSLAM and
its three modules can be found in [9]. Figure 1. The main RatSLAM modules: LocalView, PoseCell, and ExperienceMap, including the
external modules RobotOdometry and RobotVision. Dashed arrows indicate messages. Figure 1. The main RatSLAM modules: LocalView, PoseCell, and ExperienceMap, including the
external modules RobotOdometry and RobotVision. Dashed arrows indicate messages. 3. The Proposed Extensible RatSLAM Framework The proposed xRatSLAM framework is designed to address computational issues in
previous works and foreseen future needs, yielding the following guidelines: 1. xRatSLAM is implemented as a library, so it can be easily incorporated into different
robotic applications; 2. Due to its modularity, it must be easy for developers to change the code implementa-
tion of any RatSLAM module without the need to recode any other modules or the
external relations, as data input or output related ones; 3. The sensor generalisation provides a facility for integration with other sensor inputs
beyond images; 4. The module implementations should run in multithreading mode, enabling paralleli-
sation; 5. The framework input parameters are compatible with the configuration parameters
used by the original RatSLAM implementation. 2.1. PoseCell Module The PoseCell is a continuous attractor network (CAN) configured in a three-dimensional
matrix with fixed hard-coded weights between its cells. The weights are set to excite nearby
cells and inhibit distant ones. This structure aims to keep track of clusters of excitatory cells
called activity packets. The network’s total activity level is kept constant. Hence, as distant
cells inhibit each other, distant activity packets compete with each other. By contrast,
the excitation between nearby cells fuses activity packets near each other. The network uses
periodic boundary conditions, i.e., the cells on the network’s boundary are neighbours. The PoseCell elements are not meant to represent each possible robot pose. Therefore,
the possible robot pose representation is limited by PoseCell dimension sizes. Instead,
the PoseCell network serves to resolve conflicts between competing sensor readings. The
activity packet remains stable in the absence of external excitation. Two external inputs can
modify its dynamics: odometry readings and sensory data. The odometry indicates how
the robot’s pose has changed at each time step. If the odometry can be trusted, this pose
variation is used to find the PoseCell element capable of representing the next robot pose. Then, this element is injected with some excitation energy. The influence of the sensory
data is discussed in the following section. 4 of 15 Sensors 2022, 22, 8305 3.1. xRatSLAM as a Library Design As xRatSLAM can be used as a C++ library, it can be ported to any C++/11 standard
robotic compliant system. This approach was motivated by the possibility of subdividing
complex problems into different components implemented in independent libraries: au-
tonomous navigation, obstacle avoidance, learning the environment, mapping, etc. In this
scenario, it is easier for the designer to build the robotic application using libraries dedicated
to each specific robotic task. 2.2. LocalView Sensory data influence PoseCell elements through the LocalView module. The Lo-
calView structure stores features of sensory data perceived at each robot pose and checks if
the current sensor data are new or have been perceived before. In the original RatSLAM
implementation, this structure stores image features in a list. Each time a new image is
captured, its features are extracted and compared with all stored images using a match
threshold parameter. If the current sensory input is new, it is stored and associated with the PoseCell element
representing the centre of the current activity packet. If the current input matches a stored
image, its associated PoseCell element is excited. When repeated, the excitation of PoseCell
elements by known sensory inputs leads to a growth of an activity packet in the PoseCell
network, which competes with the existing activity packet maintained by robot odometry. The coherence of sensory data determines the winner of this competition. 2.3. ExperienceMap RatSLAM was initially designed for use in a 2D space based on visual sensors in a
terrestrial robot. Therefore, the topological map of the environment is stored as a list of
nodes, called experiences, which store the robot poses and the elements of LocalView and
PoseCell that were activated when they were created. The graph links store times and
distances between the experiences. Since the experience references the activated PoseCell and LocalView elements, it
is possible to detect whether the robot is in a place it has occupied before. When this
occurs, a loop is detected, and the path correction process takes place to reorganise all
ExperienceMap node poses. 3.3. xRatSLAM Modules The xRatSLAM library interface was implemented through a single class named
xratslam::XRatSLAM. The library class diagram is shown in Figure 2. The interface class
allows the user to start and stop internal module threads, feed input data, read results,
and perform other practical computing operations. The xRatSLAM framework uses one
abstract base class for each RatSLAM module and the original proposed modules are
implemented as classes extending its respective base class OriginalLV, OriginalPC, and
OriginalEM (Figure 2). Figure 2. xRatSLAM main class diagram. Modules are designed as abstract classes, which different
possible module implementations can easily overload. The diagram shows two implementation
examples for the LocalView module: the original one uses visual sensors, and another uses magnetic
sensors. Used symbols follow standard UML. Figure 2. xRatSLAM main class diagram. Modules are designed as abstract classes, which different
possible module implementations can easily overload. The diagram shows two implementation
examples for the LocalView module: the original one uses visual sensors, and another uses magnetic
sensors. Used symbols follow standard UML. This way, the original RatSLAM modules are seen as examples of how the modules
can be implemented, allowing developers to create their module implementations. For
instance, LocalView uses generic sensor data instead of the image data used by the original
algorithm (OriginalLV). This modification allows, for example, the implementation of a
MagneticLV to deal with magnetic or another kind of sensor [28]. g
g
An important benefit of the proposed architecture is that it lets users choose dynamically
which combination of module implementations they would like to use. This is done through
the xRatSLAM methods: setLocalView(), setPoseCell(), and setExperienceMap(). 3.2. xRatSLAM as a Framework Framework modelling has a more direct impact on developing new features and
behaviour. The advantage of the xRatSLAM framework stems from the definition of
the system architecture, which is often the most challenging part of the software design,
generally neglected by other reuse techniques. The modelling by framework simplifies the
synchronisation of the execution of individual components and the communication among Sensors 2022, 22, 8305 5 of 15 5 of 15 them. It also provides highly optimised and parallelised methods to transfer data between
model grids [27]. them. It also provides highly optimised and parallelised methods to transfer data between
model grids [27]. 3.4. Module Parallelisation The OpenRatSLAM implementation uses parallelisation that is inherent in ROS
nodes [16]. In ROS, each node runs as a different system process, synchronised through the
ROS topic-publishing mechanism. By contrast, xRatSLAM parallelisation is implemented
through different threads of the same process. Each thread runs on an internal loop method
responsible for reading data from one or more synchronised queues, calling the associated
module entry point in its abstract class (Figure 3). y p
g
The synchronisation between threads is achieved through data queues. For example,
consider the case when loopExperienceMap() tries to read data from QueueOdometry. If the queue is empty, the thread is blocked until some Odometry data are inserted in
the queue. Sensors 2022, 22, 8305 6 of 15 Figure 3. The xRatSLAM main execution loop. The main loop and internal threads communicate
using data queues. Incoming odometry and sensor data are stored in its respective queue (Odometry
is replicated). Each module (LocalView, PoseCell, and ExperienceMap) has its own thread responsible
for reading incoming queue data and communicating with its respective module abstract class
to process the data and then store the result in a queue for further processing. Arrows indicate
dependencies between objects. Figure 3. The xRatSLAM main execution loop. The main loop and internal threads communicate
using data queues. Incoming odometry and sensor data are stored in its respective queue (Odometry
is replicated). Each module (LocalView, PoseCell, and ExperienceMap) has its own thread responsible
for reading incoming queue data and communicating with its respective module abstract class
to process the data and then store the result in a queue for further processing. Arrows indicate
dependencies between objects. Once data are available, the thread tries to read an Action object from QueueAc-
tion, and, again, it is blocked until there are data to be read. When both Odometry and
Action are read, the thread calls ExperienceMap::onFeed() to let the ExperienceMap mod-
ule perform its computations. The loop restarts, and the thread tries to read the next
QueueOdometry data. All threads are also blocked when trying to insert an element into a full queue. The queue’s push-and-pop blocking mechanisms ensure synchronisation among the inter-
nal loop threads. The maximum size of all queues is hard-coded and is set to 10 elements as
this size was empirically found to be sufficient for obtaining satisfactory parallelisation re-
sults. 3.5. Customised Modules 3.5. Customised Modules 3.5. Customised Modules Customised RatSLAM modules can be internally instantiated using the Factory design
pattern (Figure 4). Currently, there is no plug-and-play mechanism to insert a new module into the frame-
work. Thus, the following steps are required: 1. Create a class with the new module code extending the corresponding module base
class (LocalView, PoseCell, or ExperienceMap). The derived class must implement
at least the base class pure virtual methods. p
2. Change the Factory class code, so it knows how to instantiate the newly created
module by name. 3. As xRatSLAM uses cmake to build the code, all *.cc used by the new module should
be added to "CMakeLists.txt” file. 4. Finally, the client program should select the customised module, by its name (as a
xRatSLAM object), before calling XRatSLAM::start() as explained in Section 4. 7 of 15 7 of 15 Sensors 2022, 22, 8305 Figure 4. xRatSLAM class diagram highlighting factory project aspects. The Factory design pattern
allows for dynamically instantiating each specific module implementation based on its respective
name by knowing internal module packages independently of package implementations. Note that
the system uses just one Factory element. Used symbols follow standard UML. Figure 4. xRatSLAM class diagram highlighting factory project aspects. The Factory design pattern
allows for dynamically instantiating each specific module implementation based on its respective
name by knowing internal module packages independently of package implementations. Note that
the system uses just one Factory element. Used symbols follow standard UML. All modules are accessed by calling their onFeed() method, which receives different
parameters for each module and has the following functions: •
LocalView: receives a Sensor object (ex: Image) and generates a templateId number
identifying a sensory scene corresponding to the given sensor object. If the object was
already perceived, the templateId should be set to the id of the previously sensed
object. If not, a new template should be created with a new templateId. •
PoseCell: receives an Odometry and a Template object representing what is currently
sensed and generating an Action object. The module should decide where the agent
stores the log of odometry data and experiences. The Action indicates what should be
done by the ExperienceMap: (a) do nothing, (b) store a new pose, or (c) update a pose
already known. y
•
ExperienceMap: receives an Odometry and an Action object. 3.6. Using Different Types of Sensors In RatSLAM, loop closures are triggered by the LocalView module whenever it detects
that the current state has been sensed. In the original implementation, an image template
is created to represent a set of similar images. Each new template is compared to the
stored ones. The current template is discarded, and loop closure is triggered if already
sensed. Otherwise, the current template represents a new state and should be stored in
memory for later comparisons. Hence, a sensed state creation action is generated. Since
RatSLAM is not intrinsically restricted to using only images and can use other data, it is
worth mentioning that LocalView is the only module directly affected by the type of sensor
used. The proposed framework generalises sensor types using the Sensor class as the base
class for all implemented sensor types. 3.5. Customised Modules It uses Odometry
to update a guess about the physical location and Action to create new location
landmarks or to link existing ones. 3.8. Peripheral Computational Tools All RatSLAM implementations share some basic computational modules for general
tasks unrelated to the algorithms. These peripheral modules play an essential role in the
framework architecture. How these modules are designed determines how easily the
framework can adapt to a new situation. There are two crucial peripheral modules used to
transform how data are captured. •
Sensor reader. The framework is meant to be used by a robot capable of reading its
sensory and odometry data in real time from its hardware, but it is also meant to be
used by researchers whose experimental data are stored in files, e.g., on a desktop
computer. Therefore, the peripheral DataReader class can read data directly from
sensors or files that store previously recorded sensor data. p
y
•
Visual odometry. The RatSLAM algorithm was conceived to use robot odometry
and sensory image data. However, the odometry information can be extracted from
a sequence of images. This module can use those images to generate visual-based
odometry, replacing the robot odometry information as proposed by [9]. 3.7. Development Aspects xRatSLAM is designed to be used in production systems, algorithm development,
and test environments. When testing a new module implementation or comparing per-
formances of different module implementations, it is desirable to log or even visualise
some internal states and measure each module’s execution time performance. xRatSLAM
provides three ways to assess how the algorithm modules perform. Sensors 2022, 22, 8305 8 of 15 8 of 15 •
Execution time. A TimeLogger object can be set for each module. This object can be
used to access the time measures in the corresponding module’s past executions. This
metric measures only the execution of the module onFeed() method, excluding the
time spent waiting for module input data to become available. •
Execution time. A TimeLogger object can be set for each module. This object can be
used to access the time measures in the corresponding module’s past executions. This
metric measures only the execution of the module onFeed() method, excluding the
time spent waiting for module input data to become available. p
g
p
•
Internal state. Access to internal data structures is desirable to provide users with
graphical feedback on what is happening while the algorithm is running. From the
framework point of view, it represents a challenge because the framework is unaware
of the modules’ internal data structures. The current framework implementation,
therefore, imposes a semantic data structure for each module. This way, LocalView
implementations should return a set of Template objects. PoseCell implementations
shall return a 3D activation matrix, and ExperienceMap implementations shall return
a topological graph. Note that data access interfaces must be changed if a new module
implementation does not fit in. p
•
State storage. A common scenario in the algorithm development process is testing the
code implementation on captured data, sometimes using a benchmark data set. This
process can be inefficient when long-running experiments and errors only occur later. The xRatSLAM class has an interface to save() and load() the modules’ internal
states. Each module extension should implement such methods. If so, the xRatSLAM
algorithm can be restored by saving the algorithm state and restarting its execution
from that point. 5. Experiments This section presents three experiments that were performed to evaluate and vali-
date the proposed framework. The first one is meant to compare the maps and running
times obtained by xRatSLAM to those of OpenRatSLAM, both running standard modules. The second experiment evaluates the flexibility of xRatSLAM, since any module imple-
mentation can be changed without impacting any other part of the framework. Finally,
the third experiment compares the data collected by visual and wheel odometry, i.e., a
typical RatSLAM experiment that usually shows the behaviour of the peripheral modules. yp
p
y
p
p
We ran all experiments on an Intel(R) Core(TM) i7-2630QM CPU @ 2.00 GHz, one phys-
ical processor, four cores, eight threads with 4G of RAM running the Debian GNU/Linux
11 (bullseye) operating system. 4. xRatSLAM Usage stop ( )
// Stop
a l l xRatSLAM threads . 1 xRatSLAM slam (
configFile
)
2
3
slam . setLocalView
( moduleName )
// Optional . 4
slam . setPoseCell
( moduleName )
// Optional . 5
slam . setExperienceMap ( moduleName )
// Optional . 6
7 // Read input images from video
f i l e . 8
DataReaderVideo reader (
configFile ,
videoInput
) ;
9
10 // Use visual odometry . 11
reader . setOdometryVisual (
configFile
) ;
12
13
TimeLogger logger (
logDir
) ;
// Optional . 14
slam . setLogger (
logger
)
// Optional . 15
16
slam . s t a r t ( )
// S t a r t
a l l xRatSLAM~threads . 17
18
while
(
reader . readNext (
Sensor ,
Odometry )
)
19
{
20
slam . feed (
Sensor , Odometry )
21
drawEM( slam . getDataEM ( )
)
// Optional . 22
}
23
24
slam . stop ( )
// Stop
a l l xRatSLAM threads . 1 xRatSLAM slam (
configFile
) 3
slam . setLocalView
( moduleName )
// Optional . 4
slam . setPoseCell
( moduleName )
// Optional . 4
slam . setPoseCell
( moduleName )
// Optional . 5
slam . setExperienceMap ( moduleName )
// Optional . 6 5
slam . setExperienceMap ( moduleName )
// Optional . 6 // Optional . 4. xRatSLAM Usage The xRatSLAM parameters are read from a configuration file following the same
syntax suggested in [16]. The basic algorithm usage is shown in Listing 1. Note that the
client program can select which combination of LocalView, PoseCell, and ExperienceMap
modules are used. Default modules are also available following the implementation used
in [16]. The way to obtain Sensor and Odometry input data is not a concern for xRatSLAM. However, to support developers, two classes are made available to read image sequences
from video files (DataReaderVideo) and to read from multiple image files (DataReaderDir). These classes can also read Odometry data from an odometry file or generate odometry
information from image sequences (visual odometry). Note that the application can read
these input data directly from a robot. p
y
The client application controls the program flow by calling the start() and stop()
methods to start and stop all loop threads, respectively. At each iteration, the application
reads Sensor and Odometry input data and feeds them by the XRatSLAM::feed() method. When the stop() method is called, all threads ensure that the data of all synchronised
queues are properly processed before they terminate. Sensors 2022, 22, 8305 9 of 15 Finally, line 21 illustrates that the client can optionally read some internal data and
use them, for example, to show graphical feedback to end users. Listing 1. Simple pseudocode to illustrate xRatSLAM usage. Listing 1. Simple pseudocode to illustrate xRatSLAM usage. g
p
p
g
1 xRatSLAM slam (
configFile
)
2
3
slam . setLocalView
( moduleName )
// Optional . 4
slam . setPoseCell
( moduleName )
// Optional . 5
slam . setExperienceMap ( moduleName )
// Optional . 6
7 // Read input images from video
f i l e . 8
DataReaderVideo reader (
configFile ,
videoInput
) ;
9
10 // Use visual odometry . 11
reader . setOdometryVisual (
configFile
) ;
12
13
TimeLogger logger (
logDir
) ;
// Optional . 14
slam . setLogger (
logger
)
// Optional . 15
16
slam . s t a r t ( )
// S t a r t
a l l xRatSLAM~threads . 17
18
while
(
reader . readNext (
Sensor ,
Odometry )
)
19
{
20
slam . feed (
Sensor , Odometry )
21
drawEM( slam . getDataEM ( )
)
// Optional . 22
}
23
24
slam . 5.1. Experiment I Performance requirements concern the framework’s accomplishment of certain func-
tions under specific conditions. They can be evaluated by measuring response and running
times and storage requirements. In this section, the xRatSLAM framework is compared
to OpenRatSLAM in terms of running time for mapping under the same circumstances,
i.e., the same data input. For fairness, the code used by OpenRatSLAM for all three RatSLAM modules (Lo-
calView, PoseCell, and ExperienceMap) was inserted as modules in the xRatSLAM frame-
work to run both programs with the same inputs and module implementations. The kits iRat and Robodeck [16,21] were used to compare both implementations. The iRat (Intelligent Rat Animat Technology) robot (www.davidmichaelball.com/portfolio-
items/irat-intelligent-rat-animat-technology/ accessed on 20 October 2022) is designed
for studies in navigation, embodied cognition, and neuroscience research, equipped Sensors 2022, 22, 8305 10 of 15 10 of 15 with a camera sensor, proximity, and odometry sensors in the wheels. The Robodeck
(http://www.xbot.com.br/educacional/robodeck/ accessed on 20 October 2022) is a wheel-
based mobile robotic educational platform composed of four independent wheels, each
controlled by a steering servo motor. The data generated by Robodeck consist of a video
stream collected by a low-resolution camera during an indoor tour in a research labora-
tory [21]. y
The experience maps generated by the two implementations were very similar, but they
were not identical. The experience maps were compared using libicp software [29], which
implements the iterative closest point (ICP) algorithm. Figure 5a,b show the resulting Expe-
rienceMaps generated by the OpenRatSLAM and xRatSLAM implementations, respectively,
for the iRat experiment. Similarly, Figure 6 shows the results for the Robodeck experiment. The error obtained by ICP were 0.0117619 (iRat) and 0.488817 (Robodeck). Interestingly,
the more complicated complex mapping had the lowest ICP error. −2
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Figure 5. ExperienceMap generated in iRat experiment. ICP error obtained was 0.0117619. (a) iRat
robot path found by OpenRatSLAM. Time: 960 s. (b) iRat robot path found by xRatSLAM. Time:
245 s. −2
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(b) (a) (b) Figure 5. 5.1. Experiment I ExperienceMap generated in iRat experiment. ICP error obtained was 0.0117619. (a) iRat
robot path found by OpenRatSLAM. Time: 960 s. (b) iRat robot path found by xRatSLAM. Time:
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Figure 6. ExperienceMap generated in Robodeck experiment. ICP error obtained was 0.488817. (a) Robodeck robot path found by OpenRatSLAM. Time: 204 s. (b) Robodeck robot path found by
xRatSLAM. Time: 38 s. 0
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(b) (b) (a) Figure 6. ExperienceMap generated in Robodeck experiment. ICP error obtained was 0.488817. (a) Robodeck robot path found by OpenRatSLAM. Time: 204 s. (b) Robodeck robot path found by
xRatSLAM. Time: 38 s. The divergence could be explained by the fact that OpenRatSLAM is based on the
Robot Operating System (ROS) (https://www.ros.org/ accessed on 20 October 2022), which
does not guarantee reading all input data in the same sequence as xRatSLAM does [20]. Figure 6a was directly generated by the OpenRatSLAM plot procedure, which does not
preserve the x:y ratio. The run time required by OpenRatSLAM was about 960 s, while xRatSLAM took only
about 245 s to obtain the ExperienceMap for the iRat data set. Considering the running Sensors 2022, 22, 8305 11 of 15 11 of 15 times for generating the experience maps of the two video data (iRat and Robodeck),
xRatSLAM tended to be around four to five times faster than OpenRatSLAM. times for generating the experience maps of the two video data (iRat and Robodeck),
xRatSLAM tended to be around four to five times faster than OpenRatSLAM. Regarding the time analysis, it is essential to mention that the input data were the same
for both software executions and that both implementations were deterministic. Therefore,
only one execution was sufficient for collecting and analysing the running times. The results could ensure that, under the same circumstances, xRatSLAM reached
similar experience maps faster. It is worth mentioning that whilst OpenRatSLAM ran with
a parallel process, xRatSLAM ran with parallel threads. 5.1. Experiment I Moreover, the improvements in
parallelisation combined with some small module recoding were responsible for the gains
concerning running time. Furthermore, the run-time measures of each xRatSLAM module were further analysed
using tracing features available in the framework. The separate performance evaluation
identified which modules were more sensitive and required for eventual implementation
improvements. Figure 7 shows the average running time for each module in each iteration
step. While the PoseCell execution time per iteration was nearly constant, the LocalView
and ExperienceMap running times grew linearly as the template list became longer. 0
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LocalView
PoseCell
ExperienceMap
Figure 7. The iRat experiment analysed by xRatSLAM. Modules execution time over iterations:
LocalView average: 14.43 ms. PoseCell average: 4.99 ms. ExperienceMap average: 6.31 ms. Total
time: 245 s. ExperienceMap data LocalView
PoseCell
ExperienceMap Figure 7. The iRat experiment analysed by xRatSLAM. Modules execution time over iterations:
LocalView average: 14.43 ms. PoseCell average: 4.99 ms. ExperienceMap average: 6.31 ms. Total
time: 245 s. Analysing the RatSLAM modules revealed that the LocalView module had poor
performance. This module stores image templates of image views that were encountered in
the environment. When a new image is perceived, its template is generated and compared
to all stored visual templates, as discussed previously. The linear increase in the running
time of LocalView (Figure 7) can be explained by the linear search algorithm used in this
module, i.e., the search for a matched template is started at the first stored template and
proceeds linearly through the list of stored templates. 5.2. Experiment II As observed in Figure 7, the running time of this module had a linear asymptotic
growth. A small change in the search algorithm’s implementation in LocalView might
significantly impact performance. The new LocalView implementation started at the last
template and moved backward through the template list. If the template was stored in the
list, the running time became mainly independent of the list size because it was very likely
that a known template had been encountered recently. If the template was new, the search Sensors 2022, 22, 8305 12 of 15 12 of 15 had to proceed through the entire list, and the algorithm obviously depended on the list
size, and the running time scaled linearly. had to proceed through the entire list, and the algorithm obviously depended on the list
size, and the running time scaled linearly. Beyond the new implementation, this experiment was used to assess the impact
of a simple code change and log resources available in the xRatSLAM framework. This
experiment also demonstrated how the performance of a specific module could be measured
and how a new module implementation could be added to the framework, focusing only
on one module code. on one module code. The xRatSLAM framework was executed using the new LocalView implementation,
RecodedLV, for which the following average execution time was obtained (see Figure 8). The xRatSLAM framework was executed using the new LocalView implementation,
RecodedLV, for which the following average execution time was obtained (see Figure 8). 0
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LocalView
PoseCell
ExperienceMap
Figure 8. Average execution times of xRatSLAM using the RecodedLV module implementation. LocalView average: 4.79 ms. PoseCell average: 5.01 ms. ExperienceMap average: 6.30 ms. Total time:
121.0 s. ExperienceMap data Figure 8. Average execution times of xRatSLAM using the RecodedLV module implementation. LocalView average: 4.79 ms. PoseCell average: 5.01 ms. ExperienceMap average: 6.30 ms. Total time:
121.0 s. As expected, the average run time of the LocalView module still grew linearly, but it in-
creased much more slowly for RecodedLV—notably the execution time of the new RecodedLV
module was 4.79 ms as compared to 14.43 ms for OriginalLV module in Experiment I. The
average execution times of both PoseCell and ExperienceMap were similar to the previous
implementation (Figure 7). 5.2. Experiment II A single, small modification in the LocalView implementation
cut the total execution time in half from 245.0 s (Figure 7) to 121.0 s (Figure 8). Hence, it
is possible to interchange execution between any module implementations (see Listing 1)
without needing to inspect or recode any other part of the system. 5.3. Experiment III In this third experiment, the results obtained using wheel odometry were compared to
those obtained with visual odometry based on changes between consecutive video frames. xRatSLAM used the same visual odometry code available in [16]. Figure 9a shows the resulting experience map obtained when the iRat robot gener-
ated the odometry, whilst Figure 9b shows the experience map obtained using the visual
odometry, calculated by xRatSLAM. As expected, the visual odometry was not as good at
generating closed curves as the robot odometry. 13 of 15 13 of 15 Sensors 2022, 22, 8305 −2
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Figure 9. ExperienceMap comparison between different odometry sources. (a) ExperienceMap
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(a) (a) (b) Figure 9. ExperienceMap comparison between different odometry sources. (a) ExperienceMap
using odometry information read from iRat robot. (b) ExperienceMap using odometry information
generated by visual odometry with parameters empirically tuned. 6. Conclusions and Future Work This work proposed an extensible, parallel, open-source framework implementation
of RatSLAM. This framework aimed to facilitate the collaborative development and testing
of RatSLAM-based algorithms, allowing researchers and developers to easily share code
for RatSLAM modules and compare their performance in experiments. The xRatSLAM framework was designed to make it possible to recode or replace a
module with no impact on other modules and apply good practices to achieve high cohesion
and low coupling. Furthermore, by mixing the features of a framework and a library,
xRatSLAM allowed us to dynamically choose different module versions or even biological
behaviours. The modelling following the framework simplified the synchronisation of the
execution of individual components and the communication among them. It also provided
highly optimised and parallel data transfer between modules. Three experiments were conducted to assess and validate the proposed framework. The results showed that: (i)
xRatSLAM could produce similar maps compared with OpenRatSLAM when fed with
the same input data, running faster than OpenRatSLAM; (ii)
A module implementation could be easily changed without impacting any other part
of the framework; (iii) The framework was featured by logging capabilities that allowed a detailed analysis
of the results; (iv) In the absence of a robot mechanical generated odometry, visual odometry could
generate good approximate maps for the case studied. In the accuracy and performance contexts (item (i)), xRatSLAM has already been
used by researchers in experiments that require shorter execution times to perform time-
consuming tasks, such as parameter tuning in long-term mapping [30,31]. However,
opportunities for enhancements are highlighted below to show how the framework can be
improved: In the accuracy and performance contexts (item (i)), xRatSLAM has already been
used by researchers in experiments that require shorter execution times to perform time-
consuming tasks, such as parameter tuning in long-term mapping [30,31]. 6. Conclusions and Future Work Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: xRatSLAM code is available to download under a GPLv3 License at
https://doi.org/10.17632/rrjv728cmj.1. Data Availability Statement: xRatSLAM code is available to download under a GPLv3 License at
https://doi.org/10.17632/rrjv728cmj.1. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results. 8.
Cai, Y.; Chen, Z.; Min, H. Improving particle swarm optimization algorithm for distributed sensing and search. In Proceedings
of the 2013 Eighth International Conference on P2P, Parallel, Grid, Cloud and Internet Computing, Compiegne, France, 28–30
October 2013; pp. 373–379. 6. Conclusions and Future Work However,
opportunities for enhancements are highlighted below to show how the framework can be
improved: (i)
Other RatSLAM module implementations; (ii)
A built-in assessment module for mapping accuracy evaluation; (iii) Dynamic libraries (plug-ins) in the module inclusion mechanism; (iv) Support for 3D SLAM, as required for unmanned aerial vehicles (UAV: drones) or
uncrewed underwater vehicles (UUV); (v)
A ROS wrapper for xRatSLAM; pp
(vi) An interface for other programming languages such as Python; (vii) A module repository for sharing implementations between different user (viii)Usability improvements to suit neuroscience theorists and practitioner 14 of 15 14 of 15 Sensors 2022, 22, 8305 The fundamental point is that xRatSLAM is not a substitute for the OpenRatSLAM
framework. The xRatSLAM is an alternative for robot systems using the RatSLAM algo-
rithm as a library without ROS. Compared with OpenRatSLAM, the main drawback is that
the xRatSLAM uses a library-like interface, so only a C++ code can access it. Moreover,
xRatSLAM does not provide a mechanism to use stored input data respecting the real time
they were saved at. As OpenRatSLAM uses ROS, this mechanism is available. y
p
However, OpenRatSLAM uses the ROS node interface. Thus, not just the program
accessing the RatSLAM needs to be written in any ROS-compliant language, but also its
modules. Robot systems already using the ROS framework can easily adopt the OpenRat-
SLAM solution, while the use of xRatSLAM requires extra effort to create a ROS node to
communicate with it. Author Contributions: Conceptualization, M.E.d.S.M., M.C.M., P.R.d.A.R., A.d.A.N. and A.C.M.d.O.;
Data curation, M.E.d.S.M., S.C. and A.C.M.d.O.; Formal analysis, M.E.d.S.M., M.C.M. and E.P.d.F.;
Funding acquisition, E.P.d.F., P.R.d.A.R., A.d.A.N. and A.C.M.d.O.; Investigation, M.E.d.S.M., E.P.d.F.,
S.C., P.R.d.A.R. and A.d.A.N.; Methodology, M.E.d.S.M., M.C.M., E.P.d.F. and A.C.M.d.O.; Project
administration, M.E.d.S.M. and A.C.M.d.O.; Resources, A.C.M.d.O.; Software, M.E.d.S.M. and
M.C.M.; Supervision, M.E.d.S.M. and A.C.M.d.O.; Validation, M.E.d.S.M., M.C.M., E.P.d.F., P.R.d.A.R.,
A.d.A.N. and A.C.M.d.O.; Writing—original draft, M.E.d.S.M. and M.C.M.; Writing—review & edit-
ing, M.E.d.S.M., E.P.d.F., S.C., P.R.d.A.R., A.d.A.N. and A.C.M.d.O. All authors have read and agreed
to the published version of the manuscript. Funding: The authors acknowledge the financial support from: FAPEMA COOPI-05109/18, CAPES/
BRAZIL Finance Code 001, PNPD/CAPES/BRAZIL 88882.315469/2019-01, CNPq projects 309505/
2020-8 and 420109/2018-8, and German Research Foundation (DFG)—project number 316803389-SFB
1280, A14. Edison Pignaton de Freitas also thanks the Ruhr-Universität Bochum—Research School
PLUS, for the Visiting International Professor (VIP) grant. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. References Path integration and the neural basis of the ‘cognitive map’. Nat. Rev. Neurosci. 2006, 7, 663. [CrossRef] [PubMed] 13. Milford, M.J.; Wyeth, G.F. Mapping a Suburb with a Single Camera Using a Biologically Inspired SLAM System. IEEE Trans. Robot. 2008, 24, 1038–1053. [CrossRef] ,
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de Oliveira, A.C.M., Eds.; Springer International Publishing: Cham, Switzerland, 2022; pp. 48–63.
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https://openalex.org/W4361873325
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https://aacr.figshare.com/ndownloader/files/39846062
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English
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Supplementary Figures 1 - 3 from USP22 Regulates Oncogenic Signaling Pathways to Drive Lethal Cancer Progression
| null | 2,023
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cc-by
| 1,275
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Supplementary Figure S1
LN-Vec
3 5
4.0
t)
Chromatin IP: AR
0.8
1.0
1.2
1.4
TA1/GAPDH
MTA1
LN-Vec
LN-USP22
A.
C.
LN Vec
LN-USP22
0.5
1.0
1.5
2.0
2.5
3.0
3.5
AR binding (% Input
n.s.
DHT
0.0
0.2
0.4
0.6
Relative MT
+
-
-
+
1 4
1.6
DH
BAG1
0.0
A
PSA ‘EF’ Region
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Relative BAG1/GAPD
DHT
+
-
-
+
DHT
+
+
B.
LN-Vec
LN-Vec
LN-USP22
LN-USP22
-c-Myc
DHT
-GAPDH
-
-
+
+
1
2
3
4
Supplementary Figure S1: USP22 Specifically Promotes AR Recruitment to Target Loci. (A) RNA was extracted
for qRT-PCR analysis of mRNA levels of Myc-target genes MTA1 and BAG1. (B) LN-USP22 and control cells were
androgen deprived and stimulated with DHT or vehicle and cell lysates were immunoblotted with Myc and GAPDH
antibodies. (C) LN-USP22 and control cells were cultured in androgen-deprived media for 72 hours. Cross-linked
chromatin derived from cells was immunoprecipitated with AR-N20 antibody and analyzed using primers targeting PSA
‘EF’ Region, which represents a region of the PSA gene that does not contain AR-binding sequences. Supplementary Figure S1
LN-Vec
3 5
4.0
t)
Chromatin IP: AR
0.8
1.0
1.2
1.4
TA1/GAPDH
MTA1
LN-Vec
LN-USP22
A. C. LN Vec
LN-USP22
0.5
1.0
1.5
2.0
2.5
3.0
3.5
AR binding (% Input
n.s. DHT
0.0
0.2
0.4
0.6
Relative MT
+
-
-
+
1 4
1.6
DH
BAG1
0.0
A
PSA ‘EF’ Region
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Relative BAG1/GAPD
DHT
+
-
-
+
DHT
+
+
B. LN-Vec
LN-Vec
LN-USP22
LN-USP22
-c-Myc
DHT
-GAPDH
-
-
+
+
1
2
3
4
Supplementary Figure S1: USP22 Specifically Promotes AR Recruitment to Target Loci. (A) RNA was extracted
for qRT-PCR analysis of mRNA levels of Myc-target genes MTA1 and BAG1. (B) LN-USP22 and control cells were
androgen deprived and stimulated with DHT or vehicle and cell lysates were immunoblotted with Myc and GAPDH
antibodies. (C) LN-USP22 and control cells were cultured in androgen-deprived media for 72 hours. Cross-linked
chromatin derived from cells was immunoprecipitated with AR-N20 antibody and analyzed using primers targeting PSA
‘EF’ Region, which represents a region of the PSA gene that does not contain AR-binding sequences. Supplementary Figure S1
LN-Vec
3 5
4.0
t)
Chromatin IP: AR
C. LN Vec
LN-USP22
0.5
1.0
1.5
2.0
2.5
3.0
3.5
AR binding (% Input
n.s. 0.0
A
PSA ‘EF’ Region C. A. LN-Vec
3 5
4.0
t)
Chromatin IP: AR
LN Vec
LN-USP22
0.5
1.0
1.5
2.0
2.5
3.0
3.5
AR binding (% Input
n.s. 0.0
A
PSA ‘EF’ Region 1 4
1.6
DH
BAG1
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Relative BAG1/GAPD
DHT
+
-
-
+
DHT
+
+ PSA ‘EF’ Region B. LN-Vec
LN-Vec
LN-USP22
LN-USP22
-c-Myc
DHT
-GAPDH
-
-
+
+
1
2
3
4 B. LN-Vec
LN-Vec
LN-USP22
LN-USP22
-c-Myc
DHT
-GAPDH
-
-
+
+
1
2
3
4 B. Supplementary Figure S1: USP22 Specifically Promotes AR Recruitment to Target Loci. (A) RNA was extracted
for qRT-PCR analysis of mRNA levels of Myc-target genes MTA1 and BAG1. (B) LN-USP22 and control cells were
androgen deprived and stimulated with DHT or vehicle and cell lysates were immunoblotted with Myc and GAPDH
antibodies. (C) LN-USP22 and control cells were cultured in androgen-deprived media for 72 hours. Supplementary Figure S2
Supplementary Figure 2: Depletion of USP22 can be mediated by multiple sequences.
Supplementary Figure 2: Depletion of USP22 can be mediated by multiple sequences.
LNCaP cells were plated in 6 well plates and 24 hours later transfected with siControl, siATXN7L3
(control for another member of SAGA complex), or siUSP22 oligonucleotides using
Dharmafect. After 72 hours, cells were lysed and resultant lysates were probed with antibodies
detecting AR, USP22, ATXN7L3, or GAPDH Supplementary Figure S2
Supplementary Figure 2: Depletion of USP22 can be mediated by multiple sequences. Supplementary Figure 2: Depletion of USP22 can be mediated by multiple sequences. LNCaP cells were plated in 6 well plates and 24 hours later transfected with siControl, siATXN7L3
(control for another member of SAGA complex), or siUSP22 oligonucleotides using
Dharmafect. After 72 hours, cells were lysed and resultant lysates were probed with antibodies
detecting AR, USP22, ATXN7L3, or GAPDH Supplementary Figure S1
LN-Vec
3 5
4.0
t)
Chromatin IP: AR
0.8
1.0
1.2
1.4
TA1/GAPDH
MTA1
LN-Vec
LN-USP22
A.
C.
LN Vec
LN-USP22
0.5
1.0
1.5
2.0
2.5
3.0
3.5
AR binding (% Input
n.s.
DHT
0.0
0.2
0.4
0.6
Relative MT
+
-
-
+
1 4
1.6
DH
BAG1
0.0
A
PSA ‘EF’ Region
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Relative BAG1/GAPD
DHT
+
-
-
+
DHT
+
+
B.
LN-Vec
LN-Vec
LN-USP22
LN-USP22
-c-Myc
DHT
-GAPDH
-
-
+
+
1
2
3
4
Supplementary Figure S1: USP22 Specifically Promotes AR Recruitment to Target Loci. (A) RNA was extracted
for qRT-PCR analysis of mRNA levels of Myc-target genes MTA1 and BAG1. (B) LN-USP22 and control cells were
androgen deprived and stimulated with DHT or vehicle and cell lysates were immunoblotted with Myc and GAPDH
antibodies. (C) LN-USP22 and control cells were cultured in androgen-deprived media for 72 hours. Cross-linked
chromatin derived from cells was immunoprecipitated with AR-N20 antibody and analyzed using primers targeting PSA
‘EF’ Region, which represents a region of the PSA gene that does not contain AR-binding sequences. Cross-linked
chromatin derived from cells was immunoprecipitated with AR-N20 antibody and analyzed using primers targeting PSA
‘EF’ Region, which represents a region of the PSA gene that does not contain AR-binding sequences. Supplementary Figure S2 Supplementary Figure 2: Depletion of USP22 can be mediated by multiple sequences. Supplementary Figure 2: Depletion of USP22 can be mediated by multiple sequences. LNCaP cells were plated in 6 well plates and 24 hours later transfected with siControl, siATXN7L3
(control for another member of SAGA complex), or siUSP22 oligonucleotides using
Dharmafect. After 72 hours, cells were lysed and resultant lysates were probed with antibodies
detecting AR, USP22, ATXN7L3, or GAPDH Supplementary Figure
shLuc
EtOH
shLuc
DHT
shUSP
EtOH
shUSP
DHT
IP: HA (Ub)
IB: AR
AR
Input
IgG
AR
Input
IgG
AR
Input
IgG
AR
Input
IgG
IB: USP22
Supplementary Figure S3: AR Ubiquitylation Levels are not Altered in Response to USP22
1
2 3 4 5
6 7 8 9 10 11 12
Supplementary Figure S3: AR Ubiquitylation Levels are not Altered in Response to USP22
Depletion. LNCaP cells were infected with shUSP22-1 or control (shLuc)-encoding lentivirus for a
total of 120 hours, including androgen deprivation during the final 72 hours, followed by 1nM DHT
or vehicle stimulation for 16 hrs. Additionally, 24 hours after infections, cells were transfected with
plasmid encoding HA-Ubiquitin using Lipofectin. At the completion of the schedule, total cell lysates
and HA (Ub) immune complexes were immunoblotted with AR and USP22 antibodies. Supplementary Figure S3 shLuc
EtOH
shLuc
DHT
shUSP
EtOH
shUSP
DHT
IP: HA (Ub)
IB: AR
AR
Input
IgG
AR
Input
IgG
AR
Input
IgG
AR
Input
IgG
IB: USP22
1
2 3 4 5
6 7 8 9 10 11 12 Supplementary Figure S3: AR Ubiquitylation Levels are not Altered in Response to USP22
Supplementary Figure S3: AR Ubiquitylation Levels are not Altered in Response to USP22
Depletion. LNCaP cells were infected with shUSP22-1 or control (shLuc)-encoding lentivirus for a
total of 120 hours, including androgen deprivation during the final 72 hours, followed by 1nM DHT
or vehicle stimulation for 16 hrs. Additionally, 24 hours after infections, cells were transfected with
plasmid encoding HA-Ubiquitin using Lipofectin. At the completion of the schedule, total cell lysates
and HA (Ub) immune complexes were immunoblotted with AR and USP22 antibodies.
|
https://openalex.org/W3019580483
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https://europepmc.org/articles/pmc7230182?pdf=render
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English
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Active Pulmonary Tuberculosis Triggered by Interferon Beta-1b Therapy of Multiple Sclerosis: Four Case Reports and a Literature Review
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Medicina
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cc-by
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Received: 15 February 2020; Accepted: 22 April 2020; Published: 24 April 2020 Abstract: In this paper, we reported on four cases of severe pulmonary active tuberculosis in patients
with multiple sclerosis (MS) undergoing interferon beta-1b (IFNβ-1b) therapy. Disease-modifying
therapies (DMTs) in MS may increase the risk of developing active tuberculosis (TB) due to their
impact on cellular immunity. Screening for latent infection with Mycobacterium tuberculosis (LTBI)
should be performed, not only for the newer DMTs (alemtuzumab, ocrelizumab) but also for IFNβ-1b,
alongside better supervision of these patients. Keywords:
IFNβ-1b; multiple sclerosis; latent infection; Mycobacterium tuberculosis; active
tuberculosis; comorbidities medicina medicina Carmen Adella Sirbu 1,2
, Elena Dantes 3,*, Cristina Florentina Plesa 1,2, Any Docu Axelerad 4
and Minerva Claudia Ghinescu 1 Carmen Adella Sirbu 1,2
, Elena Dantes 3,*, Cristina Florentina Plesa 1,2, Any Docu Axelerad 4
and Minerva Claudia Ghinescu 1 1
Neurology Department, Titu Maiorescu University, 040441 Bucharest, Romania;
sircar13@yahoo.com (C.A.S.); plesacristina@yahoo.com (C.F.P.); ghinescu_minerva@yahoo.com (M.C.G.)
2
Neurology, Carol Davila Central Military Emergency University Hospital, 010242 Bucharest, Romania
3
Department of Pneumology, Faculty of Medicine, Clinical Hospital of Pneumophtisiology,
‘Ovidius’ University of Constanta, 900527 Constanta, Romania
4
Department of Neurology, Faculty of Medicine, Clinical Emergency Hospital ‘St. Apostol Andrei’,
‘Ovidius’ University of Constanta, 900527 Constanta, Romania; docuaxi@yahoo.com
*
Correspondence: elena.dantes@gmail.com 1
Neurology Department, Titu Maiorescu University, 040441 Bucharest, Romania;
sircar13@yahoo.com (C.A.S.); plesacristina@yahoo.com (C.F.P.); ghinescu_minerva@yahoo.com (M.C.G.)
2
Neurology, Carol Davila Central Military Emergency University Hospital, 010242 Bucharest, Romania
3
Department of Pneumology, Faculty of Medicine, Clinical Hospital of Pneumophtisiology,
‘Ovidius’ University of Constanta, 900527 Constanta, Romania
4
Department of Neurology, Faculty of Medicine, Clinical Emergency Hospital ‘St. Apostol Andrei’,
‘Ovidius’ University of Constanta, 900527 Constanta, Romania; docuaxi@yahoo.com
*
Correspondence: elena.dantes@gmail.com 1
Neurology Department, Titu Maiorescu University, 040441 Bucharest, Romania;
sircar13@yahoo.com (C.A.S.); plesacristina@yahoo.com (C.F.P.); ghinescu_minerva@yahoo.com (M.C.G.)
2
Neurology, Carol Davila Central Military Emergency University Hospital, 010242 Bucharest, Romania
3
Department of Pneumology, Faculty of Medicine, Clinical Hospital of Pneumophtisiology,
‘Ovidius’ University of Constanta, 900527 Constanta, Romania
4
Department of Neurology, Faculty of Medicine, Clinical Emergency Hospital ‘St. Apostol Andrei’,
‘Ovidius’ University of Constanta, 900527 Constanta, Romania; docuaxi@yahoo.com
*
Correspondence: elena.dantes@gmail.com
www.mdpi.com/journal/medicina Medicina 2020, 56, 202; doi:10.3390/medicina56040202 1. Introduction Multiple sclerosis (MS) is a chronic, neurodegenerative, demyelinating and inflammatory disease
which may be accompanied by other autoimmune or infectious diseases due to its immunological
peculiarities and the immunomodulatory nature of the treatment applied to patients [1]. Given their
impact on cellular immunity, disease-modifying therapies (DMTs) used to treat MS could increase the
risk of infections. Global surveillance, albeit with a limited follow-up period, has previously found that
highly effective DMTs (fingolimod, natalizumab) are associated with an increased risk of infections
compared to placebo or first line therapies (interferon beta and glatiramer acetate) [2]. A recent Swedish
study found an increased risk of infections of approximately 50% among people with MS who were
treated with first line DMTs compared to the general population [3]. Another randomized clinical
trial of the recently approved anti-CD20 therapy for MS, ocrelizumab, reported an increased risk of
respiratory tract infections compared with the interferon beta treatment and placebo [4,5]. The risk of
developing tuberculosis (TB) due to MS and DMTs is unknown. Moreover, we did not find any reported
cases of active TB disease in patients with multiple sclerosis undergoing interferon beta-1b (IFNβ-1b)
treatment. We found a single case of an MS patient treated with glatiramer acetate who had contact with
infected persons, and who underwent specific treatment for nine months, for whom TB was reactivated
after 16 years [6]. The risk of TB has been reported only in patients treated with newer DMT drugs,
such as alemtuzumab, natalizumab, fingolimod, mitoxantrone and dimethyl fumarate [7]. In clinical
trials, very few TB cases have been reported during treatment with cladribine and teriflunomide [8]. www.mdpi.com/journal/medicina Medicina 2020, 56, 202; doi:10.3390/medicina56040202 2 of 7 Medicina 2020, 56, 202 There is over 25 years of experience regarding the benefits, safety and tolerability of IFNβ-1b treatment,
as this was the first DMT used for the treatment of MS, beginning in 1993. IFNβ-1b binds to type I
interferon receptors. Approximately 100 immunomodulatory and antiviral proteins are activated by
their phosphorylation and dimerization. Chronic subcutaneous administration every other day, at the
recommended dose of 0.25 mg (1 mL), has been shown to induce long-term effects in the expression of
neuroprotective genes, brain repair and clinical efficacy [9]. However, the exact mechanism of action is
not known. Tuberculosis screening is recommended for patients with multiple sclerosis before starting
with certain therapies that modify the disease, which does not include IFNβ-1b. 1. Introduction DMTs may affect
interferon-gamma release test results. The QuantiFERON-TB Gold test measures the response of T
cells stimulated by Mycobacterium tuberculosis (Mtb) antigens [10]. 2. Case Reports We presented four patients diagnosed with relapsing-remitting MS (RRMS) and secondary
progressive MS (SPMS) who developed pulmonary TB while on chronic IFNβ-1b treatment. They were
selected from the patients diagnosed and treated at our center, which has 20 years of experience based
on the care of over 400 MS patients. The demographic, diagnostic and treatment data, as well as the
particularities of each case, are listed in Table 1. Table 1. Patient characteristics. Characteristics
Case 1
Case 2
Case 3
Case 4
Gender
F
M
F
F
MS diagnosis form
26, RRMS
32, SPMS
30, RRMS
37, RRMS
Age of IFNβ-1b
treatment initiation
27
45
32
39
MRI brain scan
2001, 2002, 2003, 2009,
2011, 2019
1998, 2000, 2007
2014, 2015, 2019
2006, 2008, 2015, 2018
EDSS progression
1 (2001); 5.5 (2019)
3.5 (2000); 5.5 (2019);
1 (2015); 4 (2019)
1 (2007); 1.5 (2019)
Time between
IFNβ-1b treatment
initiation and TB
12 months
48 months
36 months
84 months
Treatments other
than IFNβ-1b
before TB
Methylprednisolone,
antidepressants
Methylprednisolone,
beta-blocker,
antihypertensive,
statins, antidepressants
Methylprednisolone
Methylprednisolone
Onset of active TB,
28 y
49 y
35 y
46 y
Onset of symptoms
prior to TB
6 w
3 w
3 w
4 w
Respiratory
symptoms
Hemoptysis, asthenia,
weight and appetite loss,
a sharp twinge in left
side of chest
Asthenia, loss of
appetite, productive
cough, exertional
dyspnea
A twinge in the left
side of chest,
weight loss
Asthenia, fever,
persistent nonproductive
cough
Radiological
presentation
(Figure 1)
Nodular and caseous
cavitary lesions
Bilateral apical ulcerated
fibrocaseous lesions with
bronchogenic
disseminations
Caseous-cavitary
lesions
Infiltrative nodular and
bronchial forms
TB localization
Apical segment (Fowler)
left lower lobe
Bilateral upper lobes
Right upper lobe
Left upper lobe
Initial
bacteriological
examination of
sputum
Negative AFB smear;
positive for M. tuberculosis culture
Positive smear and
culture
Positive smear and
culture
Negative smear, positive
culture
Category of the
treatment regimen
2HRZS (5/7) +4HR (3/7)
2HRZE (7/7) +4HR (3/7)
2HRZE (7/7) + 6HR
(3/7)
3HRZOfx (7/7) +
1HROfx (7/7) +8 OfxPr
(3/7) (H-intolerance)
Treatment duration
(months)
6
6
8
12 Table 1. Patient characteristics. 12 Medicina 2020, 56, 202
Bacteriological
follow up after 3 of 7
6, 8 Table 1. Cont. Table 1. Cont. 2. Case Reports Characteristics
Case 1
Case 2
Case 3
Case 4
Bacteriological
follow up after
treatment initiation
Negative at 2, 4, 6
months
Negative at 2, 4, 6
months
Negative at 2, 4, 6,
8 months
Negative at 2, 4, 6, 8
months
Chest X-ray after
DOT treatment
Left fibronodular
sequelae
Right post-TB
fibronodular sequelae
Right post-TB
fibronodular
sequelae
Several left subclavicular
fibromicronodular
lesions
Comorbidities
Anxiety–depressive
disorder, osteopenia,
vitamin D deficiency,
Escherichia Coli
urinary tract infection
Neurocognitive disorder,
arterial hypertension,
dyslipidemia, vitamin D
deficiency
Depressive
disorder, urinary
incontinence
Depressive disorder,
vitamin D deficiency
Special
considerations and
particularities of
the case
Recurrence with
extensive
caseous–cavitary TB
lesions left lung 14 years
later. Positive sputum
smear and culture. After
11 months of Category II
treatment regimen, the
bacteriological follow
ups at 1, 3, 5, 8, and 11
months were negative
(evaluated as cured). Bladder tumor (invasive
papillary urothelial
carcinoma T2bN0M0
Grade III)
Left
ureterohydronephrosis
-
In 1996, recurrent
transient vision loss,
labeled retrobulbar optic
neuritis, treated for 1
year with oral
corticosteroids. Received
an individualized
treatment regimen due
to isoniazid intolerance
and exclusion of E due to
its ocular side effects. AFB- Acid-Fast Bacillus; F—female; M—male; IFNβ-1b—interferon beta 1 b; H—isoniazid 5mg/body weight;
R—rifampin 10 mg/body weight; Z—pyrazinamide 30 mg/body weight; S—streptomycin 15 mg/body weight;
E—ethambutol 25 mg/body weight; Ofx—ofloxacinum; Pr—protionamide 20 mg/body weight; 7/7—daily, 3/7—three
times per week; 5/7- treatment administered five days per week; TB—tuberculosis; DOT- Directly Observed Therapy;
RRMS—relapsing-remitting multiple sclerosis; SPMS—secondary progressive multiple sclerosis; EDSS—expanded
disability status scale. Chest X ray
after DOT
treatment
Left fibronodular
sequelae
Right post-TB
fibronodular sequelae
Right post TB
fibronodular
sequelae
subclavicular
fibromicronodular
lesions
Comorbidities
Anxiety–depressive
disorder, osteopenia,
vitamin D deficiency,
Escherichia Coli
urinary tract infection
Neurocognitive
disorder, arterial
hypertension,
dyslipidemia, vitamin
D deficiency
Depressive
disorder, urinary
incontinence
Depressive disorder,
vitamin D deficiency
Special
considerations
and
particularities
of the case
Recurrence with
extensive caseous–
cavitary TB lesions left
lung 14 years later. Positive sputum smear
and culture. After 11
months of Category II
treatment regimen, the
bacteriological follow
ups at 1, 3, 5, 8, and 11
months were negative
(evaluated as cured). Bladder tumor
(invasive papillary
urothelial carcinoma
T2bN0M0 Grade III)
Left
ureterohydronephrosis
-
In 1996, recurrent
transient vision loss,
labeled retrobulbar
optic neuritis, treated
for 1 year with oral
corticosteroids. Received an
individualized
treatment regimen due
to isoniazid intolerance
and exclusion of E due
to its ocular side
effects. 2. Case Reports AFB- Acid-Fast Bacillus; F—female; M—male; IFNβ-1b—interferon beta 1 b; H―isoniazid 5mg/body
weight; R―rifampin 10 mg/body weight; Z―pyrazinamide 30 mg/body weight; S―streptomycin 15
mg/body weight; E―ethambutol 25 mg/body weight; Ofx―ofloxacinum; Pr―protionamide 20
mg/body weight; 7/7—daily, 3/7—three times per week; 5/7- treatment administered five days per
week; TB—tuberculosis; DOT- Directly Observed Therapy; RRMS—relapsing-remitting multiple
sclerosis; SPMS—secondary progressive multiple sclerosis; EDSS—expanded disability status scale. AFB- Acid-Fast Bacillus; F—female; M—male; IFNβ-1b—interferon beta 1 b; H—isoniazid 5mg/body weight;
R—rifampin 10 mg/body weight; Z—pyrazinamide 30 mg/body weight; S—streptomycin 15 mg/body weight;
E—ethambutol 25 mg/body weight; Ofx—ofloxacinum; Pr—protionamide 20 mg/body weight; 7/7—daily, 3/7—three
times per week; 5/7- treatment administered five days per week; TB—tuberculosis; DOT- Directly Observed Therapy;
RRMS—relapsing-remitting multiple sclerosis; SPMS—secondary progressive multiple sclerosis; EDSS—expanded
disability status scale. weight; R―rifampin 10 mg/body weight; Z―pyrazinamide 30 mg/body weight; S―streptomycin 15
mg/body weight; E―ethambutol 25 mg/body weight; Ofx―ofloxacinum; Pr―protionamide 20
mg/body weight; 7/7—daily, 3/7—three times per week; 5/7- treatment administered five days per
week; TB—tuberculosis; DOT- Directly Observed Therapy; RRMS—relapsing-remitting multiple
sclerosis; SPMS—secondary progressive multiple sclerosis; EDSS—expanded disability status scale. Figure 1. (a) Large fibrocavitary lesion of the Fowler segment with nodular bilateral bronchogenic
disseminations on the lower lobes (case 1); (b) bilateral infiltrative nodular TB lesions upper lobes
Figure 1. (a) Large fibrocavitary lesion of the Fowler segment with nodular bilateral bronchogenic
disseminations on the lower lobes (case 1); (b) bilateral infiltrative nodular TB lesions upper lobes (case 2);
(c) caseous ulcerated lesions, right upper lobe, with homo and contralateral nodular dissemination
(case 3); (d) extensive caseous ulcerated TB left upper lobe with contralateral bronchogenic dissemination
(case 4). Figure 1. (a) Large fibrocavitary lesion of the Fowler segment with nodular bilateral bronchogenic
disseminations on the lower lobes (case 1); (b) bilateral infiltrative nodular TB lesions upper lobes
Figure 1. (a) Large fibrocavitary lesion of the Fowler segment with nodular bilateral bronchogenic
disseminations on the lower lobes (case 1); (b) bilateral infiltrative nodular TB lesions upper lobes (case 2);
(c) caseous ulcerated lesions, right upper lobe, with homo and contralateral nodular dissemination
(case 3); (d) extensive caseous ulcerated TB left upper lobe with contralateral bronchogenic dissemination
(case 4). None of these cases had any known contact with active TB patients, other immunodepressive
states, or risk factors, except for a history of pulse corticosteroid therapy with methylprednisolone. All were HIV negative. Other causes of pulmonary involvement were excluded. AFB- Acid-Fast Bacillus; F—female; M—male; IFNβ-1b—interferon beta 1 b; H—isoniazid 5mg/body weight;
R—rifampin 10 mg/body weight; Z—pyrazinamide 30 mg/body weight; S—streptomycin 15 mg/body weight;
E—ethambutol 25 mg/body weight; Ofx—ofloxacinum; Pr—protionamide 20 mg/body weight; 7/7—daily, 3/7—three
times per week; 5/7- treatment administered five days per week; TB—tuberculosis; DOT- Directly Observed Therapy;
RRMS—relapsing-remitting multiple sclerosis; SPMS—secondary progressive multiple sclerosis; EDSS—expanded
disability status scale.
weight; R―rifampin 10 mg/body weight; Z―pyrazinamide 30 mg/body weight; S―streptomycin 15
mg/body weight; E―ethambutol 25 mg/body weight; Ofx―ofloxacinum; Pr―protionamide 20
mg/body weight; 7/7—daily, 3/7—three times per week; 5/7- treatment administered five days per
week; TB—tuberculosis; DOT- Directly Observed Therapy; RRMS—relapsing-remitting multiple
sclerosis; SPMS—secondary progressive multiple sclerosis; EDSS—expanded disability status scale. 3. Discussion Active tuberculosis has not yet been reported as an infectious complication of IFNβ-1b therapy
with MS patients [7]. IFNβ-1b decreases the expression of Very Late Antigen-4 (VLA-4) adhesion
molecules and the penetration of activated lymphocytes through the blood–brain barrier, with a role in
gene regulation [11]. Recent studies on MS pathophysiology have shown an imbalance of adaptive
immunity (T regulator, B cell, cytokine, monocytes, Th1, Th17, Th2 and proinflammatory products) due
to abnormalities in over 8000 expressed genes that control immune regulation, including interferon
(IFN) signaling. Although the mechanisms are diverse and very complex, it seems that dysregulation of
the IFN pathway is associated with active forms of MS and poor prognosis [9]. Pulmonary involvement
in MS may occur as a result of disease progression to respiratory failure, secondary to respiratory
musculature weakness and abnormalities in the neural control of respiration, but also as a result of the
complications secondary to the treatment of the underlying disease [12]. However, defense against TB
requires a complex range of innate (early response) and adaptive immune mechanisms involving a
variety of immune cells and cytokines. In active TB, severity correlates with the circulating IFN-gamma
levels, which play a special role in the activation of myeloid cells and in the inhibition of bacterial
replication Tzelepis [13]. There are no data regarding a possible link between IFNβ-1b treatment
in patients with multiple sclerosis and active tuberculosis. This is why we considered it important
to present these cases. They may encourage more extensive research that will lead to new data in
this field. Despite the different results of type I IFN response to infection, it is well documented that
many intracellular, non-viral pathogens elicit a host response that results in an increased IFN-beta
production [14]. There are different signaling pathways defining different gene expressions during
active tuberculosis infection. However, the mechanism by which Mycobacterium tuberculosis infection
regulates interferon-stimulated genes in human macrophages remains unknown [15]. Researchers at
the Max Planck Institute (MPI) have patented a host cell model that functionally reproduces pulmonary
alveolar macrophages (AM). Thus, the host interactions under infection with Mtb can be studied
in vitro. The innate primary immune response of MPI cells in the presence of Mtb showed a large and
early induction of the pro-inflammatory cytokines Tumor Necrosis Factor Alpha (TNFα), interleukin 6
(IL-6), IL-1α, and IL-1β, and elimination of the bacterium by phagolysosomes [16]. 2. Case Reports When active TB
was confirmed, IFNβ-1b treatment was discontinued and resumed immediately after the cessation
of TB treatment. In all cases, after being clinically and visually detected, TB was bacteriologically 4 of 7 Medicina 2020, 56, 202 confirmed by a microscopic examination of Ziehl–Neelsen-stained smears and culture in a solid
Löwenstein–Jensen medium. TB treatment was administered according to the National Program for
Tuberculosis Prevention, Monitoring, and Control. All patients had advanced forms of drug-susceptible
secondary (post-primary) tuberculosis, which occurred after at least one year of IFNβ-1b treatment. All cases presented ulcerated caseous lesions that spread to at least one pulmonary lobe and two cases
were highly contagious forms, with the sputum smear microscopy results being positive. In all cases,
the initial indication was the standard six-month regimen consisting of isoniazid (H), rifampicin (R),
pyrazinamide (Z), and streptomycin (S) or ethambutol (E) given daily (7/7) for the first two months,
followed by four months of isoniazid and rifampicin given three times a week (2HRZS (E) 7/7/4HT3/7). The treatment was prolonged to eight months due to lesion extension (cavitary lesions) and 12 months
due to an individualized regimen (without H and E). TB relapse occurred in only one case after
14 years. All the cases were bacteriologically monitored and were considered cured. One of the patients
developed a bladder tumor 17 years after the IFNβ-1b treatment. All patients developed different
comorbidities over the course of their MS. 3. Discussion Studies have found
that vitamin B5 can stimulate epithelial cells to express proinflammatory and antibacterial cytokines in
macrophages infected with Mycobacterium tuberculosis [17]. Some Toll-like receptors on the surfaces of immune cells can identify bacteria, playing critical
roles in tuberculosis infection. The receptors 2, 4, and 9 also have a fundamental role in pathology, with
their expression levels being increased in MS. Receptors such as peptidoglycan, a major component of
mycobacterial cell walls, have been identified in the CNS endothelial cells, cerebrospinal fluid (CSF)
and glial cells of MS patients. These receptors are crucial in the primary identification of Mtb and 5 of 7 Medicina 2020, 56, 202 the proper development of immune responses to overcome the infection [18]. The microbial agent
activates the recognition receptors by initiating the innate immune response. Mycobacterium tuberculosis
RNA determines, through the SecA2 and ESX-1 secretion system, the production of interferon-beta. Until recently, these mechanisms were only known to occur in infections with viruses [19]. y
y
In childhood, our studied patients received the Bacillus Calmette–Guérin (BCG) vaccine, which is
part of the national mandatory vaccination program. BCG influences the transition from oxidative
phosphorylation at aerobic glycolysis, thus ensuring the stimulation of the immunomodulation immune
response and attenuation of mycobacterial infection. It has been shown that after BCG infection,
IFN-β can enhance antigen-presenting cell (APC) activity, and the link between the innate and the
adaptive immune systems. This could be an opportunity to find more effective vaccines in the fight
against tuberculosis [20]. However, the protection lasts for only five to ten years after vaccination
and therefore, scientists are looking for ways to improve and increase the vaccine’s efficiency [21,22]. We can speculate that IFNβ-1b therapy may impair protective immunity to Mycobacterium tuberculosis,
given the complex immune mechanisms and genetic determinants of the two conditions. Neither the
effect of interferons on humoral or cellular immunity, as is the case with other DMTs, nor the risk of
opportunistic infections such as TB, are well known. The risk of TB depends on host susceptibility
during exposure, and on their belonging to risk groups [23]. The risk of TB has not been described
in MS patients on IFNβ-1b treatment. From recent data, the risks of tuberculosis reactivation in
patients treated with alemtuzumab or teriflunomide is the highest and the recommendation is to
screen for latent infection before starting therapy. 3. Discussion For natalizumab, fingolimod, dimethyl fumarate and
mitoxantrone, screening is optional. For monoclonal antibodies targeting CD20, the risk of reactivation
is the lowest because B-cell depletion does not affect cell-mediated immunity [7]. A group of experts,
neurologists, and pulmonologists have underlined that screening for latent tuberculosis infection
(LTBI) is not required for MS patients treated with IFNβ-1b in low-prevalence countries (TB notification
rate of <100 TB cases per million population per year) [24]. In our center, no patient was screened for
LTBI before the initiation of the treatment for MS, although our country had a TB global incidence rate
of 68 cases/100,000 persons per year during 2018. Screening for LTBI using interferon-gamma release
assays (IGRAs) is considered necessary, even in low TB-endemic countries. The higher sensitivity and
specificity of IGRAs have been reported, thus replacing the tuberculin skin test, especially in countries
where the population is BCG-vaccinated [23]. However, certain treatments (fingolimod, dimethyl
fumarate, methylprednisolone) may cause false-negative or indeterminate IGRA results. The effect of
IFNβ-1b on IGRA results is not exactly known [25]. There is no test predictive of the progression from
LTBI to active disease, which is the reason prevention plays an important role. Screening for LTBI is
currently recommended before starting daclizumab and alemtuzumab therapy, but not before initiating
CD20-acting DMTs (rituximab, ocrelizumab) [26,27]. Additionally, the screening of high-risk groups in
the population and the use of infection control measures or chemoprophylaxis are indicated [23]. In the
future, however, genetic tests could be used; to date, 16 genes with predictive values for susceptibility
to developing TB have been described [28]. References 1. Karussis, D. The diagnosis of multiple sclerosis and the various related demyelinating syndromes: A critical
review. J. Autoimmun. 2014, 48–49, 134–142. [CrossRef] [PubMed] 1. Karussis, D. The diagnosis of multiple sclerosis and the various related demyelinating syndromes: A critical
review. J. Autoimmun. 2014, 48–49, 134–142. [CrossRef] [PubMed] Autoimmun. 2014, 48–49, 134–142. [CrossRef] [PubMed 2. Grebenciucova, E.; Pruitt, A. Infections in patients receiving multiple sclerosis disease-modifying therapies. Curr. Neurol. Neurosci. Rep. 2017, 17, 88. [CrossRef] [PubMed] 2. Grebenciucova, E.; Pruitt, A. Infections in patients receiving multiple sclerosis disease-modifying therapies. Curr. Neurol. Neurosci. Rep. 2017, 17, 88. [CrossRef] [PubMed] 3. Luna, G.; Alping, P.; Burman, J.; Fink, K.; Fogdell-Hahn, A.; Gunnarsson, M.; Frisell, T. Infection Risks
Among Patients with Multiple Sclerosis Treated with Fingolimod, Natalizumab, Rituximab, and Injectable
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Among Patients with Multiple Sclerosis Treated with Fingolimod, Natalizumab, Rituximab, and Injectable
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Rammohan, K.W.; Selmaj, K.; et al. OPERA I and OPERA II Clinical Investigators. Ocrelizumab versus
interferon beta-1a in relapsing multiple sclerosis. N. Engl. J. Med. 2017, 376, 221–234. [CrossRef] [PubMed] 4. Hauser, S.L.; Bar-Or, A.; Comi, G.; Giovannoni, G.; Hartung, H.P.; Hemmer, B.; Lublin, F.; Montalban, X.;
Rammohan, K.W.; Selmaj, K.; et al. OPERA I and OPERA II Clinical Investigators. Ocrelizumab versus
interferon beta-1a in relapsing multiple sclerosis. N. Engl. J. Med. 2017, 376, 221–234. [CrossRef] [PubMed] 5. Montalban, X.; Hauser, S.L.; Kappos, L.; Arnold, D.L.; Bar-Or, A.; Comi, G.; de Seze, J.; Giovannoni, G.;
Hartung, H.P.; Hemmer, B.; et al. ORATORIO Clinical Investigators. Ocrelizumab versus placebo in primary
progressive multiple sclerosis. N. Engl. J. Med. 2017, 376, 209–220. [CrossRef] 5. Montalban, X.; Hauser, S.L.; Kappos, L.; Arnold, D.L.; Bar-Or, A.; Comi, G.; de Seze, J.; Giovannoni, G.;
Hartung, H.P.; Hemmer, B.; et al. ORATORIO Clinical Investigators. Ocrelizumab versus placebo in primary
progressive multiple sclerosis. N. Engl. J. Med. 2017, 376, 209–220. [CrossRef] 6. Sanchez-Salcedo, P.; de-Torres, J.P. Immunomodulating Effects of Glatiramer Acetate and Its Potential Role
in Pulmonary Tuberculosis Reactivation. Arch. Bronconeumol. (Engl. Ed.) 2015, 51, 656–657. [CrossRef] 7. Epstein, D.; Dunn, J.; Deresinski, S. Infectious Complications of Multiple Sclerosis Therapies: Implications
for Screening, Prophylaxis, and Management. Open Forum Infect. Dis. 4. Conclusions The presented cases demonstrated the occurrence of active tuberculosis after at least one year
of IFNβ-1b treatment in young MS patients. It is the first report in the literature of this comorbidity
involving IFNβ-1b treatment. The immunological changes in MS and TB appear to be extremely
complex. The relationship between MS, IFNβ-1b and the drugs with the longest and most widespread
use for these patients and active TB requires further clinical studies to quantify this risk. This is
important, especially since the treatment of patients with MS is chronic, spanning numerous decades. Such research would help in developing guidelines around the risk of infections, including TB, in MS
patients. The appearance of other comorbidities increases the burden of the patient’s suffering and
implies the need for a better supervision of DMT-treated patients. Pre-treatment and annual screening 6 of 7 Medicina 2020, 56, 202 for latent TB infection and the control of respiratory symptoms should be compulsory in any patient
with MS when TB is endemic. Author Contributions: Conceptualization, C.A.S.; methodology, E.D.; data curation, C.A.S. and A.D.A.; validation,
C.F.P., A.D.A., M.C.G.; investigation, C.A.S.; resources, C.F.P., A.D.A., M.C.G.; writing—original draft preparation,
C.A.S., E.D., C.F.P., A.D.A. and M.C.G.; writing—review and editing, C.F.P., A.D.A., M.C.G.; visualization, C.F.P.,
A.D.A., M.C.G.; supervision, C.F.P., A.D.A., M.C.G. All authors have read and agreed to the published version of
the manuscript. Author Contributions: Conceptualization, C.A.S.; methodology, E.D.; data curation, C.A.S. and A.D.A.; validation,
C.F.P., A.D.A., M.C.G.; investigation, C.A.S.; resources, C.F.P., A.D.A., M.C.G.; writing—original draft preparation,
C.A.S., E.D., C.F.P., A.D.A. and M.C.G.; writing—review and editing, C.F.P., A.D.A., M.C.G.; visualization, C.F.P.,
A.D.A., M.C.G.; supervision, C.F.P., A.D.A., M.C.G. All authors have read and agreed to the published version of
the manuscript. Funding: This research received no external funding. Acknowledgments: We thank all our patients who gave written informed consent in accordance with the
Declaration of Helsinki. The protocol was approved by the ethics committee of our Medical University
(nr.1620/1/29.01.2020) on 29 January 2020. The study was carried out in accordance with the recommendations of
the International Committee of Medical Journal Editors. Conflicts of Interest: The authors declare no conflicts of interest. Conflicts of Interest: The authors declare no conflicts of interest. References 2018, 5, 174. [CrossRef] 8. Rommer, P.S.; Milo, R.; Han, M.H.; Satyanarayan, S.; Sellner, J.; Hauer, L.; Illes, Z.; Warnke, C.; Laurent, S.;
Weber, M.S.; et al. Immunological Aspects of Approved MS Therapeutics. Front. Immunol. 2019, 10, 1564. [CrossRef] 9. Feng, X.; Bao, R.; Li, L.; Deisenhammer, F.; Arnason, B.G.W.; Reder, A.T. Interferon-β corrects massive
gene dysregulation in multiple sclerosis: Short-term and long-term effects on immune regulation and
neuroprotection. EBioMedicine 2019, 49, 269–283. [CrossRef] 10. Pai, M.; Behr, M. Latent Mycobacterium tuberculosis Infection and Interferon-Gamma Release Assays. Microbiol. Spectr. 2016, 4. [CrossRef] 11. Jakimovski, D.; Kolb, C.; Ramanathan, M.; Zivadinov, R.; Weinstock-Guttman, B. Interferon β for Multiple
Sclerosis. Cold Spring Harb. Perspect. Med. 2018, 8. [CrossRef] [PubMed] 12. Tzelepis, G.E.; McCool, F.D. Respiratory dysfunction in multiple sclerosis. Respir. Med. 2015, 109, 671–679. [CrossRef] [PubMed] 13. Sia, J.K.; Rengarajan, J. Immunology of Mycobacterium tuberculosis infections. Microbiol. Spectr. 2019, 7. [CrossRef] [PubMed] 13. Sia, J.K.; Rengarajan, J. Immunology of Mycobacterium tuberculosis infections. Microbiol. Spectr. 2019, 7. [CrossRef] [PubMed]
14. Banks, D.A.; Ahlbrand, S.E.; Hughitt, V.K. Mycobacterium tuberculosis Inhibits Autocrine Type I IFN 14. Banks, D.A.; Ahlbrand, S.E.; Hughitt, V.K. Mycobacterium tuberculosis Inhibits Autocrine Typ
Signaling to Increase Intracellular Survival. J. Immunol. 2019, 202, 2348–2359. [CrossRef] [PubMed] 15. Zhou, X.; Yang, J.; Zhang, Z.; Zhang, L.; Zhu, B.; Lie, L.; Ma, L. Different Signaling Pathways Define Different
Interferon-Stimulated Gene Expression during Mycobacteria Infection in Macrophages. Int. J. Mol. Sci. 2019,
20, 663. [CrossRef] 7 of 7 Medicina 2020, 56, 202 16. Woo, M.; Wood, C.; Kwon, D.; Park, K.; Fejer, G.; Delorme, V. Mycobacterium tuberculosis Infection and
Innate Responses in a New Model of Lung Alveolar Macrophages. Front. Immunol. 2018, 9, 438. [CrossRef] 17. He, W.; Hu, S.; Du, X.; Wen, Q.; Zhong, X.-P.; Zhou, X.; Zhou, C.; Xiong, W.; Gao, Y.; Zhang, S.; et al. Vitamin
B5 Reduces Bacterial Growth via Regulating Innate Immunity and Adaptive Immunity in Mice Infected with
Mycobacterium tuberculosis. Front. Immunol. 2018, 9, 365. [CrossRef] 18. Faridgohar, M.; Nikoueinejad, H. New findings of Toll-like receptors involved in Mycobacterium tuberculosis
infection. Pathog. Glob. Health 2017, 111, 256–264. [CrossRef] 19. Cheng, Y.; Schorey, J.S. Mycobacterium tuberculosis-induced IFN-β production requires cytosolic DNA and
RNA sensing pathways. J. Exp. Med. 2018, 215, 2919–2935. [CrossRef] 20. El-Sahrigy, S.A.F.; Rahman, A.M.O.A.; Samaha, D.Y. The influence of interferon-β supplemented human
dendritic cells on BCG immunogenicity. J. Immunol. Methods 2018, 457, 15–21. [CrossRef] 21. Dow, C.T. References Proposing BCG Vaccination for Mycobacterium avium ss. paratuberculosis (MAP) Associated
Autoimmune Diseases. Microorganisms 2020, 8, 212. [CrossRef] [PubMed] 22. Yamazaki-Nakashimada, M.A.; Unzueta, A.; Berenise Gámez-González, L.; González-Saldaña, N.;
Sorensen, R.U. BCG: A vaccine with multiple faces. Hum. Vaccin. Immunother. 2020, 1–10. [CrossRef] 23. Lange, C.; Mandalakas, A.M.; Kalsdorf, B. Clinical Application of Interferon-γ Release Assays for the
Prevention of Tuberculosis in Countries with Low Incidence. Pathog. Immun. 2016, 1, 308–329. [CrossRef]
[PubMed] 24. Navas, C.; Torres-Duque, C.A.; Munoz-Ceron, J.; Álvarez, C.; García, J.R.; Zarco, L.; Vélez, L.A.; Awad, C.;
Castro, C.A. Diagnosis and treatment of latent tuberculosis in patients with multiple sclerosis, expert
consensus. On behalf of the Colombian Association of Neurology, Committee of Multiple Sclerosis. Mult. Scler. J. Exp. Transl. Clin. 2018, 4. [CrossRef] [PubMed] 25. Baldassari, L.E.; Feng, J.; Macaron, G.; Planchon, S.M.; Alshehri, E.; Moss, B.P.; Ontaneda, D.; Willis, M.A. Tuberculosis screening in multiple sclerosis: Effect of disease-modifying therapies and lymphopenia on the
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5. [CrossRef] 26. Graf, J.; Leussink, V.I.; Dehmel, T.; Dehmel, T.; Ringelstein, M.; Goebels, N.; Adams, O.; MacKenzie, C.R.;
Warnke, C.; Feldt, T.; et al. Infectious risk stratification in multiple sclerosis patients receiving immunotherapy. Ann. Clin. Transl. Neurol. 2017, 4, 909–914. [CrossRef] 27. Sirbu, C.A.; Budisteanu, M.; Falup-Pecurariu, C. Monoclonal antibodies—A revolutionary therapy in multiple
sclerosis. Neurol. Neurochir. Pol. 2020, 54, 21–27. [CrossRef] 28. Zak, D.E.; Penn-Nicholson, A.; Scriba, T.J.; Thompson, E.; Suliman, S.; Amon, L.M.; Mahomed, H.; Erasmus, M.;
Whatney, W.; Hussey, G.D.; et al. A blood RNA signature for tuberculosis disease risk: A prospective cohort
study. Lancet 2016, 387, 2312–2322. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Decidability Results for Dynamic Installation of Compensation Handlers
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Lecture notes in computer science
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To cite this version: Ivan Lanese, Gianluigi Zavattaro. Decidability Results for Dynamic Installation of Compensation
Handlers. 15th International Conference on Coordination Models and Languages (COORDINATION),
Jun 2013, Florence, Italy. pp.136-150, 10.1007/978-3-642-38493-6_10. hal-01486023 Distributed under a Creative Commons Attribution 4.0 International License Decidability Results for Dynamic Installation
of Compensation Handlers Ivan Lanese and Gianluigi Zavattaro Focus Team, University of Bologna & INRIA, Italy Abstract. Dynamic compensation installation allows for easier specifi-
cation of fault handling in complex interactive systems since it enables to
update the compensation policies according to run-time information. In
this paper we show that in a simple π-like calculus with static compen-
sations the termination of a process is decidable, but it is undecidable in
one with dynamic compensations. We then consider three commonly used
patterns for dynamic compensations, showing that process termination
is decidable for parallel and replacing compensations while it remains
undecidable for nested compensations. HAL Id: hal-01486023
https://inria.hal.science/hal-01486023v1
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Introduction Nowadays, applications are composed of different interacting entities, living in
environments such as the Internet or the cloud. Programming applications in this
setting is challenging, due to their own complexity, and on the unpredictability of
the environment. For instance, a communication partner may disappear during
an interaction, or a message may be lost due to an unreliable network. Never-
theless, the users expect their applications to always provide reliable services. To build reliable services in an unreliable environment coping with unexpected
events is certainly one of the main challenges. In the setting of service-oriented computing, long running transactions have
been put forward to solve this problem. A long running transaction is a com-
putation that either succeeds, or it aborts. In the second case, a compensation
is executed to undo unwanted side effects of the aborted computation. Many
languages provide nowadays support for long running transactions [25, 26], and
different proposals exist in the literature [2, 3, 8–12,16, 22]. Originally, the com-
pensation of a long running transaction was statically fixed [26]. Recent proposals
show however that being able to dynamically update the compensation as far
as the computation progresses allows the programmer to write more easily the
compensation code for complex interactions [16]. From a language design point of view, the question of whether dynamic
compensations are just syntactic sugar, and thus need not be implemented in the
core language, or not, is relevant. Strangely, while many papers in the literature
put forward proposals of transaction constructs, very little has been done on
comparing them. A main work in this direction is [20]. In [20] it is shown that
the ability to add new compensation items to be executed in parallel with the static compensation does not increase the expressive power, while more general
patterns do. The study is carried out relying on proofs of encodability and/or
non-encodability between the different formalisms. However, there is no clear
agreement in the community on which conditions such encodings should satisfy,
and the results in [20] strongly depend both on the chosen conditions and on the
availability of suitable mechanisms in the compared languages. We want here to tackle the same problem, but with a completely different
approach. 1
Introduction In fact, we compare π-like core calculi featuring the basic mechanisms
for static and dynamic compensations according to the (un)decidability of pro-
cess termination, that is of the absence of an infinite computation starting from a
given process. Clearly, calculi where such a property is undecidable cannot be en-
coded in calculi where the same property is decidable, and this non-encodability
result is valid for all the encodings preserving the property. We show that process termination is decidable in a π-calculus with static
compensations, while it is not in one with dynamic compensations. To better
understand where this difference stems from, we limit the expressive power of
the dynamic compensation mechanism in different directions. We show that if
compensations can only be replaced, then decidability is recovered. If instead
compensations can be nested using linear patterns, we are still in an undecidable
setting. To further constrain linear patterns aiming at decidability we force the
patterns to only add new compensation items in parallel, obtaining again a
decidability result. Fig. 1. π-calculus processes. Fig. 1. π-calculus processes. P, Q ::=
Static compensation processes
. . . (π-calculus processes)
|
t[P, Q] (Transaction scope)
|
⟨P⟩
(Protected block) Fig. 2. Static compensation processes. P, Q ::=
Dynamic compensation processes
. . . (Static compensation processes)
|
X
(Process variable)
|
inst⌊λX.Q⌋.P (Compensation update)
Fig. 3. Dynamic compensation processes. P, Q ::=
Dynamic compensation processes
. . . (Static compensation processes)
|
X
(Process variable)
|
inst⌊λX.Q⌋.P (Compensation update) P, Q ::=
Dynamic compensation processes
. . . (Static compensation processes)
|
X
(Process variable)
|
inst⌊λX.Q⌋.P (Compensation update) P, Q ::= 2.1
Syntax We base our studies on a π-calculus extended with transactions and primitives
for compensation installation. We then consider different fragments, correspond-
ing to various compensation installation patterns. Our calculus is similar to the
calculus in [20]. A main difference is that we do not consider restriction. This is
forced since, if we add restriction, then process termination (and similar prop-
erties) become undecidable even in CCS (without transactions). The syntax of our calculus relies on a countable set of names N, ranged
over by lower case letters. We use x to denote a tuple x1, · · · , xn of names, and
{x} denotes the set of elements in the tuple. We use {v/x} for denoting the
substitution of names in v for names in x, and we use a similar notation for
substitutions of processes for process variables (introduced later). We start by presenting the syntax of the π-calculus, reported in Fig. 1. Pre-
fixes can be either outputs a⟨v⟩of a tuple of names v on channel a, or corre-
sponding inputs a(x), receiving a tuple of names v on channel a and applying
substitution {v/x} to the continuation. The π-calculus syntax includes the inac-
tive process 0, guarded choice P
i∈I πi.Pi, guarded replication !π.P and parallel
composition P | Q. We write a for a⟨v⟩when v is empty, and a for a(x) when
x is empty. When I is a singleton, P
i∈I πi.Pi is shortened into πi.Pi. We may
also drop trailing 0s. 2 π ::=
π-calculus prefixes
a⟨v⟩(Output prefix)
|
a(x)
(Input prefix)
P, Q ::=
π-calculus processes
0
(Inaction)
|
P
i∈I πi.Pi (Guarded choice)
|
!π.P (Guarded replication)
|
P | Q
(Parallel composition) π ::=
π-calculus prefixes
a⟨v⟩(Output prefix)
|
a(x)
(Input prefix)
P, Q ::=
π-calculus processes
0
(Inaction)
|
P
i∈I πi.Pi (Guarded choice)
|
!π.P (Guarded replication)
|
P | Q
(Parallel composition) |
a(x) Fig. 3. Dynamic compensation processes. We now extend the π-calculus with transactions and static compensations. The syntax of the extended calculus is in Fig. 2. Static compensations can be
programmed by adding just two constructs to π-calculus: transaction scope and
protected block. A transaction scope t[P, Q] behaves as process P until an error
is notified to it by an output t on the name t of the transaction scope. When
such a notification is received the transaction atomically aborts: the body P of
the transaction scope is killed and compensation Q is executed. Q is executed
inside a protected block. In this way it will not be influenced by successive
external errors. Error notifications may be generated both from the body P of
the transaction scope and from external processes. Error notifications are simply
output messages (without parameters). Protected block ⟨P⟩behaves as process
P, but it is not killed in case of failure of a transaction scope enclosing it. The calculus with dynamic compensations extends the one with static com-
pensations. The main difference is that with dynamic compensations the body
P of transaction scope t[P, Q] can update the compensation Q. Compensation
update is performed by an additional operator inst⌊λX.Q′⌋.P ′, where function
λX.Q′ is the compensation update (X can occur inside Q′). Applying such a com-
pensation update to compensation Q produces a new compensation Q′{Q/X}. Note that Q may not occur at all in the resulting compensation, and it may also
occur more than once. For instance, λX.0 deletes the current compensation. The
syntax of processes with dynamic compensations extends the one of processes 3 with static compensations with the compensation update operator and process
variables (see Fig. 3). We use X to range over process variables. We define for processes with dynamic compensations the usual notions of
free and bound names. Names in x are bound in a(x).P. Other names are free. Also, variable X is bound in λX.Q. Bound names and variables inside processes
can be α-converted as usual. We consider only processes with no free variables. Processes with static compensations are processes with dynamic compensa-
tions where the compensation update operator is never used. We will show that
dynamic compensations are very expressive, making relevant properties undecid-
able. Thus we consider different subcalculi, constraining the allowed patterns for
compensation installation. Fig. 3. Dynamic compensation processes. As a first observation, note that in a compensation
update of the form λX.Q there are no constraints on how many times X may
occur in Q. Having more than one occurrence of X, allowing to replicate the pre-
vious compensation, is rarely used in practice. Thus a meaningful restriction is
considering just linear compensations, where X occurs exactly once in Q. We call
them nested compensations, since the old compensation becomes nested inside
the new one, which acts as a context. Another relevant case is when X does not
occur at all in Q. We call compensations of this form replacing compensations,
since the new compensation completely replaces the old one, which is discarded. Finally, a relevant subcase of nested compensations are parallel compensations,
where Q has the form Q′ | X and X does not occur in Q′. In this case new and
old compensation items are in parallel in the final term. 2.2
Operational Semantics In this section we define the operational semantics of processes with dynamic
compensations. We need however an auxiliary definition. When a transaction
scope t[P, Q] is killed, part of its body P may be preserved, in particular the
protected blocks inside it. The definition of function extr(P) computing the part of process P to be pre-
served depends on the meaning of nesting of transaction scopes. In the literature,
three main approaches are considered. When the enclosing transaction scope is
killed, its subtransactions may be aborted, preserved or discarded. The aborting
semantics is used by SAGAs calculi [9], WS-BPEL [26], and others. The preserv-
ing semantics is, for instance, the approach of Webπ [22]. Finally, the discarding
semantics has been proposed by ATc [3] and TransCCS [12]. We consider all the
three possibilities, since they just differ in the definition of function extr(•). Definition 1 (Extraction function). We denote the functions corresponding
to aborting, preserving, and discarding semantics for transaction nesting respec-
tively as extra(•), extrp(•) and extrd(•). The function extra(•) is defined in Fig. 4. The definition of function extrp(•) is the same but for the clause for transaction
scope, which is replaced by the clause extrp(t [P, Q]) = t [P, Q]. The definition of
function extrd(•) instead is obtained by replacing the clause for transaction scope
by the clause extrd(t [P, Q]) = 0. 4 extra(0) = 0
extra(P
i∈I πi.Pi) = 0
extra(!π.P) = 0
extra(inst⌊λX.Q⌋.P) = 0
extra(⟨P⟩) = ⟨P⟩
extra(t [P, Q]) = extra(P) | ⟨Q⟩
extra(P | Q) = extra(P) | extra(Q) Fig. 4. Extraction function for aborting semantics. (P-Out)
a⟨v⟩.P
a⟨v⟩
−−−→P
(P-In)
a(x).P
a(v)
−−−→P{v/x}
(L-Choice)
πj.Pj
α−→P ′
j
j ∈I
X
i∈I
πi.Pi
α−→P ′
j
(L-Rep)
π.P
α−→P ′
!π.P
α
−→P ′|!π.P
(L-Par)
P
α
−→P ′
P | Q
α
−→P ′ | Q
(L-Synch)
P
x(v)
−−−→P ′
Q
x⟨v⟩
−−−→Q′
P | Q
τ−→P ′ | Q′
(L-Scope-out)
P
α
−→P ′
α ̸= λX.Q
t[P, Q]
α
−→t[P ′, Q]
(L-Recover-out)
t[P, Q]
t−→extra(P) | ⟨Q⟩
(L-Recover-in)
P
t−→P ′
t[P, Q]
τ−→extra(P ′) | ⟨Q⟩
(L-Inst)
inst⌊λX.Q⌋.P
λX.Q
−−−→P
(L-Scope-inst)
P
λX.R
−−−→P ′
t[P, Q]
τ−→t[P ′, R{Q/X}]
(L-Block)
P
α−→P ′
⟨P⟩
α−→⟨P ′⟩
Fig. 5. LTS for dynamic compensation processes. (L-Scope-out) Fig. 5. LTS for dynamic compensation processes. 2.2
Operational Semantics The operational semantics of dynamic compensations and, implicitly, of sta-
tic, replacing, parallel and nested compensation processes, is defined below. We use a(v), a⟨v⟩, τ and λX.Q as labels, and we use α to range over labels. The first three forms of labels are as in π-calculus: a(v) is the input of a tuple
of values v on channel a, a⟨v⟩is the corresponding output, and τ is an internal
action. However, an output label without parameters can also be used for error
notification, and an input without parameters for receiving the notification. The
last label, λX.Q, is specific of dynamic compensation processes and corresponds
to compensation update. We write a for a(v) and a for a⟨v⟩if v is empty. We
may use t instead of a to emphasize that the name is used for error notification. Definition 2 (Operational semantics). The operational semantics of dy-
namic compensation processes with aborting semantics for transaction nesting
is the minimum LTS closed under the rules in Fig. 5 (symmetric rules are con-
sidered for ( L-Par) and ( L-Synch)). The preserving semantics (resp. discard-
ing semantics) is obtained by replacing function extra(•) with extrp(•) (resp. extrd(•)). The first six rules are standard π-calculus rules [23], the others define the
behavior of transactions, compensations and protected blocks. 5 Auxiliary rules (P-Out) and (P-In) execute output and input prefixes, re-
spectively. The input rule guesses the received values v in the early style. Rules
(L-Choice) and (L-Rep) deal with guarded choice and replication, respectively. Rule (L-Par) allows one of the components of parallel composition to progress
while the other one stays idle. Rule (L-Synch) performs communication, syn-
chronizing an input x(v) and a corresponding output x⟨v⟩. Rule (L-Scope-out) allows the body P of a transaction scope to progress,
provided that the performed action is not a compensation update. Rule (L-
Recover-out) allows external processes to abort a transaction scope via an
output t. The resulting process is composed of two parts: the first one extracted
from the body P of the transaction scope, and the second one corresponding
to compensation Q, which will be executed inside a protected block. Rule (L-
Recover-in) is similar to (L-Recover-out), but now the error notification
comes from the body P of the transaction scope. Rule (L-Inst) requires to per-
form a compensation update. Rule (L-Scope-inst) updates the compensation
of a transaction scope. 2.2
Operational Semantics – Transaction scopes can compute:
a⟨b⟩| t[a(x).x.0, Q]
τ−→t[b.0, Q]
– Transaction scopes can be killed:
t | t[a.0, Q]
τ−→⟨Q⟩ – Transaction scopes can compute: – Transaction scopes can compute:
a⟨b⟩| t[a(x).x.0, Q]
τ−→t[b.0, Q] – Transaction scopes can compute:
a⟨b⟩| t[a(x).x.0, Q]
τ−→t[b.0, Q]
– Transaction scopes can be killed:
t | t[a.0, Q]
τ−→⟨Q⟩ a⟨b⟩| t[a(x).x.0, Q]
τ−→t[b.0, Q] ⟨⟩| [ ( )
, Q]
[
, Q]
– Transaction scopes can be killed:
t | t[a.0, Q]
τ−→⟨Q⟩ – Transaction scopes can commit suicide:
t[ t.0 | a.0, Q]
τ−→⟨Q⟩ t[ t.0 | a.0, Q] −→⟨Q⟩
– Protected blocks survive after kill:
t[ t.0 | ⟨a.0⟩, Q]
τ−→⟨a.0⟩| ⟨Q⟩
– New compensation items can be added in parallel:
t[inst⌊λX.P|X⌋.a.0, Q]
τ−→t[a.0, P|Q]
– New compensation items can be added at the beginning:
t[inst⌊λX.b.X⌋.a.0, Q]
τ−→t[a.0, b.Q]
– Compensations can be deleted:
t[inst⌊λX.0⌋.a.0, Q]
τ−→t[a.0, 0] – Protected blocks survive after kill:
t[ t.0 | ⟨a.0⟩, Q]
τ−→⟨a.0⟩| ⟨Q⟩ – New compensation items can be added
t[inst⌊λX.P|X⌋.a.0, Q]
τ−→t[a.0, P|Q] – New compensation items can be added in parallel:
t[inst⌊λX.P|X⌋.a.0, Q]
τ−→t[a.0, P|Q] [
⌊
|
⌋
, Q]
[
,
|Q]
– New compensation items can be added at the beginning:
t[inst⌊λX.b.X⌋.a.0, Q]
τ−→t[a.0, b.Q] [
⌊
⌋
, Q]
[
,
Q
– Compensations can be deleted:
t[inst⌊λX.0⌋.a.0, Q]
τ−→t[a.0, 0] [
⌊
⌋
, Q]
[
,
Q]
– Compensations can be deleted:
t[inst⌊λX.0⌋.a.0, Q]
τ−→t[a.0, 0] 2.2
Operational Semantics Finally, rule (L-Block) defines the behavior of protected
blocks. The property of protected blocks of being unaffected by external aborts
is enforced by the definition of function extr(•). In the following we consider a structural congruence ≡to rearrange the order
of parallel processes and to garbage collect process 0. Formally, ≡is the least
congruence such that P | Q ≡Q | P, P | (Q | R) ≡(P | Q) | R and P | 0 ≡P. As discussed in the Introduction, we will consider the (un)decidability of
process termination: a process P terminates if there exists no infinite sequence
of processes P1, P2, . . . , Pi, . . . such that P
τ−→P1
τ−→P2
τ−→. . . τ−→Pi
τ−→. . . . As discussed in the Introduction, we will consider the (un)decidability of
process termination: a process P terminates if there exists no infinite sequence
of processes P1, P2, . . . , Pi, . . . such that P
τ−→P1
τ−→P2
τ−→. . . τ−→Pi
τ−→. . . . Example 1. We give here a few examples of transitions. Example 1. We give here a few examples of transitions. 3
Termination Undecidability for Nested Compensations We now move to the proof of undecidability of termination in the calculus with
nested compensations. This contrasts with the decidability of termination for
static compensations (the proof of this result is deferred to Corollary 2). 6 The undecidability proof is by reduction from the termination problem in
Random Access Machines (RAMs) [24], a well-known Turing powerful formalism
based on registers containing non-negative natural numbers. The registers are
used by a program, that is a set of indexed instructions Ii of two possible kinds: – i : Inc(rj) that increments the register rj and then moves to the execution
of the instruction with index i + 1 and – i : DecJump(rj, s) that attempts to decrement the register rj; if the register
does not hold 0 then the register is actually decremented and the next in-
struction is the one with index i + 1, otherwise registers are unchanged and
the next instruction is the one with index s. We assume that given a program I1, · · · , In, it starts by executing I1. It termi-
nates when an undefined program instruction is reached. Since the computational
model is Turing complete, the termination of a RAM program is undecidable. We encode RAMs as follows. Each register rj containing the value n is en-
coded as a transaction rj[Rj, Qj] where Qj is a process u.u. · · · .u.z with exactly
n prefixes u. The process Rj is responsible for updating its compensation Qj by
performing inst⌊λX.u.X⌋every time the register must be incremented. Each in-
struction Ii will be encoded as a process !pi.Pi: the instruction will be activated
by pi and then Pi will be performed. If i : Inc(rj) is an increment instruction on
rj, Pi will interact with Rj in order to activate the update of its compensation
Qj. If i : DecJump(rj, s) is a decrement/jump instruction, on the other hand, Pi
will terminate the transaction rj so that the compensation Qj becomes active. If Qj is z then the value of the register is 0. In this case a new instance of the
register rj[Rj, z] is spawn and the jump is executed. If Qj is u. · · · .z then the
register is not 0. In this case, a new instance of the register rj[Rj, z] is spawn and
a protocol is started to initialize correctly this new register. 4. Il is undefined iffthere exists no P ′ s.t. P
τ−→P ′. 4. Il is undefined iffthere exists no P ′ s.t. P
τ−→P ′. Proof. In each case there is just one possible computation, that we describe
by listing the channels on which synchronizations happen or the installation of
compensation performed: 1. pl, incj, inst⌊λX.u.X⌋, ack: 4 transitions; 1. pl, incj, inst⌊λX.u.X⌋, ack: 4 transitions; 1. pl, incj, inst⌊λX.u.X⌋, ack: 4 transitions; 2. pl, rj, z: 3 transitions; 3. pl, rj, u, recj, then the sequence u, inst⌊λX.u.X⌋, recj repeated nj −1 times,
and finally z, ack: 3(nj −1) + 5 transitions; 3. pl, rj, u, recj, then the sequence u, inst⌊λX.u.X⌋, recj repeated nj −1 times,
and finally z, ack: 3(nj −1) + 5 transitions; ⊓⊔ j
4. no synchronization is possible. j
4. no synchronization is possible. We finally conclude with the proof of the undecidability result. Corollary 1. Termination is undecidable in π-calculus with nested compensa-
tions. Proof. By Theorem 1 each step of a RAM precisely corresponds to a finite num-
ber of steps of its encoding, thus a RAM terminates iffits encoding terminates. Thus, termination of RAMs reduces to termination in π-calculus with nested
compensations. Since termination in RAMs is undecidable then also termina-
tion in π-calculus with nested compensations is undecidable. ⊓⊔ Theorem 1. Theorem 1. Given P ≡pl|JI1K| . . . |JInK|Jr1 = n1K| . . . |Jrj = njK| . . . |Jrm = nmK we have: Theorem 1. Given P ≡pl|JI1K| . . . |JInK|Jr1 = n1K| . . . |Jrj = njK| . . . |Jrm = nmK we have: 1. Il : Inc(rj) iff 1. Il : Inc(rj) iff
P →4
≡pl+1|JI1K| . . . |JInK|Jr1 = n1K| . . . |Jrj = nj + 1K| . . . |Jrm = nmK;
2. Il : DecJump(rj, s) and nj = 0 iff
P →3
≡ps|JI1K| . . . |JInK|Jr1 = n1K| . . . |Jrj = 0K| . . . |Jrm = nmK;
3. Il : DecJump(rj, s) and nj ̸= 0 iff
P →k
≡
pl+1|JI1K| . . . |JInK|Jr1 = n1K| . . . |Jrj = nj −1K| . . . |Jrm = nmK wit
k = 3(nj −1) + 5;
4. Il is undefined iffthere exists no P ′ s.t. P
τ−→P ′. 2. Il : DecJump(rj, s) and nj = 0 iff
P →3
≡ps|JI1K| . . . |JInK|Jr1 = n1K| . . . |Jrj = 0K| . . . |Jrm = nmK;
3. Il : DecJump(rj, s) and nj ̸= 0 iff
P →k
≡
pl+1|JI1K| . . . |JInK|Jr1 = n1K| . . . |Jrj = nj −1K| . . . |Jrm = nmK with
k = 3(nj −1) + 5;
4
I i
d fi
d iffh
i
P′
P
τ P′ 3
Termination Undecidability for Nested Compensations The protocol is be-
tween the process Rj and the compensation u. · · · .z left by the previous instance
of the register. The process Rj consumes the remaining prefixes u, and for each
of them performs an inst⌊λX.u.X⌋action in order to update its compensation
accordingly. In this way, at the end of the protocol, the new register instance will
have a compensation u. · · · .z with one prefix u less w.r.t. the previous register
instance. Formally, the translation of register j storing value n is as follows: Formally, the translation of register j storing value n is as follows:
Jrj = nK ≜rj[!incj. inst⌊λX.u.X⌋.ack | !recj. u. inst⌊λX.u.X⌋.recj+z.ack
, un.z]
where un is a sequence of n prefixes u. The encoding of instructions is as follows: Ji : Inc(rj)K
≜!pi.incj.ack.pi+1
Ji : DecJump(rj, s)K ≜!pi.rj. z.(Jrj = 0K|ps) + u.(recj|Jrj = 0K|ack.pi+1)
Hence, given a RAM program I1, · · · , In with registers r1, . . . , rm with initial
values n1, . . . , nm the corresponding encoding is: p1|JI1K| . . . |JInK|Jr1 = n1K| . . . |Jrm = nmK In the proof of correctness of the encoding we use P →k
≡Q to denote the
existence of Q1, · · · , Qk such that P
τ−→Q1
τ−→. . . τ−→Qk and Qk ≡Q. 7 Definition 4. Let P, Q be two processes. We write P ⪯Q iffthere exist P ′, S,
n, m, t1, . . . , tn, P1, . . . , Pn, P ′
1, . . . , P ′
n, Q1, . . . , Qn, Q′
1, . . . , Q′
n, R1, . . . , Rm and
R′
1, . . . , R′
m such that 4
Decidability for Parallel and Replacing Compensations A state s in a WSTS terminates if there exists no infinite computation s →
s1 →s2 →. . .. The proposition below is a special case of Theorem 4.6 in [15]. Proposition 1. Termination is decidable for WSTSs. Given a process P with replacing or parallel compensations, we prove that a
transition system that includes all the derivatives of P is a WSTS. By deriva-
tives, denoted with der(P), we mean the processes that can be reached from P
via transitions labeled with τ, denoted simply with →in the following. We first
observe that given a process Q, the set of its immediate successors according
to →is finite (and computable). This follows from the limitation to τ-labeled
transitions: the labeled transition system in Fig. 5 is not finitely branching be-
cause the rule (P-In) has an instantiation for each of the infinitely many possible
vectors of values v, but if we restrict to τ transitions, only finitely many names
can be actually received because in our calculus no new names can be dynami-
cally generated. Concerning names, we also make the nonrestrictive assumption
that in process P the free names used in output actions are all distinct from
the bound names used in input actions. In this way, it is not necessary to apply
α-conversions to avoid name captures during substitutions. This guarantees that
only the names initially present in P will occur in its derivatives. We now move to the definition of our wqo. Intuitively, a process P is smaller
than a process Q if Q can be obtained from P by adding some processes in
parallel while preserving the same structure of transaction scopes and protected
blocks. Definition 4. Let P, Q be two processes. We write P ⪯Q iffthere exist P ′, S,
n, m, t1, . . . , tn, P1, . . . , Pn, P ′
1, . . . , P ′
n, Q1, . . . , Qn, Q′
1, . . . , Q′
n, R1, . . . , Rm and
R′
1, . . . 4
Decidability for Parallel and Replacing Compensations We now consider the cases in which all dynamic compensation installations fol-
low the replace or the parallel patterns. In the first case, only finitely many dif-
ferent compensation processes can be considered. In the second case, infinitely
many compensations can be reached, but all of them are parallel compositions
of finitely many distinct processes (the processes Q occurring in the updates
λX.Q | X, and static compensations R in t[P, R]). This property of the calculus
allows us to apply the theory of Well-Structured Transition Systems (WSTSs)
to prove that termination is decidable. We start by recalling some basic notions about WSTSs [1, 15]. A reflexive
and transitive relation is called quasi-ordering. A well-quasi-ordering (wqo) is a
quasi-ordering (X, ≤) such that, for every infinite sequence x1, x2, x3, · · · , there
exist i < j with xi ≤xj. From this, it follows that there exists also an in-
finite increasing subsequence xk1, xk2, xk3, · · · such that xkl ≤xkm for every 8 l < m. Given a wqo (X, ≤), we denote its extension to k-tuples as (Xk, ≤k):
⟨x1, · · · , xk⟩≤k ⟨y1, · · · , yk⟩iffxi ≤yi for 1 ≤i ≤k. Dickson’s lemma [14] states
that if (X, ≤) is a wqo, then also (Xk, ≤k) is a wqo. Given a wqo (X, ≤), we de-
note its extension to finite sequences as (X∗, ≤∗): ⟨x1, · · · , xn⟩≤∗⟨y1, · · · , ym⟩
iffthere exists a subsequence ⟨yl1, · · · , yln⟩of the latter s.t. xi ≤yli for 1 ≤i ≤n. Higman’s lemma [17] states that if (X, ≤) is a wqo, then also (X∗, ≤∗) is a wqo. We now report a definition of WSTS appropriate for our purposes. Definition 3. A WSTS is a transition system (S, →, ⪯) where ⪯is a wqo on
S which is compatible with →, i.e., for every s1 ⪯s′
1 such that s1 →s2, there
exists s′
1 →s′
2 such that s2 ⪯s′
2. Moreover, the function Succ(s), returning the
set {s′ ∈S | s →s′} of immediate successors of s, is computable. A state s in a WSTS terminates if there exists no infinite computation s →
s1 →s2 →. . .. The proposition below is a special case of Theorem 4.6 in [15]. Definition 5. Let P be a process. We define depth(P) inductively as follows: Definition 5. Let P be a process. We define depth(P) inductively as follows: Definition 5. Let P be a process. We define depth(P) inductively as follows: depth(0) = depth(X) = 0
depth(P
i∈I πi.Pi) = maxi∈I depth(Pi)
depth(!π.P) = depth(P)
depth(inst⌊λX.Q⌋.P) = max(depth(P), depth(Q))
depth(P | Q) = max(depth(P), depth(Q))
depth(t[P, Q]) = 1 + max(depth(P), depth(Q))
depth(⟨P⟩) = 1 + depth(P) It is trivial to see that the extraction functions do not increase the maximum
nesting levels in all the three considered cases. Formally, depth(extra(P)) ≤
depth(P), depth(extrp(P)) ≤depth(P) and depth(extrd(P)) ≤depth(P). We now
prove that also the labeled transitions do not increase the nesting levels. Proposition 2. Let P be a process with replacing or parallel compensations. If
P
α−→Q then depth(Q) ≤depth(P). Proof. We first observe that for every transition T
λX.S
−−−→T ′ we have that
depth(S) ≤depth(T ). In the light of this preliminary result the thesis can be
easily proved by induction on the depth of the proof of P
α−→Q. The unique in-
teresting case is when the rule (L-Scope-inst) is used. Consider the transition
t[P, Q]
λX.R
−−−→t[P ′, R{Q/X}] inferred by P
λX.R
−−−→P ′. We have that t[P ′, R{Q/X}]
does not have a greater maximum nesting level because depth(R) ≤depth(P),
for the above observation, and depth(R{Q/X}) ≤max(depth(Q), depth(R)) due
to the specificity of the replace and parallel update patterns. ⊓⊔ ⊓⊔ As a trivial corollary we have that the maximum nesting level of the derivatives
of P (i.e. processes in der(P)) is smaller or equal to depth(P). This result will
be used to define a superset of der(P) for which we will prove that ⪯is indeed
a wqo. In the definition of this superset we also need the notion of a sequential
subprocess of P, that is a subterm of P in which the top operator is not a parallel
composition, a transaction or a protection block. Definition 6. Let P be a process. The set seq(P) containing all the sequential
subprocesses of P is defined inductively as follows: seq(0) = {0}
seq(P
i∈I πi.Pi) = {P
i∈I πi.Pi} ∪S
i∈I seq(Pi)
seq(!π.P) = {!π.P} ∪seq(P)
seq(inst⌊λX.Q⌋.P) = inst⌊λX.Q⌋.P ∪seq(P) ∪seq(Q)
seq(X) = ∅
seq(P | Q) = seq(t[P, Q]) = seq(P) ∪seq(Q)
seq(⟨P⟩) = seq(P) The intuition is that no new sequential subprocesses can be generated by deriva-
tives. 4
Decidability for Parallel and Replacing Compensations , R′
m such that P ≡P ′ | Qn
i=1 ti[Pi, Qi] | Qm
j=1⟨Rj⟩
Q ≡P ′ | S | Qn
i=1 ti[P ′
i, Q′
i] | Qm
j=1⟨R′
j⟩
with Pi ⪯P ′
i and Qi ⪯Q′
i, for 1 ≤i ≤n, and Rj ⪯R′
j, for 1 ≤j ≤m. P ≡P ′ | Qn
i=1 ti[Pi, Qi] | Qm
j=1⟨Rj⟩
Q ≡P ′ | S | Qn
i=1 ti[P ′
i, Q′
i] | Qm
j=1⟨R′
j⟩
d Qi ⪯Q′
i, for 1 ≤i ≤n, and Rj ⪯R′
j, for 1 ≤j ≤ In order to prove that ⪯is indeed a wqo over the derivatives of P we need
some more notation and preliminary results. First we define the maximum nest-
ing level depth(P) of nested transactions and protected blocks in a process P. 9 Definition 5. Let P be a process. We define depth(P) inductively as follows: To be more precise, after the execution of an input action, new subprocesses The intuition is that no new sequential subprocesses can be generated by deriva-
tives. To be more precise, after the execution of an input action, new subprocesses 10 can be reached due to name substitution. But, as observed above, the names in
a derivative in der(P) already occur in P, thus they are finite. This allows us to
characterize a superset of der(P) as follows. can be reached due to name substitution. But, as observed above, the names in
a derivative in der(P) already occur in P, thus they are finite. This allows us to
characterize a superset of der(P) as follows. Definition 7. Given a process Q, we use names(Q) to denote the set of names
occurring in Q. Let P be a process and n be a natural number; we denote with
combP (n) = {Q | names(Q) ⊆names(P), depth(Q) ≤n,
∀Q′ ∈seq(Q).∃P ′ ∈seq(P).Q′ = P{v/x} for some v and x} Definition 7. Given a process Q, we use names(Q) to denote the set of names
occurring in Q. Let P be a process and n be a natural number; we denote with
combP (n) = {Q | names(Q) ⊆names(P), depth(Q) ≤n,
∀Q′ ∈seq(Q).∃P ′ ∈seq(P).Q′ = P{v/x} for some v and x} Definition 7. Given a process Q, we use names(Q) to denote the set of names
occurring in Q. Let P be a process and n be a natural number; we denote with Definition 7. Given a process Q, we use names(Q) to denote the set of names
occurring in Q. Let P be a process and n be a natural number; we denote with the set of processes with names that already occur in P, with maximum nesting
level smaller than n, and containing sequential subprocesses that already occur
in P (up-to renaming). We now prove that combP (depth(P)) is actually a superset of der(P). Proposition 3. Let P be a process with replacing or parallel compensations. Then der(P) ⊆combP (depth(P)). Proposition 3. Let P be a process with replacing or parallel compensations. Then der(P) ⊆combP (depth(P)). Proof. We first observe that P ∈combP (depth(P)). Then we consider a pro-
cess Q ∈combP (depth(P)) such that Q →Q′, and we show that also Q′ ∈
combP (depth(P)). By Proposition 2 we have that depth(Q′) ≤depth(Q) hence
also depth(Q′) ≤depth(P). Definition 5. Let P be a process. We define depth(P) inductively as follows: Moreover, it is easy to see that Q′ does not intro-
duce new sequential subprocesses (it can at most apply a name substitution to
sequential subprocesses of Q). Notice that in case the transition is a compensa-
tion update, no new sequential subprocesses can be obtained because either the
replace or the parallel pattern is used. ⊓⊔ ⊓⊔ We are finally ready to prove that (combP (depth(P)), ⪯) is indeed a wqo, by
proving a slightly more general result. Theorem 2. Let P be a process and let n be a natural number. The relation ⪯
is a wqo over combP (n). Proof. Take an infinite sequence P1, P2, . . . , Pi, . . ., with Pi ∈combP (n) for every
i > 0. We prove, by induction on n, that there exist k and l such that Pk ⪯Pl. p
,
y
,
Let n = 0. All the processes Pi do not contain neither transactions nor
protected blocks because depth(Pi) ≤0. For this reason, we have that Pi =
Qni
j=1 Pi,j with Pi,j equal to some sequential subprocess of P (up-to renaming
by using names already in P). This set is finite, then process equality = is a
wqo over this set. By Higman’s lemma we have that also =∗is a wqo over finite
sequences of such processes. Hence there exists k and l such that Pk,1 . . . Pk,nk
is a subsequence of Pl,1 . . . Pl,nl, hence we have Pk ⪯Pl. For the inductive step, let n > 0 and assume that the thesis holds for
combP (n −1). We have that the following holds for every Pi: Pi ≡
ni
Y
j=1
Pi,j |
mi
Y
j=1
ti,j[Qi,j, Ri,j] |
oi
Y
j=1
⟨Si,j⟩ 11 with Pi,j equal to some sequential subprocess of P (up-to renaming by using
names already in P), ti,j ∈names(P) and Qi,j, Ri,j, Si,j have a maximum
nesting level strictly smaller than n, hence Qi,j, Ri,j, Si,j ∈combP (n −1). We
now consider every process Pi as composed of 3 finite sequences: Pi,1 · · · Pi,ni,
⟨ti,1, Qi,1, Ri,1⟩· · · ⟨ti,mi, Qi,mi, Ri,mi⟩, and Si,1 · · · Si,oi. As observed above, =∗
is a wqo over the sequences Pi,1 · · · Pi,ni. For this reason we can extract an infi-
nite subsequence of P1, P2, . . . Definition 5. Let P be a process. We define depth(P) inductively as follows: making the finite sequences Pi,1 · · · Pi,ni increas-
ing w.r.t. =∗. We now consider the triples ⟨ti,j, Qi,j, Ri,j⟩. Consider the ordering
(combP (n −1) ∪names(P), ⊑) such that x ⊑y iffx = y, if x, y ∈names(P),
or x ⪯y, if x, y ∈combP (n −1). As names(P) is finite and due to the in-
ductive hypothesis according to which (combP (n −1), ⪯) is a wqo, we have
that also (combP (n −1) ∪names(P), ⊑) is a wqo. By Dickson’s lemma we have
that ⊑3 is a wqo over the considered triples ⟨ti,j, Qi,j, Ri,j⟩. We can apply
the Higman’s lemma as above to prove that it is possible to extract, from
the above infinite subsequence, an infinite subsequence making the finite se-
quences ⟨ti,1, Qi,1, Ri,1⟩· · · ⟨ti,mi, Qi,mi, Ri,mi⟩increasing w.r.t. (⊑k)∗. Finally,
as Si,j ∈combP (n −1) and by inductive hypothesis, we can finally apply again
Higman’s lemma to extract, from the last infinite sequence, an infinite subse-
quence making the finite sequences Si,1 · · · Si,oi increasing w.r.t. ⪯∗. It is now
sufficient to take from this last subsequence two processes Pk and Pl, with k < l,
and observe that Pk ⪯Pl. ⊓⊔ We now move to the proof of compatibility between the ordering ⪯and the
transition system →. Lemma 1. If P ⪯P ′ and P
α−→Q then there exists Q′ such that Q ⪯Q′ and
P ′
α−→Q′. Proof. By induction on the depth of the proof of P
α−→Q. ⊓⊔ roof. By induction on the depth of the proof of P
α−→Q. ⊓⊔ Proof. By induction on the depth of the proof of P
α−→Q. ⊓⊔ As the transitions →correspond to transitions labeled with τ, as a trivial corol-
lary we have the compatibility of ⪯with →. Hence, we can conclude that
given a process P with replacing or parallel (as well as static) compensations,
(combP (depth(P)), →, ⪯) is a WSTS. As a consequence, we obtain our decid-
ability result. Corollary 2. Let P be a process with replacing, parallel or static compensations. The termination of P is decidable. Proof. By definition, P terminates iffthere exists no infinite computation P
τ−→
P1
τ−→. . .. For replacing and parallel compensations, by Proposition 3, this holds
iffP terminates in the transition system (combP (depth(P)), →). But this last
problem is decidable, by Proposition 1, because (combP (depth(P)), →, ⪯) is a
WSTS. The result for static compensations follows since they form a subcalculus
of replacing/parallel compensations. ⊓⊔ 12 parallel
replacing
static
nested
[ESOP 2010]
[This paper]
dynamic
Fig. 6. Separation results for compensation mechanisms. nested [This paper] replacing parallel Fig. 6. Separation results for compensation mechanisms. 5
Related Work and Conclusion In this paper we studied decidability properties of π-calculus extended with
primitives for specifying transactions and compensations. Fig. 6 shows all the
considered calculi. Arrows denote the subcalculus relation. As already said, [20]
is the closest paper to ours. There, relying on syntactic conditions restricting
the allowed class of encodings and requiring some strong semantic properties
to be preserved, the authors proved the separation result represented by the
dotted line. The results in this paper instead, requiring only termination preser-
vation, prove the separation result represented by the dashed line. Besides sepa-
ration, [20] also showed an encoding proving the equivalence of static and paral-
lel compensations. This result, compatible with our separation result, cannot be
straightforwardly applied in our setting since it relies on the restriction operator. However, if one disallows transactions under a replication prefix, our decidability
results still hold and the encoding in [20] can be applied. It would be interesting
to look for termination-preserving encodings of dynamic into nested compensa-
tions and replacing into static compensations (such an encoding should violate
some of the conditions in [20]). The only other results comparing the expressive power of primitives for trans-
actions and compensations are in the field of SAGAs [9]/cCSP [10], but their
setting allows only for isolated activities, since it does not consider communica-
tion. There are two kinds of results: a few papers compare different variants of
SAGAs [6, 18, 7], while others use SAGAs-like calculi as specifications for π-style
processes [21, 11]. Both kinds of results cannot be easily compared with ours. Interestingly, our results have been studied in the framework of π-calculus
since it is the base of most proposals in the literature, but can similarly be stated
in CCS. Sticking to π-calculus, adding priority of compensation installation to
the calculus, as done by [20, 16, 27], does not alter the undecidability of termi-
nation for nested and dynamic compensations. For the decidability in parallel
and replacing compensations instead, the proof cannot be applied. Note however
that priority of compensation installation reduces the set of allowed traces, thus
termination without priority ensures termination with priority (but the opposite
is not true). 13 Decidability and undecidability results are a well-established tool to separate
the expressive power of process calculi. We restrict our discussion to few recent
papers. 5
Related Work and Conclusion In [5] two operators for modeling the interruption of processes are con-
sidered: P ✁Q that behaves like P until Q starts and tryP catchQ that behaves
like P until a throw action is executed by P to activate Q. Termination is proved
to be undecidable for tryP catchQ while it is decidable for P ✁Q. The undecid-
ability proof is different from the one in this paper since it exploits unbounded
nesting of try-catch constructs. The decidability proof requires to use a weaker
ordering (tree embedding) in order to deal with unbounded nesting of interrupt
operators. Such ordering is not appropriate for the calculus in the present paper
because compatibility is broken by the prefix inst⌊λX.Q⌋that synchronizes with
the nearest enclosing transaction and not with any of the outer transactions. In [13] higher-order π-calculus without restriction is considered. Despite higher-
order communication is rather different w.r.t. dynamic compensations, a similar
decidability result is proved: if the received processes cannot be modified when
they are forwarded, termination becomes decidable, while this is not the case
if they can [19]. The decidability proof is simpler w.r.t. the one in this paper
because there is no operator, like t[P, Q], that requires the exploitation of Dick-
son’s lemma. Finally, we mention [4] where a calculus for adaptable processes is
presented: running processes can be dynamically modified by executing update
patterns similar to those used in this paper. A safety property is proved to be
decidable if the update pattern does not add prefixes in front of the adapted
process, while it becomes undecidable if a more permissive pattern is admitted. The undecidability proof in the present paper is more complex because update
patterns can be executed only on inactive processes (the compensations). The
decidability proof in [4] is similar to the one in [5]: the same comments above
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and compensation. Math. Struct. Comp. Sci., 19(3):565–599, 2009. 6. R. Bruni, M. J. Butler, C. Ferreira, C. A. R. Hoare, H. C. Melgratti, and U. Mon-
tanari. Comparing two approaches to compensable flow composition. In Proc . of
CONCUR’05, volume 3653 of LNCS, pages 383–397. Springer, 2005. 7. R. Bruni, A. Kersten, I. Lanese, and G. Spagnolo. A new strategy for distributed
compensations with interruption in long-running transactions. In Proc. of WADT
2010, volume 7137 of LNCS, pages 42–60. Springer, 2010. 14 8. R. Bruni, H. C. Melgratti, and U. Montanari. Nested commits for mobile calculi:
Extending join. In Proc. of IFIP TCS’04, pages 563–576. Kluwer, 2004. 9. R. Bruni, H. C. Melgratti, and U. Montanari. Theoretical foundations for com-
pensations in flow composition languages. In Proc. of POPL ’05, pages 209–220. ACM Press, 2005. 10. M. J. Butler, C. Hoare, and C. Ferreira. A trace semantics for long-running trans-
actions. In 25 Years CSP, volume 3525 of LNCS, pages 133–150. Springer, 2004. 11. L. Caires, C. Ferreira, and H. Vieira. A process calculus analysis of compensations. In Proc. of TGC’08, volume 5474 of LNCS, pages 87–103. Springer, 2008. 12. E. de Vries, V. Koutavas, and M. Hennessy. Communicating transactions. In Proc
of CONCUR 2010, volume 6269 of LNCS, pages 569–583. Springer, 2010. 13. C. Di Giusto, J. A. P´erez, and G. Zavattaro. On the expressiveness of forwarding in
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decidability of higher-order process calculi. In Proc. of LICS’08, pages 145–155. IEEE Computer Society, 2008. 20. I. Lanese, C. Vaz, and C. Ferreira. On the expressive power of primitives for
compensation handling. In Proc. of ESOP 2010, volume 6012 of LNCS, pages
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SaCS’05, volume 3441 of LNCS, pages 282–298. Springer, 2005. 23. R. Milner, J. Parrow, and J. Walker. A calculus of mobile processes, I and II. Inf. Comput., 100(1):1–40,41–77, 1992. 24. M. Minsky. Computation: finite and infinite machines. Prentice-Hall, Englewood
Cliffs, 1967. 25. F. Montesi, C. Guidi, and G. Zavattaro. Composing services with JOLIE. In Proc. of ECOWS’07, pages 13–22. IEEE Computer Society, 2007. 26. Oasis. Web Services Business Process Execution Language Version 2.0, 2007. http:
//docs.oasis-open.org/wsbpel/2.0/OS/wsbpel-v2.0-OS.html. 27. C. Vaz, C. Ferreira, and A. Ravara. Dynamic recovering of long running trans-
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2008. 15
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Adherence comparison of concrete with unprotected
steel and hot galvanized steel Comparativo da aderência do concreto com aço sem
proteção e o aço galvanizado a quente B.F. TUTIKIAN a
bftutikian@unisinos.br
T. HILGERT a
hilgert_thai@hotmail.com
J.J. HOWLAND b
jhalbear@civil.cujae.edu.cu B.F. TUTIKIAN a
bftutikian@unisinos.br
T. HILGERT a
hilgert_thai@hotmail.com
J.J. HOWLAND b
jhalbear@civil.cujae.edu.cu Abstract Increasing the service life of structures is of great importance for civil construction, either because of economic aspects or security ones to the
users. Corrosion of reinforcement is one of the most recurring problems, especially in environments with high chloride content. One of the most
effective alternatives to protect reinforcement against corrosion is the hot-dip galvanizing of steel bars, with the addition of a zinc coating that is
consumed before steel entering in reaction, extending the service life of the structure. Nevertheless, this layer of zinc should not affect the adher-
ence of rebars with concrete. In this paper, it was investigated this connection, establishing comparisons to unprotected reinforcement, with the
analysis of three bar diameters, 8, 12.5 and 16mm through the bending test of beams, the procedure of Rilem, 1978 [1 ]. After statistical analysis,
it was observed that there was no significant loss of adherence in any of the diameters, showing that the adherence between the concrete and the
hot-dip galvanized steel is not lower than the steel without protection for these materials. Keywords: corrosion of reinforcement; hot-dip galvanization; adherence; bending test. a
ITT Performance, UNISINOS. São Leopoldo, RS, Brasil;
b
Instituto Superior Politécnico “Jose Antonio Echeverría”, CUJAE / ISPJAE, Marianao, La Havana, Cuba. Received: 19 Sep 2013 • Accepted: 03 Feb 2014 • Available Online: 03 Apr 2014 a
ITT Performance, UNISINOS. São Leopoldo, RS, Brasil;
b
Instituto Superior Politécnico “Jose Antonio Echeverría”, CUJAE / ISPJAE, Marianao, La Havana, Cuba.
Received: 19 Sep 2013 • Accepted: 03 Feb 2014 • Available Online: 03 Apr 2014 1. Introduction The galvanization is a process that creates a protective zinc film
base to the steel, isolating the surface of the bar from the exposure
environment. This protective film acts as the anode, with the steel
acting as the cathode. Thus, being zinc more electronegative, it
sacrifices itself, protecting steel from deterioration. The alloys for-
med between iron and zinc on the contact surface drive the coating
to its integration to the metal base, so that, besides protecting the
steel, zinc coating also allows the handling, transportation and ins-
tallation of galvanized parts without causing damage to the surface
(YEOMANS, 2004 [6]). According to ABNT NBR 15575:2013 [2], the service life of reinfor-
ced concrete in Brazil, for residential purposes, must be at least
50 years on the minimum level, or 75 years on upper levels. On
the other hand, BS 7543:2003 [3] stipulates 120 years of service
life for works of art, such as bridges and viaducts and 60 years for
new buildings and reformation of public buildings. Achieving these
values is no easy task, especially if the maintenance process is
not efficient. According to Yoo et al. (2011) [7], in general the average thickness
of zinc is sufficient to achieve the useful life of the structure without
maintenance for long periods. Thus, according to Pannoni (2011)
[8], it is also possible to estimate the service life of the structure
with the support of ISO 9223:2012 [9] from the thickness of galva-
nizing, as shown in Table 1. The corrosion of rebars is one of the most recurrent pathological
signs in steel-reinforced concrete structures, especially those ex-
posed to aggressive agents throughout its service life, such as
chlorides and carbon dioxide, found in abundance in large cities
and on the coast. According to Gonçalves, Andrade and Castellote (2003) [4], to
protect the reinforced concrete structures it is possible to use the
direct protection (on steel) and the indirect protection (on the con-
crete). The direct protection is more efficient because it protects
the rebars directly. Among the types of direct protection exists the
impressed current cathodic system, the cathodic galvanic type, the
physical barrier and the galvanic barrier. The first two have the
disadvantage of requiring constant maintenance and the operation
may be complex depending on the aggressiveness of the exposu-
re environment. Resumo O aumento da vida útil de estruturas é de grande importância para a construção civil, seja por aspectos econômicos quanto de segurança aos
usuários. A corrosão de armaduras é um dos problemas mais recorrentes, principalmente em ambientes com alto teor de cloretos. Uma das
alternativas mais eficientes para proteger as armaduras contra a corrosão é a galvanização a quente das barras de aço, com a incorporação
de uma camada de zinco que será consumida antes do aço entrar em reação, prolongando a vida útil da estrutura. Porém, esta camada de zin-
co não deve prejudicar a aderência das barras de aço com o concreto. Neste trabalho, foi investigada esta relação, comparativamente com a
armadura sem proteção, com a análise de três diâmetros de barras, 8, 12,5 e 16mm, através de ensaio de flexão em viga, com o procedimento
da Rilem, de 1978 [1]. Observou-se que não houve perda de aderência significativa em nenhum dos diâmetros, após análise estatística, mos-
trando que a aderência entre o concreto e o aço galvanizado a quente não é inferior do que com os aços sem proteção, para estes materiais. Palavras-chave: corrosão de armaduras; galvanização a quente; aderência; ensaio de flexão. © 2014 IBRACON Adherence comparison of concrete with unprotected steel and hot galvanized steel Adherence comparison of concrete with unprotected steel and hot galvanized stee 1. Introduction On the other hand, the physical barrier demands
skilled labor force and it is preferably used in specific situations,
due to the labor force required. For broader and more effective
response, there is the galvanic barrier produced by hot-dip gal-
vanizing of steel bars. Figure 1 shows the options for the direct
protection. The adherence between the steel and concrete ensures the pro-
per performance of reinforced concrete structures, thereby ensuring
that the materials work mutually. The galvanization of rebars cannot
affect the adherence of the set, and this is a point to be validated.i The adherence can be obtained in three ways: by superficial adhe-
rence, friction and mechanically. According to Caetano (2008) [10],
adherence friction occurs after the breaking of the bonding adhe-
rence, that is, when the sliding of the bar begins to happen. This
portion refers to the action of the frictional force between the steel
and the concrete, which varies according to the surface coefficient
bar. This factor can be harmed by the hot-dip galvanization, once
the bar gets a zinc coating, making the rebar smoother. There is no
proof of the dimension of this loss, and if it is tolerable or not, within
the parameters established by ABNT NBR 7480:2007 [11], which
specifies the steel for the reinforcement shall have a coefficient (η)
of 1.5 minimum.l The hot-dip galvanized steel has great durability and that is the
reason why its application in the market grows increasingly. Widely
used in metal structures, it can also be an option for reinforced
concrete structures (Baltazar-Zamora et al., 2012 [5]). The galva-
nization brings many advantages that go beyond the increasing of
service life, such as reducing the risks of cracks caused by the ste-
el expansion during the corrosion process, the rust stains and de-
gradation of the concrete, due to a lower frequency and magnitude
of the concrete repairs. Therefore, the initial cost to deploy such a
system in reinforced concrete structures can be counterbalanced
by the several advantages mentioned before. Regarding the influence of the friction adherence part of the ribbed
bars, there is still some disagreement among authors, whether it
influences or not. According to Lutz and Gergely (1967) [12] and
Cairins Du and Law (2007) [13] this part only exists in smooth bars,
however for most of the latest research, the adherence provoked
by friction also affects the ribbed bars. 2.1 Materials To scale the beams to be tested, it was followed the procedure
of Rilem (1978) [1] according to the specifications of Table 2. In the concrete, it was used the trace 1:6, by mass, the pro-
portion between the binder and the aggregates, with water/
cement ratio of 0.6 and abatement of the Abrams cone in the
fresh state of 100mm. This trait was set to achieve what was
proposed by the Rilem (1978) [1] method, which provides the
compressive strength of concrete at 28 days, at 25 MPa, with a
tolerance of 2.5 MPa. For a better understanding of the growth
curve of the concrete, the tests were performed in 7 and 28
days. The dosage method of the concrete was Ibracon (Brazi-
lian Institute of Concrete, 2011) [16]. To simulate all these conditions and verify adherence between the
concrete and steel without external protection and hot-dip galvani-
zation, the method that most closely matches the real situation is
the Beam Test proposed by Rilem (1978) [1] . In this experiment,
the beam is subjected to bending with the contribution of other
important factors on the steel-concrete adherence. Thus, the aim of this paper is to compare the adherence of ste-
el bars of 8.0, 12.5 and 16.0mm hot-dip galvanized and without
galvanization, traditionally used in civil construction. The test for
adherence between steel and concrete is achieved through the
Rilem (1978) [1] procedure that is the bending test on the concrete
beams to identify adherence. Concrete mix was used for the be-
ams, 1:6 of mass to ensure the compressive strength of 25MPa,
with a tolerance of 2.5 MPa, according to the procedure. After the
results, a statistical analysis to identify significant properties for this
parameter was performed. 1. Introduction Concrete mix wa
ams, 1:6 of mass to ensure the compressive s
with a tolerance of 2.5 MPa, according to the p
results, a statistical analysis to identify significan
parameter was performed. Corrosivity
category
Low-c
2
Loss in mass (g/m )
Source: ISO 9223:2012
Loss in mass
C1 – very low
C2 – low
C3 – medium
C4 – high
C5 – very high
≤10
>10 a 200
>200 a 400
>400 a 650
>650 a 1500
Table 2 – Parameters for v
Properties a
Source: Adapted of Rilem, 1978
Diameters o
Grip le
Thickness of co
Height of conc
Distance between th
Overall width
Width of t
Distance between the axis of the b
Distance between the axis of the bar and
Distance betw
Distance betw 2. Materials and method chanical resistance of carbon steel varies from 32 to 66kg/mm². According to Fusco (1995) [14] and ACI (2003) [15], the rebars still
suffer, in addition to these, three other efforts, which would be com-
pressive and frictional forces on the ribs in addition to the friction
on the body of the bar. These forces act in many ways, preventing
the sliding of the bar. 1. Introduction There is also the mechanical resistance, which in its turn is the
most aggravating for a good adherence, being directly allied to the
hardness of the material. Thus, it is worth mentioning that the me- 314
IBRACON Structures and Materials Journal • 2014 • vol. 7 • nº 2
Figure 1 – Types of direct protection of steel bars
Source: Gonçalves et al. (2003) Figure 1 – Types of direct protection of steel bars Source: Gonçalves et al. (2003) Source: Gonçalves et al. (2003) IBRACON Structures and Materials Journal • 2014 • vol. 7 • nº 2 314 B.F. TUTIKIAN | T. HILGERT | J.J. HOWLAND Table 1 – Indicatives rates of corrosion for different environmental
Corrosivity
category
Zinc
Low-carbon steel
2
Loss in mass (g/m )
Source: ISO 9223:2012
2
Loss in mass (g/m )
Loss of thickness (µm)
Loss of thickness (µm)
Loss in mass per unit of surface/loss of thickness (after one year of exposition)
C1 – very low
C2 – low
C3 – medium
C4 – high
C5 – very high
≤10
>10 a 200
>200 a 400
>400 a 650
>650 a 1500
≤1,3
>1,3 a 25
>25 a 50
>50 a 80
>80 a 200
≤0,7
>0,7 a 5
>5 a 15
>15 a 30
>30 a 60
≤0,1
>0,1 a 0,7
>0,7 a 2,1
>2,1 a 4,2
>2,1 a 4,2 Table 1 – Indicatives rates of corrosion for different environmental IBRACON Structures and Materials Journal • 2014 • vol. 7 • nº
chanical resistance of carbon steel varies from
According to Fusco (1995) [14] and ACI (2003)
suffer, in addition to these, three other efforts, w
pressive and frictional forces on the ribs in add
on the body of the bar. These forces act in man
the sliding of the bar. To simulate all these conditions and verify adhe
concrete and steel without external protection a
zation, the method that most closely matches t
the Beam Test proposed by Rilem (1978) [1] . the beam is subjected to bending with the co
important factors on the steel-concrete adheren
Thus, the aim of this paper is to compare the
el bars of 8.0, 12.5 and 16.0mm hot-dip galva
galvanization, traditionally used in civil constru
adherence between steel and concrete is ach
Rilem (1978) [1] procedure that is the bending t
beams to identify adherence. 2.2 Method 2.2 Method After defined the parameters of the beams and materials, three
sets were molded for each studied diameter (8mm, 12.5mm
and 16mm) and for each condition (reference and hot-dip Table 2 – Parameters for verifying adherence between the concrete and steel bars
Properties and dimensions
Type A
Type B
Source: Adapted of Rilem, 1978
Diameters of the bars (mm)
Grip length (ld)
Thickness of concrete blocks (cm)
Height of concrete blocks (cm)
Distance between the concrete blocks (cm)
Overall width of beam (cm)
Width of the bars (cm)
Distance between the axis of the bar and the axis of the kneecap (cm)
Distance between the axis of the bar and the axis of the bottom face of the beam (cm)
Distance between loads (cm)
Distance between suports (cm)
<16
10 ø
18
37,5
5
80
100
10
5
15
65
≥16
10 ø
24
60
6
126
150
15
5
20
110 Table 2 – Parameters for verifying adherence between the concrete and steel bars IBRACON Structures and Materials Journal • 2014 • vol. 7 • nº 2 315 Adherence comparison of concrete with unprotected steel and hot galvanized steel Adherence comparison of concrete with unprotected steel and hot galvanized steel Adherence comparison of concrete with unprotected steel and hot galvanized stee Figure 2 – concrete placement set beams for testing adherence between the bars and the concrete galvanized), totalizing 18 sets, consisting of 36 parts. Figure 2
illustrates one of the concretes and six molded sets. galvanized), totalizing 18 sets, consisting of 36 parts. Figure 2
illustrates one of the concretes and six molded sets. (1)
vb =
2
5 x d
100 (1) After this, the sets were cured in a moist chamber for 28 days. The bea-
ms were instrumented at its ends, with digital dial indicators to measure
the deformations of the bars that tend to slip during the test. Points 1
mm, 0.1 mm and 0.01 mm, specified in the procedure, were measured. The principle used was the arithmetic mean of the results obtained
at both ends, moments before the breaking of the beam. For the
last reading (1mm) it was considered the first load that reached this
limit. The rate of the load application to the rods of 8.00 mm, 12.5
mm and 16.0 mm diameter was obtained according to equation 1: vb = speed of load application vb = speed of load application
d = diameter of the test bar in cm d = diameter of the test bar in cm After that, the beams were placed and subjected to the bending
test being double supported and receiving the load application Figure 3 – Execution of the beam test Figure 3 – Execution of the beam test IBRACON Structures and Materials Journal • 2014 • vol. 7 • nº 2 316 B.F. TUTIKIAN | T. HILGERT | J.J. HOWLAND Table 3 – Results of compressive strength at 7 and 28 days
Diameter of the bars (mm)
Trace of concrete, in mass
w/c ratio
fc 7 days (MPa)
fc 28 days (MPa)
8
12,5
16
1:6
1:6
1:6
0,6
0,6
0,6
16,7
15,5
14,5
26,4
23,2
22,5 Table 3 – Results of compressive strength at 7 and 28 days Table 3 – Results of compressive strength at 7 and 28 days 3.1 Compressive strength The results of the 7 and 28 days of compressive strength of the
concrete are shown in Table 3. It is observed that the concrete had compressive strength within
the range recommended by Rilem (1978) [1], allowing the tests
within the specified time. Figure 4 – Behavior of load versus for each bar diameter
for each condition, with 3 beams tested for each case Figure 4 – Behavior of load versus for each bar diameter
for each condition, with 3 beams tested for each case Figure 4 – Behavior of load versus for each bar diameter
for each condition, with 3 beams tested for each case IBRACON Structures and Materials Journal • 2014 • vol. 3.1 Compressive strength 7 • nº 2 317 Adherence comparison of concrete with unprotected steel and hot galvanized steel Table 4 – Values obtained from adherence tests
Type and
diameter of
the bars
Left measure
Wright measure
Adherence
strength
( t – bu – MPa)
Average adherence
strength
( t – bu – MPa)
Deflection
(mm)
Deflection
(mm)
Load
(kN)
Load
(kN)
0,01
0,10
0,53*
0,01
0,10
0,23*
0,01
0,10
0,53*
0,01
0,10
0,52*
0,01
0,10
1,00
0,01
0,10
0,81*
0,01
0,10
1,00
0,01
0,10
0,98*
0,01
0,10
0,87*
0,01
0,10
1,00
0,01
0,10
1,00
0,01
0,10
1,00
0,01
0,10
1,00
0,01
0,10
0,65*
0,01
0,10
1,00
0,01
0,10
0,28*
0,01
0,10
1,00
0,01
0,10
0,43*
0,01
0,10
0,40*
0,01
0,10
1,00
0,01
0,10
1,00
0,01
0,10
1,00
0,01
0,07**
–
0,01
0,10
1,00
0,01
0,10
0,30*
0,01
0,10
1,00
0,01
0,10
1,00
0,01
0,10
0,76*
0,01
0,10
1,00
0,01
0,10
0,46*
0,01
0,10
1,00
0,01
0,10
1,00
0,01
0,10
0,43*
0,01
0,10
1,00
0,01
0,10
0,11*
0,01
0,10
1,00
8
11
32
8
8,5
–
8
8
–
8
10
–
8
8,5
8,5
8
8
16,3
11
23
40,3
17
28
–
15,5
28,3
–
19
30
48,8
25,8
36
47
23
35,7
46,5
24
39
64,5
8,0
49,5
67,0
20,5
28,5
51,2
36,5
50,8
–
30,0
42,7
63,2
32,5
43,5
62,5
8
14
32
8
8
12
8
13,2
16,5
8
9
15,5
8
–
–
8
9
13,5
9
23,3
–
14,5
28,5
46,5
18
29,5
44,5
21
35,5
–
21,5
36
47
18
31,5
–
29,5
41,2
58,2
18,5
37,8
65,2
22,5
40,2
58,0
32,5
42,3
63,0
52,5
62,0
64,0
54
56
60,7
7,77
7,46
10,26
9,64
5,28
(resultado
desprezado)
8,39
10,26
11,84
11,33
12,43
11,97
11,84
11,44
12,16
9,55
11,75
11,79
11,32
–
8,50
–
–
9,01
–
–
11,14
–
–
12,08
–
–
11,05
–
–
11,62
–
8mm
reference
8mm
reference
8mm
reference
8mm
hot-dip
galvanized
8mm
hot-dip
galvanized
8mm
hot-dip
galvanized
12,5mm
reference
12,5mm
reference
12,5mm
reference
12,5mm
hot-dip
galvanized
12,5mm
hot-dip
galvanized
12,5mm
hot-dip
galvanized
16mm
reference
16mm
reference
16mm
reference
16mm
hot-dip
galvanized
16mm
hot-dip
galvanized
16mm
hot-dip
galvanized
* Beam breakup occurred before completing 1,00 mm deflection
** Beam breakup occurred before completing 0,10mm deflection
*** Beam with problems during the tests – despised result Table 4 – Values obtained from adherence tests IBRACON Structures and Materials Journal • 2014 • vol. 3.1 Compressive strength 7 • nº 2 318 B.F. TUTIKIAN | T. HILGERT | J.J. HOWLAND Table 5 – Tests results of adherence
Parameters
SQ
GL
MQ
Fcal
P
Interception
Diameter of bar (mm)
Type of bar
Diameter of bar (mm) and type of bar
1855,494
25,557
1,884
0,146
1
2
1
2
1855,494
12,779
1,884
0,073
1900,878
13,091
1,931
0,075
0,000000
0,001233
0,192179
0,928198
SQ – sum of squares; GL – degree of freedom ; MQ – mean square; Fcal – Fisher parameter for the test of significance of effects Table 5 – Tests results of adherence 3.2 Adherence between the concrete and hot-dip
galvanized and unprotected rebars 3.2 Adherence between the concrete and hot-dip
galvanized and unprotected rebars But when comparing the adherence strength between the different
types of steel, it is observed that the galvanized bars and rods have
always obtained higher values in comparison to the unprotected
ones, 6% for the 8mm diameter, 8.4% for the 12, 5mm diameter
and 5.2% for the 16mm. Therefore, the zinc layer which protects
the reinforcement against corrosion did not damage the adherence
between the steel and the concrete, and there may even be a small
gain in certain situations. For each type of bar diameter (8mm, 12.5mm and 16mm), and
each condition (reference and hot-dip galvanized) was examined
by the proposed method of Rilem (1978) [1], measuring the load
required in kN for deformed bars of 0.01 mm, 0.1 mm and 1 mm,
both the left and the right of the application of the load, when the
test is considered ended. Figure 4 shows the load versus deflec-
tion curves for each diameter and provided with three measure-
ments for each condition. 4. Conclusions Therefore, for these materials under these conditions, it is possible
to specify the hot-dip galvanized steel without concerning for adhe-
rence strength between the concrete and the steel. 8.4 and 5.2% for diameters of 8, 12.5 and 16mm respectively. Therefore, for these materials under these conditions, it is possible
to specify the hot-dip galvanized steel without concerning for adhe-
rence strength between the concrete and the steel. 4. Conclusions After the experimental work, it is possible to conclude that:il It is noticeable that for beams with diameter of 8.0 mm the limit of
1.0 mm has always occurred to the right side of the beam, getting
a smaller load for most of them, with a beam having a different
performance of others. In beams with bars of 12.5mm diameters,
achieved loads were higher than in the previous beams, as it was
to be expected. For both bars, from 8mm to 12mm, the adherence
between the concrete and steel was higher in beams with hot-dip
galvanized reinforcements. The same performance was observed
on the beams with 16mm bars, showing that for larger diameters,
the adherence between the concrete and hot-dip galvanized steel
was also higher. It is important to highlight that the parameters of
the beams with 16mm bars is different from the earlier ones, accor-
ding to the Rilem procedure (1978). It is noticeable that for beams with diameter of 8.0 mm the limit of
1.0 mm has always occurred to the right side of the beam, getting
a smaller load for most of them, with a beam having a different
performance of others. In beams with bars of 12.5mm diameters,
achieved loads were higher than in the previous beams, as it was
to be expected. For both bars, from 8mm to 12mm, the adherence
between the concrete and steel was higher in beams with hot-dip
galvanized reinforcements. The same performance was observed
on the beams with 16mm bars, showing that for larger diameters,
the adherence between the concrete and hot-dip galvanized steel
was also higher. It is important to highlight that the parameters of
the beams with 16mm bars is different from the earlier ones, accor-
ding to the Rilem procedure (1978). n the diameter of the bars has a significant influence on the adhe-
rence strength between the concrete and the steel; n the type of steel, hot-dip galvanized or without protection, does
not have significant influence on the adherence strength betwe-
en the concrete and the steel; n the hot-dip galvanized bars and rods were resistant to adherence
between the concrete and the upper unprotected steel bars, in 6, 8.4 and 5.2% for diameters of 8, 12.5 and 16mm respectively. 8.4 and 5.2% for diameters of 8, 12.5 and 16mm respectively. 5. References Based on the collected data, it was calculated the values of adhe-
rence strength, expressed in Table 4. [01] RILEM, FIP; CEB. Essai portanto sur I’ adherence dês arma-
tures du béton. 1. Essai per flexion (7-II-28D). Recommen-
dation Provisoires. Matériaux et Constructions. Paris, v. 6, n. 32, p. 96-101, 1978. Based on the bond tension, statistical analysis (ANOVA) was used,
in the software STATISTICA to verify the relationship between
adherence and the type and diameter of the bars. The results are
shown in Table 5. If Fcal is higher than the tabulated value of F, the null hypothe-
sis will be rejected. So that means there is significant difference
between the group means and, consequently, the study variable
influences the dependent variable. In this way, it is noticeable that
the kind of bar (galvanized or not) does not have a significant level,
because P is greater than 0.5, commonly used in civil engineering,
unlike the diameter of the bar, which has a significant level. There-
fore, it is observed that the diameter of the bar influences the bond
strength between the concrete and the steel, but whether the bar is
hot-dip galvanized or not, it does not affect the final adherence to
the results obtained in this paper. Figure 5 – Adherence strength
versus diameter of the bars Figure 5 – Adherence strength
versus diameter of the bars Finally, Figure 5 shows the values of the arithmetic mean among
the three results of bond strength for each type and diameter of
the bar. It is observed that the adherence strength increases considerably
at 31% for the unprotected steel and 34% for the hot-dip galva-
nized steel to diameters between 8.0 and 12.5 mm. As for the
16mm diameter was reduced from 1% for the regular steel to
4% for the hot-dip galvanized one, showing some stabilization in
larger diameters. IBRACON Structures and Materials Journal • 2014 • vol. 7 • nº 2 319 Adherence comparison of concrete with unprotected steel and hot galvanized steel Adherence comparison of concrete with unprotected steel and hot galvanized steel [02] ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. Edi-
ficações habitacionais - desempenho. NBR 15575, Rio de
Janeiro, 2013. [03] BRITISH STANDARDS INSTITUTION. Guide to durability of
buildings and building elements, products and components. BS 7543, London, 2003. [04] GONÇALVES, A.; ANDRADE, C.; CASTELLOTE, M. IBRACON Structures and Materials Journal • 2014 • vol. 7 • nº 2 Adherence comparison of concrete with unprotected steel and hot galvanized steel 5. References Manu-
al de Inspección, Evaluación y Diagnóstico de Corrosión en
Estructuras de Concreto Armado. Red Durar, CYTED Pro-
gram, Rio de Janeiro, 1997. [05] BALTAZAR-ZAMORA, M.A.; BANDALA, E.M.; TELLO, M.U.;
HURTADO, G.S.; COCA, F.J.; CEDANO, A.O.; BARRIOS,
C.P.; NUÑEZ, R.E.; ZAMBRANO, P.; TIBURCIO, C.; CAL-
DERÓN, F. Efficicency of galvanized steel embedded in
concrete previously cantamineted with 2, 3 and 4% of NaCl. In: Internacional Journal of Electrochemical Science, n°7, p. 2997-3007, 2012. [06] YEOMENS, S.R. Galvanized steel reinforcement in concre-
te: an overview. Elsevier, 2004. [07] YOO, Y.; NAM, T.; CHOI, Y.; KIM, J.; CHUNG, L. A galvanic
sensor system for detecting the corrosion damage of the ste-
el embedded in concrete structures: laboratory tests to de-
termine the cathodic protection and stray-current. In: Metals
and Materials International, v.17, issue 4, p.623-629, 2011. [08] PANNONI, F. D. Princípios da proteção de estruturas metá-
licas em situação de corrosão e incêndio. In: Coletânea do
uso do aço. Gerdau, 2011. [09] INTERNATIONAL ORGANIZATION FOR STANDARDIZA-
TION. Corrosion of metals and alloys – corrosivity of atmos-
pheres – classification, determination and estimation. ISO
9223:2012, Genève, Switzerland. [10] CAETANO, L.F. Estudo do comportamento da aderência em
elementos de concreto armado submetidos à corrosão e
elevadas temperaturas. Universidade Federal do Rio Gran-
de do Sul. Programa de Pós Graduação em Engenharia Ci-
vil (dissertação de mestrado). Porto Alegre, 2008. [11] ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. Aço
destinado a armaduras para estruturas de concreto armado
- Especificação. NBR 7480, Rio de Janeiro, 2007. [12] LUTZ, L.A.; GERGELY, P. Mechanics of bond and slip of
de formed bars in concrete. ACI Journal Proceedings, v.64,
n.11, p.711-721, nov. 1967.l [13] CAIRINS, J.; DU, Y.; LAW, D. Influence of corrosion on the
friction characteristics of the steel / concrete interface. In:
Construction and Buildings Materials. Vol. 21, n°1, p. 190-
197, 2007. [14] FUSCO, P.B. Técnicas de armar as estruturas de concreto. 1°Ed. São Paulo: PINI, 1995. [15] AMERICAN CONCRETE INSTITUTE, ACI. Bond and deve-
lopment of straight reinforcing bars in tension. Comitê 408,
2003. [16] TUTIKIAN, B; HELENE, P. Dosagem de concretos de cimen-
to Portland. In: Concreto: Ciência e Tecnologia. G.C. Isaia. 1°ed. São Paulo, IBRACON, 2011. 2v. IBRACON Structures and Materials Journal • 2014 • vol. 7 • nº 2 320
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https://josr-online.biomedcentral.com/track/pdf/10.1186/s13018-020-02022-9
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English
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Correlation of the anatomical sacral slope with pelvic incidence in female patients with developmental hip dysplasia: a retrospective cross-sectional study
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Journal of orthopaedic surgery and research
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cc-by
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RESEARCH ARTICLE Open Access Imai et al. Journal of Orthopaedic Surgery and Research (2020) 15:486
https://doi.org/10.1186/s13018-020-02022-9 Imai et al. Journal of Orthopaedic Surgery and Research (2020) 15:486
https://doi.org/10.1186/s13018-020-02022-9 Correlation of the anatomical sacral slope
with pelvic incidence in female patients
with developmental hip dysplasia: a
retrospective cross-sectional study Norio Imai1*, Hayato Suzuki2, Atsushi Sakagami2, Yuki Hirano1,2 and Naoto Endo1,2 * Correspondence: imainorio2001@yahoo.co.jp * Correspondence: imainorio2001@yahoo.co.jp
1Division of Comprehensive Musculoskeletal Medicine, Niigata University
Graduate School of Medical and Dental Sciences, 1-757, Asahimachi-dori,
Chuo ku, Niigata City, Niigata Prefecture 951-8510, Japan
Full list of author information is available at the end of the article * Correspondence: imainorio2001@yahoo.co.jp
1Division of Comprehensive Musculoskeletal Medicine, Niigata University
Graduate School of Medical and Dental Sciences, 1-757, Asahimachi-dori,
Chuo ku, Niigata City, Niigata Prefecture 951-8510, Japan
Full list of author information is available at the end of the article Abstract Background: The anatomical sacral slope is considered as an anatomical pelvic parameter independent of femoral
head centers for measurement of anatomical sacral slope and was previously described to strongly correlate with
pelvic incidence on a two-dimensional examination of healthy subjects. However, the correlation between
anatomical sacral slope and pelvic incidence was unclear in patients with developmental dysplasia of the hip. This
study aimed to examine the correlation between anatomical sacral slope and other spinopelvic parameters by
analyzing plain radiographs of female patients with developmental dysplasia of the hip. Methods: Eighty-four women with developmental dysplasia of the hip were examined. Lumbar lordosis, thoracic
kyphosis, pelvic incidence, sacral slope, and anatomical sacral slope (the angle formed by the straight line of the S1
superior endplate and a line at a right angle to the anterior pelvic plane) were determined by analyzing plain
radiographs. The correlations were examined by Pearson’s correlation coefficients, and intra- and inter-rater
intraclass correlation coefficients were evaluated for reliability. Results: A strong correlation was observed between pelvic incidence and anatomical sacral slope (r = 0.725, p <
0.001). In addition, the correlation between anatomical sacral slope and lumbar lordosis was similar to that between
pelvic incidence and lumbar lordosis (r = 0.661, p < 0.001, and r = 0.554, p < 0.001, respectively). The intra-rater
intraclass correlation coefficient values were 0.869 and 0.824 for anatomical sacral slope and pelvic incidence,
respectively. Furthermore, the inter-rater intraclass correlation coefficient values were 0.83 and 0.685 for anatomical
sacral slope and pelvic incidence, respectively. Conclusions: We observed that the strong correlation between anatomical sacral slope and pelvic incidence in
patients with developmental dysplasia of the hip was equal to that in normal healthy subjects. The correlation
between anatomical sacral slope and lumbar lordosis was equal to that between pelvic incidence and lumbar
lordosis. Additionally, the intraclass correlation coefficient values for the anatomical sacral slope were slightly higher
than those for pelvic incidence. Thus, we conclude that anatomical sacral slope can be considered as a helpful
(Continued on next page) * Correspondence: imainorio2001@yahoo.co.jp
1Division of Comprehensive Musculoskeletal Medicine, Niigata University
Graduate School of Medical and Dental Sciences, 1-757, Asahimachi-dori,
Chuo ku, Niigata City, Niigata Prefecture 951-8510, Japan
Full list of author information is available at the end of the article Background Eighty-four women with bilateral DDH, who had under-
gone curved periacetabular osteotomy [15] for treating
early-stage hip osteoarthritis due to DDH from April 1,
2010, to July 30, 2017, were examined in our hospital. The inclusion criterion was a center-edge angle of the
hip joints less than 20°, obtained from the anteroposter-
ior view of the hips on plain radiographs. This is because
these patients seemed to have a common morphological
characteristic of DDH in their pelvis and might have a
common functional alignment in the pelvis and spine. We excluded subjects who had undergone any hip joint
surgery, those who were evaluated to have hip dysplasia
of Crowe stages 2–4 [16] regarding subluxation, or those
in which arthritic change was evaluated as Tonnis grades
2–3 [17] observed on plain radiographs of the hip. Based on previous studies, it was considered that pelvic
morphology, as well as pelvic incidence (PI), influences
sagittal spinal alignments, such as lumbar lordosis (LL),
sacral slope (SS) [1–3], and standing posture [4–6]. A
larger PI is considered to be a risk factor for spondylo-
listhesis because it seemingly leads to anterior deviation
of the sagittal vertical axis [7, 8]. Additionally, the dis-
crepancy between PI and LL leads to spinal deformity in
adults [9]. In sagittal spinal malalignment, maintaining a
suitable balance is considered difficult, and it may lead
to “hip-spine syndrome” [10]. Consequently, PI is con-
sidered as one of the most important clinical parameters
and should be evaluated. Generally, many surgeons evaluate sagittal thoracol-
umbar spinal alignment and pelvic parameters by analyz-
ing two-dimensional (2D) plain radiographs captured in
the standing position [11, 12]. SS and pelvic tilt (PT) are
defined as functional parameters, since these angles are
influenced by the anteroposterior tilt of the pelvis, that
is, anterior or posterior tilt in the sagittal plane in the
standing position. On the contrary, PI is deemed to be
an anatomical parameter as it is not influenced by the
anteroposterior tilt of the pelvis. SS and PT are related
to PI in geometrical relation by the formula PI = SS +
PT. The Ethical Review Board of our institution approved
this study and waived the need for informed consent be-
cause of the retrospective cross-sectional design of the
study. Background Measurements of pelvic and thoracolumbar parameters
The pelvic parameters, such as PI, SS, and a-SS, and
the thoracolumbar parameters, such as thoracic ky-
phosis (TK) and LL, were measured using thoracic
and lumbar plain radiographs including the pelvis in
the standing position. PI was established as the angle
formed by the line at a right angle to the superior
endplate of S1 at its middle point and the line con-
necting this point to the axis linking the bilateral
femoral heads (Fig. 1) [11]. The SS was established as
the angle formed by the straight line of the S1 super-
ior endplate and a leveled line at a right angle to the
gravitational force direction. PT was established as
the angle formed by the straight line connecting the
middle point of the S1 endplate to the hip axis and
the vertical line parallel to the direction of gravita-
tional force (Fig. 1). Further, LL was established as
the angle from the line of T12 inferior endplate and
the line of the S1 superior endplate (Fig. 2). TK was
defined as the angle formed by the line of the T1 su-
perior endplate and the line of the T12 inferior end-
plate (Fig. 2). We recently described a correlation between PI and
anatomical sacral slope (a-SS), SS relative to anterior
pelvic plane (APP), in normal healthy subjects using
2D and three-dimensional (3D) measurements [1, 13,
14]. The a-SS was considered as an anatomical par-
ameter that does not require femoral head measure-
ments, as is the case when determining the PI. This
is advantageous as the femoral head center is some-
times difficult to establish. Previously, we observed a
close correlation between PI and a-SS among normal
healthy subjects and patients with developmental dys-
plasia of the hip (DDH) measured using only the 3D
method [13]. However, the correlation between PI
and a-SS has not been examined in patients with
DDH, wherein pelvic and/or spinal morphological fea-
tures observed using 2D measurements may be differ-
ent from those of normal subjects. This study aimed to examine the correlation between
PI and a-SS using plain radiographs of patients with
DDH. Similarly, we examined the correlation between a-
SS and LL using 2D measurements. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the
data made available in this article, unless otherwise stated in a credit line to the data. Imai et al. Journal of Orthopaedic Surgery and Research (2020) 15:486 Page 2 of 6 (Continued from previous page)
anatomical pelvic parameter that is a substitute for pelvic incidence not only in normal healthy subjects, but also in
patients with developmental dysplasia of the hip. Keywords: Anatomical sacral slope, Developmental dysplasia of the hip, Lumbar lordosis, Pelvic incidence Statistical analysis We analyzed the data using SPSS software (version 24;
SPSS, Inc., Chicago, IL). The correlation of PI, SS, a-SS, Imai et al. Journal of Orthopaedic Surgery and Research (2020) 15:486 Page 3 of 6 Imai et al. Journal of Orthopaedic Surgery and Research Fig. 1 Anatomical and functional parameters of the pelvis. a Anatomical parameters. b functional parameters. PI, pelvic incidence; SS, sacral slope;
APP, anterior pelvic plane; L- and R-ASIS, left and right anterior superior iliac spine Fig. 1 Anatomical and functional parameters of the pelvis. a Anatomical parameters. b functional parameters. PI, pelvic incidence; SS, sacral slope;
APP, anterior pelvic plane; L- and R-ASIS, left and right anterior superior iliac spine LL, and TK were evaluated with Pearson’s correlation
coefficients according to Guilford’s definition [18]. Simi-
larly, we evaluated the statistical power (type II (β) error)
using a post hoc analysis, with 0.3 as the effect size (d)
and 0.05 as type I (α) error, for the correlation analysis. We evaluated the validity of this study by calculating the
mean absolute difference (MAD), the variability by the
standard deviation (SD), and the intra- and inter-rater
reliabilities with interclass correlation coefficients (ICCs)
(with 95% confidence intervals) by two-tailed analysis. We measured 1-week intervals twice to determine the
intra-rater reliability and drew a parallel between the
measurements examined by two other observers to as-
sess the inter-rater reliability. Values of p below 0.05
were considered statistically significant. Fig. 2 Sagittal thoracolumbar spinal parameters. Lumbar lordosis
(LL) was established by the T12 inferior end plate and the S1
superior end plate. Thoracic kyphosis (TK) is measured between the
T1 superior end plate and the T12 inferior end plate Results The average age and body mass index of the participants
were 35.0 ± 9.2 years (range 20–52 years) and 22.0 ± 2.9
kg/m2 (range 16.2–27.8 kg/m2), respectively. Table 1 shows the details of the parameters. A close
correlation was observed between PI and a-SS (r =
0.725, p < 0.001) (Fig. 3), as defined by Guilford [18]
(Table 2). The regression formula calculated from this
correlation is as follows: PI = 0.8 × a-SS + 21. Regarding the correlation between pelvic and thoracol-
umbar parameters, a strong correlation was observed be-
tween SS and LL (r = 0.827, p < 0.001). Concerning the
anatomical parameters, the correlation between a-SS and
LL was equal to that between PI and LL (r = 0.554, p <
0.001, and r = 0.661, p < 0.001, respectively) (Table 2). However, no correlation was observed between TK and
PI, SS, or a-SS. The power analysis of the correlation Fig. 2 Sagittal thoracolumbar spinal parameters. Lumbar lordosis
(LL) was established by the T12 inferior end plate and the S1
superior end plate. Thoracic kyphosis (TK) is measured between the
T1 superior end plate and the T12 inferior end plate Imai et al. Results The
mean values in patients with DDH were as follows: PI,
54.2°; SS, 38.5°; and LL, 55.43°. In normal adults, the
measured values of PI, SS, and LL have been described
to range between 44.6 and 57.7°, 32.5 and 41.4°, and 48.2
and 57.2°, respectively [19, 20]. Formerly, PI had been
reported to be strongly correlated to SS and also to LL
in normal women [8]. Our findings were similar to those
of previous results [13]. Therefore, the results of our
study can be considered valid. showed a power value of 0.803. Intra-rater MADs ranged
from 2.6° for SS to 3.7° for PI, and the smallest ICC was
0.708 for TK (Table 3). As regards the MADs, intra-
rater MADs were slightly smaller than the inter-rater
MADs (the largest MAD was 4.5° for PI), and the smal-
lest ICC was 0.685 for PI (Table 3). Results Journal of Orthopaedic Surgery and Research (2020) 15:486 Page 4 of 6 Table 1 The details of spinopelvic and spinal parameters of the
84 patients with developmental dysplasia of the hip
PI
54.2 ± 10.6 (31.0–77.0°)
SS
38.5 ± 10.8 (10.0–69.0°)
PT
15.7 ± 7.0 (−8.0 to 27.0°)
a-SS
40.8 ± 9.4 (20.0–61.0°)
TK
35.0 ± 10.7 (7.0–83°)
LL
55.4 ± 18.4 (3.0–83.0°)
Mean ± standard deviation (range)
PI Pelvic incidence, SS Sacral slope, PT Pelvic tilt, a-SS Anatomical sacral slope,
TK Thoracic kyphosis, LL Lumbar lordosis Table 1 The details of spinopelvic and spinal parameters of the
84 patients with developmental dysplasia of the hip
PI
54.2 ± 10.6 (31.0–77.0°)
SS
38.5 ± 10.8 (10.0–69.0°)
PT
15.7 ± 7.0 (−8.0 to 27.0°)
a-SS
40.8 ± 9.4 (20.0–61.0°)
TK
35.0 ± 10.7 (7.0–83°)
LL
55.4 ± 18.4 (3.0–83.0°)
Mean ± standard deviation (range)
PI Pelvic incidence, SS Sacral slope, PT Pelvic tilt, a-SS Anatomical sacral slope,
TK Thoracic kyphosis, LL Lumbar lordosis Table 2 Pearson’s correlation coefficients of pelvic and sagittal
spinal parameters Table 2 Pearson’s correlation coefficients of pelvic and sagittal
spinal parameters
SS
PT
a-SS
LL
TK
PI
0.632*
0.341*
0.725*
0.554*
−0.017
SS
−0.229
0.698*
0.827*
0.141
PT
0.128
0.034
−0.068
a-SS
0.661*
0.057
PI Pelvic incidence, SS Sacral slope, PT Pelvic tilt, a-SS Anatomical sacral slope,
TK Thoracic kyphosis, LL Lumbar lordosis
*p < 0.05 Mean ± standard deviation (range)
PI Pelvic incidence, SS Sacral slope, PT Pelvic tilt, a-SS Anatomical sacral slope,
TK Thoracic kyphosis, LL Lumbar lordosis measurements [1]. Consequently, the relationships be-
tween a-SS and LL and between PI and LL were similar
among patients with DDH and normal subjects. The
mean values in patients with DDH were as follows: PI,
54.2°; SS, 38.5°; and LL, 55.43°. In normal adults, the
measured values of PI, SS, and LL have been described
to range between 44.6 and 57.7°, 32.5 and 41.4°, and 48.2
and 57.2°, respectively [19, 20]. Formerly, PI had been
reported to be strongly correlated to SS and also to LL
in normal women [8]. Our findings were similar to those
of previous results [13]. Therefore, the results of our
study can be considered valid. measurements [1]. Consequently, the relationships be-
tween a-SS and LL and between PI and LL were similar
among patients with DDH and normal subjects. Discussion In this study, a strong correlation was observed between
PI and a-SS; consequently, PI was considered feasible of
being estimated from a-SS. Moreover, the correlation
between a-SS and LL was similar to that between PI and
LL (r = 0.661, p < 0.001, and r = 0.554, p < 0.001, re-
spectively). These results were similar to those of a study
in patients with DDH using 3D measurements [13] and
results in normal healthy subjects, obtained by 2D y
PI-LL discrepancy (PI-LL ≥11°) has been described to
likely lead to disability in patients with spinal deformities
[9]. Following spinal fusion surgery for lumbar degenera-
tive diseases, PI-LL discrepancy reportedly leads to re-
sidual symptoms, such as lumbago and other disabilities Fig. 3 The relationship between pelvic incidence and anatomical-sacral slope. Pelvic incidence was strongly related to anatomical-sacral slope Fig. 3 The relationship between pelvic incidence and anatomical-sacral slope. Pelvic incidence was strongly related to anatomical-sacral slope p between pelvic incidence and anatomical-sacral slope. Pelvic incidence was strongly related to anatomical-sacral slope 3 The relationship between pelvic incidence and anatomical-sacral slope. Pelvic incidence was strongly related to anatomica Imai et al. Journal of Orthopaedic Surgery and Research (2020) 15:486 Page 5 of 6 Page 5 of 6 Page 5 of 6 Table 3 Intra- and interrater reliabilities of the measured values
Intra-rater reliability
Inter-rater reliability
PI
3.7 ± 2.8° (0.824*)
4.5 ± 3.6° (0.685*)
SS
2.6 ± 2.2° (0.869*)
3.5 ± 2.8° (0.712*)
PT
2.9 ± 2.8° (0.842*)
4.0 ± 3.2° (0.697*)
a-SS
2.9 ± 2.6° (0.868*)
3.7 ± 2.7° (0.835*)
TK
3.4 ± 3.0° (0.708*)
3.9 ± 3.8° (0.698*)
LL
3.0 ± 2.4° (0.823*)
3.6 ± 3.8° (0.714*)
Mean absolute difference ± standard deviation (intraclass
correlation coefficient)
PI Pelvic incidence, SS Sacral slope, PT Pelvic tilt, a-SS Anatomical sacral slope,
TK Thoracic kyphosis, LL Lumbar lordosis
*p < 0.05 participants. Previously, differences in sagittal thoracol-
umbar spinal and pelvic parameters among races had
been described [23]. Therefore, our findings may be dif-
ferent from those in other races. Further studies are
needed to investigate the generalizability of this result to
other populations. Third, only female patients were in-
cluded. DDH is predominant in females, with a female
to male ratio of 9:1 [24]. Furthermore, < 20 male pa-
tients have undergone periacetabular osteotomy during
the last 10 years in our hospital. Funding Not applicable. Acknowledgements We would like to thank Editage (www.editage.com) for English language
editing and publication support. Discussion Finally, we exclusively
evaluated Crowe type 1 hip dysplasia; however, since the
number of patients with high dislocation was less com-
mon, the influence seems not to be significant. [21, 22]. Therefore, measuring the exact PI is essential. However, measuring PI requires identifying femoral head
centers, which is sometimes arduous, especially in pa-
tients with aspherical femoral heads and with sublux-
ation
following
osteoarthritis
of
the
hip. In
these
patients, a new parameter, independent of the femoral
head, seems to be required. Moreover, placing the ace-
tabular component within a moderate level might be re-
quired to avoid increasing PI after total hip arthroplasty. Authors’ contributions Conceptualization and formulation were done by NI, HS, AS, YH, and NE. Investigation and data collection were done by all the authors. NI carried out
the statistical analysis. The study was performed under supervision of HS and
NE. The authors read and approved the final manuscript. Conclusions
l [21, 22]. Therefore, measuring the exact PI is essential. However, measuring PI requires identifying femoral head
centers, which is sometimes arduous, especially in pa-
tients with aspherical femoral heads and with sublux-
ation
following
osteoarthritis
of
the
hip. In
these
patients, a new parameter, independent of the femoral
head, seems to be required. Moreover, placing the ace-
tabular component within a moderate level might be re-
quired to avoid increasing PI after total hip arthroplasty. In this study, we observed that PI was strongly corre-
lated to a-SS on 2D radiologic measurements. These
findings were similar to those of several previous studies
that described a strong correlation between PI and a-SS
on 3D measurements in patients with DDH [13]. From
these results, PI could be estimated from a-SS using the
following regression formula: PI = 0.8 × a-SS + 21. Moreover, this formula is similar to that used in normal
healthy subjects: PI = 0.8 × a-SS + 18 [1]. Anatomical sacral slope (a-SS), a novel parameter, can
be considered convenient and can be examined by 2D
plain radiographs. Additionally, the correlation between
a-SS and LL was similar to that between PI and LL, and
the 2D and 3D findings were similar in patients with
DDH [13]. Thus, we believe that a-SS, which does not
require the femoral head center for measurement, is a
useful and a new suggested anatomical pelvic parameter
that may be available instead of PI. Further large-scale
studies are required to evaluate the validity and useful-
ness of a-SS as an anatomical parameter. In this study, we observed that PI was strongly corre-
lated to a-SS on 2D radiologic measurements. These
findings were similar to those of several previous studies
that described a strong correlation between PI and a-SS
on 3D measurements in patients with DDH [13]. From
these results, PI could be estimated from a-SS using the
following regression formula: PI = 0.8 × a-SS + 21. Moreover, this formula is similar to that used in normal
healthy subjects: PI = 0.8 × a-SS + 18 [1]. Abbreviations 2D: Two-dimensional; 3D: Three-dimensional; APP: Anterior pelvic plane; a-
SS: Anatomical sacral slope; DDH: Developmental dysplasia of the hip;
ICCs: Interclass correlation coefficients; LL: Lumbar lordosis; MAD: Mean
absolute difference; PI: Pelvic incidence; PT: Pelvic tilt; SS: Sacral slope;
TK: Thoracic kyphosis Additionally, the findings showing a significant correl-
ation between a-SS and LL were similar to those for PI
and LL in patients with DDH and normal healthy sub-
jects [13]. Therefore, a-SS may be useful to estimate PI
in normal healthy subjects and patients with DDH and
may be considered as a new anatomical pelvic parameter
that is independent of the femoral head center for mea-
surements. This is because measurements involving the
femoral head were occasionally unsuitable, such as in
patients with aspherical, flattening, or dislocated femoral
heads. Authors’ information Norio Imai, PhD, Professor of Division of Comprehensive Musculoskeletal
Medicine, Niigata University Graduate School of Medical and Dental Sciences,
Japan. Hayato Suzuki, PhD, Assistant professor of Division of Orthopedic
Surgery, Department of Regenerative and Transplant Medicine, Niigata
University Graduate School of Medical and Dental Sciences, Japan. Additionally, intra-rater and inter-rater MADs of PI,
which included the femoral head center for measure-
ments, were larger than those of a-SS, equal to those in
normal subjects [13]. Therefore, a-SS may lead to higher
reliability
than
PI
when
using
2D
radiological
measurements. Availability of data and materials All data generated or analyzed during this study are included in this
published article. This study had several limitations. First, the sample
size was small; however, the power value in the correl-
ation analysis was 0.803; therefore, the sample size of
this study was considered sufficient by power analysis. Second, this study only included Japanese individuals as Author details
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Graduate School of Medical and Dental Sciences, 1-757, Asahimachi-dori,
Chuo ku, Niigata City, Niigata Prefecture 951-8510, Japan. 2Division of
Orthopedic Surgery, Department of Regenerative and Transplant Medicine,
Niigata University Graduate School of Medical and Dental Sciences, 1-757,
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https://openalex.org/W2950090179
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https://research-information.bris.ac.uk/ws/files/197739850/Published_Article.pdf
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English
| null |
Influence of Fixation Methods on Prosthetic Joint Infection Following Primary Total Knee Replacement: Meta-Analysis of Observational Cohort and Randomised Intervention Studies
|
Journal of clinical medicine
| 2,019
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cc-by
| 10,120
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Kunutsor, S. K., Wylde, V., Whitehouse, M. R., Beswick, A. D.,
Lenguerrand, E., & Blom, A. W. (2019). Influence of Fixation Methods
on Prosthetic Joint Infection Following Primary Total Knee
Replacement: Meta-Analysis of Observational Cohort and
Randomised Intervention Studies. Journal of Clinical Medicine, 8(6),
Article 828. https://doi.org/10.3390/jcm8060828 Publisher's PDF, also known as Version of record
License (if available):
CC BY
Link to published version (if available):
10.3390/jcm8060828 Publisher's PDF, also known as Version of record
License (if available):
CC BY
Link to published version (if available):
10.3390/jcm8060828 This is the final published version of the article (version of record). It first appeared online via MDPI at
https://www.mdpi.com/2077-0383/8/6/828. Please refer to any applicable terms of use of the publisher. Received: 21 May 2019; Accepted: 6 June 2019; Published: 11 June 2019 Abstract: The type of fixation used in primary total knee replacement (TKR) may influence the risk
of prosthetic joint infection (PJI). We conducted a systematic review and meta-analysis to assess
published evidence linking type of fixation (cemented, uncemented, or hybrid) with the risk of
PJI following primary TKR. Randomised controlled trials (RCTs) and observational cohort studies
comparing fixation methods and reporting PJI incidence following primary TKR were identified
in MEDLINE, Embase, Web of Science, and Cochrane Library up until November 2018. Summary
measures were relative risks (RR) with 95% confidence intervals (CIs). We identified 32 eligible articles
(24 observational studies and 8 RCTs) involving 1,161,292 TKRs. In pooled analysis of observational
studies, uncemented fixation was associated with a decreased overall PJI risk when compared with
cemented fixation at 0.76 (0.64–0.89). Comparing antibiotic-loaded cemented fixation with plain
cement, there was no significant difference in overall PJI risk at 0.95 (0.69–1.31), but PJI risk was
increased in the first 6-month postoperative period to 1.65 (1.12–2.43). Limited data from RCTs showed
no differences in PJI risk among the fixation types. Observational evidence suggests uncemented
fixation may be associated with lower PJI risk in primary TKR when compared with cemented
fixation. In the early postoperative period, antibiotic-loaded cemented fixation may be associated with
increased PJI risk when compared with plain cement. This may either reflect appropriate selection of
higher risk patients for the development of PJI to cemented and antibiotic-loaded cement or may
reflect a lower PJI risk in uncemented TKR due to factors such as shorter operative time. Keywords: fixation; cemented; uncemented; hybrid; antibiotic-loaded cement; prosthetic joint
infection; total knee replacement; meta-analysis University of Bristol – Bristol Research Portal
General rights This document is made available in accordance with publisher policies. Please cite only the
published version using the reference above. Full terms of use are available:
http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ Journal of
Clinical Medicine Journal of
Clinical Medicine Setor K. Kunutsor 1,2,*, Vikki Wylde 1,2, Michael R. Whitehouse 1,2
, Andrew D. Beswick 2,
Erik Lenguerrand 2 and Ashley W. Blom 1,2 1
National Institute for Health Research Bristol Biomedical Research Centre, University Hospitals Bristol NHS
Foundation Trust and University of Bristol, Bristol BS8 2BN, UK; V.Wylde@bristol.ac.uk (V.W.);
Michael.Whitehouse@bristol.ac.uk (M.R.W.); Ashley.Blom@bristol.ac.uk (A.W.B.) National Institute for Health Research Bristol Biomedical Research Centre, University Hospitals Bristol NHS
Foundation Trust and University of Bristol, Bristol BS8 2BN, UK; V.Wylde@bristol.ac.uk (V.W.); y
p
Foundation Trust and University of Bristol, Bristol BS8 2BN, UK; V.Wylde@bristol.ac.uk (V.W.);
Michael.Whitehouse@bristol.ac.uk (M.R.W.); Ashley.Blom@bristol.ac.uk (A.W.B.)
2
Translational Health Sciences, Bristol Medical School, Musculoskeletal Research Unit, University of Bristol,
Learning & Research Building (Level 1), Southmead Hospital, Bristol BS10 5NB, UK;
Andy.Beswick@bristol.ac.uk (A.D.B.); Erik.Lenguerrand@bristol.ac.uk (E.L.)
*
Correspondence: setor.kunutsor@bristol.ac.uk; Tel.: +44-7539589186 (
)
y
(
)
2
Translational Health Sciences, Bristol Medical School, Musculoskeletal Research Unit, University of Bristol,
Learning & Research Building (Level 1), Southmead Hospital, Bristol BS10 5NB, UK;
Andy.Beswick@bristol.ac.uk (A.D.B.); Erik.Lenguerrand@bristol.ac.uk (E.L.) y
g
*
Correspondence: setor.kunutsor@bristol.ac.uk; Tel.: +44-7539589186 Received: 21 May 2019; Accepted: 6 June 2019; Published: 11 June 2019 Influence of Fixation Methods on Prosthetic Joint
Infection Following Primary Total Knee Replacement:
Meta-Analysis of Observational Cohort and
Randomised Intervention Studies Setor K. Kunutsor 1,2,*, Vikki Wylde 1,2, Michael R. Whitehouse 1,2
, Andrew D. Beswick 2,
Erik Lenguerrand 2 and Ashley W. Blom 1,2 Setor K. Kunutsor 1,2,*, Vikki Wylde 1,2, Michael R. Whitehouse 1,2
, Andrew D. Beswick 2,
Erik Lenguerrand 2 and Ashley W. Blom 1,2 1. Introduction Total knee replacement (TKR) is one of the most common elective surgical procedures performed
worldwide. In 2017 alone, 102,777 TKRs were performed in England, Wales, Northern Ireland, and the
Isle of Man, as recorded in the National Joint Registry (NJR) [1]. In a primary TKR, the knee implants
(femoral and tibial components) may be secured to the bone with (cemented) or without (uncemented)
bone cement (i.e., type of fixation). The TKR construct fixation is referred to as cemented if the femoral J. Clin. Med. 2019, 8, 828; doi:10.3390/jcm8060828 www.mdpi.com/journal/jcm www.mdpi.com/journal/jcm J. Clin. Med. 2019, 8, 828 2 of 15 and tibial implants are bonded to the bone using cement; in uncemented fixation, the femoral and
tibial implants use press-fit into the bone for initial stability and then bone ingrowth into coatings on
the structure of the implant without cement; in hybrid fixation, there is a mixture of fixations, with one
of the implants being cemented and one being uncemented. Although TKR is often a successful intervention for alleviating pain and improving function
in joint disease, such as osteoarthritis [2], some patients experience complications, such as aseptic
loosening, prosthetic joint infection (PJI), chronic pain, instability, malalignment, and wear [1]. PJI is a
rare but dreaded complication of TKR; it affects between 0.4–1.5% of primary TKRs [3]. PJI and its
management has devastating effects on patients [4] and it is associated with significant morbidity [5–7],
as well as with high healthcare costs [8,9]. With increasing life expectancy and number of people who
will be affected by osteoarthritis, there will be a rise in the numbers of TKRs and the number of patients
affected by PJI is also expected to increase in a proportionate manner [10,11]. In England and Wales,
over a thousand revision operations are performed annually due to PJI of the knee [12]. p
p
y
Patient-, surgery-, and health-system-related factors influence the risk of developing PJI following
a knee replacement [13,14]. Whether surgery-related factors such as fixation methods influence the risk
of developing a PJI following total joint replacement has been a controversial issue, as the evidence has
been inconsistent. In a recent review, our group showed that compared with other fixation methods,
uncemented and antibiotic-loaded cemented fixations carry the lowest risk for PJI following total hip
replacement (THR) [15]. Data on whether fixation methods affect PJI rates differentially following TKR
remains uncertain, as the literature is conflicting. 2.1. Data sources and Search Strategy This review was conducted according to PRISMA and MOOSE guidelines [16,17] (Tables S1 and
S2) and was based on a pre-defined protocol, which has been registered in the PROSPERO International
prospective register of systematic reviews (CRD42018114592). We searched MEDLINE, Embase,
and The Cochrane Library for studies comparing two or more of the following fixation types: cemented,
uncemented, and hybrid, and reported PJI outcomes after primary TKR from inception to November
2018. The computer-based searches combined free and MeSH search terms and combinations of
keywords related to the target population (e.g., “total knee replacement”, “total knee arthroplasty”,
“total joint replacement”), the intervention (e.g., “fixation”, “cemented”, “uncemented”, “cementless”,
“hybrid”), and outcome (e.g., “prosthetic joint infection”, “deep infection”, “infection”). The search was
limited to human studies with no restrictions on language. The detailed search strategy is reported in
Table S3. All titles and abstracts of studies retrieved from the databases were initially screened to assess
their suitability for inclusion. Full-text evaluation of articles potentially meeting eligibility criteria was
conducted independently by two authors (S.K.K. and V.W.) for study selection. Any disagreements
regarding eligibility of an article were discussed and consensus was reached with involvement of a
third author (M.R.W) when necessary. Reference lists of eligible articles and relevant review articles
were manually scanned for additional studies not identified by our original search. Citations of key
studies were checked in Web of Science. 1. Introduction In this context, we aimed to evaluate the body of
evidence linking cemented, uncemented, and hybrid fixation methods with the risk of PJI following
primary TKR, using a systematic review and meta-analysis of both observational and randomised
trial evidence. Our specific objectives were: (i) to compare the nature and magnitude of potential
associations of different fixation methods with risk of PJI; and (ii) to assess if the associations varied by
study and individual level characteristics. 2.3. Data Extraction and Quality Assessment One reviewer (S.K.K.) initially conducted the data extraction using a standardised data collection
form. A second reviewer (V.W.) independently checked the extracted data with that in the original
articles. Data on the following were extracted: first author’s name, study publication date, country and
geographical location of study, study design, baseline year, mean age, duration of follow-up, sample
size, intervention and control, number of PJI outcomes, risk estimates (relative risks (RRs), hazard
ratios (HRs), or odds ratios (ORs)), and degree of covariate adjustment (univariable or multivariable). We assessed the methodological quality of observational studies using the Newcastle-Ottawa Scale
(NOS) [18]. This scale is used for assessing the quality of non-randomised studies and uses a star system
that rates the quality of evidence from a score of zero to nine, based on three domains: selection of
participants; comparability of study groups; and ascertainment of outcomes of interest. The Cochrane
Collaboration’s risk of bias tool was used to assess the quality of RCTs [19]. 2.2. Eligibility Criteria Studies were eligible if they were comparative observational cohort designs, case-control designs,
or randomised controlled trials (RCTs) that: (i) recruited participants undergoing primary TKR; 3 of 15 J. Clin. Med. 2019, 8, 828 (ii) compared any two or more of the following fixation types: cemented, uncemented, and hybrid
fixation; and (ii) reported PJI outcomes after a period of follow-up following primary TKR. No
restrictions were imposed on the follow-up duration. We excluded the following studies: (i) the
intervention was based on only revision TKR; (ii) studies of only bilateral TKR or studies reporting
paired fixations (e.g., cemented and uncemented TKR in the same patient); (iii) compared fixation
methods of only one component (tibial or femoral) and did not provide information on the fixation
type of the other component; (iv) and those conducted in selected populations (e.g., patients with
diabetes only). 2.4. Data Synthesis and Analysis The risk ratios, expressed as RRs with 95% confidence intervals (CIs), were used as the summary
measures of association across studies. Since PJI is considered a rare outcome, reported HRs and ORs
were assumed to approximate the same measure of RR following Cornfield’s rare disease outcome
assumption [20]. Fully multivariable adjusted risk estimates were used when reported, otherwise
crude RRs were calculated from studies that reported raw counts for intervention and control arms. To minimize the effect of heterogeneity, the inverse variance-weighted method was used to pool RRs
using random-effects models. We reported RRs of the associations for the overall duration of follow-up. Sub-analyses were also conducted for specific post-operative periods (e.g., first 3–6 months of follow-up)
for studies that reported these data. Heterogeneity across studies was assessed using the Cochrane
χ2 statistic and the I2 statistic [21]. We explored sources of heterogeneity and assessed for interactions
on the associations by pre-defined study-level characteristics using stratified analyses and univariable
meta-regression [22]. For pooled analysis involving 10 or more studies, publication bias was assessed by
visually inspecting a funnel plot and applying Egger’s regression symmetry test bias [23]. We also adjusted
for the effect of publication bias by the use of the Duval and Tweedie’s nonparametric trim-and-fill method,
which imputes hypothetical small missing null or negative studies [24]. All statistical analyses were
performed with Stata release 15 (StataCorp, College Station, TX, USA). 3.1. Study Identification and Selection The literature search strategy, manual scanning of reference lists, and citation check of Web of
Science identified 665 potentially relevant articles. After the initial screening of titles and abstracts,
51 articles remained for full text evaluation. Following detailed full text evaluation, 19 articles were
excluded because: (i) the outcome was not relevant (n = 8); (ii) intervention was not relevant (n = 7);
(iii) population was not relevant (n = 2); and (iv) duplicate studies (n = 2). The remaining 32 articles
based on 24 unique observational cohort studies and eight RCTs met the inclusion criteria and were
included in the review (Figure 1; Table 1; Table S4). 4 of 15 J. Clin. Med. 2019, 8, 828 Table 1. Characteristics of studies included in review. Author,
Year of
Publication
Year of
Study
Country
Indication for Total Hip
Replacement
Average
Age
(Years)
Design, Source of
Data
Fixation Types
Compared
Mean/Median
Follow-Up
Duration, Years
No. of
Participants/
Knees
Infection Outcome
Reported (Definition)
No. of
PJIs
Study
Quality
Wilson, 1990
1973–1987
U.S. NR
NR
Observational
cohort, Hospital
Uncemented,
cemented, hybrid
Up to 6.0 years
4171
Deep infection (Purulent
material obtained from
joint and positive
bacterial culture)
67
4
Duffy, 1998
1985–1987
U.S. Uncemented (OA 76.4%; RA
16.4%; PTA 5.5%; ankylosing
spondylitis 1.8%); Cemented
(OA 82.4%; RA 11.8%; ancient
sepsis 3.9%; osteonecrosis 2.0%)
59.6
Retrospective cohort
Uncemented,
cemented
10.0
106
Revision for infection
(NR)
1
4
McCaskie,
1998
1987–1990
U.K. Cemented (OA 84%);
Uncemented (OA 86%)
68.8–70.2
RCT, Hospital
Uncemented,
cemented
5.0
113
Infection (NR)
1
NA
Pecina, 2000
1985–1991
Croatia
OA 68.3%; RA 31.7%
61.0
Observational
cohort
Uncemented,
cemented, hybrid
7.3
142
Revision for infection
(NR)
5
5
Eveillard,
2003
1995–1999
France
NR
NR
Observational
cohort, Hospital
Antibiotic loaded
cement, plain
cement
At least 1 year
167
Infection (Isolation of
organisms from tissue
sample; confirmed by
surgeon)
9
5
Baker, 2007
1987–1997
U.K. 3.1. Study Identification and Selection OA 91.4%; RA 7.6%; other 1.0%
70.5
RCT
Uncemented,
cemented
8.7–8.9
396
Revision for infection
(NR)
11
NA
Beaupre,
2007
1996–2000
Canada
Non-inflammatory arthritis 100%
63.4
RCT
Uncemented, hybrid
5.0
81
Infection (NR)
6
NA
Jamsen, 2009
1997–2004
Finland
Primary OA 87.9%; secondary
OA 2.7%; RA 7.6%; other arthritis
1.0%; other 0.8%
71.0
Retrospective cohort,
FAR and FHDR
Uncemented,
cemented, hybrid
3.1
40,135
Revision for infection
(NR)
387
7
Dowsey, 2009
1998–2005
Australia
OA 91.8%; RA 7.8%;
osteonecrosis 0.2%; trauma 0.2%
72.0
Retrospective cohort,
Institutional
database
Antibiotic loaded
cement, plain
cement
1.0
1214
PJI (CDC criteria)
18
6
Ghandi, 2009
1998–2006
Canada
Primary or secondary OA; RA
66.1
Retrospective cohort,
Hospital
Antibiotic loaded
cement, plain
cement
1.0
1625
Deep infection
(CDC criteria)
43
5
Namba, 2009
2003–2007
U.S. OA 92.4%; other 7.6%
68.0
Retrospective cohort,
community-based
registry
Antibiotic loaded
cement, plain
cement
NR
22,889
Deep infection
(CDC criteria)
182
8
Demey, 2011
2004–2005
France
OA (96.9%);
chondrocalcinosis (3.1%)
72.3
RCT
Hybrid, cemented
2.7–2.8
130
Deep infection (NR)
1
NA
Namba, 2013
2001–2009
U.S. OA 96.8%; PTA 1.2%; RA 2.2%;
osteonecrosis 0.4%; other 0.9%
67.4
Retrospective cohort,
Registry
Antibiotic loaded
cement, plain
cement
NR
56,216
Deep SSI (CDC criteria)
404
8
Lass, 2013
2003–2007
Austria
Idiopathic arthritis 88.3%; PTA
5.0%; RA 3.3%; avascular
necrosis 0.8%
66.9
Observational
cohort
Uncemented, hybrid
5.0
120
Revision for infection
(NR)
1
5 Table 1. Characteristics of studies included in review. 5 of 15 J. Clin. Med. 2019, 8, 828 Table 1. Cont. Table 1. Cont. Author,
Year of
Publication
Year of
Study
Country
Indication for Total Hip
Replacement
Average
Age (Years)
Design, Source of
Data
Fixation Types
Compared
Mean/Median
Follow-Up
Duration, Years
No. of
Participants/
Knees
Infection Outcome
Reported (Definition)
No. of
PJIs
Study
Quality
Pelt, 2013
NR
U.S. Hybrid (OA 95%; RA 2%; PTA
3%; other 0%); Cemented (OA
90%; RA 7%; PTA 2%; other 1%)
59.3–65.9
Observational
cohort
Hybrid, cemented
3.2–4.1
304
Revision for sepsis (NR)
5
5
Hinarejos,
2013
2005–2010
Spain
NR
75.9
RCT
Antibiotic loaded
cement, plain
cement
3.2
2948
Deep and superficial
infection (CDC criteria)
85
NA
Qadir, 2014
2000–2010
U.S. NR
68.1
Retrospective cohort,
Institutional registry
Antibiotic loaded
cement, plain
cement
1.0
2511
Infection (CDC criteria)
17
6
Gutowski,
2014
2000–2002;
2004–2007
U.S. 3.1. Study Identification and Selection NR
65.8
Retrospective cohort,
Hospital
Antibiotic loaded
cement, plain
cement
Over a 3.0-year
period
7878
PJI (MSIS criteria)
63
5
Bohm, 2014
2003–2008
Canada
OA 100%
70.0
Retrospective cohort,
CIHI and CJRR
Antibiotic loaded
cement, plain
cement
2.0
36,681
Revision for infection
(NR)
36
6
Choy, 2014
2002–2004
Korea
OA 100%
67.8
RCT
Uncemented, hybrid
9.5
168
SSI (NR)
2
NA
Lizaur-Utrilla,
2014
1999–2007
Spain
OA (92.5%); PTA (7.5%)
51.7
RCT
Uncemented, hybrid
7.1
93
Deep wound infection
(NR)
1
NA
Petursson,
2015
1999–2012
Norway
Primary OA 90%; other 10%
69.0
Observational
cohort, NAR
Hybrid, cemented
11.0
24,680
Revision for infection
(NR)
217
7
Wang, 2015
2003–2012
China
OA 87.8%; other 12.2%
64.8
Retrospective cohort,
Hospital
Antibiotic loaded
cement, plain
cement
1.0
2293
Deep infection (CDC
criteria)
10
6
Fricka, 2015
2010–2012
U.S. NR
58.6–60.2
RCT
Uncemented,
cemented
2.0
100
PJI (NR)
1
NA
Tayton, 2016
1999–2012
New
Zealand
OA 95%; AVN 0.3%; Trauma
1.2%; RA 3.4%; other 0.2%
<55 to >75 *
Prospective cohort,
New Zealand Joint
Registry
Antibiotic-loaded
cement, plain
cement
1.0
64,566
Revision for infection
(NR)
179
7
Wu, 2016
2009–2013
Taiwan
OA, RA, PTA
69.7
Retrospective cohort
Antibiotic-loaded
cement, plain
cement
1.0–5.0
3152
SSI (CDC criteria)
48
6
Prudhon,
2017
2003–2006
France
OA 88.5%; post-traumatic OA
3.0%; RA 4.5%; patellofemoral
OA 4.0%
73.0
Observational
cohort
Uncemented,
cemented
12.1–13.7
200
Infection (NR)
1
5
Sanz-Ruiz,
2017
2009–2012
Spain
NR
76.1–76.4
Prospective cohort
Antibiotic-loaded
cement, plain
cement
2.0 (minimum)
1250
Infection (MSIS criteria)
30
4
Vertullo,
2018
1999–2015
Australia
OA 100%
69.0
Observational
cohort, Registry
Hybrid, cemented
13.0
39,623
Revision for infection
(NR)
215
7 J. Clin. Med. 2019, 8, 828 6 of 15 Table 1. Cont. Author,
Year of
Publication
Year of
Study
Country
Indication for Total Hip
Replacement
Average
Age
(Years)
Design, Source of
Data
Fixation Types
Compared
Mean/Median
Follow-Up
Duration, Years
No. of
Participants/
Knees
Infection Outcome
Reported (Definition)
No. of
PJIs
Study
Quality
Gwam, 2018
2015
U.S. OA 100%
65.8
Retrospective cohort,
NIS database
Uncemented,
cemented
NR
167,930
SSI (NR)
NR
5
Lenguerrand,
2018
2003–2013
U.K. OA (97.3%); other (2.7%)
69.0
Prospective cohort,
Registry
Uncemented,
cemented
4.6
679,010
Revision for infection
(NR)
3227
7
Miller, 2018
2013–2014
U.S. Study Characteristics and Study Quality
3.2. Study Characteristics and Study Quality Study Characteristics and Study Quality
3.2. Study Characteristics and Study Quality Table 1 provides key characteristics of eligible observational cohort studies and RCTs included
he review. Overall, the 32 studies included 1,161,292 TKRs and 5706 PJI outcomes. The 24
ervational cohort studies included 1,157,263 TKRs and 5598 PJI outcomes. Of the 24 studies, 11
e conducted in North America (USA and Canada), eight in Europe (Austria, Croatia, Finland,
nce, Norway, Spain, and the United Kingdom), three in the Pacific region (Australia and New
land), and two in Asia (China and Taiwan). Observational studies were published between 1990
2018. The population sources from which these studies were based included arthroplasty and
mmunity registries, hospitals, and institutional databases. The mean/median baseline age of
icipants ranged from 59 to approximately 76 years. PJI outcomes were reported in a variety of
ys and included revision for infection, deep infection, and surgical site infection (Table 1). Majority
tudies, especially the registry studies, did not provide any detailed information on the diagnoses
JI. For studies reporting the diagnoses of infection, the definitions varied but were mostly based
riteria developed by Centers for Disease Control Prevention (CDC) [25] and the Musculoskeletal
ction Society (MSIS) [26]. For registry studies, a previously published and related study has
cated that reporting of infection as the cause of revision in registry studies reflects the surgeon’s
nion based on clinical information and findings at surgery [27]. The average overall follow-up for
Table 1 provides key characteristics of eligible observational cohort studies and RCTs included in
the review. Overall, the 32 studies included 1,161,292 TKRs and 5706 PJI outcomes. The 24 observational
cohort studies included 1,157,263 TKRs and 5598 PJI outcomes. Of the 24 studies, 11 were conducted
in North America (USA and Canada), eight in Europe (Austria, Croatia, Finland, France, Norway,
Spain, and the United Kingdom), three in the Pacific region (Australia and New Zealand), and two
in Asia (China and Taiwan). Observational studies were published between 1990 and 2018. The
population sources from which these studies were based included arthroplasty and community
registries, hospitals, and institutional databases. The mean/median baseline age of participants ranged
from 59 to approximately 76 years. PJI outcomes were reported in a variety of ways and included
revision for infection, deep infection, and surgical site infection (Table 1). Majority of studies, especially
the registry studies, did not provide any detailed information on the diagnoses of PJI. 3.1. Study Identification and Selection NR
64.4
Institutional
database
Uncemented,
cemented
2.4–5.3
400
Infection (NR)
1
5
Note: *, age range of participants; CDC, Centres for Disease Control Prevention; CIHI, Canadian Institute for Health Information; CJRR, Canadian Joint Replacement Registry; FAR, Finnish
Arthroplasty Register; FHDR, Finnish Hospital Discharge Register; MSIS, Musculoskeletal Infection Society; NA, not applicable; NAR, Norwegian Arthroplasty Register; NIS, National
Inpatient Sample; NR, not reported; OA, osteoarthritis; PJI, prosthetic joint infection; PTA, post-traumatic arthritis; RA, rheumatoid arthritis; RCT, randomised controlled trial; SSI, surgical
site infection. Note: *, age range of participants; CDC, Centres for Disease Control Prevention; CIHI, Canadian Institute for Health Information; CJRR, Canadian Joint Replacement Registry; FAR, Finnish
Arthroplasty Register; FHDR, Finnish Hospital Discharge Register; MSIS, Musculoskeletal Infection Society; NA, not applicable; NAR, Norwegian Arthroplasty Register; NIS, National
Inpatient Sample; NR, not reported; OA, osteoarthritis; PJI, prosthetic joint infection; PTA, post-traumatic arthritis; RA, rheumatoid arthritis; RCT, randomised controlled trial; SSI, surgical
site infection. 7 of 15
17 J. Clin. Med. 2019, 8, 828
n. Med. 2019, 8, x FOR PEE 665 Potentially relevant citations identified
From MEDLINE, EMBASE, Web of Science,
Cochrane Library, and reference list of
relevant studies
614 excluded on the basis of title
and/ or abstract
19 Articles excluded due to:
8 Outcome not relevant
7 Intervention not relevant
2 Population not relevant
2 Duplicate studies
32 Articles included in pooled analysis
Based on 24 observational cohort
studies and 8 RCTs
51 Full-text articles retrieved for more
detailed evaluation
Identification
Screening
Eligibility
Included
Figure 1. PRISMA flow diagram; Note: RCT, randomised controlled trial
Figure 1. PRISMA flow diagram; Note: RCT, randomised controlled trial. 665 Potentially relevant citations identified
From MEDLINE, EMBASE, Web of Science,
Cochrane Library, and reference list of
relevant studies 614 excluded on the basis of title
and/ or abstract 51 Full-text articles retrieved for more
detailed evaluation 32 Articles included in pooled analysis
Based on 24 observational cohort
studies and 8 RCTs ure 1. PRISMA flow diagram; Note: RCT, randomised controlled trial
Figure 1. PRISMA flow diagram; Note: RCT, randomised controlled trial. Study Characteristics and Study Quality
3.2. Study Characteristics and Study Quality 3.3. Fixation Types and PJI Risk Figure 2 reports RRs (95% CIs) for overall PJI, comparing various fixation types for all studies. In observational studies, compared with cemented fixation, uncemented fixation was associated with
a lower risk of PJI (8 studies, 892,094 TKRs, and 4118 PJIs) RR 0.76 (95% CI: 0.64–0.89) (Figure S2). There was no significant evidence of heterogeneity between contributing studies (I2 = 14%; 95% CI:
0–57%; p = 0.318). When the largest study, which was based on the NJR [14], was excluded from
the analysis, the pooled RR was 1.45 (95% CI: 0.85–2.48). There was no significant difference in PJI
risk when hybrid fixation was compared with cemented RR 0.98 (95% CI: 0.80–1.21) or uncemented
fixation RR 0.93 (95% CI: 0.13–6.25) (Figure 2; Figure S3). In pooled analysis of 12 observational
studies (200,442 TKRs, and 1039 PJIs), there was no significant difference in overall PJI risk when
antibiotic–loaded cemented fixation was compared with plain cemented fixation RR 0.95 (95% CI:
0.69–1.31) (Figure 2; Figure S4). There was evidence of significant heterogeneity between contributing
studies (I2 = 71%; 95% CI: 47–84%; p < 0.001), which was partly explained by geographical location
(p for meta-regression < 0.001) and population source (joint registries versus other data sources) (p for
meta-regression = 0.035) (Figure 3). Antibiotic-loaded fixation was associated with decreased PJI risk
in Asian populations, with no difference in risk in other geographical locations. In further analysis
limited to PJI outcomes at 6 months of follow-up in studies providing these data (3 studies, [28–30]
74,955 TKRs, 147 PJIs), antibiotic-loaded cemented fixation was associated with an increased risk of PJI
when compared with plain cemented fixation RR 1.65 (95% CI: 1.12–2.43). There was no evidence of
heterogeneity between contributing studies (I2 = 0%; 95% CI: 0–90%; p = 0.561). In pooled analysis
of 4 studies (48,961 TKRs, 177 PJIs) restricted to PJI diagnosed at 24 or more months of follow-up,
there was no significant difference in PJI risk when antibiotic-loaded cemented fixation was compared
with plain cemented fixation RR 0.73 (95% CI: 0.33–1.63). In RCTs, there was no difference in PJI risk when uncemented fixation was compared with
cemented or hybrid fixation and when hybrid fixation was compared with cemented fixation (Figure 2;
Figure S5). 3.3. Fixation Types and PJI Risk In one trial based on 2948 TKRs and 85 PJIs, [31], there was no difference in PJI risk
when antibiotic-loaded cemented fixation was compared with plain cemented fixation RR 1.22
(95% CI: 0.80–1.86). Study Characteristics and Study Quality
3.2. Study Characteristics and Study Quality For studies
reporting the diagnoses of infection, the definitions varied but were mostly based on criteria developed
by Centers for Disease Control Prevention (CDC) [25] and the Musculoskeletal Infection Society
(MSIS) [26]. For registry studies, a previously published and related study has indicated that reporting
of infection as the cause of revision in registry studies reflects the surgeon’s opinion based on clinical
information and findings at surgery [27]. The average overall follow-up for PJI outcomes ranged from J. Clin. Med. 2019, 8, 828 8 of 15 one year to approximately 14 years. Some studies also reported early postoperative follow-up results
within periods of 30 days to 6 months. The NOS methodological quality of included observational
studies ranged from 4–8. Of the eight RCTs, five were conducted in Europe (France, Spain, and the United Kingdom),
two in North America (Canada and the United States), and one in Asia (Korea). These trials were
published between 1998 and 2015. Altogether, the RCTs comprised 4029 TKRs and 108 PJI outcomes,
with sample sizes ranging from 81 to 2948 TKRs. The average duration of follow-up for PJI outcomes
ranged from 2 to 9.4 years. Using the Cochrane risk of bias tool, all trials demonstrated a high risk of
bias within 1–5 areas of study quality (random sequence generation, allocation concealment, blinding
of participants and personnel, blinding of outcome assessment, and other bias). All trials had a low
risk of bias for incomplete outcome data and selective reporting. Four trials had an unclear risk of bias
in allocation concealment (Figure S1). 3.4. Publication Bias Di
i Univariate
Multivariate
Study quality
> 5
114,678
85,764
189,522
377
662
894
Figure 3. Comparison of all antibiotic-loaded cemented fixation with plain cemented fixation in
primary total knee replacement and the risk of prosthetic joint infection in observational studies,
grouped according to several study characteristics. Note: CI, confidence interval (bars); PJI, prosthetic
joint infection; RR, relative risk; *, p-value for meta-regression. 4 P bli
ti
Bi
Figure 3. Comparison of all antibiotic-loaded cemented fixation with plain cemented fixation in primary
total knee replacement and the risk of prosthetic joint infection in observational studies, grouped
according to several study characteristics. Note: CI, confidence interval (bars); PJI, prosthetic joint
infection; RR, relative risk; *, p-value for meta-regression. 3.4. Publication Bias Fixation types in primary total knee replacement and risk of prosthetic joint infec
observational studies and randomised controlled trials. Note: CI, confidence interval (bar
Figure 2. Fixation types in primary total knee replacement and risk of prosthetic joint infection in
observational studies and randomised controlled trials. Note: CI, confidence interval (bars); PJI,
prosthetic joint infection; RR, relative risk. Figure 2. Fixation types in primary total knee replacement and risk of prosthetic joint infection in
observational studies and randomised controlled trials. Note: CI, confidence interval (bars); PJI,
prosthetic joint infection; RR, relative risk. (
prosthetic joint infection; RR, relative risk. Location
North America
Europe
Asia
Pacific
Population source
Registries
Others
Follow-up (years)
> 1.0
1.0
PJI outcome
Revision for infection
Infection
Statistical adjustment
Univariate
Multivariate
Study quality
> 5
≤ 5
Subgroup
127,800
1417
5445
65,780
184,113
16,329
128,233
72,209
101,247
99,195
114,678
85,764
189,522
10,920
No. of participants/knees
745
39
58
197
848
191
772
267
215
824
377
662
894
145
No. of PJIs
Location
North America
Europe
Asia
Pacific
Population source
Registries
Others
Follow-up (years)
> 1.0
1.0
PJI outcome
Revision for infection
Infection
Statistical adjustment
Univariate
Multivariate
Study quality
> 5
≤ 5
Subgroup
127,800
1417
5445
65,780
184,113
16,329
128,233
72,209
101,247
99,195
114,678
85,764
189,522
10,920
No. of participants/knees
745
39
58
197
848
191
772
267
215
824
377
662
894
145
No. of PJIs
1.31 (0.99, 1.72)
0.33 (0.15, 1.72)
0.36 (0.19, 0.68)
1.17 (0.86, 1.59)
1.25 (0.91, 1.72)
0.65 (0.38, 1.11)
0.91 (0.55, 1.50)
1.03 (0.80, 1.34)
1.36 (0.89, 2.07)
0.82 (0.54, 1.24)
0.90 (0.61, 1.33)
1.01 (0.55, 1.85)
1.13 (0.80, 1.62)
0.63 (0.34, 1.17)
RR (95% CI)
< 0.001
0.035
0.962
0.208
0.636
0.102
P-value*
1
0.15 0.25
0.5
1
2.5
5
RR (95% CI)
Figure 3. Comparison of all antibiotic-loaded cemented fixation with plain cemented fixation in
primary total knee replacement and the risk of prosthetic joint infection in observational studies,
grouped according to several study characteristics. Note: CI, confidence interval (bars); PJI, prosthetic
joint infection; RR, relative risk; *, p-value for meta-regression. 3.4. Publication Bias
Figure 3. Comparison of all antibiotic-loaded cemented fixation with plain cemented fixation in primary
total knee replacement and the risk of prosthetic joint infection in observational studies, grouped
according to several study characteristics. Note: CI, confidence interval (bars); PJI, prosthetic joint
infection; RR, relative risk; *, p-value for meta-regression. 3.4. Publication Bias A funnel plot for the comparison that involved 12 studies (antibiotic-loaded cement vs. plain
cemented fixation) showed visual evidence of publication bias (Figure S6), which was consistent with
Egger’s regression symmetry test (p = 0.038). Using the trim-and-fill method did not impute any
artificial studies into the meta-analysis. 9 of 15 J. Clin. Med. 2019, 8, 828
Clin Med 2019 8
FO lin. Med. 2019, 8, x FOR PEER REVIEW
Observational cohort studies
Uncemented vs cemented
Uncemented vs hybrid
Hybrid vs cemented
Antibiotic-loaded cement vs plain cement
Randomised controlled trials
Uncemented vs cemented
Uncemented vs hybrid
Hybrid vs cemented
Antibiotic-loaded cement vs plain cement
Fixation comparisons
8
2
6
12
3
3
1
1
No. of studies
892,094
4291
109,055
200,442
609
342
130
2948
No of patients/knees
4118
4
892
1039
13
9
1
85
No. of PJIs
0.76 (0.64, 0.89)
1.08 (0.16, 7.51)
0.98 (0.80, 1.21)
0.95 (0.69, 1.31)
0.81 (0.28, 2.36)
0.90 (0.26, 3.11)
3.00 (0.12, 72.32)
1.22 (0.80, 1.86)
RR (95% CI)
1
0.1
0.25
0.751
2.5
5
15 25
50 75
RR (95% CI)
Figure 2. Fixation types in primary total knee replacement and risk of prosthetic joint infectio
observational studies and randomised controlled trials. Note: CI, confidence interval (bars);
Figure 2. Fixation types in primary total knee replacement and risk of prosthetic joint infection in
observational studies and randomised controlled trials. Note: CI, confidence interval (bars); PJI,
prosthetic joint infection; RR, relative risk. J. Clin. Med. 2019, 8, x FOR PEER REVIEW
11 of 17
Observational cohort studies
Uncemented vs cemented
Uncemented vs hybrid
Hybrid vs cemented
Antibiotic-loaded cement vs plain cement
Randomised controlled trials
Uncemented vs cemented
Uncemented vs hybrid
Hybrid vs cemented
Antibiotic-loaded cement vs plain cement
Fixation comparisons
8
2
6
12
3
3
1
1
No. of studies
892,094
4291
109,055
200,442
609
342
130
2948
No of patients/knees
4,118
4
892
1,039
13
9
1
85
No. of PJIs
0.76 (0.64, 0.89)
1.08 (0.16, 7.51)
0.98 (0.80, 1.21)
0.95 (0.69, 1.31)
0.81 (0.28, 2.36)
0.90 (0.26, 3.11)
3.00 (0.12, 72.32)
1.22 (0.80, 1.86)
RR (95% CI)
1
0.1
0.25
0.751
2.5
5
15 25
50 75
RR (95% CI)
Figure 2. Fixation types in primary total knee replacement and risk of prosthetic joint infection in
observational studies and randomised controlled trials. Note: CI, confidence interval (bars); PJI,
prosthetic joint infection; RR, relative risk. Figure 2. 4.2. Comparison with Previous Work To our knowledge, no previous reviews have evaluated the associations of all fixation types with
the risk of PJI following primary TKR; therefore, it is difficult to make a head-to-head comparison in the
context of previously published work beyond the papers included in this analysis. However, a number
of reviews have compared uncemented versus cemented fixations or antibiotic-loaded cemented versus
plain cemented fixations. Two reviews compared cemented fixation with uncemented fixation in terms
of implant survival but did not compare infection outcomes between the fixation methods [32,33]. In a
pooled analysis of five studies, Wang and colleagues showed no difference between cemented and
uncemented fixation with respect to infection [34]. Consistent with our findings, several published
meta-analyses of observational studies and RCTs have also not demonstrated any difference in the
overall incidence of infection between antibiotic-loaded cement and plain cement fixations in primary
TKR [35–37]. However, based on a larger number of studies and more detailed analyses, our review
presents new observational findings which show that uncemented fixations are associated with lower
PJI risk when compared with cemented fixations (albeit on inclusion of the large NJR study [14]) and
the effects of antibiotic-loaded and plain cemented fixations seem to depend on the timing of the
postoperative period following primary TKR, geographical location, and the source of the data. In our
recent review conducted in primary THR patients, we have also shown that uncemented fixations are
associated with lower PJI risk when compared with cemented fixations [15]. Consistent with the data
in THR patients, the evidence from RCTs in knee patients is also limited and inconclusive. p
cemented fixation
E
’
i
4.1. Key Findings 1
.15
.25
.5
1
2.5
5
RR (95% CI)
Figure 3 Comparison of all antibiotic-loaded cemented fixation with plain cemented fixation i
Egger’s regression symmetry test (p = 0.038). Using the trim-and-fill method did not impute any
artificial studies into the meta-analysis. 4 Di
i
Based on a systematic review and meta-analysis of observational and interventional evidence,
we have evaluated the body of evidence linking cemented, uncemented, and hybrid fixation methods 10 of 15 J. Clin. Med. 2019, 8, 828 with the risk of PJI following primary TKR. Pooled evidence from observational studies suggests that
uncemented fixation is associated with lower overall PJI risk when compared with cemented fixation. This reduction in risk, however, lost significance when the largest study [14] was excluded. There were
no differences in PJI risk when hybrid fixation was compared with cemented or uncemented fixation. There was no significant difference in overall PJI risk when antibiotic-loaded cemented fixation was
compared with plain cemented fixation. However, in analysis limited to the first 6 postoperative months
of follow-up, antibiotic-loaded cement was associated with an increased PJI risk when compared
with plain cemented fixation. Subgroup analyses involving the comparison between antibiotic-loaded
cemented and plain cemented fixation showed evidence of effect modification by geographical location
and population source. Antibiotic-loaded cement compared with plain cemented fixations was
associated with decreased PJI risk in Asian populations. However, given the limited number of studies
available for these subgroup analyses, the results need to be interpreted with caution. Finally, limited
data from RCTs showed no differences in PJI risk between fixation types. 4.4. Implications of our Findings Though prosthesis design and materials are constantly evolving, the ideal fixation method for TKR
is still under considerable debate [41], and this is because of surgical preferences and inconsistencies
in clinical outcomes reported. Cemented fixation in TKR has been regarded as the gold standard
for several decades, given the extensive evidence on its good clinical outcomes. Discouraging initial
results for uncemented TKRs [48,49] led to a decline in use. However, with the development of
new materials and prosthetic designs, the use of uncemented fixation is becoming an attractive
option among surgeons [50]. Emerging evidence suggests that modern uncemented knee prostheses
have comparable survivorship and clinical outcomes to cemented prostheses. In addition, cemented
fixations have drawbacks, which include longer surgical time, possibility of thermal osteonecrosis,
and complex revisions in the event of a failure [51]. Uncemented fixation, commonly used in younger
patients and those with good bone quality, is approximately three times more expensive than cemented
fixation; however, they have many advantages, which include shorter operative time, providing
a biologic interface between bone and implant leading to a durable fixation, preservation of bone
stock, reduced risk of cement-related complications, such as third body wear from retained loose
fragments, ease of revision in the event of a failure [41,51], and in addition, a lower risk of PJI. There
is a changing demography in the TKR population, as the population with osteoarthritis is getting
younger [52]. Given the large projected increases in the numbers of TKRs that will be performed [11],
the incidence of PJI is also expected to rise. It appears cemented fixation may be associated with
an increased risk of PJI when compared to uncemented fixation. If equivalent outcomes in terms
of revision for other indications can be achieved with modern uncemented TKR when compared to
cemented TKR, then it may be reasonable to recommend surgeons to use an expensive uncemented
fixation (especially for patients at high risk of revision), as it gives comparable clinical outcomes
to cemented fixation, has several advantages, and is also associated with lower PJI risk. However,
the current evidence suggests a higher overall rate of revision for uncemented TKR compared to
cemented TKR [1]. The efficacy of antibiotic-loaded cement compared with plain cement in decreasing
infection has been demonstrated in studies of primary THR [15]. 4.3. Possible Explanations for Findings Compared to uncemented prostheses, cemented prostheses may cause an increased risk of infection
via a number of pathways. Evidence from studies conducted in THR patients suggest the bone necrosis
caused by direct toxicity or generation of heat during the cemented polymerization process [38] may
create conditions conducive for bacterial growth [39,40]; although in TKR, it is unlikely that the cement
mantle thickness reaches the threshold required to lead to osteonecrosis. Compared to uncemented
TKR, cemented TKR has a longer operating room time [41], which may increase the likelihood of
perioperative contamination [42]. One would expect that antibiotic-loaded bone cement should confer a
lower risk of infection compared with plain bone cement, due to the elution of antibiotics from the bone
cement [43]. However, when the overall evidence was considered, antibiotic-loaded cemented fixations
seemed to be associated with increased PJI risk in the early postoperative period but not at longer-term
follow-up. The elution of antibiotics may only achieve effective concentrations against certain bacteria
or for very short postoperative time periods [43]. Emergence of antimicrobial resistance could be
an explanation; in vitro data suggests that prolonged exposure of micro-organisms to subinhibitory
concentrations of antibiotics promotes mutations that confer resistance [44,45]. Evidence from both
animal and human studies show high rates of antibiotic-resistant infections in antibiotic-loaded 11 of 15 J. Clin. Med. 2019, 8, 828 cement [46,47]. Given that our findings are limited to the observational studies included, it could be
that our observations reflect appropriate selection of low risk patients only to receive plain cement
with higher risk patients receiving antibiotic-loaded cement, with this selection occurring on the
basis of factors not included in, or adjusted for, in the studies included. Other possible explanations
include biases in study designs, such as misdiagnosis of PJI and inability to account for important risk
factors, such as age, sex, and comorbidities, including diabetes mellitus, nature of prostheses, surgical
environment, nature of prior surgical procedures, and other patient factors. 4.4. Implications of our Findings Apart from reasons such as the
development of antibiotic resistance, biases of the study designs, and chance findings, it is difficult to
explain the contrasting findings we have demonstrated in the early postoperative period for TKRs. However, there is a possibility that antibiotic-loaded cement fixation has no effect on the prevention of
PJI after primary TKA. Wang and colleagues, in their combined analysis of 2293 patients and critical
review of seven articles, concluded that antibiotic-loaded bone cement had no effect on PJI prevention
compared with plain cement in primary TKR [53]. The authors recommended that since previous
studies were based on short-term follow-up (12 months), further studies with long-term follow-up
were needed. Even though the evidence base is limited, antibiotic-loaded cement is commonly used for
primary TKR throughout Europe, whereas in the United States it is mostly used for treating revision
for infection in TKR. Recommendations against its use for primary joint surgery in North America are
based on concerns regarding high costs, allergic reactions, toxicity, and antibiotic resistance [29,54–56]. We acknowledge that much of the evidence is based on observational data, which are limited by biases
such as selection bias and reporting, residual confounding, and uncertainty with coding of fixation
types; hence, the results should be interpreted with caution. Definitive RCTs with long-term follow-ups
are warranted to confirm or refute these findings. J. Clin. Med. 2019, 8, 828 12 of 15 12 of 15 4.5. Study Strengths and Limitations We have conducted a comprehensive systematic review and meta-analysis that evaluates the
relationships of cemented, uncemented, and hybrid fixation methods with PJI risk following primary
TKR. We employed a comprehensive search strategy across multiple databases, as well as manual
searches of relevant articles, thereby identifying several additional observational and intervention
studies conducted on the topic. We were able to harmonize the data to a consistent comparison to
enable pooling, and this enhanced interpretation of our findings. In addition, we extracted detailed
data that enabled reporting of estimates for specific time periods, exploration of heterogeneity, and
the assessment of effect modification where possible. Finally, a detailed assessment of the quality of
the included studies (including risk of bias) was conducted using established and validated tools. There were several limitations to the current study, the majority of which were related to the studies
included in the review, and these should be taken into account when interpreting the results. The lack
of reporting or the heterogeneous definition of PJI employed by included studies could have limited
the validity of the findings. In registry studies, it is well known that PJI diagnosis reflects the
clinical judgement of the surgeon and there are issues relating to under-reporting of revision for PJI,
thus yielding potentially lower incidence estimates of PJI [57]. There were a limited number of studies
(<10) for most of the comparisons reported and these precluded assessment of heterogeneity and effect
modification. The large registry study [14] contributed to the beneficial effect seen in uncemented
fixations. Most of the evidence comes from observational study designs, which are unable to directly
prove causation. In addition, the majority of reported risk estimates were confounded as they were
estimated from the raw data. Finally, some of the studies were conducted decades ago and inclusion of
these data do not take into account the evolving nature of prosthetic materials, surgical procedures,
as well as contemporary antibiotic prophylaxis. References Bozic, K.J.; Katz, P.; Cisternas, M.; Ono, L.; Ries, M.D.; Showstack, J. Hospital resource utilization for primary
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R f] [P bM d] 8. Bozic, K.J.; Katz, P.; Cisternas, M.; Ono, L.; Ries, M.D.; Showstack, J. Hospital resource utilization for primary
and revision total hip arthroplasty. J. Bone Jt. Surg. Am. Vol. 2005, 87, 570–576. [CrossRef] [PubMed] 8. Bozic, K.J.; Katz, P.; Cisternas, M.; Ono, L.; Ries, M.D.; Showstack, J. Hospital resource utilization for primary
and revision total hip arthroplasty. J. Bone Jt. Surg. Am. Vol. 2005, 87, 570–576. [CrossRef] [PubMed]
9. Ong, K.L.; Mowat, F.S.; Chan, N.; Lau, E.; Halpern, M.T.; Kurtz, S.M. Economic burden of revision hip and
knee arthroplasty in Medicare enrollees. Clin. Orthop. Relat. Res. 2006, 446, 22–28. [CrossRef] [PubMed]
10
K
t
S M
L
E
O
K
Zh
K
K ll
M
B
i
K J F t
ti
t d
d f
i 9. Ong, K.L.; Mowat, F.S.; Chan, N.; Lau, E.; Halpern, M.T.; Kurtz, S.M. Economic burden of revision hip and
knee arthroplasty in Medicare enrollees. Clin. Orthop. Relat. Res. 2006, 446, 22–28. [CrossRef] [PubMed] 10. Kurtz, S.M.; Lau, E.; Ong, K.; Zhao, K.; Kelly, M.; Bozic, K.J. Future young patient demand for primary
and revision joint replacement: National projections from 2010 to 2030. Clin. Orthop. Relat. Res. 2009, 467,
2606–2612. [CrossRef] [PubMed] 11. Kurtz, S.; Ong, K.; Lau, E.; Mowat, F.; Halpern, M. Projections of primary and revision hip and knee
arthroplasty in the United States from 2005 to 2030. J. Bone Jt. Surg. Am. 2007, 89-A, 780–785. [CrossRef] 12. Lenguerrand, E.; Whitehouse, M.R.; Beswick, A.D.; Toms, A.D.; Porter, M.L.; Blom, A.W.; National Joint
Registry for England, Wales, Northern Ireland and the Isle of Man. 5. Conclusions are members of the National Joint Registry lot 2 contract
(statistical analysis) team. Triathlon total knee replacement. M.R.W. and A.W.B. are members of the National Joint Registry lot 2 contract
(statistical analysis) team. 5. Conclusions Aggregate observational evidence suggests uncemented primary TKR may be associated
with lower PJI risk compared with cemented primary TKR. In the early postoperative period,
antibiotic-loaded cemented fixation may be associated with increased PJI risk when compared
with plain cement. There are no differences in PJI risk when hybrid fixations are compared with
cemented or uncemented fixations. Data from RCTs is limited and uncertain. Supplementary Materials: The following are available online at http://www.mdpi.com/2077-0383/8/6/828/s1,
Table S1: PRISMA checklist, Table S2: MOOSE checklist, Table S3: Literature search strategy, Table S4: Reference list
of included studies, Figure S1: Assessment of risk of bias in randomised controlled trials, Figure S2: Comparison
of all uncemented fixation with cemented fixation and the risk of prosthetic joint infection in observational
studies, Figure S3: Comparison of hybrid fixation with uncemented or all cemented fixations and the risk
of prosthetic joint infection in observational studies, Figure S4: Comparison of antibiotic-loaded cemented
fixations with plain cemented fixations and the risk of prosthetic joint infection in observational studies, Figure S5:
Comparison of uncemented fixation with cemented or hybrid fixations and the risk of prosthetic joint infection in
randomised controlled trials, Figure S6: Assessment of small study effects by funnel plots and Egger’s regression
symmetry tests. Author Contributions: Conceptualization, S.K.K., V.W., M.R.W., A.D.B., E.L. and A.W.B.; data curation, S.K.K.,
V.W., M.R.W., A.D.B., M.R.W. and E.L.; formal analysis, S.K.K.; funding acquisition, A.D.B. and A.W.B.;
investigation, M.R.W.; methodology, S.K.K. and V.W.; supervision, A.W.B.; writing—original draft, S.K.K.;
writing—review & editing, S.K.K., V.W., M.R.W., A.D.B., E.L. and A.W.B. Funding: This article presents independent research funded by the National Institute for Health Research (NIHR)
under its Programme Grants for Applied Research program (RP-PG-1210-12005). This study was supported by
the NIHR Biomedical Research Centre at University Hospitals Bristol NHS Foundation Trust and the University
of Bristol (BRC-1215-20011). The views expressed in this publication are those of the author(s) and not necessarily
those of the NHS, the NIHR, or the Department of Health and Social Care. Conflicts of Interest: M.R.W. undertakes teaching on basic sciences for Orthopaedic trainees preparing for the
Fellowship of the Royal College of Surgeons. His institution receives market rate payment for this teaching from
Heraeus. M.R.W., V.W. and A.W.B. are co-applicants on a grant from Stryker investigating the outcome of the 13 of 15 13 of 15 J. Clin. Med. 2019, 8, 828 Triathlon total knee replacement. M.R.W. and A.W.B. References 1. NJR Steering Commitee. National Joint Registry for England, Wales, Northern Ireland and the Isle of Man: 15th
Annual Report, 2017; National Joint Registry Centre: Hemel Hempstead, UK, 2018. 1. NJR Steering Commitee. National Joint Registry for England, Wales, Northern Ireland and the Isle of Man: 15th
Annual Report, 2017; National Joint Registry Centre: Hemel Hempstead, UK, 2018. 2. Lenguerrand, E.; Wylde, V.; Gooberman-Hill, R.; Sayers, A.; Brunton, L.; Beswick, A.D.; Dieppe, P.; Blom, A.W. Trajectories of Pain and Function after Primary Hip and Knee Arthroplasty: The ADAPT Cohort Study. PLoS ONE 2016, 11, e0149306. [CrossRef] [PubMed] 2. Lenguerrand, E.; Wylde, V.; Gooberman-Hill, R.; Sayers, A.; Brunton, L.; Beswick, A.D.; Dieppe, P.; Blom, A.W. Trajectories of Pain and Function after Primary Hip and Knee Arthroplasty: The ADAPT Cohort Study. PLoS ONE 2016, 11, e0149306. [CrossRef] [PubMed] 3. Schwarz, E.M.; Parvizi, J.; Gehrke, T.; Aiyer, A.; Battenberg, A.; Brown, S.A.; Callaghan, J.J.; Citak, M.;
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impact of periprosthetic knee infection. BMC Musculoskelet. Disord. 2018, 19, 352. [CrossRef] 4. Mallon, C.M.; Gooberman-Hill, R.; Moore, A.J. Infection after knee replacement: A qualitative study of
impact of periprosthetic knee infection. BMC Musculoskelet. Disord. 2018, 19, 352. [CrossRef] 5. Cahill, J.L.; Shadbolt, B.; Scarvell, J.M.; Smith, P.N. Quality of life after infection in total joint replacement. J. Orthop. Surg. 2008, 16, 58–65. [CrossRef] [PubMed] 5. Cahill, J.L.; Shadbolt, B.; Scarvell, J.M.; Smith, P.N. Quality of life after infection in total joint replacement. J. Orthop. Surg. 2008, 16, 58–65. [CrossRef] [PubMed] 6. Andersson, A.E.; Bergh, I.; Karlsson, J.; Nilsson, K. Patients’ experiences of acquiring a deep surgical site
infection: An interview study. Am. J. Infect. Control 2010, 38, 711–717. [CrossRef] [PubMed] 7. Moore, A.J.; Blom, A.W.; Whitehouse, M.R.; Gooberman-Hill, R. Deep prosthetic joint infection: A qualitative
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cement in primary total knee arthroplasty. J. Arthroplast. 2009, 24, 44–47. [CrossRef] [PubMed] 56. Hinarejos, P.; Guirro, P.; Puig-Verdie, L.; Torres-Claramunt, R.; Leal-Blanquet, J.; Sanchez-Soler, J.; Monllau, J.C. Use of antibiotic-loaded cement in total knee arthroplasty. World J. Orthop. 2015, 6, 877–885. [CrossRef] [PubMed] 57. Gundtoft, P.H.; Overgaard, S.; Schonheyder, H.C.; Moller, J.K.; Kjaersgaard-Andersen, P.; Pedersen, A.B. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). References The “true” incidence of surgically treated deep prosthetic joint infection after 32,896 primary total hip
arthroplasties. Acta Orthop. 2015, 86, 326–334. [CrossRef] [PubMed] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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A historical and proteomic analysis of botulinum neurotoxin type/G
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BMC Microbiology
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* Correspondence: jbarr@cdc.gov
† Contributed equally
1Centers for Disease Control and Prevention, National Center for
Environmental Health, Division of Laboratory Sciences, 4770 Buford Hwy, N.
E., Atlanta, GA 30341, USA
Full list of author information is available at the end of the article © 2011 Terilli 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. A historical and proteomic analysis of botulinum
neurotoxin type/G Rebecca R Terilli1,2,3†, Hercules Moura1†, Adrian R Woolfitt1, Jon Rees1, David M Schieltz1 and Abstract Background: Clostridium botulinum is the taxonomic designation for at least six diverse species that produce
botulinum neurotoxins (BoNTs). There are seven known serotypes of BoNTs (/A through/G), all of which are potent
toxins classified as category A bioterrorism agents. BoNT/G is the least studied of the seven serotypes. In an effort
to further characterize the holotoxin and neurotoxin-associated proteins (NAPs), we conducted an in silico and
proteomic analysis of commercial BoNT/G complex. We describe the relative quantification of the proteins present
in the/G complex and confirm our ability to detect the toxin activity in vitro. In addition, we review previous
literature to provide a complete description of the BoNT/G complex. Results: An in-depth comparison of protein sequences indicated that BoNT/G shares the most sequence similarity
with the/B serotype. A temperature-modified Endopep-MS activity assay was successful in the detection of BoNT/G
activity. Gel electrophoresis and in gel digestions, followed by MS/MS analysis of/G complex, revealed the presence
of four proteins in the complexes: neurotoxin (BoNT) and three NAPs–nontoxic-nonhemagglutinin (NTNH) and two
hemagglutinins (HA70 and HA17). Rapid high-temperature in-solution tryptic digestions, coupled with MS/MS
analysis, generated higher than previously reported sequence coverages for all proteins associated with the
complex: BoNT 66%, NTNH 57%, HA70 91%, and HA17 99%. Label-free relative quantification determined that the
complex contains 30% BoNT, 38% NTNH, 28% HA70, and 4% HA17 by weight comparison and 17% BoNT, 23%
NTNH, 42% HA70, and 17% HA17 by molecular comparison. Conclusions: The in silico protein sequence comparisons established that the/G complex is phenetically related to
the other six serotypes of C. botulinum. Proteomic analyses and Endopep-MS confirmed the presence of BoNT and
NAPs, along with the activity of the commercial/G complex. The use of data-independent MSE data analysis,
coupled to label-free quantification software, suggested that the weight ratio BoNT:NAPs is 1:3, whereas the molar
ratio of BoNT:NTNH:HA70:HA17 is 1:1:2:1, within the BoNT/G progenitor toxin. Terilli et al. BMC Microbiology 2011, 11:232
http://www.biomedcentral.com/1471-2180/11/232 Open Access Background agents [2,3]. BoNTs target the Soluble NSF Attachment
Protein Receptors (SNARE) complex of proteins in the
synaptic vesicle and plasma membranes, preventing
acetylcholine from being released causing botulism (Fig-
ure 1) [3]. Seven immunologically distinct BoNT sero-
types (/A through/G) have been described [1,3]. Clostridium botulinum is the taxonomic designation for
at least six diverse species that produce botulinum neu-
rotoxins (BoNTs). This heterologous species is further
classified into six metabolically distinct groups (I-VI). The groups include the toxin-forming strains of C. botu-
linum, C. butyricum, C. baratii, and C. argentinense [1]. C. botulinum is a spore-forming anaerobic bacteria
which produces toxins that are lethal to humans and
animals, and are classified as category A bioterrorism Botulinum neurotoxin G (BoNT/G) is the least stu-
died of the seven serotypes. BoNT/G-producing organ-
isms were first isolated by Gimenez and Ciccarelli in
1969 from soil samples taken from a cornfield in the
Mendoza Province of Argentina [4]. The investigators
indicated that a novel strain of bacterium produced an
antigenically specific, heat-labile botulinum-like toxin
that was not neutralized by any of the known botulinum
antisera. The antitoxin developed using this strain only * Correspondence: jbarr@cdc.gov
† Contributed equally
1Centers for Disease Control and Prevention, National Center for
Environmental Health, Division of Laboratory Sciences, 4770 Buford Hwy, N. E., Atlanta, GA 30341, USA
Full list of author information is available at the end of the article Page 2 of 12 Terilli et al. BMC Microbiology 2011, 11:232
http://www.biomedcentral.com/1471-2180/11/232 Figure 1 Graphical representation of the cell and peptide
targets of Botulinum neurotoxin. 1(A) is a representation of the
Synaptic cleft where BoNT enters the eukaryotic nerve cell. 1(B)
displays the position on the synaptobrevin-2 (VAMP-2) protein
where BoNT/G cleaves, stopping the synaptic vesicle from releasing
acetylcholine, inhibiting nerve impulse and causing muscle paralysis. In a healthy cell, synaptobrevin-2 on the synaptic vesicle must
interact with syntaxin and synaptosomal-associated protein-25
(SNAP-25) on the neuronal membrane for fusion to occur. Fusion
allows the nerve impulse to be delivered across the synaptic
junction. These strains were often reported to cause serological
cross-reactions with type/G producing organisms and
the BoNT/G protein in ELISA and Fluorescence Reso-
nance Energy Transfer (FRET) detection assays [5,8,9]. The C. argentinense species can be distinguished from
other asaccharolytic, proteolytic clostridia by a biochem-
ical test that detects the production of a unique deriva-
tive of indole [5]. Background However, to avoid confusion among
the medical and scientific communities, C. argentinense
type/G producing organisms are still referred to as C. botulinum type/G [7]. Type/G toxin is produced in culture as a relatively
large protein complex (L complex ~500 kDa) consisting
of a neurotoxin (BoNT) and three neurotoxin-associated
proteins (NAPs): two hemagglutinins (HA17 and HA70)
and a nontoxic-nonhemagglutinin (NTNH) component. In addition, there is a gene expression protein (P21) that
is responsible for regulating the expression of the four
complex proteins. P21, however, is not associated with
the toxin complex itself [10,11]. The function of the
NAPs has been shown to protect the neurotoxin in
harsh environments in order to allow the toxin to enter
the synaptic membrane. Once inside the vesicle, the
toxin can cleave its specific SNARE complex protein
[3,12]. BoNT/G is known to cleave the Synaptobrevin
protein (VAMP-2) in the SNARE complex (Figure 1B). It is the only toxin known to cleave at a single Ala81-
Ala82 peptide bond [13] (Table 1). Figure 1 Graphical representation of the cell and peptide
targets of Botulinum neurotoxin. 1(A) is a representation of the Figure 1 Graphical representation of the cell and peptide
targets of Botulinum neurotoxin. 1(A) is a representation of the
Synaptic cleft where BoNT enters the eukaryotic nerve cell. 1(B)
displays the position on the synaptobrevin-2 (VAMP-2) protein
where BoNT/G cleaves, stopping the synaptic vesicle from releasing
acetylcholine, inhibiting nerve impulse and causing muscle paralysis. In a healthy cell, synaptobrevin-2 on the synaptic vesicle must
interact with syntaxin and synaptosomal-associated protein-25
(SNAP-25) on the neuronal membrane for fusion to occur. Fusion
allows the nerve impulse to be delivered across the synaptic
junction. Figure 1 Graphical representation of the cell and peptide
targets of Botulinum neurotoxin. 1(A) is a representation of the
Synaptic cleft where BoNT enters the eukaryotic nerve cell. 1(B)
displays the position on the synaptobrevin-2 (VAMP-2) protein
where BoNT/G cleaves, stopping the synaptic vesicle from releasing
acetylcholine, inhibiting nerve impulse and causing muscle paralysis. In a healthy cell, synaptobrevin-2 on the synaptic vesicle must
interact with syntaxin and synaptosomal-associated protein-25
(SNAP-25) on the neuronal membrane for fusion to occur. Fusion
allows the nerve impulse to be delivered across the synaptic
junction. Figure 1 Graphical representation of the cell and peptide
targets of Botulinum neurotoxin. 1(A) is a representation of the
Synaptic cleft where BoNT enters the eukaryotic nerve cell. Background 1(B)
displays the position on the synaptobrevin-2 (VAMP-2) protein
where BoNT/G cleaves, stopping the synaptic vesicle from releasing
acetylcholine, inhibiting nerve impulse and causing muscle paralysis. In a healthy cell, synaptobrevin-2 on the synaptic vesicle must
interact with syntaxin and synaptosomal-associated protein-25
(SNAP-25) on the neuronal membrane for fusion to occur. Fusion
allows the nerve impulse to be delivered across the synaptic
junction. Type/G-forming organisms have a relatively low toxi-
genicity, producing only small amounts of toxin in cul-
ture. This characteristic makes it difficult to identify
type/G organisms in the presence of other species [14]. The toxin requires tryptic activation to be successfully
detected in vitro; this requirement is also associated
with toxins produced by non-proteolytic types/B and/F,
as well as all strains of type/E [14]. Regardless of BoNT/
G’s low toxigenicity in vitro, Rhesus monkeys, chickens,
and guinea pigs have demonstrated susceptibility to
non-activated toxin when BoNT/G has been adminis-
tered by various routes [15]. In addition, it has been
reported that the ability to produce BoNT/G can be lost
from toxigenic strains after several culture passages [16]. neutralized its homologous toxin and showed no activity
on any of the other known types of BoNT [4]. Overall,
nine strains of type G producing organisms have been
isolated, two from Argentina and seven from Switzer-
land; none of which have ever been clearly implicated as
the cause of paralytic illness or death in humans or ani-
mals [5]. Type G organisms are historically associated with the
C. botulinum species, because of their ability to produce
botulinum neurotoxin [3,4]. However, it is well known
that botulinal toxin production is a poor parameter on
which to base species identification and that the C. botulinum species is a taxonomic collection of several
distinct species [3,5-7]. Type/G producing organisms are
classified as Clostridium argentinense [5]. This species
includes 12 strains of bacteria from the genus Clostri-
dium: nine toxigenic strains and three non-toxigenic
strains. These strains are genetically and phenotypically
distinct from all other strains of C. botulinum and other
clostridial species [5]. Table 1 Peptide Cleavage Products for BoNT/B and/G. BoNT/B and/G Substrate
Masses
Intact
LSELDDRADALQAGASQFESAAKLKRKYWWKNLK
4025
/B-NT
LSELDDRADALQAGASQ
1759
/B-CT
FESAAKLKRKYWWKNLK
2283
/G-NT
LSELDDRADALQAGASQFESA
2281
/G-CT
AKLKRKYWWKNLK
1762
The predicted cleavage products and the masses of the substrate and product The predicted cleavage products and the masses of the substrate and product
peptides for both/B and/G are shown. The predicted cleavage products and the masses of the substrate and product
peptides for both/B and/G are shown. The substrate peptide was derived from
the human Synaptobrevin-2 (VAMP-2) protein. Note that/B and/G cleave 4
amino acids apart. Background The substrate peptide was derived from Two of the three non-toxigenic strains were once clas-
sified as C. subterminale, and the third as C. hastiforme. Terilli et al. BMC Microbiology 2011, 11:232
http://www.biomedcentral.com/1471-2180/11/232 Page 3 of 12 Terilli et al. BMC Microbiology 2011, 11:232
http://www.biomedcentral.com/1471-2180/11/232 The application of a rapid digestion method, coupled
with nano ultra-pressure liquid chromatography tandem
mass spectrometry (nUPLC-MS/MS), was successful at
obtaining a greater percentage of amino acid sequence
coverage of each protein associated with the/G complex
than was previously reported. In addition, we describe
the characterization and relative quantification of the
proteins present in the/G complex. We also compare
BoNT/G to other BoNT serotypes and discuss the pre-
vious literature reports to provide a complete descrip-
tion of the BoNT/G complex. The loss is thought to occur because the complete
nucleotide sequence of the BoNT/G gene, and the
NAPs, are found on a 81-MDa plasmid and not on the
chromosome [16,17] (Figure 2). Of the seven serotypes,
the BoNT/G nucleotide sequence has the most similar-
ity to that of BoNT/B, as previously described [17]. y
p
y
Although BoNT/G is the least studied serotype of C. botulinum, previous reports have described a digestion
method, two protein detection assays, and an activity
detection assay. Hines et al. were the first to apply a
proteomics approach for BoNT/G. The authors used a
16-hour digestion method, followed by high-pressure
liquid chromatography (HPLC) coupled to mass spectro-
metry (MS). The method returned limited recovery of
peptides and protein sequence coverage. However, it
provided enough information to distinguish the proteins
associated with the BoNT/G complex [18]. Glasby and
Hatheway described the potential use of fluorescent-
antibody reagents to identify C. botulinum type/G pro-
ducing strains, but they encountered cross-reactivity
issues with similar species of non-toxigenic clostridia
[9]. Lewis et al. reported an ELISA BoNT/G protein
detection assay that was able to detect low concentra-
tions of the BoNT/G proteins. The assay, however, also
suffered from issues of cross-reactivity with similar non-
toxigenic Clostridium species [8]. Finally, we have pre-
viously described a mass spectrometry-based activity
detection assay, the Endopep-MS method, which was
developed to detect the activity of BoNTs in vitro
against toxin-specific substrate peptides. This method
was successful at detecting all seven BoNT serotypes
[19]. Amino acid sequence comparisons confirmed BoNT/G
and/B similarity Phenetic analysis of the seven available toxin sequences
compared revealed that BoNT/G was the most similar
to the BoNT/B Okra and the least similar to BoNT/C
Stockholm, with a 58.2% and a 32.9% sequence similar-
ity, respectively (Figure 3A, additional file 1). To deter-
mine the extent to which the/G sequence is shared
among toxins in the/B family,/G was compared with 22
different/B strains, including subtypes of/B1,/B2,/B3,
bivalent (Bv/A and Bv/F), and non-proteolytic/B (np/B). Of the 22 sequences,/G shared the most sequence
homology with the/B2 Prevot 25 NCASE strain, with an
overall 58.9% sequence similarity (additional file 2). In a
focused look at the similarities between/G and the/B2
strain, the individual domains of the toxin proteins were
compared. The percent similarity returned for each
domain were as follows: peptidase (light chain) 60.9%,
translocation (heavy chain) 63.8%, binding N-terminal
(NT) (heavy chain) 55.3%, and binding C-terminal (CT)
(heavy chain) 52.4% (Figure 3B). Additional comparison
of BoNT/G NAPs with the NAPs of the other six sero-
types indicated that not only is the type/G toxin
sequence the most similar to/B, but the NAPs sequences
for both serotypes do as well. The percent similarity
returned for the NAPs were as follows: NTNH 78.3%,
HA70 73.1% and HA17 58.7% (Figure 3C-D, additional
files 3, 4, and 5). Proteomics has been used to study changes after treat-
ment with BoNT/A on suprachiasmatic nucleus [20], on
the thyroarytenoid muscle [21], and of C3 exoenzyme
from C. botulinum [22], but there are very few reports
on the BoNT proteome. In the present report, we detail
proteomics methods that were successfully applied to
the analysis of BoNT/G complex and thus further the
understanding of the serotype. We confirmed the detec-
tion of toxin activity by use of the Endopep-MS method. Figure 2 Schematic of Type G 81 MDa Plasmid. This is a visual display of the order and direction in which the genes within the BoNT/G
complex are associated along the 81 MDa plasmid. NCBI does not have the gene listed under one accession number but rather is split into
two: the NAPs X87972 and the toxin X74162. Figure 2 Schematic of Type G 81 MDa Plasmid. This is a visual display of the order and direction in which the genes within the BoNT/G
complex are associated along the 81 MDa plasmid. Gel LC-MS/MS Analysis identified the four main proteins
within the BoNT complex The results of the Endopep-MS experiments conducted
through use of various dilutions of BoNT/G indicated
that the optimum temperature for/G activity is 42°C,
not 37°C as observed with other BoNT serotypes. Addi-
tionally, the experiments indicated that the toxin is the
most active, or best activated, when first exposed to a
short 10 min pulse at 47°C and then continuously incu-
bated at 42°C for 120 hrs. The detection of the 2281 m/
z (NT) and 1762 m/z (CT) product ions in each experi-
ment confirmed that the lots of commercial toxin used
were active. Six of the 17 gel slices, tryptically digested overnight and
analyzed by use of nLC-MS/MS, returned protein
matches with high sequence coverage and a 99% identity
confidence when searched by use of PLGS v2.3 and vali-
dated with Scaffold v2.1. The four main proteins asso-
ciated with the botulinum neurotoxin complex were
identified in various bands from the gel: BoNT/G,
NTNH, HA70, and HA17 (Figure 4). Amino acid sequence comparisons confirmed BoNT/G
and/B similarity NCBI does not have the gene listed under one accession number but rather is split into
two: the NAPs X87972 and the toxin X74162. Terilli et al. BMC Microbiology 2011, 11:232
http://www.biomedcentral.com/1471-2180/11/232 Page 4 of 12 Figure 3 In-depth protein sequence comparisons of the seven BoNT and NAPs. This figure displays the phenetic grouping of: (A) the seven
serotypes, most common strains, toxin sequences; (B) individually compared toxin domains of/G and the/B2 Prevot strain, the toxin sequence in
the/B family that shares the most similarities with/G; (C) the seven serotypes, most common strains, NTNH sequences; (D) the seven serotypes,
most common strains, HA70 sequences; and (E) the seven serotypes, most common strains, HA17 sequences. Of the seven serotypes,/G shares
the most similarity with the/B serotype. The percent identity shared between each/G and/B protein or domain is highlighted above1. Figure 3 In-depth protein sequence comparisons of the seven BoNT and NAPs. This figure displays the phenetic grouping of: (A) the seven
serotypes, most common strains, toxin sequences; (B) individually compared toxin domains of/G and the/B2 Prevot strain, the toxin sequence in
the/B family that shares the most similarities with/G; (C) the seven serotypes, most common strains, NTNH sequences; (D) the seven serotypes,
most common strains, HA70 sequences; and (E) the seven serotypes, most common strains, HA17 sequences. Of the seven serotypes,/G shares
the most similarity with the/B serotype. The percent identity shared between each/G and/B protein or domain is highlighted above1. Endopep-MS Analysis confirmed toxin activity
Th
l
f h
E d
MS
i Relative quantification of type G toxin and NAPs was
determined by use of MSE The results of the six digests of BoNT/G from both ana-
lytical instruments (QTof-Premier and LTQ-Orbitrap)
were compiled to determine the greatest percent of
sequence coverage of each protein identified: BoNT/G
[NCBI, CAA52275], NTNH [NCBI, CAA61228], HA70
[NCBI, CAA61225], and HA17 [NCBI, CAA61226] (Fig-
ure 5A-D). The percent recovery was determined by
combining all unique peptides identified by both nLC-
MS/MS instruments and calculating the ratio of amino
acids identified vs. total amino acids in the protein
sequence. Label-free relative protein quantification was obtained
for each component of the type G toxin complex (Table
2). When calculated by weight, the BoNT/G complex
contained 30% of toxin, 38% of NTNH, 28% of HA70,
and 4% of HA17. These percentages and nanogram
amounts indicate that the overall weight ratio of BoNT:
NAPs present within the complex is 1:3. The percen-
tages of each molecule present in the complex are as
follows: 17.2% of toxin, 23.1% of NTNH, 42.0% HA70, Page 5 of 12 Terilli et al. BMC Microbiology 2011, 11:232
http://www.biomedcentral.com/1471-2180/11/232 Figure 4 1D SDS-PAGE and in gel digestion analysis of/G
complex. This image depicts the All Blue standard (Bio-Rad, CA)
and the/G complex after staining with GelCode™Blue Safe Protein
Stain (Pierce, IL). The lane of interest was cut into 17 segments,
digested overnight, analyzed on a nanoLC-MS/MS system, and
identified by use of PLGS protein database searching. The proteins
identified were BoNT/G (band 4), NTNH (5); HA70 was identified in
three bands (7, 9, and 13) and HA17 in band 14. general, past analyses [7,10,23] have included a compari-
son at the gene level; this study focuses solely on pro-
tein level. While comparisons of toxin and NTNH
proteins to select serotypes have been previously
described [23], a complete comparison of all/G complex
proteins (toxin, NTNH, HA70, and HA17) with the
other six serotypes has not been previously reported. Phenetic analysis confirmed that the BoNT/G complex
of proteins shared the most similarity with the/B sero-
type (Figure 3C-E), as previously reported [10,23]. To determine the extent of/G’s homology to the/B
toxin serotype, we completed an in-depth comparison of
six/B subtypes, 22 different accession numbers (Figure
3B, additional files 2). The comparison of individual
domains–translocation domain, binding domain NT,
binding domain CT, and peptidase–revealed the area of
the toxin in which/G shares the greatest (translocation
domain) and least (binding domain CT) similarity. Relative quantification of type G toxin and NAPs was
determined by use of MSE Over-
all, each domain compared, between the two toxins, is
greater than 50% similar. This comparison helped to
determine which substrate peptide would be optimal to
test the activity of/G. Although there are no direct indi-
cations that sequence similarity would imply overall
identical functionality, similar sequences would allow
similar crystal structures to form, suggesting similar
functionality [24]. It is currently known that both
BoNT/B and/G cleave the Synaptobrevin protein;/B
cleaves a Gln76-Phe77 bond and/G an Ala81-Ala82 bond
five amino acids downstream (Table 1). Because the
cleavage sites of both toxins are relatively near one
another–thus the similarity of their binding domain
sequences and therefore structures–the same peptide
substrate currently used to test/B activity was used to
test/G activity [19]. Figure 4 1D SDS-PAGE and in gel digestion analysis of/G Figure 4 1D SDS-PAGE and in gel digestion analysis of/G
complex. This image depicts the All Blue standard (Bio-Rad, CA)
and the/G complex after staining with GelCode™Blue Safe Protein
Stain (Pierce, IL). The lane of interest was cut into 17 segments,
digested overnight, analyzed on a nanoLC-MS/MS system, and
identified by use of PLGS protein database searching. The proteins
identified were BoNT/G (band 4), NTNH (5); HA70 was identified in
three bands (7, 9, and 13) and HA17 in band 14. Figure 4 1D SDS-PAGE and in gel digestion analysis of/G
complex. This image depicts the All Blue standard (Bio-Rad, CA)
and the/G complex after staining with GelCode™Blue Safe Protein
Stain (Pierce, IL). The lane of interest was cut into 17 segments,
digested overnight, analyzed on a nanoLC-MS/MS system, and
identified by use of PLGS protein database searching. The proteins
identified were BoNT/G (band 4), NTNH (5); HA70 was identified in
three bands (7, 9, and 13) and HA17 in band 14. and 17.8% HA17. These percentages and femtomole
amounts indicate a 1:1:2:1 BoNT:NTNH:HA70:HA17
ratio, or a 1:4 BoNT:NAPs ratio, of molecules within the
complex. In order to confirm that the commercial BoNT/G com-
plex was active and therefore could be considered analo-
gous to the toxin complex found in clinical samples,
various dilutions of the commercial toxin were tested
using the Endopep-MS method previously described (Fig-
ure 6) [19]. In addition to confirming the toxin’s activity,
the Endopep-MS experiments indicated a new optimum
temperature for/G activity. Relative quantification of type G toxin and NAPs was
determined by use of MSE When reactions were pulsed
at 47°C for 10 min, followed by incubation at 42°C for at
least eight hours–as opposed to 37°C for a minimum of
17 hr–an increase in activity and in the quality of mass
spectra produced was observed. Other serotypes of
BoNT (/C and/D) are often associated with botulism in
animals, avians, equines, and bovines, whose body tem-
peratures are higher than those of humans. BoNT/G has
yet to be associated with botulism in a particular organ-
ism; however, it is possible that/G would be more effec-
tive at causing disease in an organism with a higher body
temperature than that of humans, similar to BoNT/C
and/D. Discussion BoNT/G is the least-studied and the most recently
reported of the seven serotypes produced by C. botuli-
num. Although BoNT/G is associated with a distinct
species and metabolic group, the toxin shares multiple
characteristics with the other six progenitor toxins. The
seven serotypes have similar biochemical and molecular
mechanisms of cell entry and membrane translocation. They cause disease by inhibiting synaptic transmission
as a result of the enzymatic cleavage of the SNARE pro-
tein complex. In the present work, we detail the in silico
comparison of BoNT/G progenitor toxin proteins to the
other six serotypes of C. botulinum, as well as methods
for the digestion, detection, and relative quantification
of BoNT/G and its NAPs. The comparison of the BoNT/G progenitor toxin with
the other six serotypes was completed to determine/G’s
phenotypic relationship with the other BoNTs. In Terilli et al. BMC Microbiology 2011, 11:232
http://www.biomedcentral.com/1471-2180/11/232 Page 6 of 12 Figure 5 Sequence coverage returned from in solution tryptic digests. The four main proteins that are associated with the BoNT/G
complex and the percent of each sequence that was returned after digestion are highlighted above. The percent recovery was determined by
combining all unique peptides returned from two nanoLC-MS/MS instruments and calculated by use of the number of amino acids recovered
vs. total amino acids in the protein sequence. (A) BoNT/G 66% [NCBI, CAA52275] (B) NTNH 57% [NCBI, CAA61228] (C) HA17 99% [NCBI,
CAA61226] (D) HA70 91% [NCBI, CAA61225] Figure 5 Sequence coverage returned from in solution tryptic digests. The four main proteins that are associated with the BoNT/G
complex and the percent of each sequence that was returned after digestion are highlighted above. The percent recovery was determined by
combining all unique peptides returned from two nanoLC-MS/MS instruments and calculated by use of the number of amino acids recovered
vs. total amino acids in the protein sequence. (A) BoNT/G 66% [NCBI, CAA52275] (B) NTNH 57% [NCBI, CAA61228] (C) HA17 99% [NCBI,
CAA61226] (D) HA70 91% [NCBI, CAA61225] complex [11]. HA33, a hemagglutinin component, is not
found on the/G plasmid. The lack of evidence of the
protein’s presence further endorsed the theory that,
unlike the other serotypes, HA33 is not associated with
the/G complex [10]. Two gel slices (Figure 4; #6 and 11)
out of 17 visually had protein but did not return any
identifiable peptides when digested and analyzed. The proteins identified in the/G complex, NCBI accession numbers, and average masses are shown, in addition to the calculated amounts on column, femtomoles
and nanograms, and the percent of each protein, by weight and molarity, within the BoNT complex. Discussion The sequences are listed
in Table 1. *Indicates double charged ion of the intact substrate peptide. allow for identification. The SDS-Page gel and in gel
digestions confirmed visually and analytically which pro-
teins are present in the commercial toxin complex and
allowed us to continue to in solution digestions with
some prior knowledge of which proteins should be
identified. previously been reported. This sequence coverage lends
insight into the complex proteins being studied. A high
percentage of sequence coverage indicates that there are
few PTMs associated with the proteins, as well as no
truncation. The presence of PTMs has been known to
compromise protein identification, and truncated pro-
teins do not function as expected. As anticipated, the same proteins that were identified
with the in gel digestions were also identified in the ana-
lysis of the in solution digestions. The four main com-
plex components– BoNT, NTNH, HA70, and HA17–
were all identified with high confidence, and returned a
large number of peptides. Hines et al. reported the use
of a reduction and alkylation overnight digestion
method that produced sequence coverages of 16% for
BoNT, 10% for NTNH, 38% for HA70, and 49% for
HA17 [18]. The method used in our study allowed the
recovery of more than four times the sequence coverage
for BoNT at 66%, more than five times for NTNH at
57%, and more than double for both HA70 and HA17
at 91% and 99%, respectively. In addition to providing enhanced sequence coverage,
the use of data-independent MSE analysis and label-free
quantification software allowed us to relatively quantify
the amount of each protein present in the BoNT/G
complex (Table 2). This quantification method has the
advantage of being able to provide accurate estimates of
relative protein abundance (often within 30% of the
known values on most identified proteins in a mixture,
without the much more rigorous requirements of tar-
geted protein quantification methods. A percentage of
abundance (by weight and molecules, separately) of each
protein within the complex was determined, as well as
an overall weight ratio of BoNT:NAPs and a molecular
ratio of BoNT:NTNH:HA70:HA17. Analysis of the indi-
vidual proteins within the complex illustrated that the
weight of the toxin (30.4%) is almost equivalent to that
of HA70 (27.8%) and about eight percent less than that
of NTNH (38%); whereas HA17 makes up only a minute
portion of the overall weight at just 3.7%. Discussion This
could be due to a number of factors: the protein was
relatively difficult to digest, there was not a sufficient
amount of protein to digest, the sequence was not pre-
sent in the database used, or post-translational modifica-
tions (PTMs) altered the protein sequence and did not Proteomic strategies and analyses used in this study
were important to help define the characteristics of pro-
teins associated with the BoNT/G complex. The 1D-
SDS PAGE analysis confirmed the presence of the four
expected complex proteins (BoNT, NTNH, HA70, and
HA17), with relatively high sequence coverage for in gel
digestion (Figure 4). As expected the proteins, P21 and
HA33, were not identified. P21, a positive regulator of
gene expression, lies just upstream of NTNH on the
toxin plasmid (Figure 2) [10]. The purpose of P21, in
complex development, is not completely understood and
previous reports have not identified it as part of the/G Table 2 Relative quantification of Type G toxin and NAPs. Protein Description
Accession #
Avg Mass (kDa)
Amount OnColumn
% in the Complex
femtomoles
nanograms
molecules
weight
BoNT/G
CAA52275
149034
110.0
16.4
17.2
30.4
NTNH type G
CAA61228
139083
147.6
20.5
23.1
38.1
HA-70 (III) type G
CAA61225
55791
268.5
14.9
42.0
27.8
HA-17 (II) type G
CAA61226
17372
113.8
1.9
17.8
3.7
The proteins identified in the/G complex, NCBI accession numbers, and average masses are shown, in addition to the calculated amounts on column, femtomoles
and nanograms, and the percent of each protein, by weight and molarity, within the BoNT complex. Table 2 Relative quantification of Type G toxin and NAPs. The proteins identified in the/G complex, NCBI accession numbers, and average masses are shown, in addition to the calculated amounts on column, femtomoles
and nanograms, and the percent of each protein, by weight and molarity, within the BoNT complex. Terilli et al. BMC Microbiology 2011, 11:232
http://www.biomedcentral.com/1471-2180/11/232 Page 7 of 12 Figure 6 Endopep-MS method confirmation of commercial BoNT/G activity. This is a representative spectrum indicating BoNT/G activity on
a specific substrate peptide. 1Intact substrate, 2C-Terminus product mass 1762.9, and 3N-Terminus product mass 2281.8. The sequences are listed
in Table 1. *Indicates double charged ion of the intact substrate peptide. Figure 6 Endopep-MS method confirmation of commercial BoNT/G activity. This is a representative spectrum indicating BoNT/G activity on
a specific substrate peptide. 1Intact substrate, 2C-Terminus product mass 1762.9, and 3N-Terminus product mass 2281.8. Discussion Conversely,
analysis using molecular amounts indicated that the
complex contains an equivalent amount of the toxin,
NTNH, and HA17, whereas HA70 is almost twice as
abundant. The nanogram and femtomole on column
data sets signify a likely overall ratio of 1:3 BoNT:NAPs
weight ratio and a 1:1:2:1 BoNT:NTNH:HA70:HA17
molar ratio. As stated earlier, the function of the NAPs
has been shown to protect the neurotoxin in harsh
environments [12]. Due to this protective ability, in the-
ory, a larger ratio of NAPs:BoNT, ie the greater the
number of molecules of NAPs to BoNT, would protect BoNT complexes are difficult to digest in solution
[18]. This rapid high-temperature digestion method
does not involve reduction and alkylation, unlike classi-
cal methods; instead, it uses an acid labile surfactant to
solubilize the hydrophobic proteins. The increased solu-
bility allows a denatured protein to be more susceptible
to tryptic digestion, thereby increasing the rate of diges-
tion and the number of tryptic peptides produced [25]. It has also been previously reported that the use of high
temperature for a short period of time is the best condi-
tion for the enzymatic activity of trypsin [26]. This BoNT complex digestion method, in addition to
analysis of the samples on two different electrospray
(ESI) MS instruments using data-dependent (DDA) and
data-independent MSE analysis, allowed for the detec-
tion of a greater number of peptides for each protein,
leading to a greater overall sequence coverage than had Terilli et al. BMC Microbiology 2011, 11:232
http://www.biomedcentral.com/1471-2180/11/232 Page 8 of 12 Terilli et al. BMC Microbiology 2011, 11:232
http://www.biomedcentral.com/1471-2180/11/232 MegAlign®–DNA Star Inc; Madison, WI). The align-
ments followed the Clustal W method [28]. We
obtained the toxin protein sequences used for phenetic
analysis of the seven BoNT serotypes, the 22 sequences,
covering six subtypes, of/B toxin family, and the NAPs
(NTNH, HA70 and HA17) of the seven BoNT serotypes
from the NCBI protein database (March 2010). For the
complete listing of all the accession numbers used in
the toxin,/B subtypes, and the NAPs comparison, see
additional files 1, 2, 3, 4, and 5. more effectively the toxin from the acidic environment
of the stomach. This potentially would increase the tox-
in’s effectiveness at penetrating the mucosa of the intes-
tine and entering the blood stream, increasing the
toxin’s chances of entering the synaptic cell and causing
disease. Tryptic Digestion We purchased the BoNT/G complex from C. argenti-
nense strain 89 from Metabiologics (Madison, WI). The company provided the complex at 1 mg/mL in 50
mM sodium citrate buffer, pH 5.5 and quality control
activated. The toxin activity in mouse LD50 or units
(U) of specific toxicity obtained from the provider was
as follows: [3.3-3.6 × 10^6]. We acquired all chemicals
from Sigma-Aldrich (Saint Louis, MO), unless other-
wise stated. Los Alamos National Laboratory (Los Ala-
mos, NM) synthesized the substrate peptide used in
the Endopep-MS assay. The peptide sequence is listed
in Table 1 along with the targeted cleavage products. We followed standard safety handling and decontami-
nation procedures, as described for botulinum neuro-
toxins [27]. We needed only very low toxin amounts
for this work. We lyophilized the individually cut and stored gel slices
for 30 min by use of a Centrivap concentrator (Lab-
conco; Kansas City, MO). We added 10 μL of mass
spectrometry-grade trypsin (Promega; Madison, WI) to
each sample and incubated each sample at room tem-
perature for 5 min. We then added 25 μL of digestion
buffer (50 mM ammonium bicarbonate:1 mM CaCl2) to
each sample and incubated the samples at 37°C
overnight. Post-Digestion We added 5 μL of 0.1% formic acid to the samples for
acidification, followed by 2-3 min of sonication to
release peptides. We then centrifuged the samples at 12,
100 × g for 10 min to remove insoluble material. We
collected the soluble peptide mixtures for nLC-MS/MS
analysis. Discussion Knowledge of the stoichiometry of proteins
within the BoNT complexes would be useful to further
understanding of NAPs significance and toxin potency. Conclusions We have presented a detailed in silico comparison of
the/G complex of proteins to the other six serotypes in
an effort to compare, contrast, and further define the
complex’s relationship relative to the/B serotype and
subtypes within the botulinum toxins. Proteomic ana-
lyses, consisting of gel electrophoresis, in gel and in
solution digestions, and Endopep-MS, confirmed the
presence of BoNT, NTNH, HA70, and HA17 proteins
and the activity of the commercial/G complex. We were
successful at obtaining high sequence coverage for all
four complex proteins by using a rapid, high-tempera-
ture digestion method and analysing with two different
nLC-MS/MS instruments. The efficiency of this method
allowed for a greater recovery of protein sequence and
further insight into the complex proteins. The use of
data-independent MSE data analysis coupled to label-
free quantification software suggested that relative quan-
tification of the proteins within BoNT progenitor toxins
could be determined and would be very informative to
further analysis of C. botulinum potency. Sample Excision We cut the sample lane of interest from a previously
run 1D SDS-PAGE gel into 1 × 2 mm slices–17 slices
total–and stored the slices at -80°C prior to tryptic
digestion. One-dimensional sodium dodecyl sulphate/
polyacrylamide gel electrophoresis (1D SDS-PAGE) We added a 4 μL aliquot of [1 μg/μL] commercial
BoNT/G complex to 2 μL of NuPAGE® LDS sample
buffer and 1 μL NuPAGE® Reducing agent (Invitrogen;
Carlsbad, CA) and reduced it by heating at 70°C for 10
min. We cooled and loaded the sample onto a 4-12%
NuPAGE® Novex® Bis-Tris mini polyacrylamide gel
(Invitrogen) and analyzed it alongside 10 μL of Precision
Plus: All Blue and Kaleidoscope protein pre-stained
molecular weight markers (Bio-Rad, CA). We performed
electrophoresis at 200 V for 35 min, then rinsed the gel
3 × 5 min with dH2O and stained it with GelCode™
Blue Safe Protein Stain (Pierce; Rockford, IL) for 1 hr
before de-staining overnight in dH2O. nLC-MS/MS analysis We carried out all in silico work, including the sequence
alignments, sequence identities, and phylogenetic trees,
using Lasergene software (Protean, EditSeq, and We obtained data by using a nanoAcquity ultra-perfor-
mance liquid chromatography (nUPLC) coupled to a Page 9 of 12 Terilli et al. BMC Microbiology 2011, 11:232
http://www.biomedcentral.com/1471-2180/11/232 QTof-Premier MS system (Waters Corp; Milford, MA). We loaded protein digests onto a capillary reverse phase
Symmetry C18 trapping column and a BEH C18 analyti-
cal column (100 μm I.D. × 100 mm long, 1.7Å packing;
Waters Corp) at a flow rate of 1.2 μL/min. Each sample
was separated by use of a 90 min gradient. The mobile
phase solvents were (solvent A) 0.1% formic acid (FA;
Thermo Scientific; Rockford, IL) in water (Burdick and
Jackson; Muskegon, MI) and (solvent B) 0.1% FA in
acetonitrile (ACN; Burdick and Jackson). The gradient
profile consisted of a ramp from 1%B to 85%B over 82
min, followed by a second ramp to 1%B over 8 min,
with data acquired from 5 to 50 min. We analyzed pep-
tides by nano-electrospray on a QTof-Premier hybrid
tandem mass spectrometer. The QTof used an MSE (or
Protein Expression) method, which involved acquiring
data-independent alternating low- and high-collision
energy scans over the m/z range 50-1990 in 0.6 sec,
along with lockmass data on a separate channel to
obtain accurate mass measurement. use of a trap column. The peptide separation was per-
formed over a 120 minute gradient at a flow rate of 400
nl/min. The mobile phase solvents were: (solvent A)
0.2% FA, 0.005% trifluoroacetic acid (TFA) in water, and
(solvent B) 0.2% FA, 0.005% TFA in ACN. The gradient
was set at 5% B for 5 minutes, followed by a ramp to
30% B over 100 minutes, then a ramp up to 90% B in 5
min and held at 90% B for 2 min before returning to 5%
B in 2 min and re-equilibration at 5% B for 20 min. Peptides were analyzed by nano-electrospray on an LTQ
Orbitrap hybrid tandem mass spectrometer. The mass
spectrometer was programmed to perform data-depen-
dent acquisition by scanning the mass range from m/z
400 to 1600 at a nominal resolution setting of 60, 000
for parent ion acquisition in the Orbitrap. Then, tandem
mass spectra of doubly charged and higher charge state
ions were acquired for the top 10 most intense ions. Endopep-MS analysis of toxin activity In solution Tryptic Digestion for nLC-MS/MS analysis
We completed the tryptic digestions as previously
described [25] with few modifications. In all cases, 5 μg
of commercial BoNT/G complex was digested, ending
with a final digestion volume of 50 μL. All digestions
were initially treated with an acid-labile surfactant (ALS)
and performed at 52°C for 3 min following the addition
of trypsin (Promega; Madison, WI). After acidification,
the samples were centrifuged at 12, 100 × g for 10 min
to remove insoluble material. The soluble peptide mix-
tures were then collected for nLC-MS/MS analysis. Once the method was optimized, the experiment was
repeated three times for two lots of commercial toxin
(six digests total) to confirm that the results were con-
sistent with the proteins that are expected in the toxin
complex. The reactions were performed as described previously [19]
with a few modifications. In all cases, the final reaction
volume was 20 μL; the final concentration of reaction buf-
fer was 0.02 M Hepes (pH 7.4), 10 mM dithiothreitol, 0.2
mM ZnCl2, and 1 mg/mL bovine serum albumin (BSA);
and the final concentration of the peptide substrate was 50
picomles/μL. For all experiments, 2 μL [1 μg/μL] of
BoNT/G complex was diluted with dH2O to various unit
(U) concentrations; 1 μL of each dilution was subsequently
spiked into 20 μL of reaction buffer and incubated at 37°C,
42°C, or 47°C for 10 min, followed by 42°C for 120 hrs. Time points to gauge the progress of the reaction were
taken at 6, 8, 24, 72, and 120 hrs (although in a few cases,
a 96 or 144 hr point was taken as a substitute for 120 hrs). 2 μL of each reaction was mixed with 18 μL of a-cyano-4-
hydroxycinnamic acid (CHCA) matrix and spotted for
analysis by matrix-assisted laser desorption/ionization-
time of flight (MALDI-TOF) MS. MS Acquisition p
nLC-MS/MS analysis The in solution tryptic digests were analysed by use of
two analytical instruments, a QTof-Premier and an
LTQ-Orbitrap (Thermo-Finnigan; San Jose, CA), to help
to improve the overall protein coverage of the BoNT/G
complex. The analyses of digests that used the QTof-
Premier were performed initially as described above in
the GeLC-MS/MS methods section. nLC-MS/MS analysis All
tandem mass spectra were recorded by use of the linear
ion trap. This process cycled continuously throughout
the duration of the gradient. MS Acquisition The Endopep-MS reactions were run on a 4800
MALDI-TOF (Applied Biosystems; Framingham, MA). Mass spectra of each sample well were obtained by
scanning from 1000 to 4400 m/z in MS positive-ion
reflector mode. The instrument uses a Nd:YAG laser at
337 nm with a 200 MHz repetition rate, and each spec-
trum generated was an average of 2400 laser shots. Pre-
ceding each run, the instrument was tuned and
calibrated for accurate MS analysis by use of a mixture
of five peptides: des-Arg1-Bradykinin (m/z 904.47),
angiotensin I (m/z 1, 296.69), Glu1-fibrinopeptide B (m/
z 1, 570.68), ACTH (1-17)(m/z 2093.08), ACTH (18-39)
(m/z 2, 465.20). Additional material Additional file 1: Protein sequence comparisons of toxin from the 7
BoNT serotypes. The seven BoNT serotypes toxin sequences (A-G; most
common strains) were compared and it was determined that the BoNT/B
serotype shared the most sequence similarity to/G. This figure depicts
the percent of identity (top to bottom) and percent of divergence (left
to right) of the protein sequences compared. Identity equals the percent
of similarity the toxin sequences share and divergence the percent of
difference between the toxin sequences. Scaffold (Proteome Software Inc.; Portland, OR; v2.1.03)
was used to validate all MS/MS-based peptide and protein
identifications. Peptide identifications were accepted if
they could be established at greater than 95.0% probability,
as specified by the Peptide Prophet algorithm [29]. Protein
identifications were accepted if they could be established
at greater than 99.0% probability and if they contained at
least two identified peptides. Protein probabilities were
assigned by the Protein Prophet algorithm [30]. Proteins
that contained similar peptides and that could not be dif-
ferentiated on the basis of MS/MS analysis alone were
grouped to satisfy the principles of parsimony. With the
stringent parameters of Peptide Prophet and Protein Pro-
phet, the false discovery rate was zero. Additional file 2: In-depth comparison of BoNT/G and/B subtypes. An in-depth comparison of/G and 22/B strains was completed to
determine how similar/G was to the/B family. This figure depicts the
percent of identity (top to bottom) and percent of divergence (left to
right) of the protein sequences compared. Identity equals the percent of
similarity the toxin sequences share and divergence the percent of
difference between the toxin sequences. Additional file 3: Protein sequence comparisons of NTNH from all 7
BoNT serotypes. The seven NTNH serotype toxin sequences (A-G; most
common strains) were compared to determine which serotype shared
the most sequence similarity to/G. This figure depicts the percent of
identity (top to bottom) and percent of divergence (left to right) of the
protein sequences compared. Identity equals the percent of similarity the
toxin sequences share and divergence the percent of difference between
the toxin sequences. Additional file 4: Protein sequence comparisons of HA70 from all 7
BoNT serotypes. The seven HA70 serotype toxin sequences (A-G; most
common strains) were compared to determine which serotype shared
the most sequence similarity to/G. This figure depicts the percent of
identity (top to bottom) and percent of divergence (left to right) of the
protein sequences compared. nLC-MS/MS and Endopep-MS data processing
nLC-MS/MS data Data obtained from the QTof-Premier were processed by
use of Waters’ ProteinLynx Global Server (PLGS v2.3; Mil-
ford, MA) and searched against a curated C. botulinum
database consisting of 22, 000 NCBI entries, including the
protein standard Alcohol dehydrogenase (ADH, Waters
Corp; Milford, MA) and contaminants such as trypsin. Tandem mass spectra were analyzed by use of the follow-
ing parameters: variable modification of oxidized M, 1%
false positive rate, a minimum of three fragment ions per
peptide and seven fragment ions per protein, a minimum
of 1 peptide match per protein, and with up to two missed
cleavages per peptide allowed. Root mean square mass
accuracies were typically within 8 ppm for the MS data
and within 15 ppm for MS/MS data. Tandem mass spectra, obtained from the LTQ-Orbi-
trap, were extracted by Mascot Distiller (Matrix Science;
London, UK; v2.2.1.0) and subsequently searched by use
of Mascot (Matrix Science; v2.2.0) against a NCBI data-
base consisting of seven million entries. All files gener-
ated by Mascot Distiller were searched with the
following parameters: 200 ppm parent MS ion window,
0.8 Da MSMS ion window, and up to 2 missed cleavages
allowed. Variable modifications for the Mascot searches
were deamidation and oxidation. Endopep-MS data The MS Reflector data, obtained from the Endopep-MS
reactions, were analyzed by hand. A visual comparison
(by an expert researcher) of the intact substrate and its
cleavage products was enough to confirm a positive or
negative reaction. Additional file 5: Protein sequence comparisons of HA17 from all 7
BoNT serotypes. The seven BoNT serotype HA17 sequences (A-G; most
common strains) were compared to determine which serotype shared
the most sequence similarity to/G. This figure depicts the percent of
identity (top to bottom) and percent of divergence (left to right) of the
protein sequences compared. Identity equals the percent of similarity the
toxin sequences share and divergence the percent of difference between
the toxin sequences. LTQ-Orbitrap Data were obtained by use of an Eksigent 2D nanoLC
system (Eksigent Technologies; Dublin, CA) coupled to
an LTQ-Orbitrap tandem mass spectrometer. A 365 μm
O.D. × 75 μm I.D. fused silica pulled needle capillary
(New Objective; Woburn, MA) was packed in house
with 10 cm of 5 μm Symmetry 300 reverse phase pack-
ing material (Waters Corp). The tryptic digests were
loaded directly onto the analytical column without the Terilli et al. BMC Microbiology 2011, 11:232
http://www.biomedcentral.com/1471-2180/11/232 Page 10 of 12 Page 10 of 12 Page 10 of 12 Terilli et al. BMC Microbiology 2011, 11:232
http://www.biomedcentral.com/1471-2180/11/232 Premier operated in data independent acquisition mode
[31,32]. The relative protein quantification of individual
replicates was determined based on the average MS sig-
nals of the three most intense tryptic peptides per pro-
tein, through use of the PLGS IdentityE software. Once
processed, the data sets were exported from PLGS and
clustered according to digestion number for further eva-
luation by use of Excel (Microsoft Corporation, Red-
mond, WA). The femtomole and nanograms on column
values (Table 2) were calculated by averaging the techni-
cal replicates, excluding outliers with 30% or greater
variation. These values were then averaged on the basis
of lot grouping. The lot grouping averaged values were
used to determine a percent by weight, nanograms on
column, and a percent of molecules, femtomole on col-
umn, of each protein within the BoNT/G complex. In
addition, a molar ratio of BoNT:NTNH:HA70:HA17,
and BoNT:NAPs, by weight, was determined. nLC-MS/MS and Endopep-MS data processing
nLC-MS/MS data Additional material Identity equals the percent of similarity the
toxin sequences share and divergence the percent of difference between
the toxin sequences. Author details
1C
f
Di 16. Eklund MW, Poysky FT, Mseitif LM, Strom MS: Evidence for plasmid-
mediated toxin and bacteriocin production in Clostridium botulinum
type G. Appl Environ Microbiol 1988, 54:1405-1408. 1Centers for Disease Control and Prevention, National Center for
Environmental Health, Division of Laboratory Sciences, 4770 Buford Hwy, N. E., Atlanta, GA 30341, USA. 2Association of Public Health Laboratories, 8515
Georgia Avenue, Suite 700, Silver Spring, MD 20910, USA. 3Oak Ridge
Institute for Scientific Education, P.O. Box 117, Oak Ridge, TN 37831, USA. 17. Zhou Y, Sugiyama H, Nakano H, Johnson EA: The genes for the
Clostridium botulinum type G toxin complex are on a plasmid. Infect
Immun 1995, 63:2087-2091. Terilli et al. BMC Microbiology 2011, 11:232
http://www.biomedcentral.com/1471-2180/11/232 Terilli et al. BMC Microbiology 2011, 11:232
http://www.biomedcentral.com/1471-2180/11/232 clostridium botulinum (Clostridium argentinense) type G and
nonproteolytic Clostridium botulinum type B. Curr Microbiol 1997,
35:207-214. Acknowledgements clostridium botulinum (Clostridium argentinense) type G and
nonproteolytic Clostridium botulinum type B. Curr Microbiol 1997,
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Spectrometry Laboratory at the National Center for Environmental Health,
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preparation and in silico proteomics analysis, created dendrograms and
drafted the manuscript. HM initiated the project, conceived the whole study
and experimental design, carried out experiments and contributed to
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Charles PD, Reddy AB, Kyriacou CP, Lilley KS, et al: Proteomic analysis
reveals the role of synaptic vesicle cycling in sustaining the
suprachiasmatic circadian clock. Curr Biol 2009, 19:2031-2036. Received: 3 December 2010 Accepted: 18 October 2011 Received: 3 December 2010 Accepted: 18 October 2011 Published: 18 October 2011 Published: 18 October 2011 21. Welham NV, Marriott G, Tateya I, Bless DM: Proteomic changes in rat
thyroarytenoid muscle induced by botulinum neurotoxin injection. Proteomics 2008, 8:1933-1944. References 1. Hill K, Xie G, Foley B, Smith T, Munk A, Bruce D, Smith L, Brettin T, Detter J:
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indicating cellular changes after treatment of neuronal cells with the C3
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285:1059-1070. 23. Campbell K, Collins MD, East AK: Nucleotide sequence of the gene coding
for Clostridium botulinum (Clostridium argentinense) type G neurotoxin:
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http://www.biomedcentral.com/1471-2180/11/232 31. References Silva J, Denny R, Dorschel C, Gorenstein M, Li G-Z, Richardson K, Wall D,
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Escherichia coli proteome: a sweet tale. Mol Cell Proteomics 2006,
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peptide precursor and product ions from data independent LC-MS with
data dependant LC-MS/MS. Proteomics 2009, 9:1683-1695. doi:10.1186/1471-2180-11-232
Cite this article as: Terilli et al.: A historical and proteomic analysis of
botulinum neurotoxin type/G. BMC Microbiology 2011 11:232. 31. Silva J, Denny R, Dorschel C, Gorenstein M, Li G-Z, Richardson K, Wall D,
Geromanos S: Simultaneous qualitative and quantitative analysis of the
Escherichia coli proteome: a sweet tale. Mol Cell Proteomics 2006,
5:589-607. 32. Geromanos S, Vissers JPC, Silva J, Dorschel C, Li G-Z, Gorenstein M,
Bateman R, Langridge J: The detection, correlation, and comparison of
peptide precursor and product ions from data independent LC-MS with
data dependant LC-MS/MS. Proteomics 2009, 9:1683-1695. doi:10.1186/1471-2180-11-232
Cite this article as: Terilli et al.: A historical and proteomic analysis of
botulinum neurotoxin type/G. BMC Microbiology 2011 11:232. 31. Silva J, Denny R, Dorschel C, Gorenstein M, Li G-Z, Richardson K, Wall D,
Geromanos S: Simultaneous qualitative and quantitative analysis of the
Escherichia coli proteome: a sweet tale. Mol Cell Proteomics 2006,
5:589-607. 32. Geromanos S, Vissers JPC, Silva J, Dorschel C, Li G-Z, Gorenstein M,
Bateman R, Langridge J: The detection, correlation, and comparison of
peptide precursor and product ions from data independent LC-MS with
data dependant LC-MS/MS. Proteomics 2009, 9:1683-1695. doi:10.1186/1471-2180-11-232
Cite this article as: Terilli et al.: A historical and proteomic analysis of
botulinum neurotoxin type/G. BMC Microbiology 2011 11:232. Submit your next manuscript to BioMed Central
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Figure S1
A G0/G1
S
G2/M
Cell proportion (%)
control
irono 100
irono 500
control
irono 100
irono 500
control
irono 100
irono 500
control
irono 100
irono 500
0
50
100
150
DMSO
irono. 50 nM
irono. 100 nM
irono. 500 nM
% viabilty
Days
OCI-AML3
2
4
7
0
50
100
MOLM-13
Days
2
4
7
% viabilty
MV4;11
Days
2
4
7
% viabilty
0
50
100
0
50
100
ns
***
***
***
**
***
***
******
**
*
***
***
***
***
***
******
ns
***
***
***
***
**
******
Number of cells
10⁴
MFI
DMSO
24h irono
48h irono
BODIPY C11
Figure S1
ns
% of PI+ cells
0
20
40
60
MOLM-13
OCI-AML3
RSL-3
ironomycin
Liproxstatin -
- +
- -
-
-
+
+
-
-
-
-
+
-
+
-
+
-
+
+
-
-
+
-
-
-
lysosomal Fe²+
100 nM
10⁵
Number of cells
MFI
DMSO
6h irono
24h irono
Number of cells
10⁵
MFI
500 nM
0
50
100
% of viable cells
(normalized to control)
***
ns
ns
48h
24h
untreated
irono. veneto. Z-VAD
-
+
-
+
-
+
A
0
50
100
Cell proportion (%)
HL-60
OCI-AML3
Cell proportion (%)
0
50
100
G0/G1
S
G2/M
DMSO
DMSO
irono. irono. ***
**
B
+
C
D
E
G
H
ns
LMP
LMP
LMP
2%
3%
37%
32%
5%
5%
25%
90%
95%
PI
Lysotracker
DMSO
24h
48h
ironomycin
PI-/Lyso+
PI-/Lyso- (= LMP)
PI+
F
Z-VAD-fmk
Cell death inhibitors
***
***
ns
-
- +
- -
-
-
+
+
-
-
-
-
+
-
+
-
+
-
+
+
-
-
+
-
-
-
***
***
ns
ns A DMSO
irono. 50 nM
irono. 100 nM
irono. 500 nM
% viabilty
Days
OCI-AML3
2
4
7
0
50
100
MOLM-13
Days
2
4
7
% viabilty
MV4;11
Days
2
4
7
% viabilty
0
50
100
0
50
100
ns
***
***
***
**
***
***
******
**
*
***
***
***
***
***
******
ns
***
***
***
***
**
****** 0
50
100
% of viable cells
(normalized to control)
***
ns
ns
48h
24h
untreated
irono. veneto. Z-VAD
-
+
-
+
-
+
0
50
100
Cell proportion (%)
HL-60
OCI-AML3
Cell proportion (%)
0
50
100
G0/G1
S
G2/M
DMSO
DMSO
irono. irono. Figure S1
A ***
**
B
+
C
Z-VAD-fmk 0
50
100
% of viable cells
(normalized to control)
***
ns
ns
48h
24h
untreated
irono. veneto. Z-VAD
-
+
-
+
-
+
1
+
C
Z-VAD-fmk C B G0/G1
S
G2/M
Z-VAD-fmk
necros. ferros. Cell proportion (%)
DMSO
control
irono 100
irono 500
control
irono 100
irono 500
control
irono 100
irono 500
control
irono 100
irono 500
0
50
100
150
Number of cells
10⁴
MFI
DMSO
24h irono
48h irono
BODIPY C11
ns
% of PI+ cells
0
20
40
60
MOLM-13
OCI-AML3
Ferrostatin-1
RSL-3
ironomycin
Liproxstatin -
- +
- -
-
-
+
+
-
-
+
-
-
-
-
+
+
-
-
-
-
+
-
+
-
+
-
+
+
-
-
+
-
-
-
lysosomal Fe²+
100 nM
10⁵
Number of cells
MFI
DMSO
6h irono
24h irono
Number of cells
10⁵
MFI
500 nM
0
48h
24h
Z-VAD
-
+
-
+
-
+
0
C
C
0
DMSO
DMSO
irono. irono. +
D
E
G
H
ns
LMP
LMP
LMP
2%
3%
37%
32%
5%
5%
25%
90%
95%
PI
Lysotracker
DMSO
24h
48h
ironomycin
PI-/Lyso+
PI-/Lyso- (= LMP)
PI+
F
Cell death inhibitors
***
***
ns
-
- +
- -
-
-
+
+
-
-
+
-
-
-
-
+
+
-
-
-
-
+
-
+
-
+
-
+
+
-
-
+
-
-
-
***
***
ns
ns Number of cells
10⁴
MFI
DMSO
24h irono
48h irono
BODIPY C11
lysosomal Fe²+
100 nM
10⁵
Number of cells
MFI
DMSO
6h irono
24h irono
Number of cells
10⁵
MFI
500 nM
48h
24h
D
D
D
E
LMP
LMP
LMP
2%
3%
37%
32%
5%
5%
25%
90%
95%
PI
Lysotracker
DMSO
24h
48h
ironomycin
PI-/Lyso+
PI-/Lyso- (= LMP)
PI+
F Number of cells
10⁴
MFI
DMSO
24h irono
48h irono
BODIPY C11
LMP
LMP
LMP
2%
3%
37%
32%
5%
5%
25%
90%
95%
PI
Lysotracker
DMSO
24h
48h
ironomycin
PI-/Lyso+
PI-/Lyso- (= LMP)
PI+
F lysosomal Fe²+
100 nM
10⁵
Number of cells
MFI
DMSO
6h irono
24h irono
Number of cells
10⁵
MFI
500 nM
D
E
PI E D F Number of cells PI Lysotracker G0/G1
S
G2/M
Z-VAD-fmk
necros. ferros. Figure S1
A Cell proportion (%)
DMSO
control
irono 100
irono 500
control
irono 100
irono 500
control
irono 100
irono 500
control
irono 100
irono 500
0
50
100
150
H
ns
Cell death inhibitors G0/G1
S
G2/M
Z-VAD-fmk
necros. ferros. Cell proportion (%)
DMSO
control
irono 100
irono 500
control
irono 100
irono 500
control
irono 100
irono 500
control
irono 100
irono 500
0
50
100
150
ns
% of PI+ cells
0
20
40
60
MOLM-13
OCI-AML3
Ferrostatin-1
RSL-3
ironomycin
Liproxstatin -
- +
- -
-
-
+
+
-
-
+
-
-
-
-
+
+
-
-
-
-
+
-
+
-
+
-
+
+
-
-
+
-
-
-
G
H
ns
Cell death inhibitors
***
***
ns
-
- +
- -
-
-
+
+
-
-
+
-
-
-
-
+
+
-
-
-
-
+
-
+
-
+
-
+
+
-
-
+
-
-
-
***
***
ns
ns ns
% of PI+ cells
0
20
40
60
MOLM-13
OCI-AML3
Ferrostatin-1
RSL-3
ironomycin
Liproxstatin -
- +
- -
-
-
+
+
-
-
+
-
-
-
-
+
+
-
-
-
-
+
-
+
-
+
-
+
+
-
-
+
-
-
-
G
***
***
ns
-
- +
- -
-
-
+
+
-
-
+
-
-
-
-
+
+
-
-
-
-
+
-
+
-
+
-
+
+
-
-
+
-
-
-
***
***
ns
ns H G Cell death inhibitors Cell proportion (%) Figure S1| (related to Figure 1). Ironomycin induces potent cell death in acute myeloid
leukaemia through a non-classical cell death pathway. A, Cell viability assessed by FACS
(PI) in OCI-AML3, MOLM-13 and MV4;11 cell lines treated with ironomycin (n=3 biological
replicates, means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001). B, Cell cycle analysis using DAPI
on OCI-AML3 and HL60 cell lines after 24 hours of DMSO or 500 nM (n=3 biological
replicates, means ± SD; **p < 0.01, ***p < 0.001). C, Effect on cell death of the pan-caspase
inhibitor Z-VAD-fmk. MV4;11 cells were pretreated with 50 µM Z-VAD-fmk for 30 minutes
and treated with 500 nM ironomycin or 50 nM venetoclax. Figure S1
A Cell death was assessed by FACS
(PI, n=3 biological replicates, means ± SD, *** indicates p<.0005). D, FACS plots showing
lysosomal Fe2+ using a lysosomal FACS turn-on probe (60) upon ironomycin treatment in
MOLM-13 cells. Fe2+ specifically reduces Rhonox-M to a Rhodamine B derivative, which
fluoresces. One representative experiment is shown. E, FACS plot showing lysosomal
membrane permeabilization (LMP) using lysotracker (Lyso) in MOLM-13 cell line. We treated
cells with DMSO or 500 nM ironomycin. LMP is associated with a loss of lysotracker staining. We show one single representative experiment F, FACS plots showing lipid peroxidation using
BODIPY C11 staining at 24 and 48 hours of 500 nM ironomycin treatment and DMSO control
(48 h) in MOLM-13 cells. One representative experiment is shown. G, Effect on cell viability
of the ferroptosis inhibitors ferrostatin-1 or liproxstatin. MOLM-13 cell line was pretreated with
20 µM ferrostatin-1 or 10 µM liproxstatin for 30 minutes and treated with 500 nM ironomycin
or 30 nM RSL-3 for the indicated times. Cell death (PI positive cells) was assessed by FACS
staining (n=3 biological replicates, means ± SD, ***p < 0.001). H, Cell cycle analysis using
DAPI staining by FACS in AML cell lines without treatment or after 24 hours of 500 nM
ironomycin. Cells were pretreated with 50 µM Z-VAD-fmk, 10 µM necrostatin-1 or 20 µM
ferrostatin-1 for 30 minutes before ironomycin treatment (n=3 biological replicates in OCI-
AML3, HL-60 and MOLM-13 cell lines).
|
https://openalex.org/W2949750514
|
https://europepmc.org/articles/pmc6566137?pdf=render
|
English
| null |
Cultural Variation in Reactions to a Group Member’s Vicarious Choice and the Role of Rejection Avoidance
|
Frontiers in psychology
| 2,019
|
cc-by
| 12,208
|
Edited by:
Glenn Adams,
University of Kansas,
United States Reviewed by:
Joanna Schug,
College of William & Mary,
United States
Yu Niiya,
Hosei University, Japan Keywords: agency, culture, rejection avoidance, personal choice, vicarious choice “It is our choices, Harry, that show what we truly are” (Harry Potter and the Chamber of
Secrets, 1998). Rowling’s (1998) line reveals how central choice is for an individual’s psychology
and life course. The provision of choice and self-determination are crucial for autonomy and
human motivation and make individuals happier and healthier (Zuckerman et al., 1978; Deci
and Ryan, 2008). On the other hand, evidence on social influence suggests that individuals tend
to adjust themselves to the thought of the majority in a group pressure situation (Asch, 1952;
Cialdini and Goldstein, 2004). Although both the pursuit of personal choice and seeking to fit
in the group underlie individuals’ behaviors, which of these two aspects is emphasized might
be moderated by cultural differences in the weight on self and social relationships. Indeed, a
growing research stream has documented that Western individualistic cultures generally promote
a stronger desire for personal choice, but a smaller influence of interpersonal concerns on decisions
than East Asian collectivistic cultures (e.g., Kitayama et al., 2004; Savani et al., 2008, 2010).f “It is our choices, Harry, that show what we truly are” (Harry Potter and the Chamber of
Secrets, 1998). Rowling’s (1998) line reveals how central choice is for an individual’s psychology
and life course. The provision of choice and self-determination are crucial for autonomy and
human motivation and make individuals happier and healthier (Zuckerman et al., 1978; Deci
and Ryan, 2008). On the other hand, evidence on social influence suggests that individuals tend
to adjust themselves to the thought of the majority in a group pressure situation (Asch, 1952;
Cialdini and Goldstein, 2004). Although both the pursuit of personal choice and seeking to fit
in the group underlie individuals’ behaviors, which of these two aspects is emphasized might
be moderated by cultural differences in the weight on self and social relationships. Cultural Variation in Reactions to a
Group Member’s Vicarious Choice
and the Role of Rejection Avoidance
Charis Eisen1* and Keiko Ishii2
1Department of Social Sciences Darmstadt University of Applied Sciences Darmstadt Germany 2Department of Psychology 1 Department of Social Sciences, Darmstadt University of Applied Sciences, Darmstadt, Germany, 2 Department of Psychology,
Graduate School of Informatics, Nagoya University, Nagoya, Japan Extending the literature on culture and the personal or interpersonal construction of
choices, this research investigates consequences of an ingroup member’s vicarious
decision for the entire group and the mechanism behind cultural variation. In Study 1,
Japanese people showed, compared to Germans, greater acceptance of vicarious choice
and evaluated the ingroup member who had chosen on their behalf more positively. Using
mediation analyses and priming methods, Studies 2 and 3 identified rejection avoidance
to partly explain culturally diverse reactions to vicarious choices. These findings suggest
that the mechanism behind cultural differences in choice is related to variation in strength
of the motivation to maintain social approval. Indeed, a
growing research stream has documented that Western individualistic cultures generally promote
a stronger desire for personal choice, but a smaller influence of interpersonal concerns on decisions
than East Asian collectivistic cultures (e.g., Kitayama et al., 2004; Savani et al., 2008, 2010).f *Correspondence:
Charis Eisen
charis.eisen@h-da.de Specialty section:
This article was submitted to
Cultural Psychology,
a section of the journal
Frontiers in Psychology Received: 29 January 2019
Accepted: 20 May 2019
Published: 07 June 2019 g
y
In this research, drawing on the literature on culture and choice, we examine cultural differences
in reactions to a group member’s vicarious choice in Germans and Japanese. Based on previous
work suggesting that East Asians are more motivated than Westerners to avoid rejection by
group members (Sato et al., 2014; Hashimoto and Yamagishi, 2016), we hypothesized that
Japanese would be more likely than Germans to react positively to vicarious choice and that
cultural differences in rejection avoidance would account for the cultural influence on response
to vicarious choice. ORIGINAL RESEARCH
published: 07 June 2019
doi: 10.3389/fpsyg.2019.01311 VICARIOUS CHOICE Consistently, previous research illustrated that whereas Asians
and Asian Canadians showed no cognitive dissonance in a
condition with the standard free-choice paradigm, dissonance
was observable in a condition where interpersonal concerns
and worries were induced by presenting eyes of others (Kitayama
et al., 2004) and when they were to make a choice for their
friends (Hoshino-Browne et al., 2005). Citation: Eisen C and Ishii K (2019) Cultural
Variation in Reactions to a Group
Member’s Vicarious Choice and the
Role of Rejection Avoidance. Front. Psychol. 10:1311. doi: 10.3389/fpsyg.2019.01311 June 2019 | Volume 10 | Article 1311 1 Frontiers in Psychology | www.frontiersin.org Cultural Variation in Vicarious Choice Eisen and Ishii VICARIOUS CHOICE On
the contrary, many East Asian contexts promote a rather conjoint
model of agency, in which actions are responsive to others’
obligations and expectations, and good actions promote
interdependence with and adjustment to others (Markus and
Kitayama, 2003; Kitayama and Uchida, 2005; Markus et al., 2006). Mirroring these divergent models of agency, previous research
has illustrated sociocultural variation in emphases on personal
and interpersonal aspects in choice. Research identified cultural
differences in whether choice is considered individual, personal
decision-making or whether multiple people can be involved
in a more interpersonally constructed form of choice (Markus
and Kitayama, 2003; Mesquita and Markus, 2004; Savani et al.,
2010). People in many cultures base their choices not merely
on their personal preferences, but rather seek advice and include
others’ opinions in their choices without feeling constricted
or burdened (Savani et al., 2008, 2010; Eisen et al., 2016). Consistently, previous research illustrated that whereas Asians
and Asian Canadians showed no cognitive dissonance in a
condition with the standard free-choice paradigm, dissonance
was observable in a condition where interpersonal concerns
and worries were induced by presenting eyes of others (Kitayama
et al., 2004) and when they were to make a choice for their
friends (Hoshino-Browne et al., 2005). Cultures promote different implicit frameworks of normative
behavior, called models of agency (Markus and Kitayama, 2003). North American and Western European contexts promote a
rather disjoint model of agency, which characterizes good actions
by their independence of social circumstances and their contingency
on one’s own preferences, goals, intentions, and motives. On
the contrary, many East Asian contexts promote a rather conjoint
model of agency, in which actions are responsive to others’
obligations and expectations, and good actions promote
interdependence with and adjustment to others (Markus and
Kitayama, 2003; Kitayama and Uchida, 2005; Markus et al., 2006). y
y
Mirroring these divergent models of agency, previous research
has illustrated sociocultural variation in emphases on personal
and interpersonal aspects in choice. Research identified cultural
differences in whether choice is considered individual, personal
decision-making or whether multiple people can be involved
in a more interpersonally constructed form of choice (Markus
and Kitayama, 2003; Mesquita and Markus, 2004; Savani et al.,
2010). People in many cultures base their choices not merely
on their personal preferences, but rather seek advice and include
others’ opinions in their choices without feeling constricted
or burdened (Savani et al., 2008, 2010; Eisen et al., 2016). Frontiers in Psychology | www.frontiersin.org VICARIOUS CHOICE greater psychological reactance when they had to give up their
personal freedom to use the respective good. As described in
reactance theory (Brehm, 1966), the denial of personal choice
by another person’s vicarious choice likely threatens Westerners’
freedom and therefore elicits negative responses (reactance)
from them. Moreover, Jonas et al. (2009) manipulated independent
and interdependent orientations and found that people primed
with independent orientation reported more reactance than
did those primed with interdependent orientation when they
had to give up personal freedom. The self has been featured as being independent and separate
from other people in Western cultures such as in Germany,
whereas it has been featured as being interdependent and connected
with others in East Asian cultures such as in Japan (Markus
and Kitayama, 1991). Against the backdrop of the greater emphasis
on independence, the pursuit of personal choice is crucial for
Westerners, as choice enables them to express their individual,
autonomous selves through showing their preferences, attitudes,
values, and feelings (Kim and Sherman, 2007). On the other
hand, Eastern cultures place greater emphasis on social adjustment
and accommodation to others (Morling et al., 2002), while self-
expression through choice is relatively unimportant.f In this research, we pay attention to situations in which
someone else decides on behalf of a group, for example, when
selecting or saying something on the others’ behalf, and thereby
restricts the others in their expression of personal preferences
and ideas. Studies have examined how choices made by ingroup
(e.g., mother) or outgroup (e.g., experimenter) choosers on
behalf of the individual affect this individual’s performance
and judgments (Iyengar and Lepper, 1999; Hoshino-Browne
et al., 2005; Savani et al., 2008). However, vicarious choice
situations in which an equal-status ingroup member decides
on behalf of the entire group have not received sufficient
attention – in spite of being frequent daily life occurrences. Examinations of the consequences of vicarious choices made
for the whole group could add to the understanding of how
agency is understood in interdependent contexts. Therefore,
the present research focuses on these vicarious choices and
investigates reactions in Eastern and Western cultures. Cultures promote different implicit frameworks of normative
behavior, called models of agency (Markus and Kitayama, 2003). North American and Western European contexts promote a
rather disjoint model of agency, which characterizes good actions
by their independence of social circumstances and their contingency
on one’s own preferences, goals, intentions, and motives. THE ROLE OF REJECTION
AVOIDANCE IN THE RESPONSE
TO VICARIOUS CHOICE While negative reactions to the denial of freedom are associated
with independence of the self (Savani et al., 2008; Jonas et al.,
2009), a positive reaction to the denial of freedom might
be associated with interdependence of the self. Although this
possibility has been suggested by Iyengar and Lepper (1999),
who found an association between personal choice and intrinsic
motivation in European American children and an association
between a choice made by a close other (e.g., their mothers)
and intrinsic motivation in Asian American children, the
underlying psychological mechanism of such a cultural difference
has not been fully tested.f Although evidence is limited, the consequence of the denial
of choice also differs across cultures. For instance, in Savani
et al.’s (2008) Study 5, the experimenter usurped participants
of their personal pen choices by choosing on their behalf. The
researchers found that, compared to an own choice condition,
North Americans indicated less liking of the pen in the usurped
choice condition, while Indian participants rated the pens equally
likable in both conditions. In addition, Jonas et al. (2009)
asked participants to imagine that a colleague they briefly knew
requests the abandonment of a certain good participants assumed
as theirs, or that an authority prohibited certain products for
health reasons, and measured self-reported negative reactions
to these scenarios. They found that compared to people from
collectivistic cultures, people from individualistic cultures showed y
To explore the underlying mechanism of a cultural difference
in the response to vicarious choice, we focus on how being
afraid of social rejection leads to avoidant behavior. People
care deeply about social rejection, as they want to connect
with other people in their own group. The need to belong
has been shown to play a significant role across cultures (Fiske
and Yamamoto, 2005). Previous research illustrated that
experience of rejection leads to rejection sensitivity, which in
turn promotes rejection avoidance behaviors (e.g., Feldman
and Downey, 1994). Molden et al. (2009) showed that when
people recalled or underwent experiences of being rejected,
they showed prevention-focused responses. Similarly, studies
suggest that rejection experiences bring to mind broader social June 2019 | Volume 10 | Article 1311 2 Cultural Variation in Vicarious Choice Eisen and Ishii THE PRESENT RESEARCH In contrast, a society like Germany
allows its members to find alternative interaction partners easily,
and therefore, social rejection is not as deleterious. Sato et al. (2014) have provided empirical evidence for the claim that
being rejected is more threatening to East Asian people than
for Westerners because the cost of being disliked and eventually
excluded by others is greater in these societies, where finding
alternative relationships is rather difficult. Other studies have
consistently found that East Asians exhibit more pronounced
rejection avoidance tendencies than Westerners (Yamaguchi
et al., 1995; Garris et al., 2011). Consistently, empirical research
has shown that Asians oftentimes behave in a way that allows
them to avoid any disruption of harmonious relationships:
Compared to Westerners, East Asians more frequently engage
in self-criticism (Heine et al., 2000), are less willing to seek
social support (Kim et al., 2008; Ishii et al., 2017), and more
frequently inhibit their desire to express disagreement (Hashimoto
et al., 2012) in order to prevent social disapproval. These findings
suggest that although rejection poses a threat and experiences
of rejection lead to avoidance behaviors to people regardless
of their cultural background, structural factors (e.g., whether
a society is maintained by a mutual monitoring and sanctioning
system) promote these avoidance behaviors to varying degrees. The concern for others’ appraisals might lead individuals
across cultures to feel a threat of rejection from the group
they belong to, particularly in situations in which all group Given that rejection avoidance reflects a concern for social
disapproval, the impact on responses to vicarious choice would
be clearly demonstrated in a group situation such as when
people are working in a team. Thus, we developed a set of
group scenarios that one group member first chooses and
proposes a collective behavior without asking and considering
individual preferences and opinions. Little is known about
cultural differences in reactions to vicarious choice in a group
setting, as previous findings that the consequences of vicarious
choice depend on culture are mainly based on the examinations
at the dyadic level (e.g., mother; Iyengar and Lepper, 1999). Testing with these group scenarios, we also explore the cultural
differences in reactions to vicarious choice can be generalized
even in a condition where group pressure can be estimated
and perceived. THE PRESENT RESEARCH Other studies have
consistently found that East Asians exhibit more pronounced
rejection avoidance tendencies than Westerners (Yamaguchi
et al., 1995; Garris et al., 2011). Consistently, empirical research
has shown that Asians oftentimes behave in a way that allows
them to avoid any disruption of harmonious relationships:
Compared to Westerners, East Asians more frequently engage
in self-criticism (Heine et al., 2000), are less willing to seek
social support (Kim et al., 2008; Ishii et al., 2017), and more
frequently inhibit their desire to express disagreement (Hashimoto
et al., 2012) in order to prevent social disapproval. These findings
suggest that although rejection poses a threat and experiences
of rejection lead to avoidance behaviors to people regardless
of their cultural background, structural factors (e.g., whether
a society is maintained by a mutual monitoring and sanctioning
system) promote these avoidance behaviors to varying degrees. The concern for others’ appraisals might lead individuals
across cultures to feel a threat of rejection from the group
they belong to, particularly in situations in which all group
members form a mutual commitment relationship and can
observe individuals’ behavior. Choosing based on one’s inner
attributes while ignoring the social context or failing to
incorporate others’ preferences could be seen as incongruent
to social standards and thus potentially elicit rejection by the
other group members. However, as structural factors in Japan
promote the prevention of social rejection more strongly than
structural factors in Germany, Japanese people might respond
more positively to vicarious choice by ingroup members, while
Germans would be more likely to risk rejection and claim
personal choice. We hypothesized, accordingly, that the cultural
differences in reactions can be partly explained by variation
in levels of rejection avoidance. However, the literature also suggests cultural differences in
the significance of the strategy to avoid rejection in order to
live a good life. Hashimoto and Yamagishi (2013, 2016) argued
that a society like Japan, which is maintained by mutual
monitoring and sanctioning within fixed group boundaries,
promotes heavy dependency of individuals on others. As groups
are closed to outsiders and mutual commitment relationships
are prevalent, rejection by group members and exclusion from
the community-based cooperation system is very harmful. It
is therefore wise to be sensitive to the needs and expectations
of other members of the group and not to offend them in
order to avoid social rejection. Participants One hundred and ten Japanese (55 women, Mage = 44.9,
SDage = 14.35) and 99 German adults (50 women, Mage = 43.0,
SDage = 13.95) were recruited through an online crowdsourcing
service in each country (Macromill in Japan, Respondi AG in
Germany). The participants were paid according to local
standards. We determined the sample size by referring to Jonas
et al. (2009, Study 1), showing a small/medium effect size for
the main effect of culture (ds = 0.33–0.61). We estimated that
the sample size was 99 for each culture, assuming the effect
size (d) as 0.4 and a value for desired power as 0.80. Frontiers in Psychology | www.frontiersin.org THE PRESENT RESEARCH connections, for example, social groups one belongs to (Knowles
and Gardner, 2008) and promotes to seek out group settings
(Maner et al., 2007), to adhere to group norms (Kerr et al.,
2009), and to increase contribution to group efforts (Williams
and Sommer, 1997). Taken together, these findings suggest
that the experience of being rejected generally leads to increases
of rejection sensitivity and avoidance at the individual level.f The present research conducted three studies among German
(representing a Western culture) and Japanese (representing
an East Asian culture) participants to explore cultural differences
in responses to vicarious choice. We hypothesized that Germans
would be more likely than Japanese to demand choice as a
reaction to vicarious choice and evaluate the chooser more
negatively, whereas Japanese would be more likely than Germans
to accept vicarious choices and evaluate the chooser more
positively. Following Study 1, which tests the cultural differences,
Studies 2 and 3 hypothesized that Japanese would be higher
in rejection avoidance than Germans and that higher rejection
avoidance would lead people to accept vicarious choice and
demand less personal choice.l and Sommer, 1997). Taken together, these findings suggest
that the experience of being rejected generally leads to increases
of rejection sensitivity and avoidance at the individual level. However, the literature also suggests cultural differences in
the significance of the strategy to avoid rejection in order to
live a good life. Hashimoto and Yamagishi (2013, 2016) argued
that a society like Japan, which is maintained by mutual
monitoring and sanctioning within fixed group boundaries,
promotes heavy dependency of individuals on others. As groups
are closed to outsiders and mutual commitment relationships
are prevalent, rejection by group members and exclusion from
the community-based cooperation system is very harmful. It
is therefore wise to be sensitive to the needs and expectations
of other members of the group and not to offend them in
order to avoid social rejection. In contrast, a society like Germany
allows its members to find alternative interaction partners easily,
and therefore, social rejection is not as deleterious. Sato et al. (2014) have provided empirical evidence for the claim that
being rejected is more threatening to East Asian people than
for Westerners because the cost of being disliked and eventually
excluded by others is greater in these societies, where finding
alternative relationships is rather difficult. Method
Ethics Statementh The study was reviewed and approved by the Experimental
Research Ethics Committee at the Graduate School of Humanities,
Kobe University. Participants provided a written informed
consent at the beginning of the study. All responses
were confidential. 1We admit that dominant can also be seen as a positive feature of a person
in some contexts. However, we do not think that this context provokes positive
associations with someone who is dominant and therefore treat dominant as
a negative evaluation of the chooser. Procedure We composed a questionnaire in Japanese and translated it
into German. A bilingual third-party back-translated the June 2019 | Volume 10 | Article 1311 3 Cultural Variation in Vicarious Choice Eisen and Ishii choice situations independent of how frequent such situations
occur, we controlled for this variable in the following analyses2. questionnaire into Japanese, and we compared this back-
translation to the original to assure that all questions were
equal in meaning. We asked participants to imagine the following
three scenarios: Reaction We calculated participants’ likeliness to accept the vicarious
choice (αGer = 0.87, αJap = 0.84) and demand personal choice
(αGer = 0.74, αJap = 0.71) in all three scenarios and conducted
ANCOVAs to investigate cultural differences. As predicted,
Japanese participants (M = 4.57, SD = 0.97) were significantly
more likely than German participants (M = 3.65, SD = 1.29)
to accept the choices on their behalf: F(1, 206) = 24.03,
p < 0.001, h2
p = 0.10. In addition, German participants
(M = 4.91, SD = 1.18) were significantly more likely than
Japanese participants (M = 3.76, SD = 0.97) to demand personal
choice: F(1, 206) = 57.74, p < 0.001, h2
p = 0.22. The results
are illustrated in Figures 1, 2. 1. You finished a big project at work and your colleagues and
you go to celebrate this success with a business meal. At
the restaurant, you find a menu, but one of your colleagues
orders
for
the
whole
team
without
asking
for
individual preferences. 2. You plan an event together with your coworkers, and there
are many tasks to share. Someone needs to take care of
the finances, someone needs to do advertising, someone
needs to invite and take care of the guests, and someone
needs to do the paper work. One of your coworkers takes
the lead and tells you and the others what to do without
asking for individual preferences. 3. You are in a meeting and your boss asks for feedback about
a new policy that he introduced last week. One of your
colleagues answers in detail, representing the whole team
without asking individual opinions. Evaluation We calculated how positively (αGer = 0.84, αJap = 0.68) and
negatively (αGer = 0.83, αJap = 0.80) the participants would
evaluate the ingroup member who had chosen for the entire
group across scenarios and compared German and Japanese
respondents. As predicted, Japanese evaluated the chooser as
more likable and sociable (M = 3.77, SD = 0.80) and less
egoistic and dominant (M = 4.42, SD = 1.01) than Germans
did [positive: M = 3.04, SD = 1.23, F(1, 206) = 15.82, p < 0.001,
h2
p = 0.07; negative: M = 5.27, SD = 1.23, F(1, 206) = 28.94,
p < 0.001, h2
p = 0.12]. For each scenario, participants indicated on three items how
likely they would be to accept the vicarious choice (e.g., “accept
the decision of the colleague”) and on two items how likely
they would be to demand personal choice (e.g., “choose something
to eat and drink from the menu yourself”) on Likert-type
scales ranging from 1 (very unlikely) to 7 (very likely), respectively. In the following analyses, we created an index for acceptance
and an index for choice demand over all three scenarios. However, the results were the same when we analyzed
them separately. Ethics Statementh If vicarious choices are, as expected, more positively connoted
in Japan as compared to Germany, it would be natural if
vicarious choice situations were more frequent occurrences in
Japan than in Germany. Indeed, across scenarios (αGer = 0.82,
αJap = 0.76), Japanese participants (M = 4.30, SD = 1.10)
indicated that such situations are frequent daily life situations
more than German participants (M = 3.57, SD = 1.61,
t(207) = 3.86, p < 0.001, d = 0.53, 95% CI = [0.36, 1.10]). However, as we were interested in consequences of vicarious The study was reviewed and approved by the Experimental
Research Ethics Committee at the Graduate School of Humanities,
Kobe University. Participants provided a written informed consent
at the beginning of the study. All responses were confidential. 2Please note that when we ran the analyses without this covariate, the results
were the same. For acceptance: t(207) = 5.89, p < 0.001; for choice demand:
t(207) = −7.74, p < 0.001; for positive evaluation: t(207) = 5.11, p < 0.001;
for negative evaluation: t(207) = −5.52, p < 0.001. STUDY 1B Furthermore, participants indicated how positively (i.e., likable
and sociable) and negatively (i.e., egoistic and dominant)1 they
would evaluate the ingroup member who had chosen for the
entire group in such a situation on 7-point scales (1 = strongly
disagree, 7 = strongly agree). Moreover, participants indicated
their agreement to the statement “a situation like this occurs
frequently” on a 7-point scale (1 = strongly disagree, 7 = strongly
agree) for each scenario. In Study 1a, we constructed the scenarios such that the person
choosing for the entire group is a colleague and not an individual
who is higher or lower in the social hierarchy. However, we did
not make the equal status explicit and participants might have
assumed that the chooser is someone of higher social status. Therefore, we conducted an additional study to eliminate the
possibility that cultural differences are side effects of differences
in perceived social status. 3Please note that when we ran the analyses without this covariate, the results
were the same. For acceptance: t(208) = 6.26, p < 0.001; for choice demand:
t(208) = −7.13, p < 0.001; for positive evaluation: t(208) = 4.18, p < 0.001;
for negative evaluation: t(208) = −2.14, p < 0.05. Perceived Rank Across scenarios (αGer = 0.87, αJap = 0.65), Japanese participants
(M = 3.98, SD = 1.00) and German participants (M = 3.69,
SD = 1.51) perceived the rank of the chooser similarly,
t(208) = 1.67, p = 0.10, 95% CI = (−0.64, 0.05). Thus, the
participants in both cultures understood the information of rank
correctly, as we intended. In contrast, perceived rank correlated
with reactions to vicarious choice (German sample: acceptance
r(98) = 0.53, p < 0.001; choice demand r(98) = −0.19, p = 0.051. Japanese sample: acceptance r(108) = 0.20, p = 0.035; choice
demand r(108) = −0.05, p = 0.629). To preclude the possibility
that perceptions of rank influenced participants’ reactions,
we statistically controlled for this variable in the following analyses3. Evaluation We calculated how positively (αGer = 0.89, αJap = 0.85) and
negatively (αGer = 0.88, αJap = 0.83) participants would evaluate
the ingroup member who had chosen for the group across
scenarios. As predicted, Japanese people evaluated the chooser
as more likable and sociable (M = 3.68, SD = 1.08) and less
egoistic and dominant (M = 4.71, SD = 1.02) than Germans
did [positive: M = 3.00, SD = 1.26, F(1, 207) = 14.48, p < 0.001,
h2
p = 0.07; negative: M = 5.06, SD = 1.38, F(1, 207) = 5.45,
p = 0.020, h2
p = 0.03]. As in Study 1a, participants indicated how likely they would
be to accept the vicarious choice/demand personal choice and
how positively/negatively they would evaluate the ingroup
member who had chosen for the entire group. Participants One hundred and ten Japanese (55 women, Mage = 44.5,
SDage = 13.96) and 100 German adults (50 women, Mage = 46.4, One hundred and ten Japanese (55 women, Mage = 44.5,
SDage = 13.96) and 100 German adults (50 women, Mage = 46.4, 1We admit that dominant can also be seen as a positive feature of a person
in some contexts. However, we do not think that this context provokes positive
associations with someone who is dominant and therefore treat dominant as
a negative evaluation of the chooser. June 2019 | Volume 10 | Article 1311 Frontiers in Psychology | www.frontiersin.org 4 Cultural Variation in Vicarious Choice Eisen and Ishii SDage = 15.15) were recruited through online crowdsourcing
services (as in Study 1a). FIGURE 1 | Cultural differences in acceptance of vicarious choice in Studies
1a, 1b, and 2. Error bars show 95% confidence intervals. FIGURE 2 | Cultural differences in personal choice demand as a reaction to
vicarious choice in Studies 1a, 1b, and 2. Error bars show 95% confidence
intervals. FIGURE 1 | Cultural differences in acceptance of vicarious choice in Studies
1a, 1b, and 2. Error bars show 95% confidence intervals. FIGURE 2 | Cultural differences in personal choice demand as a reaction to
vicarious choice in Studies 1a, 1b, and 2. Error bars show 95% confidence
intervals. FIGURE 1 | Cultural differences in acceptance of vicarious choice in Studies
1a, 1b, and 2. Error bars show 95% confidence intervals. SDage = 15.15) were recruited through online crowdsourcing
services (as in Study 1a). SDage = 15.15) were recruited through online crowdsourcing
services (as in Study 1a). SD = 0.93) were significantly more likely than German
participants (M = 3.38, SD = 1.24) to accept choices on their
behalf, F(1, 207) = 36.77, p < 0.001, h2
p = 0.15, and German
participants (M = 4.94, SD = 1.13) were significantly more
likely than Japanese participants (M = 3.89, SD = 1.02) to
demand personal choice, F(1, 207) = 47.68, p < 0.001, h2
p = 0.19. The results are illustrated in Figures 1, 2. Procedure We asked participants to imagine the same three vicarious
choice scenarios as in Study 1a, but added the information
that the colleagues were all of the same status. We included
an item that asked participants to indicate how strongly they
agree to the statement “the colleague, who chose for the group,
is high in rank” (1 = strongly disagree, 7 = strongly agree). STUDY 2 Study 1 investigated how Germans and Japanese respond to
situations in which an ingroup member has made a choice,
specifically what to eat, which task to perform, and how to
respond, for an entire group. Eliminating possible confounding
effects of perceived frequency and social status, the results
support the hypothesis that vicarious choice is more accepted
and more positively connoted in Japan as compared to Germany. Study 2 aimed at shedding light on the mechanism behind
the observed cultural variation. Specifically, we tested whether
individual rejection avoidance tendencies mediate cultural
differences in reactions to vicarious choices. Reaction We calculated participants’ likeliness to accept vicarious choice
(αGer = 0.87, αJap = 0.83) and demand personal choice (αGer = 0.75,
αJap = 0.79) and conducted ANCOVAs to investigate cultural
differences. As predicted, Japanese participants (M = 4.33, Ethics Statementh The study was reviewed and approved by the Experimental
Research Ethics Committee at the Graduate School of Humanities,
Kobe University. Participants provided a written informed consent
at the beginning of the study. All responses were confidential. June 2019 | Volume 10 | Article 1311 Frontiers in Psychology | www.frontiersin.org 5 Cultural Variation in Vicarious Choice Eisen and Ishii (M = 3.42, SD = 1.26) to accept the vicarious choices,
t(430) = 8.58, p < 0.001, d = 0.83, 95% CI = (−1.14, −0.71). In addition, German participants (M = 5.03, SD = 1.14) were
significantly more likely than Japanese participants (M = 3.88,
SD = 0.97) to demand personal choice, t(430) = 11.24, p < 0.001,
d = 1.09, 95% CI = (0.95, 1.35). The results are illustrated
in Figures 1, 2. Thus, we replicated the findings of Study 1. Procedure In a questionnaire, we asked participants to imagine the same
scenarios as in Study 1 and to indicate their likely reactions
to these situations. Thereafter, participants answered a scale
measuring rejection avoidance tendencies (Hashimoto and
Yamagishi, 2016). The specific items were “I find myself feeling
anxious if people are watching me,” “I find myself being
concerned about what others think of me,” “I often feel anxious
about the nature of my relationships with others and their
status as compared to mine,” “I sometimes get so anxious
about what other people might think that I am prevented from
doing what I really want to do,” and “I often behave in a way
that will keep others from disliking me.” Participants indicated
how well each of these statements describes them on Likert-
type scales (1 = does not describe me at all, 7 = describes me
very much)4. Next, we conducted mediation analyses to investigate the
hypothesis that the cultures provoke different levels of rejection
avoidance, which in turn affect reactions to choices on one’s
behalf. We dummy coded German culture as 0 and Japanese
culture as 1. Regressing culture on acceptance tendencies,
we found that culture is a significant predictor, b = 0.93,
SE = 0.11, t(430) = 8.58, p < 0.001, 95% CI = [0.72, 1.14]. Culture
also
predicted
rejection
avoidance
tendencies
significantly, b = 0.77, SE = 0.12, t(430) = 6.53, p < 0.001,
95% CI = [0.54, 1.00]. Importantly, individual rejection
avoidance tendencies in turn affected reactions to vicarious
choice b = 0.36, SE = 0.04, t(430) = 8.82, p < 0.001, 95%
CI = (0.28, 0.44) and the predictive power of culture was
significantly reduced to b = 0.65, SE = 0.11, t(430) = 6.23,
p < 0.001, 95% CI = (0.45, 0.86) when we controlled for
rejection avoidance. Bootstrap analyses revealed a significant
indirect effect (1,000 bootstrap samples): 95% CI [0.17, 0.41]. Hence, the cultural difference in acceptance of vicarious choice
was partially mediated by individual variation in rejection
avoidance (Figure 3). Mediation Analyses To analyze whether individual rejection avoidance tendencies
constitute a factor underlying the cultural differences in reactions
to vicarious choice, we first calculated rejection avoidance
tendencies for each participant by merging the five items
measuring this construct (αGer = 0.82, αJap = 0.85). In line
with previous research, we found that compared to Germans
(M = 3.63, SD = 1.33), Japanese (M = 4.40, SD = 1.11) were
more anxious about being rejected by others, t(430) = 6.53,
p < 0.001, d = 0.63, 95% CI = (−1.00, −0.54). Correlation
patterns between rejection avoidance and acceptance were
rGer(210) = 0.45, p < 0.001, rJap(218) = 0.31, p < 0.001 and
between
rejection
avoidance
and
choice
demand
rGer(210) = −0.36, p < 0.001, rJap(218) = 0.07, p = 0.33. Results
Reaction We calculated participants’ likeliness of acceptance (αGer = 0.87,
αJap = 0.85) and personal choice demand (αGer = 0.76, αJap = 0.72)
over all three scenarios. A t-test to investigate cultural differences
revealed that Japanese participants (M = 4.35, SD = 0.97)
were significantly more likely than German participants 4We also measured the other three facets of independence and interdependence
proposed by Hashimoto and Yamagishi. Compared to the Japanese sample,
the German sample indicated higher mean values in engaging independence
(= self-expression; MGer = 5.32, SDGer = 1.15, MJap = 4.09, SDJap = 1.09,
t(430) = 11.53, p < 0.001), engaging interdependence (= harmony seeking;
MGer = 5.42, SDGer = 1.02, MJap = 4.66, SDJap = 0.97, t(430) = 7.94, p < 0.001),
and disengaging independence (= uniqueness; MGer = 4.94, SDGer = 0.95,
MJap = 4.28, SDJap = 0.92, t(430) = 7.36, p < 0.001). Simple regression analyses
revealed that rejection avoidance (b = 0.44, SE = 0.04, p < 0.001), self-expression
(b = −0.38, SE = 0.04, p < 0.001), and uniqueness (b = −0.28, SE = 0.06,
p < 0.001) significantly predicted acceptance, whereas harmony seeking (b = 0.08,
SE = 0.06, p = 0.153) did not. Concerning personal choice demand, rejection
avoidance (b = −0.32, SE = 0.04, p < 0.001), self-expression (b = 0.61, SE = 0.04,
p < 0.001), harmony seeking (b = 0.27, SE = 0.05, p < 0.001), and uniqueness
(b = 0.56, SE = 0.05, p < 0.001) significantly predicted choice demand. Although
other facets of independence and interdependence as proposed by Hashimoto
and Yamagishi had effects on reactions to vicarious choice, this paper focuses
on the effect of rejection avoidance, given that researchers have paid little
attention to the effect of this concept across cultures. FIGURE 3 | Individual levels of rejection avoidance as a mediator of the
cultural differences in acceptance of vicarious choice in Study 2. Unstandardized coefficients and standard errors are shown. Coefficients
indicating the relationship between culture (coded as German culture = 0 and
Japanese culture = 1) and acceptance of vicarious choice after controlling for
rejection avoidance tendencies are given in parentheses. ***p < 0.001. FIGURE 3 | Individual levels of rejection avoidance as a mediator of the
cultural differences in acceptance of vicarious choice in Study 2. Unstandardized coefficients and standard errors are shown. Participants (M = 3.42, SD = 1.26) to accept the vicarious choices,
t(430) = 8.58, p < 0.001, d = 0.83, 95% CI = (−1.14, −0.71). In addition, German participants (M = 5.03, SD = 1.14) were
significantly more likely than Japanese participants (M = 3.88,
SD = 0.97) to demand personal choice, t(430) = 11.24, p < 0.001,
d = 1.09, 95% CI = (0.95, 1.35). The results are illustrated
in Figures 1, 2. Thus, we replicated the findings of Study 1. p
Crowdsourcing services recruited two community samples of
220 Japanese (110 women, Mage = 44.26, SDage = 13.81) and
212 Germans (96 women, Mage = 43.58, SDage = 14.08) and
reimbursed them according to their standard. We determined
the sample size by referring to Jonas et al. (2009, Study 1),
showing a small/medium effect size for the main effect of
culture, for the expected direct effect. We referred to Hashimoto
and Yamagishi (2016), who found a medium effect (d = 0.46)
for cultural differences in rejection avoidance, for the expected
effect of culture on rejection avoidance. Based on Fritz and
MacKinnon (2007), we estimated that the sample size should
be 404 in total to detect the expected mediated effect using
bootstrap analyses with a desired power of 0.80. 4We also measured the other three facets of independence and interdependence
proposed by Hashimoto and Yamagishi. Compared to the Japanese sample,
the German sample indicated higher mean values in engaging independence
(= self-expression; MGer = 5.32, SDGer = 1.15, MJap = 4.09, SDJap = 1.09,
t(430) = 11.53, p < 0.001), engaging interdependence (= harmony seeking;
MGer = 5.42, SDGer = 1.02, MJap = 4.66, SDJap = 0.97, t(430) = 7.94, p < 0.001),
and disengaging independence (= uniqueness; MGer = 4.94, SDGer = 0.95,
MJap = 4.28, SDJap = 0.92, t(430) = 7.36, p < 0.001). Simple regression analyses
revealed that rejection avoidance (b = 0.44, SE = 0.04, p < 0.001), self-expression
(b = −0.38, SE = 0.04, p < 0.001), and uniqueness (b = −0.28, SE = 0.06,
p < 0.001) significantly predicted acceptance, whereas harmony seeking (b = 0.08,
SE = 0.06, p = 0.153) did not. Concerning personal choice demand, rejection
avoidance (b = −0.32, SE = 0.04, p < 0.001), self-expression (b = 0.61, SE = 0.04,
p < 0.001), harmony seeking (b = 0.27, SE = 0.05, p < 0.001), and uniqueness
(b = 0.56, SE = 0.05, p < 0.001) significantly predicted choice demand. Although
other facets of independence and interdependence as proposed by Hashimoto
and Yamagishi had effects on reactions to vicarious choice, this paper focuses
on the effect of rejection avoidance, given that researchers have paid little
attention to the effect of this concept across cultures. 4We also measured the other three facets of independence and interdependence
proposed by Hashimoto and Yamagishi. Compared to the Japanese sample,
the German sample indicated higher mean values in engaging independence
(= self-expression; MGer = 5.32, SDGer = 1.15, MJap = 4.09, SDJap = 1.09,
t(430) = 11.53, p < 0.001), engaging interdependence (= harmony seeking;
MGer = 5.42, SDGer = 1.02, MJap = 4.66, SDJap = 0.97, t(430) = 7.94, p < 0.001),
and disengaging independence (= uniqueness; MGer = 4.94, SDGer = 0.95,
MJap = 4.28, SDJap = 0.92, t(430) = 7.36, p < 0.001). Simple regression analyses
revealed that rejection avoidance (b = 0.44, SE = 0.04, p < 0.001), self-expression
(b = −0.38, SE = 0.04, p < 0.001), and uniqueness (b = −0.28, SE = 0.06,
p < 0.001) significantly predicted acceptance, whereas harmony seeking (b = 0.08,
SE = 0.06, p = 0.153) did not. Concerning personal choice demand, rejection
avoidance (b = −0.32, SE = 0.04, p < 0.001), self-expression (b = 0.61, SE = 0.04,
p < 0.001), harmony seeking (b = 0.27, SE = 0.05, p < 0.001), and uniqueness
(b = 0.56, SE = 0.05, p < 0.001) significantly predicted choice demand. Although
other facets of independence and interdependence as proposed by Hashimoto
and Yamagishi had effects on reactions to vicarious choice, this paper focuses
on the effect of rejection avoidance, given that researchers have paid little
attention to the effect of this concept across cultures. Frontiers in Psychology | www.frontiersin.org STUDY 3 Study 2 provided the first evidence to show the mediating role
of rejection avoidance in culturally different responses to vicarious
choice. Assessing people’s chronic rejection avoidance tendencies,
however, Study 2 was based on self-reports and might
be compromised by people’s inability to accurately report on
their cultural beliefs. To avoid the problems inherent to trait
measures and to clarify the causal direction between rejection
avoidance and reactions to vicarious choice, we tested in Study
3 whether priming social rejection sensitivity would cause divergent
reactions to vicarious choice in Japanese and Germans. Reflecting
the importance of belonging across individuals and cultures,
previous research illustrated that rejection experiences promote
avoidance behavior (Molden et al., 2009). Therefore, we hypothesized
that priming rejection sensitivity by asking people to recall a
rejection episode would promote rejection avoidant behavior (i.e.,
acceptance of vicarious choice) regardless of culture. p
y
y
j
(
g
)
As an alternative possibility, based on the claim that rejection
avoidance is more connected to structural factors in Japan than
in Germany, the association between rejection avoidance and
responses to vicarious choices could be evident in the Japanese
sample, but not in the German sample. We tested this possibility
by comparing two regression models, one that includes culture
and rejection avoidance to predict acceptance of vicarious choice
(model 1) and one that includes also the interaction of culture
and rejection avoidance (model 2). Although both models were
significant [model 1: R2 = 0.277, F(2, 429) = 82.22, p < 0.001;
model 2: R2 = 0.283, F(3, 428) = 56.20, p < 0.001], the variance
explained by these models did not differ significantly [R2
change = 0.006, F(1, 428) = 3.29, p = 0.071] and the interaction
term was not significant (b = 0.15, SE = 0.08, p = 0.071). The
effect of rejection avoidance was significant in both samples
[German sample: b = 0.42, SE = 0.05, 95% CI (0.32, 0.52);
Japanese sample: b = 0.27, SE = 0.06, 95% CI (0.15, 0.40)],
suggesting that if someone has a strong tendency to avoid
rejection, this person is likely to accept vicarious choice regardless
of his/her cultural background. When we performed this analysis
with choice demand as dependent variable, both models were
significant [model 1: R2 = 0.270, F(2, 429) = 79.53, p < 0.001;
model 2: R2 = 0.287, F(3, 428) = 59.84, p < 0.001]. Results
Reaction Coefficients
indicating the relationship between culture (coded as German culture = 0 and
Japanese culture = 1) and acceptance of vicarious choice after controlling for
rejection avoidance tendencies are given in parentheses. ***p < 0.001. FIGURE 3 | Individual levels of rejection avoidance as a mediator of the
cultural differences in acceptance of vicarious choice in Study 2. Unstandardized coefficients and standard errors are shown. Coefficients
indicating the relationship between culture (coded as German culture = 0 and
Japanese culture = 1) and acceptance of vicarious choice after controlling for
rejection avoidance tendencies are given in parentheses. ***p < 0.001. June 2019 | Volume 10 | Article 1311 Frontiers in Psychology | www.frontiersin.org 6 Cultural Variation in Vicarious Choice Eisen and Ishii German sample (b = −0.31, SE = 0.05, 95% CI [−0.42, −0.21])
than in the Japanese sample [b = −0.06, SE = 0.06, 95% CI
(−0.18, 0.06)]. This indicates that in Germany, people with
strong tendencies to avoid rejection would rather not demand
personal choice, compared to people with weaker rejection
avoidance tendencies. In Japan, people would be unlikely to
demand
personal
choice,
regardless
of
their
rejection
avoidance tendencies. Next, we tested whether rejection avoidance tendencies
likewise mediated the cultural difference in choice demand. Culture was a significant predictor for choice demand tendencies,
b = −1.15, SE = 0.10, t(430) = −11.24, p < 0.001, 95%
CI = (−1.35, −0.95). When we entered rejection avoidance
simultaneously to an analysis of regression, the effect of culture
was significantly reduced, b = −0.99, SE = 0.10, t(430) = −9.50,
p < 0.001, 95% CI = (−1.19, −0.78) and rejection avoidance
predicted choice demand, b = −0.21, SE = 0.04, t(430) = −5.05,
p < 0.001, 95% CI = [−0.28, −0.12]. The indirect effect was
significant [1,000 bootstrap samples, 95% CI (−0.26, −0.07)];
suggesting that the cultural difference in choice demand was
partially mediated by rejection avoidance (Figure 4). Ethics Statementh The study was reviewed and approved by the Experimental
Research Ethics Committee at the Graduate School of Humanities,
Kobe University. Participants provided a written informed consent
at the beginning of the study. All responses were confidential. STUDY 3 We found
a significant change in R2 = 0.016, F(1, 428) = 9.84, p = 0.002
and the interaction term was significant (b = −0.25, SE = 0.08,
p = 0.002). However, quite the contrary to the assumption that
rejection avoidance plays a crucial role in Japan but not in
Germany, the effect of rejection avoidance was stronger in the Participants We recruited 105 German and 87 Japanese students. However,
because some participants failed to report a situation in which
they were strongly rejected, the final sample consisted of 92
German (77 women, Mage = 21.64, SD = 2.45) and 80 Japanese
students (47 women, Mage = 19.87, SD = 0.87). Although the
German sample consisted of more female participants than
the Japanese sample, χ2(2, N = 172) = 13.64, p = 0.001, a
preliminary analysis showed that there were no gender differences
in acceptance [t(170) = 0.58, p = 0.561] or personal choice
demand [t(170) = −0.73, p = 0.464]. Gender was thus not
included in the following analyses. Accordingly, data from 81
participants in the social rejection condition (43 Germans and
38 Japanese) and 91 participants in the control condition
(49 Germans and 42 Japanese) were analyzed. Based on past
research showing a main effect of manipulated social rejection
(ds = 0.35 and 0.45 in Pickett et al., 2004; d = 0.67 in Knowles
and Gardner, 2008), we assumed a medium effect size (d = 0.50),
set the desired power to 0.80, and estimated that the sample
size was 63 for each condition. FIGURE 4 | Individual levels of rejection avoidance as a mediator of the
cultural differences in personal choice demand as a reaction to vicarious
choice in Study 2. Unstandardized coefficients and standard errors are
shown. Coefficients indicating the relationship between culture (coded as
German culture = 0 and Japanese culture = 1) and acceptance of vicarious
choice after controlling for rejection avoidance tendencies are given in
parentheses. ***p < 0.001. FIGURE 4 | Individual levels of rejection avoidance as a mediator of the
cultural differences in personal choice demand as a reaction to vicarious
choice in Study 2. Unstandardized coefficients and standard errors are
shown. Coefficients indicating the relationship between culture (coded as
German culture = 0 and Japanese culture = 1) and acceptance of vicarious
choice after controlling for rejection avoidance tendencies are given in
parentheses. ***p < 0.001. FIGURE 4 | Individual levels of rejection avoidance as a mediator of the
cultural differences in personal choice demand as a reaction to vicarious
choice in Study 2. Unstandardized coefficients and standard errors are
shown. GENERAL DISCUSSION As in Studies 1 and 2, we calculated participants’ likeliness
to accept vicarious choice (αGer = 0.69, αJap = 0.74) and demand
personal choice (αGer = 0.52, αJap = 0.62) over the scenarios. We ran two ANOVAs to test whether culture and priming
condition had an influence on acceptance and choice demand,
respectively. Regarding acceptance, Japanese students (M = 4.13,
SD = 0.91) were significantly more likely than German students
(M = 3.78, SD = 0.89) to accept the choices on their behalf,
F(1, 168) = 6.42, p = 0.012, h2
p = 0.037. Importantly, regardless
of culture, participants primed with sensitivity toward social
rejection (M = 4.10, SD = 0.86) were more likely to accept Aligning with previous findings, over the studies presented
here, Germans indicated to be likely to demand personal choice
when a group member had chosen vicariously, while Japanese
were found to likely accept vicarious choices. This indicates
that Westerners likely perceive vicarious choice as a threat to
their rather independently oriented selves, thereby promoting
reactance. However, the emphasis on interdependence would
lead East Asians to avoid social rejection by meeting others’
intentions and expectations. Hence, the mechanism behind
cultural variation in choice seems to be related to the extent
to which people are motivated to avoid social disapproval.hi FIGURE 5 | Cultural and group differences in acceptance of vicarious choice
in Study 3. Error bars show 95% confidence intervals. FIGURE 6 | Cultural and group differences in choice demand as a reaction
to vicarious choice in Study 3. Error bars show 95% confidence intervals. FIGURE 5 | Cultural and group differences in acceptance of vicarious choice
in Study 3. Error bars show 95% confidence intervals. FIGURE 5 | Cultural and group differences in acceptance of vicarious choice
in Study 3. Error bars show 95% confidence intervals. This research investigated a specific form of interpersonal
choice: psychological consequences of situations in which one
group member decides for the entire ingroup without consulting
its individual members. This is unique, as previous studies
(e.g., Iyengar and Lepper, 1999; Hoshino-Browne et al., 2005;
Savani et al., 2008) largely ignored group processes. The focus
on groups, however, allowed us to test whether previous findings
extent to this very common form of vicarious choice, in which
group pressure can be anticipated and perceived. Participants Coefficients indicating the relationship between culture (coded as
German culture = 0 and Japanese culture = 1) and acceptance of vicarious
choice after controlling for rejection avoidance tendencies are given in
parentheses. ***p < 0.001. June 2019 | Volume 10 | Article 1311 Frontiers in Psychology | www.frontiersin.org Frontiers in Psychology | www.frontiersin.org 7 Cultural Variation in Vicarious Choice Eisen and Ishii the ingroup member’s choice than the control group [M = 3.80,
SD = 0.94, F(1, 168) = 4.60, p = 0.033, h2
p = 0.027]. The
interaction of priming and culture was not significant,
F(1, 168) = 1.13, p = 0.289. Similarly, German students (M = 4.61,
SD = 0.86) were more likely to demand personal choice than
Japanese students [M = 3.79, SD = 0.98, F(1, 168) = 34.81,
p < 0.001, h2
p = 0.172]. Furthermore, as predicted, rejection-
primed participants (M = 4.02, SD = 0.98) were less likely to
demand personal choice than the control group [M = 4.41,
SD = 0.99, F(1, 168) = 7.45, p = 0.007, h2
p = 0.042]. The
interaction was not significant, F(1, 168) = 0.76, p = 0.385. The results are illustrated in Figures 5, 6. Procedure We adopted a priming method which successfully manipulated
social rejection in previous studies (Pickett et al., 2004; Knowles
and Gardner, 2008) and asked half of the participants to recall
a situation in which they were strongly rejected, while the
other half of the participants recalled their walk to campus
that day. Next, participants answered to the three scenarios
of Studies 1 and 2 (slightly adjusted to fit students’ daily life
experiences) and indicated how likely they would be to accept
this vicarious choice or demand personal choice. Finally, they
answered demographic questions and were rewarded with 700
Yen or the equivalent of 6 Euros, respectively. June 2019 | Volume 10 | Article 1311 GENERAL DISCUSSION However, it is possible that the
differences observed pertain to norms about work settings in
particular, as opposed to more general cultural differences. In
addition, the scenarios described situations in which someone
chooses food on behalf of the group, situations in which someone
chooses which task each team member has to accomplish, and
situations in which a team member responds on behalf of the
whole group. This is a very wide understanding of vicarious
choice and goes beyond the common definition of choice. Future
research needs to explore the generalizability of our findings. Despite providing insights into the mechanism behind
culturally diverse reactions to vicarious choice, rejection avoidance
tendencies cannot fully explain sociocultural differences. Additional testing for mediators, such as self-esteem (Heine
et al., 1999; Schmitt and Allik, 2005), self-monitoring (Gudykunst
et al., 1989), relationship and group-based trust (Yuki et al.,
2005) will be necessary to specify the precise factors and their
interactions to completely understand the underlying mechanism
behind diverse reactions to choice situations. incorporate the social context more strongly or to be even more
likely to decide merely based upon their own preferences. Indeed,
previous research suggested that the importance of a decision
is a relevant factor to consider (Savani et al., 2010; Li et al.,
2014). Furthermore, our scenarios are all work-related and
included diverse vicarious choices. Choosing work-related contexts
enabled us to create scenarios in which an equal-status ingroup
member chose vicariously. However, it is possible that the
differences observed pertain to norms about work settings in
particular, as opposed to more general cultural differences. In
addition, the scenarios described situations in which someone
chooses food on behalf of the group, situations in which someone
chooses which task each team member has to accomplish, and
situations in which a team member responds on behalf of the
whole group. This is a very wide understanding of vicarious
choice and goes beyond the common definition of choice. Future
research needs to explore the generalizability of our findings. were not concerned about social rejection as much and were,
therefore, more likely to reject vicarious choices and demand
personal choice in order to regain their sense of autonomy. While individual differences in rejection avoidance tendencies
affected the likeliness to accept vicarious choice in Japanese
and German participants, rejection avoidance did not affect
how likely Japanese participants in Study 2 were to demand
personal choice. GENERAL DISCUSSION A possible explanation for this is that the
norm of not standing out would be so strong that it covered
the effect of rejection avoidance at the individual level. However,
when we temporarily made rejection avoidance salient with a
priming method in Study 3, German and Japanese participants’
tendency to demand personal choice decreased.hi The finding that the described vicarious choice situations
occur more frequently in Japan than in Germany gives some
insight as to the instantiation of cultural differences in daily
life. It supports the claim that sociocultural contexts affect
individuals by providing them with particular kinds of regularly
encountered
situations,
and
the
experiences
in
these
socioculturally shaped situations lead to habitual ways of thinking
about oneself and the world (Kitayama et al., 1997). As
sociocultural contexts foster specific situations that demand
specific behaviors and ways of being, individuals learn to
construct themselves in order to match these sociocultural
expectations. If so, frequently encountered situations might
have shaped individuals’ understandings of the self and agency,
thereby (unconsciously) advising them on either emphasizing
personal,
autonomous
choice
or
social
connectedness
and conformity.i Despite providing insights into the mechanism behind
culturally diverse reactions to vicarious choice, rejection avoidance
tendencies cannot fully explain sociocultural differences. Additional testing for mediators, such as self-esteem (Heine
et al., 1999; Schmitt and Allik, 2005), self-monitoring (Gudykunst
et al., 1989), relationship and group-based trust (Yuki et al.,
2005) will be necessary to specify the precise factors and their
interactions to completely understand the underlying mechanism
behind diverse reactions to choice situations. Another future research direction could be to examine more
automatic and unconscious responses to vicarious choice and
to investigate its neural mechanism. For instance, given that
the dorsal anterior cingulate cortex (dACC) plays a crucial
role in the detection of a behavioral conflict, previous research
found that cognitive dissonance, particularly conflict evoked
by difficult choice, is linked to strong activation of dACC
(Kitayama et al., 2013). If Westerners evaluate the group member
who chooses vicariously more negatively than East Asians,
they might feel that the denial of personal choice caused by
the group member’s vicarious choice causes a conflict. If so,
the cultural differences in response to vicarious choice would
be reflected as cultural differences in activation of dACC.f Whereas previous findings were based on studies conducted
primarily with North American samples, we examined German
people’s behavior and found that they responded negatively to
vicarious choice. Frontiers in Psychology | www.frontiersin.org GENERAL DISCUSSION This is in line with previous findings illustrating
that
compared
to
East
Asians,
Germans
considered
recommendations by ingroup members less in their workplace
choice (Eisen et al., 2016) and that Western Europeans showed
strong psychological reactance when they had to give up their
personal freedoms (Jonas et al., 2009; Graupmann et al., 2012). Although some previous findings proposed differences in the
emphasis on individual achievement and self-promotion between
North Americans and Western Europeans (Kitayama et al.,
2009), the findings related to choice and agency would not
suggest differences between these cultures in the tendency to
condemn social influence. However, as this study does not
include a North American sample, a direct comparison remains
to be addressed in future research. lf
Finally, it is important to explore whether cultural differences
in reaction to vicarious choice mediated by rejection avoidance
can be observed even in children. Given that Iyengar and
Lepper (1999) tested children ranging in age from 7 to 9 years
and found cultural differences in intrinsic motivation toward
tasks chosen by either children themselves or others, future
work is needed to test children in elementary schools and
investigate whether socialization may impact on reaction to
vicarious choice as well as rejection avoidance across cultures. This investigation will contribute to our understanding of how
children learn and acquire culturally proper forms of choice
through socialization. In our studies, we used vicarious choice stimuli that are
likely to happen in everyday life. While we eliminated concerns
that culturally different reactions are only side effects of these
situations occurring more or less frequent in the two cultures
(Study 1a), our findings can be generalized only to everyday
choice situations and not to more consequential choices. It is
possible that Japanese people are more likely to demand personal
choice if the decision is important to them, or alternatively,
that increased importance makes them even more likely to
reflect upon social approval and accept choices on their behalf. Similarly, more consequential decisions might lead Germans to To conclude, the present research adds to previous evidence
on culture and choice by showing that responses to vicarious
choice differ across cultures. It also provided the first evidence
that the cultural influence on responses to vicarious choice
can be explained by cultural differences in rejection avoidance. GENERAL DISCUSSION Despite this
group pressure, our German participants indicated that they
would reject vicarious choices and thereby risk negative social
consequences. This gives an idea about the strength of German
people’s desire for personal choice. In addition, the results
suggest that cultural differences in reactions to an ingroup
member’s vicarious choice are not limited to a closely related
other like a mother or best friend. Rather, they would extent
to colleagues, who are ingroup members but not as closely related. FIGURE 5 | Cultural and group differences in acceptance of vicarious choice
in Study 3. Error bars show 95% confidence intervals. FIGURE 6 | Cultural and group differences in choice demand as a reaction
to vicarious choice in Study 3. Error bars show 95% confidence intervals. FIGURE 6 | Cultural and group differences in choice demand as a reaction
to vicarious choice in Study 3. Error bars show 95% confidence intervals. Moreover, focusing on rejection avoidance, these studies
contribute to the literature by partly revealing the mechanism
behind cultural differences. Elucidating the mechanism behind
cultural differences contributes greatly to the field and advances
the knowledge of culture and choice by explaining existing
differences rather than merely describing them. In addition,
identifying the driving motivations behind variation in behaviors
informs psychology in general about the processes underlying
individual conducts (Heine and Norenzayan, 2006). Our findings
would suggest that interdependently oriented Japanese people
are acceptant of choices that imply others’ expectations because
falling short of these expectations would result in social rejection
or exclusion. In contrast, independently oriented German people FIGURE 6 | Cultural and group differences in choice demand as a reaction
to vicarious choice in Study 3. Error bars show 95% confidence intervals. June 2019 | Volume 10 | Article 1311 Frontiers in Psychology | www.frontiersin.org Frontiers in Psychology | www.frontiersin.org 8 Cultural Variation in Vicarious Choice Eisen and Ishii incorporate the social context more strongly or to be even more
likely to decide merely based upon their own preferences. Indeed,
previous research suggested that the importance of a decision
is a relevant factor to consider (Savani et al., 2010; Li et al.,
2014). Furthermore, our scenarios are all work-related and
included diverse vicarious choices. Choosing work-related contexts
enabled us to create scenarios in which an equal-status ingroup
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gy
Fritz, M. FUNDING The research was supported by Grant-in-aid for Scientific
Research 26380843 and 15KK0120 from the Ministry of
Education, Culture, Sports, Science and Technology of Japan. AUTHOR CONTRIBUTIONS vicarious choice has implications for fields such as marketing
and politics particularly in the globalizing world today where
people with different cultural backgrounds are urged to work
together as a group. Also, the present research presents interesting
questions, which should be addressed in future research. We believe that additional insight provided by further
investigations suggested in the present research will enhance
our understanding of cultural mechanisms behind responses
to vicarious choice. CE and KI designed the research. CE and KI performed the
research. CE analyzed the data. CE and KI wrote the paper. ACKNOWLEDGMENTS The study was reviewed and approved by the Experimental
Research Ethics Committee at the Graduate School of Humanities,
Kobe University. Participants provided written informed consent
at the beginning of the study. All responses were confidential. Research Ethics Committee at the Graduate School of Humanities,
Kobe University. Participants provided written informed consent
at the beginning of the study. All responses were confidential. We thank Tilmann Betsch, Anne Staude, Jana Winkelbach, and
Sakura Yahata for their help with data collection and translations. Kobe University. Participants provided written informed consent
at the beginning of the study. All responses were confidential. GENERAL DISCUSSION This evidence for the mechanism that underlies responses to June 2019 | Volume 10 | Article 1311 June 2019 | Volume 10 | Article 1311 9 Eisen and Ishii Cultural Variation in Vicarious Choice REFERENCES Self and dissonance in two cultures. Psychol. Sci. 15,
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10.1037/a0012958 Conflict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or financial relationships that could be constructed
as a potential conflict of interest. Conflict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or financial relationships that could be constructed
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influence in the United States and adjustment in Japan. Personal. Soc. Psychol. Bull. 28, 311–323. doi: 10.1177/0146167202286003 Copyright © 2019 Eisen and Ishii. Frontiers in Psychology | www.frontiersin.org June 2019 | Volume 10 | Article 1311 REFERENCES This is an open-access article distributed under
the terms of the Creative Commons Attribution License (CC BY). The use, distribution
or reproduction in other forums is permitted, provided the original author(s) and
the copyright owner(s) are credited and that the original publication in this journal
is cited, in accordance with accepted academic practice. No use, distribution or
reproduction is permitted which does not comply with these terms. Pickett, C. L., Gardner, W. L., and Knowles, M. (2004). Getting a cue: the
need to belong and enhanced sensitivity to social cues. Personal. Soc. Psychol. Bull. 30, 1095–1107. doi: 10.1177/0146167203262085 Rowling, J. K. (1998). Harry Potter and the chamber of secrets. (London:
Bloomsbury). June 2019 | Volume 10 | Article 1311 Frontiers in Psychology | www.frontiersin.org 11
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Internal Hernia Through Foramen of Winslow a Rare Cause of Small Bowel Obstruction: A Case Report
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Contents lists available at Vilnius University Press
Lietuvos chirurgija
ISSN 1392–0995 eISSN 1648–9942
2023, vol. 22(2), pp. 88–92
DOI: https://doi.org/10.15388/LietChirur.2023.22.82 Contents lists available at Vilnius University Press
Lietuvos chirurgija
ISSN 1392–0995 eISSN 1648–9942
2023, vol. 22(2), pp. 88–92
DOI: https://doi.org/10.15388/LietChirur.2023.22.82 Contents lists available at Vilnius University Press ISSN 1392–0995 eISSN 1648–9942
DOI: https://doi.org/10.15388/LietChirur.2023.22.82 Lietuvos chirurgija
2023, vol. 22(2), pp. 88–92 Received: 2023/02/03. Accepted: 2023/03/15.
Copyright © 2023 Vipin Venugopal Nair, Malik Parmjit, Pawan Sharma, Sarali Santhosh Raja, Atreya Aprajita. Published by Vilnius University Press. This is an Open Access
article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited. Atreya Aprajita Department of Surgery, AFMC Pune, Maharashtra, India
E-mail: draprajitaatreya@gmail.com
ORCID: https://orcid.org/0000-0002-4181-7522 Department of Surgery, AFMC Pune, Maharashtra, India
E-mail: draprajitaatreya@gmail.com
ORCID: https://orcid.org/0000-0002-4181-7522 Abstract. Internal hernia through foramen of Winslow (FoW) is rare condition as there are only 200 cases reported so far in the literature. Our patient a 78 years man presented with a clinical picture suggestive of small bowel obstruction for 5 days. Patient underwent emergency
laparotomy following suspicion of internal hernia on imaging. On exploratory laparotomy there was grossly dilated bowel loops and
a small segment of terminal ileum and omentum was found herniating through FoW in to the lesser sac. The bowel segment was reduced
with gentle traction and herniated segment of omentum was excised due to questionable viability. Opening of FoW was unusually large
and to prevent hernia recurrence it was closed partially. Postoperative period was uneventful. This unusual case presented to us diagnostic
confusion and management challenge considering the previous history, multiple comorbidities and geriatric profile. Key words: foramen of Winslow hernia, internal hernias, intestinal obstructions. Internal Hernia Through Foramen of Winslow a Rare
Cause of Small Bowel Obstruction: A Case Report Vipin Venugopal Nair
Department of Surgery, AFMC Pune, Maharashtra, India
E-mail: vipinvenugopalnair@gmail.com
ORCID: https://orcid.org/0000-0001-6903-6368 Vipin Venugopal Nair
Department of Surgery, AFMC Pune, Maharashtra, India
E-mail: vipinvenugopalnair@gmail.com
ORCID: https://orcid.org/0000-0001-6903-6368 Received: 2023/02/03. Accepted: 2023/03/15. p
enugopal Nair, Malik Parmjit, Pawan Sharma, Sarali Santhosh Raja, Atreya Aprajita. Published by Vilnius University Press. This is an Open Access
he terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the
e are credited. Malik Parmjit Department of Surgery, AFMC Pune, Maharashtra, India
E-mail: paramjeetmalik84@gmail.com
ORCID: https://orcid.org/0000-0002-8265-4491 Introduction Majority of bowel obstructions occur in the small intestine (80–90%) and are commonly due to adhesions,
abdominal wall hernias, or neoplasm. Internal hernia, defined as the protrusion of abdominal viscera through
a peritoneal or mesenteric aperture into a compartment in abdominal or peritoneal cavity is one of the rare
cause (<5%) of small bowel obstruction. They are more common after laparoscopic gastric bypass surgeries 88 Klinikinė praktika / Vipin Venugopal Nair, Malik Parmjit, Pawan Sharma, Sarali Santhosh Raja, Atreya Aprajita. Internal Hernia Through Foramen of Winslow a Rare Cause of Small Bowel Obstruction: A Case Report [1, 2] internal hernia through foramen of Winslow (FoW) is a rare condition. Since the f [1, 2] internal hernia through foramen of Winslow (FoW) is a rare condition. Since the first report in 1834
by Blandin in autopsies, <200 cases have been reported in the medical literature [3]. gi
p
by Blandin in autopsies, <200 cases have been reported in the medical literature [3]. by Blandin in autopsies, <200 cases have been reported in the medical literature [3]. We report a case of acute intestinal obstruction in an elderly individual due to herniation of small bowel
through FoW. The diagnostic and management challenges were highlighted in this article. Case report A 78-year-oldmale, a known case of hypothyroidism on irregular thyroxine supplementation, presented with
complaints of diffuse pain abdomen, obstipation and progressive abdominal distension for last five days to
the surgical emergency room. There was nausea and multiple episodes of bilious vomiting for one day. He
also gave a past history of laparotomy twice in 2017 and 2019 for intestinal obstruction. On arrival to accident and emergency he had tachycardia (pulse – 110 bpm) and was dehydrated. Rest
parameters were normal. He was afebrile. Abdominal was grossly distended with generalised guarding on
palpation and hyper-tympanic note on percussion. Bowel sounds were absent. Rest of systemic examination
was unremarkable On further evaluation following findings were noted. His lab parameters were: Hemoglobin – 15.3 gm/dl
(13–16 gm/dl), Total Leukocyte Count – 6 800/cmm (4 000–11 000/cmm), Platelet Counts – 268 000/cmm
(1.5–400 000/cmm), PT/INR – 13.1 sec/1.06 (13–16 sec/1 to 1.3), Urea – 51 mg/dl (15–45 mg/dl), Creati
nine – 0.9 mg/dl (0.5–1.3 mg/dl), Na+ – 134 meq/L (135–145 meq/L), K+ – 4.2 meq/L (3.5–4.5 meq/L),
4.5 meq/L, Bilirubin – 1.1 mg/dl (0.1–1.2 mg/dl), AST/ALT – 44 IU/L, 75 IU/L (15–45 IU/L, 15–60 IU/L),
Amylase/Lipase – 22 IU/L, 110 IU/L (20–85 IU/L, <300 IU/L), Proteins (Total/Albumin) – 7.2 gm/dl,
4.0 gm/dl (6–8 gm/dl, 3–4.5 gm/dl), T3/T4/TSH – 40 ng/ml, 4.13 ug/dl, 9.39 mIU/L (0.2–2.0 ng/ml,
6–12 ug/dl, 0.3–4.0 mIU/L), HIV – 1&2/HbsAg, HCV – negative. On X-ray abdomen erect and supine with both domes of diaphragm multiple air fluid levels were seen
suggestive of intestinal obstruction. There was no gas under diaphragm (Figure 1A). Figure 1. A, B Figure 1. A, B Figure 1. A, B Figure 1. A, B Figure 1. A, B 89 ISSN 1392–0995 eISSN 1648–9942 Lietuvos chirurgija Underwent CECT abdomen on suspicion of small bowel obstruction. The finding were suggestive of
small bowel obstruction with a definitive transition zone at distal ileum and four centimetre dilatation of
proximal small bowel loops (Figure 1B). In view of clinical presentation of intestinal obstruction, he was taken up for emergency exploratory
laparotomy under general anaesthesia. Intraoperative, there was grossly dilated bowel loops of questionable viability. On evisceration of the in
testinal loops, there was no faecal or biliary contamination. On further manipulation of the gut, we were able
to find a loop of ileum herniating through the lesser sac (Figure 2A). Discussion There are eight main types of internal hernias, categorized depending on where the hernia occurs. Foramen
of Winslow hernia can be defined as peculiar variant of internal abdominal hernia, since it is a normal peri
toneal orifice kept closed by normal intra-abdominal pressure that may be permeated by the intra-abdominal
viscera. Hernias through the foramen of Winslow are rare and constitute only 8% of internal hernias and
0.08% of total hernias [4, 5].h There are multiple anatomical abnormalities reported as possible predisposing factors for a visceral hernia
tion through this foramen (i) abnormally enlarged foramen; (ii) the presence of an unusually long small-bowel
mesentery or persistence of the ascending mesocolon; (iii) an elongated right hepatic lobe, which could be
directing the mobile intestinal loop into the foramen; (iv) a lack of fusion between caecum or ascending colon
to the parietal peritoneum; (v) a defect in the gastro-hepatic ligament; (vi) incomplete intestinal rotations
or malrotation [6].hi The rate of preoperative diagnosis has been reported to be <10% of the intraoperatively confirmed cases. A delay in diagnosis and treatment is often observed and may be responsible for the high mortality rate of up
to 49% associated with this hernia type. Internal hernia is often revealed by intestinal obstruction associated
with non-viable bowel at the time of operation [3]. Symptoms are usually related to bowel obstruction. The
severity of the pain is related to the presence of bowel strangulation with subsequent necrosis. In some very
particular cases, the internal hernia through the Winslow hiatus is revealed by an obstructive jaundice due
to direct compression of the hepatic pedicle [2]. Clinical examination is non-specific and laboratory findings are rarely helpful. The key to diagnosis relies
on prompt radiologic studies and the CT scan is nowadays considered the investigation of choice. Various,
more or less specific findings have been reported, such as an air-fluid collection in the lesser sac or signs of
small bowel obstruction associated with the presence of mesenteric vessels stretching anterior to the inferior
vena cava and posterior to the portal vein; the absence of the ascending colon in the right gutter and an
antero-lateral displacement of the stomach [7, 8].h The treatment invariably requires urgent surgery, and even if symptoms are limited, it should be consi
dered in order to assess intestinal viability because of the risk of intestinal strangulation. Case report The loops of intestine were so dilated
that it prevented the reduction of this internal hernia. We used hot saline mops to reduce bowel oedema. The
patient was given 100% oxygen to improve tissue oxygenation. Once the bowels were eviscerated and secured
safely an attempt to reduce the hernia was tried. However, the bowel was stuck to the lesser sac. Widening
of the sac was done using limited kocherisations. Once the neck of sac was wide enough the herniated loop
of intestine was reduced (Figure 2B). The associated necrosed omental segment was excised and tissue send
for histopathology (Figure 2C). Figure 2. A, B, C, D
After reduction the herniated loop of bowel seemed to be dusky. The bowel was kept in warm saline mops
and patient was administered 100% oxygen. There was return of peristalsis and the colour turned pink after
5 minutes (Figure 2D). The decision was taken to close the hernia defect with interrupted Polyglactin (™Vicryl Figure 2. A, B, C, D Figure 2. A, B, C, D Figure 2. A, B, C, D Figure 2. A, B, C, D After reduction the herniated loop of bowel seemed to be dusky. The bowel was kept in warm saline mops
and patient was administered 100% oxygen. There was return of peristalsis and the colour turned pink after
5 minutes (Figure 2D). The decision was taken to close the hernia defect with interrupted Polyglactin (™Vicryl After reduction the herniated loop of bowel seemed to be dusky. The bowel was kept in warm saline mops
and patient was administered 100% oxygen. There was return of peristalsis and the colour turned pink after
5 minutes (Figure 2D). The decision was taken to close the hernia defect with interrupted Polyglactin (™Vicryl After reduction the herniated loop of bowel seemed to be dusky. The bowel was kept in warm saline mops
and patient was administered 100% oxygen. There was return of peristalsis and the colour turned pink after
5 minutes (Figure 2D). The decision was taken to close the hernia defect with interrupted Polyglactin (™Vicryl 90 inikinė praktika / Vipin Venugopal Nair, Malik Parmjit, Pawan Sharma, Sarali Santhosh Raja, Atreya Aprajita. Internal Hernia Through Foramen of Winslow a Rare Cause of Small Bowel Obstruction: A Case Report 3.0) sutures. Rest of bowel inspected and no significant abnormality noted. The dilated bowels were preven
ting abdominal closure. Case report The contents of small intestine were milked retrograde to the stomach and aspirated
from the Nasogastric tube through suction. Once the bowel was successfully decompressed the laparotomy
wound was closed in layers. The patient was started on clear oral fluids from second postoperative day and subsequently on soft diet. He was discharged on postoperative day five. Presently patient is doing well on third month of follow-up. The histopathology of the excised omentum revealed normal matured adipocytes with few inflammatory
infiltrates. No evidence of giant cell granuloma, atypical or malignant cells seen. References 1. Sikiminywa-Kambale P, Anaye A, Roulet D, Pezzetta E. Internal hernia through the foramen of Winslow: a diag
nosis to consider in moderate epigastric pain. J Surg Case Rep 2014; 2014(6): rju065. 1. Sikiminywa-Kambale P, Anaye A, Roulet D, Pezzetta E. Internal hernia through the foramen of Winslow: a diag
nosis to consider in moderate epigastric pain. J Surg Case Rep 2014; 2014(6): rju065. 2. Maingot’s abdominal operations. 13th edition. Chapter 38. Small bowel obstruction. 3. Osvaldt AB, Mossmann DF, Bersch VP, Rohde L. Intestinal obstruction caused by a foramen of Winslow her
nia. Am J Surg 2008; 196(2): 242–244. 3. Osvaldt AB, Mossmann DF, Bersch VP, Rohde L. Intestinal obstruction caused by a foramen of Winslow her
nia. Am J Surg 2008; 196(2): 242–244. 4. González Conde R, Pardo Rojas P, Valeiras Domínguez E, Pérez López C, Santos Lloves R, Gómez Lorenzo FJ. Correct preoperative diagnosis of herniation through the Foramen of Winslow: two case reports. Hernia 2013; 17(3):
409–414. 4. González Conde R, Pardo Rojas P, Valeiras Domínguez E, Pérez López C, Santos Lloves R, Gómez Lorenzo FJ. Correct preoperative diagnosis of herniation through the Foramen of Winslow: two case reports. Hernia 2013; 17(3):
409–414. 5. Takeyama N, Gokan T, Ohgiya Y, Satoh S, Hashizume T, Hataya K, Kushiro H, Nakanishi M, Kusano M, Mune
chika H. CT of internal hernias. Radiographics 2005; 25(4): 997–1015. 5. Takeyama N, Gokan T, Ohgiya Y, Satoh S, Hashizume T, Hataya K, Kushiro H, Nakanishi M, Kusano M, Mune
chika H. CT of internal hernias. Radiographics 2005; 25(4): 997–1015. 6. Moris D, Tsilimigras DI, Yerokun B, Seymour KA, Guerron AD, Fong PA, Spartalis E, Sudan R. Foramen of
Winslow Hernia: a Review of the Literature Highlighting the Role of Laparoscopy. J Gastrointest Surg 2019; 23(10):
2093–2099. DOI: 10.1007/s11605-019-04353-3. 6. Moris D, Tsilimigras DI, Yerokun B, Seymour KA, Guerron AD, Fong PA, Spartalis E, Sudan R. Foramen of
Winslow Hernia: a Review of the Literature Highlighting the Role of Laparoscopy. J Gastrointest Surg 2019; 23(10):
2093–2099. DOI: 10.1007/s11605-019-04353-3.h 7. Huang Y, Qin L, Wu L, Huang Q. An Adolescent with Ileum Herniation Through Foramen of Winslow: A Case
Report and Literature Review. Nigerian Journal of Clinical Practice 2022; 25(8): 1372–1376. DOI: 10.4103/njcp. njcp_1778_21. 7. Huang Y, Qin L, Wu L, Huang Q. An Adolescent with Ileum Herniation Through Foramen of Winslow: A Case
Report and Literature Review. Discussion Treatment is based
on careful inspection with subsequent hernia reduction that is frequently possible with simple and gentle
traction. Occasionally, this can be difficult; in these situation the gastrocolic or gastro-hepatic ligaments must
be opened or, alternatively, a wide Kocher manoeuvre performed. In the case of massive colonic dilatation
a colotomy for decompression with a suction device can be useful [1, 9]. In the case of overt intestinal necrosis
an adequate resection is obviously mandatory; nevertheless there is no clear and established consensus on
surgical management when the herniated contents are grossly viable. In our case limited kocherisation for
reduction of hernia was performed followed by partial closure of the defect. Furthermore, in order to prevent recurrent herniation, some surgeons decide to definitively close the
foramen of Winslow. This option can however lead to meaningful complications such as accretions and/or 91 ISSN 1392–0995 eISSN 1648–9942 Lietuvos chirurgija portal vein thrombosis. Thus, leaving the foramen open may be justifiable since the inflammatory postopera
tive adhesions will most often obliterate the foramen entrance with no evidence of recurrent herniation [4]. Conflicts of interest. The author declares no competing interest. portal vein thrombosis. Thus, leaving the foramen open may be justifiable since the inflammatory postopera
tive adhesions will most often obliterate the foramen entrance with no evidence of recurrent herniation [4]. C
fli
f i
Th
h
d l
i
i Conflicts of interest. The author declares no competing interest. 9. Akyildiz H, Artis T, Sozuer E, Akcan A, Kucuk C, Sensoy E, Karahan I. Internal hernia: complex diagnostic and
therapeutic problem. Int J Surg 2009; 7(4): 334–337. References Nigerian Journal of Clinical Practice 2022; 25(8): 1372–1376. DOI: 10.4103/njcp. njcp_1778_21. 8. Doishita S, Takeshita T, Uchima Y, Kawasaki M, Shimono T, Yamashita A, Sugimoto M, Ninoi T, Shima H,
Miki Y. Internal Hernias in the Era of Multidetector CT: Correlation of Imaging and Surgical Findings. Radiographics
2016; 36(1): 88–106. 9. Akyildiz H, Artis T, Sozuer E, Akcan A, Kucuk C, Sensoy E, Karahan I. Internal hernia: complex diagnostic and
therapeutic problem. Int J Surg 2009; 7(4): 334–337. 92 92
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TYPE
Original Research
08 September 2022
10.3389/fpls.2022.971274
PUBLISHED
DOI
OPEN ACCESS
EDITED BY
Pietro Gramazio,
University of Tsukuba, Japan
REVIEWED BY
Vidya Sagar,
Indian Institute of Vegetable Research
(ICAR), India
Jietang Zhao,
South China Agricultural University,
China
*CORRESPONDENCE
Dasen Xie
xiedasen@126.com
Yujuan Zhong
zhongyujuan@gdaas.cn
A time-course transcriptome
analysis of wax gourd fruit
development reveals
predominant genes regulating
taste and nutrition
Shudan Xue1,2 , Xiaotong Wan1,2 , Sen Lu1,2 , Yujuan Zhong1,2*
and Dasen Xie1,2*
1
Guangdong Key Laboratory for New Technology Research of Vegetables, Vegetable Research
Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, China, 2 Guangdong
Laboratory for Lingnan Modern Agriculture, Guangzhou, China
SPECIALTY SECTION
This article was submitted to
Crop and Product Physiology,
a section of the journal
Frontiers in Plant Science
RECEIVED 17
June 2022
ACCEPTED 22 August 2022
PUBLISHED 08 September 2022
CITATION
Xue S, Wan X, Lu S, Zhong Y and Xie D
(2022) A time-course transcriptome
analysis of wax gourd fruit
development reveals predominant
genes regulating taste and nutrition.
Front. Plant Sci. 13:971274.
doi: 10.3389/fpls.2022.971274
COPYRIGHT
© 2022 Xue, Wan, Lu, Zhong and Xie.
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.
Wax gourd, which belongs to Cucurbitaceae, is an excellent plant resource
with the concomitant function of both medicine and foodstuff. Its unique
taste and rich nutrition are deeply accepted by consumers. However, the
main flavor and nutrients are still unclear, which restricts the quality breeding
process of wax gourd. Here, we discovered that monosaccharides, malic acid
and citrulline affect the flavor and nutrition of wax gourd and clarified the
dynamic accumulation process of these metabolites. To gain insights into the
underlying predominant genes regulating accumulation of these metabolites,
we performed a time-course transcriptome analysis using RNA-sequencing
analysis and compared the expression of screened genes among twentyfour germplasms with different metabolites levels. In addition, the expression
abundance among the homologous genes were also analyzed. Finally, a total
of 8 genes related to sugar [AGA2 (Bhi03G001926), SUS (Bhi12G001032)],
malic acid [MDH (Bhi12G001426, Bhi01G000427), PEPC (Bhi12G000721,
Bhi09G002867), ME (Bhi01G002616)] and citrulline [ASS (Bhi02G000401)],
respectively were determined. In summary, understanding the core genes
influencing taste or nutrition will provide a theoretical basis for fruit quality
improvement in wax gourd.
KEYWORDS
wax gourd, taste, nutrition, monosaccharides (glucose and fructose), malic acid,
citrulline, transcriptome
Introduction
Wax gourd [Benincasa hispida (Thunb.) Cogn.], known as ash gourd or winter
melon, is one of the most valuable plants in Cucurbitaceae family. Fruits of wax
gourd are valued for nutritional and medicinal purposes, which are rich in water
and valuable phytochemicals (sugars, organic acids, amino acid, vitamins, etc.)
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10.3389/fpls.2022.971274
dehydrogenase (MDH), and malic enzyme (ME). However,
there are no studies on the acid composition and associated key
enzyme genes involved in wax gourd.
Citrulline is naturally present in many cucurbits. Its
nutraceutical and therapeutic potentials have previous reported
in many researches. Citrulline, as the main amino acid of wax
gourd, has a significant effect on vasodilation, widely used to
treat high blood pressure (Huang et al., 2004; Nakashima et al.,
2011). The citrulline homeostasis is the composite outcome
of its synthesis and metabolism, inseparable from arginine
metabolism. Based on the current researches information
and gene annotations, the genes involved in the citrulline
biosynthesis include carbamoyl phosphate synthetase (),
NO synthase (NOS), Ornithine transcarbamoylase (OTC),
N- acetylglutamate synthase (NAGS), N-acetylglutamate
kinase (NAGK), N-acetylglutamatyl-5-P reductase (NAGPR),
N-acetylornithine aminotransferase (NAOAT), acetylornithine
deacetylases (NAOD), while genes involved in citrulline
catabolism consist of argininosuccinate synthases (ASS)
and arginosuccinate lyase (ASL) (Winter et al., 2015; Joshi
and Fernie, 2017). Other than watermelons, there is limited
information available about the citrulline content and closely
related genes in wax gourd.
Most wax gourd cultivars bear a giant fruit,which can weigh
normally over 20 kg (Xie et al., 2019) or in some cases up to 50 kg
(Dhillon et al., 2016). Thus, the main type of carbohydrate in
wax gourd catches our attention and this study will focus on the
types and proportion of sugar, acid and citrulline and the related
genes. For sugar, organic acid and citrulline, many studies
have previously reported about their biosynthesis, degradation
and developmental regulation of the genes/enzymes in some
Cucurbitaceae family members, but comprehensive metabolite
profiling of sugars, organic acids and citrulline in wax
gourd is lacking. Nowadays the high-quality wax gourd draft
genome sequence and the available databases of genomic
information [Cucurbit Genomics Database (CuGenDB)] would
likely provide a genetic information foundation for exploring
metabolism networks of flavor and nutrition metabolites (Xie
et al., 2019). Therefore, we performed comparative analysis
of primary metabolites and transcriptome changes during
the fruit developmental progress under significantly different
varieties, which would provide clear insights into the synthesis,
catabolism of sugar, organic acid and citrulline in wax gourd.
Furthermore, our study establishes a foundation for flavor and
nutritional quality improvement in wax gourd breeding.
(Palamthodi and Lele, 2014; Sun et al., 2018). Various studies
indicated that wax gourd had numerous therapeutic activities,
including antihypertensive, antioxidant, anti-obesity, antiviral,
anti-inflammatory and antitumor activities (Huang et al., 2004;
Kumar and Vimalavathini, 2004; Nakashima et al., 2011; Doshi
et al., 2015).
Wax gourd fruit stores a large amount of biomass in a
very short growth cycle, along with changes in carbohydrate
biosynthesis and metabolism (sugars and organic acid). Sugars
and organic acids also majorly directly affect the organoleptic
fruit quality and consumer acceptability. Soluble sugars, which
mainly composed of disaccharide sucrose and its two hydrolysis
products, the hexoses, such as glucose and fructose, are the
source of sweetness in the fruits of Cucurbitaceae, and different
sugar components result in different sweetness (Pangborn, 1963;
Yamaguchi et al., 1970; Doty, 1976). The different sweetness
of Cucurbitaceae family members mainly depends on the
type and composition of sugar. Among them, watermelon,
melon, pumpkin all have more sweetness than cucumber
owing to with more sucrose. (Corrigan et al., 2000; Hu
et al., 2009; Dai et al., 2011; Zhong et al., 2017; Gao et al.,
2018; Muhammad Jawad et al., 2020; Umer et al., 2020). For
several Cucurbitaceae species, many sugar genes associated with
unloading and metabolism during development have previous
reported, which composed of raffinose synthase and stachyose
synthase (Rennie and Turgeon, 2009), α-galactosidase (Liu et al.,
2022), soluble/insoluble acid invertase and neutral invertase
(Chrost and Schmitz, 1997), sucrose phosphate synthase (Li
et al., 2018), sucrose synthase, UDP-glucose 4-epimerase, UDPgalactose/glucose pyrophosphorylase, hexokinase, fructokinase,
phosphoglucomutase and phosphoglucoisomerase and so on
(Dai et al., 2011; Umer et al., 2020). Although genes related
to fruit sugar quality have been reported in some gourds, few
attempts have been made to detect the key genes that influence
wax gourd sugar phenotypic changes.
Organic acids as crucial factors influence organoleptic
quality and consumer demand. The types and quantity of
organic acids varies considerably among different Cucurbitaceae
family members. Citric acid is the predominant organic acid
found in melon, with levels of about only 0.2% fresh weight,
followed by malic acid levels (Leach et al., 1989; Wang et al.,
1996; Flores et al., 2001; Burger et al., 2003; Tang et al., 2010).
It was recognized that malic acid and citric acid serve as main
organic acids in the mature watermelon fruit (Gao et al., 2018;
Aslam et al., 2020; Muhammad Jawad et al., 2020; Umer et al.,
2020). Three organic acids (citric, malic, and fumaric) have been
identified in the pumpkin fruit with varied content in particular
species and cultivars (Nawirska-Olszanska et al., 2014). Acid
homeostasis is the outcome of acid synthesis and metabolism.
Organic acids are intermediates in the tricarboxylic acid (TCA)
cycle, associated with series of enzymes. These enzymes consist
of phosphoenolpyruvate carboxylase (PEPC), citrate synthase
(CS), aconitase (ACO), isocitrate dehydrogenase (IDH), malate
Frontiers in Plant Science
Materials and methods
Plant materials and fruit samples
Two varieties (B and G) with different quality phenotype
(flavor and nutrition) were employed in this study, which were
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Xue et al.
10.3389/fpls.2022.971274
citric acid and fumaric acid purchased from Sigma-Aldrich
(St. Louis, MO, United States) were used as calibration
standards.
HPLC-PDA equipped with a C18 column (Phenomenex,
Gemini C18, 250 × 4.6mm, 3 µm partial size) was carried
out to detect citrulline (Jayaprakasha et al., 2011). A sample of
20 mg lyophilized powder was extracted with MeOH/1N HCl
in a 30:1 v/v combination and the supernatants were filtered
through a 0.22-µm membrane filter. The column temperature
was set at 25◦ C and detection was performed at 207 nm, using
0.03mM phosphoric acid as the mobile phase with a flow rate
of 0.7 ml/min. L-citrulline purchased from Sigma-Aldrich (St.
Louis, MO, United States) were used as calibration standards.
planted in the research experiment field of Vegetable Research
Institute, Guangdong Academy of Agricultural Sciences. To
explore the dynamic changes of wax gourd fruits sugar, organic
acids and citrulline at time-course developmental stages, the
flesh samples on the middle section of two varieties fruits were
collected at 0, 5, 10, 20, 30 days after pollination (commercial
maturity stage), respectively. Twenty-four germplasms were
used to analyze the correlation between the expression of key
genes and the content of metabolites. Three or six individual
fruits from different plants were chosen at each time point
or different germplasms and all the samples were divided
into two subsets. One subset was freeze-dried to a powder
for sugar, organic acid and citrulline content determinations.
The other subset was immediately frozen in liquid nitrogen
and stored at −80◦ C for transcriptome analysis or RTPCR verification.
RNA-seq and read mapping
Total RNA was extracted from the frozen wax gourd flesh
at 0, 5, 10, 20, 30 DAP from B variety and 30 DAP from G
variety using a Huayueyang RNA extraction kit (Huayueyang,
Beijing, China) according to the manufacturer’s instructions.
At each time point, six fruit harvested from different plants
growing in consistent conditions were divided into two
biological replicates. The quantity, quality, and integrity of
the twelve RNA samples were determined with an Agilent
2100 Bioanalyzer and a Nanodrop NanoPhotometer. RNAseq was performed on an Illumina Nova Seq 6000 Platform
at Novogene Corporation Inc., and low-quality reads and
adapters were removed by the company. High-quality reads
were mapped to the wax gourd B227 reference genome1 (Xie
et al., 2019) using Hisat2 (v.2.0.5) (Kim et al., 2015) with
default settings. Gene expression was estimated in terms of
fragments per kilobase of transcript per million mapped reads
(FPKM).
Measurement of sugar, organic acid
and citrulline content in wax gourd
fruit flesh
The frozen fruit samples were lyophilized and used for
the following preparation. Determination of soluble sugars
was carried out according to Obando-Ulloa et al. (2009),
with slight modifications (Obando-Ulloa et al., 2009). Highperformance liquid chromatography (Alliance e2695 HPLC
system,Waters, Milford, MA, United States) coupled with a
refractive index detector (HPLC-RID) was applied to determine
the sugar profile of samples. The lyophilized powder (20 mg)
was extracted with 50% acetonitrile water and soluble sugar
concentration was determined in a medium polarity NH2
column (Waters Xbridge BEH Amide-4.6 × 250 mm, 5 µm
particle size), using acetonitrile/deionized water in a 80:20 v/v
combination as the mobile phase with a flow of 1 ml/min.
Glucose, fructose and sucrose purchased from Sigma-Aldrich
(St. Louis, MO, United States) were used as calibration
standards.
Organic acids, including malic acid, shikimic acid, citric
acid and fumaric acid, were detected according to Scherer et al.
(2012) with slight modifications (Scherer et al., 2012). Highperformance liquid chromatography (Alliance e2695 HPLC
system, Waters, Milford, MA, United States) coupled with
Waters 2998 photodiode array detector (HPLC-PDA) was
applied to determine organic acids. A sample of 20 mg
lyophilized powder was extracted with distilled deionized water.
Organic acids were determined in reverse-phase C18 column
(Waters Atlantis T3 C18 column, 250 mm × 4.6 mm i.d.,
5 µm particle size) operated at 25◦ C, using 5 g/l (NH4)2 HPO4
solution (pH 2.5)/methanol in a 97:3 v/v combination as the
mobile phase with a flow of 0.6 ml/min. Eluted compounds
were detected by UV absorbance at 214 nm and quantitated
by external linear calibration. Malic acid, shikimic acid,
Frontiers in Plant Science
RT-qPCR analysis
Total RNA was isolated using a Huayueyang RNA extraction
kit (Huayueyang) and then reverse transcribed using SuperReal
PreMix Plus (Tiangen, beijing) following the manufacturers’
protocol. RT-qPCR was conducted in 96-well plates with a
CFX connect Real-Time PCR Detection System (Bio-Rad,
United States) using the Ultra SYBR Mix kit (CWBIO, Beijing,
China). Three biological replicates and three technical replicates
were performed for each combination of cDNA samples
and primer pairs. Relative quantitative analysis of data was
performed by the 2−11Ct method with internal control gene
β-actin. The gene-specific RT-qPCR primers are listed in
Supplementary Table 9.
1
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Xue et al.
10.3389/fpls.2022.971274
FIGURE 1
The developmental process of two specific significantly different varieties (G and B) at 0, 5, 10, 20, 30 days after pollination, respectively.
Bar = 3 cm.
Statistical analyses
The sugar content of the two varieties was significantly different
at 30 DAP, showing that G was significantly higher than B.
The study found that the main organic acid in wax gourd
fruit is malic acid, and its accumulation pattern is "V"-shaped,
showing a trend of first decreasing from 0 to 10 DAP, and then
increasing steadily as the fruit matured. For fumaric acid, citric
acid, and shikimic acid, these acids are higher in the young fruit
stage and lower in the commercial maturity stage. The malic acid
content of the G variety at the later stage of development was
significantly lower than that of the B variety (Figure 2).
As indicated in Figure 2, there was an increase of citrulline
gradually during the course of fruit development. With fruit
enlargement and development, a maximum of citrulline content
was reached the final values of nearly 11.97 and 8.52 mg·g−1 dry
weight at 30 DAP, respectively for B and G.
The experimental data were analyzed using SPSS 23.0
(company, city, state abbrev if United States, country). For
weighted gene co-expression network analysis (WGCNA),
differentially expressed genes (DEGs) (coefficient of variation
(CV) > 0.5) were used to generate co-expression network
modules by WGCNA package in R. The co-expression modules
were obtained using automatic network construction function
(blockwiseModules) with default parameters, apart from the soft
threshold power of 10, TOMtype was signed, mergeCutHeight
was 0.25 and minModuleSize was 30. And the visio2021 and
Mev 4.2 software were used for drawing pathway and heat map
analysis of gene expression.
Result
The generation of fruit development
time course transcriptome data
Metabolism of sugar, acid and citrulline
in wax gourd during fruit development
To gain insights into wax gourd fruit development, we
generated time-coursed transcriptome data by sampling the
middle section flesh at 0 DAP, 5 DAP, 10 DAP, 20 DAP, 30
DAP from a representative wax gourd variety (B). To further
narrow down the key genes related to fruit flavor and nutrition,
another variety (G) at the commercial maturity stage 30 DPA
was also sampled.
A total of 559 million high-quality reads was generated and
mapped to the wax gourd B227 reference genome using Hisat2
(v.2.0.5) (Kim et al., 2015; Xie et al., 2019). About 7 Gb of clean
bases for each individual sample was obtained with more than
a 94% Q30 base percentage. And the average GC content was
Sugar increases gradually in the developmental stage of wax
gourd fruit (Figure 1). The main components of sugar in the
young fruit stage are fructose, glucose and sucrose, but in the
commodity maturity stage, fructose and glucose are the main
components, with sucrose reduced to an undetectable level. As
shown in the Figure 2, for B variety, with fruit enlargement
and development, fructose and glucose reached the maximum at
20 days after pollination, and decreased slightly at 30 days after
pollination. For G variety, hexoses (fructose, glucose) gradually
accumulated and reached the peak at 30 days after pollination.
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FIGURE 2
Soluble sugars (glucose, sucrose, and fructose), organic acids (malic acid, fumaric acid, citric acid, and shikimic acid) and citrulline in wax gourd
fruit at 0, 5, 10, 20, and 30 days after pollination between two specific significantly different varieties. Each value indicates mean ± standard
deviation (± SD) values of three biological replicates. The concentrations were expressed in milligram per gram dry weight (DW). Dark green and
light green represent B, G varieties with diverse genetic backgrounds.
43.14% for all libraries. An average of 92% reads were uniquely
mapped (Supplementary Table 1) and used to calculate
normalized gene expression level as fragments per kilobase of
transcript per million mapped reads (FPKM). A comparison of
the biological replicates showed that the expression values were
highly associated (Supplementary Figure 1), indicating a high
repeatability of the collection process. Therefore, the average
FPKM value was used for expression analysis. In addition,
six genes related with sugar, organic acid, citrulline metabolic
pathways were randomly selected for qRT-PCR. Most of the
genes showed a consistent expression profile between qRT-PCR
and RNA-seq (Supplementary Figure 2), which indicated that
the transcriptome data were of high quality and reliable.
Based on the pairwise comparison with | log2(fold
change)| ≥ 0 and FDR value < 0.05 as the threshold, a total of
937 (546 up- and 391 down-regulated), 6019 (3073 up- and 2946
down-regulated), 11691 (5664 up- and 6027 down-regulated),
Frontiers in Plant Science
10605 (5060 up- and 5545 down-regulated), 2519 (1301 upand 1218 down-regulated), 10222 (4927 up- and 5295 downregulated), 8269 (3852 up- and 4417 down-regulated), 7058
(3228 up- and 3830 down-regulated), 4544 (1753 up- and 2791
down-regulated), 7489 (3473 up- and 4016 down-regulated)
and 4718 (2356 up- and 2362 down-regulated) differentially
expressed genes (DEGs) were identified in B_5d vs. B_0d, B_10d
vs. B_0d, B_20d vs. B_0d, B_30d vs. B_0d, B_10d vs. B_5d,
B_20d vs. B_5d, B_30d vs. B_5d, B_20d vs. B_10d, B_30d vs.
B_10d, B_30d vs. B_20d and G_30d vs. B_30d, respectively
using DESeq version 2 (Supplementary Tables 2, 3).
GO enrichment analysis of wax gourd fruit developmental
progress was conducted, and the result indicated the
differentially expressed genes were mainly enriched in
cellular carbohydrate metabolic/biosynthetic process, cellular
glucan metabolic process, cellular polysaccharide metabolic
process, cellulose metabolic/biosynthetic process, amide
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FIGURE 3
Developmental expression profiles of genes of sugar metabolism in developing wax gourd fruit. AGA/GLA: alkaline/acid α-galactosidase; AI/NI:
acid/neutral invertase; SPP: sucrose-phosphatase; SPS: sucrose phosphate synthase; SUS: sucrose synthase; FK: fructokinase.
and sucrose synthase (Bhi11G001031, Bhi12G001032)
positively regulate monosaccharide content. In addition,
during fruit expansion and development, sucrose gradually
decreased, and the expression of sucrose-phosphatase genes
(Bhi11G002060, Bhi07G000567), which positively regulate
sucrose accumulation, also decreased with development.
Fructokinase is also an enzyme that negatively regulates
monosaccharides, among which Bhi11G001594 gradually
decreases with the developmental stage, and its expression level
in G variety is lower than that in B variety (Figure 3).
The taste of wax gourd and the composition and pattern
of sugar accumulation are similar to those of cucumber. In
the research of cucumber, the key enzymes regulating the
accumulation of hexose are mainly α-galactosidase, sucrose
synthase or invertase (Liu et al., 2022). Therefore, the
expressions of the screened 8 genes associated with wax
gourd monosaccharide from the above were compared among
thirteen germplasms with different sugar levels. Finally, only
BhAGA2 (Bhi03G001926) expression was positively correlated
with monosaccharide content, with a correlation coefficient of
0.695(P ≤ 0.01) (Figure 4A and Supplementary Figure 3).
In addition, by comparing the expression abundance of
these 8 genes in the homologous genes, it was also found
that, BhAGA2 (Bhi03G001926) has the highest expression
biosynthetic/metabolic process, peptide biosynthetic/metabolic
process during fruit development, as shown in Supplementary
Table 4 in detail.
Candidate genes associated with sugar
accumulation of wax gourd fruit
Wax gourd is rich in monosaccharides, which affect its
flavor and taste. In order to explore the key genes involved in
regulating sugar accumulation in wax gourd, we mapped the
KEGG pathway of sugar anabolism in wax gourd (Figure 3).
Based on transcriptome data and gene annotation, we
searched for all the homologous genes of key enzymes in
the pathway nodes (Supplementary Table 5). Based on
the expression difference between the two varieties at the
commercial stage of 30 DAP and the expression difference
in the developmental period of one variety, we found that
the enzymes that positively regulate the accumulation of
fructose and glucose are α-galactosidase, invertase, sucrose
synthase, but different homologous genes of the same
enzyme may have specific functions, of these, α-galactosidase
(BhAGA2 (Bhi03G001926), Bhi03G000736, Bhi04G000582),
invertase (Bhi12G000699, Bhi06G001349, Bhi01G000796)
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FIGURE 4
A total of 8 genes related to sugar [AGA2 (Bhi03G001926), SUS (Bhi12G001032)], malic acid [MDH (Bhi12G001426, Bhi01G000427), PEPC
(Bhi12G000721, Bhi09G002867), ME (Bhi01G002616)] and citrulline [ASS (Bhi02M000401)], respectively were determined, expression of which
have high correlation with metabolite contents. (A) Genes related to sugar; (B) genes related to citrulline; (C) genes related to malic acid.
abundance among the homologous genes and is a key
gene regulating the accumulation of monosaccharides in wax
gourd. Incredibly, one SUS-related gene (Bhi12G001032) is
Frontiers in Plant Science
highly negatively correlated with monosaccharides among
thirteen germplasm resources (r = −0.800, P ≤ 0.01)
(Figure 4A).
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Candidate genes associated with
organic acids accumulation of wax
gourd fruit
found that only Bhi02G001226 and Bhi06G000195 were the key
genes positively regulating fumaric acid (Figure 5).
Malic acid is the predominant acid component that affects
the taste of wax gourd, so the above eight screened genes
associated with malic acid, ME (Bhi01G002616, Bhi01G000110),
MDH (Bhi12G001426, Bhi01G000427, Bhi02G000222) and
PEPC (Bhi12G000721, Bhi09G002867, Bhi05G001633), were
compared about their expression among thirteen germplasm
resources with different malic acid content (ranging from
14.16 mg/g dry weight to 50.97 mg/g dry weight, showing
in Figure 4). Finally we found that the expression of MDH
(Bhi12G001426, Bhi01G000427) and PEPC (Bhi12G000721,
Bhi09G002867) were positively correlated with malic acid
content (correlation coefficient, r = 0.817, 0.731, 0.642, 0.769,
P ≤ 0.01, ≤0.01, ≤0.05, ≤0.01, respectively), the expression
of ME (Bhi01G002616) is negatively correlated with malic acid
content (correlation coefficient, r = −0.752, P ≤ 0.01). These five
genes are the most decisive genes regulating the accumulation
of malic acid in wax gourd (Figure 4C and Supplementary
Figure 4).
The main type of organic acid in wax gourd fruit is malic
acid, which shows a V-shaped accumulation trend during fruit
growth and development. In order to explore the key genes
involved in regulating the accumulation of organic acids in
wax gourd, we also draw the organic acid KEGG pathway
and compare the pathway associated genes (Figure 5 and
Supplementary Table 6). The study showed that malic enzyme
(ME) and malate dehydrogenase (MDH) are the key enzymes
involved in the regulation of malic acid, among which ME
is the enzyme responsible for cleaving malic acid. There are
6 ME homologous genes with different expression trends.
Among them, expressions of Bhi01G002616 and Bhi01G000110
present inverted V-shaped trend accompanied by negatively
correlation with malic acid during fruit developmental stages,
moreover Bhi01G002616 was found with the highest expression
abundance among the ME homologous genes, therefore it
is speculated as the core ME gene to regulate malic acid
accumulation (Figure 6). Malate dehydrogenase (MDH) is a
pivotal enzyme that positively regulates the accumulation of
malate. It has 7 homologous genes with different expression
trends. According to the difference in expression between the
two varieties in the commercial period of 30 days, it is found
that Bhi12G001426, Bhi01G000427 and Bhi02G000222 are the
regulators for promoting malic acid accumulation. Among
them, the expression of Bhi12G001426 is highly expressed
in MDH homologous genes (Figure 6). The homeostasis of
malic acid is regulated by both biosynthesis and catabolism.
Phosphoenolpyruvate carboxylase (PEPC) catalyzes the direct
conversion of phosphoenolpyruvate (PEP) to oxaloacetate
(OAA), thereby reducing the carbon flux to pyruvate and
other pathways, and is an upstream core gene that regulates
the accumulation of organic acids. PEPC has 3 homologous
genes (Bhi12G000721, Bhi09G002867, Bhi05G001633), mainly
participate in organic acids metabolism (Figure 5).
The accumulation of citric acid in wax gourd showed a
decreasing trend during the time course development, and the
young fruit stage was significantly higher than the commercial
fruit stage. Citrate synthase (CS) was the key enzyme affecting
the accumulation of citric acid. Among them, the expression
of Bhi10G000779 was positively correlated with the citric acid
content during development (Figure 5).
Fumaric
acid
showed
a
decreasing
trend
during development, among which nine succinate
dehydrogenase gene orthologs (SDH) were found to
be differentially expressed, of these, the expression of
Bhi02G001226,Bhi02G000310,Bhi06G000195 was positively
correlated with the accumulation of fumaric acid. In addition,
by comparing the trends of those gene expression and fumaric
acid content between 30 PDA of two materials (B and G), it was
Frontiers in Plant Science
Candidate genes associated with
citrulline accumulation of wax gourd
fruit
Citrulline showed an increasing trend during fruit
development. The pathway was drawn based on the clear
citrulline metabolism, and putative all homologous genes
likely to be involved in the citrulline metabolic pathway were
identified based on the wax gourd genomic and transcriptomic
database (Figure 7 and Supplementary Table 7). Based on
the expression difference between the two varieties at the
commercial stage of 30 days and the expression difference in the
developmental period of one variety, we found that the enzyme
genes that positively regulate the accumulation of citrulline
are N-acetylglutamate synthase (NAGS: Bhi03G000800),
N-acetylornithine aminotransferase (N-AOAT: Bhi09G000417),
and carbamoyl phosphate synthetase (CPS: Bhi09G001538)
(Figure 7). But gene expression was significantly uncorrelated
with citrulline content among natural germplasms with different
citrulline (ranging from 5.6 mg/g dry weight to 18.63 mg/g
dry weight) (Supplementary Figure 5). The breakdown of
citrulline in plants is triggered by two catabolic enzymes,
argininosuccinate synthases (ASS) and argininosuccinate
lyases (ASL) (Figure 7). Of these, only the expression of
one homologous gene ASS (Bhi02M000401) were negatively
correlated with citrulline content among germplasm resources
with different citrulline content (correlation coefficient,
r = −0.634, P ≤ 0.05) (Figure 4B and Supplementary
Figure 5). In addition, ASS (Bhi02M000401) had the highest
expression abundance, whereas the other two ASS-related genes
remained at low levels during all development stages (Figure 6).
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FIGURE 5
Developmental expression profiles of genes of organic acid biosynthesis and catabolism in developing wax gourd fruit. PEPC:
phosphoenolpyruvate carboxylase; PEPCK: phosphoenolpyruvate carboxykinase; MDH: malate dehydrogenase; ME: malic enzyme; PK:
pyruvate kinase; SDH: succinate dehydrogenase; FH: fumarate hydratase; CS: citrate synthase.
Co-expression network construction
metabolites, we screened out 80 TFs with highly correlated
with sugar gene AGA2 (Bhi03G001926) in the yellow module
and formed a correlation network (R > 0.80), among which,
genes with correlation coefficients greater than 0.95 include
14 TFs (2 C2H2s, 3 Dofs, 4 NACs, 1 ZF-HD, 2 MYBs,
1 GATA, 1 B3). And we identified 19 TFs (3 NACs, 4
MYBs, 1 ERF, 1 GATA, 2 Dofs, 1 SRS, 1 bHLH, 1 WRKY,
1 C2H2,1 LBD, 1 NF-YB, 1 bZIP) highly correlated with
malic acid genes MDH (Bhi01G000427) in the “brown”
module and 5 TFs (2 ERFs, 1 EIL, 1 RAV) highly correlated
with malic acid gene ME (Bhi01G002616) in the “purple”
module(R > 0.80), but there is no transcription factor
with a correlation coefficient greater than 0.8 with the ASS
gene in the “black” module (Figure 8 and Supplementary
Table 8). To sum up, these TFs, as highly connected
genes, may have regulatory effects on accumulation of
the taste and nutrition metabolites in wax gourd fruit
development.
To further reveal the gene regulatory network of taste
and nutrition metabolites in wax gourd fruits, weighted gene
co-expression network analysis (WGCNA) was performed on
the transcriptome data to investigate highly coordinated gene
sets during fruit development. A total of 21 co-expression
modules (each labeled with a different color) were identified
based on their similar expression patterns (Supplementary
Figure 6). In the above study, the key enzyme genes screened
based on metabolic pathways belong to different modules,
namely, sugar gene AGA2 (Bhi03G001926) belongs to the
“yellow” module, malic acid genes MDH (Bhi12G001426,
Bhi01G000427) belongs to the “darkred and brown” module,
malic acid gene ME (Bhi01G002616) belongs to the “purple”
module, and citrulline gene ASS (Bhi02G000401) belongs to
the “black” module. To further characterize the transcription
factors (TFs) that putatively regulate taste and nutrition
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FIGURE 6
RNA-Seq expression profiles of AGA/GLA, SUS, MDH, ME, ASS family genes at different developmental stages in B fruit from 0 to 30 DAP, G fruit
at 30 DAP.
Discussion
amino acid of wax gourd, was widely used in traditional
Chinese medicine to treat high blood pressure related to
the NO pathway. (Huang et al., 2004; Nakashima et al.,
2011).
Wax gourd, which belongs to Cucurbitaceae, is an excellent
plant resource that has the concomitant function of both
medicine and foodstuff. Elucidation of the key genes influencing
the fruit quality of wax gourd becomes essential because
of higher consumer demand for quality. Sensory quality is
mainly determined by fruit sugar and organic acid levels, in
addition to the volatile aromatic components. The sweetness
of wax gourd depends on fructose and glucose, and the acidity
depends on malic acid. These three substances generally affect
the flavor and taste of wax gourd. Citrulline, as the main
Frontiers in Plant Science
AGA2 as the key gene regulating sugar
accumulation in wax gourd
In wax gourd, the total sugar accumulated gradually
with the developmental period, which was consistent with
the sugar accumulation pattern of cucumber, watermelon
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FIGURE 7
Developmental expression profiles of genes of citrulline biosynthesis and catabolism in developing wax gourd fruit. NAGS: N-acetylglutamate
synthase; NAGK: N-acetylglutamate kinase; NAGPR: N-acetylglutamatyl-5-P reductase; NAOAT: N-acetylornithine aminotransferase; NAOD:
N-acetylornithine deacetylase; OTC: ornithine transcarbamylase; ASS: argininosuccinate synthase; ASL: argininosuccinate lyase; GS: glutamine
synthetase; CPS: carbamoyl phosphate synthetase; NOS; putative nitric-oxide synthase.
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FIGURE 8
Coexpression networks of TFs and enzyme genes involved in taste and nutrition metabolites. Center circles represent key enzyme genes
involved in soluble sugars (pink) and malic acid (dark-pink and orange) metabolism during fruit development. Outer circles with different colors
represents different families of transcription factors identified in the same module whose transcripts are correlated with expression of enzyme
genes. Red lines represent correlations higher than 0.95; dotted blank lines represents correlation between 0.8 and 0.95.
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showed an obviously negative correlation among natural
germplasms at commercially available ripe stage with different
sugar content Explanations for this may be that the different
regulation mechanism between young and mature fruit stage,
which needs further study.
and other cucurbit crops. Cucurbitaceae fruit sugar is first
assimilated by the source organ, transported to the sink organ
through the phloem, and finally accumulated in the fruit
(Chrost and Schmitz, 1997),and raffinose and stachyose are
the principal sugars for translocation from the source to
the sink in Cucurbitaceae rather than sucrose (Zhang et al.,
2012). Alpha-galactosidase is the main enzyme hydrolyzing
stachyose and raffinose and determining sink strength in
cucurbit plants (Gao and Schaffer, 1999). In Cucurbitaceae
two species of a-galactosidase have been identified: acid αgalactosidase activity with acid pH optimum and alkaline αgalactosidase activity with a distinct alkaline pH optimum
(Pharr and Sox, 1984). As the initial step in catabolism of
the raffinose oligosaccharides, the distribution and function
of these α-galactosidases seem to be different. In this study,
based on the expression differences of transcriptome data,
the expression trends of all α-galactosidase family genes in
the wax gourd genome were clarified, including four alkaline
α-galactosidases and six acid α-galactosidases. Among them,
the expression abundance of BhAGA2 (Bhi03G001926) gene
is extremely higher than that of the other 9 homologous
genes. The expression is highly correlated with sugar content
in developmental stages and in differentiated germplasm. It is
speculated to be a key factor regulating the sugar accumulation
in wax gourd fruit. Alkaline α-galactosidase which contributes
to sugar accumulation in fruit flesh has been reported in
some Cucurbitaceae, such as cucumber (Cucumis sativus
L.) and watermelon (Citrullus lanatus L.) and muskmelon
(Cucumis melo L.). In watermelon, based on a genome-wide
association study (GWAS) and functional analysis, the alkaline
α-galactosidase ClAGA2 functions in the hydrolysis of raffinose
family oligosaccharides (RFOs) and regulates the accumulation
of fruit sugar (Ren et al., 2021). In cucumber, CsAGA2 (alkaline
α-galactosidase 2) was involved in phloem unloading (Li et al.,
2021), meanwhile overexpression of CsAGA2 could alter the
sugar metabolism resulting in substantial accumulation of
monosaccharides, such as glucose and fructose (Liu et al.,
2022). In melon, predominantly CmAGA2, Homologous gene
of wax gourd BhAGA2, account for all of a-gal gene expression
in the fruit tissue functioning in key processes of galactosyloligosaccharide metabolism (Carmi et al., 2003; Dai et al.,
2011).
In contrast, the monosaccharides presented a noticeable
positive correlation with sucrose synthase (SUS: Bhi12G001032)
expression during the fruit time-course development, but
showed an obviously negative correlation among natural
germplasms at commercially available ripe stage with different
sugar content Explanations for this may be that the different
regulation mechanism between young and mature fruit stage,
which needs further study.
In contrast, the monosaccharides presented a noticeable
positive correlation with sucrose synthase (SUS: Bhi12G001032)
expression during the fruit time-course development, but
Frontiers in Plant Science
Five genes related-PEPC, MDH, ME as
the core genes regulating organic acid
accumulation in wax gourd
Fruit acidity is due to the presence of organic acids,
among which malic and citric acids are the main acids
found in most ripe fruits (Etienne et al., 2013). As the
main organic acid in watermelon, patterns of malic acid
accumulation from 10 to 34 DAP in fruit development
showed an inverted V shape, with decreasing trend in the
commercial maturity stage (Gao et al., 2018; Aslam et al.,
2020; Muhammad Jawad et al., 2020). Citric acid was the
main organic acid in melon fruit, and its accumulation showed
an increasing trend in the development process, but with
slightly decreasing in the ripening period (Flores et al., 2001;
Burger et al., 2003; Tang et al., 2010). Whether watermelon
or melon, the research on organic acids in young fruit stages
before 10 DAP is rarely reported. In our present study,
the accumulation of organic acids was well monitored at
successive developmental stages after ovary pollination (0, 5,
10, 20, 30 DAP). We found that wax gourd has a high
malic acid content at 0 DAP, and then gradually decreased,
reaching to minimum content around 10 ∼20 DAP, and
then increased steadily as the fruit matured. With wax gourd
fruit enlargement, the stage that biomass accumulates rapidly
is around 10 ∼20 DAP. At this stage, the cell division of
wax gourd is extremely fast, and cell skeleton structures
such as cell walls are produced in large quantities. The
cell wall is composed of a diversity of polysaccharides, such
as cellulose, hemicellulose, or pectin (Caffall and Mohnen,
2009). So the carbon flux to polysaccharides is dominant,
with less malic acid accumulated in the fruit at 10 ∼20
DAP. In addition, wax gourd fruit organic acid continued
to accumulate until maturity without reducing, which is
different from the accumulation pattern of other crops, such as
watermelon, melon (Burger et al., 2003; Muhammad Jawad et al.,
2020).
The processes involved in the metabolism and accumulation
of organic acid in fruit are under genetic control. Many studies
have helped decipher some of the mechanisms that control
acidity. MDH catalyzes the reversible conversion of malate
into OAA, the most likely direction being the synthesis
of malic acid (Sweetman et al., 2009; Etienne et al., 2013).
Based on our research, two MDH genes (Bhi12G001426,
Bhi01G000427) critical for regulating malate accumulation,
especially MDH (Bhi12G001426), which have more expression
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Transcription factors were identified
from coexpression network with key
enzyme genes related to taste and
nutrition metabolites
abundance among all homologous genes and were highly
positive correlated with malic acid content in different
wax gourd germplasms. In apple, the overexpression of
MdMDH gene (GenBank Accession No. DQ221207), the
homolog of wax gourd Bhi12G001426, contributed to
malate accumulation in the apple callus, which supporting
the involvement of MdMDH directly in malate synthesis
(Yao et al., 2011; Yu et al., 2021). Phosphoenolpyruvate
carboxylase (PEPC) was also indicated as the key enzyme in
fruit malic acid synthesis (Etienne et al., 2013). In our study,
the expression of two PEPC-related genes (Bhi12G000721,
Bhi09G002867) was positively correlated with the malic
acid content of different germplasm resources. Homeostasis
and accumulation of organic acids depend on synthesis
and degradation. Malic enzyme (ME) is considered an
important enzyme involved in malic acid degradation,
of which, ME gene (Bhi01G002616) with the highest
expressed abundance is highly negatively correlated with
malate content among diverse germplasm resources. It was
concluded that MDH (Bhi12G001426), two PEPC-related
genes (Bhi12G000721, Bhi09G002867) and ME (Bhi01G002616)
may participate in the regulation of malic acid and its
accumulation.
Weighted gene co-expression network analysis is useful
for identifying networks of co-expressed genes. By correlating
the patterns of transcript accumulation and metabolic enzyme
genes associated with the taste and nutrition metabolites,
we identified 80, 24 TFs in the “yellow,” “brown or purple”
modules, respectively with correlation coefficient > 0.80,
among which, TFs (Dof, NAC) in the “yellow” module with
correlation coefficients greater than 0.95 have been previously
reported to strongly influence sugar accumulation in pitayas
and watermelon (Wang et al., 2021; Mou et al., 2022). And TFs
(MYB, ERF, bHLH, NAC) in the “brown or purple” modules
with correlation coefficients greater than 0.80 were shown
previously involved in malic or citric acid accumulation (Li
et al., 2016, 2017; Hu et al., 2017; Yu et al., 2021). To further
characterize the transcription factors that putatively regulate
taste and nutrition metabolites, the potential binding affinity for
the promoters of metabolic enzyme genes need to be further
validated.
One argininosuccinate synthases as
catabolism pathway gene dominates
the citrulline accumulation
Conclusion
This study clarified the taste and nutritional composition
of wax gourd, as well as the accumulation patterns of these
substances during fruit development. Furthermore, based on the
time-course transcriptome analysis, the expression correlation
verification analysis among twenty-four germplasms with
different metabolites contents, and expression abundance of
homologous genes, finally dominant genes regulating wax gourd
monosaccharides, organic acids, and citrulline were identified.
Understanding the core genes that influence the concentration
of these substances deciding the taste and nutrition in fruit cells
is thus of primary importance for fruit quality improvement.
Citrulline is widely found in cucurbit crops (Fish and
Bruton, 2010; Hartman et al., 2019; Joshi et al., 2019). Based
on the comparative analysis of citrulline in seven cucurbit
crops (watermelon, melon, cucumber, bitter gourd, pumpkin,
loofah, and wax gourd), it was found that the content of
citrulline in wax gourd is rich, ranking in the forefront of
cucurbitaceae, second only to watermelon (unpublished data).
Citrulline in wax gourd accumulates gradually with the fruit
developmental period, which is consistent with the findings in
watermelon (Guo et al., 2013; Joshi et al., 2019). According
to the RNAseq profiles of Guo et al. (2013), the expression
of citrulline synthesis gene OTC remained steady throughout
the fruit development, however, genes involved in citrulline
degradation, two ASS genes were highly downregulated during
watermelon flesh development. The highly negative correlation
between catabolic genes and citrulline concentration suggests
that citrulline accumulation depended on decreased activities of
citrulline degradation rather than increased synthesis (Joshi and
Fernie, 2017). In our study, the expression of one ASS-related
gene (Bhi02M000401) was negatively correlated with citrulline
content of different germplasm resources. In addition, it had the
highest expression abundance among ASS homologous genes.
Therefore, it is inferred as the most core gene to regulate the
accumulation of citrulline.
Frontiers in Plant Science
Data availability statement
The original contributions presented in this study
are publicly available. This data can be found here:
NCBI, PRJNA857066.
Author contributions
SX, YZ, and DX conceived and designed the experiments. SX
and XW performed the experiments. SX, XW, SL, YZ, and DX
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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.
analyzed the data. SX wrote the manuscript. XW, SL, YZ, and
DX reviewed and revised the manuscript. All authors have read
and approved the final version of the manuscript.
Funding
Supplementary material
This work was supported by the Laboratory of Lingnan
Modern Agriculture Project (NT2021004), the Key Realm
R&D Program of Guangdong Province (2020B020220003
and 2018B020202007), the Agricultural Competitive Industry
Discipline Team Building Project of Guangdong Academy of
Agricultural Sciences (202103TD), the Science and Technology
Program of Guangzhou of China (202201010231), and
the Innovation Fund projects of Guangdong Academy of
Agricultural Sciences (202106) and Food nutrition and health
Collaborative Innovation Center of GDAAS (XTXM202205).
The Supplementary Material for this article can be
found online at: https://www.frontiersin.org/articles/10.3389/
fpls.2022.971274/full#supplementary-material
SUPPLEMENTARY FIGURE 1
Principle component analysis (PCA) of time-course transcriptome data.
SUPPLEMENTARY FIGURE 2
Validation of RNA-Seq results by qRT-PCR. The relative expression levels
of six genes related with sugar, organic acid, citrulline metabolic
pathways in five development stages of B variety by RNA-Seq using
FPKM method and by qRT-PCR using the 2−MMCt method.
SUPPLEMENTARY FIGURE 3
The expression of other genes related to sugar were compared among
numerous germplasm resources with different sugar content.
Conflict of interest
SUPPLEMENTARY FIGURE 4
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.
The expression of other genes related to malic acid were compared
among numerous germplasm resources with different
malic acid content.
SUPPLEMENTARY FIGURE 5
The expression of other genes related to citrulline were compared
among numerous germplasm resources with different citrulline content.
Publisher’s note
SUPPLEMENTARY FIGURE 6
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
Dendrogram showing co-expression modules (clusters) identified by
weighted correlation network analysis (WGCNA) across fruit
developmental stages. The major tree branches constitute 21 modules
labeled with different colors.
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Infecção hospitalar em unidade de tratamento intensivo de um hospital universitário brasileiro
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Original Article Original Article Rev. Latino-Am. Enfermagem
Mar-Apr 2010; 18(2):233-9
www.eerp.usp.br/rlae 1 RN, Faculty, Escola de Enfermagem, Universidade Federal de Minas Gerais, MG, Brazil. E-mail: adrianacoliveira@gmail.com.
2 RN, Faculty, New York University, College of Nursing, NY, USA. E-mail: nepircs@hotmail.com.
3 Nursing Undergraduate Student, Escola de Enfermagem, Universidade Federal de Minas Gerais, MG, Brazil. E-mail: rafeudes@
yahoo.com.br. ulty, Escola de Enfermagem, Universidade Federal de Minas Gerais, MG, Brazil. E-mail: adrianacoliveira@gmail.com 1 RN, Faculty, Escola de Enfermagem, Universidade Federal de Minas Gerais, MG, Brazil. E-mail: adrianacoliveira@gmail.com.
2 RN, Faculty, New York University, College of Nursing, NY, USA. E-mail: nepircs@hotmail.com.
3 Nursing Undergraduate Student, Escola de Enfermagem, Universidade Federal de Minas Gerais, MG, Brazil. E-mail: rafeudes@ Universidade Federal de Minas Gerais. Escola de Enfermagem ulty, Escola de Enfermagem, Universidade Federal de Minas Gerais, MG, Brazil. E-mail: adrianacoliveira@gmail.com
ulty, New York University, College of Nursing, NY, USA. E-mail: nepircs@hotmail.com. Corresponding Author:
Adriana Cristina de Oliveira
Universidade Federal de Minas Gerais. Escola de Enfermagem
Av. Alfredo Balena s/n
Bairro Santa Efigênia
CEP: 30130-100 Belo Horizonte, MG, Brasil
E-mail: adrianacoliveira@gmail.com 1 RN, Faculty, Escola de Enfermagem, Universidade Federal de Minas Gerais, MG, Brazil. E-mail: adrianacoliveira
2 RN, Faculty, New York University, College of Nursing, NY, USA. E-mail: nepircs@hotmail.com. ,
g
,
,
,
@g
, New York University, College of Nursing, NY, USA. E-mail: nepircs@hotmail.com. graduate Student, Escola de Enfermagem, Universidade Federal de Minas Gerais, MG, Brazil. E-mail: rafeudes@ 3 Nursing Undergraduate Student, Escola de Enfermagem, Universidade Federal de Minas Gerais, MG, Brazil. E-mail: rafeudes@
yahoo.com.br. Infecção hospitalar em unidade de tratamento intensivo de um hospital
universitário brasileiro Este estudo prospectivo objetivou determinar a incidência da infecção hospitalar (IH)
em uma unidade de terapia intensiva (UTI), sua associação com características clínicas
do paciente e sítios de ocorrência. Inclui-se 1.886 pacientes de UTI de um hospital
universitário, entre agosto de 2005 e janeiro de 2008. Utilizou-se, neste estudo, o teste
exato de Fisher e Risco Relativo. Foram identificadas 383 (20,3%) IH: 144 (37,6%) do
trato urinário, 98 (25,6%) pneumonia, 58 (15,1%) sepses, 54 (14,1%) do sítio cirúrgico
e 29 (7,7%) outras. A permanência média foi de 19,3 dias para pacientes com IH e 20,2
dias para colonizados com microrganismos resistentes. Registrou-se 39,5% óbitos entre
pacientes com IH (RR: 4,4; 3,4-5,6). A IH esteve associada a pacientes provenientes
de outras unidades da instituição/unidade de emergência, permanência superior a 4
dias de internação, infecção comunitária à internação, colonizados por microrganismos
resistentes, em uso de procedimentos invasivos e óbitos resultantes de IH. Descritores: Vigilância Epidemiológica; Infecção Hospitalar; Unidades de Terapia
Intensiva. Nosocomial Infection in an Intensive Care Unit in a
Brazilian University Hospital Adriana Cristina de Oliveira1
Christine Tassone Kovner2
Rafael Souza da Silva3 Adriana Cristina de Oliveira1
Christine Tassone Kovner2
Rafael Souza da Silva3 This prospective study aimed to determine the nosocomial infection (NI) incidence in an
Intensive Care Unit (ICU), its association with clinical characteristics and occurrence sites. It was carried out among 1.886 patients admitted in an ICU of a University Hospital, from
August 2005 to January 2008. Data analysis was done using Fisher’s test and Relative
Risk (RR). There were 383 NIs (20.3%). The infections were in the urinary tract (n=144;
37.6%), pneumonia (n=98; 25.6%), sepsis (n=58; 15.1%), surgical site (n=54; 14.1%)
and others (n=29; 7.7%). Hospitalization average was 19.3 days for patients with NI and
20.2 days for those with colonization by resistant microorganisms. The mortality was 39.5%
among patients with NI (RR: 4.4; 3.4-5.6). The NI was associated with patients originated
from other units of the institution/emergency unit, more than 4 days of hospitalization,
community infection, colonized by resistant microorganisms, using invasive procedures and
deaths resulting from NI. Descriptors: Epidemiologic Surveillance; Cross Infection; Intensive Care Units. 234 Infección hospitalaria en unidad de tratamiento intensivo de un
hospital universitario brasileño Este estudio prospectivo tuvo como objetivo determinar la incidencia de infección
hospitalaria (IH) en una Unidad de Terapia Intensiva (UTI), su asociación con características
clínicas del paciente y sitios de ocurrencia. Fueron incluidos 1886 pacientes de la UTI de
un hospital universitario, entre agosto de 2005 y enero de 2008. Se utilizó el test exacto
de Fisher y Riesgo Relativo. Fueron identificadas 383 (20,3%) IH: 144 (37,6%) del
tracto urinario, 98 (25,6%) neumonía, 58 (15,1%) sepsis, 54 (14.1%) sitio quirúrgico
y 29 (7,7%) otras infecciones. La permanencia promedio fue 19,3 días para pacientes
con IH y 20,2 días para colonizados con microorganismos resistentes. Se registró 39.5%
óbitos entre pacientes con IH (RR: 4,4; 3,4-5,6). La IH estuvo asociada a pacientes
provenientes de otra unidad de la institución/unidad de emergencia, internación mayor
que 4 días, con infección comunitaria, colonizados por microorganismos resistentes, uso
de procedimientos invasivos y óbitos resultantes de IH. Descriptores: Vigilancia Epidemiológica; Infección Hospitalaria; Unidades de Terapia
Intensiva. Introduction ICU nosocomial infections are primarily related to
the patient’s health status, invasive device utilization such
as venous central line, long term urinary catheterization
and mechanical ventilation, use of imunosupressors,
prolonged hospitalization, colonization by resistant
microorganisms, antibiotics prescription and the setting
itself which propitiate bacterial natural selection(2-5). The 21st century reveals a new healthcare scenario
as result of scientific and technological progress. New
infectious agents are documented and infections resurged
with new strength(1), especially in Intensive Care Units
(ICU). Nosocomial infections (NI) are more severe in
these high technology hospital units which hold acutely
ill patients needing intensive life support(1-3). www.eerp.usp.br/rlae 235 Oliveira AC, Kovner CT, Silva RS. ICU nosocomial infection rate varies from 18 to
54%, five to ten times higher than other hospital units’
rates. It is responsible for 5 to 35% of all NI and for
approximately 90% of all outbreaks of diseases in an
ICU(2-3,6-7). The ICU high mortality rates, commonly
ranging from 9 to 38%, can reach 60% due to nosocomial
infection occurrence(5,8). colonization/infection by resistant microorganisms is a
routine at the study hospital according to this criteria:
patients from the emergency unit, patients transferred
from other hospitals with hospitalization greater or
equal than 72 hours or patients from the own hospital
transferred to the ICU with prior stay greater or equal
than seven days(12); - Nosocomial infection: any notified infection in an ICU,
after 48 hours of admission in the Unit, or 48 hours after
patient’s discharge. Urinary tract infections which appear
up to seven days after discharge and are associated with
long term catheterization are considered NI as well. This study aimed to determine the nosocomial
infection incidence in an ICU, its association with
clinical
characteristics
(gender,
age,
provenance,
medical profile, ICU length-of-stay (days), community
infection, colonization by resistant microorganisms,
invasive devices use, and deaths) and occurrence sites. As secondary objective, the study aimed to identify the
more common microorganisms responsible for NI and
their resistance status. Before data collection, the research was approved
by the institutional review board, protocol 267/2003,
in compliance with Resolution 196/96 of the National
Health Council, which regulates research involving
human subjects. Data were collected by a trained
nursing undergraduate student, from medical charts and
patients’ microbial count. Results of the present study contribute to support
results of other studies on NI and may serve as
comparison with other health establishments’ NI rates. Introduction The study adds knowledge on ICU infection rate and
emphasizes the importance of performing the control of
its related outcomes. The collected data included gender, age, origin,
medical profile, ICU length-of-stay, community infection,
colonization during the stay in the ICU by resistant
microorganisms, invasive device utilization, nosocomial
infection, deaths and NI´ occurrence sites. www.eerp.usp.br/rlae Material and methods Data were entered in the Statistical Program for
Social Sciences (SPSS, version 13.0) and analyzed using
Fisher’s Exact Test to verify association of the NI with
clinical characteristics and demographic variables, and
Relative Risk (RR). Confidence Interval (CI) of 95% and
statistical significance of 0.05 was established. The study design was prospective, descriptive and
epidemiological. Data was collected in an adult ICU of
a University Hospital. The Unit has 18 beds, among
which two are specially equipped for patient isolation. Staff team is composed by 10 medical doctors and
four residents, four physical therapists, 12 registered
nurses and 71 licensed nurses, a total of 101 healthcare
professionals. Almost all (90%) of all admissions are
paid by the Unified National Health System and the
remainder are paid by private health insurances or by
the own patient. Demographic characteristics of the sample A total of 1.889 patients were admitted in the ICU
during data collection, and 1.886 (99.3%) were eligible
for the study. Table 1 presents demographic and clinical
data of the sample. The sample included all the patients who were
admitted in the ICU from August 2005 to January
2008 (N=1.889). Exclusion criteria was uncompleted
medical records (n=03). Definitions from the National
Nosocomial Infection Surveillance System (NISS) were
used(9-11) as follows: The overall average for the ICU stay was 5.7 days
(median= 3 days). The average for patients who did not
acquire NI was 3.7 days (median= 3 days) and 19.3
days (median= 13 days) for those who had NI. For the
non-colonized patients, the average ICU stay was 3.8
days (median= 3 days) and 20.2 days (median= 14
days) for those colonized by resistant microorganisms. The majority (n=1.407; 74.6%) used invasive
device, among which urinary catheter (68.5%), venous
central line (49.6%) or mechanical ventilation (49.9%). The averages of days under the devices were 5.2, 6.6
and 5.3, respectively. The overall average for the ICU stay was 5.7 days
(median= 3 days). The average for patients who did not
acquire NI was 3.7 days (median= 3 days) and 19.3
days (median= 13 days) for those who had NI. For the
non-colonized patients, the average ICU stay was 3.8
days (median= 3 days) and 20.2 days (median= 14
days) for those colonized by resistant microorganisms. - Community infections: all notified infections at the
patient’s ICU admittance, whether the infection have
appeared in another hospital, another hospital ward or
outside any hospital establishment; - Community infections: all notified infections at the
patient’s ICU admittance, whether the infection have
appeared in another hospital, another hospital ward or
outside any hospital establishment; The majority (n=1.407; 74.6%) used invasive
device, among which urinary catheter (68.5%), venous
central line (49.6%) or mechanical ventilation (49.9%). The averages of days under the devices were 5.2, 6.6
and 5.3, respectively. - Surgical patients: patients who underwent any
surgical procedure, with incision and suture, including
videolaparoscopy, in an Operation Room; - Colonization by resistant microorganisms: isolation
identified by laboratorial culture. The notification of - Colonization by resistant microorganisms: isolation
identified by laboratorial culture. The notification of Rev. Latino-Am. Enfermagem 2010 Mar-Apr; 18(2):233-9. 236 istribution of the sample according to the study variables, (Aug. 2005 – Jan. 2008). Demographic characteristics of the sample Brazil, 2008 Table 1 – Distribution of the sample according to the study variables, (Aug. 2005 – Jan. 2008). Brazil, 2008
Variables
Categories
ICU (N=1.886)
N
%
SD
Gender
Male
999
53.0
-
Female
887
47.0
-
Age
Mean (years)/Median
53.3
55
17.2
Without infection
53
55
17.3
With infection
54.7
56
16.0
Source of admission
Other hospital units
917
48.6
-
Community
797
42.3
-
Emergency department
158
8.4
-
Other hospitals
14
0.7
-
Patient medical profile
Medical
550
29.2
-
Surgical
1336
70.8
-
Number of days in ICU
Mean (days)/Median
5.7
3
9.7
Without infection
3.7
3
3.9
With infection
19.3
13
20.3
Community infection
No
1545
81.9
-
Yes
341
18.1
-
Colonized patient during ICU stay
No
1660
88.0
-
Yes
226
12.0
-
Invasive device used
No
479
25.4
-
Yes
1407
74.6
-
Patients with nosocomial infections
No
1640
87.0
-
Yes
246
13.0
-
Outcomes
Discharges from ICU*
1691
89.7
-
Deaths
195
10.3
-
* Home discharge, discharge to other wards or hospital transference. SD: Standard deviation mple according to the study variables, (Aug. 2005 – Jan. 2008). Brazil, 2008 Urinary infection was the commonest type of
NI with 144 (37.6%) cases, followed by pneumonia
(n=98; 25.6%), sepsis (n= 58; 15.1%), surgical site
(n=54; 14.1%) and others (n=29; 7.7%) (vascular,
eye, ear, mouth, nose and throat, skin, reproductive and
gastrointestinal systems), as depicted in Figure 1. Nosocomial infections incidence and patients
outcomes The ICU nosocomial infection rate was twenty percent
(383 NIs); there were 246 (13.0%) patients infected and
approximately 10% (n=195) of the patients died. Subtitle: UTI: Urinary Tract Infections; PNM: Pneumonia; SSI: Surgical Site Infections; CVS:
Cardiovascular; EENT: Eye, Ear, Nose and Throat, REPR: Reproductive; GI: Gastrointestinal. Figure 1 – Distribution of the Intensive Care Unit Nosocomial Infections, according to the type of infection, (Aug. 2005 – Jan. 2008). Brazil, 2008
N
%
140
120
100
80
60
40
20
0
UTI
PNM
Sepsis
SSI
CVS
EENT
Skin
REPR
GI
Nosocomial infection sites
Infections frequencies Figure 1 – Distribution of the Intensive Care Unit Nosocomial Infections, according to the type of infection, (Aug. 2005 – Jan. 2008). Brazil, 2008 Figure 1 – Distribution of the Intensive Care Unit Nosocomial Infections, according to the type of infection, (Aug. 2005 – Jan. 2008). Brazil, 2008 www.eerp.usp.br/rlae 237 Oliveira AC, Kovner CT, Silva RS. Oliveira AC, Kovner CT, Silva RS. those who came from the community. The distribution of the patients with and without
infection is shown in Table 2. Most patients (n=1.075)
were hospitalized at the studied hospital prior to
admission in the ICU, and among them 177 (16.5%)
developed NI. Those transferred from the hospital
emergency unit were more likely to acquire infection
(RR: 2.6; CI: 1.8-3.7, p<0.05), than those who came
from the community. Also, a relative risk of 1.9 (CI:
1.4-2.4, p<0.05) was verified for those who came from
another units within the hospital, when compared with As reported by other studies in the field, the
length-of-stay for more than four days, the episode
of community infection, the colonization by resistant
microorganisms, and the use of invasive devices were
significantly associated with the occurrence of NI, with
high relative risks indexes (more than 2.4). Furthermore,
among the 195 deaths in the ICU during the study, 77
patients (39.5%) had developed NI. Table 2 – Comparison of the patients with and without nosocomial infection, (Aug. 2005 – Jan. 2008). www.eerp.usp.br/rlae Nosocomial infections incidence and patients
outcomes Brazil, 2008
Variables
Category
Intensive Care Unit
Nosocomial infection
P*
R.R**
N = 246
No (%)
Yes (%)
Gender
Female
766 (86.4)
121 (13.6)
1.0
Male
874 (87.5)
125 (12.5)
0.46
0.9[0.7-1.2]
Age
11-55
840 (87.9)
116 (12.1)
1.0
56-99
800 (86.0)
130 (14.0)
0.26
1.2[0.9-1.5]
Provenance
Community
729 (91.5)
68 (8.5)
1.0
Other hospital units
774 (84.4)
143 (15.6)
<0.01
1.9[1.4-2.4]
Emergency department
124 (78.5)
34 (21.5)
<0.01
2.6[1.8-3.7]
Other hospital
13 (92.9)
1 (7.1)
0.75
0.9[0.1-5.7]
Medical profile
Medical
480 (87.3)
70 (12.7)
1.0
Surgical
1160 (86.8)
176 (13.2)
0.85
1.0[0.8-1.3]
ICU length-of-stay (days)
1-3
1606 (92.4)
132 (7.6)
1.0
>4
34 (23.0)
114 (77.0)
<0.01
10.2[8.5-12.3]
Community infection
No
1383 (89.5)
162 (10.5)
1.0
Yes
257 (75.4)
84 (24.6)
<0.01
2.4[1.9-3.0]
Colonization by resistant
No
1552 (93.5)
108 (6.5)
1.0
microorganisms
Yes
88 (38.9)
138 (61.1)
<0.01
9.5[7.7-11.7]
Invasive devices use
No
472 (98.5)
7 (1.5)
1.0
Yes
1168 (83.0)
239 (17.0)
<0.01
11.6[5.5-24.4]
Deaths
No
1522 (90.0)
169 (10.0)
1.0
Yes
118 (60.5)
77 (39.5)
<0.01
4.4[3.4-5.6]
*p-value
**Relative Risk Table 2 – Comparison of the patients with and without nosocomial infection, (Aug. 2005 – Jan. 20 son of the patients with and without nosocomial infection, (Aug. 2005 – Jan. 2008). Brazil, 2008 parison of the patients with and without nosocomial infection, (Aug. 2005 – Jan. 2008). Brazil, 2008 Discussion a risk factor for infection development (p<0.01; RR:
10.2; CI: 8.5-12.3). For a period from four to 11 days
after the 17th ICU hospitalization day, the risk for NI
development increased. Nosocomial infection rate
for patients hospitalized from 17 to 21 days was 1.1,
while for those hospitalized from 43 to 134 days the
infection rate was 3.1, result similar to Latin-American
and European studies, which demonstrated relationship
between longer hospitalization and infection(7,9,15). a risk factor for infection development (p<0.01; RR:
10.2; CI: 8.5-12.3). For a period from four to 11 days
after the 17th ICU hospitalization day, the risk for NI
development increased. Nosocomial infection rate
for patients hospitalized from 17 to 21 days was 1.1,
while for those hospitalized from 43 to 134 days the
infection rate was 3.1, result similar to Latin-American
and European studies, which demonstrated relationship
between longer hospitalization and infection(7,9,15). Patients who were derived within and hospitalized
prior to ICU admission had a risk of 1.93 (CI: 1.48-2.49)
higher for acquiring NI in the ICU when compared to
those admitted from the community. Consistent with our
findings, results of studies developed in North America,
using similar sample, showed double risk for developing
ICU nosocomial infection for patients transferred from an
emergency room or other hospital unit than those who
came from the community, or from another hospital(13-14). Out of the 341 patients who came from the community,
84 (24.6%) developed NI (p<0.01 RR: 2.4 CI: 1.9-3.0). Studies report that patients with any infection diagnosis by Among the 151 patients who had hospital length
of stay greater than four days, 114 (77%) developed
NI, indicating that prolonged hospitalization constitutes 238 Rev. Latino-Am. Enfermagem 2010 Mar-Apr; 18(2):233-9. the ICU admission had higher risk of developing NI in the
Unit compared with those who did not have prior infection
(RR: 0.25; CI: 0.07-0.86; p <0.05)(16). nosocomial pneumonia is the second commonest NI and
the most common NI cause of death. This is consistent
with results of the present study, where pneumonia was
present in 25.6% (n=98) of NI, however less common
than UTI(8,17-18). Patients colonized by resistant microorganisms
evidenced a high NI rate (61.6%) and had a relative
risk of 9.5 (CI: 7.7-11.7; p <0.05) when compared with
those who did not have colonization by the resistant
microorganisms. Conclusion Nosocomial infection rate was 20.3% in 246 patients. The ICU NI rate in the present study was consistent
with the literature. The commonest infection was in the
Urinary tract, followed by pneumonia, sepsis, surgical site
infection and others (vascular, eye, ear, mouth, nose and
throat, skin, reproductive and gastrointestinal systems). The average hospitalization was 19.3 days for
patients with NI and 20.2 days for those with colonization
by resistant microorganism. The mortality rate was
39.5% among patients with NI (RR: 4.4; 3.4-5.6). The
NI was associated with patients originated from other
units of the institution/emergency unit, more than 4
days of hospitalization, community infection, colonized
by resistant microorganisms, using invasive devices and
deaths resulting from NI. Nosocomial infection rate was 20.3% in 246 patients. The ICU NI rate in the present study was consistent
with the literature. The commonest infection was in the
Urinary tract, followed by pneumonia, sepsis, surgical site
infection and others (vascular, eye, ear, mouth, nose and
throat, skin, reproductive and gastrointestinal systems). The more frequent non resistant microorganisms
responsible for the NI were Candida albicans (18.5%),
Escherichia coli (15.1%), Pseudomonas aeruginosa
(8.9%), Enterobacter cloacae (8.2%), and Enterococcus
faecalis (8.2%). Among the more frequent multi-drug
resistant microorganisms causing NI were identified
Acinetobacter
baumannii
(35.1%),
Pseudomonas
aeruginosa (21.6%), and MRSA, Klebsiella pneumonia
and Escherichia coli (10.8%). The average hospitalization was 19.3 days for
patients with NI and 20.2 days for those with colonization
by resistant microorganism. The mortality rate was
39.5% among patients with NI (RR: 4.4; 3.4-5.6). The
NI was associated with patients originated from other
units of the institution/emergency unit, more than 4
days of hospitalization, community infection, colonized
by resistant microorganisms, using invasive devices and
deaths resulting from NI. Most of the patients used at least one invasive device. Approximately 70% (n=1.292) of the patients used long
term urinary catheter, 49.9% (n=942) used mechanical
ventilation and 49.6% (n=935) venous central line,
demonstrating high device utilization in the Unit. The most common non resistant microorganisms
responsible for the NI were Candida albicans, Escherichia
coli and Pseudomonas aeruginosa. And those identified
as most frequent multi-drug resistant microorganisms
causing
NI
were
Acinetobacter
baumannii
and
Pseudomonas aeruginosa. Researchers report that urinary tract infection
(UTI) associated with long term urinary catheterization
accounts for 8 to 35% of all ICU infections. The most
prevalent urinary infection found in the studies were
asymptomatic bacteriuria(4,8,14). Discussion In the sample were isolated 343 cases
of colonization (an average of 1.5 per patient), by 13
different resistant microorganisms; the five most common
represented more than 90% of cases (Acinetobacter
baumannii, 36.3%; Pseudomonas aeruginosa, 21.9%;
MRSA, 14.7%; Klebsiella pneumonia, 11.0%; and
Escherichia coli, 7.8%). However, this does not mean
that the NI was caused by the isolated resistant
microorganisms, only that the colonization in many
situations was associated with the presence of the same
or other microorganisms in the NI. Among the 195 deaths (10.3%), 39.5% (n=77)
were patients who developed NI, a datum consistent
with findings of others researches that found a
positive relation between higher mortality rates and NI
diagnosis(5,7-8). Based on our findings, we suggest periodical
discussions among the healthcare team about NI rates,
resistant microorganism profile at the institution, and
mortality rate associated with them, in seminars, clinical
meetings, or training sessions. Moreover, to encourage team
participation and raise awareness about the importance of
NI prevention campaigns. Those activities can contribute to
healthcare management, allow an overview of tendencies
and fluctuations of infections, and provide information for
the creation and review of protocols. Indeed, in some cases the resistant microorganisms
identified as causing the NI were different. In 146 of 383
cases the NI was caused by 25 different non resistant
microorganisms, in 74 cases the diagnosed NI was
caused by at least 9 different resistant microorganisms,
and in 163 cases of NI it was not clear from the records
what was the specific microorganism neither the
resistance status. www.eerp.usp.br/rlae References epidemiologia, prevenção e controle. Rio de Janeiro: Guanabara
Koogan; 2005. p. 15-33. 1. Lima ME, Andrade D, Haas VJ. Avaliação prospectiva da
ocorrência de infecção em pacientes críticos de Unidade de
Terapia Intensiva. Rev Bras Ter Intensiva 2007; 19(3):342-7. 1. Lima ME, Andrade D, Haas VJ. Avaliação prospectiva da
ocorrência de infecção em pacientes críticos de Unidade de
Terapia Intensiva. Rev Bras Ter Intensiva 2007; 19(3):342-7. 2. Allen S. Prevention and control of infection in the ICU. Curr
Anaesth Crit Care. 2005; 16(4):191-9. 11. Azevedo FM, Paiva LFR. O laboratório no controle de infecção
hospitalar. In: Martins MA. Manual de infecção hospitalar:
epidemiologia, prevenção e controle. Belo Horizonte: Medsi;
2001. p .435-48. 2. Allen S. Prevention and control of infection in the ICU. Curr
Anaesth Crit Care. 2005; 16(4):191-9. 3. Gusmão MEN, Dourado I, Fiaccone Rl, Salvador C. Nosocomial
pneumonia in the intensive care unit of a brazilian university
hospital: na analysis of the time span from admission to disease
onset. Am J Infect Control 2004; 32(4):209-14. 12. Oliveira AC, Horta B, Martinho GH, Dantas LV, Ribeiro MM. Nosocomial infections and bacterial resistance in patients from a
Teaching Hospital Intensive Care Unit. Online Braz J Nurs, 2007;
6:1-132. 4. Wagenlehner FME, Loibl E, Vogel H, Naber KG. Incidence of
nosocomial urinary tract infections on a surgical intensive care
unit implications for management. Int J Antimicrob Agents 2006;
28(1):86-90. 13. Urli T, Perone G, Acquarolo A, Zappa S, Antonini B, Candiani A. Surveillance of infections acquired in intensive care: usefulness
in clinical practice. J Hosp Infect 2002; 52(2):130-5. 14. McCusker ME, Périssé ARS, Roghmann MC. Severity-of-
illness markers as predictors of nosocomial infection in adult
intensive care unit. Am J Infect Control 2002; 30(3):139-44. 5. Colpan A, Akinci E, Erbay A, Balaban N, Bodur H. Evaluation
of risk factors for mortality in intensive care units: a prospective
study from a referral hospital in Turkey. Am J Infect Control
2005; 33(1):42-7. 15. Orsi GB, Scorzolini L, Franchi C, Mondillo V, Rosa F. Denditti
M. Hospital-acquired infection surveillance in a neurosurgical
intensive care unit. J Hosp Infect 2006; 64(1):23-9. 6. Espósito S, Leone S. Antimicrobial treatment for Intensive Care
Unit (ICU) infections including the role of the infectious disease
specialist. Int J Antimicrob Agents 2007; 29(5):494-500. 16. Beyersmann J, Gastmeier P, Grundmann H, Bärwolff S,
Geffer C, Behnke M, et al. Conclusion In the present study,
UTI were responsible for 37.6% (n=144) of all reported
ICU infections, among which 76.4% (n=110) were
asymptomatic and 33.6% (n=34) were symptomatic. The results of this study contribute to support other
studies on NI, and reaffirm the importance of an effective
program for infection control with the involvement
of healthcare workers. The study adds knowledge on
ICU infection rate and emphasizes the importance of
performing the control of its related outcomes such
as patient risk, mortality and occurrence of resistant
microorganisms. NNIS (National Nosocomial Infections Surveillance
System) data indicates that pneumonias account for
31% of all ICU infections. Some authors believe that www.eerp.usp.br/rlae 239 Oliveira AC, Kovner CT, Silva RS. References Transmission-associated nosocomial
infections: prolongation of intensive care unit stay and risk
factor for analysis using multistate models. Am J Infect Control
2008; 36(2):98-103. 7. Rosenthal VD, Guzman S, Orellano PW. Nosocomial
infections in medical-surgical intensive care units in Argentina:
attributable motality and lenght of stay. Am J Infect Control
2003; 31(5):291-5. 8. Vincent JL. Nosocomial infections in adult intensive care units. Lancet 2003; 361(9374): 2068-77. 17. Zeitoun SS, Barros ALBL, Diccinia S. Incidência de pneumonia
associada à ventilação mecânica em pacientes submetidos à
aspiração endotraqueal pelos sistemas aberto e fechado: estudo
prospectivo – dados preliminares. Rev Latino-am Enfermagem
2001; 9(1):46-52. 9. Martins MA. Conceitos gerais e terminologia básica em
epidemiologia hospitalar e controle de infecção. In: Martins
MA. Manual de infecção hospitalar: epidemiologia, prevenção e
controle. Belo Horizonte: Medsi; 2001. p. 16-26. 10. Rezende EM, Santos AAM, França E. Vigilância epidemiológica
das infecções hospitalares. In: Oliveira AC. Infecções hospitalares: 9. Martins MA. Conceitos gerais e terminologia básica em
epidemiologia hospitalar e controle de infecção. In: Martins
MA. Manual de infecção hospitalar: epidemiologia, prevenção e
controle. Belo Horizonte: Medsi; 2001. p. 16-26. 18. Mesiano ERAB, Merchán-Hamann. Bloodstream infections
among patients using central venous catheters in intensive care
units. Rev Latino-am Enfermagem 2007; 15(3):453-9. 10. Rezende EM, Santos AAM, França E. Vigilância epidemiológica
das infecções hospitalares. In: Oliveira AC. Infecções hospitalares: 10. Rezende EM, Santos AAM, França E. Vigilância epidemiológica
das infecções hospitalares. In: Oliveira AC. Infecções hospitalares: www.eerp.usp.br/rlae
Received: Mar. 30th 2009
Accepted: Oct. 13rd 2009 Received: Mar. 30th 2009
Accepted: Oct. 13rd 2009 www.eerp.usp.br/rlae
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https://openalex.org/W3112807204
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https://revistas.uam.es/bajopalabra/article/download/bp2020_24_024/12876, https://dialnet.unirioja.es/descarga/articulo/7695895.pdf
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La significación desde la autoexpresión del Logos divino en Buenaventura: Entender la Unidad en la Pluralidad
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Bajo palabra
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cc-by
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Significance from the self-expression of the divine
Logos in Bonaventure: Understanding Unity in Plurality
La significación desde la autoexpresión
del Logos divino en Buenaventura:
Entender la Unidad en la Pluralidad
Manuel Lázaro Pulido
Universidad Nacional de Educación a Distancia, España
Universidad Bernardo O’Higgins, Chile
mlazarop@fsof.uned.es
DOI: https://doi.org/10.15366/bp.2020.24.024
Bajo Palabra. II Época. Nº 24. Pgs: 475-492
Recibido: 26/02/2019
Aprobado: 12/08/2020
Resumen
Abstract
El cristianismo realizó una lectura
cristiana del logos clásico situándolo en
el prólogo del evangelio de san Juan. En
este estudio exponemos el modo en que
Buenaventura realiza una reflexión sobre
el logos en tanto que verbum que es en
realidad una teología del logos divino y,
por lo tanto, una teología cristológica
dentro del misterio del ser y la bondad
de la divina Trinidad. Esto supone que la
teología del logos se manifiesta ad intra
en la unidad desde la teología trinitaria,
usando los parámetros propios de la filosofía emanativa (de emanatione) y ad
extra en la pluralidad desde la teología
de la creación, usando la filosofía ejemplarista (de exemplaritate). La metafísica
bonaventuriana intenta así explicar los
planos metafísicos y el dinamismo de la
unidad y la multiplicidad llevando a la
consumación (de consumatione) como el
lugar definitivo de la expresión del logos,
completando así la estructura metafísica
de Buenaventura en tanto que entitativa
(quidditas) y significativa (significatio).
Christianity made a Christian reading
of the classical logos by placing it in the
prologue of the Gospel of Saint John. In
this study we expose the way in which
Buenaventura makes a reflection on the
logos as verbum that is actually a theology of the divine logos and, therefore, a
Christological theology within the mystery of the being and the goodness of
the divine Trinity. This supposes that the
theology of the logos manifests ad intra
in unity from Trinitarian theology, using
the parameters proper to the emanative
philosophy (de emanatione) and ad extra in the plurality from the theology of
creation, using exemplary philosophy (de
exemplaritate). Bonaventurian metaphysics thus tries to explain the metaphysical planes and the dynamism of unity
and multiplicity leading to the consummation (de consummatione) as the definitive place of the expression of the logos,
thus completing the metaphysical structure of Buenaventura as entitive (quidditas) and significant (significatio).
Keywords: Logos, st. Bonaventure, mePalabras clave: logos, san Buenaventura, metafísica, filosofía medieval, ejempla- taphysics, medieval philosophy, exemplarism.
rismo
476 —
L
a teología del logos es uno de los capítulos más brillantes e intensos del desarrollo teológico del cristianismo con relación al discurso propiamente teológico, es decir, reclamando para sí el logos –entendido como discurso– sobre
Dios 1. Y revindicar el discurso de Dios en tanto que logos supone en y desde la
época del helenismo –momento en el que se forja la teología del logos– una reformulación del logos definida como una realidad fundante del paganismo 2. Esto lo
podemos ver tanto en Justino 3 como, especialmente en su carácter polémico, en
el Contra Celso de Orígenes 4, donde el alejandrino tiene que defender el carácter
racional del logos cristiano frente a la caracterización del logos platónico tradicional del cual Celso se siente heredero y defensor 5. Celso ubica el logos en un lugar
inferior al “Primer Dios” entendido este como un Dios totalmente absoluto 6. En
este sentido se establece la lucha teológica de la racionalidad entre el henoteísmo
del platonismo medio y el monoteísmo trinitario del cristianismo 7. La verdad es
que el cristianismo desde el inicio se tomó muy en serio la doctrina de la encarnación y supo asimilar (encarnar) aquellas doctrinas que expresaran mejor el Misterio
divino. Desde entonces la historia de la formulación del logos en el discurso teológico recorre los caminos del dinamismo teológico, partiendo de la intimidad del
Misterio trinitario y pasando al Mundo desde la caracterización del logos, que en
el cristianismo se identifica –a partir de la lectura del texto evangélico del capítulo
Vigo, A. G. “Cristianismo y opción por el «lógos»”, Scripta Theologica 39 (2007), pp. 853-861.
Lilla, S., “Ellenismo e cristianismo”, en Dizionario Patristico e di Antichità Cristiane, vol. 1, Di Bernardino, A. di
(ed.), Casale Monteferrato, Casa Editrice Marietti, 1983, p. 1138; Behr, J., “The World of Word in the Second
Century”, Pro Ecclesia 9 (2000), pp. 85-107.
3
Como afirma José Antonio Sayés su teología es fundamentalmente una teología del logos. Sayés, J. A., Teología
de la creación, Madrid, Palabra, 2002, p. 66. Esta tematización aparece en la obra clásica de Aloys Grillmeier.
Grillmeier, A., Christ in Christian Tradition. I. From the Apostolic Age to Chalcedon (451), Atlanta, Westminster
John Knox Press, 1975, 2ª ed., p. 92.
4
Orígenes, Contre Celse, intr, text., trad. y not. M. Borret, Paris, Les éditions du Cerf, 1967.
5
Cf. Corsini, E., (ed.), Commento al Vangelo di Giovanni di Origene, Torino, Unione Tipografico-Editrice Torinese, 1968; Zañartu, S., “Y el Logos era Dios. Comentario de Orígenes al evangelio de Juan”, Teología 108
(2012), pp. 91-100.
6
Bader, R., Der Alethes Logos des Kelsos, Stuttgart-Berlin, Kohlhammer, 1940; Andresen, C., Logos und Nomos. Die
Polemik des Kelsos wider das Christentum, Berlin, Walter de Gruyter, 1955; Watson, G., “Celsus and the philosophical Opposition to Christianity”, Irish theological Quarterly 58/3 (1992), pp. 165-179.
7
Cf. Dorrie, H., Die platonische Theologie des Kelsos in ihrer Auseinandersetzung mit der christlichen Theologie,
Göttingen, Vandehoeck & Ruprecht, 1967; Fédou, M., Christianisme et religions païennes dans le Contre Celse
d’Origène, Paris, Éditions Beauchesne, 1988, pp. 562-600; López, A., ¿Dioses o Cristo? Momentos claves del enfrentamiento pagano al cristianismo, Pamplona, Servicio de publicaciones de la Universidad de Navarra, 2009.
1
2
— 477
primero de san Juan– en la formulación del Verbo (Verbum) de Dios, es decir, en
clave cristocéntrica 8.
Buenaventura, diez siglos más tarde, tendrá que afrontar estos planos metafísicos
de la intimidad trascendente del Padre, la autodonación expresiva trinitaria y la expresión del mundo creado que caracterizan, a su vez, una teología del monoteísmo
trinitario fundamentado en la metafísica del dinamismo del ser, cuando el discurso
filosófico irrumpa con fuerza en las aulas de teología de la mano del platonismo de
san Agustín y Dionisio y de la metafísica del ser y la filosofía natural de Aristóteles 9.
De esta forma, cuando Buenaventura habla del logos lo hace en su versión latina
de verbum definiéndolo, siguiendo a Agustín, como “lo que expresa y manifiesta la
res” 10.
La reflexión de Buenaventura sobre el logos en tanto que verbum es en realidad una teología del logos divino y, por lo tanto, una teología cristológica dentro
del misterio del ser y la bondad de la divina Trinidad. La teología del logos hace
relación a todas las procesiones ad intra y ad extra, expresando al Padre y a las creaturas. En términos filiales, de relación personal, se expresa en la teología del Hijo
que comparte la esencia del Padre, implicando una relación más esencial a partir
de la filiación divina. De esta forma la teología del logos entra en el ámbito de la
expresión.
El Seráfico desarrolla su pensamiento sobre el logos en la figura de la Segunda
Persona de la Trinidad manifestando la mediación del logos como la consumación
de toda metafísica 11:
Este es el medio metafísico que reduce y esta es toda nuestra metafísica: de la
emanación, de la ejemplaridad, de la consumación, es decir, iluminar por rayos
espirituales y reducir a lo sumo. Así serás un verdadero metafísico 12.
Andreu, A., El Logos alejandrino, Madrid, Siruela, 2009, pp. 19-25.
Lázaro, M., La creación en Buenaventura. Acercamiento filosófico al la metafísica del ser finito, Grotaferrata (Roma),
Fratri Editori di Quaracchi, 2005, pp. 35-60.
10
“…verbum est, quo res manifestatur et exprimitur”; Buenaventura, In I librum Sententiarum, d. 27, p. 2, q. 1,
vol. I, p. 481b (Usamos la Edición crítica Opera omnia, 10 vols., Quaracchi, Ad Claras Aquas, 1882-1902 y
señalamos el volumen, la página y la columna).
11
No se trata aquí solo de un pensamiento relacionado con el lenguaje, aunque tampoco podemos alejarlo como
señala Oppes, S. “Verbum”, en Dizionario bonaventuriano. Filosofía, teología, spiritualità, Caroli, E. (ed.), Padova, Editrice Franciscana, 2008, pp. 832-838.
12
“Haec est medium metaphysicum reducens, et haec est tota nostra metaphysica: de emanatione, de exemplaritate, de consummatione, scilicet illuminari per radios spirituales et reduci ad summum. Et sic eris verus metaphysicus”; Buenaventura, Collationes in Hexaëmeron, col.1, n.17, vol. V, p. 332b. Un comentario de la filosofía
natural de Buenaventura teniendo en cuenta este texto en Cullen, C. M., Bonaventure, Oxford / New York,
Oxford University Press, 2006, pp. 60-90. doi: https://doi.org/10.1093/acprof:oso/9780195149258.003.0004.
La relación entre metafísica y cristología que subyace a la propia filosofía y teología del Logos y que expresa el
texto de las colaciones cf. Hayes, Z., “Christology and Metaphysics in the Thought of Bonaventure”, The Journal
of Religion 58 (1978), pp. 82-96. doi: https://doi.org/10.1086/jr.58.41575982.
8
9
478 —
Tenemos pues tres radios de acción del Logos divino:
1. De emanatione. La autoexpresión ad intra de Dios. La vida íntima de la trinidad divina. La autoexpresión desde la unidad de Dios: quidditas.
2. De ejemplaritate. La expresión ad extra de Dios. La vida manifestada por la
trinidad divina. La expresión de la pluralidad de Dios: significatio.
3. De consumatione. La encarnación del Logos divino. La mediación entre la Unidad y la Pluralidad desde Dios: quidditas y significatio.
1. La autoexpresión ad intra de Dios. Doctrina trinitaria
El papel del logos en la relación intratrinitaria es fundamental, pues el maestro
franciscano, como hemos señalado, parte en su concepción trinitaria del texto del
prólogo del evangelio de san Juan en el que la relación entre el Padre y el Hijo es
semejante a la relación entre quien habla y lo hablado 13. El hablante es el Padre y la
palabra es Palabra dotada de una infinita potencia de tal magnitud que es, a su vez,
quien escucha el Padre. La Palabra del Padre es, también, la escucha. La teología del
logos no es una alternativa a la doctrina de la filiación divina, sino que es una confirmación en la expresión de lo que la esencialidad implica: de la relación entre Dios
Padre y Dios Hijo 14. La relación Paterno-Filial se explicita en la expresión del logos,
pues el logos es precisamente comunicación. De esta forma, el Padre expresa lo que
es, sabe y puede en el Logos, en la Palabra, en el Hijo y el Hijo es todo lo que dice
el Padre como Palabra 15. Esta relación Padre-Palabra es íntima a la propia Primera
Persona de la Trinidad y se da desde la unidad, se expresa en el hecho de que el amor
entre el Padre y el Hijo es Uno: el Espíritu Santo que es “nexo entre Padre e Hijo” 16.
Buenaventura vierte la doctrina rubricada por el magisterio conciliar en momentos en los que, como hemos señalado, estos temas estaban encima de la mesa, especialmente en la lectura exagerada del neoplatonismo realizada por la herejía arriana.
Buenaventura, Commentarium in Evangelium Ioannis, c.1, p.1, q.1, vol. VI, p. 247b. Buenaventura, Collationes
in Hexaëmeron, col. 3, n. 4, vol. V, p. 343b. Cf. Bougerol, J.-G., “Verbum”, en Léxique Saint Bonaventure,
Bougerol, J.-G. (dir.), Paris, Éditiones franciscaines, 1969 p. 131.
14
Buenaventura, In I librum Sententiarum, d. 27, p. 2, q. 2, ad 5, vol. I, p. 486b y Buenaventura, Collationes in
Hexaëmeron, col. 1, nn. 11, 17, vol. V, pp. 331a, 332a.
15
Buenaventura, In I librum Sententiarum, d. 27, p. 2, q. 2, vol. I, p. 485ab; Buenaventura, Quaestiones disputatae
de misterio Trinitatis, q. 4, a. 2, ad 8, vol. V, p. 87a.
16
Buenaventura, In I librum Sententiarum, d. 11, q. 1, vol. I, pp. 209a-2013b. Cf. Iammarrone, L., “La struttura
della vita trintaria come amore in san Bonaventure”, Miscellanea Francescana 89 (1989), pp. 315-334.
13
— 479
El concilio de Nicea, luchando contra el arrianismo, asentó la teología del logos y
de su dimensión intratrinitaria 17. No hacía el concilio sino asimilar las doctrinas de
la época. Buenaventura se ve abocado también a realizar desde la teología universitaria una reformulación desde las fuentes. La llegada de Aristóteles supuso recordar
el hecho de que “logos” significa “razón, palabra, pensamiento y comunicación” 18.
Aristóteles no hizo sino indicar el carácter divino del logos que hay en el hombre,
expresión del logos universal que lo rige todo y del cual hablaba Heráclito, reflejándose, en cierta forma, en el pensamiento de los estoicos, quienes afirmaban como
tesis fundamental la existencia de un logos eterno que dirige el mundo y que es su
ley, su providencia. La presencia del pensamiento estoico en la teología del logos se
abría a la teología de la imagen, no en vano los estoicos ven el logos humano como
una participación del Logos divino 19.
Ni que decir tiene que la base filosófica neoplatónica basada en la interpretación
metafísica del demiurgo creador del Timeo platónico 20 está también muy presente.
Después del Uno definido por su inaccesibilidad e inefabilidad el logos aparece
como hipóstasis intermediaria entre la unidad trascendente y la diversidad de lo
existente 21.
Estos ítems filosóficos –puestos por la teología sistemática– alimentan la aproximación hermenéutica del citado texto de Juan como una anticipación pagana, de la
verdad cristiana 22. Y lo que los Padres —desde Orígenes y la Escuela de Alejandría 23
a san Agustín 24, pasando por los Padres Capadocios— desarrollarán, sobre todo,
desde la cuestión ad intra, se reflejará en Buenaventura.
La teología del logos –que es teología de la Segunda Persona– se manifiesta desde el Logos increado como expresión creadora del Padre 25. Buenaventura afronta
el tema recordando la doctrina patrística que relacionaba el Logos increado con la
difusión intratrinitaria y las procesiones divinas. Un ejemplo de ello lo tenemos en
Agustín. La tematización que realiza de la Segunda Persona de la Trinidad a partir
Cf. Grillmeier, A., Christ in Christian Tradition: From the Apostolic Age to Chalcedon (451), op. cit., pp. 249-341.
Una tematización que ha recorrido trasversalmente la filosofía hasta la era contemporánea. Cf. Cassin, B., Aristote et le Logos. Contes de la phénoménologie ordinaire, Paris, PUF, 1997.
19
Garrido, J. J., El pensamiento de los Padres de la Iglesia, Madrid, Akal, 1997, p. 8.
20
Cf. Meyer-Abich, K. M., “Eikos Logos. Platons Theorie der Naturwissenschaft”, en Einheit und Vielheit. Festschrift für Carl Friedrich von Weizsaecker zum 60 Geburstag, Von Sheibe, E. (ed.), Göttingen, Vandenhoeck &
Ruprecht, 1973, p. 20-44.
21
Cf. O’Meara D. J. (ed.), Neoplatonism and Christian Thought, Albany, N. Y., State University of New York
Press, 1982.
22
Garrido, J. J., El pensamiento de los Padres de la Iglesia, op. cit., p. 8.
23
Cf. Aeby, G., Les missions divines de St. Justin à Origène, Fribourg, Editions Universitaires, 1958.
24
Cf. Maier, D.-L., Les missions divines selon Saint Augustin, Fribourg, Editions Universitaires, 1960.
25
Cf. Gerken, A., La théologie du Verbe. La relation entre l’Incarnation et la création selon S. Bonaventure, Paris,
Éditions Franciscaines, 1969, pp. 49-167.
17
18
480 —
del desarrollo de una teología del logos se manifiesta y desarrolla desde la función
expresiva del ser divino 26. Esta relación se sustenta en una metafísica que desarrolla
el dinamismo de la teología del logos, que no es otra que la doctrina ejemplarista
donde la creatura participa del ser divino 27. Agustín une la metafísica teológica
de inspiración neoplatónica 28 con la filosofía estoica 29, Cristo, el Hijo es el Logos
imagen del Padre y su expresión que adquiere la condición de arquetipo de los seres
creados. El logos se manifiesta como pura comunicación por lo que las comunicaciones intra y extratrinitarias pertenecen al Verbo 30.
Una teología del logos debe establecer un equilibrio entre la unidad y la pluralidad habida cuenta que ante nosotros aparece lo mudable, pero este movimiento
descansa en una verdad inmutable que es Dios y por apropiación Cristo 31. Esto
supone que el logos es expresión de la unidad y el ejemplar de lo múltiple. Y ello
acontece por el hecho de que el logos es el Logos del Padre como forma subsistente
en Dios. De modo que en Dios la expresión es infinita 32. Siendo así aún en la Unidad –como definición propia del Padre– y sin perder un ápice de su esencialidad –al
contrario en cuanto que se define como unidad de ser infinito– su expresión ha de
ser infinita, por lo que el logos se ha de expresar por definición en objetos infinitos 33. El Logos no es ajeno ni inferior al Padre, sino que es expresión pronunciada
por el Padre y espirada por el Espíritu. El Logos es autoexpresión intratrinitaria que
nace de la unidad divina.
La autoexpresión intratrinitaria del Logos nace de la relación del Padre como
procesión que expresa en toda su dimensión “lo que Es” en su omnipotencia –que
se manifiesta en la propia expresión del Logos–, por lo que expresa de forma omnipotente, infinita, sin restricción 34. La procesión del Hijo, del Logos, nace en su
esencialidad “según razón natural, no al modo de liberalidad nacida de la volun Cf. Agustín, De Trinitate, lib. 15, c. 14, n. 23, en Patrologia Latina, vol. 42, Paris, J.-P. Migne, 1865, col. 1076.
Gillet, R., “Temps et exemplarisme chez saint Augustin”, en Augustinus Magister. Congrès International Augustinien Paris, 21-24 Septembre 1954. Vol. 3. Communications, Paris, Études Augustiniennes, 1954, pp. 933-941.
28
Solignac, A., “Réminiscences plotiniennes et porphyriennes dans le debut du ‘De ordine’ de saint Augustin”,
Archives de philosophie, 19 (1936), pp. 148-156; Solignac, A., “Notes”, Saint Augustin, Les Confessions, ed. y
trad. E. Tréhorel y G. Bouissou, Desclée de Brouwer, Paris, 1962, vol. I, pp. 682-689.
29
Cf. Testard, M., Saint Augustin et Cicéron. Tome I. Cicéron dans la formation et dans l’oeuvre de Saint Augustin.
Tome II. Répertoire des textes, Paris, Études augustiniènnes, 1958; Spanneut, M., “Le Stoïcisme et saint Augustin”, en Forma futuri. Studi in onore del cardinale Michele Pellegrino, Torino, Bottega d’Erasmo, 1975, pp.
895-914.
30
Agustín, De diversis quaestionibus LXXXIII, n. 69, 1, Patrologia Latina, vol. 40, Paris, J.-P. Migne, 1865, col. 74.
Cf. la cita del texto Col 1, 15.
31
Berubé, C., De la philosophie à la sagesse chez Bonaventure et Roger Bacon, Roma, Istituto Storico Cappuccini,
1974, p. 37.
32
Buenaventura, Quaestiones disputatae de misterio Trinitatis, q. 4, a. 2, ad 8, vol. V, p. 87a.
33
Buenaventura, Quaestiones disputatae de misterio Trinitatis, q. 4, a. 2, ad 9, vol. V, p. 87ab.
34
Bonaventura, Quaestiones disputatae de scientia Christi, q. 1, concl., vol. V, p. 5a.
26
27
— 481
tad” 35. La teología del logos desde la naturaleza de la quididad supone que el Hijo
es emanación del Padre no en cuanto degradación ontológica, sino en cuanto que
es la propia expresión de Dios, y expresión es comunicación, es salir del Padre e
implica dinamismo de pluralidad en la unidad. Y, por eso, el Hijo es Logos, puesto
que el Logos expresa tanto al Padre como a sí mismo 36. El Logos divino es el propio
Dios que se comunica, lugar de la emanación intratrinitaria entendiéndose como
emanación de las personas en una misma naturaleza. Así se entiende la explicación
de Buenaventura en las Cuestiones sobre el Misterio de la Trinidad de que “por cuanto
en la emanación de las personas la naturaleza es lo que se comunica y la persona es
la que produce, por razón de la misma emanación se concluye a la pluralidad de
personas y a la unidad de naturaleza” 37.
En la naturaleza divina no cabe división ni partición, lo que supone que es idéntica en el producido y en el producente. En este sentido, la emanación de la persona
se da en lo producido y lo producente como expresión o representación del logos
en tanto que –como ha afirmado en otros lugares el Seráfico– el logos se manifiesta
en la relación de quien habla y lo hablado. De ahí que Buenaventura declare que
el logos “representa todas las cosas tal como son producidas en el ser” 38. Desde la
teología del logos, el Hijo “expresa al Padre como principio que origina de sí, y de
este modo explica y representa la producción del Espíritu Santo y la suya o los de
los eternos” 39. En este sentido, Alexander Gerken concluye que “por el Verbo, la
estructura trinitaria de Dios se convierte, ella misma expresión, porque es lo propio
del Verbo es ser, en Dios expresión” 40. El Padre es el todo originante y emanante y
el Hijo desde la autoexpresión del Logos es el todo expresivo y ejemplar. Desde la
intimidad quiditativa nace la propia expresión, desde la emanación omnipotente la
ejemplaridad universal. La unidad del ser es en sí pluralidad personal.
La segunda persona de Dios trinitario participa del don originante como causa
ejemplar. Desde la teología del Logos Buenaventura explica la co-esencialidad del
Verbo. “Como Verbo increado —escribe Bougerol— el Hijo de Dios es similitud
“…secundum rationem naturae, non tamen secundum liberalitarem coluntatis”; Buenaventura, In I librum
Sententiarum, d. 2, q. 4, ad 2, vol. I, p. 58b.
36
Buenaventura, Collationes in Hexaëmeron, col. 9, n. 2, vol. V, pp. 372b-373a.
37
“Quoniam in emanatione personarum natura se habet in ratione communicabilis, persona se habet in ratione
communicabilis, persona se habet ratione producentis; propter eandem emanationem necesse est, plualitatem
esse in personis et unitatem in natura”; Bonaventura, Quaestiones disputatae de scientia Christi, q. 2, a. 2, concl.,
vol. V, p. 65b.
38
“Verbum sic repraesentat res, ut in esse producuntur”; Buenaventura, Collationes in Hexaëmeron, col. 3, n. 6,
vol. V, p. 344a.
39
“Et Verbum exprimit Patrem ut principium principians de se, et sic explicans et repraesentans productionem
Spiritus sancti et suam sive aeternorum”; Buenaventura, Collationes in Hexaëmeron, col. 3, n. 7, vol. V, p. 344ab.
40
A. Gerken, La théologie du Verbe, op. cit., p. 73.
35
482 —
hipostática del Padre, por la que el Padre se conoce a sí mismo” 41. Buenaventura
concluye en el Comentario a las Sentencias de Pedro Lombardo “que el Hijo procede
bien, no como un ejemplariado, sino más bien siendo él mismo el ejemplar, es
decir, la razón misma de expresar a los demás seres” 42. El Verbo es propiamente el
ejemplar al ser expresión del Padre en la Trinidad. Dios Trinitario es ejemplar en
la medida en la que es la ratio cognoscendi del universo, y es la ratio emanandi en la
comunicación como Verbo.
La emanación que hace referencia al origen, da lugar en sí a la expresión que hace
referencia al ejemplar y, desde allí, a la autoexpresión ad extra de Dios. En palabras
de Von Balthasar: el Cristo-Logos es “la auto-expresión de Dios del Padre al interior
de la Trinidad, y la expresión de todo lo que Dios puede producir hacia fuera” 43.
2. La autoexpresión ad extra de Dios. Doctrina de la creación
Buenaventura conecta y articula la teología del Logos en relación con la creación, puesto que todo es producido por el Verbo increado 44: “El Verbo expresa tanto al Padre, como a sí mismo, como al Espíritu Santo y todas las demás cosas” 45. A
partir de la lectura del Evangelio de san Juan (Jn 1,3) y de la Epístola a los Hebreos
(Heb 1,2) Buenaventura establece una hermenéutica desde el Logos que desemboca
en el acto creador 46. La autoexpresión del Logos increado del Padre desempeña un
papel significativo como co-originante de la creación. Junto a la labor emanativa
del Padre, Él aparece en su dimensión expresiva como ejemplar. Y como ejemplar
expresivo, el Logos explicita el carácter creador de Dios trinitario. El Verbo es la
expresión divina que hace referencia a la creatura 47.
En el sermón universitario Christus unus omnium magister, Buenaventura muestra el Verbo encarnado, como la vía de acceso a la sabiduría. El Verbo “como Arte
La expresión de que el Padre se conoce en el Hijo ha tenido matices interpretativos por parte de Bissen, Peter o
Gerken. Cf. J. F. Bartos entiende que, cuando se afirma que el Padre se conoce en y por el Verbo, se está insistiendo en una dimensión terminal y no formal de la expresión, es decir, el Padre se conoce en el Verbo que es el
fruto de su actividad cognoscitiva infinita. Cf. Bissen, J.-M., L’Exemplarisme divin selon Saint Bonaventure, Paris,
J. Vrin, 1929, p. 109; A. Gerken, La théologie du Verbe, op. cit., p. 52.
42
Mathieu, L., La Trinité créatrice d’après saint Bonaventure, Paris, Les Éditions Franciscaines, 1992, 157.
43
Balthasar, H. U. von, La Dramatique divine. Le dénouement, vol. IV, Namur, Culture et Vérité, 1993, p. 54.
44
Buenaventura, Collationes in Hexaëmeron, col. 3, n. 2, vol. V, p. 343a.
45
“…quia Verbum et Patrem et se ipsum et Spiritum sanctum exprimit et omnia alia”; Buenaventura, Collationes
in Hexaëmeron, col. 9, n. 2, vol. V, pp. 372b-373a.
46
San Buenaventura se pregunta e indica esta relación en su Comentario al Evangelio de San Juan en un texto
explicitado en líneas precedentes (Buenaventura, Commentarium in Evangelium Ioannis, c. 1, p. 1, q. 1, vol.
VI, p. 247b).
47
Cf. Buenaventura, In I librum Sententiarum, d. 31, p. 2, q. 2, concl., vol. I, p. 542b.
41
— 483
del Padre es, a la vez, Verdad y Luz, principio del ser, como causa ejemplar en la que
las creaturas tienen su ser inmutable, y principio del saber, como causa normativa
de la certeza intelectual” 48. El Verbo es la expresión de la sabiduría divina como
camino de acceso al Dios trinitario y como ratio intelligendi; es, pues, expresión de
la verdad de Dios: “Dios es por la inteligencia la ratio intelligendi suprema, la fuente
creadora del espíritu, y la residencia iluminadora de todas sus operaciones” 49.
De este modo, el Verbo (hipóstasis del Padre) es el ejemplar de toda creatura en
cuanto comunicación de la potencia del Padre 50. No solo es la ratio emanandi, sino
que siendo ratio exemplandi se convierte en ratio cognoscendi de todas las creaturas 51. El Logos autoexpresivo del Padre que hace intrínsecamente y en primer lugar
mención al Padre, es el medio de toda la creación como expresión de la potencia
todopoderosa del Padre, es decir, como ejemplar del universo 52. Dios-Hijo es expresión perfecta de todo lo que el Padre se ha propuesto hacer fuera de él. Todo
ha sido explicado en su Hijo en una representación única y por eso es verdaderamente razón de ser de las cosas, pues ninguna creatura es producida, si no es por el
Verbo, en quien el Padre ha dispuesto todo 53. Así, podemos decir que la Segunda
persona de la Trinidad es el significado de la expresión de la potencia del Padre;
pero es, también, el Verbo increado como significante expresivo y ejemplar que
se comunica. Y si la ratio emanandi señalaba la expresión de la quididad –de la res
expresada–, la ratio exemplandi es expresión significativa manifestada en la creación.
Efectivamente, una de las características de Dios es la Unidad en la Trinidad, en la
pluralidad, una unidad de los elementos diferentes de naturaleza quiditativa. Esta
unidad en la pluralidad se da, mediante el Logos expresado ejemplarmente en la
creación, de una forma atenuada, significativa, de modo que la creación es unidad
compuesta de pluralidad de planos, gradual, auténtica multiplicidad a su vez, y esto
es en su dimensión significativa y sígnica –al modo del ejemplar– y expresada desde
la teología de la imagen 54.
Toda la realidad es expresión, logos en toda su dimensión, como reflejo de la
Trinidad. El Logos es el ejemplar de la realidad del mundo. Y si el plano divino
Madec, G., “Notes complementaires”, en Le Christ Maître, Paris, J. Vrin, 1990, p. 85. Buenaventura, Collationes
in Hexaëmeron, col. 1, n. 13, vol. V, p. 331b.
49
J.-M. Bissen, L’Exemplarisme divin selon Saint Bonaventure, op. cit., p. 175.
50
Buenaventura, In I librum Sententiarum, d. 32, a. 2, q. 1, concl., ad. 4, vol. I, p. 563a-b.
51
Buenaventura, In I librum Sententiarum, d. 6, q. 3, ad. 4 in fine, vol. I, p. 130b. San Buenaventura sigue la doctrina de Guillermo de Auxerre en su Summa aurea, lib.1, in fine, Ribailler, J. Ed., Paris-Grottaferrata, CNRS,
Collegium S. Bonaventurae ad Claras Aquas, 1985.
52
San Buenaventura sigue a su maestro Alejandro de Hales en la Summa Halensis. Alejandro de Hales, Summa
universis theologiae, n. 425, vol. I, p. 618b (4 vols., Collegii S. Bonaventurae, Roma, 1924-1948).
53
Bougerol, J.-G., Introduction à l’étude de Saint Bonaventure, Tournai, Desclée, 1961, p. 80.
54
Un desarrollo desde la pregunta metafísica en Lázaro, M., “Más allá de la quiditas: reflexiones sobre el proyecto
metafísico bonaventuriano”, Cauriensia 14 (2019), pp. 49-80.
48
484 —
aparece como unidad que se expresa en la pluralidad, el mundo aparece en la pluralidad que puede ser contemplada y conocida en la unidad. De ahí que la realidad
trinitaria divina se autoexpresa al mundo como estructura trinitaria y expresión de
forma simbólica, en cuanto que el mundo es imagen del Creador. Desde la autoexpresión del Logos ad extra en los parámetros de la razón ejemplar –de su causalidad
y el esquema metafísico del que Buenaventura es un maestro–, aparece una comprensión significativa del universo al que se le suma la teología de la imagen. Este
plano de ejemplaridad del Logos nos lleva a una epistemología del mundo desde su
significación, desde los grados de significación. Pero aún se nos escapa un espacio
de conocimiento capaz de reducir la pluralidad a la unidad desde la intimidad de
la realidad significada, donde quididad y significación sean posibles, de modo que
podamos afirmar con Gilson que el sistema bonaventuriano es una visión simbólica
del universo 55.
3.
De consumatione. La encarnación del Logos divino: quidditas y significatio.
El mundo se ofrece a la significación, pues el logos hace relación a Dios y a
las creaturas. El logos es originante de la naturaleza creada en el tiempo y como
mediador entre Dios y sus creaturas se ofrece, de una manera particular al hombre,
en el Verbo encarnado. Buenaventura en el Sermón de la Navidad afirma que la encarnación del Logos hace visible la realidad significada y anteriormente invisible, no
solo se hace significativa la realidad quiditativa, sino que se hace sensible. El Logos
no solo manifiesta la razón inteligible, audible, sino se manifiesta visiblemente. El
niño de Belén, el Hijo de Dios hecho hombre, es el Logos encarnado: expresión
simbólica que une quidditas y significatio 56.
Aunque no podemos detenernos en ello, la teología del Logos en su configuración de Logos encarnado lleva en sí un componente reparador, de redención de
segunda creación muy importante y esencial 57. Más allá de esta teorización y para
lo que nos interesa ahora, es preciso señalar que esta reparación que se relaciona estrechamente con la acción creadora del Logos supone una continuidad de la acción
y apunta así a la consumación. La encarnación en su función reparadora señala la
quidditas de la significatio del mundo, el trasfondo simbólico de la realidad creada
como acto de continuidad de la teología de la creación. El esquema cristológico bo Gilson, E., La filosofía de san Buenaventura, Buenos Aires, Desclée de Brouwer, 1948, pp. 227-228.
Buenaventura, Sermones de tempore. Nativitas Domini, serm. 1, vol. IX, p. 103a.
57
Esto es algo que Tomás de Aquino también desarrollará, cf. Ramos, A. “A metaphysics of the logos in St. Thomas Aquinas: creation and knowledge”, Cauriensia 9 (2014), p. 97.
55
56
— 485
naventuriano sigue dependiendo de Anselmo en el sentido de que el acto reparador
es fundamental en la encarnación del Logos, pero esta continuidad del acto creador
parece a su vez señalar un protagonismo del acto creador del Logos, que apunta ya
a lo que generaciones más tarde Escoto afirmará sin rodeos sobre una cristología
dependiente de la teología de la creación como formulación de una teología de la
gracia. Lo que Buenaventura está expresando es un razonamiento que parte de la
razón de congruencia (ratio congruantiae) 58 en la que el mundo queda desde el Logos encarnado consumado en su acto creador, afirmando la realidad bondadosa de
la realidad divina sobre la razón y la conveniencia del acto reparador, anticipando
de nuevo a Escoto 59. En su Logos, Dios ha llamado a toda creatura a la existencia.
Y en la encarnación continúa su obra creadora por la reparación en Él, como pura
donación de amor y expresión divina por gracia 60.
La encarnación del Logos es la consumación del acto creador, la tercera parte de
toda la metafísica. Una nueva creación que implica que si la actividad creadora del
Logos en cuanto ejemplar hace referencia a la producción significativa en el ser, la
reparación de las cosas se relaciona con la ordenación al bien 61. La encarnación del
Hijo de Dios, del Logos, eleva la naturaleza, en especial la naturaleza humana (el
concepto de encarnación era “extraño” al pensamiento grecorromano), pues beatifica la naturaleza humana y afirma la bondad de la creatura. La bondad creatural se
reafirma en el mismo propósito de la historia de la salvación: la carne –la naturaleza
creada– es buena en virtud de su vocación a salvarse como cuerpo humano, en
cuanto reparación de la humanidad en Cristo (Verbo encarnado). La realidad natural significada se dota de realidad simbólica desde la gracia. El ejemplo es el propio
Jesús, naturaleza divina mostrada al hombre en su significado en sí, paradigma de
realidad simbólica: quidditas y significatio.
Existe entre la creatura y Dios una distancia de orden y de ser. La creatura se define por su vanidad frente a Dios que es ser, bien, uno y verdad; pero esta distancia
ontológica se aproxima por la iniciativa divina reflejada en la encarnación del Logos. La contingencia existente en el interior del mundo dotada de significación por
la realidad Trinitaria y por el Logos ejemplar, mantiene en su interior la expresión
de la gracia trinitaria y, por lo tanto, en su realidad simbólica, muestra la huella de
Dios que se manifiesta en el acto de la reparación. El Logos encarnado –venido al
Buenaventura In III librum Sententiarum, d. 1, a. 2, q. 1 concl., vol. III, p. 20a.
Osborne, K. B., “The Trinity in Bonaventure”, en Phan, P. C. (ed.), The Cambridge Companion to the Trinity, Cambridge, Cambridge University Press, 2011, p. 122. doi: https://doi.org/10.1017/CCOL9780521877398.007
60
Buenaventura, Sermones de Beata Virgine Maria. De annuntiatione Beata Virginis Mariae, serm. 3, I, vol. IX, p.
667b. Cf. Lázaro, M., “Comprensión desde la filosofía de la afirmación «Dios es amor» en san Buenaventura”,
Cauriensia 2 (1997), pp. 179-210.
61
Buenaventura, Breviloquium, p. 4, c. 1, vol. V, p. 241a.
58
59
486 —
mundo para la reparación del hombre– es la expresión teológica de las categorías
metafísicas de regreso (“reditus”) hacia el Uno (el bien) que queda tematizado en las
Collationes in Hexaëmeron con el Verbo inspirado 62. El Logos (increado, encarnado
e inspirado) se muestra como imagen y mediación perfecta de la Trinidad (creadora-Padre, reparadora-Hijo, reveladora-Espíritu Santo) 63.
En la encarnación del Logos nos encontramos la acción mediadora de la Segunda persona de la Trinidad y su acción dinámica como expresión divina. El Verbo
representa el centro de reparación y de cumplimiento de la creación, ejerciendo de
punto de unión con Dios Trinitario (mediación). Como señala A. Gerken, “Cristo, en cuanto Verbo encarnado, no puede imaginarse de modo independiente a la
creación, ya que esta relación es la que le hace el jefe, cabeza, de esta creación” 64.
El Verbo encarnado es el epicentro de este esquema comunicativo que reúne así a
todas las creaturas 65. Y es, también, el centro teológico (centro de la economía de la
salvación): centro de la creación, centro del tiempo y centro de la salvación.
La mediación del Verbo y su carácter comunicativo (inspirado) expresan la
unión y la progresión del Verbo increado al Verbo encarnado como dos aspectos
del mismo sujeto: la Palabra que crea, que repara y que es sabia. La segunda persona
de la Trinidad perfecciona, así, el esquema metafísico de la expresión. La autoexpresión del Logos encarnado llama a la reducción, a la consumación de la realidad
quiditativa y de la significación existencial –en el acto de continuidad de la creación
y de la reparación respectivamente–, manifestando, así, una teología del símbolo 66
que se suma al esquema metafísico del ejemplarismo. De esta forma se entiende
que el simbolismo bonaventuriano tenga un valor de mediación como reflejo de la
mediación del Logos. La propia creatura lleva en sí la expresión de la pluralidad en
la unidad de Dios. Se entiende la reflexión epistemológica y ontológica expresada
en el Itinerarium en el binomio “por creaturas” y “en las creaturas” que Francisco
de Asís Chavero Blanco categorizaba como una verdadera ontología semántica en la
que se identifican las funciones de ser y significar 67. Y ello es así por medio de una
metafísica de la imagen y una teología simbólica dependiente de la teología del Lo Cf. Maranesi, P., Verbum inspiratum. Chiave ermeneutica dell’Hexaëmeron di san Bonaventura, Roma, Istituto
storico dei cappuccini, 1996.
63
Buenaventura, Collationes in Hexaëmeron, col. 3, n. 2, vol. V, p. 343a.
64
Gerken, A., La théologie du Verbe, op. cit., p. 323.
65
“In dispensationem plenitudinis temporum: recapitulare omnia in Christo” (Ef 1,10); “Qui est imago Dei invisibilis
primogenitus omnis creaturae” (Col 1, 15); san Buenaventura retoma el tema paulino de Cristo como el nuevo
Adán como signo de recapitulación del universo, cf. Chavero, F. de A., “Novus Adan. Significado de la tipología
de Adán en san Buenaventura (Leg. Maior, cap. 5)”, Verdad y Vida 50 (1992), pp. 137-171.
66
Sobre el simbolismo bonaventuriano cf. Piazza, L., Mediazione simbólica in san Bonaventura Vicenza, LIEF,
1948; Zas, R., La teología del símbolo de san Buenaventura, Roma, Pontificia Università Gregoriana, 1997.
67
Chavero, F. de A. “Ser y significar. Aproximación al simbolismo bonaventuriano”, Thémata. Revista de filosofía
5 (1988), p. 65.
62
— 487
gos, (1) de su autoexpresión ad intra emanativa de la realidad, (2) ad extra ejemplar
que dota significación al mundo y (3) de consumación en el paradigma creador y
reparador del Logos encarnado, capaz de establecer la expresión de la realidad simbólica en la potencialidad del Logos como medio que es como señala Buenaventura
“el principio del ser y del conocer” 68.
68
“Nam idem est principium essendi et cognoscendi”; Buenaventura, Collationes in Hexaëmeron, col. 1, n. 13,
vol. V, p. 331b.
488 —
Referencias Bibliográficas
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Migne, 1865
Agustín, De Trinitate, en Patrologia Latina, vol. 42, Paris, J.-P. Migne, 1865.
Agustín, Les Confessions, ed. y trad. E. Tréhorel y G. Bouissou, Desclée de
Brouwer, Paris, 1962.
Alejandro de Hales, Summa universis theologiae, 4 vols., Roma, Collegii S. Bonaventurae, 1924-1948.
Andreu, A., El Logos alejandrino, Madrid, Siruela, 2009.
Balthasar, H. U. von, La Dramatique divine. Le dénouement, vol. IV, Namur,
Culture et Vérité, 1993.
Berubé, C., De la philosophie à la sagesse chez Bonaventure et Roger Bacon, Roma,
Istituto Storico Cappuccini, 1974.
Bissen, J.-M., L’Exemplarisme divin selon Saint Bonaventure, Paris, J. Vrin, 1929.
Bougerol, J.-G., Introduction à l’étude de Saint Bonaventure, Tournai, Desclée,
1961.
Bougerol, J.-G., “Verbum”, en Léxique Saint Bonaventure, Bougerol, J.-G. (dir.),
Paris, Éditiones franciscaines, 1969 p. 131.
Buenaventura, Opera omnia, 10 vols., Quaracchi, Ad Claras Aquas, 1882-1902.
Buenaventura, Le Christ Maître, Paris, J. Vrin, 1990.
Chavero, F. de A. “Ser y significar. Aproximación al simbolismo bonaventuriano”, Thémata. Revista de filosofía 5 (1988), pp. 51-171
Chavero, F. de A., “Novus Adan. Significado de la tipología de Adán en san Buenaventura (Leg. Maior, cap. 5)”, Verdad y Vida 50 (1992), pp. 137-171.
Cullen, C. M., Bonaventure, Oxford / New York, Oxford University Press, 2006,
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Garrido, J. J., El pensamiento de los Padres de la Iglesia, Madrid, Akal, 1997.
Gerken, A., La théologie du Verbe. La relation entre l’Incarnation et la création
selon S. Bonaventure, Paris, Éditions Franciscaines, 1969.
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Gilson, E., La filosofía de san Buenaventura, Buenos Aires, Desclée de Brouwer,
1948.
Guillermo de Auxerre, Summa aurea, lib.1, in fine, Ribailler, J. Ed., Paris-Grottaferrata, CNRS, Collegium S. Bonaventurae ad Claras Aquas, 1985
Hayes, Z., “Christology and Metaphysics in the Thought of Bonaventure”, The Journal of Religion 58 (1978), pp. 82-96. doi: https://doi.org/10.1086/
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san Buenaventura”, Cauriensia 2 (1997), pp. 179-210.
Lázaro, M., “Más allá de la quiditas: reflexiones sobre el proyecto metafísico
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Cerf, 1967.
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Piazza, L., Mediazione simbólica in san Bonaventura Vicenza, LIEF, 1948.
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Juan”, Teología 108 (2012), pp. 91-100.
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Gregoriana, 1997.
DOI: https://doi.org/10.15366/bp.2020.24.024
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Altered spring phenology of North American freshwater turtles and the importance of representative populations
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Ecology and evolution
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Altered spring phenology of North American freshwater turtles
Altered spring phenology of North American freshwater turtles
and the importance of representative populations
and the importance of representative populations Fredric J. Janzen
Iowa State University p g
Part of the Ecology and Evolutionary Biology Commons, Forest Sciences Commons, and the
Integrative Biology Commons Part of the Ecology and Evolutionary Biology Commons, Forest Sciences Commons, and the
Integrative Biology Commons Fredric J. Janzen, Luke A. Hoekstra, Ronald J. Brooks, David M. Carroll, J Whitfield Gibbons, Judith L.
Greene, John B. Iverson, Jacqueline D. Litzgus, Edwin D. Michael, Steven G. Parren, Willem M. Roosenburg,
Gabriel F. Strain, John K. Tucker, and Gordon R. Ultsch This article is available at The Research Repository @ WVU: https://researchrepository.wvu.edu/faculty_publications/
1672 Digital Commons Citation
Digital Commons Citation gg
Janzen, Fredric J.; Hoekstra, Luke A.; Brooks, Ronald J.; Carroll, David M.; Gibbons, J Whitfield; Greene,
Judith L.; Iverson, John B.; Litzgus, Jacqueline D.; Michael, Edwin D.; Parren, Steven G.; Roosenburg,
Willem M.; Strain, Gabriel F.; Tucker, John K.; and Ultsch, Gordon R., "Altered spring phenology of North
American freshwater turtles and the importance of representative populations" (2018). Faculty & Staff
Scholarship. 1672. https://researchrepository.wvu.edu/faculty_publications/1672 This Article is brought to you for free and open access by The Research Repository @ WVU. It has been accepted
for inclusion in Faculty & Staff Scholarship by an authorized administrator of The Research Repository @ WVU. For
more information, please contact researchrepository@mail.wvu.edu. This article is available at The Research Repository @ WVU: https://researchrepository.wvu.edu/faculty_publications/
1672 Abstract Globally, populations of diverse taxa have altered phenology in response to climate
change. However, most research has focused on a single population of a given taxon,
which may be unrepresentative for comparative analyses, and few long-term studies
of phenology in ectothermic amniotes have been published. We test for climate-
altered phenology using long-term studies (10–36 years) of nesting behavior in 14
populations representing six genera of freshwater turtles (Chelydra, Chrysemys,
Kinosternon, Malaclemys, Sternotherus, and Trachemys). Nesting season initiation oc-
curs earlier in more recent years, with 11 of the populations advancing phenology. The onset of nesting for nearly all populations correlated well with temperatures
during the month preceding nesting. Still, certain populations of some species have
not advanced phenology as might be expected from global patterns of climate
change. This collection of findings suggests a proximate link between local climate
and reproduction that is potentially caused by variation in spring emergence from
hibernation, ability to process food, and thermoregulatory opportunities prior to Ecology and Evolution. 2018;8:5815–5827.
| 5815
www.ecolevol.org
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.
© 2018 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.
Fredric J. Janzen and Luke A. Hoekstra contributed equally to the work and are joint first authors. Altered spring phenology of North American freshwater turtles
and the importance of representative populations Correspondence
Luke A. Hoekstra, Department of Ecology,
Evolution, & Organismal Biology, Iowa State
University, Ames, IA. Email: lhoek@iastate.edu Correspondence
Luke A. Hoekstra, Department of Ecology,
Evolution, & Organismal Biology, Iowa State
University, Ames, IA. Email: lhoek@iastate.edu Authors
Authors Received: 25 October 2017 | Revised: 22 March 2018 | Accepted: 29 March 2018 Received: 25 October 2017 | Revised: 22 March 2018 | Accepted: 29 March 2018
DOI: 10.1002/ece3.4120 Ecology and Evolution. 2018;8:5815–5827. Received: 25 October 2017 | Revised: 22 March 2018 | Accepted: 29 March 2018
DOI: 10.1002/ece3.4120
O R I G I N A L R E S E A R C H
Altered spring phenology of North American freshwater turtles
and the importance of representative populations
Fredric J. Janzen1 | Luke A. Hoekstra1
| Ronald J. Brooks2 | David M. Carroll3 |
J. Whitfield Gibbons4 | Judith L. Greene4 | John B. Iverson5 |
Jacqueline D. Litzgus6 | Edwin D. Michael7 | Steven G. Parren8 | Willem M. Roosenburg9 |
Gabriel F. Strain10 | John K. Tucker11 | Gordon R. Ultsch12
1Department of Ecology, Evolution & Organismal Biology, Iowa State University, Ames, Iowa
2Department of Integrative Biology, University of Guelph, Guelph, ON, Canada
3Warner, New Hampshire
4Savannah River Ecology Laboratory, Aiken, South Carolina
5Department of Biology, Earlham College, Richmond, Indiana
6Department of Biology, Laurentian University, Sudbury, ON, Canada
7Division of Forestry and Natural Resources, West Virginia University, Morgantown, West Virginia
8Hinesburg, Vermont
9Department of Biological Sciences, Ohio University, Athens, Ohio
10GAI Consultants, Inc., Bridgeport, West Virginia
11Jerry F. Costello National Great Rivers Research and Education Center Confluence Field Station, East Alton, Illinois
12Department of Biology, University of Florida, Gainesville, Florida O R I G I N A L R E S E A R C H he terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
y cited 1 | INTRODUCTION 1 are necessarily representative of the entire species. Evolutionarily,
however, marginal populations may be the least suited to respond
to steepening environmental gradients because of genetic drift as
well as gene flow from populations in other environments (Peischl,
Kirkpatrick, & Excoffier, 2015; Polechová & Barton, 2015). All
these factors challenge the assumption that conspecific popula-
tions will respond similarly to climate change and thus can be rep-
resented by a point estimate. Global climate has warmed substantially and at an accelerating rate
in recent decades (IPCC, 2014), although some regions have warmed
more slowly (Pan et al., 2004). Diverse biotas are responding to this
climatic change in various ways (Bell et al., 2015; Gibbs & Breisch,
2001; Li, Cohen, & Rohr, 2013; Parmesan & Yohe, 2003; Root et al.,
2003; Thackeray, Jones, & Maberly, 2008). Emerging from large-
scale analyses of longitudinal field studies of these phenomena is the
conclusion that altered phenology (i.e., timing of life-cycle events)
is a key biotic response to climate change. Populations of numer-
ous taxa, from birds to butterflies to angiosperms, are advancing
the annual onset of fundamental biological activities, occasionally
with documented effects on fitness (Benard, 2015; Pike, Antworth,
& Stiner, 2006). Reviews of biotic responses to climate change have incorporated
a wealth of data from a variety of species, but the data sets still con-
tain notable taxonomic gaps. In particular, few studies of long-term
phenology of ectothermic amniotes (=nonavian reptiles) have been
available for comparison (Table S1). Although such studies are begin-
ning to appear in the literature (Urban, Richardson, & Freidenfelds,
2014), this paucity nonetheless may reflect the noteworthy chal-
lenges in accurately observing life-history events in these often-
secretive taxa over many years (Frazer, Greene, & Gibbons, 1993). Moreover, this group exhibits numerous biological features linked
strongly to temperature (e.g., many have temperature-dependent
sex determination (Bull, 1980; Janzen & Paukstis, 1991) and a num-
ber of species are already imperiled (Turtle Taxonomy Working
Group, 2017; Ihlow et al., 2012)), thus illuminating both the scientific
importance and practical urgency of the issue. Many reports of phenological shifts, however, document the
response of single populations often near the edge of a species’
range. Summaries of these individual studies typically assume that
conspecific populations will respond similarly to climate change
and, therefore, use a single datapoint per species (Brown et al.,
2016; Parmesan, 2007; Parmesan & Yohe, 2003). K E Y W O R D S advancing phenology, climate, nesting, phenotypic plasticity, representative population,
reptile Funding information Division of Environmental Biology, Grant/
Award Number: 1242510; Natural Sciences
and Engineering Research Council of
Canada; U.S. Department of Energy, Grant/
Award Number: FC09-07SR22506 and
FC09-96SR18546; University of Georgia
Research Foundation; NSERC Discovery;
Canadian Wildlife Federation; Ontario
Ministry of Natural Resources (OMNR);
Algonquin Provincial Park; Toronto Zoo;
University of Guelph and Laurentian
University; the Joseph Moore Museum; the
Dr. Ned and Sally Test Fund; and the Cope
Museum Fund of Earlham College | 5815
www.ecolevol.org 5816 | 5816 |
JANZEN et al. nesting. However, even though all species had populations with at least some evi-
dence of phenological advancement, geographic variation in phenology within and
among turtle species underscores the critical importance of representative data for
accurate comprehensive assessments of the biotic impacts of climate change. K E Y W O R D S
advancing phenology, climate, nesting, phenotypic plasticity, representative population,
reptile 5816 JANZEN et al. nesting. However, even though all species had populations with at least some evi-
dence of phenological advancement, geographic variation in phenology within and
among turtle species underscores the critical importance of representative data for
accurate comprehensive assessments of the biotic impacts of climate change. 1 | INTRODUCTION TA B LE 1 List of species, locations, years sampled, and phenological trait(s) reported
Species
Locality
Latitude, longitude
Years (N)a
Trait
Chelydra serpentina
Algonquin Provincial Park, ON
45.54N, 78.27W
1976–2011 (36)
First nest
Chelydra serpentina
Thomson Causeway Recreation
Area, IL
41.95N, 90.11W
1989–2012 (23)
First nest
Chelydra serpentina
Crescent Lake National Wildlife
Refuge, NE
41.73N, 102.3W
1981–2013 (23)
First nest
Chelydra serpentina
Sand Run Lake, WV
39.07N, 79.38W
1988–2006 (18)
First emergence
Chelydra serpentina
Sand Run Lake, WV
39.07N, 79.38W
1988–2007 (19)
First hibernationb
Chelydra serpentina
Savannah River Site, SC
33.34N, 81.74W
1977–1998 (9)
First nest
Chrysemys picta
Algonquin Provincial Park, ON
45.54N, 78.27W
1985–2011 (26)
First nest
Chrysemys picta
Thomson Causeway Recreation
Area, IL
41.95N, 90.11W
1989–2013 (25)
First nest
Chrysemys picta
Crescent Lake National Wildlife
Refuge, NE
41.73N, 102.3W
1986–2013 (20)
First nest
Chrysemys picta
Two Rivers National Wildlife
Refuge, IL
38.99N, 90.55W
1995–2010 (15)
First nest
Clemmys guttata
Warner, NH
43.29N, 71.83W
1988–2012 (25)
First emergence
Glyptemys insculpta
Monkton, VT
44.27N, 73.12W
1986–2012 (19)
First basking
Kinosternon flavescens
Crescent Lake National Wildlife
Refuge, NE
41.73N, 102.3W
1982–2013 (17)
First nest
Kinosternon subrubrum
Savannah River Site, SC
33.34N, 81.74W
1977–2003 (10)
First nest
Malaclemys terrapin
Patuxent River, MD
38.50N, 76.70W
1987–2005 (18)
First gravidc
Malaclemys terrapin
Poplar Island, MD
38.76N, 76.38W
2004–2013 (10)
First nestc
Sternotherus odoratus
Two Rivers National Wildlife
Refuge, IL
38.99N, 90.55W
1995–2011 (13)
First nest
Trachemys scripta
Two Rivers National Wildlife
Refuge, IL
38.99N, 90.55W
1994–2012 (19)
First nest
Trachemys scripta
Savannah River Site, SC
33.34N, 81.74W
1977–2003 (16)
First nest
aRange of years sampled with total number of years sampled in parentheses. Note that some studies were not contiguous. bFirst hibernation is the date the first turtle was observed to enter hibernation. cThese data were combined for analyses. See Methods for justification. TA B LE 1 List of species, locations, years sampled, and phenological trait(s) reported aRange of years sampled with total number of years sampled in parentheses. Note that some studies were not contiguous. bFirst hibernation is the date the first turtle was observed to enter hibernation. cThese data were combined for analyses. See Methods for justification. We collected long-term nesting data on one population of
Kinosternon flavescens, one population of K. 1 | INTRODUCTION This practice
obscures intraspecific variation in phenological responses to cli-
mate change and potentially inhibits mechanistic understanding of
phenological shifts that population comparisons afford. Boundary
populations may differ greatly from conspecific populations to-
ward the center of the geographic range (Angert & Schemske,
2005). One reason is that boundary populations are more likely
to be limited by abiotic factors than are more central populations. For example, in the northern temperate zone, populations at the
northern edge of their species’ range are more thermally limited
than are conspecific populations farther from the range boundary
(Gilman, Wethey, & Helmuth, 2006; Root, 1988). Niche modeling
of 108 reptile species endemic to the United States supports the
idea that climatic factors are the primary cause of poleward range
limits, whereas southern ranges of these species are more likely
limited by nonclimatic factors (Cunningham, Rissler, Buckley, &
Urban, 2015). Because climate warming is occurring more rapidly
toward the polar regions (IPCC, 2014; Karl & Trenberth, 2003),
populations closer to the poles may exhibit more substantive
phenotypic responses than conspecific populations located to-
ward the center of the range (Mazaris, Kallimanis, Pantis, & Hays,
2013; Rosenblatt, Crowley, & Schmitz, 2016) and, hence, neither We combine long-term field data on nesting behavior in 14
populations representing six genera of North American freshwater
turtles, along with spring emergence data from three populations
representing three genera, to investigate effects of accelerating
climate change on phenology. Because of the biological signifi-
cance of nesting behavior and for ease of comparison among in-
dependent field studies, we focused on date of the first nesting
event in a population in a given year as a measure of phenology. We used these data first (i) to document annual variation in nest-
ing phenology and identify populations and species with advanc-
ing nesting phenology (i.e., initiating the nesting season earlier in
more recent years). We then (ii) assessed the extent to which ge-
ography contributed to the observed patterns, with special focus
on assessing the biophysical and climatological prediction that
populations at the northern boundary of a species’ range in the
northern hemisphere should exhibit the most significant tempo-
ral responses. In this context, we also (iii) explored local climatic
thermal cues that might be mechanistically related to annual vari-
ation in nesting phenology. To evaluate mechanisms (phenotypic 5817 JANZEN et al. 1 | INTRODUCTION subrubrum, four popula-
tions of Chelydra serpentina, four populations of Chrysemys picta, one
population of Sternotherus odoratus, two neighboring populations
of Malaclemys terrapin, and two populations of Trachemys scripta
(Table 1, Figure S1). The primary nesting phenology data set encom-
passed 280 monitor-years at six research sites between 1976 and
2013, with individual efforts encompassing periods of field study
from 10 to 36 years (mean = 24; Table 1). plasticity vs. genetic adaptation) that underpin within-population
patterns of annual variation in nesting phenology, we (iv) interpret
our findings in light of available population-level data for annual
variation in key prenesting activities (i.e., phenological traits re-
lated to spring emergence from hibernation) and individual-level
data for annual variation in onset of nesting (e.g., is earlier nesting
in more recent years driven by older females [within-generation
~ plasticity] or by primiparous females [across-generations ~
adaptation]?). At each of the six field sites, three of which were near the north-
ern edge of the range for the genera Kinosternon, Chelydra, Chrysemys,
and Trachemys (see Turtle Taxonomy Working Group, 2017 for spe-
cies’ range maps), experienced personnel monitored the areas prior
to onset of the nesting season (Carroll & Ultsch, 2007; Gibbons,
1990; Iverson, 1991; Iverson & Smith, 1993; Pfau & Roosenburg,
2010; Riley & Litzgus, 2013; Schwanz & Janzen, 2008; Schwarzkopf
& Brooks, 1985; Strain, Anderson, Michael, & Turk, 2012; Tucker,
Dolan, Lamer, & Dustman, 2008). Onset was indicated when the first
gravid turtle was observed nesting, which we recorded as day of the 2.1 | Data collection We focused on six genera from three families of North American
freshwater turtles whose reproductive biology has been studied
intensively in multiple populations from Nebraska, Illinois, South
Carolina, Maryland, and Ontario over at least a 10-year period
(Table 1). 5818 JANZEN et al. Smith, 2009) and the sample-size-corrected Akaike information
criteria (AICc). Because we wanted to estimate potential temporal
and climatic effects on phenology for each species and population,
and because the populations sampled were unlikely to represent
random samples of their species distributions, when justified we
fit population and species as fixed effects. For all analyses, when
estimating rates of change for multiple sites (i.e., fitting a common
slope), we also compared our reported estimates (Tables S2–S6;
Table 2) to those from varying intercept mixed models with site
fit as a random effect. These estimates were always well within
error of each other. Due to potential interactions between year
and species, we then used ANCOVA to test for heterogeneity of
slopes. When possible, we fit a common slope to estimate the
rate of change at the highest justifiable grouping of populations. When we could not fit a common slope for all populations, we split
populations by species. When we could not fit a common slope to
all populations within a species, we estimated separate slopes for
each population. In particular, we combined data on Malaclemys
populations from Patuxent, Maryland, and Poplar Island, Maryland
after ANCOVA tests failed to find a significant effect of site (i.e.,
the populations have responded similarly to temporal and climatic
variation). There was minimal autocorrelation in our time series
(Durbin–Watson test, p > .2 for all populations), thus we consid-
ered linear regression analyses appropriate. We inspected all data
and residuals for assumptions of normality and conducted all tests
in R version 3.1.2 (R Core Team 2015), employing a two-tailed
alpha of 0.05 (except where noted). year for statistical analyses. From 1995 to 2005, the first nesting
date for the Malaclemys population from Patuxent, Maryland was
not available, so first gravid date, as determined by palping the ingui-
nal area for shelled eggs, was used instead. For these years, we es-
timated first nesting date from the relationship between first gravid
date and first nesting date previously established for this population
between 1987 and 1994. 2.4 | Assessing the explanatory power of geography To assess whether temporal patterns in nesting phenology might be
related to geography, we compared regression slope estimates of the
relationship between first nesting date and year. For species with
distinct populations, we plotted estimates of phenological advance-
ment by latitude (Figure S2). We also calculated the Pearson’s prod-
uct moment correlation between rate of advancement and latitude
for each species and performed a one-tailed test for the significance
of this correlation based on the hypothesis that change in the onset
of nesting would be greater at higher latitudes (i.e., higher latitudes
would have a more negative slope). 2.1 | Data collection We focused on first nesting date because it
is widely available for the populations studied and we hypothesized
it would respond in a direct, linear way to climate change. Whereas
first nesting date often may be significantly correlated with median
(or mean) nesting date (Tucker et al., 2008), median nesting date
can obscure changes in the underlying population dynamics of mul-
tivoltine species (Schwanz & Janzen, 2008). Furthermore, we note
that first nesting date and the first major pulse of nesting activity
are highly correlated (e.g., R2 = .92 for our Illinois Trachemys popu-
lation). To further clarify relationships between spring climate and
phenology in North American freshwater turtles, we also examined
data from long-term studies of spring emergence from hibernation
of Chelydra in West Virginia and Clemmys guttata in New Hampshire
and of onset of spring thermoregulatory (i.e., aerial basking) behavior
of Glyptemys insculpta in Vermont. These three studies were of simi-
lar duration to our nesting studies (mean = 24 years; Table 1). We obtained air temperature data from weather stations within
1–30 km of each field site from the National Climatic Data Center
(ncdc.noaa.gov) for the USA and from Environment Canada (cli-
mate.weather.gc.ca) for Canada. We calculated heating degree-days
(HDD) as the sum of the number of degrees Fahrenheit that each
daily mean temperature fell below 65°F (~18°C; Strachey 1878) for
1–28 February, 1–31 March, 1–30 April, and 1–31 August. The base
temperature (i.e., 65°F) represents a minimum thermal threshold
below which freshwater turtles cannot perform many tasks neces-
sary for energy acquisition and allocation (Bulte & Blouin-Demers,
2010; Edwards & Blouin-Demers, 2007). Note that higher HDD
values indicate cooler temperatures. Such degree-day models can
provide useful mechanistic explanations of phenological change
(Bell et al., 2015; Cayton, Haddad, Gross, Diamond, & Ries, 2015;
Williams, Stichter, Hitchcock, Polgar, & Primack, 2014). As employed
here, this climate metric integrates thermal variation prior to onset
of the reproductive season (here, starting in late April–June), empha-
sizing spring conditions that could impact onset of the nesting sea-
son due to temporal proximity (Iverson, Higgins, Abby, & Griffiths,
1997). Relationships between first nesting date and HDD for April
were similar to those between first nesting date and mean April tem-
perature (Table S8). 2.3 To evaluate consistency in temporal changes in phenology, we re-
gressed date of first nesting (or other phenological measure) against
year. In addition to our attempts to identify congruence in the re-
sponse to climate change using ANCOVA, to aid comparison be-
tween temporal and climatic variation in phenology, we fit separate
regressions for each species and population (Tables 2 and S2, Tables
S4 and S5). This means that some slope estimates made at the spe-
cies level or higher, as noted in Table S2, were provided for illustra-
tive purposes, despite evidence of significant heterogeneity among
populations comprising these groupings. TA B LE 2 Estimates of the phenological response to climatic variation from linear regressions of first nesting date on heating degree-days
(HDD) for April. Rate of change reflects an estimate from the regression slope. “All populations” represents a regression using data from all
14 populations, with the common slope estimate justified by a comparison of slopes test (black line, Figure 3a). Separate regressions were
used to independently estimate change in nesting date for each species and population. Bold text indicates significance at α = 0.05 level TA B LE 2 Estimates of the phenological response to climatic variation from linear regressions of first nesting date on heating degree-days
(HDD) for April. Rate of change reflects an estimate from the regression slope. “All populations” represents a regression using data from all
14 populations, with the common slope estimate justified by a comparison of slopes test (black line, Figure 3a). Separate regressions were
used to independently estimate change in nesting date for each species and population. Bold text indicates significance at α = 0.05 level TA B LE 2 Estimates of the phenological response to climatic variation from linear regressions of first nesting date on heating degree-days
(HDD) for April. Rate of change reflects an estimate from the regression slope. “All populations” represents a regression using data from all
14 populations, with the common slope estimate justified by a comparison of slopes test (black line, Figure 3a). Separate regressions were
used to independently estimate change in nesting date for each species and population. Bold text indicates significance at α = 0.05 level p
y
g
g
p
p p
g
Species-site
Rate of change (days
per 100 degree-days)
SE
N
F
pc
Rb
All populationsa
4
0.5
280
62.3
<.001
.75
Chelydra serpentinaa
3.4
0.7
91
59.5
<.001
.72
Algonquin Provincial Park, ON
3.4
1.1
36
10
.002
.2
Crescent Lake National
Wildlife Refuge, NE
2.7
1
23
6.83
.008
.21
Thomson Causeway
Recreation Area, IL
4.9
1.5
23
11
.002
.31
Savannah River Site, SC
0.9
5.9
9
0.02
.444
0
Chrysemys pictaa
4.1
0.9
86
18.1
<.001
.45
Algonquin Provincial Park, ON
4.3
1.3
26
10.4
.002
.27
Crescent Lake National
Wildlife Refuge, NE
2.5
2.5
20
1.02
.163
.33
Thomson Causeway
Recreation Area, IL
4.1
1.5
25
7.69
.005
.22
Two Rivers National Wildlife
Refuge, IL
5.4
2.4
15
5
.022
.22
Trachemys scriptaa
6.2
2.3
35
13.5
.006
.42
Two Rivers National Wildlife
Refuge, IL
7
2.1
19
11.4
.002
.37
Savannah River Site, SC
2.2
6.8
16
0.1
.376
0
Kinosternon spp.a
3.3
1.9
27
69.9
.048
.84
Crescent Lake National
Wildlife Refuge, NE
2
1.2
17
2.67
.062
.09
Savannah River Site, SC
13.2
7.6
10
3.02
.06
.18
Sternotherus odoratus
Two Rivers National Wildlife
Refuge, IL
4.1
3.1
13
1.76
.106
.06
Malaclemys terrapin
Poplar Island, MDb
5
1.9
28
7.24
.006
.16
aPopulation was included as an independent variable in these models, significantly improving the statistical fit. bThis includes data from Patuxent, MD, and Poplar Island, MD. cSignificance calculated from a one-tailed t test for a positive slope. Index (SOI), and 3-months averages of the Oceanic Niño Index
(ONI)), all downloaded from the NOAA Climate Prediction Center
(cpc.ncep.noaa.gov; Table S6). Again, model selection favored mod-
els containing only HDD for April. Once we determined the optimal
covariate structure, we again employed ANCOVA and linear regres-
sion to estimate relationships between the onset of nesting and
HDD for April. 2.5 | Identifying potential climatic factors
affecting phenology To explore climatic variation that might be mechanistically related
to annual variation in nesting phenology, we adopted a similar sta-
tistical approach as above. We modeled the onset of nesting season
using measures of HDD summarizing climatic variation during the
preceding months. Model comparison using HDD for February, HDD
for March, HDD for April and all covariate combinations showed that
models containing solely HDD for April were favored by AICc. For
all populations, we also evaluated possible correlation or covariation
with climate indices (“winter” and monthly means of the Northern
Atlantic Oscillation index (NAO), monthly means of the Pacific North
American index (PNA), monthly means of the Southern Oscillation 2.2 | Statistical approach and model selection Testing for temporal trends in phenology and links to climate
primarily involved estimating the relationship (i.e., the slope) be-
tween the discrete timing of phenological events and a continuous
predictor (i.e., year or climatic factor). We determined the optimal
random and fixed components of our statistical models using the
top-down approach (described in Zuur, Ieno, Walker, Saveliev, & | 5819
JANZEN et al. TA B LE 2 Estimates of the phenological response to climatic variation from linear regressions of first nesting date on heating degree-days
(HDD) for April. Rate of change reflects an estimate from the regression slope. “All populations” represents a regression using data from all
14 populations, with the common slope estimate justified by a comparison of slopes test (black line, Figure 3a). Separate regressions were
used to independently estimate change in nesting date for each species and population. Bold text indicates significance at α = 0.05 level 5819 JANZEN et al. (Figure 2). All species studied except K. subrubrum tended to nest
earlier through time, with populations from three of seven species
doing so significantly earlier (Table S2; Figure 1) and another one
nearly so (S. odoratus, p < .10). Where it occurred, Chelydra was the
last species to initiate the nesting season in a given year compared
to the other species studied at a given location. That is, where com-
parisons can be made, the smaller turtle species (Sternotherus and
Kinosternon) tended to nest earlier at a particular site than the mod-
erately sized species (Chrysemys and Trachemys), which in turn began
nesting sooner than the larger-bodied Chelydra (Figure 2). from winter hibernation and for initial observation of spring basking. Model selection favored models containing only HDD for February
to explain variation in the onset of spring emergence and basking,
and similarly, only HDD for August to explain variation in the onset
of hibernation. 3.2 | Geographic trends Geography exerted a noticeable effect on both mean first nesting
date (Figure 2) and phenological advancement of nesting (Figure 1),
but these effects were inconsistent with expectations. Focusing
on species with at least two distinct populations, as described
above, the northern range-edge population of Trachemys in Illinois
(Figure 1c) exhibited the most striking advancement in the onset of
nesting among all populations studied (−9.0 days/decade; Table S2). By comparison, the Trachemys population in South Carolina, from
a more central position in the geographic range of this species, ex-
hibited no evidence of advancement in the onset of nesting date
(+1.7 days/decade; Table S2). Limiting the comparison of Trachemys
populations to years with overlapping samples (1994–2003) did not
qualitatively change these slope estimates. This geographic pattern
was essentially reversed for northern range-edge vs. range-center
populations of Chelydra and Chrysemys. Ontario populations of
both species only modestly advanced the onset of the nesting sea-
son in more recent years compared to the northern Illinois popula-
tions of these species that are closer to the latitudinal centers of
their respective geographic ranges. The southernmost populations
studied of these species (South Carolina and southern Illinois, re-
spectively) advanced their nesting phenology at similar rates (Table
S2). Nebraska populations of Chelydra and Chrysemys showed the
least evidence of phenological advancement for each species, with
the Chrysemys population actually trending toward later nesting,
further complicating a simple interpretation of the influence of ge-
ography (i.e., latitude). Even so, we did not detect anomalous trends
in the climatic factors identified to be important for nesting onset
at the Nebraska site that could explain this inconsistency (Table
S3). Taken together, we found no consistent latitudinal pattern in
temporal changes in the onset of nesting within species (Figure S2)
and no significant correlation between latitude and the magnitude
of phenological change in nesting among species (r8 = .07, p = .58). 2.7 | Examining the evidence for contemporary
climate change Lastly, we assessed temporal trends in HDD (i.e., climate change)
using a similar combination of ANCOVA and linear regression,
except that we also estimated the rate of change in HDD for a
subset of sites containing at least one population with evidence
of advancing nesting phenology. We evaluated the sensitivity of
this estimate to unequal sampling across sites by subsampling the
X-axis for years where at least 2 (of 6), at least 3 (of 6), at least 4
(of 6), or at least 5 (of 6) sites were represented. The reported re-
gression using the full range of data provided a relatively minimal
estimate of the rate of warming (range of slope estimates = −16.2
to −40.4 HDDs for April per decade). Of note, the greatest rate
of spring warming was estimated from recent years (1994–2011)
for which five (of six) sites were represented (−40.4 HDDs for
April per decade, R2 = .89). For species with distinct populations,
we also plotted our estimates of phenological advancement by
the rate of change in HDD for April (Figure S2b). We then cal-
culated the Pearson’s product moment correlation between the
rate of advancement in phenology and the rate of decline in HDD
for April (i.e., the rate of spring warming) for each population and
performed a one-tailed test for the significance of this correlation
based on the hypothesis that the rate of advancement would be
greater for populations that have experienced a greater decline in
HDD (i.e., more warming). 3.1 | Temporal trends All populations exhibited annual variation in date of first nesting. Eleven of the 14 populations examined displayed negative trends
with respect to time (Table S2; Figure 1), which is more than ex-
pected by chance (one-sided sign test, p = .03, Cohen’s h = 0.59), but
only three of these comparisons were individually significant (i.e.,
p < .05 without adjusting for multiple comparisons). Still, 79% of the
examined populations began the nesting season earlier than they
did at the beginning of the respective field studies. The advance in
onset of the nesting season for populations from the initial year of
fieldwork to the last year of study varied from as few as 0 day to as
many as 27 days (Table S2; Figure 1). Perhaps most notably, by 2012,
the Illinois population of Trachemys initiated the nesting season over
3 week earlier than it did in the mid-1990s (from 30 May 1994 to 3
May 2012). 2.6 | Testing the relationship between
prenesting and nesting behavior To interpret our nesting phenology findings in light of key prenesting
activities, we applied the same model selection and regression ap-
proach to evaluate temporal and climatic trends for first emergence 5820 JANZEN et al. 3.3 | Climatic cues (b) Chrysemys picta from Crescent Lake National Wildlife
Refuge, NE (dashed blue line) have a significantly different slope from the other three populations, preventing precise estimation of this
species rate of phenological change. (c) The nesting phenology of a northern Trachemys scripta population has significantly advanced,
while a more southern population has not. (d) Kinosternon flavescens from Crescent Lake National Wildlife Refuge, NE (dashed blue line)
and K. subrubrum from the Savannah River Site, SC (dashed red line) show possible latitudinal differences in the advancement of nesting
phenology, but these differences could also represent species-specific responses. (e) The single population of Sternotherus studied shows a
nonsignificant temporal trend in nesting phenology. (f) The nesting phenology of Malaclemys populations has been relatively static across
the time period studied. Note here the open symbols represent estimated first nest dates calculated from first gravid dates based on the
relationship between first nest date and first gravid date established at this site FI G U R E 1 The first nesting date of freshwater turtles has advanced in the past 36 years for most populations studied in the northern
United States and Canada, although the magnitude and significance of this advancement have varied among species and populations. Different symbols and colors represent different populations. Solid lines indicate linear regressions with significant, negative slopes (p < .05). Dashed lines represent linear regressions with slopes not significantly different from zero (p > .05). Black lines are from regressions of
multiple populations grouped at the species level (see Table S2). Colored lines are regressions from single populations, typically highlighting
populations that differed significantly in their phenological response relative to other populations of the species. (a) The solid black line was
estimated from all four populations of Chelydra serpentina, but the solid green regression line for Thomson Causeway, IL illustrates significant
variation in the magnitude of phenological advancement among these populations. (b) Chrysemys picta from Crescent Lake National Wildlife
Refuge, NE (dashed blue line) have a significantly different slope from the other three populations, preventing precise estimation of this
species rate of phenological change. (c) The nesting phenology of a northern Trachemys scripta population has significantly advanced,
while a more southern population has not. (d) Kinosternon flavescens from Crescent Lake National Wildlife Refuge, NE (dashed blue line)
and K. 3.3 | Climatic cues Nesting phenology was strongly linked to spring temperature,
as summarized by heating degree-days for April (HDD for April;
Figure 3a). Nearly all populations nested early when April was
warmer, 8 of 14 populations significantly so (Table 2). HDD for
April also significantly changed with time when all field sites were Onset of the nesting season also varied among years for each
species (Figure 1), and mean first nesting date varied among species JANZEN et al. 5821 FI G U R E 1 The first nesting date of freshwater turtles has advanced in the past 36 years for most populations studied in the northern
United States and Canada, although the magnitude and significance of this advancement have varied among species and populations. Different symbols and colors represent different populations. Solid lines indicate linear regressions with significant, negative slopes (p < .05). Dashed lines represent linear regressions with slopes not significantly different from zero (p > .05). Black lines are from regressions of
multiple populations grouped at the species level (see Table S2). Colored lines are regressions from single populations, typically highlighting
populations that differed significantly in their phenological response relative to other populations of the species. (a) The solid black line was
estimated from all four populations of Chelydra serpentina, but the solid green regression line for Thomson Causeway, IL illustrates significant
variation in the magnitude of phenological advancement among these populations. (b) Chrysemys picta from Crescent Lake National Wildlife
Refuge, NE (dashed blue line) have a significantly different slope from the other three populations, preventing precise estimation of this FI G U R E 1 The first nesting date of freshwater turtles has advanced in the past 36 years for most populations studied in the northern
United States and Canada, although the magnitude and significance of this advancement have varied among species and populations. Different symbols and colors represent different populations. Solid lines indicate linear regressions with significant, negative slopes (p < .05). Dashed lines represent linear regressions with slopes not significantly different from zero (p > .05). Black lines are from regressions of
multiple populations grouped at the species level (see Table S2). Colored lines are regressions from single populations, typically highlighting
populations that differed significantly in their phenological response relative to other populations of the species. 3.3 | Climatic cues (a) The solid black line was
estimated from all four populations of Chelydra serpentina, but the solid green regression line for Thomson Causeway, IL illustrates significant
variation in the magnitude of phenological advancement among these populations. (b) Chrysemys picta from Crescent Lake National Wildlife
Refuge, NE (dashed blue line) have a significantly different slope from the other three populations, preventing precise estimation of this
species rate of phenological change. (c) The nesting phenology of a northern Trachemys scripta population has significantly advanced,
while a more southern population has not. (d) Kinosternon flavescens from Crescent Lake National Wildlife Refuge, NE (dashed blue line)
and K. subrubrum from the Savannah River Site, SC (dashed red line) show possible latitudinal differences in the advancement of nesting
phenology, but these differences could also represent species-specific responses. (e) The single population of Sternotherus studied shows a
nonsignificant temporal trend in nesting phenology. (f) The nesting phenology of Malaclemys populations has been relatively static across
the time period studied. Note here the open symbols represent estimated first nest dates calculated from first gravid dates based on the
relationship between first nest date and first gravid date established at this site 1 The first nesting date of freshwater turtles has advanced in the past 36 years for most populations studied FI G U R E 1 The first nesting date of freshwater turtles has advanced in the past 36 years for most populations studied in the northern
United States and Canada, although the magnitude and significance of this advancement have varied among species and populations. Different symbols and colors represent different populations. Solid lines indicate linear regressions with significant, negative slopes (p < .05). Dashed lines represent linear regressions with slopes not significantly different from zero (p > .05). Black lines are from regressions of
multiple populations grouped at the species level (see Table S2). Colored lines are regressions from single populations, typically highlighting
populations that differed significantly in their phenological response relative to other populations of the species. (a) The solid black line was
estimated from all four populations of Chelydra serpentina, but the solid green regression line for Thomson Causeway, IL illustrates significant
variation in the magnitude of phenological advancement among these populations. 3.3 | Climatic cues Different colors represent different
species as in Figure S1 The solid black line represents a common f
Ch l d
i
W
t Vi
i i ) b t
th
h
i kl
i
FI G U R E 3 Spring phenologies of freshwater turtles are
positively associated with a single climatic factor. (a) First
nesting date is positively associated with heating degree-days
(HDD) for April (p < .001). Different colors represent different
species as in Figure S1. The solid black line represents a common
regression slope for all 14 populations studied from a best-fit
model that included population as an additive effect. There was
no significant effect of population on slope of the regression line
(Population × Year, p = .66). There was significant heterogeneity
in the slope of the regression line among species (Year × Species,
p < .05), however, all species-specific slope estimates were
positive and all except Sternotherus were significantly so. Table 2
enumerates variation in this relationship within and among species. (b) Spring emergence of freshwater turtles is also positively
associated with a single climatic factor, heating degree-days (HDD)
for February, which summarizes thermal variation immediately
preceding spring emergence. The solid black line represents a
common regression slope for three populations with estimates
of spring emergence, justified by a comparison of slopes test
(ANCOVA: Year × Population, p > .05). Separate regression
estimates for each population are listed in Table S5 FI G U R E 2 Mean first nesting date (±95% CI) for 14 populations
of freshwater turtles showing the relative contribution of site
(different shapes) and species (different colors). Sites are presented
in ascending order by latitude spring temperature and nesting phenology. For these two popula-
tions, HDD for April varied inversely with time (r = −.44, p = .060 and
r = −.30, p = .151, respectively) and positively with date of first nest-
ing (r = +.63, p = .004 and r = +.50, p = .011, respectively). In other
words, annual April climate warmed and this warming coincided
with an earlier onset of the nesting season in both populations. In
fact, the southern Illinois site was the locality with the greatest ev-
idence of climate warming (Table S3) and its Trachemys population
showed the greatest advancement in nesting phenology (Table S2,
Figure 1c). 3.3 | Climatic cues Furthermore, sites with little to no evidence of progres-
sively warmer springs (South Carolina and Poplar Island, Maryland)
harbored populations of freshwater turtles with no evidence of pro-
gressively earlier nesting, despite these populations having corre-
spondingly strong relationships between nesting onset and HDD for
April (Tables S2 and S3; Table 2). For the same set of populations,
we used to test the influence of latitude on the rate of phenological
advancement, the rate of change in HDD for April better predicts
temporal change in nesting phenology (Figure S2; r8 = .50, p = .07). FI G U R E 3 Spring phenologies of freshwater turtles are
positively associated with a single climatic factor. (a) First
nesting date is positively associated with heating degree-days
(HDD) for April (p < .001). Different colors represent different
species as in Figure S1. The solid black line represents a common
regression slope for all 14 populations studied from a best-fit
model that included population as an additive effect. There was
no significant effect of population on slope of the regression line
(Population × Year, p = .66). There was significant heterogeneity
in the slope of the regression line among species (Year × Species,
p < .05), however, all species-specific slope estimates were
positive and all except Sternotherus were significantly so. Table 2
enumerates variation in this relationship within and among species. (b) Spring emergence of freshwater turtles is also positively
associated with a single climatic factor, heating degree-days (HDD)
for February, which summarizes thermal variation immediately
preceding spring emergence. The solid black line represents a
common regression slope for three populations with estimates
of spring emergence, justified by a comparison of slopes test
(ANCOVA: Year × Population, p > .05). Separate regression
estimates for each population are listed in Table S5 3.3 | Climatic cues 3.4 | Prenesting activities
The phenological patterns of two additional traits (first day of
spring emergence from hibernation and first day basking) for
three separate populations and species exhibited temporal
trends (Table S4, Figure 4) that mirrored those we described
FI G U R E 2 Mean first nesting date (±95% CI) for 14 populations
of freshwater turtles showing the relative contribution of site
(different shapes) and species (different colors). Sites are presented
in ascending order by latitude
FI G U R E 3 Spring phenologies of freshwater turtles are
positively associated with a single climatic factor. (a) First
nesting date is positively associated with heating degree-days
(HDD) for April (p < .001). Different colors represent different
species as in Figure S1. The solid black line represents a common
regression slope for all 14 populations studied from a best-fit
model that included population as an additive effect. There was
no significant effect of population on slope of the regression line
(Population × Year, p = .66). There was significant heterogeneity
in the slope of the regression line among species (Year × Species,
p < .05), however, all species-specific slope estimates were
positive and all except Sternotherus were significantly so. Table 2
enumerates variation in this relationship within and among species. (b) Spring emergence of freshwater turtles is also positively
associated with a single climatic factor, heating degree-days (HDD)
for February, which summarizes thermal variation immediately
preceding spring emergence. The solid black line represents a
common regression slope for three populations with estimates
of spring emergence, justified by a comparison of slopes test
(ANCOVA: Year × Population, p > .05). Separate regression
estimates for each population are listed in Table S5 5822 |
FI G U R E 2 Mean first nesting date (±95% CI) for 14 populations
of freshwater turtles showing the relative contribution of site
(different shapes) and species (different colors). Sites are presented
in ascending order by latitude 5822 | JANZEN et al. JANZEN et al. FI G U R E 3 Spring phenologies of freshwater turtles are
positively associated with a single climatic factor. (a) First
nesting date is positively associated with heating degree-days
(HDD) for April (p < .001). 3.3 | Climatic cues subrubrum from the Savannah River Site, SC (dashed red line) show possible latitudinal differences in the advancement of nesting
phenology, but these differences could also represent species-specific responses. (e) The single population of Sternotherus studied shows a
nonsignificant temporal trend in nesting phenology. (f) The nesting phenology of Malaclemys populations has been relatively static across
the time period studied. Note here the open symbols represent estimated first nest dates calculated from first gravid dates based on the
relationship between first nest date and first gravid date established at this site considered together (Table S3 “All sites”). Larger-scale climate indi-
ces such as the NAO, PNA, SOI, and ONI did not explain substantial
variation in nesting date and including these indices as covariates did
not improve our ability to predict nesting date (Table S6). Focusing on the Illinois populations of Trachemys (northern edge
of the species’ geographic range) and Chrysemys (north-central por-
tion of the species’ geographic range but farther north than the
Trachemys population) illustrates the general relationship between 5822 |
JANZEN et al. spring temperature and nesting phenology. For these two popula-
tions, HDD for April varied inversely with time (r = −.44, p = .060 and
r = −.30, p = .151, respectively) and positively with date of first nest-
ing (r = +.63, p = .004 and r = +.50, p = .011, respectively). In other
words, annual April climate warmed and this warming coincided
with an earlier onset of the nesting season in both populations. In
fact, the southern Illinois site was the locality with the greatest ev-
idence of climate warming (Table S3) and its Trachemys population
showed the greatest advancement in nesting phenology (Table S2,
Figure 1c). Furthermore, sites with little to no evidence of progres-
sively warmer springs (South Carolina and Poplar Island, Maryland)
harbored populations of freshwater turtles with no evidence of pro-
gressively earlier nesting, despite these populations having corre-
spondingly strong relationships between nesting onset and HDD for
April (Tables S2 and S3; Table 2). For the same set of populations,
we used to test the influence of latitude on the rate of phenological
advancement, the rate of change in HDD for April better predicts
temporal change in nesting phenology (Figure S2; r8 = .50, p = .07). 4 | DISCUSSION Our long-term field studies of freshwater turtle populations in North
America occurred over a period of increasingly rising global temper-
atures (IPCC, 2014). Our assessment is among the first to provide
long-term data on intraspecific and interspecific patterns of phenol-
ogy for ectothermic amniotes. Although implying linkage between
changing climate and critical behaviors, the results of our study are
not wholly consistent with predictions that populations at a range
edge will respond to climate change differently than populations in
the center of a species’ range, highlighting prominent intraspecific
variation. We focused our analyses on date of the first observed behav-
ior to assess phenological variation. This emphasis promoted ease
of comparison among our independent research programs and is
consistent with most literature on phenological responses to climate
change. Indeed, various shorter-term studies of freshwater turtles
had already suggested that onset of nesting season might be linked
to proximate thermal conditions (Congdon, Breitenbach, Sels, &
Tinkle, 1987; Iverson et al., 1997). Interestingly, however, most work
on marine turtles has noted thermally linked temporal changes in
median nesting date, but not in onset of the nesting season (Table
S1). We therefore recognize that this trait might not reflect popu-
lation response to climatic variation for all chelonian species, much
less for all organisms. However, median nesting date has not shifted
temporally as did onset of the nesting season for the northern Illinois
Chrysemys population, a pattern resulting from increased production
of subsequent nests within the same year (Schwanz & Janzen, 2008). Although this outcome may increase offspring recruitment in the
short term, demographic costs may be incurred in the form of bi-
ased cohort sex ratios and a decline in the condition of adult females
(Tucker et al., 2008). Despite overall consistency in responses of nesting behavior
to spring temperature, not all turtle populations responded to
warmer springs to the same degree or, in one case, the same di-
rection. Variation in the onset of nesting could derive from multi-
ple sources. Life-history variation, and variation in the underlying
physiology, could have influenced responses of nesting behavior
to climate conditions. FI G U R E 4 First spring emergence or first basking of freshwater
turtles has advanced significantly in the past 25 years. The
solid black line represents a common regression slope for three
populations with estimates of spring emergence. A comparison
of slopes test justified fitting a common slope (ANCOVA:
Year × Population, p > .05). Separate regression estimates for each
population are listed in Table S4 FI G U R E 4 First spring emergence or first basking of freshwater
turtles has advanced significantly in the past 25 years. The
solid black line represents a common regression slope for three
populations with estimates of spring emergence. A comparison
of slopes test justified fitting a common slope (ANCOVA:
Year × Population, p > .05). Separate regression estimates for each
population are listed in Table S4 data. This interpretation of predominately plastic phenological
responses to local, temporally proximate conditions (vs. genetic
adaptation) is supported by other research at our field sites. Specifically, capture-mark-recapture studies in these populations
without exception identify different marked individuals as initiat-
ing the nesting season each year as opposed to new, unmarked fe-
males (Schwanz & Janzen, 2008). Thus, at least over the time frame
of our field studies, plasticity appears to be the primary mechanism
underlying the observed phenological patterns below the species
level, consistent with interpretations of most studies of responses
to climate change (Urban et al., 2014). One important conclusion of this comparative study is that in-
adequate geographic sampling could skew assessments of the biotic
impacts of climate change. Populations at higher latitudes within
a species’ range may be more likely to experience climate change
(IPCC, 2014) and could potentially be more sensitive to those ther-
mal changes (Cunningham et al., 2015). Illustrating this issue, the
Illinois populations of Trachemys at the northern edge of its species’
range exhibited a stronger phenological response to climate change
than the more northern Illinois population of Chrysemys that is more
central to its species’ range. This pattern of response is explained by
the greater degree of warming experienced at the more southern
Illinois site, but not predicted by simple latitudinal trends in climate
change prediction models. Nevertheless, disproportionate repre-
sentation of populations near range limits (either poleward or equa-
torward) in a data set could lead one to overestimate the strength of
response of a species to climate change. Moreover, the velocity of
climate warming through 2100 is generally predicted by large-scale
global climate models to be higher in continental interiors relative
to localities closer to coasts (Loarie et al., 2009), whereas regionally
downscaled climate models do not always concur (Pan et al., 2004). Thus, the choice of representative populations can affect both pat-
tern and projection. 3.4 | Prenesting activities The phenological patterns of two additional traits (first day of
spring emergence from hibernation and first day basking) for
three separate populations and species exhibited temporal
trends (Table S4, Figure 4) that mirrored those we described
above for onset of the nesting season. Spring emergence and
basking patterns were also similar to those detected for nest-
ing activities with respect to taxonomic and geographic variation. Furthermore, these two traits similarly covaried with spring tem-
perature (Table S5; Figure 3b). Spring emergence behavior did
not depend on what date the turtles entered hibernation (at least for Chelydra in West Virginia), but rather on how quickly spring
warmed. In other words, despite variation among taxa and locali-
ties, multiple thermally linked phenological traits of freshwater
turtle populations in North America commonly began sooner in
more recent years. 5823 JANZEN et al. conditions suggest that earlier nesting seasons alone will not
counteract impacts of climate change on developing reptile em-
bryos (Telemeco, Abbott et al., 2013). Moreover, assuming non-
marine taxa no longer have the capacity to migrate to suitable
habitats without anthropogenic assistance, computer simula-
tions imply that populations with TSD almost invariably go ex-
tinct via biased sex ratios if they respond to even a modest 2°C
increase in climatic temperature by employing only plasticity in
nesting behaviors rather than by genetic adaptation (Morjan &
Janzen, 2003). Based on these two theoretical exercises, plas-
ticity in nesting behavior of Chrysemys from climatically diverse
localities exhibited under common-garden conditions (Refsnider
& Janzen, 2012) may not bode well for those populations in the
coming decades, in contrast with among-population variation
in TSD in Chelydra (Ewert, Lang, & Nelson, 2005) that may re-
flect local adaptation. In contrast, others suggest that turtles
with TSD apparently have satisfactorily navigated prior climatic
disruptions without inordinate extinction (Silber, Geisler, &
Bolortsetseg, 2011) and might even benefit from female-biased
sex ratios (Hays, Mazaris, Schofield, & Laloë, 2017). However,
evidence for an abrupt thermal change at the K-Pg boundary is
lacking and skewed sex ratios induce deleterious genetic effects
by reducing the effective population size (Mitchell & Janzen,
2010). Regardless, turtles are already among the most glob-
ally endangered major taxa (Turtle Taxonomy Working Group,
2017), thus our findings have important conservation implica-
tions given the strong thermal dependence of the key phenolog-
ical traits we examined. We predict significant future alteration
of North American turtle behavior and subsequent impacts on
population biology that will challenge the persistence of these
increasingly imperiled organisms. Broadening the taxonomic scope, many aquatic amphibians
have a thermally sensitive life cycle similar to freshwater turtles
(Feder & Burggren, 1992), allowing instructive comparison concern-
ing thermal effects on phenology. Where temporal climate shifts
are substantive, amphibian phenological patterns are among those
changing most swiftly (Parmesan, 2007; Todd, Scott, Pechmann, &
Gibbons, 2011). Phenological rates of change for freshwater turtles
were typically rapid as well, ranging from 4.7 to 9.0 days per decade
for populations that exhibited significant temporal trends (Tables
S2 and S4). It is further notable that, of the phenological changes
recorded by Todd et al. (2011), none involved spring-breeding am-
phibians at their South Carolina site, which is the same locality we
also found negligible changes in nesting season onset for the three
turtle taxa we monitored there. This result highlights the likely
thermal concordance in spring activity of syntopic aquatic amphib-
ians and reptiles. 4.1 | Implications for the persistence of
freshwater turtles The preponderance of species in our study possesses an intrigu-
ing life cycle that involves offspring overwintering in the natal
nest after hatching (Costanzo, Lee, & Ultsch, 2008; Gibbons,
2013). This substantially delayed emergence from the nest may
be adaptive (Spencer & Janzen, 2014), yet also may incur di-
rect metabolic costs via warmer winters (Converse, Iverson, &
Savidge, 2005; Willette, Tucker, & Janzen, 2005) and thus may
be affected indirectly by changing phenology. If earlier emer-
gence of adults from hibernation is followed by earlier onset
of the nesting season as implied by our findings, embryonic
development during summer should also be accelerated. If em-
bryos do not succumb directly to lethal incubation temperatures
(Telemeco, Abbott, & Janzen, 2013) or suffer elevated levels
of physical abnormalities (Telemeco, Warner, Reida, & Janzen,
2013), their earlier hatching could be deleterious energetically if
they are obligated to stay in the nest until the following spring
without feeding (Muir, Dishong, Costanzo, & Lee, 2012). As such,
this notable life-history trait of within-nest overwintering should
experience strong negative selection across many parts of the
range as a consequence of increasingly earlier onset of the nest-
ing season. 4 | DISCUSSION For example, egg follicles develop in the
fall in Chelydra, but develop in both fall and spring in Chrysemys
(Rollinson, Farmer, & Brooks, 2012), thereby potentially contrib-
uting to both within-locality annual variation among species and
among-locality annual variation within species in the onset of
nesting season because nesting cannot commence until follicles
are fully developed and then shelled (Ewert, 1979). Additional vari-
ation in phenology could be driven by plastic responses to other
environmental factors, including water temperature, cloudiness,
and precipitation events (Bowen, Spencer, & Janzen, 2005), al-
though note that we did not find a link between hibernation entry
and hibernation departure for the one population with available 5824 JANZEN et al. fieldwork at all sites over the past four decades; much of the
fieldwork could not have been completed without the assis-
tance of several generations of dedicated students. All work was
carried out under approved university Animal Care Committee
protocols. disturbances. Ecological Applications, 15, 2171–2179. https://doi. org/10.1890/04-0431 disturbances. Ecological Applications, 15, 2171–2179. https://doi. org/10.1890/04-0431 Costanzo, J. P., Lee, R. E., & Ultsch, G. R. (2008). Physiological ecology
of overwintering in hatchling turtles. Journal of Experimental Zoology
Part a-Ecological Genetics and Physiology, 309a, 297–379. https://doi. org/10.1002/(ISSN)1932-5223 Cunningham, H. R., Rissler, L. J., Buckley, L. B., & Urban, M. C. (2015). Abiotic and biotic constraints across reptile and amphibian ranges. Ecography, 39, 1–8. CONFLICT OF INTEREST Edwards, A. L., & Blouin-Demers, G. (2007). Thermoregulation as a func-
tion of thermal quality in a northern population of painted turtles,
Chrysemys picta. Canadian Journal of Zoology, 85, 526–535. https://
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and genetic adaptation mediate amphibian and reptile responses
to climate change. Evolutionary Applications, 7, 88–103. https://doi. org/10.1111/eva.12114 Willette, D. A. S., Tucker, J. K., & Janzen, F. J. (2005). Linking climate
and physiology at the population level for a key life-history stage
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et al. Altered spring phenology of North American freshwater
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R. B. (2014). Phenological advancement of Lycaenid butterflies in
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Complex Autism Spectrum Disorder with Epilepsy, Strabismus and Self-Injurious Behaviors in a Patient with a De Novo Heterozygous POLR2A Variant
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Complex Autism Spectrum Disorder with Epilepsy, Strabismus
and Self-Injurious Behaviors in a Patient with a De Novo
Heterozygous POLR2A Variant Daniel R. Evans 1, Ying Qiao 2,3,4, Brett Trost 5
, Kristina Calli 2,3,4, Sally Martell 2,3,4, Steven J. M. Jones 2,4,6
,
Stephen W. Scherer 5 and M. E. Suzanne Lewis 2,3,4,* 1
Department of Family Practice, University of British Columbia (UBC), Victoria, BC V8R 1J8, Canada;
dre542@mun.ca 2
Medical Genetics, University of British Columbia (UBC), Vancouver, BC V6H 3N1, Canada;
yqiao@mail.ubc.ca (Y.Q.); kcalli@mail.ubc.ca (K.C.); sally.martell@ubc.ca (S.M.); sjones@bcgsc.ca (S.J.M.J.)
3
BC Children’s Hospital Research Institute, Vancouver, BC V6H 3N1, Canada 2
Medical Genetics, University of British Columbia (UBC), Vancouver, BC V6H 3N1, Canada;
yqiao@mail.ubc.ca (Y.Q.); kcalli@mail.ubc.ca (K.C.); sally.martell@ubc.ca (S.M.); sjones@bcgsc.ca (S.J.M.J.)
3
BC Children’s Hospital Research Institute, Vancouver, BC V6H 3N1, Canada
4
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The Centre for Applied Genomics and McLaughlin Centre, Hospital for Sick Children, University of Toronto
Toronto, ON M5G 0A4, Canada; brett.trost@sickkids.ca (B.T.); stephen.scherer@sickkids.ca (S.W.S.)
6 5
The Centre for Applied Genomics and McLaughlin Centre, Hospital for Sick Children, University of Toronto,
Toronto, ON M5G 0A4, Canada; brett.trost@sickkids.ca (B.T.); stephen.scherer@sickkids.ca (S.W.S.)
6
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Michael Smith Genome Sciences Centre, Vancouver, BC V6H 3N1, Canada
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Michael Smith Genome Sciences Centre, Vancouver, BC V6H 3N1, Canada 6
Michael Smith Genome Sciences Centre, Vancouver, BC V6H 3N1, Canada *
Correspondence: suzanne.lewis@ubc.ca Abstract: Autism spectrum disorder (ASD) describes a complex and heterogenous group of neu-
rodevelopmental disorders. Whole genome sequencing continues to shed light on the multifactorial
etiology of ASD. Dysregulated transcriptional pathways have been implicated in neurodevelopmen-
tal disorders. Emerging evidence suggests that de novo POLR2A variants cause a newly described
phenotype called ‘Neurodevelopmental Disorder with Hypotonia and Variable Intellectual and Be-
havioral Abnormalities’ (NEDHIB). The variable phenotype manifests with a spectrum of features;
primarily early onset hypotonia and delay in developmental milestones. In this study, we investigate
a patient with complex ASD involving epilepsy and strabismus. Whole genome sequencing of the
proband–parent trio uncovered a novel de novo POLR2A variant (c.1367T>C, p. Val456Ala) in the
proband. The variant appears deleterious according to in silico tools. We describe the phenotype in
our patient, who is now 31 years old, draw connections between the previously reported phenotypes
and further delineate this emerging neurodevelopmental phenotype.
Citation: Evans, D.R.; Qiao, Y.; Trost,
B.; Calli, K.; Martell, S.; Jones, S.J.M.;
Scherer, S.W.; Lewis, M.E.S. Complex
Autism Spectrum Disorder with
Epilepsy, Strabismus and
Self-Injurious Behaviors in a Patient
with a De Novo Heterozygous
POLR2A Variant. Genes 2022, 13, 470. https://doi.org/10.3390/genes
13030470 Keywords: POLR2A; autism spectrum disorder; neurodevelopmental disorder with hypotonia and
variable intellectual and behavioral abnormalities (NEDHIB); variant Complex Autism Spectrum Disorder with Epilepsy, Strabismus
and Self-Injurious Behaviors in a Patient with a De Novo
Heterozygous POLR2A Variant This study sheds new insights
into this neurodevelopmental disorder, and more broadly, the genetic etiology of ASD. genes
G C A T
T A C G
G C A T genes
G C A T
T A C G
G C A T genes
G C A T
T A C G
G C A T 1. Introduction Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. ASD describes a group of complex neurodevelopmental disorders. ASD is a clinical
diagnosis established by the DSM-5 criteria, and characterized by deficits in social com-
munication and interaction, as well as restrictive, repetitive behavior patterns [1]. The
Canadian prevalence of ASD is 1 in 66 children [2]. ASD is a heterogenous disorder, with
a multifactorial etiology involving both environmental and genetic factors. The genetic
contribution to ASD is substantial, with heritability estimated to be between 64% to 91% [3]. Both monogenic and complex genetic traits have been implicated in ASD. Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). g
p
g
p
Genes implicated in ASD are involved in a variety of biological pathways, particularly
in brain function and development, synaptogenesis, transcription regulation and chromatin
remodeling [4–7]. There is genetic overlap between ASD and other neurodevelopmental
disorders such as schizophrenia, epilepsy and intellectual disability [8]. The importance of
chromatin remodeling, transcriptional regulation and alternative splicing in ASD has been
proposed by prior studies [9,10]. https://www.mdpi.com/journal/genes Genes 2022, 13, 470. https://doi.org/10.3390/genes13030470 Genes 2022, 13, 470 2 of 9 The RNA polymerase II (pol II) complex is a well-studied and essential enzyme
responsible for transcribing all protein-coding and some non-coding genes [11]. The largest
subunit of pol II is RPB1 which is encoded by POLR2A. RPB1 and other subunits form the
DNA-binding domain of pol II [12]. The RPB1 trigger loop region facilitates incorporation
of incoming nucleotides during the elongation cycle, and residues within the trigger loop
have been shown to control the rate of RNA synthesis [13,14]. Recently, POLR2A was implicated in an emerging neurodevelopmental disorder abbre-
viated NEDHIB (Neurodevelopmental Disorder with Hypotonia and Variable Intellectual
and Behavioral Abnormalities) (OMIM # 618603). NEDHIB was described by Haijes and
colleagues who identified a cohort of 16 patients with ultra-rare de novo POLR2A variants
ascertained through GeneMatcher [15]. Their analysis yielded 11 probably disease-causing
POLR2A variants, 4 possibly disease-causing variants and 1 variant was unresolved. 1. Introduction Indi-
viduals with these variants demonstrated a suite of phenotypic features; the most striking of
which was profound early onset general hypotonia and delayed developmental milestones. Additional features included poor speech, intellectual impairment, seizures, strabismus and
others. In vitro variant modeling showed that RPB1 mutants introduced into Saccharomyces
cerevisiae (S. cerevisiae) with genetic backgrounds lacking transcription factors dst1 and sub1
resulted in aberrant growth; suggesting reduced transcriptional fidelity [15]. g
gg
g
p
y
Subsequently, Hansen and colleagues identified deleterious POLR2A variants in a
cohort of 12 individuals ascertained through an online data lake, a pediatrics clinic and
an online community for affected individuals [16]. Their analysis included one individual
who was previously reported by Haijes and colleagues [15]. Hansen and colleagues
identified a higher proportion of epilepsy and a lower proportion of hypotonia in their
cohort. Moreover, they described other features including ataxia, joint hypermobility,
short stature, skin abnormalities and cardiac congenital anomalies; further expanding the
potential phenotypic spectrum. Here, we describe the phenotype of a patient with confirmed ASD who was further
assessed genetically through the Autism Spectrum Interdisciplinary Research (ASPIRE)
Program based at BC Children’s Hospital Research Institute in British Columbia (BC),
Canada. Genetic investigation using whole genome sequencing of parent–offspring trios
revealed a novel de novo heterozygous variant in POLR2A (NM_000937.5: c.1367T>C;
NP_000928.1: p. Val456Ala). This variant is deleterious according to multiple in silico tools
and is not reported in population databases. We describe the phenotype in our patient,
draw connections between previously reported phenotypes and further delineate this
emerging neurodevelopmental disorder. Our study further emphasizes the phenotypic
spectrum of disorders characterized by deleterious POLR2A variants and emphasizes the
role of this gene in ASD. 2.1. Patient Recruitment The patient presented to the Provincial Medical Genetics Programme (PMGP) Clinic
in British Columbia, Canada for assessment of ASD and was invited to participate for
research-based genomic testing through the ASPIRE Program [17]. The patient and her
parents provided their respective informed consent to participate. The patient was as-
sessed by M.E.S.L. and detailed clinical genetic phenotyping beyond the pre-existing ASD
psychometric indices was also performed (see Results). 2.3. Whole Genome Sequencing Pipeline and Variant Analysis 2.3. Whole Genome Sequencing Pipeline and Variant Analysis DNA was extracted from whole blood using standard ethanol-based protocols. Parent–
offspring trio analysis using whole genome sequencing was performed through ASPIRE’s
iTARGET Autism Initiative (www.itargetautism.ca/, accessed on 1 March 2019) in collabo-
ration with the Center for Applied Genomics (TCAG; Sick Kids Hospital, Toronto, Ontario,
Canada) and the MSSNG project (Autism Speaks; www.mss.ng/, accessed on 1 April 2018). The pipeline for whole genome sequencing (WGS) has been described elsewhere [18] and
is briefly summarized. Whole genome sequencing was performed using Illumina HiSeq
X WGS platform by the TCAG at the Hospital for Sick Children. Raw data were aligned
to the human reference genome (GRCh38). The depth of WGS was 30x. Variants were
detected by importing vcf and bam files into VarSeq (GoldenHelix, Inc., Bozeman, MT, USA,
https://www.goldenhelix.com, accessed on 7 July 2020) for (single nucleotide variants
(SNVs) and insertion-deletions (indels) and copy number variant (CNV) analysis. CNVs
were generated with VarSeq using CNValgorithm with a minimum 10 kb binned region
coverage. SNVs/Indels were filtered using quality controls prior to variant annotation
with VarSeq. Quality control criteria included read depth ≥10 and genotype quality ≥20. For homozygous recessive and compound heterozygous variants, a minor allele frequency
(MAF) threshold of ≤0.05 was selected. Alternatively, a MAF ≤0.01 was applied for de
novo variants, as well as X-linked variants, and variants in candidate ASD genes, imprinted
genes, loss of function variants, as well as those in the 59 actionable ACMG genes, and
finally, those in custom in-house gene lists of candidate genes involved in ASD, ID, seizure,
hearing loss, overgrowth and others. Variant interpretation was performed using a custom
internal pipeline using VarSeq which incorporates data from over 20 different databases. The in silico tools used in our analysis included Sorting Intolerant from Tolerant (SIFT) [19],
Polyphen-2 [20], MutationTaster [21], Functional Analysis through Hidden Markov Mod-
els (FATHMM) [22], Combined Annotation Dependent Depletion (CADD) [23], Genomic
Evolutionary Rate Profiling (GERP) [24] and Grantham scores [25]. The WGS data from
this study are available online (https://research.mss.ng/, accessed on 7 July 2020). 2.4. Dynamic Molecular Simulations The amino acid sequence of the protein product of wild-type POLR2A (NCBI accession
number NP_000928.1), as well as the same sequence with the p.Val456Ala or p.Ile457Thr
variant, were used as input to AlphaFold v2.0.1 (DeepMind Technologies Limited, London,
United Kingdom) [26] in order to predict their three-dimensional structures. The two
predicted structures were visualized using PyMol v2.5.2 (Schrödinger, New York, NY,
USA) [27] after superposing them using the “align” command. The predicted structures
were also used as input to the molecular dynamics web server CABS-flex 2.0, accessed on
27 February 2022 [28] to assess whether any gross changes in amino acid contacts were
predicted. We searched gnomAD [29] to identify common (allele frequency > 1%) missense
variants within 20 amino acid residues of the mutation of interest (residues 436–476) that
could be used as controls in our structural comparisons, but none were found, consistent
with the high missense constraint observed in POLR2A (gnomAD missense Z-score = 7.13;
observed/expected = 0.42). 2.2. Psychometric Analysis of ASD The patient was formally diagnosed with ASD prior to the research study at age 5. Psychometric analyses for ASD were performed using Childhood Autism Rating Scale
(CARS). Cognitive and learning assessments were performed using Wechsler Preschool and
Primary Scale of Intelligence (WPPSI) and Leiter. Language was assessed using Peabody
picture vocabulary test. Adaptive skills and behavior were assessed using the Vineland and Genes 2022, 13, 470 3 of 9 Beery test of visual motor integration, according to the Diagnostic and Statistical Manual of
Mental Disorders, fourth edition (DSM IV). Beery test of visual motor integration, according to the Diagnostic and Statistical Manual of
Mental Disorders, fourth edition (DSM IV). 3. Results The proband is a female who was born at 41 weeks via induction to a healthy
34-year-old mother. The prenatal and perinatal histories were uncomplicated. Her post-
natal history was unremarkable until 18 months, when she suffered a febrile seizure. She
was diagnosed with a petit mal seizure disorder and was treated with anticonvulsant
therapy from age 5 through to age 8, after which medications were discontinued given a
seizure-free period. Her developmental milestones were considered delayed relative to her Genes 2022, 13, 470 4 of 9
zure-
ib siblings; however, they were generally achieved within the upper limit of normal for age. She walked at 17 months. Her speech was late with first words occurring after two years,
with only babbling prior. She began linking words together after 2 and a half years. She
developed self-injurious behaviors primarily manifesting as skin picking, without any head
banging or hair pulling. At age 3 she developed temper tantrums when people moved
into her physical space. She had selective sensitivity to certain sounds and demonstrated
stereotyped movements, facial motor tics and marked sensitivity to touch. Given concerns
surrounding dependency, communication, adaptive skills, self-injurious behaviors and
hyperacusis, she underwent psychometric testing which identified a definitive diagnosis of
ASD at age 5. She walked at 17 months. Her speech was late with first words occurring after two years,
with only babbling prior. She began linking words together after 2 and a half years. She
developed self-injurious behaviors primarily manifesting as skin picking, without any
head banging or hair pulling. At age 3 she developed temper tantrums when people
moved into her physical space. She had selective sensitivity to certain sounds and demon-
strated stereotyped movements, facial motor tics and marked sensitivity to touch. Given
concerns surrounding dependency, communication, adaptive skills, self-injurious behav-
iors and hyperacusis, she underwent psychometric testing which identified a definitive
diagnosis of ASD at age 5. Psychometric testing using CARS demonstrated ASD in the mild to moderate range. Psychometric testing using CARS demonstrated ASD in the mild to moderate range. WPSSI1 was limited due to lack of cooperation and apparent inability to comprehend task
requirements. She scored in the borderline range for object assembly. Subsequently, WPSSI2
testing revealed uneven performance subtests. Geometric designs and mazes, both of which
involve graphomotor components, were not administered due to fine motor delays. Testing
on the verbal scale was discontinued due to severe communication difficulties. 3. Results WPSSI1 was limited due to lack of cooperation and apparent inability to comprehend task
requirements. She scored in the borderline range for object assembly. Subsequently,
WPSSI2 testing revealed uneven performance subtests. Geometric designs and mazes,
both of which involve graphomotor components, were not administered due to fine motor
delays. Testing on the verbal scale was discontinued due to severe communication diffi-
culties. The proband was assessed in the PMGP medical genetics clinic at age 13. Her height
was 163 cm (75th percentile), weight was 59.5 cm (75–90th percentile) and head circum-
ference was 56.4 cm (97th percentile; +1.91 standard deviations and proportionate). Both
her parents were macrocephalic (> 98th percentile). This was felt to represent familial
benign macrocephaly in the overall context of the investigations below [30]. She had no
obvious dysmorphisms aside from a slight right-sided facial prominence (Figure 1). She
had significant far sightedness secondary to strabismus (intermittent left exotropia) which
was corrected with glasses by age 13. The proband was assessed in the PMGP medical genetics clinic at age 13. Her height
was 163 cm (75th percentile), weight was 59.5 cm (75–90th percentile) and head circum-
ference was 56.4 cm (97th percentile; +1.91 standard deviations and proportionate). Both
her parents were macrocephalic (> 98th percentile). This was felt to represent familial be-
nign macrocephaly in the overall context of the investigations below [30]. She had no ob-
vious dysmorphisms aside from a slight right-sided facial prominence (Figure 1). She had
significant far sightedness secondary to strabismus (intermittent left exotropia) which was
corrected with glasses by age 13. Figure 1. Clinical features of the proband with complex ASD involving epilepsy and strabismus. The photograph depicts the patient aged 13 years old. There are no dysmorphic features aside from
subtle right-sided facial prominence. Her strabismus was corrected with glasses (not shown), and
eventually was surgically corrected by age 24. Figure 1. Clinical features of the proband with complex ASD involving epilepsy and strabismus. The photograph depicts the patient aged 13 years old. There are no dysmorphic features aside from
subtle right-sided facial prominence. Her strabismus was corrected with glasses (not shown), and
eventually was surgically corrected by age 24. Figure 1. Clinical features of the proband with complex ASD involving epilepsy and strabismus. The photograph depicts the patient aged 13 years old. There are no dysmorphic features aside from
subtle right-sided facial prominence. 3. Results Her strabismus was corrected with glasses (not shown), and
eventually was surgically corrected by age 24. Figure 1. Clinical features of the proband with complex ASD involving epilepsy and strabismus. The photograph depicts the patient aged 13 years old. There are no dysmorphic features aside from
subtle right-sided facial prominence. Her strabismus was corrected with glasses (not shown), and
eventually was surgically corrected by age 24. Routine genetic investigations including karyotype, fragile X testing, chromosomal
microarray and targeted fluorescence in situ hybridization (FISH) for 22q11 and 22q13
were normal. Brain magnetic resonance imaging (MRI) demonstrated grossly normal
Routine genetic investigations including karyotype, fragile X testing, chromosomal
microarray and targeted fluorescence in situ hybridization (FISH) for 22q11 and 22q13 were
normal. Brain magnetic resonance imaging (MRI) demonstrated grossly normal structures
and several foci of increased white matter intensities, consistent with Virchow-Robin spaces
and considered a variant of normal. The patient and her parents subsequently enrolled in the ASPIRE iTARGET Autism
Initiative to elucidate the genetic etiology of her complex ASD phenotype. Whole genome
sequencing of the parent–offspring trio uncovered a de novo missense variant in POLR2A
(NM_000937.5: c.1367T>C; NP_000928.1: p.Val456Ala; chr17:7499070). The POLR2A
p.Val456Ala variant is deleterious according to SIFT, Polyphen-2 and MutationTaster while
FATHMM and MutationAssessor are discordant (Table 1). A highly deleterious effect is
predicted by the CADD score (29.6). Furthermore, the variant is novel, absent in gnomAD
and ClinVar. The impacted amino acid residue is situated within an active site in the Genes 2022, 13, 470 5 of 9 RPB1 domain 2 [14]. Our filtering pipeline examined published candidate ASD genes
based on high-ranking scores within the SFARI database, and identified three variants with
high SFARI scores, but unconvincing evidence for pathogenicity (Supplementary Material
Results). Table 1. Bioinformatic Tools Predict the POLR2A p. V456A Variant is Deleterious. Tool
Score
Prediction
SIFT
0.01
Deleterious
Polyphen-2 (HDIV)
0.998
Probably damaging
MutationTaster
0.999889
Damaging
FATHMM
−1.09
Tolerated
MutationAssessor
0.94726
Functional
CADD PHRED
29.6
Deleterious
GERP ++ RS
5.93
Conserved
Grantham
64
Similar Table 1. Bioinformatic Tools Predict the POLR2A p. V456A Variant is Deleterious. We used AlphaFold [26] to predict the structures of both the wild-type and mutant
protein and then visualized the superposed structures using PyMol [27]. We observed
no gross change in the three-dimensional structure, either overall (Supplementary Figure
S1A) or in the vicinity of the mutation (Supplementary Figure S1B). 3. Results Further support for
this interpretation was derived by visualizing amino acid contacts using the CABS-flex
2.0 server [28], with no gross changes in contacts observed between the two structures
(Supplementary Figure S1C). A damaging impact of the mutation may instead be due to
weakened hydrophobic interactions in the catalytic pocket due to the decreased side chain
bulk of alanine relative to valine, such as between residue 456 and a nearby hydrophobic
residue, L505 (Supplementary Figure S1D). The patient was seen in follow up at age 31. She remains healthy with a high level of
functioning and has had no recurrence of seizures. Her intermittent left exotropia eventually
led to a progressive deterioration in her distance vision such that she elected to have surgical
recession of the left medial and lateral extraocular rectus muscles at age 24, without any
complications. There were no further co-morbidities aside from anxiety and depression. Today she is living with family, enjoying a good quality of life and is independent to
activities of daily living. She recently obtained her driver’s license. Through assistance
with an individualized education program, she completed grade 12 equivalency. She briefly
enrolled in college before opting to work a part-time occupation instead. Targeted review
of systems, focusing on key clinical features identified by Hansen and colleagues [16] were
negative for ataxia, joint hypermobility, skin abnormalities, recurrent fever of unknown
etiology, congenital heart disease, immune dysfunction or developmental dysplasia of the
hip. 4. Discussion This study explores the genetic etiology of a patient who presented with complex
ASD involving epilepsy, delayed communication, self-injurious behaviors and strabismus. Genomic investigation identified a novel de novo POLR2A variant (p.Val456Ala), which
resides in the catalytic domain of RPB1 and is predicted to be deleterious by multiple in
silico tools. Prior functional studies modelling de novo POLR2A variants in S. cerevisiae
demonstrate a reduced fitness thought to be caused by malfunctioning pol II enzyme [15]. Our report provides unique insights into the emerging phenotypic spectrum caused by
de novo deleterious POLR2A variants. This refines our understanding of the underlying
structure and function of pol II and expands our understanding of the variable presentation
of this neurodevelopmental phenotype. Our case report demonstrates a patient with milder Genes 2022, 13, 470 6 of 9 features compared to existing literature and provides helpful health guidance, phenotype
and management insights over a 31-year natural history thus far. features compared to existing literature and provides helpful health guidance, phenotype
and management insights over a 31-year natural history thus far. g
g
y
y
Table 1 shows that the novel p.Val456Ala POLR2A variant is deleterious according to
SIFT, PolyPhen-2 and MutationTaster. Although FATHMM and MutationAssessor were
discordant with these predictions, the high CADD score of 29.6 argues for the deleterious
nature of this variant. CADD scores above 20 indicate a variant is among the top 1% of the
most deleterious substitutions that can occur in the genome; whereas a score of 30 indicates
a variant is within the top 0.1% of the most deleterious substitutions that are predicted to
occur [23]. This variant at position 456 resides in the RPB1 domain 2, which is a functionally
important domain containing an active site [14]. The valine at this position shows a high
degree of evolutionarily conservation as indicated by the GERP score of 5.93. Our dynamic
molecular simulations did not show any gross change in the three-dimensional structure
resultant from this variant (Supplementary Figure S1). Valine and alanine are both non-
polar amino acids of similar structure as demonstrated by Grantham score. We propose that
a damaging impact could arise due to weakened hydrophobic interactions in the catalytic
pocket resulting from the decreased side chain bulk of alanine relative to valine. 4. Discussion Support for
this is demonstrated by molecular dynamic modeling of the adjacent residue (p.Ile457Thr)
(Supplementary Figure S2) which has previously been reported as deleterious by Haijes and
colleagues, which also does not grossly alter the protein structure, but results in aberrant
growth in S. cerevisiae with background dst1 and sub1 knockout [15]. We therefore propose
future in vitro studies could explore whether this p.Val456Ala alters transcription efficiency. Finally, p.Val456Ala is novel and not previously reported in GnomAD or ClinVar. y p
p
y
p
Interestingly, there is a patient reported by Haijes and colleagues with a POLR2A
variant in the active site at the adjacent amino acid residue (p. Ile457Thr) (individual
2) [15]. A notable contrast can be drawn between this individual and the one described
in our study, as they were more severely affected compared to our patient. For example,
individual 2 was hospitalized on day 3 of life for feeding difficulties and cyanotic spells
with oxygen desaturations. He was followed by physiotherapy at 7 weeks of age for
general hypotonia and he developed three respiratory tract infections requiring antibiotics
by 8 months. He was managed in a tertiary care center for developmental delays. He had
slow development in gross and fine motor skills, dystrophic muscle mass and significant
muscle weakness. He sat without support at 23 months, walked by > 55 months and had
MRI changes showing delayed myelination and wide ventricles. There were no concerns for
autistic behaviors reported by 4 years of age. This contrasts with the phenotype presented
in our patient, which was predominantly characterized by complex ASD with epilepsy
and strabismus, and behavioral abnormalities. Her milestones were within an upper limit
of normal (walking at 17 months), and she primarily experienced speech delays, which
gradually improved. At age 31, she is independent to activities of daily living, working
part-time and maintains a good quality of life and functional status. To our knowledge, she
is the oldest patient reported in the literature with this phenotype, and our detailed clinical
information spans a longer interval. p
g
More broadly, our patient presents with a milder phenotype compared to most pre-
viously reported individuals with de novo POLR2A variants. For example, Haijes and
colleagues describe the most striking feature in their cohort was infantile onset hypotonia
in 14 out of 16 individuals [15]. Moreover, hypotonia was reported as profound in nine of
these individuals. 4. Discussion Conversely, they only described two individuals in their cohort with-
out hypotonia. Of these two individuals, one reportedly had autistic behaviors (without
evidence of psychometric testing), and little information was available for the second
individual. Interestingly, Hansen and co-authors observed a lower prevalence of hypotonia
in their cohort (8 out of 12 individuals) compared to Haijes and colleagues’ previously
reported cohort with POLR2A variants (p = 0.076309) [16]. In their study, five individuals
were without hypotonia, however full phenotypic information was available for only one
individual (individual eight). Genes 2022, 13, 470 7 of 9 This person (individual eight) was a 14-year-old girl who was striking for her mild
phenotype compared to other individuals in their cohort. She had seizures and speech
delay like our patient, however, her seizure onset was from 13 years old, and she had
an abnormal MRI showing polymicrogyria involving large portions of the left cerebral
hemisphere among other findings [16]. She had hypertonia (age eight) with spasticity in the
right extremities, with motor delays, ataxia, intellectual disability, and no reported autistic
features. Thus, her phenotype was more severe than our patient. Remarkably, individual
eight inherited the distal POLR2A variant (p.Gln1814Valfs99ter) from her mother, who was
reported to have speech delay and only mild learning difficulties. The paucity of available
clinical information regarding milder phenotypes underscores the importance in detailing
the complete phenotypic spectrum of patients with de novo POLR2A variants. Milder
phenotypes and the potential for inherence of POLR2A variants raises important questions
about other individuals who could be diagnosed through genetic testing. The different methods of ascertainment stand out as a distinct feature in our case
report compared to previous studies. Our patient presented to the BC PMGP clinic for
genetic assessment due to her diagnosis of ASD, whereas previously published cohorts
were ascertained through GeneMatcher, Hadoop Architecture Lake of Exomes (HARLEE)
or a pediatrics clinic. Four individuals were described with autistic behaviors by Haijes and
colleagues [15] while Hansen and co-authors reported six individuals with ASD in their
cohort [16]. Unfortunately, information surrounding the autistic features in previous studies
are limited by a lack of reported information, such as whether cases had confirmed with
psychometric testing and what functional impact these diagnoses had on the patients. Author Contributions: Conceptualization, M.E.S.L. and S.W.S.; methodology, Y.Q., S.M. and K.C.;
software, Y.Q. and B.T.; validation and formal analysis, D.R.E.; investigation, D.R.E.; writing—original
draft preparation, D.R.E.; writing—review and editing, M.E.S.L., K.C., Y.Q., D.R.E., S.W.S. and S.J.M.J.;
All authors have read and agreed to the published version of the manuscript. 4. Discussion Genes 2022, 13, 470 8 of 9 Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki and approved by the University of British Columbia, and the Children’s & Women’s
Health Centre of BC joint Research Ethics Board, study protocol: H01-70507. Originally approved in
2001, latest approval date is 15 January 2021. Informed Consent Statement: Informed consent was obtained from all subjects involved in the
study. Written informed consent has been obtained from the patient(s) to publish this paper. 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 confidentiality. 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 confidentiality. Acknowledgments: We would like to extend sincere thanks and appreciation to the patient and their
family for participating in the ASPIRE program and facilitating our work. Acknowledgments: We would like to extend sincere thanks and appreciation to the patient and their
family for participating in the ASPIRE program and facilitating our work. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 4. Discussion Our
case identifies a patient with psychometrically confirmed high-functioning ASD diagnosed
at age five, which was primarily marked by temper tantrums, dependency and skin picking,
without any documented intellectual disability. Re-visited at age 31, our patient enjoys
an excellent quality of life as described above. Thus, the milder phenotypic presentation
identified in this study could have profound implications with respect to identifying other
patients around the world who harbor POLR2A variants. This study emphasizes the
importance of future studies investigating POLR2A variants specifically in the context
of ASD. Future studies should explore whether patients with mild autistic features +/−
epilepsy in the absence of intellectual disability might harbor POLR2A variants. p
p y
y
g
In conclusion, we identified and characterized a novel heterozygous de novo POLR2A
variant which provides key insights into an emerging neurodevelopmental phenotype
and its clinical variability. Our study and previously published cohorts of patients with
POLR2A variants demonstrate the diagnostic utility of next-generation sequencing as it
pertains to ASD and other neurodevelopmental disorders. These findings expand our
understanding of the clinical and genetic heterogeneity of phenotypes caused by POLR2A
variants and highlight the importance of this gene in the context of contributing to ASD. Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/genes13030470/s1, Figure S1: Dynamic molecular simulations
observe no gross structural changes predicted between wild type and p.V456A mutant; Figure S2:
Dynamic molecular simulations observe no gross structural changes predicted between wild type
and p.I457T mutant; Table S1: Variants detected in our patient reported as strong candidate ASD
genes according to SFARI ASD genes. Author Contributions: Conceptualization, M.E.S.L. and S.W.S.; methodology, Y.Q., S.M. and K.C.;
software, Y.Q. and B.T.; validation and formal analysis, D.R.E.; investigation, D.R.E.; writing—original
draft preparation, D.R.E.; writing—review and editing, M.E.S.L., K.C., Y.Q., D.R.E., S.W.S. and S.J.M.J.;
All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by funding from Genome British Columbia (Strategic Initiative
B22ITG). iTARGET is supported by Genome British Columbia (Strategic Initiative B22ITG). The
ASPIRE BC work was supported by funding from the Canadian Foundation for Innovation (CFI-LEF). MESL is a senior clinician scientist supported by the B.C. Children’s Hospital Research Institute
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Numerical modelling challenges for clinical electroporation ablation technique of liver tumors
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Mathematical modelling of natural phenomena
| 2,020
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cc-by
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To cite this version: Olivier Gallinato, Baudouin Denis de Senneville, Olivier Seror, Clair Poignard. Numerical modelling
challenges for clinical electroporation ablation technique of liver tumors. Mathematical Modelling of
Natural Phenomena, 2020, 15, 10.1051/mmnp/2019037. hal-02493795 Numerical modelling challenges for clinical
electroporation ablation technique of liver tumors
Olivier Gallinato, Baudouin Denis de Senneville, Olivier Seror, Clair Poignard 1. Introduction Electroporation consists in applying high voltage short pulses to cells (typically several hundreds of volts
per centimeter during one hundred microseconds) in order to create defects in the cell membrane. Since the
last decades, the phenomenon has been widely studied both in vivo and in vitro experiments. Interestingly it
has been shown that this method selectively affects the cell membrane, which becomes permeable if the high
voltage is high enough. It thus makes it possible to either introduce non permeant molecules (ions, cytotoxic
drugs like bleomycin, DNA plasmids) into living cells, which is referred to as reversible electroporation [3, 46] or
to kill directly the cells in the targeted region (tumor) by a nonthermal mechanism (this is named irreversible
electroporation [11, 12, 15]. NUMERICAL MODELLING CHALLENGES FOR CLINICAL
ELECTROPORATION ABLATION TECHNIQUE OF LIVER TUMORS Olivier Gallinato1, Baudouin Denis de Senneville1,
Olivier Seror2,3 and Clair Poignard1,* Abstract. Electroporation ablation is a promising non surgical and minimally invasive technique of
tumor ablation, for which no monitoring is currently available. In this paper, we present the recent
advances and challenges on the numerical modeling of clinical electroporation ablation of liver tumors. In particular, we show that a nonlinear static electrical model of tissue combined with clinical imaging
can give crucial information of the electric field distribution in the clinical configuration. We conclude
the paper by presenting some important questions that have to be addressed for an effective impact of
computational modeling in clinical practice of electroporation ablation. Mathematics Subject Classification. 35J15, 35J87, 92B. Received February 10, 2019. Accepted September 2, 2019. Received February 10, 2019. Accepted September 2, 2019. 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. * Corresponding author: clair.poignard@inria.fr HAL Id: hal-02493795
https://inria.hal.science/hal-02493795v1
Submitted on 28 Feb 2020 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
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lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Mathematical Modelling of Natural Phenomena
www.mmnp-journal.org Math. Model. Nat. Phenom. 15 (2020) 11
https://doi.org/10.1051/mmnp/2019037 1.1. Electroporation-based therapies: an alternative to non surgical a From the clinical point of view, electroporation-based therapies (EBTs) provide interesting alternatives to
standard ablative techniques. Actually radiotherapy, microwave and radiofrequency ablation, or cryotherapy
for instance, are widely used in treatment of patients with deep-seated solid tumors, when surgery cannot
be used. Such methods rely upon the indiscriminate use of thermal energy to induce necrosis of the affected c⃝The authors. Published by EDP Sciences, 2020 Keywords and phrases: computational tissue electroporation, clinical electroporation ablation, nonlinear electrical tissue model.
1 INRIA Bordeaux-Sud-Ouest, CNRS, Bordeaux INP, Univ. Bordeaux, IMB, UMR 5251, 33400 Talence, France.
2 Department of Radiology, Hˆopital Jean Verdier, Hˆopitaux Universitaires Paris-Seine-Saint-Denis, Assistance Publique
Hˆopitaux de Paris, Avenue du 14 juillet, 93140 Bondy, France.
3 UMR 1162, G´enomique Fonctionnelle des Tumeurs solides, INSERM, Paris, France. Keywords and phrases: computational tissue electroporation, clinical electroporation ablation, nonlinear electrical tissue model. p
,
j
,
y,
3 UMR 1162, G´enomique Fonctionnelle des Tumeurs solides, INSERM, Paris, France. 1 INRIA Bordeaux-Sud-Ouest, CNRS, Bordeaux INP, Univ. Bordeaux, IMB, UMR 5251, 33400 Talence, France.
2 Department of Radiology, Hˆopital Jean Verdier, Hˆopitaux Universitaires Paris-Seine-Saint-Denis, Assistance Publique
Hˆopitaux de Paris, Avenue du 14 juillet, 93140 Bondy, France.
3 UMR 1162, G´enomique Fonctionnelle des Tumeurs solides, INSERM, Paris, France.
* Corresponding author: clair.poignard@inria.fr Keywords and phrases: computational tissue electroporation, clinical electroporation ablation, nonlinear electrical tissue model.
1 INRIA Bordeaux-Sud-Ouest CNRS Bordeaux INP Univ Bordeaux IMB UMR 5251 33400 Talence France c⃝The authors. Published by EDP Sciences, 2020 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. 2 O. GALLINATO ET AL. 2 cells (cancerous or not). In particular, due to the thermal diffusion in tissues, these techniques may result in
dramatic damages to the nearby structures including blood vessels, vital organs close to the target, bile ducts,
and nerves. Compared to these standard ablative therapies, EBTs – mainly irreversible electroporation (IRE)
and electrochemotherapy (ECT) – have several advantages: cells (cancerous or not). In particular, due to the thermal diffusion in tissues, these techniques may result in
dramatic damages to the nearby structures including blood vessels, vital organs close to the target, bile ducts,
and nerves. Compared to these standard ablative therapies, EBTs – mainly irreversible electroporation (IRE)
and electrochemotherapy (ECT) – have several advantages: – As electroporation is not based on thermal mechanisms – since pulses duration is too short for Joule
heating– the tissue scaffold is preserved: this makes such therapies suitable for tumors in proximity of
vital organs [4, 38, 39], and it improves the healing. – As electroporation is not based on thermal mechanisms – since pulses duration is too short for Joule
heating– the tissue scaffold is preserved: this makes such therapies suitable for tumors in proximity of
vital organs [4, 38, 39], and it improves the healing. – It has been proved that electroporation preserves the integrity of the main vessels, while it disrupts
neovascularization [46], leading to tumor hypoxia. – It has been proved that electroporation preserves the integrity of the main vessels, while it disrupts
neovascularization [46], leading to tumor hypoxia. [
],
g
yp
– Due to a vascular lock effect [23], electroporation greatly reduces bleeding. – Due to a vascular lock effect [23], electroporation greatly reduces bleeding. – As proteins are not denatured, antigens may stimulate an immune response against remaining cancer cells,
including metastasis located far from the target tissue. Note that this question is still unclear with contro-
versial publications: in [1], no evidence of immune response has been found (unlike for electrochemotherapy
as reported in [6] while Thomson et al. [49] claim that some evidence of an immune response has been
shown in animals. 1.2. Objectives and outline of the paper The aim of this paper is to present our numerical strategies that can provide highly valuable answers to
the above clinical questions. In the next section, we present briefly the clinical workflow which is routinely
performed at the University Hospital J. Verdier at Bondy. Then we present the most used numerical model
of tissue electroporation we investigate numerical the sensitivity to needle location and to conductivity value. Section 5 proposes a strategy which has been presented in [20]. In conclusion, we draw some clinically relevant
perspectives to increase the impact of the numerical modeling on the clinical practice of EBTs. c⃝The authors. Published by EDP Sciences, 2020 In spite of these advantages, that have been well-established in the experiments, the clinical use of EBT is still
mainly limited to cutaneous and subcutaneous tumors. It is worth noting that the hospital CHU J. Verdier
has become a leader in the use of IRE for liver tumors, in particular hepatocellular carcinoma (HCC) to the
liver: we thus benefit from this knowledge to focus our proposal on HCC. A first reason of the limitation in the
use of EBTs comes from the technical difficulties raised by such therapies: unlike standard ablative techniques
which mainly deal with one needle, EBTs need at least two and usually three to four needles (even more for
complex tumor shapes): the a priori determination of the treated zone is thus trickier than for standard ablative
techniques. In addition, these therapies suffer from the lack of appropriate post treatment protocols to evaluate
the tumor response. For standard ablative techniques, the MRI control is usually performed several weeks after
the treatment, while recent publications suggest [8, 38] that for IRE, early post procedure MRI at 1 day could
improve the understanding and the evaluation of the treatment. Another reason for the limitation of IRE in
clinic resides in the remaining lack of knowledge of the effect of IRE on cells. More precisely, there is currently
no standard criterium to evaluate the procedure on early MRI. Indeed, the image exhibits a white enhancement
in the vicinity of the needle location on the T2 weighted MRI modality, which raises several crucial questions
(see Fig 1). For instance, is it a reversibly permeabilised or irreversibly permeabilised region? How to assess the
success of the procedure on this early image? It is possible to predict this white enhancement with to the needle
location? 2. Clinical electroporation of liver tumors As previously described, the needles are percutaneously inserted around the tumor by
the interventional radiologist with free-hand technic under combination of real-time ultrasound (US) and
3D Virtual Target Fluoroscopic Display such that the electric field covers the target region. The needle
positioning is performed as parallel as possible and the needles location is recoverd by cone beam CT
(CBCT) imaging (see Fig. 2). Then the pulse parameters are set in the pulse generator (number and
duration of pulses, voltage per needle pairs, etc.). Clin. Step 3 Three days after the procedure, an MRI is performed to assess the treatment efficacy (Fig. 1
(Right)). Clin. Step 3 Three days after the procedure, an MRI is performed to assess the treatment efficacy (Fig. 1
(Right)). The clinical question can be summarized as follows. Is it possible, thanks to the pretreatment imaging and
the data acquired during the procedure to interpret the posttreatment imaging? In the following, we investigate
a model of tissue electroporation that can be used to address the clinical question. 3. Standard tissue electroporation model At the tissue scale, the electroporation modeling is in progress, since the phenomenon is still unclear. The
most current models are based on nonlinear static descriptions of the electric field. 2. Clinical electroporation of liver tumors The clinical workflow as routinely performed at the University Hospital J. Verdier at Bondy has been detailed
and validated by Sutter et al. [47, 48]. It is somehow the minimal clinical procedure, which can be performed in
most of interventional radiology operative room equipped with new generation of angiographic suit including
3D cone beam CT acquisition capacity [48]. The clinical workflow is split into 3 steps as described in [20]. 3 NUMERICAL CHALLENGES IN CLINICAL ELECTROPORATION 3 Figure 1. Imaging of the liver. (Left): CTScan performed 1 month before the IRE procedure. The image exhibits a scar tissue left from a previous RF ablation. The patient biomarkers
indicated a suspicion fo relapse located by the radiologist by the black arrow. (Right): Imaging
of the liver of the same patient at Day3 after the IRE procedure in T2 weighed MRI modality. The treatment region exhibits a white enhancement for which radiology interpretation is still
controversial [45]. Figure 1. Imaging of the liver. (Left): CTScan performed 1 month before the IRE procedure. The image exhibits a scar tissue left from a previous RF ablation. The patient biomarkers
indicated a suspicion fo relapse located by the radiologist by the black arrow. (Right): Imaging
of the liver of the same patient at Day3 after the IRE procedure in T2 weighed MRI modality. The treatment region exhibits a white enhancement for which radiology interpretation is still
controversial [45]. Clin. Step 1 The diagnostic stage provides a preoperative CT scan or MRI where the hepatic capsule, the
tumor and the main liver structures are determined several days before the treatment (Fig. 1 (Left)). Clin. Step 2 The day of the procedure, IRE ablation is performed under general anesthesia, as described by
Martin et al. [36]. As previously described, the needles are percutaneously inserted around the tumor by
the interventional radiologist with free-hand technic under combination of real-time ultrasound (US) and
3D Virtual Target Fluoroscopic Display such that the electric field covers the target region. The needle
positioning is performed as parallel as possible and the needles location is recoverd by cone beam CT
(CBCT) imaging (see Fig. 2). Then the pulse parameters are set in the pulse generator (number and
duration of pulses, voltage per needle pairs, etc.). Clin. Step 2 The day of the procedure, IRE ablation is performed under general anesthesia, as described by
Martin et al. [36]. 3.1. The standard tissue electroporation model: a nonlinear static model The mostly used model to describe tissue electroporation consists of the classic electrostatic problem [35, 37,
44]. The tissue is described as a conductive medium, whose conductivity σ depends on the amplitude of the 4 O. GALLINATO ET AL. Figure 2. CBCT Imaging of the liver of the same patient as Figure 1. The resolution is quite
low but one can see easily the needles and the liver boundary. Figure 2. CBCT Imaging of the liver of the same patient as Figure 1. The resolution is quite
low but one can see easily the needles and the liver boundary. electric field −∇V . The model reads then −∇· (σ(∥∇V ∥)∇V ) = 0,
in Ω,
(3.1a)
∂nV |Γout = 0,
V |E± = g±,
(3.1b) (3.1a)
(3.1b) (3.1a) (3.1b) where E± denotes respectively the activated anode and cathode needles used for the pulse delivery, Γout is the
boundary of the domain Ωdeprived of the activated needles, and g± are the Dirichlet data at the needle E±. The classical description of the tissue conductivity consists of a of 4-parameter sigmoid function. For instance where E± denotes respectively the activated anode and cathode needles used for the pulse delivery, Γout is the
boundary of the domain Ωdeprived of the activated needles, and g± are the Dirichlet data at the needle E±. The classical description of the tissue conductivity consists of a of 4-parameter sigmoid function. For instance The classical description of the tissue conductivity consists of a of ∀λ ≥0,
σ(λ) = σ0 + σ1 −σ0
2
(1 + erf(kep(λ −Eth))) ,
(3.2) (3.2) where σ0 is the conductivity of the non electroporated tissue, σ1 is the tissue conductivity of the fully porated
tissue, Eth is the threshold amplitude for electroporation, and kep is the slope of the nonlinearity. Remark 3.1. The choice of the sigmoid function does not seem essential according to Ivorra et al. [28], but it
is probably because of the lack of experimental data. In the code we took a hyperbolic tangent but the function
erf can be preferred. Standard arguments lead to the well-posedness of the nonlinear static model. Standard arguments lead to the well-posedness of the nonlinear static model. Theorem 3.2. Assume that Ωis a Lipschitz domain. We assume that the boundary of Ωhas three connected
components: the outer boundary ∂Ω, and 2 inner disjoint boundaries E±. 3.1. The standard tissue electroporation model: a nonlinear static model ll
h where in the above inequality the equality holds only for w = ˜w. Fi
ll
i
h where in the above inequality the equality holds only for w = ˜w. −Finally, since one has −Finally, since one has −Finally, since one has lim
∥w∥V(Ω)→+∞J(u) = +∞, then J admits a unique minimum W which satisfies ∀h ∈V(Ω),
Z
Ω
σ(∥∇(U + W)∥)∇(U + W)∇h dx = 0. Then the solution V to (3.1) is given by V = W + U. 3.2. Numerical method dedicated to clinical applications The evaluation of the treatment efficacy is crucial information, which is still not monitored in clinical use of
IRE. Usually, the treatment planning numerical strategies aim to provide to physicians the optimal positioning of
needles to perform the electroporation ablation. Our approach changes this paradigm by letting the physician
place percutaneously the needles as best as possible, and then by using this effective clinical positioning of
the needles as a starting point of the simulation. We benefitted from a tight collaboration with Olivier Seror,
interventional radiologist at Jean Verdier Hospital, to develop the finite difference method-based software IRENA
for the IRE Numerical Assessment in clinical routine [22]. 3.1. The standard tissue electroporation model: a nonlinear static model Let g± ∈H1/2(E±) be the applied
potentials on each inner boundary. Then there exists a unique potential V in H1(Ω) solution to problem (3.1)–
(3.2). Sketch of the proof. Thanks to the monotonicity of σ one can easily show that the above nonlinear PDE is
well-posed in H1(Ω) if g± ∈H1/2(E±). Actually, denote by U a lift of the Dirichlet condition g±. For instance NUMERICAL CHALLENGES IN CLINICAL ELECTROPORATION 5 5 let U be the solution to the linear elliptic problem: let U be the solution to the linear elliptic problem: et U be the solution to the linear elliptic problem: −∆U = 0,
in Ω,
(3.3)
∂nU|Γout = 0,
U|E± = g±. (3.4) −∆U = 0,
in Ω,
∂nU|Γout = 0,
U|E± = g±. ∂nU|Γout = 0,
U|E± = g±. (3.4) ∂nU|Γout = 0,
U|E± = g±. (3.4) U obviously belongs to H1(Ω). Let V(Ω) be defined by
V(Ω) =
u ∈H1(Ω) : u|E± = 0
. U obviously belongs to H1(Ω). Let V(Ω) be defined by U obviously belongs to H1(Ω). Let V(Ω) be defined by V(Ω) =
u ∈H1(Ω) : u|E± = 0
. Define by J the functional by Define by J the functional by ∀w ∈V(Ω),
J(w) =
Z
Ω
Z ∥∇(U+w)∥
0
sσ(s) ds dx. (3.5) (3.5) −The hypotheses on g± clearly imply that for any w ∈V(Ω), |J(w)| < +∞. −Moreover J is differentiable in V(Ω), since for any (w, h) ∈(V(Ω))2, one has −The hypotheses on g± clearly imply that for any w ∈V(Ω), |J(w)| < +∞. −Moreover J is differentiable in V(Ω) since for any (w h) ∈(V(Ω))2 one has −The hypotheses on g± clearly imply that for any w ∈V(Ω), |J(w)| < +∞. −Moreover J is differentiable in V(Ω), since for any (w, h) ∈(V(Ω))2, one has J(w + h) −J(w) =
Z
Ω
Z ∥∇(U+w+h)∥
∥∇(U+w)∥
sσ(s) ds dx =
Z
Ω
σ(∥∇U + w∥)∇(U + w)∇h dx + o(∥h∥H1). −The strict convexity of J comes from the strict mononicity of σ: −The strict convexity of J comes from the strict mononicity of σ (J′(w) −J′( ˜w), w −˜w) ≥0,
∀(w, ˜w) ∈V(Ω), (J′(w) −J′( ˜w), w −˜w) ≥0,
∀(w, ˜w) ∈V(Ω) where in the above inequality the equality holds only for w = ˜w. where in the above inequality the equality holds only for w = ˜w. 3.2.1. IRENA: a finite difference method-based dedicated to clinical IRE IRENA provides a numerical tool to assess the distribution of the electric field and check whether the tumor
is included in the estimated treatment area or not. For this purpose, numerical simulations are performed from
the real clinical data: the medical images which give the position of the organ and the tumor, the real position
of the needles, and the current graphs of test pulses for tissues conductivity calibration. These data are the
input parameters (see the numerical workflow detailed in Gallinato et al. [20]). In a first step of the procedure, the software makes it possible to setup the generator (Nanoknife R
⃝) by
computing the input voltages or nominal electric field to be applied. This essential step avoids over currents and 6
O. GALLINATO ET AL. Figure 3. Scheme of the modified ghost fluid method to address the issue of the needle location. The circle corresponds to the needle/tissue interface, the grid is the numerical grid. O. GALLINATO ET AL. O. GALLINATO ET AL. 6 Figure 3. Scheme of the modified ghost fluid method to address the issue of the needle location. The circle corresponds to the needle/tissue interface, the grid is the numerical grid. makes easier the procedure. In a second step, the electric field is computed, giving the immediate assessment
of the procedure. Finally, a retrospective validation can be done afterwards, by quantitatively comparing the
numerical results with the observations on MRI a few days after the procedure. Difference in volumes and shape
indicators (Hausdorffdistance) are provided. As the description of electrical current propagation in biological
tissues remains an open issue, IRENA also provides a numerical framework for confronting the existing different
mathematical models, as discussed in Section 6.4. For now, the standard nonlinear model is implemented in
IRENA but other models will be added in a near future. The discretization is performed thanks to the finite
difference method on Cartesian grid. This is the natural choice for computing from the voxel structure of medical
imaging. This also gives a significant advantage for future code parallelization. 3.2.2. Discretization close to the needle location The value of the potential u0 is such that the
isosurface Cu0 is far enough from the needle. In practice, u0 is 1/5 of the tension applied to the
needle. linear interpolations in operator discretization ensure the first order accuracy of the electric field magnitude at
least. 3.2.2. Discretization close to the needle location Far from the interfaces, the Laplace operator is discretized with the standard 5-points stencil. As the needles
are too thin to be discretized by the grid, the computation of the operator at the point close to a needle relies
on a modified ghost fluid method. As shown in Figure 3b, computing the operator at one point ui,j of the first
layer around a needle (Fig. 3a) may involve some neighboring points inside the needle (ui,j+1), some which
are themselves in the first layer ui−1,j and ui+1,j and farther points (ui,j−1) which do not require any specific
treatment. Neighboring points inside the needle are considered as standard ghost point and are handled as in
[17] with the exact boundary condition, whereas neighboring points in the first layer are handled as specific
ghost points (Fig. 3c), even if they are in the tissue. Let consider the projection uN on the needle of the specific
ghost point. Then, in the Neumann case, the derivative in the normal direction at the specific ghost point
is a first order accurate approximate of the known normal derivative at the projected point. In the Dirichlet
case, a first order expansion about the specific ghost point towards its projection gives a second order accurate
approximate of the known value at the projected point. Hence, the Neumann and Dirichlet boundary conditions
on the needle surface are accounted for thanks to Neumann and Robin conditions at the specific ghost points,
so that the overall accuracy of the solution is preserved in [21]. The above ghost fluid methods (GFM) require to know the distances to the immersed interfaces (liver capsule,
domain bounds, needle surfaces), These interfaces are implicitly described by the level 0 of the signed distance
function, obtained from a mask and the Fast Marching algorithm. As the geometrical configuration is static, the
redistanciation step is required only in case of change in needle position (pullback, pushforward or new needle
insertion) during the procedure. As the electric field derives from the electric potential, the main PDE variable, NUMERICAL CHALLENGES IN CLINICAL ELECTROPORATION 7 7 Figure 4. Scheme of the intensity computation. The value of the potential u0 is such that the
isosurface Cu0 is far enough from the needle. In practice, u0 is 1/5 of the tension applied to the
needle. Figure 4. Scheme of the intensity computation. 4. Numerical investigation of model sensitivity It is crucial to investigate the model sensitivity to parameters. Two main types of uncertainties are crucial to
analyze. In appendix, we also illustrate the reason why 3D simulations are crucial to provide relevant results. On
the one hand, uncertainties can come from the conductivities obtained by the calibration. The second source
of uncertainties is due to the errors of the geometric data. In the numerical sensitivity tests, the Hausdorff
distance as defined in [43] between the isolines of the electric field magnitude is computed, and the volumes are
compared. We first study the influence of liver and tumor conductivities on the electric field distribution. The
tissue conductivity is patient-dependent, with more or less variability depending on the tissue and the pathology. Thus, the conductivity value ranges found in literature are wide as stated above. For both tests on liver and
tumor conductivity, σX
0 and σX
1 stand for the minimal (tissue at rest) and the maximum (fully porated tissue)
conductivity values, respectively. As a second step, we investigate the influence of a small translation (3 mm)
or inclination (5◦) of a needle on the electric field distribution. 3.2.3. Intensity computation An efficient and accurate computation of the electrical intensity which flows through one needle – say E+ – is
required to calibrate the tissues conductivity from initial test pulses and their recorded intensities (IRE current
graphs) [20]. It is defined as IE+ =
Z
E+ σ(∥∇V ∥)∂nV dx. However the direct computation of the above integral leads to numerical errors due to the fact that the intensity
is along the needle which is placed in the region where the nonlinearity has the most influence on the conductivity. Therefore a small error on the electric field leads to a large error on the conductivity and thus on IE+. To prevent
such drawbacks, we propose to compute IE+ along an potential closed isosurface Cu0, where u0 is the value of
the chosen potential. This potential is such that Cu0 is around and far enough from the needle E+, the Green’s
formula shows that it leads to the same intensity: IE+ =
Z
Cu0
σ(∥∇V ∥)∂nV dx. The surface Cu0 is approximate by the face of the mesh cells, and the fluxes are computed along these faces (see
Fig. 4). The harmonic mean of the conductivity and the normal electric field are directly computed at the center
of the faces with the known conductivity and the potential values are computed at the center of the adjacent
meshes. O. GALLINATO ET AL. 8 8 8
O. GALLINA TO ET AL. Figure 5. Sensitivity to the medium conductivity (mS cm−1). The dotted and filled lines
stand for the IRE threshold (650 V cm−1) for the lower and larger values of the conductivity
parameters, respectively. For (a) and (b), the figure on the left shows a cross section, and the
figure on the right shows the longitudinal section orthogonal to the cross section. (a): Influence
of the liver conductivity (σL
0 = 1), Dotted line: σL
1 = 2.25, filled line: σL
1 = 4.5. (b): Influence
of the tumor conductivity. Dotted: (σT
0 , σT
1 ) = (2.5, 8), filled: (σT
0 , σT
1 ) = (4.5, 16) Figure 5. Sensitivity to the medium conductivity (mS cm−1). The dotted and filled lines
stand for the IRE threshold (650 V cm−1) for the lower and larger values of the conductivity
parameters, respectively. 3.2.3. Intensity computation For (a) and (b), the figure on the left shows a cross section, and the
figure on the right shows the longitudinal section orthogonal to the cross section. (a): Influence
of the liver conductivity (σL
0 = 1), Dotted line: σL
1 = 2.25, filled line: σL
1 = 4.5. (b): Influence
of the tumor conductivity. Dotted: (σT
0 , σT
1 ) = (2.5, 8), filled: (σT
0 , σT
1 ) = (4.5, 16) 4.1. Model sensitivity to liver and tumor conductivities Regarding the sensitivity of the electric field distribution to the liver conductivity, equations (3.1)–(3.2)
suggest that only the ratio between both linear and nonlinear parts of expression (3.2) matters. Therefore σL
0
is set to the median value found in literature (σL
0 = 1 mS cm−1 [19, 24, 25, 31, 41]), and σL
1 ranges from 2.25 to
4.5 mS cm−1 [10, 26, 31]. Figure 5a shows the variation of the electric field distribution for a given position of
the needles with respect to the liver conductivity (without any tumor). As mentioned above the strict parallel
placement of the needles is not always achievable because of anatomical constraints faced by the operator using
percutaneous approaches. In this section, the choice of the needle positions is the typical positioning performed
by the physicians in the case of liver tumors located near vital regions. The Hausdorffdistance between the two
3D areas (delineated by the dotted and filled lines in the planar views of Fig. 5a) is 2.1 mm, and the difference
in volumes is 994 mm3. In order to assess the sensitivity to the tumor conductivity, the liver conductivity parameters are now fixed
to (σL
0 , σL
1 ) = (1, 4) mS cm−1. The tumor parameters (σT
0 , σT
1 ) are alternately set to the extremum values found
in literature, (2.5, 8) mS cm−1 and (4.5, 16) mS cm−1 [24, 27]. The results are shown in Figure 5b (Hausdorff
distance = 2.7 mm, difference in volumes = 415 mm3). Both results, in transverse and longitudinal views, highlight the relatively weak influence of changes in
conductivity (in the range of values found in literature for liver and HCC) on the electric field distribution. This is corroborated by the corresponding Hausdorffdistances. However, the differences in volumes highlight NUMERICAL CHALLENGES IN CLINICAL ELECTROPORATION 9 9 Figure 6. Numerical study of the sensitivity of the IRE threshold isolines (650 V cm−1) with
respect to the geometry. (a) Influence of the needle position. The dotted (filled) lines stand
for the positions 1 (2 resp.). (b) Influence of the tumor position. The dotted line corresponds
to the tumor in the center of the needles. (c) Infuence of the hepatic capsule. The dotted line
corresponds to the case without liver capsule. Figure 6. Numerical study of the sensitivity of the IRE threshold isolines (650 V cm−1) with
respect to the geometry. 4.2. Model sensitivity to the needle locations As known, the electric field is very sensitive to the needle positions. Figure 6 shows that a small translation
(3 mm) or inclination (5◦) of a needle has a significant impact on the electric field distribution. A translation of
3 mm leads to a Hausdorffdistance of 3.4 mm between the 650 V.cm−1–isolines, and the difference in volumes
is 209 mm3. An inclination of 5◦of one needle leads to a Hausdorffdistance of 4.4 mm, and the difference
in volumes is 247 mm3. For each test, the Hausdorffdistance is larger than for the tests with extreme values
of the conductivity. Figure 6a shows that a small error in the needle location may lead to large errors in the
prediction of the region affected by the electric field. This aspect shows that the discrepancy between the
treatment planning and the clinical procedure may lead to treatment failure. In liver, the actual layout can then
vary substantially (sometimes with much larger variations than an inclination of 5◦or a translation of 3 mm). The second step of the numerical workflow proposes a way to overcome this drawbacks by reconstructing the
geometrical framework of the actual clinical procedure. Regarding the tumor location, it has been shown previously that changes in conductivities have only a small
influence. However, due to its high conductivity, the tumor behaves as a potential well, which attracts the
electric field lines. As a result, the change in the malignant tissue position leads to a local change in the electric
field distribution, as shown in Figure 6b. More generally, any structure substantially more conductive than the
liver tissue (surgical clips, scar tissue) and located in the vicinity of the target lesion has to be accounted for
by the numerical simulations. Among the structures that may influence the electric field, the liver capsule (Glisson’s capsule) behaves like
a barrier to the current. It is composed of a fine dense connective tissue layer, which makes it a thin insulating
membrane. If the ablation zone is located in the upper part of the right lobe, the lung is adjacent to the capsule. The air contained in the lung then reinforces the insulating property of this part of the capsule, which is modeled
by imposing a Neumann condition. However, the influence of the capsule depends strongly on the proximity of
the needles and vanishes when the distance increases (Fig. 6c). 4.1. Model sensitivity to liver and tumor conductivities (a) Influence of the needle position. The dotted (filled) lines stand
for the positions 1 (2 resp.). (b) Influence of the tumor position. The dotted line corresponds
to the tumor in the center of the needles. (c) Infuence of the hepatic capsule. The dotted line
corresponds to the case without liver capsule. two points: the influence slightly impacts the whole isosurface resulting in a significant difference in volumes,
and changes in liver conductivity have a stronger impact than changes in tumor conductivity, as previously
highlighted in [30]. 10 O. GALLINATO ET AL. Note that an underestimation of the tumor conductivity could result in an overestimation of the treated
region (see area below the IRE threshold in Fig. 5b), which may lead to a treatment failure. However, only one
pulse per pair of needles has been simulated. The accumulation of pulses in clinical procedure leads to a better
coverage of the tumor. In addition, the radiologist may also perform a pullback to enlarge the treated region. 5. How to provide clinically relevant simulations? The above numerical sensitivity analysis shows that for deep-seated tumors, the numerical treatment plan-
ning solution, for the needles are exactly parallel and at the same depth cannot be used for the treatment
efficiency assessment. Indeed, a small inclination of the needle due to anatomic constraint modifies substantially
the isosurfaces of the electric field, and thus the electroporation ablation zone. In [20], we propose a numer-
ical workflow to provide a numerical evaluation of the clinical electroporation ablation as performed by the
interventional radiologist during the procedure. 5.2. The issue of image registration For the successful completion of the proposed numerical workflow, several concepts, such as delineation
propagation, rely on establishing a spatial coherence between pre-/post-operative images and images acquired
at different time instants over the course of the therapy. To meet this need, image-based motion estimation
and compensation can relie on fast, automatic, accurate and precise registration algorithms. In the current
study we used the evolution algorithm. The latter aims at minimizing a variationnal functional composed by a
regularization term (assuming that displacements in neighboring voxels is moderate) added to a data fidelity
term (i.e. a measure of the similarity between input images). The data fidelity term here quantifies the angle
between the image gradient orientations. Parallel and anti-parallel gradients are favored, in order to match
similar local contrast patterns. 5.1. Numerics for clinical electroporation ablation: radiologists at the center of the
procedure The novelty of this workflow is to mimic the steps of the clinical procedure, in order to the all the information
provided by the clinical data. Num. Step 1 Segmentation of the Regions of Interest (ROIs) from the preoperative image (CT-scan or MRI)
thanks to a semi-automatic segmentation before the day of the procedure. Num. Step 2-a Needles segmentation from the CBCT of the day of the procedure (see Fig. 2), and ROIs
registration on the CBCT to get the patient-dependent geometrical framework. 11 NUMERICAL CHALLENGES IN CLINICAL ELECTROPORATION 11 Figure 7. Measured (left) and simulated (right) intensities of the first test-pulse of each pair
of probes, from [20]. Figure 7. Measured (left) and simulated (right) intensities of the first test-pulse of each pair
of probes, from [20]. Num. Step 2-b Calibration of the electroporation model parameters from the test pulse chronograms (pre-
simulations), and simulation/prediction of the treated region. Num. Step 3 Registration of the numerical simulation on the early postoperative MRI. 5.3. Estimation of the conductivities from recorded intensities and feedback
to physicians (a), (b) and (c) Simulated 3D
isosurfaces 500 V cm−1 at each step of the procedure. the electroporation ablation, it provides to the physicians a visualization of the treated region, enabling possible
treatment optimization. the electroporation ablation, it provides to the physicians a visualization of the treated region, enabling possible
treatment optimization. 5.3. Estimation of the conductivities from recorded intensities and feedback
to physicians Literature provides approximations of liver and tumor conductivities. However, as the conductivity param-
eters are patient-dependent, it is once again possible to use clinical data to adjust them. At the beginning of
the procedure, test-pulses are recommended by the manufacturer, to check whether or not the device has been
correctly setup. As the device provides current graphs with the intensities measured during the pulses, these
tests can be advantageously used to estimate the conductivity parameters from the intensity measurements. The values used for simulations are obtained thanks to a preliminary simulation, by a rough fitting of the
simulated intensities on chronograms, as shown in Figure 7 by a trial error method –starting from conductivity
values available in the literature and changing them handly step by step to obtain the fitting–, to fit the numerical
intensities with the intensities recorded by Nanoknife R
⃝. The tolerance criterium of the fitting procedure is set
such that during the first pulse of each pair combination, the relative error between the maximal value of
the recorded intensity and the simulated intensity is less than 10%. It is worse noting that the calibration is
performed once, with the initial needle location, and not modified afterwards. They are consistent with literature
and are reported in Table 1. The calibration enables to provide to physician the effective distribution of the electric field, as performed
during the procedure (see Fig. 8). This new insights to the interventional radiologist could improve drastically 12 12 O. GALLINATO ET AL. Table 1. Estimate of conductivity parameters computed from test-pulse current graphs, from
[20]. Parameter
Value (mS cm−1)
Reference
σL
0
1.2
[25]
σL
1
4.2
[26]
σS
0
6.0
[18]
σS
1
6.0
–
Figure 8. Isovalues of the maximum electric field magnitude. (a), (b) and (c) Simulated 3D
isosurfaces 500 V cm−1 at each step of the procedure. Parameter
Value (mS cm−1)
Reference
σL
0
1.2
[25]
σL
1
4.2
[26]
σS
0
6.0
[18]
σS
1
6.0
– Figure 8. Isovalues of the maximum electric field magnitude. (a), (b) and (c) Simulated 3D
isosurfaces 500 V cm−1 at each step of the procedure. Figure 8. Isovalues of the maximum electric field magnitude. (a), (b) and (c) Simulated 3D
isosurfaces 500 V cm−1 at each step of the procedure. Figure 8. Isovalues of the maximum electric field magnitude. 5.4. Retrospective validation The early postoperative MRI shows an impact of the treatment 3 days after the procedure. This area can be
segmented and then, the images can be registered on the CBCT, for a retrospective study. Then, the preoperative
ROIs and simulations can be superimposed on the registered MRI (Fig. 9) for comparison between observations
and simulations. In Figure 9, the 2D area delineated by the isosurface 500 V cm−1 seems to match roughly
with the contour of the observed area. In the 3D representation of Figure 10, the shape of the simulated area
looks correct and is included in the observed area. This is consistent with the assumption that the observation
contains the ablation area, and a margin in which irreversible and reversible phenomena coexist (margin between
reversible and irreversible thresholds). Finally, it can be retained that the tumor is inside the area where the
maximum electric field is beyond 500 V cm−1, and inside the observed area, which gives a rather favorable
tendency to the success of the procedure chosen for the proof of concept. It should be noted that the mismatch between simulated and observed areas would indicate a model error
or the omission of a crucial influence. As a result, even if what is observed on the MRI is not fully understood
yet, this retrospective validation is essential to check the consistency of the simulation, and to give a second
assessment of the procedure. NUMERICAL CHALLENGES IN CLINICAL ELECTROPORATION 13 Figure 9. Superimposition of simulation results on the registered postoperative MRI. Figure 9. Superimposition of simulation results on the registered postoperative MRI Figure 10. 3D comparison between the simulation (isosurface 500V cm−1) and the registered
ROI of the treatment area (delineation in light color). Figure 10. 3D comparison between the simulation (isosurface 500V cm−1) and the registered
ROI of the treatment area (delineation in light color). 6. Discussion and still open questions Each step of the numerical workflow raises crucial numerical questions which are described below and that
need further investigations. 6.1. The importance of the image registration algorithm Regarding the image registration, it is important to use non rigid registration algorithms. Indeed, it is worth
noting that between two medical exams, the location of the liver may change. This is particularly true when
needles are inserted: the insertion imposes a deformation to the liver, and therefore a rigid registration lead
to non relevant results. In [20] we propose to use the EVolution algorithm proposed by Denis de Senneville
et al. [14]. Roughly speaking, the velocity field between the two gradient of the images is regularized by a O. GALLINATO ET AL. 14 diffusion term. The minimization is perfomed by solving the reaction-diffusion equation on the velocity field. This approach has been validated on clinical data, for soft tissue registration: bladder, liver, etc. However the
use of constant in space diffusion coefficients is very restrictive. In particular, in a rigid body such a bone has
to be registered, the algorithm fails to provide relevant results, because the bone would be deformed as the soft
tissues. Therefore it is necessary to develop new strategies for clinical image registration. 6.3. Automatic model calibration procedure The strategy of the model calibration lies in the fact that the model parameters impact the value of the
electrical current that flows through the needle. Since the recording intensities are the only electrical data of
tissue, it is natural to calibrate the model by fitting the numerical intensity on the recorded intensity. Here we
present the results of [20] obtained thanks to a trial–error calibration. However it is important to develop an
appropriate calibration strategy to improve and to automate this step. Several approaches will be tested in a
near future, from simple Monte-Carlo approach, to more advanced calibration such as variational or sequential
approaches. In particular, the sequential strategy of Chapelle, Moireau et al. [9, 33, 34] could be the most
appropriate way to perform the joint state-parameter estimation. It which consists in solving a dynamical system
obtained by incorporating in the original model a correction taking into account the discrepancy between the
current simulation and the data. 6.2. The issue of the choice of boundary conditions When several needles (more than 2) are used, the choice of the boundary conditions on the inactivated needles
depends on the procedure. The interventional radiologist has the possibility to isolate the inactivated needles
with sheathing. Then the standard Neumann boundary condition has to be imposed on the inactivated needles. However, due to a lack of time, the inactivated needles are sometimes left not insulated. The device imposes a
high impedance so the net flux through the needle is zero, however Neumann condition is not appropriate to
account for this situation. The floating potential boundary condition should be used: the potential needle is set
at a constant value chosen such that the total net flux through the needle is zero [2]. Then the isosurface of
the eletric field are modified compared with the homogeneous Neumann conditions. Even though this situation
often happens in clinical application, there is still a lack of appropriate numerical study of the influence of such
boundary conditions. 6.4. Dynamical model of electroporation Even though tissue electroporation has been discovered for several decades, there is currently a lack of
model that can describe all the features of the phenomenon. Indeed, the model presented in equation (3.1) is
standardly used, but is is a static model, which cannot described the dynamical behavior of the increase of
tissue conductivity. Remark 6.1. We would like to make clear a misunderstanding which appeared in some previous studies. In
some papers (see for instance [7, 35, 37]) this model is referred to as dynamic. We emphasize that the model
is static since time is not involved in the equations. Due to the nonlinearity, the numerical solution is obtained
iteratively, but the iterations have nothing to do with any time evolution. Langus et al. proposed in [31] a dynamical model of tissue electroporation that they use for clinical applica-
tion [40]. They adopted a numerical view point by introducing a discretized time step in the equations. However
it seems difficult to write their model in a continuous way (see equation (11) of [31]) and their solution should
hardly depend on their time step ∆t, which raises questions regarding the numerical stability. Voyer et al. proposed in
[50] a dynamical two-phase model based on the electric circuit approach, which is written in a
continuous way. The model can be written as follows: ∇· (σe∇φe + Jc) = 0,
(6.1a)
εm∂tJc + (σm(t, Em) + σc)Jc = σcσm∇φe,
(6.1b) ∇· (σe∇φe + Jc) = 0,
εm∂tJc + (σm(t, Em) + σc)Jc = σcσm∇φe, (6.1a)
(6.1b) εm∂tJc + (σm(t, Em) + σc)Jc = σcσm∇φe, NUMERICAL CHALLENGES IN CLINICAL ELECTROPORATION 15 where Em = ∥∇φe −σ−1
c Jc∥, and where Em = ∥∇φe −σ−1
c Jc∥, and (6.1c) σm(t, Em) = σm
0 + σm
1 X1(t, Em) + σm
e X2(t, Em). (6.1c) σm(t, Em) = σm
0 + σm
1 X1(t, Em) + σm
e X2(t, Em). The functions X1 and X2 are the respective degrees of poration and degrees of permeabilisation s described by
Legu`ebe et al. [32]. The well-posedness of this model is still under investigation, but interestingly the model
has been validated on experimental data [50]. Fitting the dynamical model on the clinical data is a challenging
numerical problem which will be addressed in forthcoming studies. The functions X1 and X2 are the respective degrees of poration and degrees of permeabilisation s described by
Legu`ebe et al. [32]. Statement of ethics board approval This retrospective study is in accordance with ethical principals of the Declaration of Helsinki and has been
approved by the local committee on human research of the University Hospital J. Verdier. 6.4. Dynamical model of electroporation The well-posedness of this model is still under investigation, but interestingly the model
has been validated on experimental data [50]. Fitting the dynamical model on the clinical data is a challenging
numerical problem which will be addressed in forthcoming studies. 7. Conclusion In this paper, we have shown how numerical modeling combined with clinical imaging can assist the clinical
procedure of IRE ablation in liver tumor. The 3D sensitivity analysis shows that the needle location is a crucial datum, which has to be precisely
extracted from the clinical imaging to obtain realistic distribution of the elecric field. The proof of concept
on a specific clinical case, which is detailed in [20] has been summarized here. It gives an interesting basis
for confronting models to clinical cases in order to improve understanding and modeling. The electric field
distribution in realistic geometric configuration (and for a simplistic model) shows that it possible to get an
estimation of treatment success or failure from the computation. The IRENA software offers a sufficiently
accurate computational framework for electric field computation from clinical data. In particular, this gives the
possibility to the physician to visualize the estimated area of edema (see Sect. 1.1) during the procedure itself. Obviously, this simulation result has to be validated afterwards thanks to the postoperative imaging. But this is
a first (and only) valuable information obtained in real-time, which can help the operator in his decision-making. Future implementations in IRENA will be related to new models, specific far field boundary conditions on the
computational domain bounds, automatic calibration from test pulse current graphs, and a GUI for using the
software in clinical routine. Forthcoming investigations will deal with more advanced modeling of tissue IRE,
for which an energy-based approach could be adopted. As a conclusion, numerical modeling of IRE has a highly
valuable potential impact in the clinical practice of IRE ablation. However to achieve this goal, several issues,
from the high performance computing to the parameter estimation and from the mathematical modeling to the
image registration have to be addressed and solved. Appendix A. On the importance of 3D-simulations Most of numerical studies are devoted to better understanding and modeling the electric field distribution
during electroporation and its impact at cell or tissue level [5, 13, 16, 27, 29, 30, 36, 42]. For simplicity, these
studies generally consider simple geometries, in two dimensions, with two parallel needles. The disadvantage of the two-dimensional framework lies in the strong assumptions imposed on the geometric
configuration: this supposes to consider electrodes parallel to the study plane, with infinite active lengths. Figure A.1 shows 2D and 3D simulations of the maximum electric field in a homogeneous tissue, after one
pulse delivered by each pair of electrodes. For each 3D simulation, the area where the maximum electric field is
above the IRE threshold is compared to this computed in the corresponding 2D simulation. If the 2D simulation
is performed on the axial plane passing through the middle of the needle active tip, then the 2D simulation
gives a result very close to what is observed on the same plane in the equivalent 3D simulation (Fig. A.1a). However, the results are different if the 2D simulation is performed in another plane (Fig. A.1b). Moreover, if
the needles are parallel but inclined on the 2D simulation plane, then the results differ significantly as shown in
Figure A.1c. These configurations impose the hypothesis of parallel needles, inserted at the same depth. Even O. GALLINATO ET AL. 16 16
O. GALLINATO ET AL. Figure A.1. Difference between 2D and 3D computation of the electric field magnitude after
1 pulse for each needle pair (V = 3000 V, σ0 = 1 mS cm−1, σ1 = 4 mS cm−1). The dotted and
filled lines stand for the IRE threshold (650 V cm−1) in 2D and 3D simulations, respectively. The median plane passes through the middle of the active part of the needles. Figure A.1. Difference between 2D and 3D computation of the electric field magnitude after
1 pulse for each needle pair (V = 3000 V, σ0 = 1 mS cm−1, σ1 = 4 mS cm−1). The dotted and
filled lines stand for the IRE threshold (650 V cm−1) in 2D and 3D simulations, respectively. The median plane passes through the middle of the active part of the needles. Figure A.2. Fluoroscopic image acquisition of the needles position. One can see that the
needles are neither in exact parallel configuration nor at the exact same depth. Figure A.2. NUMERICAL CHALLENGES IN CLINICAL ELECTROPORATION NUMERICAL CHALLENGES IN CLINICAL ELECTROPORATION As clinical configurations are sometimes far from the ideal parallel configuration (see for instance Fig. A.2),
a 3D framework is therefore mandatory. Note also that nonparallel needles do not compromise the clinical
procedure. The distribution of the electric field is certainly less intuitive, but it is computationally predictable. This is one of the reasons why 3D numerical simulation is essential for clinical procedures. As clinical configurations are sometimes far from the ideal parallel configuration (see for instance Fig. A.2),
a 3D framework is therefore mandatory. Note also that nonparallel needles do not compromise the clinical
procedure. The distribution of the electric field is certainly less intuitive, but it is computationally predictable. This is one of the reasons why 3D numerical simulation is essential for clinical procedures. Acknowledgements. This study has been carried out within the frame of the LABEX TRAIL, ANR-10-LABX-0057 with
financial support from the French State, managed by the French National Research Agency (ANR) in the frame of the
“Investments for the future” Programme IdEx (ANR-10-IDEX-03-02). The authors are supported by the INCA Aviesan
Plan Cancer projects DYNAMO (ref. PC201615) and NUMEP (ref. PC201615). Appendix A. On the importance of 3D-simulations Fluoroscopic image acquisition of the needles position. One can see that the
needles are neither in exact parallel configuration nor at the exact same depth. though the generator manufacturer (NanoKnife) recommends parallel needles, this is not always possible in
clinical practice, especially for treatment of liver deep-seated tumors. The presence of ribs, vena cava, hepatic
arteries or important bile ducts may require a more complex needle placement. Then, if the needles are not
parallel as in Figure A.1d, the 2D simulation can be very far from the results in three dimensions, and even may
incorrectly suggest a total treatment of the tumor and therefore provide a wrong assessment of the procedure. though the generator manufacturer (NanoKnife) recommends parallel needles, this is not always possible in
clinical practice, especially for treatment of liver deep-seated tumors. The presence of ribs, vena cava, hepatic
arteries or important bile ducts may require a more complex needle placement. Then, if the needles are not
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Cortical presentation of language functions in patients after total laryngectomy: a fMRI study
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https://doi.org/10.1007/s00234-020-02407-x
Neuroradiology (2020) 62:843–849 https://doi.org/10.1007/s00234-020-02407-x
Neuroradiology (2020) 62:843–849 FUNCTIONAL NEURORADIOLOGY Abstract Purpose The aim of this study is to use functional magnetic resonance (fMRI) to analyse the cortical presentation of selected
language functions in patients after a total laryngectomy. g
g
p
y g
y
Methods Eighteen patients after total laryngectomy treated with electrolarynx speech and 18 volunteers were included. The mean
number of patients’ post-operative speech rehabilitation sessions was five (range of 3–8 sessions). Four paradigms were used,
including noun generation, pseudoword reading, reading phrases with pseudowords, and nonliteral sign reproduction. Results In noun, the most significant difference between the groups was the stronger activation of both lingual gyri in the
volunteers. Pseudoword reading resulted in stronger activations in patients than in volunteers in the lingual gyri, the right
cerebellum, the right Broca’s area, and the right parietal operculum. Reading phrases with pseudowords involved different parts
of the Brodmann area 40. During nonliteral sign reproduction, there was a stronger activation of the left Broca’s area in volunteers
and a stronger activation of the left premotor cortex in patients. Methods Eighteen patients after total laryngectomy treated with electrolarynx speech and 18 volunteers were included. The mean
number of patients’ post-operative speech rehabilitation sessions was five (range of 3–8 sessions). Four paradigms were used,
including noun generation, pseudoword reading, reading phrases with pseudowords, and nonliteral sign reproduction. Results In noun, the most significant difference between the groups was the stronger activation of both lingual gyri in the
volunteers. Pseudoword reading resulted in stronger activations in patients than in volunteers in the lingual gyri, the right
cerebellum, the right Broca’s area, and the right parietal operculum. Reading phrases with pseudowords involved different parts
of the Brodmann area 40. During nonliteral sign reproduction, there was a stronger activation of the left Broca’s area in volunteers
and a stronger activation of the left premotor cortex in patients. Conclusion This study provides evidence of altered cortical activation in response to language tasks in patients after a laryngec-
tomy compared with healthy volunteers, which may be considered brain plasticity in response to a laryngectomy. Keywords Laryngectomy . Magnetic resonance imaging . Gray matter Cortical presentation of language functions in patients after total
laryngectomy: a fMRI study Aleksandra Wypych1 & Małgorzata Wierzchowska2 & Paweł Burduk2 & Elżbieta Zawada3,4 & Katarzyna Nadolska3 &
Zbigniew Serafin3 Received: 4 December 2019 /Accepted: 13 March 2020
# The Author(s) 2020
/ Published online: 6 April 2020 1
The Interdisciplinary Center for Modern Technologies, Nicolaus
Copernicus University, Toruń, Poland * Zbigniew Serafin
serafin@cm.umk.pl 1
The Interdisciplinary Center for Modern Technologies, Nicolaus
Copernicus University, Toruń, Poland
2
Department of Otolaryngology, Oncology and Oral and
Maxillofacial Surgery, Nicolaus Copernicus University, Collegium
Medicum, Bydgoszcz, Poland
3
Department of Radiology and Diagnostic Imaging, Nicolaus
Copernicus University, Collegium Medicum, Bydgoszcz, Poland
4
Department of Geriatrics, Nicolaus Copernicus University,
Collegium Medicum, Bydgoszcz, Poland Introduction profound effect on contemporary understanding of speech. Sakai et al. [3] commented that human language is a unique
faculty of the mind. The grammaticality of a sentence, they
assert, needs to be made explicit by the adoption of an appro-
priate theoretical framework for linguistic structures. Grammatical rules arise from the human brain so that lan-
guage must be considered a subsystem of the mind, with the
language system being a distinct module, which in turn pos-
sesses its own modularity or subsystems such as phonology,
semantics, and syntax, which interact systematically with each
other through the information flow between them [3]. The ability to speak is one of the sophisticated features that
make a distinction between humans and other animals. No
wonder that how the brain controls speech has remained a
subject of investigation for centuries. The pioneers of research
on cerebral language functions were Pierre Paul Broca and
Karl Wernicke [1, 2]. Their studies and theories had a However, despite the huge progress in neuroimaging, the
definite location of these areas still remains controversial [4,
5]. Some authors define it as a unimodal auditory association
in the superior temporal gyrus anterior to the primary auditory
cortex (the anterior part of BA 22) [6]. This is the site most
consistently implicated in auditory word recognition by func-
tional brain imaging experiments [7]. Others also include ad-
jacent parts of the heteromodal cortex in Broca’s area (BA) 39
and BA40 in the parietal lobe [8]. Recently, Ardila et al. sug-
gested that grammar correlates with the ability to internally
represent actions (verbs) depending on the functioning of BAs 3
Department of Radiology and Diagnostic Imaging, Nicolaus
Copernicus University, Collegium Medicum, Bydgoszcz, Poland 4
Department of Geriatrics, Nicolaus Copernicus University,
Collegium Medicum, Bydgoszcz, Poland Neuroradiology (2020) 62:843–849 844 women at mean age 61 ± 8 years) and 18 healthy volunteers
(15 men and 3 women at mean age 57 ± 7 years). All the
patients were treated with ELS and were clinically considered
subjects who successfully completed post-operative speech
rehabilitation. The mean number of speech rehabilitation ses-
sions was five (range of 3–8 sessions). Exclusion criteria were
as follows: psychiatric or neurological disorders, previous
brain or ear surgery, pregnancy, claustrophobia, metal foreign
objects or metal implants (heart stimulator, cardioverter, insu-
lin pump, cochlear implant, CNS stimulation, prosthesis). All
participants were adults and gave informed consent to partic-
ipate. Scanning and language tasks The study was conducted using a 3T MRI scanner (GE
Discovery MR 750, General Electric Healthcare, Waukesha,
IL, USA) and an 8-channel neurovascular head coil (HD 8-
CH Neurovascular Array, General Electric Healthcare,
Waukesha, Il, USA). For each participant, scanning started
with a high-resolution 3D BRAVO sequence (3D T1W1, TR
8.2 ms, TE 3.2 ms, FA 12°, thickness 1.0 mm, no interslice
gap, 176 slices, voxel size 1 × 1 × 1 mm). Then, the functional
experiment was performed using the BOLD technique in the
axial plane using an EPI sequence (TR 2000 ms, TE 28 ms,
FA 90°, thickness 3.0 mm, no interslice gap, 44 slices, voxel
size 3 × 3 × 3 mm). In the fMRI experiment, four paradigms
were used: noun generation, pseudoword reading, reading
phrases with pseudowords, and nonliteral sign reproduction. The tasks were presented in the subjects’ mother tongue
(Polish) using NNL VisualSystem goggles (NordicNeuroLab
AS, Bergen, Norway). Paradigms were created in Presentation
(http://www.neurobs.com), a stimulus delivery and
experiment control program for neuroscience. Considering the complicated and not fully understood corti-
cal control of speech, it is obvious that a sudden inability to
speak in an adult, caused by a total laryngectomy, is a vast
disablement to the patient. Moreover, speech rehabilitation
methods, as described above, seem to stimulate cortical func-
tion remodelling. Remodelling of both connectivity and grey
matter areas is a well-known phenomenon after brain injury and
the loss of peripheral functions [11, 14]. Several different lan-
guage paradigms were proposed to localize speech-related
brain areas [18, 19]. Word generating is one of the most com-
monly used paradigms that results in activation mostly at the
left inferior frontal gyrus and the bilateral motor cortex [20]. One may suppose that after laryngectomy, only the activation of
motor cortex may be affected. Similarly, more or less sophisti-
cated conceptual paradigms, including pseudoword reading and
repeating, and nonliteral sign reproduction [21] cortical pro-
cessing should not be influenced by laryngectomy as well. Each language activation comprised one fMRI run per par-
adigm in block design experiments. Stimuli were presented as
six 30 s active blocks separated by six 30 s rest pauses. Each
session lasted 6 min, 10 s, including 5 dummy scans. Tasks
were always presented in the same order and were of the same
duration. Introduction We obtained permission to conduct the study from the
local bioethical committee. 44 and 45 and the brain circuits related to them [9]. Grammar
is also thought to be associated with the ability to quickly
carry out the sequencing of the articulatory movements re-
quired for speaking (speech praxis) [10]. Meta-analytic stud-
ies which aimed to analyse the specific contribution of differ-
ent BAs to the language system identified some areas poten-
tially related to the adoption and comprehension of language
(the lexical and semantic system) and areas related to language
production (the grammatical system) [9, 11]. Total laryngectomy is still the method of choice for the
treatment of advanced laryngeal cancer when radiotherapy
fails to preserve the organ [12]. However, after a laryngecto-
my, patients not only lose the ability to communicate but also
fall into a kind of social exclusion [12, 13]. Voice rehabilita-
tion is a very important part of both pre-operative and post-
operative treatment. The most technically advanced speech
generation technique, ELS, uses an electronic device that gen-
erates sound regulated by vibrations of the patient’s neck or
cheek muscles [11]. It seems to be the most comparable to a
healthy person’s speech considering acoustic parameters in-
cluding fundamental frequency, maximum phonation time,
and intensity of the voice [11, 14–17]. Scanning and language tasks Participants were instructed before the MRI scan-
ning about the structure of the study and they were given
examples of tasks that were different from the paradigms pre-
sented in the scanner. For each task, the volunteers were asked
only to think about a word: We hypothesise that a similar process takes place after a
laryngectomy. To our knowledge, there are no published stud-
ies that analyse brain cortical function related to a laryngecto-
my. The aim of this study was to use fMRI to analyse the
cortical presentation of selected language functions in patients
after a total laryngectomy. In task 1, they were asked to generate nouns beginning
with a given letter (e.g. T, P, R, S, K). In task 1, they were asked to generate nouns beginning
with a given letter (e.g. T, P, R, S, K). In task 2, pseudonouns were presented in strings. The list
of words was arranged gradually from the easiest to the
most difficult in terms of structure (C means a consonant
and V means a vowel): (i) two-syllable words with the
CVC-CVC recording scheme, e.g. “chesnut”; (ii) Methods In task 2, pseudonouns were presented in strings. The list
of words was arranged gradually from the easiest to the
most difficult in terms of structure (C means a consonant
and V means a vowel): (i) two-syllable words with the
CVC-CVC recording scheme, e.g. “chesnut”; (ii) Results None of participants terminated the MRI examination and
none of exams were rejected due to artefacts. In task 3, participants were asked to read phrases
consisting of single words with converted or shifted let-
ters, e.g. “raed veihcle”, “lietr of waeter”. Results showed a stronger activation of the left visual cor-
tex V3Vand come part of the right visual cortex V2 in controls
compared with patients during a noun generation task. Conversely, laryngectomy patients presented stronger activa-
tion of the right visual cortex V1, another part of the right
visual cortex V2, the right inferior parietal lobule, the left
cingulum, and the right premotor cortex (Table 1). On the
other hand, in response to task 2, a stronger activation in
volunteers was seen for visual cortex V3 and a stronger acti-
vation in patients for the right visual cortex V2, the left visual
cortex V4, and the right Broca’s area (Table 2). In task 4, nonliteral sign reproduction (e.g. !, +,?,:) were
presented. Participants were asked to repeat the name of
graphic sign in their mind. Data processing and analysis Data pre-processing and analysis were performed using an
FSL v. 5.0 (The FMRIB Software Library) toolkit. Images
were controlled for field distortion, spike artefacts, and tem-
poral signal-to-noise ratio. Then, EPI scans were subject to
movement correction, co-registration, normalisation, segmen-
tation, and spatial smoothing. Movement correction was con-
ducted using a MCFLIRT package [10]. For structural and
functional image registration, a FLIRT library (FMRIB’s
Linear Image Registration Tool) and the MNI152 standard-
space T1-weighted average structural template image were
used [9]. Spatial smoothing was performed with a Gaussian
filter (FWHM 4.0 mm). We also used a high-pass filter in a
time domain (Gaussian-weighted least-squares straight line
fitting, sigma = 50.0 S). Time series analysis was conducted
using FILM software (FMRIB’s Improved Linear Model)
with local correlation correction [22, 23]. The fMRI data anal-
ysis was conducted using a FEAT library v. 6.00 (FMRI
Expert Analysis Tool). Task 3 resulted in the strongest cortical activation in pa-
tients. The left Broca’s area, the left anterior intra-parietal
sulcus, and visual cortex (V3, V4) were strongly activated in
patients, whereas V1 and V2 visual cortex, the left primary
somatosensory cortex, and the left premotor cortex presented
stronger response in controls (Table 3, Fig. 1). Finally, during
nonliteral sign reproduction, controls activated more the left
Broca area than patients, while patients activated more the left
premotor cortex than controls (Table 4). Participants The study group consisted of 36 right-handed subjects, includ-
ing 18 patients after a total laryngectomy (15 men and 3 Neuroradiology (2020) 62:843–849 845 stronger in patients than in controls. Significant clusters were
labelled based on Juelich Histological Atlas [24]. stronger in patients than in controls. Significant clusters were
labelled based on Juelich Histological Atlas [24]. polysyllable words consisting of a different number of
syllables with a CV and CVC recording scheme, e.g. “chesstboard”; (iii) two-syllable words with a CCV-CV
recording scheme, e.g. “prima”. Each string was
displayed for 30 s and consisted of five words. The sub-
jects were instructed to read and repeat the pseudonouns
in their minds until the next string of words appeared. polysyllable words consisting of a different number of
syllables with a CV and CVC recording scheme, e.g. “chesstboard”; (iii) two-syllable words with a CCV-CV
recording scheme, e.g. “prima”. Each string was
displayed for 30 s and consisted of five words. The sub-
jects were instructed to read and repeat the pseudonouns
in their minds until the next string of words appeared. Discussion To our knowledge, this is the first study published presenting
differences between patients after a laryngectomy and healthy
volunteers in cortical activation in response to language tasks. Tasks resulted in activations at different levels of the visual
cortex but no clear pattern could be defined in both volunteers
and patients. The only difference was activation of the V3
visual cortex in volunteers in most of the experiments. Despite intensive investigation, the precise location and func-
tion of the third visual cortex remains a matter of debate [25,
26]. Generally, it is considered to play a role in the processing
of motion, either global or coherent [25]. Based on the con-
struction of tasks used in this study, the observed stronger
activation of the V3 cortex in healthy subjects remains
unclear. Z statistic images (Gaussianized T/F) were set in terms of
clusters for Z > 2.3. The cluster significance (with correction)
was set at p = 0.05. The first five EPI scans were discarded in
each data package to achieve signal balance. Cluster correc-
tion was performed on both the first-level and the second-level
analysis. The first-level statistical analysis (individual data)
was conducted using a general linear model (GLM). The
second-level statistical analysis (group analysis) for difference
assessment between healthy controls and laryngectomy pa-
tients was conducted using a FLAME library (FMRIB’s
Local Analysis of Mixed Effects), which is a type of variance
test for modelling and estimating the random-effects compo-
nent of the measured inter-session mixed-effects variance in a
full Bayesian network. FLAME 1 option with mixed-effects
model was applied. Statistical analysis was made by the un-
paired sample t test. Each task was analysed in two variants:
activation stronger in controls than in patients and activation Noun generation (task 1) requires semantic categorisation
and, thus, makes great demands on semantic processing [27]. The most significant difference between the groups was the
stronger activation of both lingual gyri in the volunteers. These areas are responsible for semantic categorisation, word
retrieval, word generation, and single letter processing
[28–31] so their activation presents a proper function. Discussion On Neuroradiology (2020) 62:843–849 846 Table 1
Response to task 1
Juelich histological atlas
Z value
P value
x
y
z
Activation stronger in healthy volunteers than in laryngectomy patients
Left visual cortex V3V
2.86
< 0.0000
53
22
33
Right visual cortex V2
2.79
0.0002
41
22
33
Right visual cortex V3
2.53
0.0031
29
21
32
Right cingulum
2.46
0.0042
42
40
49
Left secondary somatosensory cortex / parietal operculum OP4
2.43
0.0369
67
58
42
Visual cortex V1
2.43
0.0369
40
21
38
Right Broca’s area
2.40
0.0435
24
72
39
Right secondary somatosensory cortex/parietal operculum OP1
2.39
0.0435
18
50
47
Activation stronger activated in laryngectomy patients than in healthy volunteers
Right visual cortex V1
2.63
0.0002
37
17
38
Right visual cortex V2
2.61
0.0027
35
21
32
Right inferior parietal lobule Pga
2.47
0.0028
18
35
52
Left cingulum
2.42
0.0032
48
55
52
Right premotor cortex
2.37
0.0036
37
55
67
Left premotor cortex
2.32
0.0413
51
56
69 phonation. The left premotor cortex is also responsible for
speech initiation and speech motor programming [15, 38],
which again require more effort after a laryngectomy. the other hand, patients after a laryngectomy presented a
stronger activation of the right angular gyrus, the left anterior
cingulate gyrus, and the bilateral premotor cortex. The angular
gyrus is surrounded by secondary somatosensory, visual and
auditory cortical areas and is essential in the multimodal, high-
ly complex synthesis of information [32]. Thus, it can be
considered an adjuvant cortical area activated in patients. The anterior cingulum was linked to many different functions,
including semantic and phonological verbal fluency [33]. However, in patients after a laryngectomy, activation of this
area may be also linked to its role in cognitive and motor
inhibition, motor imagery as well as in motor preparation
and planning [34, 35]. The premotor cortex, which is also
connected with multiple functions, was activated for word
generation in other studies [27]. Basic functions of this area
include motor sequencing, movement planning, and imagina-
tion of movement [36, 37]. These functions may require stron-
ger activation when oral speech has to be replaced by artificial Pseudoword reading (task 2) resulted in stronger activations in
patients than in volunteers in the lingual gyri, the right cerebel-
lum, the right Broca’s area, and the right parietal operculum
(OP1). Discussion On the other
Yang et al. concluded that there is flexible involvement
sensory-motor system in abstract concept processing, whi
pends on semantic features of the language stimuli and
between abstract and literal meanings [12]. In our study, th
significant differences between the groups again included a
ger activation of the left Broca’s area in volunteers and a st
activation of the left premotor cortex in patients. Some limitations of the current study have to b
dressed. Firstly, the time of rehabilitation after a lary
tomy varied in our study group between three and
learning sessions. We believe that the extent and th
ciency of speech processing plasticity in the brain m
cal presentation of the brain areas that were significantly stronger activated in laryngectomy patients than in healthy volunteers in re
ponse to task 3
gical atlas
Z value P value
x
y
z
onger in healthy volunteers than in laryngectomy patients
area
3.10
< 0.0000 71 75 42
intra-parietal sulcus hip2 2.57
< 0.0000 67 44 54
cortex V3
2.53
< 0.0000 34 23 32
cortex V4
2.43
0.0006
29 25 31
parietal lobule pft
2.39
0.0032
72 50 54
cortex V2
2.32
0.0034
37 17 38
onger activated in laryngectomy patients than in healthy
ortex V1
2.73
0.0010
49 15 37
cortex V2
2.65
0.0002
37 21 32
somatosensory cortex
2.58
0.0035
71 55 58
ortex V2
2.46
0.0028
49 16 33
r cortex
2.46
0.0020
51 56 69
tor cortex
2.38
0.0252
38 55 71
somatosensory cortex
2.38
0.0392
70 52 60
y (2020) 62:843–849 Neuroradiology (2020) 62:843–849 Neuroradiology (2020) 62:843–849 847 Reading phrases with pseudowords (task 3) was a more com-
plicated paradigm and involved different parts of the Brodmann
area 40 L that is responsible for more elaborate semantic pro-
cessing [42]. Apart from the activations discussed above, an
interesting observation was a much stronger left Broca’s area
activation in volunteers than in patients, which again underlines
altered speech processing after a laryngectomy. On the other
hand, a stronger response from the left primary somatosencory
cortex and the bilateral premotor cortex was observed in patients. Cortical representation of nonliteral sign reproduction (task 4)
remains a matter of debate. A meta-analysis by Rapp et al. Discussion Previous studies indicated that pseudoword reading in-
volved the left fusiform gyrus, the left angular gyrus, and the left
middle temporal gyrus for lexical and semantic processing. Furthermore, spelling-sound conversion was located in the left
inferior parietal gyrus, and phonological output in the left inferior
frontal gyrus [39]. Hauck et al. also found significant activation
in the left inferior parietal gyrus, which confirms our results. In
general, our results show more complicated processing of
pseudoword reading after a laryngectomy. Activation of OP1
has been linked to literal sentence comprehension, and word
imageability [10, 40]. The contribution of the cerebellum to
pseudoword processing is less clear. Guediche et al. postulated Table 2
Response to task 2
Juelich histological atlas
Z value
P value
x
y
z
Activation stronger in healthy volunteers than in laryngectomy patients
Visual cortex V3
2.79
0.0042
34
23
32
Activation stronger activated in laryngectomy patients than in healthy volunteers
Right visual cortex V2
2.75
< 0.0000
38
23
33
Left visual cortex V4
2.55
< 0.0000
56
24
33
Right Broca’s area
2.45
0.0009
18
74
39
Right secondary somatosensory cortex/parietal operculum OP1
2.35
0.0019
17
51
47
Left visual cortex V1
2.33
0.0282
51
18
37
Left visual cortex V2
2.32
0.0313
49
16
33 network between the cerebellum and language-
ns in the temporal and parietal lobes contributing
otor adaptation. They stated that cerebro-cerebellar
may support supervised learning mechanisms that
ory prediction error signals in speech perception
ore, cerebellar activation in our patients may be a
ain plasticity in response to a laryngectomy. Reading phrases with pseudowords (task 3) was a more
plicated paradigm and involved different parts of the Brod
area 40 L that is responsible for more elaborate semant
cessing [42]. Apart from the activations discussed abo
interesting observation was a much stronger left Broca
activation in volunteers than in patients, which again und
altered speech processing after a laryngectomy. On the
hand, a stronger response from the left primary somatos
cortex and the bilateral premotor cortex was observed in pa
Cortical representation of nonliteral sign reproduction (
remains a matter of debate. A meta-analysis by Rapp et a
cated that a predominantly left lateralised network, includi
left and right inferior frontal gyrus, the left, middle, and su
temporal gyrus, and medial prefrontal, superior frontal, c
lar, parahippocampal, precentral, and inferior parietal re
was important for nonliteral expressions [43]. Discussion indi-
cated that a predominantly left lateralised network, including the
left and right inferior frontal gyrus, the left, middle, and superior
temporal gyrus, and medial prefrontal, superior frontal, cerebel-
lar, parahippocampal, precentral, and inferior parietal regions,
was important for nonliteral expressions [43]. On the other hand,
Yang et al. concluded that there is flexible involvement of the
sensory-motor system in abstract concept processing, which de-
pends on semantic features of the language stimuli and links
between abstract and literal meanings [12]. In our study, the most
significant differences between the groups again included a stron-
ger activation of the left Broca’s area in volunteers and a stronger
activation of the left premotor cortex in patients. Left visual cortex V1
2.73
0.0010
49 15 37
Right visual cortex V2
2.65
0.0002
37 21 32
Left primary somatosensory cortex
2.58
0.0035
71 55 58
Left visual cortex V2
2.46
0.0028
49 16 33
Left premotor cortex
2.46
0.0020
51 56 69
Right premotor cortex
2.38
0.0252
38 55 71
Left primary somatosensory cortex
2.38
0.0392
70 52 60 a functional network between the cerebellum and language-
related regions in the temporal and parietal lobes contributing
to sensorimotor adaptation. They stated that cerebro-cerebellar
interactions may support supervised learning mechanisms that
rely on sensory prediction error signals in speech perception
[41]. Therefore, cerebellar activation in our patients may be a
feature of brain plasticity in response to a laryngectomy. Some limitations of the current study have to be ad-
dressed. Firstly, the time of rehabilitation after a laryngec-
tomy varied in our study group between three and eight
learning sessions. We believe that the extent and the effi-
ciency of speech processing plasticity in the brain may be Fig. 1 Graphical presentation of the brain areas that were significantly stronger activated in laryngectomy patients than in healthy volunteers in res
to task 3 Fig. References 1. Bogen JE, Bogen GM (1976) Wernicke’s region-where is it? Ann N
Y Acad Sci 280:834–843 In conclusion, this study provides the first evidence of al-
tered cortical activation in response to language tasks in pa-
tients after a laryngectomy compared with healthy volunteers,
which may be considered brain plasticity in response to a
laryngectomy. 2. Bramati IE, Rodrigues EC, Simões EL, Melo B, Höfle S, Moll J,
Lent R, Tovar-Moll F (2019) Lower limb amputees undergo long-
distance plasticity in sensorimotor functional connectivity. Sci
Rep 9:2518 3. Hurren A, Miller N (2017) Voice outcomes post total laryngectomy. Curr Opin Otolaryngol Head Neck Surg 25:205–210 3. Hurren A, Miller N (2017) Voice outcomes post total laryngectomy. Curr Opin Otolaryngol Head Neck Surg 25:205–210 Funding information This study was not supported by any funding. 4. Fridriksson J, Fillmore P, Guo D, Rorden C (2015) Chronic Broca’s
aphasia is caused by damage to Broca’s and Wernicke’s areas. Cereb Cortex 25:4689–4696 4. Fridriksson J, Fillmore P, Guo D, Rorden C (2015) Chronic Broca’s
aphasia is caused by damage to Broca’s and Wernicke’s areas. Cereb Cortex 25:4689–4696 Discussion Therefore, further investigation
is necessary involving more sizeable and carefully com-
posed study groups to validate the current findings. Informed consent
Informed consent was obtained from all individual
participants included in the study. 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/. Discussion 1 Graphical presentation of the brain areas that were significantly stronger activated in laryngectomy patients than in healthy volunteers in response
to task 3 Neuroradiology (2020) 62:843–849 848 Table 4
Response to task 4
Juelich histological atlas
Z value
P value
x
y
z
Activation stronger in healthy volunteers than in laryngectomy patients
Left visual cortex V2
2.65
< 0.0000
52
15
33
Left visual cortex V1
2.53
< 0.0000
48
15
37
Right visual cortex V3
2.53
< 0.0000
30
22
32
Left Broca’s area
3.42
0.0035
68
67
47
Right inferior parietal lobule Pga
3.41
0.0002
23
35
50
Right visual cortex V1
2.40
< 0.0000
35
26
38
Left inferior parietal lobule pgp
2.39
0.0132
62
27
50
Activation stronger activated in laryngectomy patients than in healthy volunteers
Left visual cortex V2
2.56
0.0057
55
13
33
Left premotor cortex
2.48
0.0028
57
56
69
Right secondary somatosensory cortex/parietal operculum OP1
2.42
0.0433
23
49
47
Left visual cortex V1
2.41
0.0008
46
18
37
Right visual cortex V2
2.39
0.0068
37
17
32 Ethical approval
All procedures performed in studies involving human
participants were in accordance with the ethical standards of the institu-
tional and/or national research committee and with the 1964 Helsinki
declaration and its later amendments or comparable ethical standards. The study was approved by the Institutional Review Board. dependent on the duration of rehabilitation and therefore
might influence the results. This hypothesis still needs
confirmation. On the other hand, the number of sessions
to finish rehabilitation depended on individual abilities of
patients. At inclusion to the study, all the patients had
finished their speech rehabilitation with a positive out-
come. Therefore, they were clinically diagnosed as posi-
tively rehabilitated. Considering this diagnosis, the group
may be considered homogenous. Secondly, study partici-
pants were not selected according to education and pro-
fession, which also may have an impact on language pro-
cessing. Thirdly, a more detailed analysis of inter-subject
variability would be necessary to find other possible co-
variates of the outcome. For instance, differences in acti-
vations may be related to cognitive abilities of subjects,
which are crucial to understand if changes in cortical ac-
tivations are compensatory or maladaptive. As results of
the current preliminary study appeared encouraging to us,
we are now starting larger program, including cognitive
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https://openalex.org/W2061301971
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https://www.hal.inserm.fr/inserm-00711527/file/journal.pgen.1002390.pdf
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English
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Identification of Evolutionarily Conserved Exons as Regulated Targets for the Splicing Activator Tra2β in Development
|
PLOS genetics
| 2,011
|
cc-by
| 15,486
|
To cite this version: Sushma Grellscheid, Caroline Dalgliesh, Markus Storbeck, Andrew Best, Yilei Liu, et al.. Identification
of evolutionarily conserved exons as regulated targets for the splicing activator tra2β in development.. PLoS Genetics, 2011, 7 (12), pp.e1002390. 10.1371/journal.pgen.1002390. inserm-00711527 Identification of evolutionarily conserved exons as
regulated targets for the splicing activator tra2β in
development. Sushma Grellscheid, Caroline Dalgliesh, Markus Storbeck, Andrew Best, Yilei
Liu, Miriam Jakubik, Ylva Mende, Ingrid Ehrmann, Tomaz Curk, Kristina
Rossbach, et al. Abstract Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2b (Sfrs10) is essential for mouse embryogenesis and implicated in
spermatogenesis. Here we find that Tra2b is up-regulated as the mitotic stem cell containing population of male germ cells
differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2b binds a high
frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have
analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse
(Sfrs10fl/fl; Nestin-Cretg/+). This mouse has defects in brain development and allowed correlation of genuine physiologically
Tra2b regulated exons. These belonged to a novel class which were longer than average size and importantly needed
multiple cooperative Tra2b binding sites for efficient splicing activation, thus explaining the observed splicing defects in the
knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone
that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new
pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily
conserved, suggesting they might control fundamental developmental processes. Tra2b protein isoforms lacking the RRM
were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the
N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2b. Versions of Tra2b lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length
Tra2b protein. Citation: Grellscheid S, Dalgliesh C, Storbeck M, Best A, Liu Y, et al. (2011) Identification of Evolutionarily Conserved Exons as Regulated Targets for the Splicing
Activator Tra2b in Development. PLoS Genet 7(12): e1002390. doi:10.1371/journal.pgen.1002390 itor: Wendy A. Bickmore, Medical Research Council Human Genetics Unit, United Kingdom Editor: Wendy A. Bickmore, Medical Research Council Human Genetics Unit, United Kingdom Received May 11, 2011; Accepted October 5, 2011; Published December 15, 2011 Copyright: 2011 Grellscheid 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. Identification of Evolutionarily Conserved Exons as
Regulated Targets for the Splicing Activator Tra2b in
Development Sushma Grellscheid1.*, Caroline Dalgliesh1., Markus Storbeck2,3,4., Andrew Best1, Yilei Liu1¤a, Miriam
Jakubik2,3,4, Ylva Mende2,3,4, Ingrid Ehrmann1, Tomaz Curk5, Kristina Rossbach2,3,4, Cyril F. Bourgeois6¤b, James Ste´venin6, David Grellscheid7, Michael S. Jackson1, Brunhilde Wirth2,3,4, David J. Elliott1* 1 Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom, 2 Institute of Human Genetics, University of Cologne, Cologne, Germany, 3 Institute of
Genetics, University of Cologne, Cologne, Germany, 4 Center for Molecular Medicine, University of Cologne, Cologne, Germany, 5 University of Ljubljana, Faculty of
Computer and Information Science, Ljubljana, Slovenia, 6 Department of Functional Genomics and Cancer, Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire
(IGBMC), INSERM U 964, CNRS UMR 7104, Universite´ de Strasbourg, Illkirch, France, 7 Institute for Particle Physics Phenomenology, Durham University, Durham, United
Kingdom Abstract Funding: This work was supported by the Wellcome Trust (Grant numbers WT080368MA and WT089225/Z/09/Z to DJE), the BBSRC (Grant numbers BB/D013917/
1 and BB/I006923/1 to DJE), the Breast Cancer Campaign (to DJE), the Center of Molecular Medicine Cologne (CMMC) to BW [D7], the Deutsche
Forschungsgemeinschaft to BW [Wi 945/12-3], European Commission FP7 EURASNET (to JS), and the Slovenian Research Agency to TC (grant number Z7-3665). 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. Competing Interests: The authors have declared that no competing interests exist. * E-mail: David.Elliott@ncl.ac.uk (DJE); sushma@cantab.net (SG) ¤a Current address: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America
¤b Current address: INSERM 1052 / CNRS UMR 5286, Centre de Recherche en Cance´rologie de Lyon, Centre Le´on Be´rard, Lyon, Fran . These authors contributed equally to this work. alternatively spliced to target mRNAs for degradation through
Nonsense Mediated Decay (NMD) [4,5,6,7,8]. HAL Id: inserm-00711527
https://inserm.hal.science/inserm-00711527v1
Submitted on 25 Jun 2012 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
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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. PLoS Genetics | www.plosgenetics.org Tra2b is ubiquitously expressed but up-regulated at the
onset of meiosis in male germ cells arginine-serine rich (RS1 and RS2) domains [13,14]. The N-
terminal Tra2 RS1 domain is longer and contains more RS
dipeptides than RS2. The reason for this unique modular
organisation is unknown, but is conserved in vertebrate and
invertebrate Tra2 proteins and different from the classical SR
super-family which have a single C-terminal RS domain [15]. Also
unlike classical SR proteins, Tra2 proteins do not restore splicing
activity to S100 extracts [12]. arginine-serine rich (RS1 and RS2) domains [13,14]. The N-
terminal Tra2 RS1 domain is longer and contains more RS
dipeptides than RS2. The reason for this unique modular
organisation is unknown, but is conserved in vertebrate and
invertebrate Tra2 proteins and different from the classical SR
super-family which have a single C-terminal RS domain [15]. Also
unlike classical SR proteins, Tra2 proteins do not restore splicing
activity to S100 extracts [12]. We analysed the expression of Sfrs10 mRNA in different adult
mouse (Mus musculus) tissues by RT-PCR using primers in exons 1
and 4. An RT-PCR product derived from Sfrs10 mRNA in which
exons 1 and 3 were directly spliced (skipping poison exon 2) was
seen in every tissue indicating the Sfrs10 gene is ubiquitously
expressed (Figure 1A and 1B). A larger Sfrs10 RT-PCR product
made from mRNAs including poison exon 2 was detected at high
levels in just two tissues, testis and muscle, indicating that
expression of Tra2b is particularly tightly controlled in these
tissues [22]. Similar levels of expression of Hprt mRNA were
observed in each tissue by multiplex RT-PCR. A single Tra2 protein is conserved in fruit flies, where it is
essential for spermatogenesis and sex determination [16]. There
are two mammalian Tra2 proteins called Tra2a (encoded by the
Tra2a gene on mouse chromosome 6) and Tra2b (encoded by the
Sfrs10 gene on mouse chromosome 16) which share 63% amino
acid identity and similar RNA binding specificities [12]. NMR
analyses have recently shown that the optimal core RNA target
sequence for binding full length Tra2b protein is an AGAA motif,
with each of the nucleotide residues being specifically recognized
by the Tra2b RRM [17,18]. A polyclonal antiserum raised to Tra2b protein identified a
single endogenous protein of around 40 KDa in both transfected
and untransfected
HEK293 cells corresponding in size to
endogenous Tra2b (Figure 1C). Tra2b is ubiquitously expressed but up-regulated at the
onset of meiosis in male germ cells A Tra2b-GFP fusion protein
was additionally detected within transfected cells, but no cross-
reaction was detected with a Tra2a-GFP fusion indicating high
specificity of the antiserum. We were also able to detect a GFP-
fusion protein containing Tra2bDRS1, but not endogenous
Tra2bDRS1 protein suggesting that this particular isoform is
expressed at low levels in these cells. Further probing of the same
filter indicated that all the GFP fusion proteins were expressed at
similar levels (Figure 1C, lower panel). A key priority to understand the biological functions of Tra2b is
to identify target RNAs which are functionally regulated within
animal cells, and associated pathways of gene activity. Mice with
ubiquitous deficiency of the Sfrs10 gene die at around 7.5 to 8.5
days of gestation [19]. Splicing of some Tra2b candidate target
exons have been investigated using minigenes, but recently a well
known regulated splice target exon (SMN2 exon 7) was found to
have the same splicing pattern within wild type mice and Smn2/2;
SMN2tg/tg; Sfrs102/2 mouse cells which do not express Tra2b
protein [19]. These data suggest Tra2b is not the key protein
regulating physiological inclusion of SMN2 exon 7 within animal
cells. We used indirect immunohistochemistry to determine the cell
type distribution of full length Tra2b in the adult testis (Figure 1D
and 1E). Tra2b was detected as a nuclear protein (Figure 1E upper
panel), and all staining was prevented by pre-incubation of the
antisera with the immunising peptide (Figure 1E lower panel). Tra2b was most highly expressed during mouse male germ cell
development at the meiotic stage in spermatocytes (abbreviated
Spc), and afterwards in round spermatids (abbreviated Rtd). Less
intense Tra2b staining was detected within spermatogonia which
contain the mitotically active stem cell population. No immuno-
staining was detected in elongating spermatids (abbreviated Spd). This regulated expression pattern predicts that Tra2b might play a
role in regulating meiotic and post-meiotic exon inclusion during
male germ cell development. Outside the germline, Tra2b protein
expression was detected in Sertoli cells (indicated by red arrows on
Figure 1E). The Sfrs10 gene itself is alternatively spliced to five mRNA
isoforms encoding at least 2 protein isoforms [20,21,22]. The
major isoform encodes full length Tra2b protein. Full length
Tra2b protein regulates its own levels through activating splicing
inclusion of a poison exon (exon 2) into a second mRNA isoform,
preventing protein translation (Figure 1A) [22]. Introduction Almost all transcripts from genes encoding multiple exons are
alternatively spliced, and correct patterns of alternative splicing are
important for health and normal development [1,2,3]. Alternative
splicing introduces new coding information into mRNAs, thereby
increasing genome capacity to encode an expanded number of
mRNAs and proteins from a finite number of genes [3]. Poison
exons which introduce premature stop codons can also be Alternative splice events are controlled in part by trans- acting
RNA binding proteins which help establish patterns of alternative
splicing through deciphering a splicing code embedded within the
pre-mRNA sequence [9,10,11]. Tra2 proteins bind directly to
target exons thereby activating splicing inclusion [12], and have a
modular organisation comprising a single central RNA recognition
motif (RRM) which binds to target RNA sequences, flanked by December 2011 | Volume 7 | Issue 12 | e1002390 1 New Roles and Splicing Targets for Tra2b distinct function has been assigned to the Tra2bDRS1 isoform
compared to full length Tra2b [17], although this isoform is
conserved in invertebrates so likely important. Tra2bDRS1
expression is tissue specific in both flies and mammals, and is
up-regulated by expression of Clk kinases and neural stimulation
[20,21,22,23]. Author Summary Alternative splicing amplifies the informational content of
the genome, making multiple mRNA isoforms from single
genes. Tra2 proteins bind and activate alternative exons,
and in mice Tra2b is essential for embryonic development
through unknown target RNAs. Here we report the first
target exons that are physiologically regulated by Tra2b in
developing mice. Normal activation of these regulated
exons depends on multiple Tra2b binding sites, and
significant mis-regulation of these exons is observed
during mouse development when Tra2b is removed. As
expected, Tra2b activates splicing of some target exons
through direct RNA binding via its RNA Recognition Motif. Surprisingly, for some exons Tra2b can also activate
splicing independent of direct RNA binding through two
domains enriched in arginine and serine residues (called RS
domains). The
N-terminal
RS1
domain
of
Tra2b
is
absolutely essential for splicing activation of physiological
target exons, explaining why this domain is conserved
between vertebrates and invertebrates. Surprisingly, Tra2b
proteins without RS1 operate as splicing repressors,
suggesting the possibility that endogenous Tra2b protein
isoforms may differentially regulate the same target exons. Male germ cell development is one of the few developmental
pathways to continue into the adult. In the fly testis, Tra2 regulates
splicing of Exuperentia and Att pre-mRNAs in male germ cells, as
well as its own alternative splicing pathway [24,25]. Tra2b has
been implicated in mammalian spermatogenesis through interac-
tion with RBMY protein which is genetically deleted in some
infertile men [26,27], and regulates the splicing of the human
testis-specific HIPK3-T exon through a switch-like mechanism
[28,29]. Given its important role in Drosophila spermatogenesis and
established interactions with proteins implicated in human male
fertility we predicted that Tra2b-regulated alternative splicing
events would control fundamental pathways in mammalian male
germ cell development. We have tested this prediction here using a
transcriptome-wide approach. PLoS Genetics | www.plosgenetics.org December 2011 | Volume 7 | Issue 12 | e1002390 Tra2b is ubiquitously expressed but up-regulated at the
onset of meiosis in male germ cells A third mRNA
isoform encodes just the C-terminus of the protein (containing the
RRM, glycine linker and the RS2 domain) giving rise to the
protein isoform Tra2beta-3 or Tra2bDRS1 [20,21,22]. No December 2011 | Volume 7 | Issue 12 | e1002390 2 New Roles and Splicing Targets for New Roles and Splicing Targets for Tra2b PLoS Genetics | www.plosgenetics.org
3 3
December 2011 | Volume 7 | Issue PLoS Genetics | www.plosgenetics.org December 2011 | Volume 7 | Issue 12 | e1002390 3 New Roles and Splicing Targets for Tra2b Figure 1. Tra2b is a nuclear protein highly expressed in mouse germ cells. (A) Diagram showing in silico PCR of the mouse Sfrs10 mRNA
redrawn from the UCSC mouse genome browser [69]. Two different RT-PCR products are amplified using primers in exons 1 and 4. The smaller
product (438 nucleotides) represents the amplified product when exon 1 is directly spliced to exon 3 and then exon 3 to exon 4 (upper Sfrs10 mRNA
isoform). The larger product (714 nucleotides) represents when the poison exon 2 is spliced resulting in the non-translated isoform Tra2b4 (lower
Sfrs10 mRNA isoform). (B) Capillary gel electrophoresis image showing levels levels of Sfrs10 mRNA assayed by multiplex RT-PCR using RNA purified
from adult mouse tissues. Primers were used for amplification complementary to exons 1 and 4 as described in (A) above. Within a multiplex RT-PCR,
primers were included to detect Hprt as a parallel loading control to ensure equivalent amounts of RNA were used in each lane. (C) Immunoblotting
experiment to confirm the specificity of the polyclonal antisera used for immunohistochemistry. HEK293 cells were transfected with plasmids
expressing the indicated proteins. Proteins were then isolated and analysed by SDS-PAGE and Western blotting. The same blot was probed
sequentially with an affinity purified antisera ab31353 raised against Tra2b (top panel) and then with a polyclonal specific for GFP to detect overall
expression of each of the fusion proteins (lower panel). The ab31353 a-Tra2b antisera detected a single band in HEK293 cells corresponding to
endogenous Tra2b protein, and in transfected cells additionally detected the Tra2b-GFP fusion protein and Tra2bDRS1-GFP. No cross reaction with
Tra2a-GFP was observed, indicating that this purified antisera is highly specific to Tra2b. (D) Flow chart summarising major developmental stages in
male germ cell development. (E) Tra2b is a nuclear protein expressed during and after meiosis. Tra2b is ubiquitously expressed but up-regulated at the
onset of meiosis in male germ cells Paraffin embedded adult mouse testis sections were
stained with an affinity purified antibody raised against Tra2b (brown staining), and counterstained with haematoxylin (blue). Abbreviations: Spg –
spermatogonia (mitotically active population which includes stem cells); Spc –spermatocyte (meiotic cells); Rtd –round spermatid (post-meiotic
haploid cell); Spd –elongating spermatid (differentiating haploid cell with condensed nuclei). The scale bar is equivalent to 20 mm. The red arrows
indicate Sertoli Cells. Based on these immunohistochemistry results, the Tra2b protein expression levels during mouse germ cell development are
summarised also on the flow chart in part (D). doi:10.1371/journal.pgen.1002390.g001 downstream of the GAA motif (position 5), and an A at the next
nucleotide position downstream (position 6). This results in an
extended AGAAGA consensus, in agreement with the sequence of
the 3 top hexamers. Interestingly, when only a GAA triplet but not
an AGAA core is present within a CLIP tag, 89% of the tags have
a G residue immediately downstream (GAAG), consistent with the
important contribution of the G5 residue for efficient binding of
Tra2b to its natural RNA targets. No further strong sequence bias
was noticed in the sequences upstream and downstream of the
AGAAGA hexamer. A similar consensus was obtained previously
for SRSF1 protein [31]. However since SRSF1 has 2 RRMs with
different RNA binding capacities and only one RS domain, it is
most likely that its global specificity of RNA recognition and
binding are broader than that for Tra2b and also depends on
other ESEs within its individual target exons. Tra2b primarily binds AGAA-rich target sequences in
mouse germ cells To identify endogenous cellular RNA targets for Tra2b we
carried out high throughput sequencing cross linking immuno-
precipitation (HITS-CLIP) [30]. Adult mouse testis cells were used
according to published procedures (see methods for details) to
retrieve an average tag length of 40 nucleotides. These recovered
CLIP tags correspond to specific RNA sequences bound and
subsequently cross-linked to endogenous Tra2b protein within the
testis. To identify frequent physiological Tra2b binding sites in mouse
testis we searched for frequently occurring 6-mers in the retrieved
CLIP tags, and normalised these to their background occurrence
in the mouse genome and transcriptome using custom-written
Python scripts (Table S1 and Table S2). Each of the most
frequently recovered 6-mers was significantly enriched in the CLIP
dataset compared to their representation in the mouse genome or
mouse testis transcriptome. Strikingly, purine-rich sequences were
preferentially recovered in our CLIP tags. In fact, 14 hexamers out
of the top 30 recovered genome corrected hexamers in Table S1
have only purine residues, and 13 have only one pyrimidine. More
specifically and consistent with the known RNA binding site for
Tra2b
[17,18],
GAA-containing
sequences
were
frequently
observed. The distribution of GAA-containing 6-mers in the
overall population of CLIP tags was visualised by plotting the
genomic ranking of 6-mer recovery (X axis) against their
representation in the CLIP population (Y axis) (Figure 2A:
GAA-containing 6-mers are shown in red, with all other 6-mer
sequences in blue). Of the 30 most frequently recovered 6-mers, 27
had a core GAA motif and the other 3 an AGA motif. The most
frequent 6-mer (the AGAAGA motif, 10u on the X axis of
Figure 2A -equivalent to 1) was found in almost 20% of the
recovered CLIP tags. The ten most frequently recovered 6-mers
were found in more than 40% of the CLIP tags. PLoS Genetics | www.plosgenetics.org Tra2b binds a high frequency of exonic sequences To identify specific endogenous target transcripts CLIP tags
were mapped onto the mouse genome sequence (a full bed file of
Tra2b CLIP tags is provided as Dataset S1) [32]. Overall, the
distribution of Tra2b CLIP tags was predominantly intragenic:
Around 69% of Tra2b binding sites were located within protein
coding genes, even though genes contribute just 25% of the
genome (Figure 2C). Network analyses indicated the main
functional properties associated withTra2b target transcripts were
post-translational modification, the cell cycle, gene expression,
RNA post-transcriptional modification and cell death (Figure 2D). Top physiological systems associated with Tra2b target transcripts
included reproductive system and nervous system development,
and there was significant enrichment of signalling pathways in the
top detected pathways (Table S3). Most intragenic CLIP tags
mapped to transcripts in the sense orientation, but 7.5% of
retrieved CLIP tags were antisense to known annotated genes. Only 1.3% of the mouse genome encodes exons (59 UTR, ORF
and 39 UTR, based on mm9 annotation version ensembl59). For
Tra2b some 29% of Tra2b CLIP tags mapped within exons of
protein coding genes (Figure 2C) which indicates the presence of
numerous
Tra2b-specific
target
exons. Similar
CLIP-based
transcriptome-wide analyses found that the SR protein SRSF1
also frequently binds to exonic sequences, while Nova and PTB
target sites are mainly intronic in distribution [30,31,33]. Next
we
aligned
full
length
CLIP
tags
to
generate
a
transcriptome-wide consensus sequence. We anchored this line-
up between CLIP tags using the trinucleotide GAA from the core
binding motif which is essential for efficient RNA protein
interactions [17] (Figure 2B). Within this consensus alignment,
an A residue followed by a T residue (and less frequently a G
residue) was usually found upstream of the GAA motif (position 1
in Figure 2B), consistent with reported in vitro RNA-protein
binding
data
between
the
RRM
of
Tra2b
and
synthetic
oligonucleotides
[17]. Furthermore,
a
G
residue
(and
less
frequently an A residue) was preferentially selected at the position Non-exonic Tra2b binding sites were found within deep
intronic regions, within locations annotated as intergenic and
within noncoding RNAs (ncRNAs) [34]. Within ncRNAs Tra2b
binding sites were found within the small subunit rRNA (also December 2011 | Volume 7 | Issue 12 | e1002390 4 New Roles and Splicing Targets for Tra2b Figure 2. Identification of binding sites for Tra2b in the mouse transcriptome. Tra2b binds a high frequency of exonic sequences (A) Nucleotide sequences enriched in the Tra2b CLIP tags are
enriched in the core motif GAA. The percentage of CLIP tags was plotted against the order of retrieval of individual 6-mers on a logarithmic scale to
identify the most frequently occurring 6-mer sequences within the CLIP tags. CLIP tag sequences which contain GAA are indicated in red. All other
CLIP tags are shown in blue. (B) Consensus binding site for Tra2b derived from alignment of full length CLIP tags. The consensus was constructed by
anchoring CLIP tags around GAA and then performing an alignment The positions 1 6 which are particularly conserved are shown underneath and
New Roles and Splicing Targets for Tra2b Figure 2. Identification of binding sites for Tra2b in the mouse transcriptome. (A) Nucleotide sequences enriched in the Tra2b CLIP tags are
enriched in the core motif GAA. The percentage of CLIP tags was plotted against the order of retrieval of individual 6-mers on a logarithmic scale to
identify the most frequently occurring 6-mer sequences within the CLIP tags. CLIP tag sequences which contain GAA are indicated in red. All other
CLIP tags are shown in blue. (B) Consensus binding site for Tra2b derived from alignment of full length CLIP tags. The consensus was constructed by
anchoring CLIP tags around GAA and then performing an alignment. The positions 1–6 which are particularly conserved are shown underneath and
discussed in the main text. (C) Pie chart showing percentage of retrieved CLIP tags mapping to different inter- and intragenic locations within the Figure 2. Identification of binding sites for Tra2b in the mouse transcriptome. (A) Nucleotide sequences enriched in the Tra2b CLIP tags are
enriched in the core motif GAA. The percentage of CLIP tags was plotted against the order of retrieval of individual 6-mers on a logarithmic scale to
identify the most frequently occurring 6-mer sequences within the CLIP tags. CLIP tag sequences which contain GAA are indicated in red. All other
CLIP tags are shown in blue. (B) Consensus binding site for Tra2b derived from alignment of full length CLIP tags. The consensus was constructed by
anchoring CLIP tags around GAA and then performing an alignment. The positions 1–6 which are particularly conserved are shown underneath and
discussed in the main text. Tra2b binds a high frequency of exonic sequences (C) Pie chart showing percentage of retrieved CLIP tags mapping to different inter- and intragenic locations within the PLoS Genetics | www.plosgenetics.org December 2011 | Volume 7 | Issue 12 | e1002390 5 New Roles and Splicing Targets for Tra2b mouse transcriptome. (D) Summary of the top 5 molecular and cellular functions for Tra2b determined by Ingenuity Pathway Analysis. (E) Distribution
of Tra2b binding sites relative to the different classes of alternative events annotated on the mouse genome. Alternative events are shown in red, and
the constitutive events as yellow boxes (exons) or black lines (introns). Alternative events are annotated according to the UCSC genome browser
track Alternative Events (URL: http://genome.ucsc.edu/cgi-bin/hgTrackUi?g = knownAlt&hgsid = 212031267). doi:10.1371/journal.pgen.1002390.g002 identified as a binding site for SRSF1 [31]) and 7SK RNA. There
were also Tra2b binding sites within the ncRNA Malat1 which is
known to be localised in nuclear splicing speckles enriched in pre-
mRNA splicing components (Malat1 is also bound by SRSF1 [31]),
and within microRNAs. These identified targets suggest that
Tra2b might in fact be a somewhat multifunctional post-
transcriptional regulator. Similarly diverse classes of target RNA
(including both coding and ncRNAs) have been identified for a
number
of
other
RNA
binding
proteins
by
HITS-CLIP
[30,31,33,35,36]. identified as a binding site for SRSF1 [31]) and 7SK RNA. There
were also Tra2b binding sites within the ncRNA Malat1 which is
known to be localised in nuclear splicing speckles enriched in pre-
mRNA splicing components (Malat1 is also bound by SRSF1 [31]),
and within microRNAs. These identified targets suggest that
Tra2b might in fact be a somewhat multifunctional post-
transcriptional regulator. Similarly diverse classes of target RNA
(including both coding and ncRNAs) have been identified for a
number
of
other
RNA
binding
proteins
by
HITS-CLIP
[30,31,33,35,36]. The Sfrs10 locus encodes a second endogenous protein isoform
called Tra2bDRS1 [20,21,22] which lacks the RS1 domain. Surprisingly, after co-expression of a Tra2b-GFPDRS1 protein
isoform we observed significant splicing repression of both the
Tra2a poison exon and Nasp-T exon (Figure 3B–3D) indicating
that this protein isoform behaves as a potent splicing repressor,
and of the same target exons recognised by full length Tra2b
protein. Analysis of endogenous target exons indicate that
isoforms of Tra2b can activate, co-activate, and repress
exon inclusion Analysis of endogenous target exons indicate that
isoforms of Tra2b can activate, co-activate, and repress
exon inclusion Tra2b bound to both constitutive and alternative exons and also
to each different class of alternative events annotated on the mouse
genome browser at UCSC. In particular, Tra2b binding sites
mapped preferentially to cassette exons (this is also the most
frequent class of alternative splicing event in metazoans [37])
(Figure 2E). To test for splicing regulation of these identified target
exons by Tra2b, a panel of seven cassette exons with high numbers
of mapped CLIP tags, together with flanking intronic sequences,
were cloned into an exon trap vector (see Materials and Methods). The resulting minigenes were then transfected into HEK293 cells
with expression constructs encoding either GFP, Tra2b-GFP, or
GFP-tagged Tra2b deletion variants. Western blots indicated each
of the GFP-fusion proteins were efficiently expressed in HEK293
cells (Figure 3A), although the fusion protein without the RS1
domain was expressed at higher levels. Splicing patterns of pre-mRNAs were analysed using RT-PCR. We observed particularly strong splicing activation of a poison
exon in the Tra2a gene in response to co-expression of Tra2b-GFP
(Figure 3B). Ectopic expression of both Tra2a and Tra2b were
equally able to activate splicing of the Tra2a poison exon
indicating that these two proteins are functionally equivalent in
this assay (Figure 3B, lanes 2 and 3). No splicing activation of the
Tra2a poison exon was observed with either Tra2bDRRM-GFP or
GFP alone, indicating a requirement for RRM-dependent binding
by full length Tra2b proteins for splicing activation (Figure 3B,
lanes 1 and 4). Tra2b binds a high frequency of exonic sequences p
Two further exons, Creb exon 2 and Lin28b exon 2, did not
detectably respond to ectopic expression of full length Tra2b or
any of its derivatives (Figure 3G and 3H) and were already
included at high levels in the absence of ectopically expressed
Tra2b protein. No strong splicing repression of Creb exon 2 and
Lin28b
exon
2
was
observed
on
co-expression
of
Tra2b-
GFPDRS1. Full length Tra2b weakly but significantly activated
splicing of two other target exons, Krba1 exon 9 and Pank2 exon 3
(Figure 3E and 3F) and splicing of these exons was also not
significantly repressed by Tra2b-GFPDRS1 (compare lanes 1 and
3: notice slight repression which was not statistically significant). We also looked at two other exons which are spliced in the testis
and which we independently characterised as being regulated by
Tra2b. Minigene experiments indicated both the Crebc and Fabp9
exons [38,39] were moderately activated by Tra2b, and were also
co-ordinately moderately repressed by the Tra2bDRS1 isoform
(Figure 3I and 3J, lanes 1 and 4). Taken together these data are
consistent with full length Tra2b protein activating specific target
exons, and the Tra2bDRS1 protein isoform specifically repressing
exons which are at least moderately to strongly activated by full
length Tra2b, but not acting as a general repressor of cellular
splicing. PLoS Genetics | www.plosgenetics.org December 2011 | Volume 7 | Issue 12 | e1002390 Tra2b directly binds to target transcripts identified by
CLIP, and binding efficiency correlates with splicing
activity We carried out further in silico and molecular analyses to
correlate Tra2b binding with the observed patterns of exon
regulation. We
firstly
looked
for
the
occurrence
of
over-
represented transcriptome-wide enriched 6-mer sequences (k-
mers) [40] to identify putative Tra2b binding sites in the analysed
target exons in silico (Figure S1). Both the Nasp-T and Tra2a poison
exon had a high predicted content of 6-mers corresponding to
putative Tra2b binding sites and consistent with their strong
Tra2b regulation observed in vitro. Full length Tra2b also mediated statistically significant splicing
activation of a cassette exon annotated Nasp-T in the Nasp gene. Surprisingly, equally strong and highly statistically significant Nasp-
T exon splicing activation was also observed in response to ectopic
expression of Tra2bDRRM-GFP protein (Figure 3C, lanes 2 and
3). Because of the high levels of splicing inclusion observed for the
wild type Nasp-T exon at endogenous cellular concentrations of
Tra2b (Figure 3C), we also repeated these experiments using a
mutated exon which is less efficiently spliced (mutant M3+M4 –see
below) and again observed significant splicing activation by
Tra2bDRRM-GFP protein (Figure 3D –in this case the effect of
Tra2bDRRM-GFP is clearer because of the lower levels of splicing
inclusion of this mutated exon at endogenous cellular Tra2b
protein concentrations). Together these data indicate that for some
exons including Nasp-T, Tra2b can activate splicing through
RRM independent interactions as well as being a direct splicing
activator as previously described. We then directly measured Tra2b binding affinities using
Electromobility Shift Assays (EMSAs) (Figure 4: the positions of
predicted binding sites within the RNA probes are shaded as in
Table S1. Notice the dark green corresponds to the top 5 most
frequently
recovered
6-mers,
and
lighter
shades
of
green
correspond to less frequently recovered 6-mers). Both Nasp-T
and Tra2a poison exon probes were very efficiently shifted by even
very low concentrations of Tra2b protein (the Nasp-T probe was
shifted into the well by only 50 ng of added Tra2b protein
indicating formation of very large Tra2b protein-RNA complexes,
and increasing molecular weight Tra2a RNA-protein complexes
were observed with increasing concentrations of full length Tra2b
protein). The Tra2a poison and Nasp-T cassette exons are
phylogenetically conserved and show high levels of
splicing inclusion in mouse testis An
important
measure
of
the
functional
importance
of
individual alternative splice events is evolutionary conservation
[1,2,37,41,42]. Although many testis-specific exons are species-
specific, phastcons analysis (which measures phylogenetic conser-
vation of sequences on a scale of 0 to 1, with 1 being most
conserved) indicated very high levels of phylogenetic conservation
for the Tra2a poison exon along with flanking intronic sequences
(Figure 5A–5C). Similar high levels of nucleotide conservation
have been reported for poison exons in other genes encoding
splicing regulator proteins including Sfrs10 itself [4,5,22]. To experimentally test the need for individual Tra2b binding
sites in splicing regulation, individual sites were mutated within the
minigenes
without
creating
Exonic
Splicing
Silencer
(ESS)
sequences (Figure 6A) [28], and the splicing effect monitored. Mutation of single Tra2b binding sites had only a minor effect on
Nasp-T splicing inclusion at endogenous cellular concentrations of
Tra2b. However, pre-mRNAs
containing
double
mutations
affecting Tra2b binding sites (M2+M3, M1+M2 and M3+M4)
had strongly reduced Nasp-T exon splicing inclusion compared to
their wild type counterparts at normal endogenous cellular
concentrations of Tra2b (Figure 6B). Mutation of different Tra2b
binding sites within Nasp-T also had distinct outcomes on exon
inclusion, indicating underlying combinatorial effects between
different patterns of Tra2b binding. In particular, mutant M3+M4
reduced exon inclusion levels to 20% of wild type at endogenous
cellular levels of Tra2b, whereas double mutations comprising M2
and M3 reduced Nasp-T exon inclusion to just below 60%
(Figure 6B). The Tra2a poison exon, which is 306 nucleotides long,
introduces stop codons into the reading frame of the Tra2a
mRNA which encodes Tra2a protein. Despite the lack of protein
coding capacity, 48% of nucleotides within the Tra2a poison exon
are in fact conserved in all vertebrates (Figure S2A: the nucleotide
positions universally conserved in sequenced vertebrate genomes
are shown in red). As a group, the 24 top most frequently
recovered 6-mers from the entire transcriptome-wide screen were
enriched in the nucleotide positions conserved between all
vertebrates at levels much higher than would be expected by
chance (Figure S2A, p = 0.0075, Fisher exact test: p = 0.0003, Chi
Squared test). These data are consistent with maintenance of
multiple Tra2b-binding sites within the Tra2a poison exon since
the radiation of vertebrates. When analysed by RT-PCR, the
Tra2a
poison
exon
was
found
to
be
particularly
strongly
alternatively spliced in the testis, with zero or much lower levels
in other adult tissues (Figure 5A–5C). Efficient splicing activation of the testis-specific Nasp-T
by Tra2b depends on multiple Tra2b binding sites To experimentally address the function of multiple Tra2b
binding sites in Nasp-T we used a combination of in silico and
experimental analyses, and focused on an upstream portion of the
exon (from positions 117 to 271). Using octamers predictive of
splicing enhancers and silencers [44,45,46], we firstly identified 3
strong putative ESEs (Exonic Splicing Enhancers, ESE1 to ESE3)
which we selected for further analysis, as well as other putative
moderate ESEs (Z score around 4) of which only one designated
ESE4 was further studied (Figure 6A). Each of these putative ESEs
directly overlapped with Tra2b binding sites initially identified
through 6-mers derived from the transcriptome-wide CLIP
analysis. Tra2b directly binds to target transcripts identified by
CLIP, and binding efficiency correlates with splicing
activity A series of increased molecular weight complexes also formed
on the Crebc exon RNA probe (corresponding exon regulated in PLoS Genetics | www.plosgenetics.org December 2011 | Volume 7 | Issue 12 | e1002390 December 2011 | Volume 7 | Issue 12 | e1002390 6 New Roles and Splicing Targets for Tra2b PLoS Genetics | www plosgenetics org
7
December 2011 | Volume 7 | Issue 12 | PLoS Genetics | www.plosgenetics.org
7
December 2011 | Volume 7 | Issue 12 | e1002390 December 2011 | Volume 7 | Issue 12 | e1002390 PLoS Genetics | www.plosgenetics.org New Roles and Splicing Targets for Tra2b New Roles and Splicing Targets for Tra2b Figure 3. Different protein isoforms of Tra2b can act as specific splicing activators, co-activators, and repressors of a target exons
identified by HITS-CLIP. (A) Efficient protein expression levels of different GFP fusion proteins used in these experiments (upper panel). Levels of
actin were measured in parallel (lower panel). (B)–(J). Upper panels: Bar charts showing percentage splicing inclusion (PSI) of a panel of exons
identified through HITS-CLIP in response to GFP and Tra2b-GFP fusion proteins. All data used to make the bar charts was from at least 3 biological
replicates, and the error bars are shown as standard errors. Lower panels: Representative capillary gel electrophoresis image from each RT-PCR
analysis. Probability (p) values were calculated using an independent two-sample T-test between the PSI levels for cells co-transfected with GFP and
each of the different Tra2b-GFP constructs (* p#0.05, **p#0.01). doi:10.1371/journal.pgen.1002390.g003 also preferentially included but in other tissues it was frequently
skipped (Figure 5F). cellulo by Tra2b) and on the Krba1 RNA probe (weakly responsive
in cellulo to Tra2b splicing activation). A single higher molecular
weight complex formed on the Lin28 probe (exon splicing not
activated in vitro by Tra2b, and contains a single predicted Tra2b
binding site). Much less efficient binding was observed for the non
Tra2b-responsive Creb exon 2 (which formed a single molecular
weight complex only with 200 ng added Tra2b protein, compared
with 50 ng for the Crebc probe). PLoS Genetics | www.plosgenetics.org The Tra2a poison and Nasp-T cassette exons are
phylogenetically conserved and show high levels of
splicing inclusion in mouse testis (A) EMSAs of Creb exon 2, Creb exon c and the wild type Nasp-T exon. (B) EMSA
of Krba1 exon 9, Lin28 exon 2 and the Tra2a poison exon. Electrophoretic Mobility Shift Assays (EMSAs) were carried out with full length Tra2b prote
and short radioactive RNA probes from pre-mRNAs identified by CLIP and which contained predicted Tra2b protein binding sites from th
transcriptome-wide 6-mer analysis. The RNA probes are shown to the right of the gel panels, and the sequences are highlighted for differen
categories of 6-mers as in Table S1. Exon sequences are shown in upper case, and any flanking intron sequence in lower case (the Lin28b exon is ve
short). doi:10.1371/journal.pgen.1002390.g004 Figure 4. Tra2b CLIP targets bind to full length Tra2b protein. (A) EMSAs of Creb exon 2, Creb exon c and the wild type Nasp-T exon. (B) EMSAs
of Krba1 exon 9, Lin28 exon 2 and the Tra2a poison exon. Electrophoretic Mobility Shift Assays (EMSAs) were carried out with full length Tra2b protein
and short radioactive RNA probes from pre-mRNAs identified by CLIP and which contained predicted Tra2b protein binding sites from the
transcriptome-wide 6-mer analysis. The RNA probes are shown to the right of the gel panels, and the sequences are highlighted for different
categories of 6-mers as in Table S1. Exon sequences are shown in upper case, and any flanking intron sequence in lower case (the Lin28b exon is very
short). Figure 4. Tra2b CLIP targets bind to full length Tra2b protein. (A) EMSAs of Creb exon 2, Creb exon c and the wild type Nasp-T exon. (B) EMSAs
of Krba1 exon 9, Lin28 exon 2 and the Tra2a poison exon. Electrophoretic Mobility Shift Assays (EMSAs) were carried out with full length Tra2b protein
and short radioactive RNA probes from pre-mRNAs identified by CLIP and which contained predicted Tra2b protein binding sites from the
transcriptome-wide 6-mer analysis. The RNA probes are shown to the right of the gel panels, and the sequences are highlighted for different
categories of 6-mers as in Table S1. Exon sequences are shown in upper case, and any flanking intron sequence in lower case (the Lin28b exon is very
short) doi:10.1371/journal.pgen.1002390.g004 The Tra2a poison and Nasp-T cassette exons are
phylogenetically conserved and show high levels of
splicing inclusion in mouse testis Although they showed decreased exon inclusion at normal
cellular concentrations of Tra2b, each of the double mutated
Nasp-T exons gave at least 80% splicing inclusion after Tra2b
protein was ectopically expressed. This suggested a requirement
for higher levels of ectopic Tra2b protein for splicing inclusion. To test this, we co-transfected cells with minigenes containing
either wild type Nasp-T exon or the M3+M4 mutant derivative,
and a concentration gradient of Tra2b (Figure 6C). Splicing
inclusion of the wild type Nasp-T exon was already 90% without
over-expression of Tra2b and was maximal after co-transfection
of no more than 30 ng Tra2b expressing plasmid. In contrast,
levels of inclusion of the M3+M4 NaspT exon derivative increased
more slowly over the whole concentration gradient, indicating
decreased splicing sensitivity to Tra2b after removal of just two
binding sites. This is particularly striking since the M3+M4 NaspT
exon retains multiple other Tra2b binding sites (both experi-
mentally confirmed sites in the case of ESEs 1–4, and further
predicted sites throughout the exon shown in Figure S1). We used
EMSAs to directly analyse RNA-protein interactions using both
wild type and mutated versions of the Nasp-T RNA probe Phastcons analyses also showed the Nasp-T cassette exon,
which is also particularly long at 975 nucleotides, has been
conserved since the last common ancestor of all vertebrates
(Figure 5D–5F). However neither the nucleotide or the peptide
sequence encoded by Nasp-T are particularly highly conserved
over the full length of the exon (Figure 5E).The Nasp gene
encodes a histone chaperone essential for mouse development
[43], and the Nasp-T exon introduces a peptide-encoding cassette
exon generating a longer version of the Nasp protein. Similar to
the Tra2a poison exon, 6-mers predicting Tra2b binding site
sequences were found throughout the Nasp-T exon, and high
frequency 6-mers mapped closely adjacent to CLIP tags (Figure
S2B). Within mammalian Nasp-T exons multiple Tra2b binding
sites have been conserved. Extremely high levels of Nasp-T exon
inclusion were detected by RT-PCR in the testis and heart. In
gut, muscle and ovary, the Nasp-T exon inclusion isoform was PLoS Genetics | www.plosgenetics.org December 2011 | Volume 7 | Issue 12 | e1002390 8 New Roles and Splicing Targets for Tra2b (Figure 7). While wild type Nasp-T and the single mutant M2
Levels of neuronal Tra2b protein are depleted in a
Figure 4. Tra2b CLIP targets bind to full length Tra2b protein. PLoS Genetics | www.plosgenetics.org Levels of neuronal Tra2b protein are depleted in a
Nestin-Cre mouse model and are functionally buffered
by the Sfrs10 poison exon High levels of splicing inclusion were detected in the
mouse testis, and lower levels of inclusion in other tissues. Multiple CLIP tags mapped to an evolutionarily conserved cassette exon in the Nasp gene. The Phastcons alignment of the Nasp-T exon from multiple vertebrates is shown. Phastcons analyses in parts (B) and (E) are shown as downloads from
UCSC [69]. The key for both parts (A) and (D) are indicated in (D). doi:10.1371/journal.pgen.1002390.g005 Figure 5. The Tra2a poison exon and Nasp-T cassette exon are conserved in vertebrates and spliced at high levels of inclusion in the
mouse testis. (A) The structure of annotated alternative Tra2a transcripts (purple) and predicted PCR products (black) are shown above. (B)
Comparative genomic analysis with supporting EST information confirm splicing inclusion of these Tra2a poison exons indicate they are found in
vertebrates as distantly related as humans, mice, zebrafish and frog. (C) Expression of the Tra2a poison exon in different mouse tissues was monitored
using RT-PCR (primers in exons 1 and 4) followed by capillary gel electrophoresis, and a representative capillary gel electrophoresis image is shown. (D) Multiple Tra2b CLIP tags mapped to a poison exon in the Nasp-T gene. The structure of annotated alternative Nasp transcripts (purple) and
predicted PCR products (black) are shown above. (E) Underneath the Phastcons alignment of the Nasp-T exon from multiple vertebrates is shown. (F)
Incorporation of the Nasp-T exon was monitored by RT-PCR and capillary gel electrophoresis. High levels of splicing inclusion were detected in the
mouse testis, and lower levels of inclusion in other tissues. Multiple CLIP tags mapped to an evolutionarily conserved cassette exon in the Nasp gene. The Phastcons alignment of the Nasp-T exon from multiple vertebrates is shown. Phastcons analyses in parts (B) and (E) are shown as downloads from
UCSC [69]. The key for both parts (A) and (D) are indicated in (D). doi:10.1371/journal.pgen.1002390.g005 neuronal specific Sfrs10-depleted mouse by crossbreeding Sfrs10fl/fl
mice with Sfrs10fl/+ mice carrying the Nestin-Cre transgene
(Nestin-Cretg/+). In Sfrs10fl/fl; Nestin-Cretg/+ offspring the Cre
recombinase would be specifically activated in neuronal and glial
precursor cells from embryonic day 11 [47] to generate animals
with a homozygous Sfrs10 knockout in the developing central
nervous system (CNS). structures (Figure 8A, right panel and data not shown) whereas
heterozygous knockout mouse embryos (Sfrs10fl/wt; Nestin-Cretg)
had normal brain morphology (Figure 8A, left panel). Levels of neuronal Tra2b protein are depleted in a
Nestin-Cre mouse model and are functionally buffered
by the Sfrs10 poison exon (Figure 7). While wild type Nasp-T and the single mutant M2
RNA were efficiently shifted, the average size of the M3+M4
RNA-protein complex was only slightly smaller (the average size
of the shifted complexes is indicated by a red asterisk on Figure 7). Hence
even
a
moderate
change
in
in
vitro
RNA-protein
interactions translates to a detectable change in splicing inclusion
within cells. (Figure 7). While wild type Nasp-T and the single mutant M2
RNA were efficiently shifted, the average size of the M3+M4
RNA-protein complex was only slightly smaller (the average size
of the shifted complexes is indicated by a red asterisk on Figure 7). Hence
even
a
moderate
change
in
in
vitro
RNA-protein
interactions translates to a detectable change in splicing inclusion
within cells. Mice with clearly reduced expression levels of Sfrs10 would be a
prerequisite to enable detection of altered splicing patterns in
Tra2b- targeted transcripts identified by CLIP. Since ubiquitous
Sfrs10 deletion leads to embryonic lethality [19], we generated a PLoS Genetics | www.plosgenetics.org December 2011 | Volume 7 | Issue 12 | e1002390 PLoS Genetics | www.plosgenetics.org 9 New Roles and Splicing Targets for Tra2b Figure 5. The Tra2a poison exon and Nasp-T cassette exon are conserved in vertebrates and sp
mouse testis. (A) The structure of annotated alternative Tra2a transcripts (purple) and predicted PC
Comparative genomic analysis with supporting EST information confirm splicing inclusion of these Tra2a Figure 5. The Tra2a poison exon and Nasp-T cassette exon are conserved in vertebrates and spliced at high levels of inclusion in the
mouse testis. (A) The structure of annotated alternative Tra2a transcripts (purple) and predicted PCR products (black) are shown above. (B)
Comparative genomic analysis with supporting EST information confirm splicing inclusion of these Tra2a poison exons indicate they are found in
vertebrates as distantly related as humans, mice, zebrafish and frog. (C) Expression of the Tra2a poison exon in different mouse tissues was monitored
using RT-PCR (primers in exons 1 and 4) followed by capillary gel electrophoresis, and a representative capillary gel electrophoresis image is shown. (D) Multiple Tra2b CLIP tags mapped to a poison exon in the Nasp-T gene. The structure of annotated alternative Nasp transcripts (purple) and
predicted PCR products (black) are shown above. (E) Underneath the Phastcons alignment of the Nasp-T exon from multiple vertebrates is shown. (F)
Incorporation of the Nasp-T exon was monitored by RT-PCR and capillary gel electrophoresis. PLoS Genetics | www.plosgenetics.org Levels of neuronal Tra2b protein are depleted in a
Nestin-Cre mouse model and are functionally buffered
by the Sfrs10 poison exon This
indicates Tra2b protein is functionally very important for brain
development in the mouse. As the liquid filled ventricles make up
the majority of the whole brain volume, the brain morphology is
heavily altered and the proportion of intact tissue is heavily
reduced. Immunohistochemical analysis of whole brain paraffin-
embedded cross-sections showed strongly decreased expression of
Tra2-b with some Tra2-b positive cell areas in the cortical plate
zone (Figure 8A, right panel). These residual Tra2-b positive cells
likely do not express Cre from the Nestin promoter and are likely of Homozygous neuronal Sfrs10 mice died immediately after birth
at postnatal day 1 (PND1) whereas heterozygote mice had normal
lifespans. Neuronal specific Sfrs10-depleted embryos showed
severe malformations of the brain including strong dilation of
the third and lateral ventricles as well as degeneration of cortical December 2011 | Volume 7 | Issue 12 | e1002390 10 New Roles and Splicing Targets for Tra2b PL S G
ti
|
l
ti
11
D
b
2011 | V l
7 | I
12 |
100 PLoS Genetics | www.plosgenetics.org December 2011 | Volume 7 | Issue 12 | e1002390 December 2011 | Volume 7 | Issue 12 | e1002390 PLoS Genetics | www.plosgenetics.org 11 New Roles and Splicing Targets for Tra2b Figure 6. The splicing response to Tra2b is mediated through binding to four independent sites. (A) z-score plot predicting the splicing
control sequences according to [45] in the upstream portion of the Nasp-T cassette exon. Investigated exonic regions with z-scores above the
threshold value for exonic splicing enhancers are labelled ESE1–4. The z-score plots of the wild type Nasp exon is shown in black, superimposed with
z-score plots for each of the point mutants which affected individual ESEs (shown as blue coloured lines, with the changed nucleotide indicated as a
broken line). Individual mutants are shown as M1–M4. Local CLIP tag coverage is shown as black lines, and the relative positions of local 6-mers
identified at a high frequency in the CLIP screen as green lines. (B) Effect of Tra2b on splicing inclusion of different Nasp-T cassette exons (wild type
and mutants) co-expressed in HEK293 cells in the presence of endogenous Tra2b or with constant levels of Tra2b (500 ng, ectopically expressed). (C)
Percentage exon inclusion of the wild type and Nasp-T exon derivative M3+M4 obtained after transfection of increasing levels of each of Tra2b. Levels of neuronal Tra2b protein are depleted in a
Nestin-Cre mouse model and are functionally buffered
by the Sfrs10 poison exon Error
bars are shown as the standard error of the mean. Probability (p) values were calculated using an independent two-sample T-test between the PSI
levels for cells co-transfected with GFP and Tra2b-GFP (black asterisks), or between endogenous PSI for each of the Nasp-T constructs at endogenous
Tra2b concentrations (just transfected with GFP, red asterisks). P value scores are indicated as * p#0.05 and **p#0.01. doi:10.1371/journal.pgen.1002390.g006 Tra2-b levels [22]. Isoform specific qRT-PCR indicated a highly
significant down-regulation of both individual mRNA splice
isoforms and total length Sfrs10 mRNA in neuronal specific
Sfrs10-depleted mice Sfrs10fl/flNestin-Cretg/+) compared to con-
trols at 16.5 dpc (Figure 8C). In contrast, in heterozygous
knockout animals (Sfrs10fl/+Nestin-Cretg/+) down-regulation of
the functional isoform (2 exon 2) was less effective than for the
non-functional (+ exon 2) isoform indicating the involvement of
the autoregulatory feedback loop which counteracts any decrease
in functional Tra2b protein in neuronal cells. non-neuronal origin, or may represent mosaicism of Nestin-Cre
expression. Furthermore, Western blots from whole brain also
demonstrated a clear down-regulation of Tra2-b in neuronal
specific Sfrs10-depleted embryos compared to controls and
heterozygous knockout animals at 16.5 dpc (Figure 8B). In control
animals the Sfrs10 mRNA levels remained largely unchanged
during development (16.5 dpc, 18.5 dpc and PND1) (Sfrs10fl/fl
n = 10; Sfrs10fl/+ n = 6; data not shown). Expression analysis of whole brain RNA from neuronal Sfrs10-
depleted embryos at 16.5 dpc and 18.5 dpc and mice at PND1
showed clearly reduced Sfrs10 mRNA levels compared with
brains of control littermates (Sfrs10fl/fl, Sfrs10fl/+ or Sfrs10fl/+;
Nestin-Cretg/+) (Figure 8C). Regardless of the developmental stage
the majority of Sfrs10fl/fl pups exhibited somewhat reduced Sfrs10
expression levels compared with heterozygously floxed mice,
which suggested that the integration of the floxed allele has a
slightly negative influence on Sfrs10 expression. Therefore for
statistical analysis the expression levels of splice isoforms of
Sfrs10fl/fl; Nestin-Cretg/+ mice were always compared with Sfrs10fl/+
and not Sfrs10fl/fl mice. Tra2b physiologically regulates splicing inclusion of the
Tra2a poison and Nasp-T cassette exons in mouse brain
development We next set out to determine whether the Tra2a poison exon
and Nasp-T cassette exon were true physiological target exons
regulated by Tra2b in vivo. Correlating with an important
regulatory role for Tra2b protein, splicing inclusion of the poison
exon into the Tra2a mRNA was reduced 3-fold in neuronal Sfrs10-
depleted
mouse
brains
compared
to
controls
at
16.5 dpc
(Figure 8E). Surprisingly, this decrease in poison exon inclusion
could not be detected at later developmental stages like 18.5 dpc
or PND1 (data not shown). Tra2-b regulates its own expression level via alternative splice
regulation in an autoregulatory feedback-loop. Inclusion of poison
exon 2 into Sfrs10 transcripts introduces a premature stop codon
which leads to a non-functional protein and thus a reduction in Figure 7. Point mutants in the Nasp-T exon within candidate Tra2b binding sites are still able to bind to Tra2b. RNA-protein
interactions were monitored by EMSAs. The average position of the slowest migrating complex in the lane containing 10 ng of added Tra2b protein is
indicated by an asterisk, and the RNA probes used were as in Figure 4 but containing the appropriate point mutation. doi:10.1371/journal.pgen.1002390.g007 Figure 7. Point mutants in the Nasp-T exon within candidate Tra2b binding sites are still able to bind to Tra2b. RNA-protein
interactions were monitored by EMSAs. The average position of the slowest migrating complex in the lane containing 10 ng of added Tra2b protein is
indicated by an asterisk, and the RNA probes used were as in Figure 4 but containing the appropriate point mutation. doi:10.1371/journal.pgen.1002390.g007 December 2011 | Volume 7 | Issue 12 | e1002390 12 New Roles and Splicing Target New Roles and Splicing Targets for Tra2b PLoS Genetics | www.plosgenetics.org
13
December 2011 | Volume 7 | Issue 12 | e1002390 PLoS Genetics | www.plosgenetics.org
13
December 2011 | Volume 7 | Issue 12 | e1002390 PLoS Genetics | www.plosgenetics.org December 2011 | Volume 7 | Issue 12 | e1002390 December 2011 | Volume 7 | Issue 12 | e1002390 13 New Roles and Splicing Targets for Tra2b Figure 8. Tra2b protein levels are drastically reduced in the brains of neuronal specific Sfrs10 knockout mice and correlate with
defects in splicing of the Nasp-T cassette and Tra2a poison exon. (A) Whole brain sections derived from 16.5 dpc Sfrs10fl/wt; Nestin-Cretg (left
panel) and Sfrs10fl/fl; Nestin-Cretg (right panel) stained with antibodies against Tra2b. Tra2b physiologically regulates splicing inclusion of the
Tra2a poison and Nasp-T cassette exons in mouse brain
development Brains of heterozygous knockout animals (left panel) appear
normal and Sfrs10 is expressed throughout all cortical layers. Brains of neuronal specific knockout animals (right panel) show a vast dilation of the
lateral ventricles and disturbed cortical patterning. Tra2b expression is not detectable in the majority of intact tissue areas but is clearly retained in
some cells of the cortical plate region. Scale bars represent 200 mm. Abbreviations are mz: marginal zone; cp: cortical plate zone; sp: subplate zone; iz:
intermediate zone; svz: subventricular zone; vz: ventricular zone; lv: lateral ventricle. (B) Western blot analysis indicates that Tra2b expression is
reduced in neuronal specific knockout mice. Proteins were isolated from whole brains of 16.5 dpc embryos and Tra2b was specifically detected by
Western blotting. The Tra2b protein level is drastically reduced in Sfrs10fl/fl; Nestin-Cretg animals compared to controls or heterozygous knockout
animals. b-actin was used as a loading control. The relative levels are shown underneath as a bar chart (a.u. = arbitrary units). (C) Expression of the
Sfrs10 mRNA in different mouse genotypes used in this study. Levels of the Sfrs10 mRNA isoforms in different mouse genotypes were independently
measured by qRT-PCR from whole brain RNA isolated at 16.5 dpc (Sfrs10fl/fl, n = 4; Sfrs10fl/+, n = 5; Sfrs10fl/+; Nestin-Cretg/+, n = 4; Sfrs10fl/fl; Nestin-Cretg/+,
n = 4). Levels of Sfrs10 mRNA isoforms are consistent with use of the poison exon for autoregulation of transcript levels in vivo at 16.5 dpc. Isoform-
specific qRT -PCR for Sfrs10 on whole brain RNA revealed a coordinate downregulation of both the functional (278%) and the non-functional (288%)
isoform in neuronal specific knockout animals at a highly significant level. The decrease of Sfrs10 transcripts was also detectable in heterozygous
knockout animals, in which the functional and non-functional isoform were decreased by 24% and 61%, respectively. (D) Splicing of the Nasp-T
cassette exon is misregulated in Sfrs10fl/fl; Nestin-Cretg/+,mice. Levels of the different mRNA isoforms were measured by qRT-PCR from brain RNA
samples isolated at 16.5 dpc (Sfrs10fl/fl, n = 2; Sfrs10fl/+, n = 3; Sfrs10fl/+; Nestin2Cretg/+, n = 5; Sfrs10fl/fl; Nestin-Cretg/+, n = 2). (E) Splicing of the Tra2a
poison exon is misregulated in Sfrs10fl/fl; Nestin-Cretg/+mice. Levels of the different mRNA isoforms were measured by qRT-PCR from brain RNA
samples (Sfrs10fl/fl, n = 2; Sfrs10fl/+, n = 3; Sfrs10fl/+; Nestin-Cretg/+, n = 5; Sfrs10fl/fl; Nestin-Cretg/+, n = 2). (C–E) Error bars represent the s.e.m. Tra2b physiologically regulates splicing inclusion of the
Tra2a poison and Nasp-T cassette exons in mouse brain
development Statistical
significance was monitored using the T-test, and the significance values are as indicated. doi:10.1371/journal.pgen.1002390.g008 To determine whether low Tra2b levels directly affect the
splicing of the Nasp-T exon, qRT-PCR was carried out on whole
brain RNA of 16.5 dpc and PND1 pups. The levels of the T-exon
isoform of Nasp mRNA (Nasp-T) were 4-fold reduced in brains of
neuronal Sfrs10-depleted mice compared to controls at 16.5 dpc
(Figure 8D) and PND1 (data not shown). Given the 4-fold
reduction of the Nasp-T isoform in Sfrs10-depleted tissue, we
conclude that Tra2b protein is likely to be an important in vivo
activator of Nasp-T exon inclusion during mouse development. survival [43]. Nasp functions in chromatin remodelling after DNA
repair, and links chromatin remodelling to the cell cycle
machinery after S phase [48]. The T exon is also spliced in
embryos, and within the testis alternative splicing inclusion of the
Nasp-T cassette exon generates the testis-enriched tNASP protein
isoform. Timing of tNASP protein expression during male adult
germ cell development [48,49] exactly parallels the expression of
Tra2b protein. The tNASP protein isoform localises to the
synaptonemal complex of meiotic chromosomes where it may help
monitor double strand DNA break repair [43,48,50]. These data correlate a defect in splicing regulation of Nasp-T
and Tra2a with Sfrs10 depletion but do not necessarily imply a
causal relationship, because of the differences in cell types present
after
Sfrs10
depletion
which
result
from
the
physiological
importance of Tra2b for brain development. To address this
further we compared overall patterns of expression of the Nasp and
Tra2a genes in wild type and knockout mice, by quantifying levels
of the somatic Nasp and Tra2a mRNA isoforms. Consistent with no
significant changes in overall Tra2a gene expression resulting from
changes in the cell type population of the knockout brains, no
statistically significant changes in functional Tra2a or Nasp
expression were seen when comparing brain RNA of Sfrs10fl/+
mice with RNA of Sfrs10fl/fl; Nestin-Cretg/+ mice (Figure 8D and
8E). These results are consistent with essentially similar patterns of
Nasp and Tra2a gene expression in the mutant and wild type brains
despite any differences in cellular composition, while in contrast
the Tra2b-regulated splice isoforms from these same genes are
very different between the wild type and mutant mice. Tra2a and Tra2b are very similar proteins, and are inter-
changeable in our in cellulo splicing assays. Tra2b physiologically regulates splicing inclusion of the
Tra2a poison and Nasp-T cassette exons in mouse brain
development Tra2b protein helps
regulate overall Tra2 protein levels through both activating
splicing inclusion of a poison exon into its own Sfrs10 mRNA,
and also activating splicing inclusion of a poison exon into Tra2a
mRNA which encodes Tra2a protein. In vivo experiments
described here show that reduced inclusion of the poison exon
does indeed help buffer the effect of decreased gene dosage in
Sfrs10 heterozygote mice. However, down-regulation of Tra2a
poison exon inclusion in Sfrs102/2 cells does not lead to an
increase in Tra2a mRNA levels sufficient to restore splicing
patterns of Tra2b target exons, perhaps suggestive of unique
functions for the Tra2a and Tra2b proteins. In flies, auto-
regulation of splicing by Tra2 protein of its own pre-mRNA has
been shown to be critical for spermatogenesis, indicating that it
might be a highly conserved feature for germ cells to tightly
maintain expression levels of this class of splicing regulator
[24,25,51]. Since Tra2a regulates Tra2a poison exon in cellulo, it is
likely that it also autoregulates its own mRNA levels in vivo through
activation of this same poison exon. PLoS Genetics | www.plosgenetics.org Discussion Here we have identified (for the first time to the best of our
knowledge) physiological target exons regulated by Tra2b during
mouse development. Identification is based on the criteria of in vivo
cross-linking
of
endogenous
RNAs
and
proteins,
in
cellulo
experiments using transfected minigenes and proteins, RNA-
protein interaction assays and genetic analysis using a newly
derived conditional mouse strain which does not express Tra2b
protein in neurons and has significant abnormalities in brain
development. Our analyses reveal important pathways regulated
by Tra2b protein in vivo which likely contribute both to prenatal
death in Sfrs102/2 embryos and also to normal germ cell
development [19]. Nasp protein is a histone chaperone required
for nuclear import of histones at the G1-S phase transition of the
cell cycle, and is essential for cell proliferation and embryonic An important current question is how RNA binding proteins
like Tra2b achieve sequence specificity in target sequence selection
despite having fairly short target sequences [15]. Here we have
found a short consensus binding motif for Tra2b (AGAAGA,
Figure 2A) which matches perfectly with specific motifs obtained
both by classical SELEX analysis [12] and from identification of
Tra2b specific ESEs in various genes [22,29,52,53,54,55,56,57]. Parallel genome-wide mapping showed that Tra2b primarily binds
to exonic sequences. An explanation for exonic enrichment despite
the short binding site would be if Tra2b binds to exons
cooperatively with adjacent exonic RNA binding proteins. In the
case of SMN2 exon 7, the Tra2b binding site is flanked by
cooperative binding sites for SRp30c and hnRNP G [17,53,58]. For Nasp-T and Tra2a there are instead arrays of exonic Tra2b December 2011 | Volume 7 | Issue 12 | e1002390 14 New Roles and Splicing Targets for Tra2b binding sites. Removal of more than one binding site negatively
affects exon activation by Tra2b, indicating for Nasp-T and Tra2a
adjacent binding and assembly of homotypic Tra2b protein
activation complexes play important roles in splicing activation. which genome sequences are available; have known functional
roles; and like other phylogenetically conserved exons are spliced
at high levels in at least some tissues [4,37,41]. The tNASP protein
has been identified immunologically after the leptotene stage of
meiosis in both rabbits and mice, indicating that this exon is
meiotically expressed in both species [48,49]. Materials and Methods Detection of RNA and proteins in different mouse tissues
mRNA levels were detected in total RNA isolated from different
mouse tissues using RT-PCR and standard conditions. RT-PCR
products were analysed both by normal agarose gel electrophoresis
(not shown) and capillary gel electrophoresis [62,63]. Sfrs10
primers were specific to sequences in exons 1 and 4 respectively
(59-GAGCTCCTCGCAAAAGTGTG-39
and
59-CAACAT-
GACGCCTTCGAGTA-39). Tra2b protein was detected using
immunohistochemistry in the mouse brain as previously described
[64] and in the mouse testis using Abcam polyclonal Tra2b
antibody ab31353 [28] as previously described [26]. p
p
g
g
We also found that full lengthTra2b protein activates splicing of
the Nasp-T exon at a lower level through its RS1 and RS2 domains
only (i.e. without the RRM and so without direct RNA binding). Mechanistically the RS domains of Tra2b might activate splicing
by
helping
assemble
other
RS-domain
containing
splicing
regulators and components of the spliceosome into functional
splicing complexes. Although both RS domains could co-activate
splicing when present together, removal of the RS1 domain
completely disabled Tra2b-mediated splicing activation of the
physiological target exons identified here. The observed functional
importance of RS1 provides a mechanistic explanation why this
N-terminal RS domain structure is maintained for Tra2 proteins
in
both
vertebrates
and
invertebrates. Surprisingly
Tra2b
molecules without the RS1 domain were not just neutral for
splicing inclusion in cellulo, but for some exons actually functioned
as potent splicing repressors. Since the Tra2bDRS1 isoform
contains a functional RRM sequence, splicing repression could be
due to competitive inhibition through this shorter Tra2b protein
binding to the same RNA targets, but then being unable to
assemble functional splicing complexes with other Tra2b proteins
in the absence of the RS1 domain. Detection of such a competitive
inhibitory function might have been helped by the increased levels
of the Tra2bDRS1 isoform expressed in our experiments. In vivo,
the Tra2b-3 protein which lacks the N-terminal RS1 domain
might also operate as a natural splicing repressor isoform
[20,21,22], depending on its level of expression being enough in
specific cell types or tissues. Tra2bDRS1 actually activates SMN2
exon 7 rather than being a repressor as seen for the physiological
target exons we describe in this report [17]. Discussion In addition, although
a high frequency of alternative splicing events in the testis are
species-specific [61], the high conservation of binding sites in the
Tra2a poison-exon suggests regulation by Tra2b has been
conserved since the radiation of vertebrates. Overall our data
indicate maintenance of ancient patterns of splicing regulation
controlled by this RNA binding protein, consistent with its
observed key role in development [19]. A model of splicing activation for the Nasp-T and the Tra2a
poison exon which depends largely on sole binding of Tra2b
protein might explain why these exons are particularly sensitive to
depletion of Tra2b in vivo compared with SMN2 exon 7 (splicing of
which is not affected after deletion of Sfrs10, and which has a single
Tra2b binding site, Figure S1). The human testis-specific HIPK3-
T exon [50] also requires multiple Tra2b binding sites to enable
splicing activation of a weak 59 splice site in vitro [28], and the
Sfrs10 poison exon also has multiple Tra2b binding sites [22]. Other than Tra2a and Nasp-T, the remaining target exons we
analysed using minigenes here have less dense coverage of Tra2b
binding sites (Figure S1). These remaining exons also responded
less robustly to Tra2b protein expression in vitro in transfected cells,
and it is likely that RNA binding proteins other than Tra2b might
also be more important for their splicing regulation in vivo. Materials and Methods Although the biology
of SMN2 exon 7 has been an area of controversy in the literature
[59,60], a possible mechanistic explanation for this difference
might be if Tra2b binding to SMN2 exon 7 blocked the action of
an adjacent Exonic Splicing Silencer, rather than directly
activating splicing by itself. Different Tra2a mRNA isoforms mRNA were detected by
multiplex RT-PCR using Tra2aF (59-GTTGTAGCCGTCGC-
CTTC T-39), Tra2aB (59-TGGGATTCAGAATGTTTGGA-39)
and Tra2a poison (59-TTCAAGTGCTTCTATCTGACCAA-39). Different Nasp-T mRNA isoforms were detected by RT-PCR
using Nasp-TF (59-AATGGAGTGTTGGGAAATGC-39), Nasp-
TB (59-TTGGTGTTTCTTCAGCCTTG-39) and Nasp-TC (59-
TGCTTTGAAGTCGGTTCAACT-39). Hprt
expression
was
detected
using
primers
HrptF
(59-
CCTGCTGGATTACATTAAAGCACTG-39) and HprtR (59-
GTCAAGGGCATATCCAACAACAAAC-39). Hprt
expression
was
detected
using
primers
HrptF
(59-
CCTGCTGGATTACATTAAAGCACTG-39) and HprtR (59-
GTCAAGGGCATATCCAACAACAAAC-39). PLoS Genetics | www.plosgenetics.org HITS-CLIP For the poison
exon-containing isoform we used the same forward oligonucleo-
tide as for the functional isoform and 59-CTTGATTTATCTTC-
CACATTCTTGG-39 (reverse) at 3 mM MgCl2 and 64uC
annealing producing a 206 bp amplicon. All quantification data
was normalized against Gapdh. Amplification was performed using
the
oligonucleotides
59-GGCTGCCCAGAACATCATCC-39
(forward)
and
59-GTCATCATACTTGGCAGGTTTCTC-39
(reverse) at 3 mM MgCl2 and 63uC annealing producing a
169 bp amplicon. Agarose gel electrophoresis and basic melting
curve analysis was performed to confirm PCR product specificity. For quantification a dilution series of cDNA was used to generate a
standard curve for each isoform. Therefore the cycle threshold was
plotted versus the logarithm of the concentration and the standard
curve was determined by linear regression. This curve was then
utilized to calculate the template concentration of unknown
samples. All samples were measured in duplicates. Individuals of a
genotype were averaged using the arithmetic mean. Fluctuations
are displayed by the standard error of the mean, and these are
indicated on the bar charts by error bars. The significance of
differences between genotypes was verified using student’s t-test. GAGACTCTCTGCCCTCGAAG-39 (reverse) at 3 mM MgCl2
and 66uC annealing resulting in a 155 bp product. For the poison
exon-containing isoform we used the same forward oligonucleo-
tide as for the functional isoform and 59-CTTGATTTATCTTC-
CACATTCTTGG-39 (reverse) at 3 mM MgCl2 and 64uC
annealing producing a 206 bp amplicon. All quantification data
was normalized against Gapdh. Amplification was performed using
the
oligonucleotides
59-GGCTGCCCAGAACATCATCC-39
(forward)
and
59-GTCATCATACTTGGCAGGTTTCTC-39
(reverse) at 3 mM MgCl2 and 63uC annealing producing a
169 bp amplicon. Agarose gel electrophoresis and basic melting
curve analysis was performed to confirm PCR product specificity. For quantification a dilution series of cDNA was used to generate a
standard curve for each isoform. Therefore the cycle threshold was
plotted versus the logarithm of the concentration and the standard
curve was determined by linear regression. This curve was then
utilized to calculate the template concentration of unknown
samples. All samples were measured in duplicates. Individuals of a
genotype were averaged using the arithmetic mean. Fluctuations
are displayed by the standard error of the mean, and these are
indicated on the bar charts by error bars. The significance of
differences between genotypes was verified using student’s t-test. by subtracting the background (genome and transcriptome
frequencies respectively) from the signal (frequency in CLIP
dataset). CLIP
reads
were
filtered
to
remove
duplicates
including overlapping reads. HITS-CLIP Statistical significance was deter-
mined using a Chi-squared test. The weblogo was derived from
tags containing a GAA sequence by analysing the sequence
composition surrounding the fixed sequence, using custom
written scripts to generate an input for the freely available
program weblogo (http://weblogo.berkeley.edu/). Minigene splicing experiments Candidate alternatively spliced exons identified by HITS-CLIP
and approximately 240 nucleotides of intronic flanking region at
each end were amplified from mouse genomic DNA with the
primer sequences given below. PCR products were digested with
the appropriate restriction enzyme and cloned into the Mfe1 site in
pXJ41 [68], which is exactly midway through the 757 nucleotide
rabbit b-globin intron 2. PCR products were made using the
following primers: Generation of neuronal specific Sfrs10 knock-out mice for
in vivo splicing analysis In our in vivo splicing study we utilized a previously established
Sfrs10 mouse model on pure C57BL/6 background as described
[19]. Genotyping was performed using tail DNA according to
established protocols [19]. To induce a conditional Sfrs10 knock-
out in the central nervous system we crossbred Sfrs10fl/fl mice with
a Nestin-Cretg/+ mouse line. These mice express Cre recombinase
under control of the rat nestin (Nes) promoter and enhancer [47]. Therefore Cre recombinase is expressed in neuronal and glia cell
precursors from embryonic day 11 as well as in neurogenic areas
of the adult brain [47,67]. For our analyses the presence of the
Nestin transgene was determined by a standard PCR using the
oligonucleotides 59–CGCTTCCGCTGGGTCACTGTCG-39 (for-
ward) and 59–TCGTTGCATCGACCGGTAATGCAGGC-39 (re-
verse) at an annealing temperature of 58uC producing a 300 bp
amplicon. HITS-CLIP HITS-CLIP was performed as previously described [30] using
an antibody specific to Tra2b [65]. The specificity of the antibody
to Tra2b was confirmed by the experiment shown in Figure S3, as
well as the additional characterization already described [65]. In
short, for the CLIP analysis mouse testis was sheared in PBS and
UV crosslinked. After lysis, the whole lysate was treated with
DNase and RNase, followed by radiolabelling and linker ligation. After immunoprecipitation with purified antisera specific to Tra2b
[65], RNA bound Tra2b was separated on SDS-PAGE. A thin
band at the size of 55 kDa (Tra2b migrates at around 40 kDa and
MW of 50 nt RNA is about 15 kDa) was cut out and subject to
protein digestion. RNA was recovered and subject to sequencing
which was carried out on the Newcastle University Roche 454 GS-
FLX platform. Mapping was done with Bowtie [66], allowing for
two mismatches (parameter –v 2). 297070 reads were processed, of
which 177457 (59.74%) aligned successfully to the mouse genome
(Mm9). 74476 (25.07%) failed to align, and 45137 (15.19%) reads
were suppressed due to multiple hits on the mouse genome. K-mer
analysis was carried out using custom written scripts in Python. Briefly, we calculated the frequency of occurance of each possible
6-mer sequence in the following: our CLIP dataset, the mouse
genome (mm9) and in the mouse testis transcriptome (http://www. ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSM475281). The
genome and transcriptome corrected frequencies were obtained Our analysis shows that the RNA targets identified for Tra2b in
developing adult germ cells can predict patterns of splicing
regulation by Tra2b in the developing brain. However, our data
further suggest that splicing regulation by Tra2b is temporally
restricted during development and also differentially regulated
between various Tra2b targets. This is highlighted by Tra2a
poison-exon splicing, which is affected by neuronal specific Sfrs10
knockout only at a defined developmental stage, while Nasp-T
exon inclusion is perturbed by Sfrs10 knockout in all analyzed
situations. Both the Nasp-T and the Tra2a poison exon are
biologically important: they are conserved in all vertebrates for December 2011 | Volume 7 | Issue 12 | e1002390 15 New Roles and Splicing Targets for Tra2b GAGACTCTCTGCCCTCGAAG-39 (reverse) at 3 mM MgCl2
and 66uC annealing resulting in a 155 bp product. Quantitative analysis of Sfrs10 expression and Tra2b
targeted transcripts g
p
Whole brain RNA was isolated from 16.5 dpc, 18.5 dpc and
PND1 mice using the RNeasy Lipid Tissue Mini Kit (Qiagen,
Hilden, Germany). RNA concentration was determined by
Quant-iT
RiboGreen
RNA
Reagent
and
Kit
(Invitrogen,
Darmstadt, Germany) and equal amounts of RNA were used for
first strand cDNA synthesis utilizing the QuantiTect reverse
Transcription Kit (Qiagen, Hilden, Germany). Quantitative real-
time PCR was carried out using the Roche LC FastStart DNA
Master SYBR green Kit (Roche, Mannheim, Germany) on the
Roche LightCycler 1.5. For realtime quantification total Sfrs10
transcripts were amplified using the oligonucleotides 59-TA-
GAAGGCATTATACAAG-39 (forward) and 599-CTCAACC-
CAAACACGC-39 (reverse) at 3 mM MgCl2 and an annealing
temperature of 63uC producing a 186 bp bp amplicon. To
quantify Sfrs10 isoforms specifically we used the oligonucleotides
59-AGAACTACGGCGAGCGGGAATC-39 (forward) and 59-
CCTTGTATAATGCCTTCTAGAACTTCTTC-39 (reverse) for
the functional isoform and 59-GAACTACGGCGAGCGGGT-
TAATG-39 (forward) and 59-CAAGTGGGACTTCTGGTCT-
GATAATTAGC-39 (reverse) for the non-functional isoform. Both
were run at annealing temperatures of 64uC resulting in amplicons
of 191 bp and 161 bp, respectively. For the quantification of
different target splice variants single isoforms were amplified
separately. For the Nasp-T exon containing isoform the oligonu-
cleotides 59-GGAGTGCATGTAGAAGAGG-39 (forward) and
59-CGTCATAAACCTGTTCTCTC-39 (reverse) were used at
1 mM MgCl2 and annealing at 65uC producing a 115 bp
amplicon. The somatic isoform of Nasp was amplified using 59-
AATGGAGTGTTGGGAAATGC-39 (forward) and 59-CTG-
AGCCTTCAGTTTCATCTAC-39 (reverse) at 3 mM MgCl2,
62uC annealing while producing a product of 118 bp length. The
functional Tra2a transcript was amplified using the oligonucleo-
tides 59-GTTGTAGCCGTCGCCTTCT-39 (forward) and 59- g p
Krba1L: 59-AAAAAAAAGAATTCtggggatcctagcaggtaca -39 Krba1R: 59-AAAAAAAAGAATTCccaaggatgtgataagcagga -39 CREB2U: 59-AAAAAAAACAATTGgggaccattcctcatttcct -39 CREB2D: 59-AAAAAAAACAATTGaaggcagttgtcatcattgc -39 LIN28F: 59-AAAAAAAAGAATTCccagcctggtctttaagagagt -39 LIN28B: 59-AAAAAAAAGAATTCcatacagtgaattatttgaaaacacc
-39 LIN28B: 59-AAAAAAAAGAATTCcatacagtgaattatttgaaaacacc
39 -39 Acknowledgments We acknowledge Lilian Martinez who technically assisted and helped
Markus Storbeck and Miriam Jakubik. We thank Jernej UIe (MRC
Laboratory for Molecular Biology) for his kind advice, Bill Mattox for the
gift of the Tra2a cDNA, Philippa Saunders for advice on immunohisto-
chemistry, and Darren Hoyland for loading the Tra2b BED file so it can be
downloaded as a hub from the Newcastle University server. Figure S1
Sequence of all the exons analysed using minigenes
and some known Tra2b target exons. The Tra2b binding sites
predicted from the k-mer analysis are coloured as indicated in
Table S1. Figure S1
Sequence of all the exons analysed using minigenes
and some known Tra2b target exons. The Tra2b binding sites
predicted from the k-mer analysis are coloured as indicated in
Table S1. (
)
DOC Supporting Information Table S3
Top functions associated with Tra2b-bound mRNAs
determined from Ingenuity Pathway Analysis (IPA). (DOCX) Dataset S1
BED file containing the Tra2b CLIP tag sequences and
their location in the mouse genome (mm9). This bed file can be saved
and added as an optional track on the UCSC mouse genome browser
(http://genome.ucsc.edu/). To load this BED file on the UCSC
genome browser, use the ‘‘manage custom tracks’’ button under
genomes. Alternatively, the bed file can be visualised by up loading the
link http://research.ncl.ac.uk/ElliottGroup/UCSC/hub.txt into the
My Hubs textbox in the UCSC Track Hubs menu. (TIF) Tra2aGSR
59-AAAAAAAAGAATTCTCATTAGCCTTCT-
TTTATCTTGATTTA-39 Table S1
Properties of the 30 most frequently retrieved 6-mers
in the Tra2b CLIP tags. The 6-mers are ordered from the most
frequently recovered at the top of the table (AGAAGA) to the 34th
most frequently recovered 6-mer at the bottom (GAAGCT). The
6-mers are arranged in colour blocks of 5 according to their
frequency of retrieval, and compared and corrected with their
frequencies in both the total mouse genome and mouse testis
transcriptome. The same colour code of the different 6-mer
categories are also used to illustrate the occurrence of these 6-mers
within the Tra2b target exons in Figures S1 and S2. (DOC) Lin28GSF 59-AAAAAAAAGGTACCCTTGAACTCTCTGA-
TTTTAGGTTCTTC-39 Lin28GSR 59-AAAAAAAAGAATTCAACAGACTAACCTG-
GGGCTGA-39 CrebcF 59-AAAAAAGGTACCTCATTGTTCTAGGTGCT-
ATCAAAGG-39 CrebcR 59-AAAAAAGAATTCCTGACATATTTTATTTT-
9 CTCATAGTAT GTCTCTC-39
Creb2F 59-AAAAAAGGTACCGTAACTAAATGACCATG- Creb2F 59-AAAAAAGGTACCGTAACTAAATGACCATG-
GAATCTGGAGCA-39 Creb2F 5 -AAAAAAGGTACCGTAACTAAATGACCATG-
GAATCTGGAGCA-39 GAATCTGGAGCA-39
Creb2R 59-AAAAAAGAATTCCTGGGCTAATGTGGCAA-
TCTGTGG-39 Table S2
List and properties of all 6-mers recovered by Tra2b
CLIP above background levels. (XLSX) Table S2
List and properties of all 6-mers recovered by Tra2b
CLIP above background levels. PankF: 59-AAAAAAAAGAATTCcacatctgtgggtgcacttt -39
PANKR: 59-AAAAAAAAGAATTCttcaaaggactatttggttaacagc -
9 PankF: 59-AAAAAAAAGAATTCcacatctgtgggtgcacttt -39
PANKR: 59-AAAAAAAAGAATTCttcaaaggactatttggttaacagc -
39 FABP9F 59-AAAAAAAACAATTGtggcattcctttctcacctt -39 gg
FABP9R 59-AAAAAAAACAATTGgagccttcctgtgtgggtat -39 CREBGammaF:
59-AAAAAAAACAATTGcaaacttctagatggta-
gaatgatagc -39 CREBGammaR: 59-AAAAAAAACAATTGtagccagagaacggaac-
cac -39 TF: 59-AAAAAAAACAATTGtccttggaggacttctgttttc-39 NaspTR: 59-AAAAAAAACAATTGggcatgcctgcttaagtgta-39 Tra2aF: 59-AAAAAAAAGAATTCattagggactaggatggaacatga - ESE mutations within Nasp-T were made by overlap PCR with
the additional primers NASPM1-S (59-GGGTGGACGATAA-
GACAT GG-39) and its complementary primer (59-CCATG-
TCTTATCGTCCAC CC-39); NASPM2-S (59-GTGAGCCT-
CAAGAGTAGCTCC-39) and its complementary primer 59-
GGAGCTACTCTTGAGGCTCAC-39; NASPM3-S (59-GAAT-
CCTCTGCATAGGCAAAAG-39) and its complementary primer
(59-CTTTTGCCTATGCAGAGGATT C-39); NASPM4-S (59-
GGACTGACTCAAGTTGAGGTCGC-39) and its complemen-
tary primer (59-GCGACCTCAACTTGAGTCAGTCC-39). PLoS Genetics | www.plosgenetics.org December 2011 | Volume 7 | Issue 12 | e1002390 16 New Roles and Splicing Targets for Tra2b Analysis of splicing of pre-mRNAs transcribed from minigenes
was carried out in HEK293 cells as previously described using
primers within the b-globin exons of pXJ41 [29]. Because of the
length of the regulated exons, additional internal primers were
included in multiplex to detect inclusion of the Nasp-T cassette
exon (59-TGCTTTGAAGTCGGTTCAACT-39) and Tra2a poi-
son exon (59-TTCAAGTGCTTCTATCTGACCAA-39). the Tra2a poison exon from mouse. (B) Sequence of Nasp-T exon
from mouse. Nucleotides in red are conserved in all vertebrates
analysed (mouse, frog, rabbit, human, rat, cow, orang-utan, chimp,
macaque, marmoset, guinea pig, dog, horse, elephant, opossum,
lizard,
zebrafinch,
tetraodon,
stickleback,
medaka,
chicken). Nucleotides conserved in all mammals are shown in blue. All other
nucleotides are shown in black. The Tra2b binding sites predicted
from the k-mer analysis are shaded as indicated in Table S1, and the
positions of CLIP tags are underlined (note that some of these
underlined regions correspond to multiple overlapping CLIP tags
which have been joined in this figure). (
)
DOC the Tra2a poison exon from mouse. (B) Sequence of Nasp-T exon
from mouse. Nucleotides in red are conserved in all vertebrates
analysed (mouse, frog, rabbit, human, rat, cow, orang-utan, chimp,
macaque, marmoset, guinea pig, dog, horse, elephant, opossum,
lizard,
zebrafinch,
tetraodon,
stickleback,
medaka,
chicken). Nucleotides conserved in all mammals are shown in blue. All other
nucleotides are shown in black. The Tra2b binding sites predicted
from the k-mer analysis are shaded as indicated in Table S1, and the
positions of CLIP tags are underlined (note that some of these
underlined regions correspond to multiple overlapping CLIP tags
which have been joined in this figure). Author Contributions Conceived and designed the experiments: DJE SG YL BW MS. Performed
the experiments: MS CD YL AB IE KR. Analyzed the data: DJE SG CD
MS AB YL TC CFB JS DG MSJ BW. Contributed reagents/materials/
analysis tools: MJ YM. Wrote the paper: SG DJE. Figure S2
Multiple Tra2b binding sites are phylogenetically
conserved in Tra2a poison exons and Nasp-T exons. (A) Sequence of 3. Stamm S (2002) Signals and their transduction pathways regulating alternative
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Experiment to confirm the specificity of the polyclonal
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10: R25. PLoS Genetics | www.plosgenetics.org December 2011 | Volume 7 | Issue 12 | e1002390 PLoS Genetics | www.plosgenetics.org 19
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English
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Development, validation and application of a Brazilian knowledge scale about sports doping in dentistry
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Brazilian Oral Research
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cc-by
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Original Research
Social/
Community Dentistry Original Research
Social/
Community Dentistry Lucas Alves JURAL(a)
Thais Rodrigues Campos SOARES(a)
Raildo da Silva COQUEIRO(b)
Tiago Braga RABELLO(c)
Matheus Melo PITHON(a)
Lucianne Cople MAIA(a) Lucas Alves JURAL(a)
Thais Rodrigues Campos SOARES(a)
Raildo da Silva COQUEIRO(b)
Tiago Braga RABELLO(c)
Matheus Melo PITHON(a)
Lucianne Cople MAIA(a) Abstract: To evaluate the knowledge of Brazilian dentists of sports
doping through the development, validation and application of
the Brazilian Knowledge Scale about Sports Doping in Dentistry
(B-KSSDD). A scale with 12 items was developed to assess a dentist’s
ability to determine whether the use of a medication characterised
sports doping according to the World Anti-Doping Agency. A
preliminary study to validate the B-KSSDD was carried out with
135 dentists, allowing the evaluation of ceiling and floor effects,
convergent and discriminant validity, test-retest reliability and
internal consistency of the instrument. A sample size calculation
using the results of the preliminary study and the B-KSSDD was
completed online using SurveyMonkey® by 270 participants from all
regions of the country. The B-KSSDD showed evidence of convergent
and discriminant validity, good temporal stability (ICC = 0.75) and
internal consistency (alpha = 0.89). In the main study, the participants
obtained an average score of 4.19/12 points on the B-KSSDD, suggesting
that these professionals have insufficient knowledge about sports
doping. The age of participants showed a negative association with
knowledge about doping, while frequency of treating athletes and
frequency of performing surgeries showed positive associations with
knowledge about doping. The dentists had insufficient knowledge
of the subject. Age of participants and frequency with which they
attend to athletes are associated with knowledge about sports doping. Professional updating and education policies on doping are necessary
for dentists, as athlete patients are at risk for severe sporting and
financial penalties. (a) Universidade Federal do Rio de Janeiro –
UFRJ, School of Dentistry, Department of
Pediatric Dentistry and Orthodontics, Rio de
Janeiro, RJ, Brazil. (b) Universidade Estadual do Sudoeste da
Bahia – UESB, Center for Studies in Aging,
Jequié, BA, Brazil. (c) Universidade Federal do Rio de Janeiro –
UFRJ, School of Dentistry, Department of
Dental Clinic, Rio de Janeiro, RJ, Brazil. Corresponding Author:
Lucianne Cople Maia
E-mail: rorefa@terra.com.br Keywords: Pharmaceutical Preparations; Dentists; Educational
Measurement; Sports Medicine; Doping in Sports. Keywords: Pharmaceutical Preparations; Dentists; Educational
Measurement; Sports Medicine; Doping in Sports. Declaration of Interests: The authors
certify that they have no commercial or
associative interest that represents a conflict
of interest in connection with the manuscript. (c) Universidade Federal do Rio de Janeiro –
UFRJ, School of Dentistry, Department of
Dental Clinic, Rio de Janeiro, RJ, Brazil. Development, validation and
application of a Brazilian knowledge
scale about sports doping in dentistry Lucas Alves JURAL(a)
Thais Rodrigues Campos SOARES(a)
Raildo da Silva COQUEIRO(b)
Tiago Braga RABELLO(c)
Matheus Melo PITHON(a)
Lucianne Cople MAIA(a) Corresponding Author:
Lucianne Cople Maia
E-mail: rorefa@terra.com.br Study design and participants This cross-sectional study included Brazilian
dentists of both genders from all regions of the country,
including dentists with professional registration
at their respective regional dentistry councils. Participants in the preliminary and main studies
answered an electronic questionnaire, hosted by
SurveyMonkey Inc. (San Mateo, USA), that tested
the professionals’ knowledge about medicines that,
when used by athletes, do or do not characterise
sports doping. In addition to the Code, which was last published
in 2015, the WADA annually publishes an updated
list of prohibited substances that can potentially be
used for doping in athletes, since they can induce an
artificial increase in athletes’ sports performance. The Code also addresses the sanctions applicable to
athletes, teams or even sports entities, ranging from
the loss of medals, points and prizes to financial
consequences.4,5 Participants in the preliminary and main studies
answered an electronic questionnaire, hosted by
SurveyMonkey Inc. (San Mateo, USA), that tested
the professionals’ knowledge about medicines that,
when used by athletes, do or do not characterise
sports doping. The document updated annually by the
WADA includes substances belonging to different
therapeutic classes, such as anabolic agents,
hormones and metabolic modulators, stimulants
and glucocorticoids, that can be banned both
during and outside the competition period.5 Many
of the drugs included in this list can be purchased
in pharmacies over the counter or by prescription
from health professionals such as doctors and
dentists, who may inadvertently prescribe their
use. Sometimes dental care may require the dentist
to prescribe medications such as glucocorticoids
and stimulants that, when used by athletes, can be
characterised as sports doping, making the athlete
susceptible to serious penalties.4,6,7 For this reason,
it is essential that dentists are knowledgeable about
sports doping and are prepared to assist athletes
without putting them at risk of positive results in
doping tests. The questionnaire consisted of questions on the
participants’ sociodemographic and professional
information; a question on self-assessment of their
knowledge about sports doping, ranging from 1
(none) to 5 (excellent); and the B-KSSDD in Brazilian
Portuguese. Methodology the International Federation of Sports Medicine
defined sports doping as the act of an athlete
intentionally or unintentionally using substances or
methods prohibited by the International Olympic
Committee (IOC).3 In 2002, the World Anti-Doping
Agency (WADA) published the World Anti-Doping
Code (Code)4, which determined doping control
guidelines and was adopted by more than 660
sports entities, including the IOC, the International
Paralympic Committee, international federations
and national anti-doping organisations. The study was conducted in accordance with
the 1964 Helsinki Declaration and approved by the
Research Ethics Committee of Clementino Fraga
Filho University Hospital, at Federal University of
Rio de Janeiro (protocol nº. 3.940.568). All participants
signed informed consent forms in order to take part
in the study. Preliminary study: development and
validation of the B-KSSDD Before beginning the main study, the questionnaire
was developed and validated in six steps: a) elaboration
of the questions; b) specialised technical evaluation;
c) general evaluation by dentists; d) evaluation of
linguistics and comprehensibility; e) finalisation of
the B-KSSDD; and f) assessment of the validity and
reliability of the B-KSSDD (test-retest). Introduction https://doi.org/10.1590/1807-3107bor-2021.vol35.0110 The term doping was formally defined in 1889, when an English
dictionary described it as a mixture of opium and narcotics in horses.1
However, the use of doping substances in horses and athletes arose in
the Roman period, when they were used to increase the capacity and
strength of these animals and gladiator athletes as the prestige of sports
grew.2 With the evolution of sports and the advent of sports medicine, Submitted: October 29, 2020
Accepted for publication: March 29, 2021
Last revision: April 27, 2021 Submitted: October 29, 2020
Accepted for publication: March 29, 2021
Last revision: April 27, 2021 1
Braz. Oral Res. 2021;35:e110 1 Braz. Oral Res. 2021;35:e110 Braz. Oral Res. 2021;35:e110 Development, validation and application of a Brazilian knowledge scale about sports doping in dentistry Linguistic and comprehensibility
evaluation The questions were submitted to a journalist
and an educator, who evaluated the linguistic and
grammatical aspects of the B-KSSDD. General evaluation by dentists Ten dentists (three oral and maxillofacial surgeons,
one stomatologist, one periodontist, one orthodontist,
one restorative dentist, two paediatric dentists
and one general dentist) evaluated the clarity and
comprehensibility of the B-KSSDD. Elaboration of the questions In view of the lack of studies assessing the
knowledge of dentists about sports doping and
substances forbidden for athletes, the objective of this
study was to report the development, validation and
application of the Brazilian Knowledge Scale about
Sports Doping in Dentistry (B-KSSDD). One graduate student in dentistry, one endodontist,
one paediatric dentist and one orthodontist consulted
the Code and Prohibited List4,5 and investigated
the absence or presence of drugs with potential
use in dental practice (such as non-steroidal anti-
inflammatory, glucocorticoids and analgesics). The scale’s questions were closed and contained Braz. Oral Res. 2021;35:e110 2 Jural LA, Soares TRC, Coqueiro RS, Rabello TB, Pithon MM, Maia LC range from 0 (no correct answers/complete lack
of knowledge) to 12 points (complete knowledge). As the professionals were Brazilian, the questions
were provided in Portuguese (Table 1). medications with potential or not to cause doping
according to WADA documents4,5 and adopted the
following model: The use of [DRUG AND ADMINISTRATION] (for
example, [COMMERCIAL NAME]), prescribed by a
dental surgeon to an athlete who is in competition,
characterises sports doping. Finalisation of the Brazilian Knowledge
Scale about Sports Doping in Dentistry
(B-KSSDD) The considerations and suggestions made by
the evaluators were analysed and incorporated
into the scale. The final version of the scale has
12 objective questions that evaluate the dentists’
knowledge about the substances forbidden for use
by athletes subject to doping control, as determined
by the WADA. Each question offers three answer
options: ‘agree’, ‘disagree’ or ‘don’t know’. For
each correct answer, 1 point was added to the
participant’s final score, while for each incorrect
answer or ‘don’t know’, 0 points were added to the
score. The final score of each participant could The level of significance adopted in all analyses
was 5% (α = 0.05). Data were tabulated and analysed
using IBM SPSS Statistics for Windows (IBM SPSS
21.0, 2012, IBM Corp., Armonk, USA). Specialised technical evaluation The questions were submitted to a dentist
specialised in Sports Dentistry. This professional
was asked whether the scale effectively assessed
the dentist’s knowledge about sports doping. After
the professional’s evaluation, his suggestions were
incorporated or modified in the instrument. The validation analysis assessed the following
data: floor and ceiling effects, by analysing the
frequency of responses for each total score of the
scale;8 convergent construct validity, by assessing the
correlation between the B-KSSDD score and the self-
reported knowledge score on sports doping using
Spearman’s correlation (correlation coefficients <
0.4 = weak magnitude correlation, ≥ 0.4 to < 0.5 =
moderate magnitude and ≥ 0.5 = strong magnitude);9
discriminant construct validity, by comparing the
B-KSSDD score between groups of dentists who
perform surgeries with lesser or greater frequency
using the Mann–Whitney test; reliability, evaluated
according to the criteria of temporal stability
(agreement between repeated test-retest measures);
and internal consistency. The intraclass correlation
coefficient (ICC) was calculated for the total score
and Cronbach’s alpha was calculated for the total
scale and for the scale with items deleted, together
with the item-total correlation coefficients. An ICC
≥ 0.70 and a Cronbach’s alpha between 0.70 and 0.95
were considered evidence of reliability.8 Validity and reliability of the questionnaire The questionnaire test was given to 135 general
dentists or specialists (from 16 different specialties),
and 15 days later, 50 of these professionals
were randomised using BioEstat 5.3 (Instituto
Mamirauá, Belém, Brazil) to complete a retest of
the questionnaire. These professionals were not
included in the main study. Answer: Agree / Disagree / Don’t know Main study Sample size calculation and application Sample size calculation considered the following
parameters: effect size = 0.10, α = 0.05, power = 0.95
and number of predictors = 9. The effect size was 3 Braz. Oral Res. 2021;35:e110 Development, validation and application of a Brazilian knowledge scale about sports doping in dentistry Table 1. Original and validated final version of the Brazilian Knowledge Scale about Sports Doping in Dentistry (B-KSSDD) and
scores added to the total score according to the participants’ responses. Leia as sentenças a seguir e informe sua concordância, discordância ou ausência de
conhecimento sobre a afirmação apresentada:
Concordo
Discordo
Não sei
1. A utilização de nimesulida via oral (por exemplo, Nisulid®, Arflex®), prescrita por
um cirurgião-dentista a um atleta que está em período de competição, caracteriza
doping esportivo. 0
1
0
2. A utilização de dexametasona via oral (por exemplo, Decadron), prescrita por
um cirurgião-dentista a um atleta que está em período de competição, caracteriza
doping esportivo. 1
0
0
3. A utilização de dipirona via oral (por exemplo, Novalgina®, Dorflex®,
Magnopirol®), prescrita por um cirurgião-dentista a um atleta que está em período
de competição, caracteriza doping esportivo. 0
1
0
4. A utilização de triancinolona por via tópica (por exemplo, Omcilon®), prescrita
por um cirurgião-dentista a um atleta que está em período de competição,
caracteriza doping esportivo. 0
1
0
5. A utilização de anestésico local com adrenalina ou epinefrina (por exemplo,
Aplhacaine) administrado por um cirurgião-dentista em um atleta que está em
período de competição caracteriza doping esportivo. 0
1
0
6. A utilização de associação de dipirona com mucato de isometepteno via oral (por
exemplo, na Neosaldina®), prescrita por um cirurgião-dentista a um atleta que está
em período de competição, caracteriza doping esportivo. 1
0
0
7. A utilização de prednisolona via oral (por exemplo, Predsim®), prescrita por
um cirurgião-dentista a um atleta que está em período de competição, caracteriza
doping esportivo. 1
0
0
8. A utilização de amoxicilina via oral (por exemplo, no Amoxil®), prescrita por
um cirurgião-dentista a um atleta que está em período de competição, caracteriza
doping esportivo. 0
1
0
9. A utilização de paracetamol via oral (por exemplo, Tylenol®), prescrita por um
cirurgião-dentista a um atleta que está em período de competição, caracteriza
doping esportivo. 0
1
0
10. Braz. Oral Res. 2021;35:e110 Main study A utilização de associação de codeína com paracetamol via oral (por exemplo,
Tylex®), prescrita por um cirurgião-dentista a um atleta que está em período de
competição, caracteriza doping esportivo. 0
1
0
11. A utilização de ibuprofeno via oral (por exemplo, Alivium®), prescrita por um
cirurgião-dentista a um atleta que está em período de competição, caracteriza
doping esportivo. 0
1
0
12. A utilização de betametasona via oral (por exemplo, Celestone®), prescrita por
um cirurgião-dentista a um atleta que está em período de competição, caracteriza
doping esportivo. 1
0
0
Escore total da B-KSSDD:
(0 a 12 pontos)
Respostas corretas são representadas por 1 enquanto as incorretas ou “não sei” são representadas por 0 pontos no escore final da B-KSSDD Escore total da B-KSSDD: Table 2. Distribution of dentists in each region of the country
(Federal Council of Dentistry, 2020).10
Country region
n
%
North
19,091
6
Northeast
56,112
17
Midwest
29,792
9
South
54,538
16
Southeast
173,63
52
Total
333,163
100
The regions of the participants included in the study sample were
stratified based on the percentage of dentists registered with the
Federal Dentistry Councils of these regions. Table 2. Distribution of dentists in each region of the country
(Federal Council of Dentistry, 2020).10 defined through preliminary analysis using data
from the 135 individuals who participated in the test
phase of the scale. A minimum sample size of 245
individuals was estimated, and with 10% added to
compensate for possible losses, the total number of
participants was 270 dentists. The sample size was
calculated using G * Power (Version 3.1.9.2, Germany). Respecting proportionality to the percentage of
dentists distributed in each region of the country10
(Table 2), general dentists or specialists were asked
to answer the validated scale. The professionals 4 Jural LA, Soares TRC, Coqueiro RS, Rabello TB, Pithon MM, Maia LC 0.87 to 0.89. The B-KSSDD scores for the test-retest
were 4.30 (SD = 3.08) and 4.80 (SD = 2.84), respectively,
and the ICC was 0.75 (p < 0.001; 95%CI: 0.56–0.86). were approached by personal contacts (co-workers,
WhatsApp groups, Facebook or e-mail).11 Statistical analysis Application of B-KSSDD in the main study Descriptive statistics were used to express the
results as relative and absolute frequencies, means,
standard deviations (SD), minimum and maximum
values and 95% confidence intervals (95%CI). Simple
and multiple linear regression analyses were conducted
to assess the relationship between sociodemographic
and professional characteristics and knowledge about
sports doping. The multiple model was built using the
backward method, in which all independent variables
were initially incorporated into the model and the
variables with the highest values of alpha (α) were
subsequently removed one by one until the minimum
value of 0.20 was reached. Thus, all independent
variables that reached α ≤ 0.20 were maintained
in the final model for purposes of adjustment. The
level of significance adopted in all analyses was 5%
(α = 0.05). Data were tabulated and analysed using
IBM SPSS Statistics for Windows (IBM SPSS 21.0,
2012, IBM Corp., Armonk, USA). The age of the 270 dentists participating in the
study ranged from 22 to 86 years (mean = 40.80;
SD = 12.88) and the stratification of dentists by
regions of the country was maintained in our sample
(Figure 2). Table 3 shows the sociodemographic and
professional characteristics. Most of the sample
was female, had more than 15 years of training,
had graduated from public institutions and had a
specialisation. In total, 22 specialties were reported
by the participants, including Sports Dentistry
(n = 4; 1.5%). The four most common specialties were
Orthodontics (n = 103; 38.1%), Paediatric Dentistry
(n = 36; 13.3%,), Periodontics and Implantology (both
with n = 21; 7.8%). Among the participants, 70% and
94.1% of professionals indicated a low frequency
(never, rarely or sometimes) of performing surgeries
and treating athletes, respectively. The average B-KSSDD score obtained by dentists
was 4.19 (SD = 3.19; 95%CI: 3.81–4.57), with 23%
scoring the minimum score (0), 74.4% scoring below
7 and only 0.4% reaching the maximum score (12). In addition, we found that, only among specialists
in Sports Dentistry, the average score presented was
9.25 (SD = 2.22; 95% CI: 5.72–12.0). Discussion After applying the multiple linear regression
backward method, only three variables (age, frequency
of surgery and frequency of treating athletes) remained
in the model for predicting knowledge about sports
doping, with the other variables (gender, service
region, geographic region within the State, time since
graduation, institution of graduation and specialisation)
were excluded because they did not reach the statistical
criterion of permanence in the model (p ≤ 0.20). Table
4 shows the results of simple and multiple linear
regressions for predicting knowledge about sports
doping in the study participants. The univariate
model showed that age was negatively associated with
B-KSSDD score. In contrast, frequency of performing
surgeries and frequency of treating athletes were
positively associated with B-KSSDD score. All these
associations were maintained in the multiple regression
model; therefore, the adjusted analysis indicated that
age (negative association) and frequency of performing
surgeries and frequency of treating athletes (positive
associations) were independent predictors of knowledge
about sports doping. B-KSSDD validation in preliminary study On a scale ranging from 1 (none) to 5 (excellent),
dentists’ self-reported knowledge about sports doping
obtained a mean score of 1.88 (SD = 0.77). A floor effect was observed, with a response
frequency of 23.7% for the minimum score; in contrast,
no ceiling effect was observed, as the response
frequency for the maximum score was only 1.5%. Figure 1. Analysis of the discriminant validity of the Brazilian
Knowledge Scale about Sports Doping in Dentistry (B-KSSDD). B-KSSDD SCORE
Lower frequency
Higher frequency
Frequency of surgery
*p-value = 0.018
12
9
6
3
0 B-KSSDD SCORE
Lower frequency
Higher frequency
Frequency of surgery
*p-value = 0.018
12
9
6
3
0 *p-value = 0.018 The convergent validity analysis showed a positive
and strong correlation (Rspearman = 0.57; p < 0.001)
between the B-KSSDD score and the self-reported
knowledge score on sports doping. The discriminant
validity showed a significant difference in the B-KSSDD
score between the groups of dentists who perform
surgeries with lower or higher frequency (Figure 1). Data obtained from the 135 scales completed
by dentists were analysed for internal consistency. Cronbach’s alpha for the total scale was 0.89. The
item-total correlation coefficients ranged from 0.47 to
0.72. The alpha values of deleted items ranged from Figure 1. Analysis of the discriminant validity of the Brazilian
Knowledge Scale about Sports Doping in Dentistry (B-KSSDD). 5 Braz. Oral Res. 2021;35:e110 Development, validation and application of a Brazilian knowledge scale about sports doping in dentistry * Mann–Whitney test. Figure 2. Distribution of sample’s dentists in each region of the country. NORTH
n = 15 (5.6%)
NORTHEAST
n = 45 (16.7%)
MIDWEST
n = 24 (8.8%)
SOUTH
n = 45 (16.7%)
SOUTHEAST
n = 141 (52.2%)
Lowest (15)
Higheast (141) MIDWEST
n = 24 (8.8%) SOUTHEAST
n = 141 (52.2%) * Mann–Whitney test. Figure 2. Distribution of sample’s dentists in each region of the country. ution of sample’s dentists in each region of the countr Braz. Oral Res. 2021;35:e110 * Mann–Whitney test. Validity and reliability of B-KSSDD –
preliminary study The validation of an instrument is a fundamental
step to ensure its accuracy in assessing the objectives
for which it is proposed to evaluate, and B-KSSDD
showed good reliability, as assessed using the criteria of
internal consistency and temporal stability (agreement
between test-retest). The fact that B-KSSDD has a floor
effect but does not have a ceiling effect suggests that
dentists have insufficient knowledge about sports
doping and the substances that characterise it, in
line with the low average score obtained for the
question that assessed the professionals’ self-reported
knowledge about sports doping. The B-KSSDD
demonstrated convergent validity, as a positive and
strong correlation was observed between the score
obtained in the B-KSSDD and the self-reported
knowledge score on sports doping, showing that
the scale is effective8 in assessing the knowledge
of dentists about the subject. In addition, the scale Braz. Oral Res. 2021;35:e110 6 6 Jural LA, Soares TRC, Coqueiro RS, Rabello TB, Pithon MM, Maia LC Table 3. Sociodemographic characteristics of the dentists
participating in the study. Variable
n
%
Gender
Female
166
61.5
Male
104
38.5
Service region
North/Northeast/Midwest
84
31.1
South/Southeast
186
68.9
Geographic region within the State
Capital
124
46.0
Inland
146
54.0
Time since graduation (years)
< 1
8
2.2
1–5
63
23.3
6–10
39
14.4
11–15
26
9.6
16–25
57
21.1
> 25
79
29.3
Graduation institution
Private
101
37.4
Public
169
62.6
Specialist
No
52
19.3
Yes
218
80.7
Frequency of surgery
Never
78
28.9
Rarely
57
21.1
Sometimes
54
20.0
Often
54
20.0
Always
27
10.0
Frequency of treating athletes
Never
42
15.6
Rarely
138
51.1
Sometimes
74
27.4
Often
12
4.4
Always
4
1.5
bl
ff
f h
l
d
l
l l Table 3. Sociodemographic characteristics of the dentists
participating in the study. presented good discriminating properties, with a
significant difference in scores between the groups
of dentists who perform surgeries less or more
frequently. The scientific literature evaluating the
domain of pharmacology and prescriptions among
the different areas of dentistry is sparse. However,
these data may indicate that, as dentists who perform
surgeries more frequently also prescribe drugs more
frequently (due to the procedures), these professionals
may have greater knowledge of the mechanisms of
action of these substances and, consequently, on their
potential capacity to increase the performance of
athletes, which characterises sports doping. Main study In general, the demographic characteristics of this
study were compatible with the national panorama of
Brazilian dentists. The highest percentage of professionals
in the main study practiced in the south and southeast
regions, which is compatible with the proportion of
dentists working in these regions (68.5%) according
to the Federal Council of Dentistry of Brazil (FCD),10
showing that this study provides a national panorama
of knowledge about sports doping by dentists in the
country. Although we can consider that the numerical
representativeness of the sample in relation to the
distribution by regions is according to the panorama of
the country, the final sample is not homogeneous enough,
since it is predominantly female. However, though the
FCD does not make available on its website the general
percentage of dentists according to sex, among experts,
the entity states that, as in our study, most are female.12
In addition, another point to be discussed is that Brazil
is a continental country, with a disproportion between
counties inland and capitals, which may explain the
predominance of dentists working inland than in the
capitals, as identified in the study by Martin et al.13 Table 4. Coefficients of the simple and multiple linear regression models for predicting knowledge about sports doping in dentists. Independent variable
B-KSSDD score
βcrude (P-value)
*βadjusted (P-value)
Age (years)
-0.032 (0.034)
-0.032 (0.034)
Frequency of surgery (ordinal)
0.400 (0.005)
0.298 (0.036)
Frequency of treating athletes (ordinal)
0.843 (< 0.001)
0.828 (< 0.001)
*p-value model < 0.001; R2 = 0.08. 7
Braz. Oral Res. 2021;35:e110 Table 4. Coefficients of the simple and multiple linear regression models for predicting knowledge about sports doping in dentists. Independent variable
B-KSSDD score
βcrude (P-value)
*βadjusted (P-value)
Age (years)
-0.032 (0.034)
-0.032 (0.034)
Frequency of surgery (ordinal)
0.400 (0.005)
0.298 (0.036)
Frequency of treating athletes (ordinal)
0.843 (< 0.001)
0.828 (< 0.001)
*p-value model < 0.001; R2 = 0.08. Braz. Oral Res. 2021;35:e110 Development, validation and application of a Brazilian knowledge scale about sports doping in dentistry The average score obtained by dentists on the
B-KSSDD in the main study shows that, in general,
these professionals have insufficient knowledge
about sports doping, increasing the risk that their
athlete patients are inadvertently exposed to drugs
that contain substances prohibited by the WADA. B-KSSDD applications This study demonstrates that the B-KSSDD is a
good instrument to use in different groups of dentists,
respecting cross-cultural adaptations, to assess their
knowledge on substances that, when prescribed to
athletes, may result in sports doping. In the face of
financial and sporting punishments applied to the
world sports community when doping tests return
positive results, applying the B-KSSDD may help the
worldwide research community and international
entities identify deficiencies in dentists’ knowledge
about prescribed drugs that can be characterised as
sports doping, preventing these professionals from
contributing to this important problem that can
generate severe punishments and losses for athletes. The results found using the B-KSSDD show that the
participants’ knowledge about substances prohibited
by the WADA is insufficient, making athletes treated
by these professionals vulnerable to the punishments
indicated in the Code. Together with the results found
by Mottram et al.,22 who applied a questionnaire on
doping that included 507 athletes, of whom only 35.1%
correctly identified items on the prohibited list, the
results of the present study are an alert to competent
sports entities to create and/or reinforce policies
to expand knowledge about sports doping among
athletes, so that they can also act as disseminators of
this information to health professionals who provide
care to them, including dentists. The simple and multiple linear regression models
for predicting knowledge about sports doping among
dentists showed that age has a negative association
with knowledge about sports doping, which may be
related to the fact that the establishment of Sports
Dentistry and the increase in training programmes
in this area are recent.14 In contrast, the frequencies
of treating athletes or performing surgeries showed
positive associations with knowledge about doping
in the preliminary study. In this sense, it is highly
recommended that, when seeking dental treatment,
athletes and clubs hire dentists with experience
in serving athletes in order to reduce the risk of
punishments applied when sports doping is identified. On the care of athletes, 5.9% of the participants
said that they attend to athletes frequently (always
or often). We believe that the low percentage can be
justified by the fact that dentists can consider only
elite athletes as “athletes”. Main study Although our sample contains a small number of
specialists in Sports Dentistry, we observed that the
average score of these professionals was approximately
two times higher than that found in the total sample,
suggesting that they are better able to provide dental
care to athletes without placing them at risk if they
are subjected to doping control. However, until
March 2021, the FCD indicated that there were only
29 professional specialists in Sports Dentistry in
the entire country, representing 0.02% of Brazilian
dentists who have a specialisation course. Thus, the
percentage of specialists in Sports Dentistry collected
by our study is compatible with the small number of
professionals in this field in the country.12 where soccer is the main national sport, although the
Brazilian Football Confederation requires that, to be
considered a training club, an entity must guarantee
constant dental care at a preventive or therapeutic
level to athletes in training, whether through their
own services, contracted or outsourced.19 However,
Rosa et al.20 and Souza et al.21 showed that young
soccer players included in their samples had poor
oral health, confirming our hypothesis that this
population’s access to dental services is naturally low. Braz. Oral Res. 2021;35:e110 Study limitation sports doping and the substances that characterise
it.23 Moreover, Woods and Moynihan,24 showed
only 9% of general practitioners in medicine said
they felt prepared to act to prevent doping. This
suggests that, at least among the classes evaluated
by the scientific community, knowledge about sports
doping is a challenge for the entire health spectrum of
professionals. For that reason, prevention programmes
and information on sports doping should be developed
and offered to athletes, doctors, coaches and family
members.25 Thus, the results obtained using the
B-KSSDD can also promote the development of
awareness and continuing education programmes
on sports doping for dentists, decreasing the risk
that athletes commit accidental sports doping as a
result of dental care. This study had limitations common in works
involving questionnaires, which include the veracity
of the information provided by the respondents and
their commitment when answering the survey, which
are also related to the comprehensibility and fluidity
of the instrument. However, these limitations were
reduced by the rigorous validation processes adopted
during the development of B-KSSDD, described in
this article. In addition, given that the scale was
developed and validated in Brazilian Portuguese, this
could be considered a limitation for its application in
other countries, which in turn could be made possible
through the cross-cultural adaptation and validation
of the version suggested for the B-KSSDD in British
English, available in Table 5. Table 5. English version (not validated) suggested for the Brazilian Knowledge Scale about Sports Doping in Dentistry (B-KSSDD)
and scores added to the total score according to the participants’ responses. Read the following sentences and inform your agreement, disagreement or lack of
knowledge about the statement presented:
Agree
Disagree
Don’t know
1. The use of oral nimesulide (for example, Nisulid®, Arflex®), prescribed by a dental
surgeon to an athlete who is in competition, characterises sports doping. 0
1
0
2. The use of oral dexamethasone (for example, Decadron), prescribed by a dental
surgeon to an athlete who is in competition, characterises sports doping. 1
0
0
3. The use of oral dipyrone (for example, Novalgina®, Dorflex®, Magnopirol®),
prescribed by a dental surgeon to an athlete who is in competition, characterises
sports doping. 0
1
0
4. The use of topical triamcinolone (for example, Omcilon®), prescribed by a dental
surgeon to an athlete who is in competition, characterises sports doping. 0
1
0
5. B-KSSDD applications However, the role of sports-
related dentistry is also intended to benefit the health
of amateurs and recreational sports practitioners,
who may also eventually be subjected to doping
control.15 In addition, international literature shows
that, even among elite athletes, oral health is poor,
suggesting that these professionals access dental
care services infrequently.16,17,18 In Brazil, for example, The results obtained using the B-KSSDD are also
compatible with a review with a systematic search
strategy, that found six articles involving health
professionals (only doctors and pharmacists were
found) and knowledge about doping, indicating that
these professionals have limited knowledge about 8 Jural LA, Soares TRC, Coqueiro RS, Rabello TB, Pithon MM, Maia LC Study limitation The use of a local anesthetic with adrenaline or epinephrine (for example,
Aplhacaine) administered by a dental surgeon in an athlete who is in a competition
period characterises sports doping. 0
1
0
6. The use of a combination of dipyrone with oral isometheptene (for example, in
Neosaldina®), prescribed by a dental surgeon to an athlete who is in competition,
characterises sports doping. 1
0
0
7. The use of oral prednisolone (for example, Predsim®), prescribed by a dental
surgeon to an athlete who is in competition, characterises sports doping. 1
0
0
8. The use of oral amoxicillin (for example, in Amoxil®), prescribed by a dental
surgeon to an athlete who is in competition, characterises sports doping. 0
1
0
9. The use of oral acetaminophen (for example, Tylenol®), prescribed by a dental
surgeon to an athlete who is in competition, characterises sports doping. 0
1
0
10. The use of an oral combination of codeine with acetaminophen (for example,
Tylex®), prescribed by a dental surgeon to an athlete who is in competition,
characterises sports doping. 0
1
0
11. The use of oral ibuprofen (for example, Alivium®), prescribed by a dentist to an
athlete who is in competition, characterises sports doping. 0
1
0
12. The use of oral betamethasone (for example, Celestone®), prescribed by a dental
surgeon to an athlete who is in competition, characterises sports doping. 1
0
0
Total score
0-12 points
Correct answers are represented by 1 and incorrect or “I don’t know” are represented by 0 points in the final B-KSSDD score. Table 5. English version (not validated) suggested for the Brazilian Knowledge Scale about Sports Doping in Dentistry (B-KSSDD)
and scores added to the total score according to the participants’ responses. Table 5. English version (not validated) suggested for the Brazilian Knowledge Scale about Sports Doping in Dentistry (B-KSSDD)
and scores added to the total score according to the participants’ responses. Correct answers are represented by 1 and incorrect or “I don’t know” are represented by 0 points in the final B-KSSDD score. Braz. Oral Res. 2021;35:e110 Development, validation and application of a Brazilian knowledge scale about sports doping in dentistry Development, validation and application of a Brazilian knowledge scale about sports doping in dentistry Development, validation and application of a Brazilian knowledge scale about sports doping in dentistry Acknowledgements We acknowledge the professionals who worked
during the initial evaluation of B-KSSDD questions. No
sources of funding were used to assist in the preparation
of this article. Lucas Alves Jural is supported by a research
Grant (147858/2020-8) from the Institutional Program for
Scientific Initiation Scholarships (PIBIC) of the Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(CNPq). Thais Rodrigues Campos Soares is supported
by a research Grant (159961/2018-1) from the CNPq. Matheus Melo Pithon is supported by a research Grant
(309800/2019-6) from the CNPq. Lucianne Cople Maia
is supported by a research Grant from the Fundação
de Amparo à Pesquisa do Estado do Rio de Janeiro
(E-26/210.208/2018) and from CNPq (310225/2020-5). The instrument proved to be valid and reliable
in assessing dentists’ knowledge about sports
doping. Dentists had insufficient knowledge about
sports doping, obtaining an average score of 4.19
out of 12 points on the B-KSSDD. Lower age of the
professional and higher frequencies of performing
surgery and treating athletes were associated
with greater knowledge about sports doping. The
development of professional updating policies on
sports doping for dentists is important to prevent
the intentional practice of doping by athletes after
dental care. References 1. Goldman B, Klatz R. Death in the locker room: drugs and sports paperback. Dacula: Elite Sports Medicine; 199 2. Voy RO, Deeter KD. Drugs, sport, and politics. Champaign: Leisure; 1991. 3. The International Federation of Sports Medicine. FIMS position statement: statement on doping in sport. Lausanne: The International
Federation of Sports Medicine; [cited 2020 Jun 16]. Available from: https://www.fims.org/files/4414/2056/2561/PS13-Statement-on-
Doping-in-Sport.pdf 3. The International Federation of Sports Medicine. FIMS position statement: statement on doping in sport. Lausanne: The International
Federation of Sports Medicine; [cited 2020 Jun 16]. Available from: https://www.fims.org/files/4414/2056/2561/PS13-Statement-on-
Doping-in-Sport.pdf 4. World Anti-doping Agency. World anti-doping code; 2012 with 2019 amendments. 2 ed. Montreal: World Anti-doping Agency; 2019
[cited 2020 Jun 26]. Available from: https://www.wada-ama.org/sites/default/files/resources/files/wada_anti-doping_code_2019_
english_final_revised_v1_linked.pdf 5. World Anti-doping Agency. The World Anti-Doping Code: international standard: phohibited list; january 2020. Montreal: World
Anti-doping Agency; 2020. Available from: https://www.wada-ama.org/sites/default/files/wada_2020_english_prohibited_list_0.pdf
6. Moore PA, Nahouraii HS, Zovko JG, Wisniewski SR. Dental therapeutic practice patterns in the U.S. II. Analgesics, corticosteroids, and
tibi ti
G
D
t 2006 54(3) 201 7
i 08 21 2 5. World Anti-doping Agency. The World Anti-Doping Code: international standard: phohibited list; january 2020. Montreal: World
Anti-doping Agency; 2020. Available from: https://www.wada-ama.org/sites/default/files/wada_2020_english_prohibited_list_0.pdf 6. Moore PA, Nahouraii HS, Zovko JG, Wisniewski SR. Dental therapeutic practice patterns in the U.S. II. Analgesi
antibiotics. Gen Dent. 2006;54(3):201-7; quiz 08, 21-2. 7. Pluim B. A doping sinner is not always a cheat. Br J Sports Med 2008;42(7):549-50.8. 7. Pluim B. A doping sinner is not always a cheat. Br J Sports Med 2008;42(7):549-50.8. 8. Terwee CB, Bot SD, Boer MR, Windt DA, Knol DL, Dekker J, et al. Quality criteria were proposed for measurem
status questionnaires. J Clin Epidemiol. 2007 Jan;60(1):34-42. https://doi.org/10.1016/j.jclinepi.2006.03.012 . Terwee CB, Bot SD, Boer MR, Windt DA, Knol DL, Dekker J, et al. Quality criteria were proposed for measurement
status questionnaires. J Clin Epidemiol. 2007 Jan;60(1):34-42. https://doi.org/10.1016/j.jclinepi.2006.03.012 9. Hulley SB, Cummings SR, Browner WS, Grady D, Hearst N, Newman TB. Delineando a pesquisa clínica: uma abordagem
epidemiológica. 2th ed. Porto Alegre: Artmed; 2003. 9. Hulley SB, Cummings SR, Browner WS, Grady D, Hearst N, Newman TB. Delineando a pesquisa clínica: uma abordagem
epidemiológica. 2th ed. Porto Alegre: Artmed; 2003. 10. Conselho Federal de Odontologia. Dados estatísticos de profissionais e entidades ativas por localidade. Brasília, DF: Conselho Federa
de Odontologia; 2020 [cited 2020 Jun 15]. Available from: https://www.website.cfo.org.br/dados-estatisticos-de-profissionais-e-
entidades-ativas-por-localidade/ 11. References Elbe AM, Jensen SN, Elsborg P, Wetzke M, Woldemariam GA, Huppertz B, et al. The urine marker test: an alternative approach to
supervised urine collection for doping control. Sports Med. 2016 Jan;46(1):15-22. https://doi.org/10.1007/s40279-015-0388-6 11. Elbe AM, Jensen SN, Elsborg P, Wetzke M, Woldemariam GA, Huppertz B, et al. The urine marker test: an alternative approach to
supervised urine collection for doping control. Sports Med. 2016 Jan;46(1):15-22. https://doi.org/10.1007/s40279-015-0388-6
12. Conselho Federal de Odontologia. Quantidade geral de cirurgiões-dentistas especialistas. Brasília, DF: Conselho Federal de 11. Elbe AM, Jensen SN, Elsborg P, Wetzke M, Woldemariam GA, Huppertz B, et al. The urine marker test: an alternative approach to
supervised urine collection for doping control. Sports Med. 2016 Jan;46(1):15-22. https://doi.org/10.1007/s40279-015-0388-6
12. Conselho Federal de Odontologia. Quantidade geral de cirurgiões-dentistas especialistas. Brasília, DF: Conselho Federal de
Odontologia; 2021 [cited 2021 Mar 8]. Available from: https://website.cfo.org.br/estatisticas/quantidade-geral-de-cirurgioes-
dentistas-especialistas/ 12. Conselho Federal de Odontologia. Quantidade geral de cirurgiões-dentistas especialistas. Brasília, DF: Conselho Federal de
Odontologia; 2021 [cited 2021 Mar 8]. Available from: https://website.cfo.org.br/estatisticas/quantidade-geral-de-cirurgioes-
dentistas-especialistas/ 13. Martin ASS, Chisini LA, Martelli S, Sartori LRM, Ramos EC, Demarco FF. Distribution of dental schools and dentists in Brazil: an overview
in relation to labor market. Rev Abeno. 2018 Mar.18(1):63-73. https://doi.org/10.30979/rev.abeno.v18i1.399 14. Saini R. Sports dentistry. Natl J Maxillofac Surg. 2011 Jul;2(2):129-31. https://doi.org/10.4103/0975-5950.94465 15. Stamos A, Mills S, Malliaropoulos N, Cantamessa S, Dartevelle JL, Gündüz E, et al. The European Association for Sports Dentistry,
Academy for Sports Dentistry, European College of Sports and Exercise Physicians consensus statement on sports dentistry integration in
sports medicine. Dent Traumatol. 2020 Dec;36(6):680-4. https://doi.org/10.1111/edt.12593 10 Braz. Oral Res. 2021;35:e110 Jural LA, Soares TRC, Coqueiro RS, Rabello TB, Pithon MM, Maia LC 16. Gallagher J, Ashley P, Petrie A, Needleman I. Oral health and performance impacts in elite and professional athletes. Community Dent
Oral Epidemiol. 2018 Dec;46(6):563-8. https://doi.org/10.1111/cdoe.12392 17. Needleman I, Ashley P, Petrie A, Fortune F, Turner W, Jones J, et al. Oral health and impact on performance of athletes
participating in the London 2012 Olympic Games: a cross-sectional study. Br J Sports Med. 2013 Nov;47(16):1054-8. https://doi.org/10.1136/bjsports-2013-092891 18. Kragt L, Moen MH, Van Den Hoogenband CR, Wolvius EB. Oral health among Dutch elite athletes prior to Rio 2016. Phys Sportsmed. 2019 May;47(2):182-8. https://doi.org/10.1080/00913847.2018.1546105 18. Kragt L, Moen MH, Van Den Hoogenband CR, Wolvius EB. Oral health among Dutch elite athletes prior to Rio 2016. Phys Sportsmed. 2019 May;47(2):182-8. https://doi.org/10.1080/00913847.2018.1546105 19. Confederação Brasileira de Futebol. References Resolução da Presidência nº 01/2029: Modifica e substitui as RDPs nº 01/2012 e 04/2015, que
estabelecem normas para a emissão de Certificado de Clube Formador (CCF). [cited 2021 Mar 8]. Available from: https://conteudo.cbf. com.br/cdn/202009/20200918145239_131.pdf 19. Confederação Brasileira de Futebol. Resolução da Presidência nº 01/2029: Modifica e substitui as RDPs nº 01/2012 e 04/2015, que
estabelecem normas para a emissão de Certificado de Clube Formador (CCF). [cited 2021 Mar 8]. Available from: https://conteudo.cbf. com.br/cdn/202009/20200918145239_131.pdf 20. Rosa AF, Costa SB, Silva PR, Roxo CD, Machado GS, Teixeira AA, et al. [Descriptive study of odontological alterations verified in four
hundred soccer players]. Rev Bras Med Esporte. 1999 Apr;5(2):55-8. Portuguese. https://doi.org/10.1590/S1517-86921999000200004 20. Rosa AF, Costa SB, Silva PR, Roxo CD, Machado GS, Teixeira AA, et al. [Descriptive study of odontological alterations verified in four
hundred soccer players]. Rev Bras Med Esporte. 1999 Apr;5(2):55-8. Portuguese. https://doi.org/10.1590/S1517-86921999000200004 21. Souza LA, Elmadjian TR, Dias RB, Coto NP. Prevalence of malocclusions in the 13-20-year-old categories of football athletes. Braz Oral
Res. 2011 Jan-Feb;25(1):19-22. https://doi.org/10.1590/S1806-83242011000100004 21. Souza LA, Elmadjian TR, Dias RB, Coto NP. Prevalence of malocclusions in the 13-20-year-old categories of football athletes. Braz Oral
Res. 2011 Jan-Feb;25(1):19-22. https://doi.org/10.1590/S1806-83242011000100004 22. Mottram D, Chester N, Atkinson G, Goode D. Athletes’ knowledge and views on OTC medication. Int J Sports Med. 2008
Oct;29(10):851-5. https://doi.org/10.1055/s-2008-1038403 22. Mottram D, Chester N, Atkinson G, Goode D. Athletes’ knowledge and views on OTC medication. Int J Sports Med. 2008
Oct;29(10):851-5. https://doi.org/10.1055/s-2008-1038403 23. Backhouse SH, McKenna J. Doping in sport: a review of medical practitioners’ knowledge, attitudes and beliefs. Int J Drug Policy. 2011
May;22(3):198-202. https://doi.org/10.1016/j.drugpo.2011.03.002 23. Backhouse SH, McKenna J. Doping in sport: a review of medical practitioners’ knowledge, attitudes and beliefs. Int J Drug Policy. 2011
May;22(3):198-202. https://doi.org/10.1016/j.drugpo.2011.03.002 24. Woods CB, Moynihan A. General practitioners knowledge, practice and training requirements in relation to doping in sport. Ir Med J. 2009 Jan;102(1):8-10. 24. Woods CB, Moynihan A. General practitioners knowledge, practice and training requirements in relation to doping in sport. Ir Med J. 2009 Jan;102(1):8-10. 25. Morente-Sánchez J, Zabala M. Doping in sport: a review of elite athletes’ attitudes, beliefs, and knowledge. Sports Med. 2013
Jun;43(6):395-411. https://doi.org/10.1007/s40279-013-0037-x 25. Morente-Sánchez J, Zabala M. Doping in sport: a review of elite athletes’ attitudes, beliefs, and knowledge. Sports Med. 2013
Jun;43(6):395-411. https://doi.org/10.1007/s40279-013-0037-x Braz. Oral Res. 2021;35:e110 11
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W2329974479.txt
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https://zenodo.org/records/2106069/files/article.pdf
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de
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Anaerobe Atmung, Alkoholgärung und Acetonbildung bei den Samenpflanzen.
|
Hoppe-Seyler's Zeitschrift für Physiologische Chemie
| 1,906
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public-domain
| 6,812
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Anaerobe Atmung, Alkoholgärung und Acetonbildung bei den
Samenpflanzen.
Von
W. Palladin und S. Kostytschew.
Mit zwei Kurvenzeiehnungen im Text.
(Pflanzenphysiologisches Institut der Universität St. Petersburg.)
(Der Redaktion zugegangen am 26. Mai 1906.)
Wir sind beide zu verschiedener Zeit und auf Grund verschiedener Erwägungen zu einem und demselben Schluß gekommen: die typische anaerobe Atmung ist mit der Alkoholgärung (Zymasegärung) nicht identisch.l) Da nun diese unsere
Anschauung mit der laufenden Vorstellung von dem Wesen der
anaeroben Atmung in offenbarem Widerspruch steht, so ist es
geboten, die Frage nach dem Chemismus der anaeroben Atmung
einer neuen experimentellen Untersuchung zu unterwerfen. Die
Resultate dieser Untersuchung bilden den Inhalt der vorliegenden
Abhandlung.
Daß voreingenommene Ansichten bei der Entwickelung
der Lehre von der anaeroben Atmung der Pflanzen eine sehr
wichtige Rolle gespielt hatten, scheint kaum zweifelhaft zu sein!
Diese Tatsache wird am besten dadurch erläutert, daß die Identität der anaeroben Atmung mit der Alkoholgärung im Verlauf
von 25 Jahren (1872—1897) durch keinen einzigen direkten Versuch nachgeprüft und trotzdem von allen Fachgenossen als festgestellt betrachtet wurde. So haben z. B.
Pfeffer 2 ) und Wortmann 3 ) ihre wohlbekannten Theorien auf
') Kostytschew, Berichte d. D. bot. Ges., 1904;, S. 207. Zentralblatt für Bakteriol., II. Abt., Bd. XIII, 1904?, S. 490.
Palladin, Diese Zeitschrift, Bd. XLVII, 1906, S. 407.
2
) Pfeffer, Landwirtsch. Jahrbücher, Bd. VU, 1878, S. 805.
8
) Wortmann, Arbeiten d. botan. Inst, zu Würzburg, Bd. II,'l880,
S. 500.
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Anaerobe Atmung, Alkoholgarung .usw. bei den Samenpflanzen.
215
der Annahme gegründet, daß der Dissoziationsprozeß der
hydrate bei der anaeroben Atmung genau nach der Gleichung
der Alkoholgärung erfolgt. Auch Diakonow 1 ) hat sich f olgender*·
maßen ausgesprochen: «Ohne Sauerstoffatmung oder die sie
vertretende Alkoholgärung findet kein Leben statt». Derselbe
Gedanke lag den Versuchen Chudiakows 2 ) zugrunde. Nur
durch die trefflichen Untersuchungen von Godlewski und
Polzeniusz 3 ) wurde zuerst der Nachweis dafür geliefert, daß
die anaerobe Atmung der Erbsensamen in allen wesentlichen
Punkten mit der Zymasegärung übereinstimmt, denn das Ver^
hältnis C02 : C2H60 bei der anaeroben Atmung der Erbsensamen entspricht der Gleichung C6H1206 *= 2 C02
2 C2H5OH,
Es ist aber den genannten Forschern nicht gelungen, die Anwesenheit eines gärungserregenden Enzyms in Erbsensameu
festzustellen. Späterhin hat Godlewski 4 ) nachgewiesen, daß
die anaerobe Atmung der Lupinensamen ebenfalls mit der AJ·koholgärung identisch ist.
Die Untersuchungen von Godlewski und Polzeniuez
wurden von Nabokich^) wiederholt und fortgesetzt. Dieser
Forscher hat gefunden, daß die anaerobe Atmung nicht immer
mit der Alkoholgärung übereinstimmt. So schwankt z. B. C02 c
C2H60 bei der anaeroben Atmung der Rizinussamen von 100 ; 50
bis zu 100 : 80. Auch bei Erbsensamen wird der Gärungsquotient durch Einwirkung stark saurer Lösungen bis auf die
Hälfte herabgedrückt.
Nach den Untersuchungen von Stoklasa 6 ) ist auch die
anaerobe Atmung der Zuckerrübe mit der Alkoholgärung identisch. Dieser Forscher hat außerdem die wichtige Tatsache
hervorgehoben, daß ihm die Darstellung der Zymase aus ver*) Diakonow, Berichte d. botan. Ges., Bd, IV, 1881, S. 1.
) G h u d i a k o w , Landw. Jahrbücher, Bd. XXIII, 1894;, S. 333.
8
) Godlewski u. Polzeniusz, Bulletin de l'Acadeniie des sciences
de Gracovie, 1897, S. 267; 1901, S. 227.
4
) Godlewski, 1. c. 1904, S. 115.
6
) Nabokich, Berichte d. botan. Gesellschaft, Bd. XXI, 1903, S. 467.
Journal für experim. Landwirtschaft, 1903, S. 696 ; 1904, S. 305 (russisch).
6
) Stoklasa, Jelinek u. Vitek, Hofmeisters Beiträge., Bd. III,
1903, S. 460.
15*
2
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216
W. Palladin und S. Kostytschew,
schiedenen pflanzlichen und tierischen Objekten gelungen sei.J)
Da aber eine ganze Reihe von Beobachtungen verschiedener
Forscher (Cohnheim, 2 ) Batelli, 3 ) Arnheim und Rosenbaum, 4 ) Landsberg, 5 ) Maze, 6 ) Maze et Perrier, 7 ) Portier 8 ) u. a.) den Angaben Stoklasas widerspricht und schlagende Einwände gegen die Methodik des bömischen Forschers
seitens Maze 6 ) und Portier 8 ) geltend gemacht worden sind,
so können wir die Frage nach dem Vorhandensein der Zymase
in Samenpflanzen und Tiergeweben noch nicht für abgeschlossen
halten.
Schließlich sei noch erwähnt, daß Hahn 9 ) im Preßsaft von
Arum maculatum eine starke Glykolyse ohne gleichzeitige
Alkoholbildung beobachtet hat. Das zuckerspaltende Agens von
Arum maculatum ist also mit der Zymase nicht identisch.
Unsere eigenen Untersuchungen sind zum größten Teil
mit Hilfe der unlängst von einem von uns10) ausführlich beschriebenen Gefriermethode ausgeführt worden. Gefrorene Versuchsobjekte wurden in geräumige U-Röhren gebracht; alsdann
wurde ein konstanter Strom von reinem Wasserstoff durch die
Röhren geleitet.n) Die von dem Versuchsmaterial ausgeschiedene
C02 wurde im Barytwasser aufgefangen und durch Titration
mit Oxalsäure bestimmt. War eine detaillierte Kenntnis des
*) Stoklasa und Cerny, Berichte d. Deutsch, ehem. Gesellsch.,
Bd. XXXVI, 1903, S. 622.
Stoklasa, Pflügers Archiv, Bd. CI, 1904,- S. 311.
Stoklasa u. Gerny, Zentralbl. f. Physiol., Bd. XVI, 1903, S. 652.
Stoklasa, ebenda, Bd. XVII, 1903, S. 465.
Stoklasa, Zentralblatt f. Bakteriologie, 1904
*) Gohnheim, Diese Zeitschrift, Bd. XXXIX, 1903, S. 336.
8
) Batelli, Gomptes rendus, Bd. CXXXVII, 1903, S. 1079.
4
) Arnheimu.Rosenbaum,DieseZeitschrift,Bd.XL,1903/4,S.220.
5
) Landsberg, ebenda, Bd. XLI, 1904, S. 505.
6
j Maze", Annales de Tlnstitut Pasteur, Bd. XVIIIr1904, S. 378 u. 535.
7
) Maze u. Perrier, ebenda, Bd. XVUI, S. 382.
8
) Portier, ebenda, Bd. XVIII, 1904, S. 633.
9
) Hahn, Ber. d. Deutsch, ehem. Ges., Bd. XXXIII, 1900, S. 3555.
10
) Palladin, Diese Zeitschrift, Bd. XLVII, 1906, S. 407.
11
) Die Wasserstoffdurchleitung wurde auch während der Nachtstunden nicht unterbrochen.
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Anaerobe Atmung, Alkoholgärung usw. bei den Samenpflanzen.
217
Ganges der enzymatischen Atmung wünschenswert, so wurde
C02 direkt in Pettenkof er sehen Röhren absorbiert; sonst
wurde die Hauptmenge der C02 in einem großen mit 500 ccm
Barytwasser beschickten Kolben zurückgehalten; das Pettenkof er sehe Rohr diente dann nur zur Kontrolle. Um einer Verdunstung des Alkohols vorzubeugen, wurde zwischen dem U-Rohr
und den Absorptionsgefäßen eine in schmelzendes Eis getauchte
und mit Wasser gefüllte Waschflasche eingeschaltet. Ein vollständiges Sterilbleiben des Versuchsmaterials wurde dadurch gesichert, daß letzteres möglichst locker im Rezipienten verteilt und
mit einer mit Toluol getränkten Watteschicht bedient wurde; es
befand sich folglich in einem fortwährend mit Toluoldämpfen gesättigten Gasstrome. Die Wasserstoffdurchleitung dauerte bis zum
vollständigen Auf hören oder wenigstens bis zu einer sehr starken
Herabminderung der Kohlensäurebildung. Alsdann schritt man
zur Bestimmung des gebildeten Alkohols. Zu diesem Zwecke wurde
das Versuchsmaterial samt dem Wasser der Waschflasche in
einen geräumigen Rundkolben gebracht, mit noch ca. 500 ccm
destillierten Wassers versetzt und alles mehrfacher Destillation
unterworfen, wobei jedesmal nicht weniger als die Hälfte der
Flüssigkeit in die Vorlage überging. Bei der ersten Destillation
wurde immer eine gewisse Menge von Toluol in der Vorlage
gefunden; derselbe ließ sich aber von der übrigen Flüssigkeit im
Scheidetrichter leicht abtrennen. Die zweite Destillation erfolgte
aus schwach saurer und die dritte aus schwach alkalischer Lösung.
Zur Ansäuerung des ersten Destillates wurde Weinsäure, zur Alkalisierung des zweiten wurde Natriumcarbonat verwendet. Ohne
Berücksichtigung dieser Vorsichtsmaßregel wird man schwerlich
ein ganz neutrales Destillat erhalten; meistens enthält es dann
eine auf Kongorot alkalisch reagierende und durch Phosphorwolframsäure fällbare Substanz. Die Menge des gebildeten
Alkohols wurde aus dem spez. Gewichte des vierten bzw. fünften
Destillates ermittelt. Das spez. Gewicht wurde mit Hilfe eines
genauen, mehr als 30 ccm fassenden Pyknometers bestimmt.
Sämtliche Füllungen des Pyknometers wurden bei 15,5° G. ausgeführt. Zur Identifizierung des Äthylalkohols dienten folgende
Reaktionen:
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218
W. Palladiü und S. Kostytschew,
1. Die Reaktion von Berthelot, 1 ) die folgendermaßen
ausgeführt wird: man schüttelt die zu untersuchende Flüssigkeit mit einer ganz geringen Menge von Benzoylchlorid und
überschüssiger Natronlauge bis zum Verschwinden des stechenden
Geruchs des Benzoylchlorids. Bei Gegenwart des Äthylalkohols
entwickelt sich der charakteristische Geruch des Benzoesäureäthylesters. Diese Probe hat den Vorzug, daß sie nur mit
Äthylalkohol positiv ausfällt; ihre Empfindlichkeit läßt nichts
zu wünschen übrig.
2* Die Jodoformprobe. Diese Reaktion haben wir immer
nach der Modifikation von Müntz 2 ) ausgeführt, die, unseren
Erfahrungen nach, die empfindlichste ist. 10 ccm des Destillates
Worden mit 2 g Natriumcarboiiat und 0,1 g Jodpulver versetzt
tiud dann auf dem Wässerbade bei 60° bis zur vollständigen
Auflösung des Jods erwärmt. Nach dem Erkalten scheiden
sich die charakterischen Krystalle des Jodoforms aus. Diese
Probe ist zwar empfindlich, doch nicht ganz zuverlässig: sie
fällt mit verschiedenen Substanzen positiv aus, von denen in
erster Reihe Aldehyde und niedere Ketotie in Betracht kommen,
da sie sich auch im Destillat vorfinden können. Ist aber die
Abwesenheit flüchtiger Aldehyde und Ketone im Destillat festgestellt, so eignet sich die Jodoformprobe zum Nachweis des
Äthylalkohols in ganz ausgezeichneter Weise. Wir haben das
Destillat jedesmal mit fuchsinschwefliger Säure geprüft, wodurch
sich minimale Spuren von Aldehyden und Aceton entdecken
lassen. Aus der nachfolgenden Darlegung wird ersichtlich sein,
daß die Jodoformprobe allein für den Nachweis des Äthyl*
alkohols ganz und gar belanglos ist. Nun gehen wir zur Beschreibung der einzelnen Versuche über.
Versuche mit Zymin.
Versuch 1.
20 g Zymin wurden mit 200 ccm destillierten Wassers
und 3 ccm Toluol in einen Kolben gebracht; alsdann wurde
ein konstanter Wasserstoifstrom durch die Flüssigkeit getrieben.
») Berthelot, Comptes rendus, Bd. LXXIII, S. 496 (Neubauer
und Vogel, Analyse des Harns, 10. Auflage, 1898).
2
) Müntz, Ann. de eh. et de phys., Ser. 5, Bd. XIII, 1878, S. 543.
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Anaerobe Atmung, Alkoholgärung usw. bei den Samenpflanzen.
219
Zeitdauer in
Stunden
C02 in
mg
C02 pro 1 Stunde in
mg
6
6
2l1/·
16
o1/*
13V2
2
99,6
261,0
475,6 *)
306,0
80,4
82,4
9,6
16,6
43,5
22,1
19,1
14,6
6,1
4,8
70'/»
1314,6 *)
Die quantitative Alkoholbestimmung ergab: G2H5OH =
1504,7 mg.
Versuch 2.
20 g Zymin wurden mit soviel destillierten Wassers versetzt, daß sich ein zähflüssiger Brei gebildet hat. Mit diesem
Brei wurden Fließpapierstreifen bestrichen, welche sodann in
einem geräumigen U-Rohr möglichst locker verteilt und mit
einer mit Toluol getränkten Watteschicht gedeckt wurden. Durch
das Rohr wurde dann ein konstanter Wasserstoffstrom geleitet.
Zeitdauer in
Stunden
G02 in
mg
C02 pro 1 Stunde in
mg
2
2 V»
6
14
8V·
14
2
116,8
243,0
511,2
419,4
131,2
73,2
6,0
58,4
97,2
85,2
29,9
15,4
5,2
3,0
49
1500,8
*) Eine beträchtliche Menge von C02 ist während der Nachtstunden,
verloren gegangen. Darum wurde in diesem Versuche das Verhältnis
C02 : C2HeOH nicht berechnet.
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220
W. Palladin und S. Kostytschew,
Die Menge des gebildeten Alkohols war 1511,3 mg. Da
das Destillat nicht acetonfrei war,1) so wurde es noch einmal
mit Natriumbisulfit und einmal mit Natriumcarbonat abdestilliert.
Im letzten Destillat, das keine Acetonreaktionen aufwies, war
die Menge des Alkohols gleich 1500,3 mg gefunden. Folglich
war der durch die Anwesenheit des Acetons verursachte Fehler
ganz unbedeutend.
C0 2 : C2H5OH = 1500,8 :1500,3 = 100 :100.
Ebendasselbe Verhältnis wurde von Buchner 2 ) für die
Zymasegärung unter Zuckerzusatz gefunden.
Aus diesen beiden Versuchen ist folgendes ersichtlich:
1. Von der Zymasegärung unter Zuckerzusatz unterscheidet sich die Selbstgärung des Zymins nicht qualitativ,
sondern nur quantitativ.
2. Durch unsere Versuchsanstellung wird eine ganz genaue
Bestimmung des gebildeten Alkohols ermöglicht: trotzdem daß
im Versuch 2 die Bedingungen zur Verdunstung des Alkohols
sehr günstig waren, fand dennoch kein Verlust des Alkohols statt.
3. Im Wasserstoffstrome kommt die Selbstgärung des Zymins
schneller zu Ende, als im Wasser. Dieses Ergebnis ist durch
die Fig. I graphisch dargestellt worden; es bildet die Grundlage der von E. Buchner und Mitscherlich 3 ) angegebenen
Methode der Darstellung des glykogenarmen Zymins. Auch hat
einer von uns^4) schon früher gefunden, daß erfrorene Weizenkeime nur in Gasmedien eine ausgiebige Kohlensäurebildung
entwickeln.
Versuche mit etiolierten Blättern und Stengelgipfeln von Vicia Faba.
Nach den Untersuchungen von Maze 5 ) und Polowzow 6 )
enthalten verschiedene frische Pflanzen eine gewisse Menge
*) Das Aceton war im lufttrockenen Zyminpräparat reichlich vorhanden.
2
) E. Buchner, H. Buchner u. M. Hahn, Zymasegärung.
s
) E. Buchner und Mitscherlich, Diese Zeitschrift, Bd. XLH,
1904, S. 554·.
4
) Palladin, ebenda, Bd. XLVH, 1906, S. 441,
5
) Maz<§, Comptes rendus, Bd. CXXVIII, 1899, S. 1608.
Maz£, Annales de Flnstitut Pasteur, Bd. XVI, 1902, S. 195.
6
) Polowzow, Untersuchung, üb. d.Pflanzenatmung, 1901 (russisch).
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Anaerobe Atmung, Alkoholgärung usw. bei den Samenpflanzen.
221
Äthylalkohol. Daher ist eine quantitative Bestimmung des
Alkoholgehaltes frischer Versuchsobjekte für unsere Zwecke
unentbehrlich. Eine solche Bestimmung wurde im folgenden
Versuche ausgeführt.
Versuch 3
<
(Kontroll\t
versuch).
i
\
2 13 g frischer
\
Stengelgipfel
von Vicia Faba wurden mit
einer beträcht•%
lichen Menge
\
\
destillierten
Wassers versetzt und in der
\
auf S. 217 be\
\
schriebenen
/
Weise mehr^
facher Destilla/
\
\
tion unterwor/
fen. Das letzte
/
v
\
Destillat gab/
\
folgende Reak\%
X
\k
tionen :
7
V< r,
1. Reaktion
\J
mit
fuchsin- 7
&
M
30
W
SO
6Q
TO
Stunde/t
schwefliger
Säure negativ.
Fig. I.
W
1/
/
2.
Jodoform- Kohlensäureaussclieidujig von 20 g Zymin im Wasser («) und
probe nach
^ Wasserstoffe &·
Müntz positiv. 3. Reaktion von Berthelot positiv.
Die quantitative Bestimmung ergab: C2H5OH = 58,2 mg
(27,3 mg auf 100 g der Frischsubstanz).
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222
W. Palladin und S. Kostytsehew,
Versuch 4.
Etiolierte Stengelgipfel von Vicia Faba wurden in zwei
Portionen geteilt: a) Blätter (120 g) und b) Gipfel ohne Blätter
(117 g). Beide Portionen wurden erfroren und in den Fettenkoferschen Apparat gebracht. Temperatur 20°.
Wasser stoff ström.
a) Erfrorene Blätter
b) Erfrorene Gipfel ohne Blätter
Zeitdauer C02 in G02 pro 1 St. in Zeitdauer G02 in C02 pro 1 St. in
in Stunden
mg
in Stunden
mg
mg
mg
2
2
4
6
13
—
48,0
57,6
41,2
20,0
14,8
24,0
28,8
10,3
3,3
—
—
27
181,6
1,1
9
24 V»
41/*
59,2
40,4
36,8
20,4
26,0
Spur
57 V«
182,8
4
3
6 l /2
14,8
13,5
5,6
2,3
1,1
—
Alkoholbestimmungen.
a) Erfrorene Blätter. Reaktionen des Destillates wie im
Versuch 3.
Die quantitative Bestimmung ergab: C2H5OH =63,9 mg
C2H5OH in 120 g frischer Blätter = 32,8 »
Von den erfrorenen Blättern wurde gebildet: C2H5OH = 31,1 »
C02: C2H5OH = 181,6 : 31,1 = 100 :17,1.
b) Erfrorene Gipfel. Das Destillat gab dieselben Reaktionen
wie im Versuch 3.
Die quantitative Bestimmung ergab: C2H5OH = 65,7 mg
C2H5OH in 117 g frischer Gipfel = 31,9 »
Von den erfrorenen Gipfeln wurde gebildet G2H5OH = 33,8.
C02 : C2H5OH = 182,8 : 33,8 = 100 : 18,5.
Dieser Versuch zeigt, daß zwischen der anaeroben Atmung
erfrorener Gipfel und Blätter gar kein Unterschied besteht.
Darum wurden in den weiter folgenden Versuchen die Blätter
nicht von den Gipfeln abgetrennt.
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Anaerobe Atmuüg, Alkohölgämng usw. bei den Samenpflanzen.
Versuch 5.
Die Gipfel der alten etiolierten Stengel von Vicia Faba
(145 g) wurden erfroren und in den Pettenkoferschen Apparat
gebracht. Temperatur 19°.
E r f r o r e n e Gipfel.
Zeitdauer in
Stunden
Wasserstoffstrom
C02 in
mg
G02 pro 1 Stunde in
mg
3
3V*
191/*
101,8
58,8
101,6
36,2
16,8
5,2
26
269,2
Alkoholbestimmung.
Das Destillat gab dieselben Reaktionen wie im Versuch 3.
Die quantitative Bestimmung ergab: C2H5OH = 40,6 mg,
C2H5OH in 145 g frischer Gipfel = 39,6 mg.
Von den erfrorenen Gipfeln wurde gebildet C2H5OH =± Spur.
Versuch 6.
209 g etiolierter Stengelgipfel von Vicia Faba wurden
erfroren und in den Pettenkoferschen Apparat gebracht.
Temperatur 19°.
Erfrorene Gipfel von Vicia Faba (Wasserstoffstrom).
Zeitdauer in
Stunden
C02 in
mg
CO, pro 1 Stunde in
mg
3
6
18
IV«
100,4
124,8
82,0
6,4
33,5
20,8
45
4,3
28V«
313,6
Alkoholbestimmung.
Reaktionen des Destillates wie im Versuch 3.
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224
W. Palladin und S. Kostytschew,
Die quantitative Bestimmung ergab: C2H5OH = 83,7 mg.
C2H5OH in 209 g frischer Gipfel = 57,1 mg.
Von den erfrorenen Gipfeln wurde gebildet C2H5OH = 26,6 mg.
C02 : C2H5OH = 313,6 : 26,6 = 100 : 8,4.
Durch diese Versuche wird der Nachweis dafür erbracht,
daß die anaerobe Atmung erfrorener Stengelgipfel von Vicia
Faba keine Alkoholgärung ist. Die von dem Versuchsmaterial
gebildeten unbedeutenden Mengen des Alkohols bleiben beinahe
in den Grenzen der Versuchsfehler. Es darf nämlich nicht
außer acht gelassen werden, daß die zum Kontrollversuch benutzten Keimlinge nicht gleichzeitig mit den übrigen gezogen
wurden. Folgender Versuch zeigt, daß die künstliche Zuckerzufuhr keinen großen Einfluß hat auf die Größe von C0 2 : C2H5OH
erfrorener Gipfel.
Versuch 7.
Die Gipfel der etiolierten Stengel von Vicia Faba wurden
im Verlauf von drei Tagen auf 10°/oiger Saccharoselösung im
diffusen Lichte kultiviert. Ein Teil der Gipfel (69 g) wurde für
den Kontrollversuch verwendet, das Übriggebliebene (178g)
wurde erfroren und in den Pettenkoferscben Apparat gebracht. Temperatur 20°.
a) Kontrollportion. 69 g frischer Gipfel wurden in der
auf Seite 217 beschriebenen Weise bearbeitet. Das letzte Destillat
gab dieselben Reaktionen wie im Versuch 3.
Die quantitative Bestimmung ergab: C2H5OH = 17,3 mg
(25,1 mg auf 100 g der Frischsubstanz).
b) Versuchsportion. Versuchsdauer26Stunden. C02 =
224,0 mg. Das erhaltene Destillat gab folgende Reaktionen:
1. Reaktion mit fuchsinschwefliger Säure schwach posittiv.
2. Jodoformprobenach Müntz positiv. 3. Reaktion von Berthelot
positiv.
Die quantitative Bestimmung ergab: C2H5OH = 118,6 mg.
C2H5OH in 178 g frischer Gipfel = 44,6 mg.
Von den erfrorenen Gipfeln wurde gebildet: C2H5OH =
74,0 mg.
C02 : C2H5OH = 224 : 74 = 100 : 33.
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Anaerobe Atmung, Alkoholgärung usw. bei den Samenpflanzen.
225
Versuche mit Samen und Keimlingen von Lupinus luteus.
Versuch 8 (Kontrollversuch).
Samen von Lupinus luteus wurden im Verlauf von
2 Tagen unter einer dünnen Wasserschicht geweicht, dann abgeschält und in zwei Portionen geteilt. Die erste Portion (a)
wurde zur Alkoholbestimmung unmittelbar, die zweite (b) nach
vorhergehender Erfrierung verwendet.
Portion a) 400 frische Lupinensamen (88 g) wurden zur
Alkoholbestimmung verwendet. Das letzte Destillat gab folgende
Reaktionen: 1. Reaktion mit fuchsinschwefliger Säure negativ.
2. Jodoformprobe nach Müntz sehr schwach. 3. Reaktion von
Berthelot sehr schwach.
Das spezifische Gewicht der Flüssigkeit war gleich 1,0000.
Die Menge des gebildeten Alkohols war also unmeßbar gering.
Portion b) 400 Lupinensamen (87 g) wurden erfroren
und dann zur Alkoholbestimmung verwendet. Das erhaltene
Destillat gab folgende Reaktionen: 1. Reaktion mit fuchsinschwefliger Säure negativ. 2. Jodoformprobe nach Müntz sehr
schwach. 3. Die Reaktion von Berthelot zweifelhaft. Die
quantitative Bestimmung ergab, daß die Menge des Alkohols
unmeßbar gering war.
Versuch 9.
Samen von Lupinus luteus wurden im Verlauf von
zwei Tagen unter einer dünnen Wasserschicht geweicht, dann
abgeschält und in zwei Portionen zu je 500 Samen geteilt. Die
eine Portion (120g) wurde unmittelbar in den Pettenkoferschen
Apparat gebracht. Die zweite Portion (104 g) wurde erst erfroren, dann ebenfalls in den Pettenkoferschen Apparat gebracht. Temperatur 20°. Wasserstoifstrom.
a) Lebende Samen. Versuchsdauer 24 Stunden. C02 =
192,0mg. Das erhaltene Destillat gab folgende Reaktionen:
1. Reaktion mit fuchsinschwefliger Säure sehr schwach. 2. Jodoformprobe nach Müntz positiv. 3. Reaktion von Berthelot
positiv.
Die quantitative Bestimmung ergab: C2H5OH = 174,0 mg.
C02 : C2H5OH = 192 : 174 = 100 : 90,6.
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226
W, Palladin und S. Kostytschew,
Dieses Resultat stimmt mit den Angaben Godlewskis 1 )
vollkommen überein, der für lebende Samen von L up in u s
luteus C02 : C2H5OH = 100 : 96 gefunden hat.
b) Erfrorene Samen. Versuchsdauer 25Stunden. C0 2 ~
84,0 mg.
Das erhaltene Destillat gab folgende Reaktionen: I.Reaktion
mit fuchsinschwefliger Säure sehr schwach. 2. Jodoformprobe
nach Müntz positiv. Reaktion von Berthelot zweifelhaft.
Die quantitative Bestimmung ergab, daß die Menge des
Alkohols unmeßbar gering war (spezifisches Gewicht = 0,99998).
C0 2 : C2H-OH = 84:0.
Versuch 10.
500 Samen von Lupinus luteus (130 g) wurden im Verlauf von zwei Tagen unter einer dünnen Wasserschicht geweicht,
dann abgeschält, erfroren und in den Pettenkofersehen Apparat
gebracht. Temperatur 20°. Wasserstoffstrom.
Versuchsdauer 24 Stunden. C02 = 152,0 mg.
Die Alkoholbestimmung ergab, daß die Menge des Alkohols
unmeßbar gering war. Reaktion von Berthelot zweifelhaft.
C02 : C2H5OH = 152 :0.
Versuch 11.
400 Lupinensamen (101 g) wurden im Verlauf von zwei
Tagen im Wasser geweicht, dann abgeschält, erfroren und in
den P e t t e n k o f er sehen Apparat gebracht. Temperatur 20°.
Wasser stoffstrom.
Versuchsdauer 46 Stunden. C02 = 238,8 mg.
Alkoholbestimmung.
Das letzte Destillat gab folgende Reaktionen: 1. Reaktion
mit fuchsinschwefliger Säure negativ. 2. Jodoformprobe nach
Müntz positiv. 3. Reaktion von Berthelot positiv.
Die quantitative Bestimmung ergab: C2H5OH = 83,6 mg.
C02 : G2H5OH = 238,8 : 83,6 = 100 : 35,0.
l
) Godlewski, Bulletin de l'Acad&nie des sciences de Cracovie,
1904, S. 115.
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Anaerobe Atmung, Alkoholgärung usw. bei den Samenpflanzen.
227
Bei diesem Versuche war die Erfrierung keine befriedigende:
die Temperatur der Kältemischung war zufälligerweise bedeutend
höher, als bei allen übrigen Versuchen. Daher läßt sich wahrscheinlich der Umstand erklären, daß in diesem Falle eine
Alkoholbildung stattgefunden hat.
Versuch 11.
8 tätige Keimlinge von Lupinus luteus (Länge des Hypokotyls 5—9 cm) wurden in drei Portionen geteilt. Die erste
Portion (200 Keimlinge = 125 g) wurde unmittelbar zur Alkoholbestimmung verwendet. Die zweite Portion (220 Keimlinge =
140g) wurde unmittelbar in den P ettenk o ferschen Apparat
gebracht. Die dritte Portion (350 Keimlinge = 217 g) wurde
erst erfroren und dann ebenfalls in den Pettenkoferschen
Apparat gebracht. Temperatur 20°. Wasserstoifstrom.
a) Kontrollportion. Das erhaltene Destillat gab folgende
Reaktionen: 1. Reaktion mit fuchsinschwefliger Säure negativ.
2. Jodoformprobe nach Müntz positiv. 3. Reaktion von Berthelot
positiv. Die quantitative Bestimmung ergab jedoch, daß nur
Spuren von Alkohol im Destillat vorhanden waren.
b) Lebende Keimlinge. Versuchsdauer 23 Stunden.
C02 = 336,0 mg. Die quantitative Bestimmung ergab, daß die
Menge des Alkohols gleich 240,1 mg war. Da aber das letzte
Destillat eine sehr starke Reaktion mit fuchsinschwefliger Säure
gab, so wurde eine abgewogene Menge der Flüssigkeit mit
Natriumbisulfit abdestilliert; das erhaltene Destillat wurde mit
Natriumcarbonat neutralisiert und wieder abdestilliert. Das
letzte Destillat gab folgende Reaktionen: 1. Reaktion mit fuchsinschwefliger Säure negativ. 2. Jodoformprobe nach Müntz
positiv. 3. Reaktion von Berthe lot positiv.
Die quantitative Bestimmung ergab: C2H5OH = 212,1 mg.
C02 : C2H-OH == 336 : 212,1 = 100 : 63,1.
c) Erfrorene Keimlinge. Versuchsdauer 23!/2 Stunden.
C02 = 46,0 mg. Das letzte Destillat gab folgende Reaktionen:
1. Reaktion mit fuchsinschwefliger Säure positiv. 2. Jodoformprobe nach Müntz positiv. Eine .abgewogene Menge der Flüssigkeit wurde erst mit Natriumbisulfit und dann mit Natriumcarbonat
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228
W. Palladia und S. Kostytschew,
abdestilliert. Das erhaltene Destillat gab folgende Reaktionen:
1. Reaktion mit fuchsinschwefliger Säure negativ. 2. Jodoformprobe nach Müntz negativ. 3. Reaktion von Berthelot negativ.
Das spezifische Gewicht der Flüssigkeit war gleich 1,000. Folglich war kein Alkohol im Destillat.
C02 : C2H5OH = 46 : 0.
Versuch 12.
500 9tägiger Keimlinge von Lupinus l u t e u s (Länge des
Hypokotyls 3—6 cm) wurden erfroren und in den Pettenkof ersehen Apparat gebracht. Temperatur 20° Wasserstoffstrom.
Versuchsdauer 29 Stunden. C02 = 56,8 mg.
Der Alkohol wurde nicht bestimmt.
Aus den Versuchen mit Lupinensamen und Lupinenkeimlingen läßt sich folgendes schließen:
Die anaerobe Atmung lebender Lupinensamen ist mit der
Alkoholgärung beinahe identisch. Die anaerobe Atmung lebender
Lupinenkeimlinge ist zwar keine typische Alkoholgärung, doch
entstammt auch in diesem Falle mehr als die Hälfte der C02
dem Prozeß der Alkoholgärung. Die anaerobe Atmung erfrorener Lupinensamen und Keimlinge hat dagegen mit der
Alkoholgärung nichts zu tun, da in diesem Falle überhaupt
keine Alkoholbildung stattfindet. Es ist danach zu schließen,
daß die Alkoholproduktion lebender Samen und Keimlinge entweder durch die Tätigkeit des Plasmas selbst erfolgt, oder es
wird die Lupinenzymase durch niedere Temperaturen zerstört;
diese letztere Ansicht scheint uns wahrscheinlicher zu sein.
Die Gefriermethode ermöglicht jedenfalls eine Trennung anaerober
Atmungsvorgänge und liefert zugleich den Beweis, daß die anaerobe Atmung auch bei nachgewiesener Abwesenheit der Zymase
möglich ist. Auf der Fig. II. sind CO^-Ausscheidung und Alkoholbildung der Lupinensamen und Lupinenkeimlinge (auf 100 g der
Frischsubstanz bezogen) graphisch dargestellt worden.
Versuche mit Samen von Ricinus communis.
Versuch 13.
Samen von Ricinus communis major wurden im Verlauf von drei Tagen im Wasser geweicht, dann abgeschält und
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Anaerobe Atmung, Alkoholgärung usw. bei den Samenpflanzen.
229
in drei Portionen zu je 200 Samen geteilt. Die erste Portion
(82 g) wurde direkt zur Alkoholbestimmung verwendet, die
zweite Portion (83 g) wurde unmittelbar in den Pet t enko ferschen
Apparat gebracht; die dritte Portion wurde erst erfroren und
dann ebenfalls in den Pettenkoferschen Apparat gebracht.
Temperatur 20°. Wasserstoffstrom.
a)Kontrollportion.
Das erhaltene Destillat
gab folgende Reaktionen: 1. Reaktion mit
fuchsinschwefliger
Säure positiv. 2. Jodoformprobe nach Müntz
positiv. Das spezifische
Gewicht des Destillates
war gleich l,0000. Eine
LX
abgewogene Menge der
Flüssigkeit wurde erst
mit Natriumbisulfit und
dann mitNatriumcarbonat abdestilliert. Das erhaltene Destillat gab folgende Reaktionen: 1.
Reaktion mit fuchsinSamen,
schwefliger Säure negaFig. .
tiv. 2. Jodoformprobe
Lupinus
luteus.
)
anaerobe
Atmung der lebenden
nach Müntz negativ.
Samen und Keimlinge, 5) Alkoholbildung der leben3. Reaktion von Ber- den Samen und Keimlinge, c) anaerobe Atmung der
erfrorenen Samen und Keimlinge.
thelot negativ. Das
spezifische Gewicht der Flüssigkeit war gleich 1,0000. Folglich
war das Destillat vollständig alkoholfrei.
Durch dieses Ergebnis wird das Unzulängliche der Jodoformprobe in überzeugender Weise klar gelegt.
b) Lebende Samen. Versuchsdauer 23 Stunden. C02 =
90,0 mg. Das erhaltene Destillat gab folgende Reaktionen:
1. Reaktion mit fuchsinschwefliger Säure positiv. 2. Jodoformprobe nach Müntz positiv. 3. Reaktion von Berthelot positiv.
Hoppe-Seyler's Zeitschrift f. physiol. Chemie. XLVHL
16
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230
W. Palladin mid S. Kostytschew,
Die quantitative Bestimmung ergab: C2H5OH = 53,9 mg.
C02 : C2H5OH = 90 : 53,9 = 100 : 59,9.
c) Erfrorene Samen. Versuchsdauer 47 Stunden. C02 =
171,6 mg. Das letzte Destillat gab dieselben Reaktionen, wie
das Destillat von lebenden Samen. Die quantitative Bestimmung
ergab: C2H5OH = 101,0 mg.
C02 : C2H5OH = 171,6 : 101 = 100 : 58,9.
Aus diesem Versuche ist ersichtlich, daß das Gefrieren
keinen Einfluß hat auf die Größe des Verhältnisses C02: C2H-OH
bei den Ricinussamen. Es darf jedoch nicht vergessen werden,
daß die gequollenen Ricinussamen viel Öl und wenig Wasser
enthalten, wodurch ein totales Gefrieren bedeutend erschwert wird.
Versuche mit Weizenkeimen.
Versuch 14.
170g geweichter und dann erfrorener Weizenkeime wurden
mit 400 ccm eines ausgekochten Weizenkeimextraktes und 8 g
NaF in einen geräumigen Kolben gebracht. Alsdann wurde ein
konstanter Wasserstoffstrom durch die Flüssigkeit getrieben.
Temperatur 19°.
Was s er s t off ström,
Zeitdauer in
Stunden
C02 in
C08 pro 1 Stunde in
8'/t
50,4
134,0
33,2
28,0
20,0
5,9
5,3
1,9
1,0
0,9
0,6
25
18
27 Yt
22
18
110
mg
9,2
mg
274,8
Alkoholbestimmung.
Das letzte Destillat gab folgende Reaktionen: 1. Reaktion
mit fuchsinschwefliger Säure positiv. 2. Jodoformprobe nach
Müntz positiv. 3. Reaktion von Berthelot positiv.
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231
Die quantitative Bestimmung ergab: C2H5OH = 111,2mg.
Das Destillat gab die Jodoformreaktion in der Kälte. Auch bei
Zusatz von NH3 und Jodtinktur wurde Jodoformbildung beobachtet (Reaktion von Gunning). Danach ist zu schließen,
daß das Destillat acetonhaltig war. Die Menge des Alkohols
wurde also vielleicht «twas zu groß gefunden; auch wurde leider
kein Kontrollversuch ausgeführt. Aus diesen Gründen wurde
bei diesem Versuche C02 : C2H5OH nicht berechnet.
Versuch 15.
Im Wasser geweichte Weizenkeime. 247 g davon wurden
unmittelbar zur Alkoholbestimmung verwendet. Eine andere
Portion (100 g) wurde erfroren und in den Pettenkoferschen
Apparat gebracht. Temperatur 19°.
Erfrorene Weizenkeime (Wasserstoffström).
Zeitdauer in
Stunden
C02 in
C02 pro 1 Stunde in
5
2
2V*
6
14
'/·'
20
23
16
187,8
76,0
56,4
100,8
118,8
72,8
138,0
94,4
56,4
37,6
38,0
22,6
16,8
97
901,4
mg
mg
8,5
8,5
6,9
4,1
3,5
Alkoholbestimmungen.
a) Kontrollportion. Das erhaltene Destillat gab folgende
Reaktionen: 1. Reaktion mit fuchsinschwefliger Säure positiv.
2. Jodoformprobe nach Müntz positiv. 3. Reaktion von Berthelot positiv. Die quantitative Bestimmung ergab: 02
==
104,9 mg. Eine abgewogene Menge der Flüssigkeit wurde erst
mit Natriumbisulfit, dann mit Natriumcarbonat abdestilliert. Das
letzte Destillat gab folgende Reaktionen: 1. Reaktion mit fuchsin16*
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232
W. Palladin und S. Kostytschew,
schwefliger Säure negativ. 2. Jodoformprobe positiv. 3. Reaktion von Berthelot positiv.
Die quantitative Bestimmung ergab: C2H5OH = 106,1 mg.
Die mit fuchsinschwefliger Säure reagierende Substanz war also
in minimaler Menge vorhanden. Der Rückstand von der unter
Zusatz von Natriumbisulfit ausgeführten Destillation wurde mit
Natriumcarbonat zerlegt und wieder abdestilliert. Das erhaltene
Destillat gab mit fuchsinschwefliger Säure rote Färbung; die
Jodoformprobe in der Kälte (Liebensche Reaktion) und die
Reaktion von Gunning fielen jedoch negativ aus.
b) Versuchsportion. Das durch Destillation mit Natriumbisulfit und Natriumcarbonat gereinigte Destillat gab folgende
Reaktionen: 1. Reaktion mit fuchsinschwefliger Säure negativ.
2. Jodoformprobe nach Müntz positiv. 3. Reaktion von Berthelot positiv.
Die quantitative Bestimmung ergab: C2H5OH = 879,2 mg.
C2H5OH in 100 g frischer Keime = 42,5 mg.
Von den erfrorenen Keimen wurde gebildet: C2H5OH =
836,7 mg.
C02 : C2H5OH = 901,4 : 836,7 = 100 : 92,8.
Die anaerobe Atmung erfrorener Weizenkeime ist also
mit der Alkoholgärung beinahe identisch.
Der Rückstand von der unter Zusatz von Natriumbisulfit
ausgeführten Destillation wurde mit Natriumcarbonat zerlegt
und wieder abdestilliert. Das erhaltene Destillat gab folgende
Reaktionen: 1. Mit fuchsinschwefliger Säure rote Färbung.
2. Mit Nitroprussidnatrium in schwach alkalischer Lösung rote
Färbung (Legalsche Reaktion). 3. Mit Jodtinktur und NH3 in
der Kälte Jodoformbildung (Gunningsche Reaktion). 4. Mit Jodjodkalium und Natronlauge ausgiebige Jodoformausscheidung in
der Kälte (Liebensche Reaktion).
Durch diese Proben wird die Anwesenheit des Acetons
im Destillat festgestellt. Für eine quantitative Bestimmung war
jedoch die Menge des Acetons zu gering. Die Acetonbildung
erfolgt auch bei Sauerstoffzutritt, wie es aus folgendem Versuche ersichtlich ist.
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233
Versuch 16.
100 g in Wasser geweichter Weizenkeime wurden erfroren,
in ein U-Rohr gebracht und mit einer mit Toluol getränkten
Wasserschicht bedeckt. Im Verlauf von 97 Stunden wurde
ein konstanter Luftstrom durch das Rohr geleitet. Alsdann
wurde das Versuchsmaterial und das Wasser der Waschflasche
in einen Destillationskolben gebracht, mit destilliertem Wasser
versetzt und mehrfacher Destillation unterworfen. Das letzte
Destillat wurde mit Natriumbisulfit versetzt und wieder abdestilliert. Der Rückstand von dieser Destillation wurde mit
Natriumcarbonat behandelt und wieder abdestilliert. Das erhaltene Destillat gab sämtliche im Versuch 15 angegebenen
Acetonreaktionen. Es bleibt noch einstweilen dahingestellt, ob
bei der Atmung erfrorener Weizenkeime direkte Acetonbildung
stattfindet: es ist ja wohl möglich, daß sich eine nicht beständige,
bei der Destillation Aceton abspaltende Substanz (wie z. B.
Acetessigsäure) im Versuchsmaterial anhäuft.
Die Acetonbildung bei der Atmung ist eine in der tierischen
Physiologie schon längst bekannte Tatsache.1) In der letzten
Zeit wurde u. a. die nicht unwahrscheinliche Vermutung ausgesprochen,2) daß Aceton bei der Oxydation des Leucins ababgespalten wird:
dug
^*"8
^"8
CH
CHa
l
^**8
CO
>
CH8
l
CHNHe
CHNH8
GOOH
COOH
In unseren Versuchen haben wir eine zweifellose Acetonbildung nur bei Weizenkeimen beobachtet.
l
) Eine gute Zusammenfassung der Literatur über das Aceton findet
man bei R. Waldvogel «Die Acetonkörper>, Stuttgart 1903.
*) v. Noorden u. Embden, Zentralblatt für die gesamte Physiologie u. Pathologie des Stoffwechsels, Bd. I, 1906, S. 2.
Embden, Salomon und Schmidt, Hofmeisters Beiträge,
Bd. VIII, 1906, S. 129.
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234
W. Palladin und S. Kostytschew,
Versuche mit Samen von Pisum sativum.
Versuch 17.
Samen von Pisum sativum wurden im Verlauf von
24 Stunden unter einer dünnen Wasserschicht geweicht, dann
abgeschält und in zwei Portionen zu je 250 Samen geteilt.
Die erste Portion (180g) wurde zur Kontrolle verwendet, die
andere (185 g) wurde erfroren und in den Pettenkoferschen
Apparat gebracht. Temperatur 19°.
Was s er stoff ström.
Zeitdauer in
Stunden
C02 in
C02 pro 1 Stunde in
IV*
2V*
3V*
12V*
3
2V*
26,1
36,8
30,9
15,0
14,4
10,9
17
7
21
22
21
39,2
92,0
108,2
186,4
43,2
27,2
30,0
80,4
36,8
79,6
56,4
48,0
117
827,4
33/4
mg
mg
8,0
4,8
5,2
3,8
2,6
2,3
Alkoholbestimmungen.
a) Kontrollportion. Das letzte Destillat gab folgende
Reaktionen: I.Reaktion mit fuchsinschwefliger Säure schwach
positiv. 2. Jodoformprobe nach Müntz positiv. 3. Reaktion von
Berthelot positiv.
Die quantitative Bestimmung gab: C2H5OH = 106,4 mg.
b) Versuchsportion. Das letzte Destillat gab dieselben
Reaktionen wie das Kontrolldestillat.
Die quantitative Bestimmung ergab: C2H5OH = 728,3 mg.
C2H4OH in 250 frischen Samen = 106,4mg.
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Anaerobe Atmung, Alkoholgärung uaw. bei den Samenpflanzen.
235
Von den erfrorenen Samen wurde gebildet: C2H5OH =
621,9 mg.
C02 : C2H-OH = 827,4 : 621,9 = 100 : 75,2.
Versuch 18.
Samen von Pisum sativum wurden im Verlauf von
24 Stunden unter einer dünnen Wasserschicht geweicht, dann
abgeschält und für 2 Stunden auf Fließpapier gelegt. Alsdann
wurden die Samen iri drei Portionen zu je 200 Stück geteilt.
Die erste Portion wurde zum Kontrollversuch verwendet; die
beiden anderen wurden erfroren und in den Pettenkoferschen
Apparat gebracht. Temperatur 20°.
Erfrorene Erbsensamen.
G08 in Milligramm
Versuchsdauer in
Stunden
2. Portion (119 g)
Luftstrom
3. Portion (121 g)
Wasserstoffstrom
98
1482,0
775,2
Alkoholbestimmungen.
a) Kontrollportion. Das erhaltene Destillat gab folgende
Reaktionen: 1. Reaktion mit fuchsinschwefliger Säure positiv.
2. Jodoformprobe nach Müntz positiv. 3. Reaktion von Berthelot
schwach positiv. 4. L egal sehe Reaktion positiv. 5. Reaktion
von Gunning negativ.
Die quantitative Bestimmung ergab: C2H5OH = Spur.
b) Luftportion. Das letzte Destillat gab dieselben Reaktionen wie im Versuch 17. Die quantitative Bestimmung
ergab: C2H5OH = 1013,4mg.
C02 : C2HäOH = 1482,0 : 1013,4 = 100 : 68,4.
c) Wasserstoffportion. Das letzte Destillat gab dieselben Reaktionen wie im Versuch 17.
Die quantitative Bestimmung ergab: C2H5OH = 552,7 mg,
C02 : C2H-OH = 775,2 : 552,7 == 100 : 71,3.
Die Versuche 17 und 18 zeigen, daß die anaerobe Atmung
erfrorener Erbsensamen zum größten Teil Alkoholgärung ist.
Das Verhältnis C0 2 : C2H5OH entspricht jedoch nicht demjenigen,
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236
W. Palladin und S. Kostytschew,
das von Godlewski und Polzeniusz 1 ) bei lebenden Erbsensamen gefunden wurde. Es scheint also nicht ganz unwahrscheinlich zu sein, daß durch die Einwirkung niedrigerer Temperatur eine totale Zerstörung der Erbsenzymase bewerkstelligt
werden könnte. Derartige Versuche (unter Anwendung der
flüssigen Luft) beabsichtigen wir zu unternehmen.
Obschon die in unseren Versuchen ausgeführte Erfrierung
für die Zerstörung der Erbsenzymase unzureichend war, waren
die Samen dennoch getötet; ein Zeugnis dafür ist die Tatsache,
daß C02 : C2H5OH erfrorener Samen bei Sauerstoffzutritt derselbe ist, wie bei Sauerstoffabschluß. Bei der Sauerstoffatmung
lebender Samen wird im Gegenteil eine nur unbedeutende Menge
Alkohol gebildet, wie aus dem folgenden Versuche ersichtlich ist.
Versuch 19.
Samen von Pisum sativum wurden im Verlauf von
24 Stunden unter einer dünnen Wasserschicht geweicht, dann
abgeschält und für 2 Stunden auf Fließpapier gelegt. Alsdann
wurden die Samen in drei Portionen zu je 300 Samen geteilt.
Die erste Portion wurde zum Kontrollversuch verwendet, die
beiden anderen Portionen wurden in den Pettenkoferschen
Apparat gebracht. Temperatur 22—24°.
Lebende Erbsensamen (Luftstrom).
2. Portion
Versuchsdauer in
C02 in
mg
Stunden
24
1204,0
3. Portion2)
Versuchsdauer in
G02 in
Stunden
mg
22
1052,8
Alkoholbestimmungen.
a) Kontrollportion. Das erhaltene Destillat gab dieselben Reaktionen wie im Versuch 17.
Die quantitative Bestimmung ergab: C2H5OH = 113,1 mg.
1
) Godlewski u. Polzeniusz, Bulletin del'Academie des sciences
de Cracovie, 1901, S. 227.
2
) Die Luftdurchleitung wurde bei der Portion 3 für die ganze
Nacht unterbrochen. Infolgedessen hat sich eine bedeutende Menge des
Alkohols gebildet.
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237
b) 2. Portion. Das erhaltene Destillat gab folgende
Reaktionen: 1. Reaktion mit fuchsinschwefliger Säure positiv.
2. Jodoformprobe nach Müntz positiv. 3. Reaktion von Berthelot
positiv.
Die quantitative Bestimmung ergab: C2H5OH = 273,0 mg.
C2H5OH in 300 frischen Samen = 113,1 mg.
Bei der Sauerstoffatmung lebender Samen wurde gebildet: C2H5OH = 159,9 mg.
C02: C2H5OH = 1204 :159,9 = 100 :16,6.
c) 3. Portion. Das Destillat gab dieselben Reaktionen
wie bei b).
Die quantitative Bestimmung ergab: C2H5OH = 879,4 mg.
C2H5OH in 300 frischen Samen = 113,1 mg.
Von den lebenden Samen wurde gebildet: 766,3 mg.
C0 2 : C2H5OH = 1052,8 : 766,3 = 100 : 72,7.
Wie aus der Anmerkung auf S. 236 ersichtlich, befand
sich die Portion 3 hauptsächlich in einer sauerstoiffreien
Atmosphäre.
Überblicken wir die Ergebnisse unserer Versuche, so ersehen wir eine vollkommene Bestätigung der von einem von
uns bereits vor 2 Jahren ausgesprochenen Voraussetzung: *)
«Wenn so häufig von der Identität der anaeroben Atmung mit
der Alkoholgärung gesprochen wird, so läßt sich dies wahrscheinlich dadurch erklären, daß . . . eine Anzahl von Übergangstypen existiert, die gleichzeitig mit der anaeroben Atmung . . .
eine mehr oder weniger typische Alkoholgärung hervorzurufen
imstande sind.»
Auch der andere von uns hat sich folgendermaßen ausgesprochen : 2 ) « . . . der in gefrorenen Blättern sich abspielende
anaerobe Prozess der Kohlensäurebildung nichts mit der Alkoholgärung gemein hat, da jener Prozess am energischsten in denjenigen Blättern verläuft, die keine Kohlehydrate enthalten, ja
sogar durch Einführung von Saccharose nur abgeschwächt
i) Kostytschew, Zentralbl. f. Bakteriol., II. Abt., Bd.
S. 401.
8
) Palladin, Diese Zeitschrift, Bd. XLVII, 1906, S. 417.
, 1904,
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238
W. Palladin und S. Kostytschew,
wird
Durch alle diese Ausführungen will ich keineswegs die Möglichkeit der Alkoholgärung bei den höheren Pflanzen
verneinen, glaube aber nur, daß sie eine Nebenrolle spielt und
nicht als ein Fundamentalprozeß bezeichnet werden kann.»
In der Tabelle S. 239 sind die wichtigsten Zahlenresultate
einzelner Versuche zusammengestellt.
Zusammenfassung der wichtigsten Ergebnisse.
1. Bei der anaeroben Atmung lebender Lupinensamen und
Lupinenkeimlinge wird eine beträchtliche Menge Alkohol gebildet. Die anaerobe Atmung dieser Objekte ist also im wesentlichen mit der Alkoholgärung identisch. Bei der an aeroben
Atmung erfrorener Lupinensamen und Lupinenkeimlinge findet
überhaupt keine Alkoholbildung statt; auch bei der anaeroben
Atmung erfrorener Stengelgipfel von Vicia Faba werden keine
nennenswerten Mengen Alkohol gebildet. Die anaerobe
Atmung erfrorener Lupinensamen, Lupinenkeimlinge
und Stengelgipfel von Vicia Faba hat also mit der
Alkoholgärung nichts zu tun.
2. Bei der anaeroben Atmung lebender und erfrorener
Erbsensamen, Ricinussamen und Weizenkeime findet eine beträchtliche Alkoholbildung statt. Die anaerobe Atmung dieser
Objekte ist also zum größten Teil Alkoholgärung. Durch das
bei unseren Versuchen in Anwendung gebrachte Gefrieren wurden
die genannten Pflanzen getötet, die in ihnen befindliche Zymase
wurde jedoch nicht zerstört.
3. Bei lebenden Erbsensamen wird eine Anhäufung des
Alkohols nur bei Sauerstoffabschluß beobachtet. Erfrorene
Erbsensamen häufen dagegen beträchtliche Mengen Alkohol bei
vollem Sauerstoffzutritt an. Dies erklärt sich dadurch, daß die
Oxydationsvorgänge in Pflanzenzellen infolge der Abtötung bedeutend abgeschwächt werden.
4. Die Meinung Mazes, Godlewskis und Stoklasas
bezüglich der Anwesenheit der Zymase bei Samenpflanzen wird
durch unseren Versuch bestätigt. Es bleibt noch freilich dahingestellt, ob die Zymase der Samenpflanzen mit der Hefezymase
identisch ist.
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239
5. Bei der normalen und anaeroben Atmung lebender und
erfrorener Pflanzen werden unter Umständen Aceton und andere
mit fuchsinschwefliger Säure reagierende Substanzen gebildet.
E r f r o r e n e Pflanzen
Frisch- Ver- Menge Menge Menge
der
des des Alge- suchs- ausge- gebil- kohols
dauer
wicht
schie- deten auf
m
in Stun- denen Alko- 100mg
C02 in lols in der
g
den
mg
mg
C02
97
9014 8367
928
165
119
121
116
98
98
827,4 621,9
1482,0 1013,4
775,2 552,7
75,2
68,4
71,3
81
47
171,6 101,0
58,9
14-5
117
209
26 2692 ' Spur
27 181,6 31,1
61V» 182,8 33,8
281/· 313,6 26,6
0
17,1
18,5
8,4
Gipfel der Stengel vonViciaFaba
nach Zucker- und Lichternährung
178
26
74,0
33,0
500 Samen von Lupinus luteus .
500
»
»
»
»
400
»
»
»
»
350 Lupinenkeimlinge
500
»
130
104
101
217
335
24 152,0
25
84,0
46 238,8
23 V* 460
24
56,8
Weizenkeime
250 Samen von Pisum sativum .
200
»
»
»
»
.
200
»
»
»
»
200 Samen von Ricinus comGipfel der etiolierten Stengel von
Dasselbe
Dasselbe
Dasselbe
120
224,0
0 ' 0
0
0
83,6 35,0
0
0
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Efficacy and safety of IVIG in CIDP: Combined data of the PRIMA and PATH studies
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Journal of the peripheral nervous system
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cc-by
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wileyonlinelibrary.com/journal/jns Ingemar S.J. Merkies1,2 | Ivo N. van Schaik3 | Jean-Marc Léger4 | Vera Bril5,6 |
Nan van Geloven7 | Hans-Peter Hartung8 | Richard A. Lewis9 | Gen Sobue10 |
John-Philip Lawo11 | Billie L. Durn11 | David R. Cornblath12 | Jan L. De Bleecker13 |
Claudia Sommer14 | Wim Robberecht15 | Mika Saarela16 | Jerzy Kamienowski17 |
Zbigniew Stelmasiak18 | Björn Tackenberg19 | Orell Mielke11 | on Behalf of the PRIMA Trial
Investigators and the PATH Study Group Ingemar S.J. Merkies1,2 | Ivo N. van Schaik3 | Jean-Marc Léger4 | Vera Bril5,6 |
Nan van Geloven7 | Hans-Peter Hartung8 | Richard A. Lewis9 | Gen Sobue10 |
John-Philip Lawo11 | Billie L. Durn11 | David R. Cornblath12 | Jan L. De Bleecker13 |
Claudia Sommer14 | Wim Robberecht15 | Mika Saarela16 | Jerzy Kamienowski17 |
Zbigniew Stelmasiak18 | Björn Tackenberg19 | Orell Mielke11 | on Behalf of the PRIMA Tria
Investigators and the PATH Study Group 6Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
7Department of Medical Statistics and Bioinformatics, Leiden University Medical Center, Leiden, The Netherlands
8Department of Neurology, UKD and Center for Neurology and Neuropsychiatry, LVR Klinikum, Medical Faculty, Heinrich Heine University, Düsseldo
9Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, California
10Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan
11CSL Behring, Marburg, Germany, and King of Prussia, Pennsylvania
12Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland
13Department of Neurology, AZ St-Lucas, Ghent, Belgium
14Department of Neurology, Universitätsklinikum Würzburg, Würzburg, Germany
15Department of Neurosciences, UZ Leuven, Leuven, Belgium
16Department of Neurology, Helsinki University Central Hospital, Helsinki, Finland
17Dolnosląski Szpital Specjalistyczny, Wrocław, Poland
18Department of Neurology, Samodzielny Publiczny Szpital Kliniczny, Lublin, Poland
19Department of Neurology, Philipps University, Marburg, Germany Correspondence
Ingemar S. J. Merkies, MD, PhD, Neurologist,
Address: St. Elisabeth Hospital, Department of
Outpatient Neurology, Breedestraat 193 (O),
Willemstad, Curaçao. Email: isjmerkies@hotmail.com
Funding information
CSL Behring, Grant/Award Number: N/A Correspondence
Ingemar S. J. Merkies, MD, PhD, Neurologist,
Address: St. Elisabeth Hospital, Department of
Outpatient Neurology, Breedestraat 193 (O),
Willemstad, Curaçao. Email: isjmerkies@hotmail.com
Funding information
CSL Behring, Grant/Award Number: N/A Correspondence
Ingemar S. J. Merkies, MD, PhD, Neurologist,
Address: St. Elisabeth Hospital, Department of
Outpatient Neurology, Breedestraat 193 (O),
Willemstad, Curaçao. Email: isjmerkies@hotmail.com
Funding information
CSL Behring, Grant/Award Number: N/A Correspondence
Ingemar S. J. Merkies, MD, PhD, Neurologist,
Address: St. Elisabeth Hospital, Department of
Outpatient Neurology, Breedestraat 193 (O),
Willemstad, Curaçao. Email: isjmerkies@hotmail.com
Funding information
CSL Behring, Grant/Award Number: N/A Intravenous immunoglobulin (IVIG) is a potential therapy for chronic inflammatory demyelinating
polyneuropathy (CIDP). R E S E A R C H R E P O R T R E S E A R C H R E P O R T 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. Journal of the Peripheral Nervous System published by Wiley Periodicals, Inc. on behalf of Peripheral Nerve Society. R E S E A R C H R E P O R T
Efficacy and safety of IVIG in CIDP: Combined data of the
PRIMA and PATH studies
Ingemar S.J. Merkies1,2 | Ivo N. van Schaik3 | Jean-Marc Léger4 | Vera Bril5,6 |
Nan van Geloven7 | Hans-Peter Hartung8 | Richard A. Lewis9 | Gen Sobue10 |
John-Philip Lawo11 | Billie L. Durn11 | David R. Cornblath12 | Jan L. De Bleecker13 |
Claudia Sommer14 | Wim Robberecht15 | Mika Saarela16 | Jerzy Kamienowski17 |
Zbigniew Stelmasiak18 | Björn Tackenberg19 | Orell Mielke11 | on Behalf of the PRIMA Trial
Investigators and the PATH Study Group
1Department of Neurology, Maastricht University Medical Center, Maastricht, The Netherlands
2Department of Neurology, St Elisabeth Hospital, Willemstad, Curaçao
3Department of Neurology, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, The Netherlands
4National Referral Center for Rare Neuromuscular Diseases, Hôpital Pitié-Salpêtrière and University Paris VI, Paris, France
5Ellen and Martin Prosserman Centre for Neuromuscular Diseases, Division of Neurology, Department of Medicine, University Health Network, University of
Toronto, Toronto, Canada
6Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
7Department of Medical Statistics and Bioinformatics, Leiden University Medical Center, Leiden, The Netherlands
8Department of Neurology, UKD and Center for Neurology and Neuropsychiatry, LVR Klinikum, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany
9Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, California
10Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan
11CSL Behring, Marburg, Germany, and King of Prussia, Pennsylvania
12Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland
13Department of Neurology, AZ St-Lucas, Ghent, Belgium
14Department of Neurology, Universitätsklinikum Würzburg, Würzburg, Germany
15Department of Neurosciences, UZ Leuven, Leuven, Belgium
16Department of Neurology, Helsinki University Central Hospital, Helsinki, Finland
17Dolnosląski Szpital Specjalistyczny, Wrocław, Poland
18Department of Neurology, Samodzielny Publiczny Szpital Kliniczny, Lublin, Poland
19Department of Neurology, Philipps University, Marburg, Germany
Correspondence
Ingemar S. J. Merkies, MD, PhD, Neurologist,
Intravenous immunoglobulin (IVIG) is a potential therapy for chronic inflammatory demyelinating
DOI: 10.1111/jns.12302 DOI: 10.1111/jns.12302 Received: 10 October 2018
Revised: 15 November 2018
Accepted: 12 January 2019 Received: 10 October 2018
Revised: 15 November 2018
Accepted: 12 January 2019 Received: 10 October 2018
Revised: 15 November 2018
Accepted: 12 January 2019
DOI: 10.1111/jns.12302 1
|
INTRODUCTION IgPro10 that were similar to those in the IVIG arm of the ICE trial. Fur-
ther evidence of the efficacy of IgPro10 in CIDP was provided by the
IVIG phase of the recent Polyneuropathy And Treatment with Hizen-
tra (PATH) study.14 Subjects were restabilized on IgPro10 after deter-
mining
individual
Ig
dependency,
prior
to
randomization
to
subcutaneous Ig (SCIG) or placebo. Chronic inflammatory demyelinating polyneuropathy (CIDP) is charac-
terized by symmetrical, proximal, and distal weakness or somatosen-
sory alterations in the arms and legs that worsens over time.1,2 The
annual incidence of CIDP is estimated to be between 0.5 and 1.6 cases
per 100 000 individuals and peak prevalence is between 40 and
60 years of age, with rates ranging from 1.6 to 8.9 per 100 000 adults
in different regions.3–8 CIDP occurs more commonly in men than
women.1 In the current analysis, results from the PRIMA and PATH studies
are combined, aiming to determine whether the findings in the smaller
PRIMA study would be validated by the much larger PATH trial, with
the final objective to confirm the efficacy and safety of IgPro10 for
the treatment in a much larger group of patients with CIDP. The value
of this combined analysis is that it leverages similarities in study
design between the two studies, for example, same dosing paradigm
and endpoints, to analyze the efficacy of IVIG in a large, combined
patient cohort. The goals of CIDP treatment are to reduce symptoms, improve
functional ability, prevent relapse, and maintain long-term remission. Immunoglobulins (Igs), corticosteroids, and plasma exchange are con-
sidered as first-line therapies.9 Intravenous Ig (IVIG) was suggested
to be efficacious vs plasma exchange, prednisolone, and placebo in
several small trials, and in a Cochrane review, including five of these
trials, totaling 235 subjects (mainly treatment-naïve) provided evi-
dence that more subjects had improvements in disability with IVIG
treatment (44%) vs placebo (18%).10 Included in these five studies
was the landmark immune globulin intravenous CIDP efficacy (ICE)
trial, which reported efficacy in a randomized trial considered large
for the CIDP disease area.11 This double-blind study, performed in
117
CIDP
subjects,
reported
a
significantly
higher
adjusted
inflammatory neuropathy cause and treatment (INCAT) improvement
rate (54%) with IVIG compared with placebo (21%). 1
|
INTRODUCTION Recently, a Japa-
nese study (n = 49) also reported IVIG efficacy with long-term treat-
ment (induction IVIG 2 g/kg bodyweight [bw] over five consecutive
days, maintenance IVIG 1.0 g/kg bw every 3 weeks for up to
52 weeks). The study reported a response rate of 78% at 28 weeks,
with a 10.5% relapse rate within the population that continued treat-
ment to Week 52.12 MERKIES ET AL. MERKIES ET AL. 49 score. Adverse drug reactions (ADRs) and ADRs/infusion were recorded. Adjusted INCAT response
rate was 60.7% in all PRIMA subjects at Week 25 (76.9% in IVIG pre-treated subjects) and 72.9% in
PATH. In the pooled cohort (n = 235), INCAT response rate was 71.5%; median time to INCAT
improvement was 4.3 weeks. No clear demographic differences were noticed between early
(responding before Week 7, n = 148) and late responders (n = 21). In the pooled cohort, median
change from baseline to last observation was −1.0 (interquartile range −2.0; 0.0) point for INCAT
score; +8.0 (0.0; 20.0) kPa for maximum grip strength; +3.0 (1.0; 7.0) points for MRC sum score. In
the pooled cohort, 271 ADRs were reported in 105 subjects (44.7%), a rate of 0.144 ADRs per infu-
sion. This analysis confirms the efficacy and safety of IgPro10, a recently FDA-approved IVIG for
CIDP, in a population of mainly pre-treated subjects with CIDP [Correction added on 14 March
2019 after first online publication: the INCAT response rate has been corrected.]. KEYWORDS
CIDP, efficacy, IVIG, PATH, PRIMA KEYWORDS
CIDP, efficacy, IVIG, PATH, PRIMA 2.1
| PRIMA PRIMA was a prospective, open-label, single-arm study, with the pur-
pose of obtaining marketing approval for IVIG in CIDP in Europe.13
The efficacy and safety of IgPro10, for both induction therapy and
maintenance therapy, were investigated. A total of 28 subjects (IVIG
pre-treated, n = 13; previously untreated, n = 15) received one induc-
tion dose of IgPro10 (2.0 g/kg bw) over 2 to 5 consecutive days, and
up to seven maintenance doses of IgPro10 (1.0 g/kg bw) given every
3 weeks on 1 day or 2 consecutive days (total treatment period,
21 weeks). Pre-treated subjects required a 10-week wash-out period
prior to enrollment. IgPro10 (Privigen; CSL Behring, King of Prussia, Pennsylvania), a
10% human IVIG, recently approved by the FDA for CIDP, was first
shown to be effective for the treatment of CIDP in the Privigen
Impact on Mobility and Autonomy (PRIMA) study.13 This single-arm
study, performed in 28 subjects, reported clinical responses to Ingemar S.J. Merkies1,2 | Ivo N. van Schaik3 | Jean-Marc Léger4 | Vera Bril5,6 |
Nan van Geloven7 | Hans-Peter Hartung8 | Richard A. Lewis9 | Gen Sobue10 |
John-Philip Lawo11 | Billie L. Durn11 | David R. Cornblath12 | Jan L. De Bleecker13 |
Claudia Sommer14 | Wim Robberecht15 | Mika Saarela16 | Jerzy Kamienowski17 |
Zbigniew Stelmasiak18 | Björn Tackenberg19 | Orell Mielke11 | on Behalf of the PRIMA Trial
Investigators and the PATH Study Group To investigate the efficacy and safety of the IVIG IgPro10 (Privigen) for
treatment of CIDP, results from Privigen Impact on Mobility and Autonomy (PRIMA), a prospec-
tive, open-label, single-arm study of IVIG in immunoglobulin (Ig)-naïve or IVIG pre-treated sub-
jects (NCT01184846, n = 28) and Polyneuropathy And Treatment with Hizentra (PATH), a
double-blind, randomized study including an open-label, single-arm IVIG phase in IVIG pre-
treated subjects (NCT01545076, IVIG restabilization phase n = 207) were analyzed separately
and together (n = 235). Efficacy assessments included change in adjusted inflammatory neurop-
athy cause and treatment (INCAT) score, grip strength and Medical Research Council (MRC) sum 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. Journal of the Peripheral Nervous System published by Wiley Periodicals, Inc. on behalf of Peripheral Nerve Society. work is properly cited. . Journal of the Peripheral Nervous System published by Wiley Periodicals, Inc. on behalf of Peripheral Nerve Society. J Peripher Nerv Syst. 2019;24:48–55. 48 being randomized to maintenance therapy with SCIG or placebo. Sub-
jects first completed a 4- to 12-week IVIG-dependency test period,
receiving no IgG therapy (wash-out period). Subjects showing a deteri-
oration (increase in adjusted INCAT by ≥1 before amendment 3; after
amendment 3: increase in adjusted INCAT by ≥1, decrease in inflam-
matory Rasch-built overall disability scale (I-RODS) by ≥4 points, or
decrease in grip strength by ≥8 kPa) progressed to the subsequent
IgPro10 restabilization period, in which subjects initially received an
IgPro10 induction dose of 2 g/kg bw, administered over 2 to 5 consec-
utive days, with a maximum of 1 g/kg bw on a single day. This was fol-
lowed by maintenance doses of 1 g/kg bw every 3 weeks, given on
1 day or over 2 consecutive days, during Weeks 4, 7, and 10. Depend-
ing on the time needed to achieve IgPro10 restabilization, a fourth
dose could be given during Week 13. in a Kaplan-Meier analysis; subjects without response were censored
at the date of their last visit. A post-hoc analysis investigated baseline
characteristics of early responders (those responding by INCAT within
7 weeks) vs those who responded after Week 7 using descriptive sta-
tistics. Week 7 cut-off was based on clinical relevance. All analyses
were done using the statistical analysis system (SAS) software package
(SAS Institute, Cary, North Carolina) version 9.2 or higher. 2.3
| Patients The inclusion and exclusion criteria were generally similar for the two
studies.13,14 The main inclusion criteria were age >18 years and defi-
nite or probable CIDP according to the European Federation of Neu-
rological
Societies/Peripheral
Nerve
Society
(EFNS/PNS)
2010
criteria.9 Subjects with polyneuropathy of other causes, diseases that
may cause neurological symptoms, or a history of thrombotic episodes
were excluded. An important difference between the studies was the
enrollment of only pre-treated subjects in PATH, compared with pre-
treated and treatment-naïve subjects in PRIMA. TABLE 1
Baseline demographics and patient characteristics of
PRIMA and PATH TABLE 1
Baseline demographics and patient characteristics of
PRIMA and PATH
Parameter
PRIMA
N = 28
PATH
N = 207
Total
N = 235
Demographic characteristics
Sex, n (%)
Female
10 (35.7)
76 (36.7)
86 (36.6)
Male
18 (64.3)
131 (63.3)
149 (63.4)
Race, n (%)
White
28 (100.0)
186 (89.9)
214 (91.1)
Asian
0
17 (8.2)
17 (7.2)
American Indian or
Alaska Native
0
1 (0.5)
1 (0.4)
Other
0
3 (1.4)
3 (1.3)
Age (years)
Mean (SD)
58.7 (14.34)
56.5 (12.76)
56.8 (12.95)
Median (range)
58.0 (22–79)
58.2 (25–83)
58.0 (22–83)
Primary disease characteristics
Diagnosis of definite
CIDPa, n (%)
23 (82.1)
185 (89.4)
208 (88.5)
Time since diagnosis of
CIDP (years), median
(range)
2.1 (0.1–13.9)
3.0 (0.1–33.5)
2.7 (0.1–33.5)
Prior IVIG treatment, n (%)
Pre-treated
13 (46.4)
207 (100.0)
220 (93.6)
Untreatedb
15 (53.6)
0
15 (6.4)
Screening INCAT total
score (points), mean
(SD)
2.9 (1.18)
2.7 (1.67)
2.8 (1.62) 3.1
| Populations The populations in PRIMA and PATH were similar in demographic and
baseline characteristics and primary disease characteristics (Table 1). The main differences between the studies were the size of the study
populations and pre-study use of IVIG (46.4% [n = 13] of subjects in the
PRIMA trial were pre-treated with IVIG, while all subjects in PATH were
pre-treated with IVIG). When data from PRIMA and PATH were pooled,
a total of 233 subjects were treated with IgPro10. Two subjects contrib-
uted data to both studies, giving a total pooled population of 235. 2.2
| PATH The same dosing regimen from PRIMA was used in 207 IVIG pre-
treated subjects during the IVIG restabilization phase (10–13 weeks)
of the PATH study.14 Study participants completed this phase before MERKIES ET AL. 50 2.4
| Outcome measures and response criteria A ≥1 point decrease (indicating improvement) from reference visit
value in adjusted INCAT disability score was considered to be a
response criterion in both studies (response had to be observed at last
observation in PRIMA to be classed as a responder; in PATH response
was recorded if the patient improved at any visit during the 13-week
observation period). Changes in maximum grip strength (using a Vig-
orimeter from Martin; Tuttlingen, Germany) and Medical Research
Council (MRC) sum score were also assessed. Treatment response for grip strength was defined in the PATH
study as an improvement of ≥8 kPa. No grip strength-related
responder criteria were prospectively defined in PRIMA, however
improvement of ≥8 kPa was used in a post-hoc analysis.15 For MRC
sum score, improvement was defined as an increase of ≥3 points in
both studies. Prior IVIG treatment, n (%)
Pre-treated
13 (46.4)
207 (100.0)
220 (93.6)
Untreatedb
15 (53.6)
0
15 (6.4)
Screening INCAT total
score (points), mean
(SD)
2.9 (1.18)
2.7 (1.67)
2.8 (1.62) Treatment-emergent adverse events (AEs) and adverse drug reac-
tions (ADRs) were assessed in both studies, in terms of the number of
events per infusion as well as percentages of subjects affected. AEs
were defined as ADRs if they were temporally associated with study
intervention (from start of infusion up to 72 hours after the end of
infusion) or considered to be causally related to IgPro10. Abbreviations: CIDP, chronic inflammatory demyelinating polyneuropathy;
INCAT, inflammatory neuropathy cause and treatment; PATH, Polyneuro-
pathy And Treatment with Hizentra; PRIMA, Privigen Impact on Mobility
and Autonomy. 3.2
| Efficacy PRIMA
IVIG pre-treated
Total
IVIG untreated Total IVIG pre-treated The response rate, based on adjusted INCAT scores in the overall
PRIMA population was 60.7% at Week 25 (95% CI: 42.4–76.4), 76.9%
in PRIMA pre-treated subjects, and 46.7% in PRIMA treatment-naïve
subjects. The response rate in PATH subjects at Week 13 was 72.9%
(95% CI: 66.5–78.5) as shown in Table 2. In the pooled cohort
(n = 235), INCAT response rate was 71.5% (95% CI: 65.9–77.3) [Cor-
rection added on 14 March 2019 after first online publication: the
INCAT response rate has been corrected.]. The median time to first INCAT response was 3.0 weeks in
PRIMA pre-treated subjects, 18 weeks in PRIMA treatment-naïve
subjects and 3.7 weeks in PATH subjects. Cumulative INCAT
response rate by time for the separate studies is presented in Figure 1
and INCAT response rate by week is shown in Table S1. In PRIMA
IVIG pre-treated subjects, 70.0% (7/10) of the responders responded
after the induction dose (Week 4), and all pre-treated responders
responded by Week 10; 25% of the treatment-naïve responders
responded by Week 4, 75% by Week 7. In the overall population of
PRIMA, 50.0% (9/18) of the responders at Week 25 responded after
the induction dose (as assessed at Week 4), and all responders at
Week 25 had responded by Week 19. In the PATH study, where all
subjects had received IVIG pre-treatment, 68.2% (103/151) of
responders responded after the induction dose (Week 4), and all
except two responders did so by Week 10. In the pooled cohort
(n = 235), median time to first INCAT improvement was 4.3 weeks. A
post-hoc analysis was undertaken in the pooled analysis to evaluate
subject baseline characteristics in early responders (those responding
by INCAT by Week 7 [n = 148]) vs late responders (those responding
after Week 7 [n = 21]). Early responders appeared to be slightly youn-
ger than late responders (55 years vs 61 years) and slightly more early
responders were diagnosed with definite CIDP (93% vs 86%; Table S2). FIGURE 1
Cumulative INCAT response rate in PRIMA and PATH. INCAT, inflammatory neuropathy cause and treatment; IVIG,
intravenous immunoglobulin; PATH, Polyneuropathy And Treatment
with Hizentra; PRIMA, Privigen Impact on Mobility and Autonomy FIGURE 1
Cumulative INCAT response rate in PRIMA and PATH. median time to first improvement in grip strength was 9.3 weeks (seen in
54.9% of subjects). 51 MERKIES ET AL. 51 70
80
60
50
40
30
20
10
0
90
100
% Responders
(adjusted INCAT score)
Ref visit
4
7
10
13
25
Time (weeks)
PRIMA
IVIG pre-treated
Total
IVIG untreated
PATH
Total IVIG pre-treated
FIGURE 1
Cumulative INCAT response rate in PRIMA and PATH. INCAT, inflammatory neuropathy cause and treatment; IVIG,
intravenous immunoglobulin; PATH, Polyneuropathy And Treatment
with Hizentra; PRIMA, Privigen Impact on Mobility and Autonomy 70
80
60
50
40
30
20
10
0
90
100
% Responders
(adjusted INCAT score)
Ref visit
4
7
10
13
25
Time (weeks)
PRIMA
IVIG pre-treated
Total
IVIG untreated
PATH
Total IVIG pre-treated 3.2
| Efficacy In the pooled cohort this was also 9.3 weeks. Median improvements in outcome measures (baseline to last
observation) for PRIMA pre-treated subjects were: INCAT, −2.0 (25%
and 75% percentile: −3.0; −1.0) points; grip strength, +5.0 (−9.0;
22.0) kPa; and MRC sum score, +5.0 (3.0; 10.0) points. For PRIMA
treatment-naïve subjects median changes were: INCAT, −1.0 (−2.0;
0.0) points; grip strength, +5.0 (−12.0, 33.0); and MRC sum score,
+6.0 (3.0; 14.0). Corresponding results for PATH subjects were:
INCAT, −1.0 (−2.0; 0.0) points; grip strength, +9.4 (1.3; 18.8) kPa; and
MRC sum score, +3.0 (0.0; 6.0) points. In the pooled cohort (n = 235)
median changes from baseline to last observation were: −1.0 (−2.0;
0.0) points for INCAT score; +8.0 (0.0; 20.0) kPa for grip strength;
and +3.0 (1.0; 7.0) points for MRC sum score. In PRIMA, the median time to first MRC sum score improvement
was 6 weeks in the overall population, 6 weeks in the treatment-naïve
population, and 3 weeks in the IVIG pre-treated population. Improve-
ment was seen in 82.1% of the overall population, 86.7% of the
treatment-naïve population, and 76.9% of the IVIG pre-treated popu-
lation. In PATH, the median time to first MRC improvement was
9.3 weeks; 56.5% of subjects improved in regards to their MRC score
during the PATH IVIG restabilization phase. In the pooled cohort,
median time to first MRC sum score improvement was also 9.3 weeks. In
PRIMA, median time to first improvement in grip strength (dominant
hand) was 7.1 weeks for pre-treated subjects and 6.1 weeks for both the
overall PRIMA population and treatment-naïve population. In PATH, the Mean changes from baseline in INCAT total score, grip strength,
and MRC sum score in PRIMA (IVIG pre-treated subjects) and PATH
are shown in Figure 2. 2.5 a According to European Federation of Neurological Societies/Peripheral
Nerve Society diagnostic criteria. No formal hypotheses were tested. Confidence intervals (CIs) were
not adjusted for multiplicity and are provided for explorative pur-
poses. Median time to first response was calculated using all subjects b Subjects with newly diagnosed CIDP (developing over ≥2 months) or
subjects with an IVIG treatment interruption for ≥1 year with a progres-
sive disease (deteriorating in the last 2 months) before enrollment. b Subjects with newly diagnosed CIDP (developing over ≥2 months) or
subjects with an IVIG treatment interruption for ≥1 year with a progres-
sive disease (deteriorating in the last 2 months) before enrollment. 4
|
DISCUSSION (
)
Grip Strength
90
100
Screening Ref visit
4
7
Deterioration
10
13
25
Time (weeks)
Grip Strength, kPa
80
70
60
50
40
Improvement
PRIMA (IVIG pre-treated, N = 13)
PATH (all IVIG pre-treated, N = 207) Grip Strength
90
100
Screening Ref visit
4
7
Deterioration
10
13
25
Time (weeks)
Grip Strength, kPa
80
70
60
50
40
Improvement
PRIMA (IVIG pre-treated, N = 13)
PATH (all IVIG pre-treated, N = 207)
MRC Sum Score
78
80
Screening Ref visit
4
7
Deterioration
10
13
25
Time (weeks)
MRC Sum Score, points
76
70
66
64
60
Improvement
PRIMA (IVIG pre-treated, N = 13)
PATH (all IVIG pre-treated, N = 207)
74
68
72
62
FIGURE 2
Mean change from baseline in INCAT total score, grip
strength, and MRC sum score in PRIMA IVIG pre-treated subjects and
PATH. INCAT, inflammatory neuropathy cause and treatment; MRC,
Medical Research Council; PATH, Polyneuropathy And Treatment
with Hizentra; PRIMA, Privigen Impact on Mobility and Autonomy In the current analysis, combination of the data originating from the
PRIMA and PATH studies resulted in the confirmation of the efficacy
of IgPro10 based on changes seen in adjusted INCAT score, grip
strength, and MRC sum score.13,14 The findings in the PRIMA trial and
findings from the larger cohort as part of the PATH trial simulta-
neously validated each other. Similar response rates and times to
response with IgPro10 in IVIG pre-treated subjects were seen in both
studies, despite PATH having a shorter study period than PRIMA. In
both studies, disability (measured by the INCAT score) and impair-
ments (muscle strength as measured by grip strength and the MRC
sum score) improved and remained stable after the IgPro10 induction
dose or at subsequent visits. Time (weeks)
MRC Sum Score
78
80
Screening Ref visit
4
7
Deterioration
10
13
25
Time (weeks)
MRC Sum Score, points
76
70
66
64
60
Improvement
PRIMA (IVIG pre-treated, N = 13)
PATH (all IVIG pre-treated, N = 207)
74
68
72
62 At least half of subjects in both studies responded after just one
induction dose of IVIG (Week 4). A total of 89% of the responders
in PRIMA and 99% of the responders in PATH responded by
Week 10 based on INCAT score (ie, after three infusions of IgPro10:
one induction dose and two maintenance doses, given at a 3 week
interval). 4
|
DISCUSSION Hence, the findings extend the knowledge that has been
extracted from the ICE trial data suggesting most subjects may
require several cycles (one induction and two maintenance doses) to
respond to therapy.16 Latov et al showed among subjects classified
as being responders that 47% had responded by Week 3 (corre-
sponding to the induction dose), and the other 53% responded by
Week 6 after a second infusion.16 The PRIMA–PATH pooled analysis
shows that a substantial number of patients become responders
after >6 weeks follow-up; however, the data also show that response
is highly unlikely after 10 weeks. This timing should be considered in
clinical practice prior to deciding if a subject is not benefitting from
treatment early in the treatment course. A recent study reported that
69% of subjects treated with IVIG for 52 weeks maintained INCAT
improvement, further supporting the acute and long-term use of
IVIGs.12 MRC Sum Score, points FIGURE 2
Mean change from baseline in INCAT total score, grip
strength, and MRC sum score in PRIMA IVIG pre-treated subjects and
PATH. INCAT, inflammatory neuropathy cause and treatment; MRC,
Medical Research Council; PATH, Polyneuropathy And Treatment
with Hizentra; PRIMA, Privigen Impact on Mobility and Autonomy 10 (76.9%) subjects (0.366/infusion), while 67 AEs were reported in
12 (80.0%) treatment-naïve subjects (0.456/infusion). A total of
284 AEs in 100 (48.3%) subjects (0.175/infusion) were reported in
the PATH safety population (n = 207). In both studies, the AE rate
per
infusion
was
lower
during
the
maintenance
treatment
(0.387/infusion in PRIMA and 0.147/infusion in PATH) than during
the induction treatment (0.493/infusion in PRIMA and 0.218/infusion
in PATH). 10 (76.9%) subjects (0.366/infusion), while 67 AEs were reported in
12 (80.0%) treatment-naïve subjects (0.456/infusion). A total of
284 AEs in 100 (48.3%) subjects (0.175/infusion) were reported in
the PATH safety population (n = 207). In both studies, the AE rate
per
infusion
was
lower
during
the
maintenance
treatment
(0.387/infusion in PRIMA and 0.147/infusion in PATH) than during
the induction treatment (0.493/infusion in PRIMA and 0.218/infusion
in PATH). This is the largest group of CIDP subjects that has undergone
well-defined outcome assessments. Previous Cochrane assessments
of a similar number of subjects were mainly on treatment-naïve sub-
jects using a variety of outcome measures, whereas the current
analysis comprised mainly pre-treated subjects with the same out-
come measures and the same IVIG induction and maintenance regi-
mens. 52 MERKIES ET AL. INCAT Total Score
1
0
Screening Ref visit
4
7
Deterioration
10
13
25
Time (weeks)
INCAT Total Score, points
2
3
4
5
6
Improvement
PRIMA (IVIG pre-treated, N = 13)
PATH (all IVIG pre-treated, N = 207) Headache was the most frequent AE in both studies, seen in
9 (32.1%) PRIMA subjects (4 pre-treated, 5 treatment-naïve subjects) and
34 (16.4%) PATH subjects (overall 42/235 subjects [18.3%]). Causally
related serious AEs were reported in 2 PRIMA subjects (hemolysis) and
7 PATH subjects (hypersensitivity, pulmonary embolism, increased blood
pressure, exacerbation of CIDP, respiratory failure, rash, migraine). INCAT Total Score INCAT Total Score, points Screening Ref visit
4
7
Deterioration
10
13
25
Time (weeks)
INCAT Total Scor
3
4
5
6
PRIMA (IVIG pre-treated, N = 13)
PATH (all IVIG pre-treated, N = 207)
Grip Strength
90
100
Screening Ref visit
4
7
Deterioration
10
13
25
Time (weeks)
Grip Strength, kPa
80
70
60
50
40
Improvement
PRIMA (IVIG pre-treated, N = 13)
PATH (all IVIG pre-treated, N = 207)
MRC Sum Score
78
80
Screening Ref visit
4
7
Deterioration
10
13
25
Time (weeks)
MRC Sum Score, points
76
70
66
64
60
Improvement
PRIMA (IVIG pre-treated, N = 13)
PATH (all IVIG pre-treated, N = 207)
74
68
72
62
FIGURE 2
Mean change from baseline in INCAT total score, grip
strength, and MRC sum score in PRIMA IVIG pre-treated subjects and
PATH. INCAT, inflammatory neuropathy cause and treatment; MRC, In the pooled population, 271 ADRs were reported in 105 subjects
(44.7%), representing a rate of 0.144 ADRs per infusion (Table 3). The
most frequent ADRs were headache, nausea, hypertension and hemo-
lysis. In PRIMA, 20 subjects (71.4%) had 71 ADRs; 85 subjects (41.1%)
had 200 ADRs in PATH. There were no deaths during PRIMA or dur-
ing the IgPro10 restabilization phase of PATH. 3.3
| Safety Across the two studies, 1879 IgPro10 infusions were administered to
233 subjects: 259 infusions to 28 subjects in PRIMA and 1620 infu-
sions to 207 subjects in PATH. In the PRIMA safety population
(n = 28), 108 AEs occurred in 22 (78.6%) subjects (0.417/infusion). In
IVIG pre-treated PRIMA subjects (n = 13), 41 AEs occurred in TABLE 2
Response rate by INCAT and MRC sum score in PRIMA and PATH
Response rate (%)
PRIMA
PATH
Pooled
Pre-treated
n = 13
Treatment-naïve
n = 15
Overall
N = 28
N = 207
Pre-treated
n = 220
Overall
N = 235
INCAT
76.9
46.7
60.7
72.9
73.2
71.5
MRC sum score
76.9
86.7
82.1
56.5
57.7
59.6
Max grip strength (dominant hand)
46.2
46.7
46.4
59.9
59.1
58.3
Abbreviations: INCAT, inflammatory neuropathy cause and treatment; MRC, Medical Research Council; PATH, Polyneuropathy And Treatment with
Hizentra; PRIMA, Privigen Impact on Mobility and Autonomy. TABLE 2
Response rate by INCAT and MRC sum score in PRIMA and PATH Abbreviations: INCAT, inflammatory neuropathy cause and treatment; MRC, Medical Research Council; PATH, Polyneuropathy And Treatment with
Hizentra; PRIMA, Privigen Impact on Mobility and Autonomy. INCAT Total Score
1
0
Screening Ref visit
4
7
Deterioration
10
13
25
Time (weeks)
INCAT Total Score, points
2
3
4
5
6
Improvement
PRIMA (IVIG pre-treated, N = 13)
PATH (all IVIG pre-treated, N = 207)
Grip Strength
90
100
Screening Ref visit
4
7
Deterioration
10
13
25
Time (weeks)
Grip Strength, kPa
80
70
60
50
40
Improvement
PRIMA (IVIG pre-treated, N = 13)
PATH (all IVIG pre-treated, N = 207)
MRC Sum Score
78
80
Screening Ref visit
4
7
Deterioration
10
13
25
Time (weeks)
MRC Sum Score, points
76
70
66
64
60
Improvement
PRIMA (IVIG pre-treated, N = 13)
PATH (all IVIG pre-treated, N = 207)
74
68
72
62
FIGURE 2
Mean change from baseline in INCAT total score, grip
strength, and MRC sum score in PRIMA IVIG pre-treated subjects and
PATH. INCAT, inflammatory neuropathy cause and treatment; MRC,
Medical Research Council; PATH, Polyneuropathy And Treatment
with Hizentra; PRIMA, Privigen Impact on Mobility and Autonomy
52 PATH STUDY GROUP Australia A Sabet, K George (Gold Coast Hospital and Health Ser-
vice, Southport, QLD). L Roberts, R Carne (St Vincent's Hospital,
Melbourne, VIC). S Blum, R Henderson (Royal Brisbane & Women's
Hospital, Herston, QLD). Belgium P Van Damme, J Demeestere
(UZ Leuven-Neurologie, Leuven). Canada S Larue, C D'Amour
(Hopital Charles LeMoyne, Recherche Sepmus, Greenfield Park,
QC). Czech Republic P Kunc, M Valis (Neurologicka klinika, Fakultni
nemocnice Hradec Kralove, Hradec Kralove). J Sussova, T Kalous
(Neurologicka klinika, Vseobecna fakultni nemocnice v Praze, Pra-
gue). R Talab, M Bednar (Privatni ordinace neurologie, Hradec Kra-
love). Estonia T Toomsoo, I Rubanovits (East Tallinn Central
Hospital, Tallinn). K Gross-Paju, U Sorro (West Tallinn Central Hos-
pital, Tallinn). Finland M Saarela, M Auranen (Helsinki University
Central Hospital, Helsinki). France J Pouget, S Attarian (Hôpital de la
Timone Neurologi, Marseille). G Le Masson, A Wielanek-Bachelet
(Hôpital Haut-Lévéque, Service de Neurologie Centre hospitalier
universitaire de Bordeaux, Bordeaux). C Desnuelle, E Delmont
(Hôpital Archet 1 Centre de référence maladies neuromusculaires,
Nice). P Clavelou, D Aufauvre (Centre hospitalier universitaire Hôpi-
tal Gabriel Montpied, Clermont-Ferrand). Germany J Schmidt, J
Zschuentzsch (Universitätsmedizin Göttingen, Göttingen). C Som-
mer, D Kramer (Universitaetsklinikum Wurzburg, Wurzburg). O
Hoffmann, C Goerlitz (St Josefs-Krankenhaus, Potsdam). J Haas, M
Chatzopoulos (Jüdisches Krankenhaus Berlin, Berlin). R Yoon, R
Gold (Klinikum der Ruhr-Universität Bochum, Bochum). P Berlit, A
Jaspert-Grehl (Alfried Krupp Krankenhaus Rüttenscheid, Essen). D
Liebetanz, A Kutschenko (Georg-August-Universitätsmedizin Göt-
tingen, Göttingen). M Stangel, C Trebst (Medizinische Hochschule The safety data from both studies indicated that there were no
unexpected AEs, almost all being categorized as mild and partially
transient, in conformity with previous reports.11,17 Hemolytic events
were observed in both trials. The trials were undertaken before the
implementation of an immunoaffinity chromatography step in the pro-
duction process, which lowers isoagglutinin levels by 75% to 88%,18
and is expected to reduce the incidence of hemolytic events seen in
future studies.19 The safety results from the combined analysis sup-
port that IgPro10 is a well-tolerated treatment option when adminis-
tered as induction and maintenance infusions to subjects with CIDP. This analysis shows the efficacy and safety of IgPro10 in a com-
bined data set of subjects with CIDP being treated with an IVIG. ACKNOWLEDGEMENTS subjects untreated with IVIG for 3 months prior to the study as
compared with the PRIMA population of which half were untreated
for 1 year, and PATH in which all subjects were pre-treated. The
response rate in treatment-naïve subjects in the PRIMA study
(46.7%) is in a similar range to the 55% INCAT response rate
reported in treatment-naïve subjects in the Cochrane review.10 A
higher response rate was observed in pre-treated subjects in our
combined analysis; the Kuwabara et al study, where all subjects
were IVIG pre-treated, had a responder rate of 77.6% at Week
28.12 It is also of note that in this combined analysis, there was a
proportion of subjects who had previously responded to IVIG who
did not respond well after the withdrawal (wash-out) and re-
establishment of IVIG treatment. This highlights both the remitting–
relapsing nature of CIDP and the importance of regular testing of Ig
dependency in clinical practice to determine if a subject is still
benefitting from IVIG treatment. subjects untreated with IVIG for 3 months prior to the study as
compared with the PRIMA population of which half were untreated
for 1 year, and PATH in which all subjects were pre-treated. The
response rate in treatment-naïve subjects in the PRIMA study
(46.7%) is in a similar range to the 55% INCAT response rate
reported in treatment-naïve subjects in the Cochrane review.10 A
higher response rate was observed in pre-treated subjects in our
combined analysis; the Kuwabara et al study, where all subjects
were IVIG pre-treated, had a responder rate of 77.6% at Week
28.12 It is also of note that in this combined analysis, there was a
proportion of subjects who had previously responded to IVIG who
did not respond well after the withdrawal (wash-out) and re-
establishment of IVIG treatment. This highlights both the remitting–
relapsing nature of CIDP and the importance of regular testing of Ig
dependency in clinical practice to determine if a subject is still
benefitting from IVIG treatment. Editorial support was provided by Meridian HealthComms, funded by
CSL Behring. 53 53 MERKIES ET AL. TABLE 3
Adverse drug reactions occurring in >5% of subjects in PRIMA (FAS) and PATH (PSDS)
Preferred term
PRIMA
PATH
Total
Number (%) of
subjects
N = 28
Number of events
(rate per infusion)
N = 259a
Number (%) of
subjects
N = 207
Number of events
(rate per infusion)
N = 1620a
Number (%) of
subjects
N = 235
Number of events
(rate per infusion)
N = 1879a
Any adverse drug
reactions
20 (71.4)
71 (0.274)
85 (41.1)
200 (0.123)
105 (44.7)
271 (0.144)
Headache
8 (28.6)
19 (0.073)
32 (15.5)
50 (0.031)
40 (17.0)
69 (0.037)
Asthenia
4 (14.3)
4 (0.015)
2 (1.0)
2 (0.001)
6 (2.6)
6 (0.003)
Hypertension
4 (14.3)
6 (0.023)
5 (2.4)
6 (0.004)
9 (3.8)
12 (0.006)
Nausea
3 (10.7)
3 (0.012)
7 (3.4)
9 (0.006)
10 (4.3)
12 (0.006)
Pain in extremity
3 (10.7)
3 (0.012)
2 (1.0)
2 (0.001)
5 (2.1)
5 (0.003)
Hemolysis
2 (7.1)
2 (0.008)
7 (3.4)
7 (0.004)
9 (3.8)
9 (0.006)
Influenza-like illness
2 (7.1)
2 (0.008)
0
0
2 (0.9)
2 (0.001)
Leukopenia
2 (7.1)
2 (0.008)
2 (1.0)
2 (0.001)
4 (1.7)
4 (0.002)
Rash
2 (7.1)
2 (0.008)
2 (1.0)
2 (0.001)
4 (1.7)
4 (0.002)
Abbreviations: FAS, full analysis set; N, number of subjects treated in the study or number of infusions; PATH, Polyneuropathy And Treatment with
Hizentra; PRIMA, Privigen Impact on Mobility and Autonomy; PSDS, pre-randomization safety data set. Temporally associated events occurred during an infusion or within 72 hours after the end of infusion and were reported as “temporally related” in the
source tables and listings of the PATH study. a Number of infusions. TABLE 3
Adverse drug reactions occurring in >5% of subjects in PRIMA (FAS) and PATH (PSDS) Abbreviations: FAS, full analysis set; N, number of subjects treated in the study or number of infusions; PATH, Polyneuropathy And Treatment with
Hizentra; PRIMA, Privigen Impact on Mobility and Autonomy; PSDS, pre-randomization safety data set. Temporally associated events occurred during an infusion or within 72 hours after the end of infusion and were reported as “temporally related” in the
source tables and listings of the PATH study. a Number of infusions 4
|
DISCUSSION It should be considered that the previous ICE study included Hannover, Hannover). P Baum, F Bergh (Universitaetsklinikum Leip-
zig, Leipzig). J Klehmet, A Meisel (Klinik und Poliklinik für Neurolo-
gie Charité-Universitätsmedizin Berlin, Berlin). F Klostermann, J
Oechtering (Charite Universitaetsmedizin Berlin). H Lehmann, M
Schroeter (Universitätsklinikum, Köln). T Hagenacker, D Mueller
(Universitätsklinikum Essen, Essen). A Sperfeld, F Bethke (Klinikum
Ibbenbüren, Ibbenbüren). Israel V Drory, A Algom (Tel Aviv Sourasky
Medical Center, Tel Aviv). D Yarnitsky, B Murinson (Rambam Health
Care Campus, Haifa). Italy A Di Muzio, F Ciccocioppo (Policlinico SS
Annunziata, Chieti Scalo). S Sorbi, S Mata (Ospedaliero Universitaria
Careggi, Firenze). A Schenone, M Grandis (Azienda Ospedaliera Uni-
versitaria San Martino di Genova, Genoa). G Lauria, D Cazzato
(Fondazione Istituto DiRicovero, Milano). G Antonini, S Morino
(Azienda Ospedaliera S Andrea Universita degli Studi di Roma “La
Sapienza”, Rome). D Cocito, M Zibetti (Azienda ospedaliero univer-
sitaria San Giovanni Battista, Torino). Japan T Yokota, T Ohkubo
(Tokyo Medical and Dental University, Tokyo). T Kanda, M Kawai
(Yamaguchi University Hospital, Yamaguchi). K Kaida, H Onoue
(National Defense Medical Hospital, Saitama). S Kuwabara, M Mori
(Chiba University Hospital, Chiba). M Iijima, K Ohyama (Nagoya Uni-
versity Hospital, Nagoya). M Baba, M Tomiyama (Aomori Prefec-
tural Central Hospital, Aomori). K Nishiyama, T Akutsu (Kitasato
University Hospital, Kanagawa). K Yokoyama, K Kanai (Juntendo
University Hospital, Tokyo). Netherlands I N van Schaik, F Eftimov
(Amsterdam University Medical Centers, University of Amsterdam,
Amsterdam). N C Notermans, N Visser (University Medical Center
Utrecht, Utrecht). C Faber, J Hoeijmakers (Maastricht University
Medical
Center,
Maastricht). Poland
K
Rejdak,
U
Chyrchel-
Paszkiewicz (Samodzielny Publiczny Szpital Kliniczny, Lublin). Spain
C Casanovas Pons, M Antonia (Universitari de Bellvitge Servicio de
Neurología, Barcelona). J Gamez, M Salvado (Hospital Universitario
Vall d'Hebron Servicio de Neurología, Barcelona). C Marquez
Infante, S Benitez (Hospital Universitario Virgen del Rocío, Seville). United Kingdom M Lunn, J Morrow (National Hospital for Neurology
and Neurosurgery, London). D Gosal, T Lavin (Salford Royal Hospi-
tal, Salford). United States I Melamed, A Testori (IMMUNOe Interna-
tional
Research
Centers,
Centennial,
CO). S
Ajroud-Driss,
D
Menichella (Northwestern University Feinberg School of Medicine,
Chicago, IL). E Simpson, E Chi-Ho Lai (Methodist Neurological Insti-
tute, Houston, TX). M Dimachkie, R J Barohn (University of Kansas
Medical Center, Kansas City, KS). S Beydoun, H Johl (University of
Southern California Keck School of Medicine, Los Angeles, CA). D
Lange, A Shtilbans (Hospital for Special Surgery, New York, NY). S
Muley, S Ladha (St Joseph's Hospital and Medical Center, Phoenix,
AZ). PRIMA STUDY GROUP Belgium
J. L. De
Bleecker,
AZ
St-Lucas,
Gent
(5
patients);
W. Robberecht, UZ Leuven, Leuven (3 patients). Finland M. Saarela,
HUS Meilahti Hospital, Helsinki (3 patients). France J. Franques, Hôpi-
tal de la Timone, Neurologie et Maladies Neuro-Musculaire, Marseille
(2 patients); J.-M. Léger, Groupe Hospitalier Pitié-Salpêtrière Unité de
Pathologie Neuro-Musculaire, Paris (1 patient); R. Juntas Morales,
CHRU Hôpital Gui de Chauliac, Montpellier (1 patient). Germany
C. Sommer, Universitätsklinikum Würzburg, Würzburg (4 patients);
A. Nguento,
ASKLEPIOS
Klinikum
Uckermark
GmbH,
Schwedt
(2 patients); J. Schmidt, Universtitätsmedizin Göttingen, Georg-
August-Universität, Göttingen (1 patient); Ch. Schrey, Facharzt für
Neurologie, Berlin (1 patient). Poland J. Kamienowski, Dolnóslàski
Szpital Specjalistyczny, Wrocław (3 patients); Z. Stelmasiak, Samod-
zielny Publiczny Szpital Kliniczny, Lublin (3 patients); G. Zwolinska,
Centrum Neurologii Klinicznej, Kraków (2 patients). PATH STUDY GROUP Our
study confirms that the majority of those with CIDP who will respond
to IVIG will do so after an induction dose plus at least 2 maintenance
doses, and that a substantial number only respond after the second
maintenance dose. Shorter regimens may not identify all who will
respond to IVIG. Longer regimens “waiting” for a response may not be
needed as late responders (after 10 weeks) are in fact unlikely. MERKIES ET AL. 54 M Freimer, J Kissel (Wexner Medical Center at the Ohio State
University, Columbus, OH). N Latov, R Chin (Weill Medical College
of Cornell University, New York, NY). E Ubogu, S Mumfrey 55 14. van Schaik IN, Bril V, van Geloven N, et al. Subcutaneous immunoglob-
ulin for maintenance treatment in chronic inflammatory demyelinating
polyneuropathy (PATH): a randomised, double-blind, placebo-controlled
phase 3 trial. Lancet Neurol. 2018;17:35-46. risk of hemolytic anemia with intravenous immunoglobulin treatment:
A hospital-based cohort study in the US. Am J Hematol. 2018;93:
E17-E20. risk of hemolytic anemia with intravenous immunoglobulin treatment:
A hospital-based cohort study in the US. Am J Hematol. 2018;93:
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English
| null |
A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum
|
PloS one
| 2,015
|
cc-by
| 10,281
|
RESEARCH ARTICLE A Metabolic Probe-Enabled Strategy Reveals
Uptake and Protein Targets of
Polyunsaturated Aldehydes in the Diatom
Phaeodactylum tricornutum Stefanie Wolfram1, Natalie Wielsch2, Yvonne Hupfer2, Bettina Mönch3, Hui-Wen Lu-
Walther4, Rainer Heintzmann4,5, Oliver Werz3, Aleš Svatoš2, Georg Pohnert1* Stefanie Wolfram1, Natalie Wielsch2, Yvonne Hupfer2, Bettina Mönch3, Hui-Wen Lu-
Walther4, Rainer Heintzmann4,5, Oliver Werz3, Aleš Svatoš2, Georg Pohnert1* 1 Bioorganic Analytics, Institute for Inorganic and Analytical Chemistry, Friedrich Schiller University, Jena,
Germany, 2 Department Mass Spectrometry/Proteomics, Max Planck Institute for Chemical Ecology, Jena,
Germany, 3 Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Friedrich
Schiller University, Jena, Germany, 4 Biomedical Imaging, Department Microscopy, Leibniz Institute of
Photonic Technology e.V., Jena, Germany, 5 Institute for Physical Chemistry, Abbe Center of Photonics,
Friedrich Schiller University, Jena, Germany * Georg.Pohnert@uni-jena.de * Georg.Pohnert@uni-jena.de OPEN ACCESS Diatoms are unicellular algae of crucial importance as they belong to the main primary pro-
ducers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes
(PUAs) that have been made responsible for chemically mediated interactions in the plank-
ton. PUA-effects include chemical defense by reducing the reproductive success of grazing
copepods, allelochemical activity by interfering with the growth of competing phytoplankton
and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologi-
cally highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and
affected metabolic pathways. By using fluorescence microscopy, we observed a substantial
uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison
to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inven-
tory of proteins covalently targeted by the α,β,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by
the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecu-
lar functions and biological processes such as photosynthesis including ATP generation
and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism
of action of PUAs involves covalent reactions with proteins that may result in protein dys-
function and interference of involved pathways. Citation: Wolfram S, Wielsch N, Hupfer Y, Mönch B,
Lu-Walther H-W, Heintzmann R, et al. (2015) A
Metabolic Probe-Enabled Strategy Reveals Uptake
and Protein Targets of Polyunsaturated Aldehydes in
the Diatom Phaeodactylum tricornutum. PLoS ONE
10(10): e0140927. doi:10.1371/journal.pone.0140927 Editor: Tilmann Harder, Universitat Bremen,
GERMANY Introduction Oceans accommodate numerous coexisting microalga species in the plankton. Their commu-
nity is shaped by different factors including nutrient limitation, predation and chemical signal-
ing. Diatoms, a class of unicellular algae, are key players in the marine food web as they are
responsible for about 40% of global marine primary productivity [1]. Some diatom species
release biologically active metabolites as mediators of interactions. An intensively studied com-
pound class in this context are oxylipins, which derive from the oxidative transformation of
polyunsaturated fatty acids [2]. Of considerable interest among oxylipins are polyunsaturated
aldehydes (PUAs), which have been reported to mediate various inter- and intraspecific inter-
actions (reviewed in [2–5]). 2,4-Decadienal (DD) is the best studied metabolite of the group of
PUAs, with attributed roles in grazer defense [6], allelophathy [7], cell to cell signaling [8], anti-
bacterial activity [7,9] and bloom termination initiation [10,11]. PUA-mediated allelopathy
[5,7,12,13] is impairing different phyla regarding growth and physiological performance. Sensi-
tivity against PUAs has been reported for the prymnesiophyte Isochrysis galbana [7], the chlor-
ophyte Dunaliella tertiolecta [7] as well as the centric diatom Thalassiosira weissflogii [14]. A
synchronized release of PUAs from intact Skeletonema marinoi cells transiently before the cul-
ture changes to the decline phase supports the idea that PUAs play a role as infochemicals in
mediating bloom termination [10]. Despite the well-documented biological functions of PUAs,
their mechanism of action and their molecular targets are almost unknown [3,4]. Only few
impaired biological processes and functions are recognized mainly involving disruption of
intracellular calcium signaling, cytoskeletal instability and induction of apoptosis (reviewed in
[2–4]). PUA activity is structure-specific, since saturated aldehydes, like decanal that lack the conju-
gated α,β,γ,δ-unsaturated aldehyde motive of PUA, are not active [15,16]. Conjugated unsatu-
rated aldehydes are reactive compounds belonging to the class of Michael acceptors. They act
as electrophiles and react with proteins [17,18] and DNA [19–21]. Model investigations
revealed that DD covalently modifies proteins by formation of imines (Schiff bases), pyridi-
nium adducts and 1,4-addition products with nucleophiles [17,18]. Thus, proteins are putative
targets of the electrophilic PUAs. PUAs also react with DNA resulting in apoptosis in copepods
(reviewed in [22]). In algae [7], sea urchin embryos [23] and copepod embryos and nauplii
[6,24] DNA laddering and chromatin dispersal or complete DNA fragmentation and disloca-
tion is observed after PUA exposure. Editor: Tilmann Harder, Universitat Bremen,
GERMANY Published: October 23, 2015 Copyright: © 2015 Wolfram et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. Data Availability Statement: All relevant data are
within the paper and its Supporting Information files. Funding: Support was provided by the Deutsche
Forschungsgemeinschaft, German Research
Foundation (DFG) and Studienstiftung des deutschen
Volkes. Competing Interests: The authors have declared
that no competing interests exist. Competing Interests: The authors have declared
that no competing interests exist. 1 / 17 PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 Uptake and Protein Targets of Polyunsaturated Aldehydes in Diatoms PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 Introduction The diatoms Phaeodactylum tricornutum [25] and Thalassiosira pseudonana [26] have
emerged as model organisms since these were the first species with sequenced genome. P. tri-
cornutum is a producer of the oxylipins 12-oxo-(5Z,8Z,10E)-dodecatrienoic acid and 9-oxo-
(5Z,7E)-nonadienoic acid [27] and was reported to be affected by DD [8,28]. Exposure to this
aldehyde altered the mitochondrial glutathione redox potential by oxidation of glutathione and
induced cell death of P. tricornutum [28]. DD also triggers intracellular calcium transients and
nitric oxide generation [8]. There is evidence for a sophisticated stress surveillance system in
which individual diatom cells sense local DD concentration thereby monitoring the stress level
of the entire population. An ortholog of the plant enzyme AtNOS1 was predicted as molecular
target of PUAs [8]. Transcriptome analysis revealed that PtNOA, a gene with similarities to
AtNOS1 [8], is upregulated in response to DD [29]. PtNOA overexpressing cell lines are hyper-
sensitive to this PUA with altered expression of superoxide dismutase and metacaspases; both
protein classes are involved in activation of programmed cell death [29]. Other studies on gene
regulation in response to PUAs focused on copepods. In Calanus helgolandicus tubulin expres-
sion [30] and primary defense systems [31] were downregulated whereas detoxification genes PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 2 / 17 Uptake and Protein Targets of Polyunsaturated Aldehydes in Diatoms like glutathione S-transferase, superoxide dismutase, and catalase remained unaffected [31] in
response to a diet of the PUA producer S. costatum compared to a control. We report here on the uptake, accumulation and molecular targets of a molecular probe
containing a DD-derived head group and a 5-tetramethylrhodamine carboxamide fluorophore
(TAMRA) reporter in P. tricornutum using an activity-based protein profiling (ABPP) strategy
(Fig 1). Such chemical probe-enabled proteome strategies have been successfully applied with
mechanism-based inhibitors [32] or protein-reactive natural products [33,34]. The utilized
probe consists of a reactive group mimicking DD and a fluorescent reporter tag for detection
[35]. By applying 2D gel electrophoresis (GE) followed by liquid chromatography/tandem
mass spectrometry (LC-MS/MS) we found specific probe-labeled proteins having important
roles regarding catalytic activity and biological functions in the alga including fucoxanthin
chlorophyll a/c proteins, ATP synthases, a ribulose-phosphate-3-epimerase (RPE) and a phos-
phoribulokinase (PRK). Materials and Methods
Uptake experiments Growth of P. tricornutum. P. tricornutum (strain UTEX 646, Segelskär, Finland) was cul-
tivated in artificial seawater prepared as described in Maier and Calenberg [36] under a 14/10
hours light/dark cycle, at 32 to 36 mmole photons s-1 m-2 and 13°C in 580 mL Weck jars
(Weck, Wehr, Germany). The 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-
buffered medium was adjusted to a pH of 7.8 before autoclaving. Nutrient levels were 14.5 mM
phosphate, 620 mM nitrate and 320 mM silicate. Incubation experiments were done under the
same conditions. Sample preparation. A P. tricornutum culture (100 μL) was pipetted on ethanol pre-
cleaned cover slips (Marienfeld 474030-9010-000, 18x18 mm², D = 0.17 mm +/- 0.005 mm;
Carl Zeiss Canada, Toronto, ON). To prevent evaporation cover slips were placed in a Petri
dish, which contained a seawater wetted filter paper and were covered. Incubation for 8 hours
allowed cells to adhere to the cover slips. Subsequently, 10μM of the substances DDY coupled
to TAMRA (TAMRA-PUA), 5-hexynal (SA) coupled to TAMRA (TAMRA-SA) or azide mod-
ified TAMRA (TAMRA-N3) were added to the cell suspensions (each 0.5 mM stocks in
DMSO), mixed gently with a pipette and incubated for one hour. Afterwards, the cell suspen-
sions were removed and the cover slips were washed seven times by carefully pipetting 200 μL
artificial seawater and incubated with 100 μL 4% [w/v] para-formaldehyde in artificial seawater
for 25 min. The cover slips were washed twice with 200 μL artificial seawater and finally the Fig 1. Synthesis of the probe TAMRA-PUA and the control TAMRA-SA. For details on the synthesis see [35]. Fig 1. Synthesis of the probe TAMRA-PUA and the control TAMRA-SA. For details on the synthesis see [35]. doi:10.1371/journal.pone.0140927.g001 PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 3 / 17 Uptake and Protein Targets of Polyunsaturated Aldehydes in Diatoms liquid was removed. A control with the same washing and fixation steps was prepared as well. Due to extensive washing steps a part of algae cells detach from cover slips. 4 μL of 2,2’-thio-
diethanol were pipetted on an ethanol pre-cleaned object slide and the treated side of the cover
slip was placed on top of the object slide and slightly pressed down. Edges were fixed with nail
polish and the slides were stored at 4°C until measurements on the next day. For each treat-
ment three individual cover slips (biological replicates) were prepared out of three different P. Materials and Methods
Uptake experiments tricornutum cultures. Those cultures have been prepared out of the same stock and kept under
identical growth condition. Fluorescence microscopy and analysis. P. tricornutum cells were observed with a struc-
tured illumination microscope (SIM), Zeiss Elyra S1 system (Carl Zeiss, Jena, Germany). Imag-
ing was performed with an oil immersion objective lens (Plan-Apo, 63X, 1.4NA, Carl Zeiss,
Jena, Germany). A 561 nm laser was used for excitation and fluorescence was filtered by a band
pass filter (BP 570–620 nm) which opens up above 750 nm. 2D wide field images were acquired
to compare fluorescence intensity of the different probes and treatments (TAMRA-PUA,
TAMRA-SA, TAMRA-N3, no probe), whereas all cells were measured using the same settings
(laser intensity, gain, exposure time). 12 cells were observed per treatment distributed over
three biological replicates (microscope slides), four cells each. Three dimensional SIM images
of treated P. tricornutum providing twofold resolution improvement were taken from selected
samples to confirm, that TAMRA-PUA and TAMRA-SA were taken up in the cells and did
not exclusively stick onto their surfaces. 15 raw images are required for reconstructing one 2D
SIM image. 3D SIM images were taken with z steps of 110 nm. All SIM images were recon-
structed on ZEN software 2010 (black edition, Carl Zeiss, Jena, Germany). For fluorescence analysis bright field and wide field fluorescence images at 561 nm excita-
tion were exported in TIF format with the ZEN software and processed with ImageJ2x 2.1.4.7
(freeware, http://www.rawak.de/ij2x/Download.html) as follows: tonal correction of the bright
field image of each cell was optimized (see S1 Folder for unmodified images), the cell was encir-
cled by hand and this selection was laid on the corresponding fluorescence picture using the
ROI manager. The mean gray value, which is defined in this software as the sum of the gray
values of all the pixels in the selection divided by the number of pixels, and the mean gray value
of the background were measured and subtracted. For the previously described issue we use
the term mean gray value per pixel. For background analysis a region of at least 1000 pixels was
used. PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 Probe labeling, 1D and 2D gel electrophoresis and identification of target
proteins In vivo labeling of P. tricornutum and sample preparation for gel electrophoresis. P. tri-
cornutum cultures were grown for 13 days in 580 mL Weck jars (1.0 × 106 to 1.5 × 106 cells
mL-1) without shaking as described before, which resulted in cells mainly sticking to the glass
bottom. The overlaying artificial seawater was almost quantitatively removed with a pipette
and cells were transferred into centrifuge tubes with the remaining medium. For 1D GE samples were incubated with 100 μM DDY or 100 μM SA (each 50 mM stocks in
DMSO) or DMSO for one hour. For 2D GE the concentrated algae suspension was treated with 14 μL (250 μM) DDY stock
solution (50 mM), which was added to 2.8 mL concentrated cell suspension (total cell number
48.7 × 106 resulting in 14 fmol DDY cell-1) and incubated for 1 hour. During this incubation
period no change in cell viability compared to untreated cells was observed by microscopy after
application of Evan’s Blue [37]; however, we observed that incubation of several hours
increases the amount of non-viable cells. The undiluted samples were centrifuged for 2 min at 4 / 17 PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 Uptake and Protein Targets of Polyunsaturated Aldehydes in Diatoms 1,800 g immediately after incubation to remove DDY and salts; the supernatant was removed
and the cells were washed twice with 1 mL buffer A (10 mM HEPES and 250 mM sucrose, pH
7.4) and twice with 1 mL buffer B (10 mM HEPES and 250 mM sucrose, pH 8.2). After each
washing step the tubes were centrifuged at 1,800 g for 2 min and the supernatant was dis-
carded. Application of Evan’s Blue [37] and microscopy ensured that cells stayed intact during
this procedure. The pellet was resuspended in buffer B and cells were treated with 1 mM dithio-
threitol (20 mM stock in buffer B) to react with possibly unremoved DDY. Cells were lysed by
sonication (ultrasonic probe: Bandelin sonotronic HD2070, power supply: Bandelin UW2070;
Bandelin electronic, Berlin, Germany) twice for 15 s on ice. The protein concentration was determined with the Roti1-Quant universal assay (Carl
Roth, Karlsruhe, Germany) based on the biuret test using a microplate reader (Mithras LB 940,
Berthold Technologies, Bad Wildbad, Germany) and bovine serum albumin as reference. Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). P. Probe labeling, 1D and 2D gel electrophoresis and identification of target
proteins tricornutum protein sam-
ples (30μL, 30 to 50 μg proteins μL-1) were diluted with 270 μL buffer B and incubated with
6 μL (0.09 mM) TAMRA-N3 solution (5 mM stock in DMSO), 18 μL (0.09 mM) ligand tris
[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amin solution (1.7 mM stock in DMSO/tert-butanol,
1/4, v/v) and 12 μL (35 mM) of a freshly prepared ascorbic acid solution (1.00 M in water). Samples were vortexed and 6 μL (0.88 mM) copper sulfate solution (50 mM in water) were
added. Samples were vortexed again and stored on ice for 1 hour. 1% Triton™X-100 and prote-
ase inhibitor cocktail (M221, Amresco Inc., Solon, OH, USA) were added according to the
manufacturer’s protocol and after 30 min on ice samples were centrifuged at 15,000g and 3°C
for 20 min. The supernatant was transferred into centrifugal filter units (vivaspin1500, 5,000
MWCO, PES, Sartorius, Göttingen, Germany) and the sample volume was reduced by centrifu-
gation at 15,000g and 3°C. 100μL buffer B was added three times and the volume was reduced
by centrifugation after each addition to give a final protein concentration of 10 to 20 μg
proteins μL-1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and in-gel fluo-
rescence detection. 20 μg of protein samples were mixed with 2× loading buffer [38] and
heated to 95°C for 6 min. A protein ladder (PageRuler unstained protein ladder, Thermo
Fischer Scientific Inc., Waltham, MA, USA) and the protein sample were loaded on a SDS mini
gel containing 12% acrylamide resolving gel and 5% stacking gel prepared according to [39]. The gel was separated in a Mini-Protean1 Tetra gel cell (Bio-Rad, Herculas, CA, USA) by
applying 80 V for 30min followed by 180 V for 65min. A fluorescent picture was taken at
312nm irradiation using a UV transilluminator (UV star, Bio-Rad), a PowerShot A640 camera
(Canon, Tokyo, Japan) and a 520 nm long pass filter. The gel was stained with RAPIDstain™
(G-Biosciences, St. Louis, MO, USA). Difference gel electrophoresis (DIGE). DIGE was conducted in triplicates. 440 to 880 μg
protein of the probe-treated sample (incubated with DDY and connected with TAMRA-N3 by
CuAAC as described before) and 50 μg protein of a control sample incubated with the N-
hydroxysuccinimide ester of the cyanine 5 fluorophore (Cy5 NHS ester) were loaded on each
of the isoelectric focusing (IEF) strips (Immobiline DryStrip, 24 cm, pH 3–11 NL, GE Health-
care Bio- Sciences, Piscataway, NJ, USA). PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 Probe labeling, 1D and 2D gel electrophoresis and identification of target
proteins LC-MS/MS analysis and data processing were conducted as described in S1 Information. Probe labeling, 1D and 2D gel electrophoresis and identification of target
proteins For Cy5 labeling of the whole proteome 20 μL of the
control sample were diluted with 48 μL buffer B. 3 μL Cy5 NHS ester (2 mM in DMSO) were
added and the mixture was incubated with shaking at 4°C for 45 min. To stop labeling 5 μL of a
10 mM solution of L-lysine in water were added. The IEF strip was rehydrated overnight (7 M urea, 2 M thiourea, 2% [w/v] CHAPS, 0.002%
[w/v] bromophenol blue, 0.5% [v/v] IPG buffer (GE Healthcare Bio-Sciences), 10 mM dithio-
threitol). Isoelectric focusing of the strips with the Ettan IPGphor II (GE Healthcare Bio- 5 / 17 PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 Uptake and Protein Targets of Polyunsaturated Aldehydes in Diatoms Sciences) was carried out according to the following protocol: 4 h at 300 V (gradient), 4 h at
600 V (gradient), 4 h at 1,000 V (gradient), 4 h at 8,000 V (gradient) and 3 h at 8,000 V (step). After isoelectric focusing the IEF strips were equilibrated for 15 min in 10 mL of equilibra-
tion buffer (6 M urea, 30% [v/v] glycerol, 2% [w/v] SDS, 75 mM tris(hydroxymethyl)amino-
methane, 0.002% [w/v] bromophenol blue) containing 1% [w/v] dithiothreitol and
subsequently for 15 min in 10 mL of equilibration buffer containing 2.5% [w/v] iodoacetamide. For separation of proteins in the second dimension, the Ettan DALT System (GE Healthcare
Bio-Sciences) was used. SDS polyacrylamide gels 12% [w/v] of 1.0 mm thickness were casted
via the Ettan DALTsix Gel caster (GE Healthcare Bio-Sciences). The separation conditions
were as follows: 1 W/gel for 1 h followed by 15 W/gel for 5 h. Proteins were visualized by ana-
lyzing the gels with a Typhoon 9410 scanner (GE Healthcare Bio-Sciences) using a resolution
of 100 μm. Proteins were fixed (10% [v/v] acetic acid, 50% [v,v] methanol in water), stained
(0,025% [w/v] Coomassie R 250, 10% [v/v] acetic acid in water) and the gels were destained
(10% [v/v] acetic acid in water; see S3 Folder for unmodified Coomassie and fluorescence
images of the gels). Gels were merged with Delta2D (DECODON, Greifswald, Germany). Protein isolation, LC-MS/MS analysis and data processing. Fluorescent TAMRA-PUA
protein spots were in-gel reduced, alkylated with iodoacetamide and digested as described by
Shevchenko et al. [40]. Tryptic peptides were extracted, dried down in a vacuum centrifuge and
dissolved in 10 μL of water containing 0.1% formic acid. DDY covalently modifies proteins of P. tricornutum We next tested for protein targets of PUAs in living cells using DDY as well as the saturated
aldehyde derivative SA. After incubation with the probes followed by cell lysis, TAMRA-N3
was coupled to the alkyne groups of DDY as well as SA via CuAAC (Fig 2). Proteins were Fig 3. Fluorescence intensity of P. tricornutum cells treated under different conditions. Cells were
either incubated with TAMRA-PUA, TAMRA-N3, TAMRA-SA for one hour or kept under identical conditions
without probe. For each treatment three microscope slides with four cells each were measured. Unmodified
raw data are available in S1 and S2 Folders. Fluorescence intensities were recorded as mean gray value per
pixel after data treatment as described in the main text. Averaged mean gray values per pixel of cells of each
treatment are presented as bars ±SD. One way Anova comparing results of different microscope slides within
one treatment revealed no statistical difference (p>0.05). Kruskal-Wallis one way analysis of variance on
ranks revealed differences in the median values among the treatment groups (H = 42.436, p<0.001) and
Tukey’s HSD test (p<0.05) allowed classification into three groups: (a) TAMRA-PUA with the highest mean
gray value per pixel of 3661±809, (b) TAMRA-SA with 800±140 and (c) TAMRA-N3 and control with almost no
emission signals (20±10 and 35±9); these controls were not significantly different to each other (Tukey’s HSD
test p>0.05). d i 10 1371/j
l
0140927 003 Fig 3. Fluorescence intensity of P. tricornutum cells treated under different conditions. Cells were
either incubated with TAMRA-PUA, TAMRA-N3, TAMRA-SA for one hour or kept under identical conditions
without probe. For each treatment three microscope slides with four cells each were measured. Unmodified
raw data are available in S1 and S2 Folders. Fluorescence intensities were recorded as mean gray value per
pixel after data treatment as described in the main text. Averaged mean gray values per pixel of cells of each
treatment are presented as bars ±SD. One way Anova comparing results of different microscope slides within
one treatment revealed no statistical difference (p>0.05). TAMRA-PUA accumulates in P. tricornutum Cell permeability and uptake of the probes by P. tricornutum was investigated by wide field
fluorescence microscopy. Living cells were treated with TAMRA-PUA, TAMRA-SA or TAM-
RA-N3 for labeling or kept as control without additional treatment. After one hour incubation
probes were removed by washing seven times with artificial seawater, once with artificial sea-
water containing 4% paraformaldehyde for cell fixation and twice with artificial seawater. Cells
were embedded in 2,2’-thiodiethanol and measured with an epifluorescence microscope in
wide field mode. Images were processed with ImageJ. Cells were encircled by hand and the
background corrected average mean gray value per pixel within each alga cell was calculated
(Fig 3). The aldehyde containing probes TAMRA-PUA and TAMRA-SA were significantly
enriched in the cells compared to TAMRA-N3 or the control. Interestingly, TAMRA-PUA
accumulation was significantly higher compared to TAMRA-SA, despite being similar in phys-
icochemical properties. Results were verified in additional independent experiments, also using a different embed-
ding medium (S1 Fig). To confirm that the probes do not only appear on the surface we con-
ducted 3D SIM showing that TAMRA-PUA and TAMRA-SA accumulate within the cells (Fig
4). Distribution of label revealed local maxima but in general nearly the entire cellular content
was affected by the probe. Probe design and labeling strategy The fluorescent α,β,γ,δ-unsaturated aldehyde-derived probe TAMRA-PUA (Fig 1) could be
used successfully to investigate uptake and accumulation of PUAs in P. tricornutum and to
monitor protein targets of these natural products. TAMRA-PUA was recently developed in our
group to monitor accumulation of PUAs in copepods [35]. The probe consists of DDY as reac-
tive group that mimics DD. The alkyne functionality allows to couple the commercially avail-
able azide modified tetramethylrhodamine TAMRA-N3 (Fig 1). To identify protein targets,
DDY was incubated with P. tricornutum cells. After work-up CuAAC allowed to covalently
link the reporter TAMRA-N3 to DDY (Figs 1 and 2). For uptake studies we employed the
probe as already coupled construct TAMRA-PUA. For comparison of the activity of α,β,γ,δ-
unsaturated aldehyde-derived probes with structurally related saturated-aldehyde-derived ones
we also applied the probe TAMRA-SA (Fig 1). Fig 2. Schematic in vivo application of the probe. Living cells of P. tricornutum were incubated with the PUA-derivative DDY. After removal of excess DDY
cell lysis followed by CuAAC enables attachment of the fluorescent reporter to covalently labeled proteins. 1D GE quickly allows detection of labeled proteins
(not shown). Identification of protein targets is enabled by 2D GE. Therefore, a second P. tricornutum sample was treated with Cy5 NHS ester to label the
whole proteome. The combined samples were separated using DIGE, labeled proteins were digested using trypsin and the resulting peptides were
separated and analyzed by LC-MS/MS. Fig 2. Schematic in vivo application of the probe. Living cells of P. tricornutum were incubated with the PUA-derivative DDY. After removal of excess DDY
cell lysis followed by CuAAC enables attachment of the fluorescent reporter to covalently labeled proteins. 1D GE quickly allows detection of labeled proteins
(not shown). Identification of protein targets is enabled by 2D GE. Therefore, a second P. tricornutum sample was treated with Cy5 NHS ester to label the
whole proteome. The combined samples were separated using DIGE, labeled proteins were digested using trypsin and the resulting peptides were
separated and analyzed by LC MS/MS doi:10.1371/journal.pone.0140927.g002 doi:10.1371/journal.pone.0140927.g002 PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 6 / 17 Uptake and Protein Targets of Polyunsaturated Aldehydes in Diatoms doi:10.1371/journal.pone.0140927.g003 DDY covalently modifies proteins of P. tricornutum Kruskal-Wallis one way analysis of variance on
ranks revealed differences in the median values among the treatment groups (H = 42.436, p<0.001) and
Tukey’s HSD test (p<0.05) allowed classification into three groups: (a) TAMRA-PUA with the highest mean
gray value per pixel of 3661±809, (b) TAMRA-SA with 800±140 and (c) TAMRA-N3 and control with almost no
emission signals (20±10 and 35±9); these controls were not significantly different to each other (Tukey’s HSD
test p>0.05). doi:10.1371/journal.pone.0140927.g003 7 / 17 PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 Uptake and Protein Targets of Polyunsaturated Aldehydes in Diatoms Fig 4. Fluorescence microscopy of P. tricornutum cells. 3D (left) and 2D (right) images of a TAMRA-PUA
(A) and a TAMRA-SA (B) treated cell. Images were taken in 3D SIM mode. A 561nm laser was used for
excitation, and fluorescence was filtered by a band pass filter (BP 570-620nm) which opens up above 750nm
Fluorescence is visible in the entire cells, which confirms that both probes were taken up. doi:10.1371/journal.pone.0140927.g004 Fig 4. Fluorescence microscopy of P. tricornutum cells. 3D (left) and 2D (right) images of a TAMRA-PUA
(A) and a TAMRA-SA (B) treated cell. Images were taken in 3D SIM mode. A 561nm laser was used for
excitation, and fluorescence was filtered by a band pass filter (BP 570-620nm) which opens up above 750nm. Fluorescence is visible in the entire cells, which confirms that both probes were taken up. doi:10 1371/journal pone 0140927 g004 Fig 4. Fluorescence microscopy of P. tricornutum cells. 3D (left) and 2D (right) images of a TAMRA-PUA
(A) and a TAMRA-SA (B) treated cell. Images were taken in 3D SIM mode. A 561nm laser was used for
excitation, and fluorescence was filtered by a band pass filter (BP 570-620nm) which opens up above 750nm. Fluorescence is visible in the entire cells, which confirms that both probes were taken up. doi:10.1371/journal.pone.0140927.g004 doi:10.1371/journal.pone.0140927.g004 separated by 1D GE and monitored for fluorescent labeling. UV-illumination revealed exclu-
sive labeling of proteins in DDY treated cells while SA and DMSO treatments did not result in
any fluorescent bands (S2 Fig). To unravel protein targets of DDY, protein extracts obtained after incubation of P. tricornu-
tum with the probe as described above were separated by DIGE in three replicates (S3 Fig, S3
Folder for unmodified pictures). For comparison samples without probe addition were incu-
bated with the Cy5 NHS ester to label the whole proteome. DDY covalently modifies proteins of P. tricornutum Separation was performed by iso-
electric focusing and SDS-PAGE as second dimension and DDY-TAMRA labeled proteins
were excised and tryptically digested. Digested peptides were separated and analyzed by
LC-MS/MS (Fig 2). More precisely separation was conducted with a nano Ultra Performance PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 8 / 17 Uptake and Protein Targets of Polyunsaturated Aldehydes in Diatoms Liquid Chromatography (nanoUPLC) and analysis by tandem mass spectrometry using data-
independent acquisition (MSE analysis). In data-independent analysis the mass spectrometer
cycles between low and high energy acquisition of data resulting in high sampling rate. A list of confident target proteins classified according to their biological processes and
molecular functions is shown in Table 1. Two ATP synthases, four different chlorophyll a/c
binding proteins, different catalytic active enzymes and some predicted proteins were found to
be modified by the probe. A full list of confident, probable and putative proteins (for evaluation
see S1 Information) as well as an overview of all protein hits for each gel is given in S1 Table. 1Proteins in this table were found in at least two of the three gels, a full list of labeled proteins can be found in S1 Information. OOO—only one protein per
excised gel spot was found, OO—identification of probe labeled protein besides other unlabeled proteins in a gel spot, O—more than one labeled protein
per excised gel spot, X—no hit.
aNames are temporarily ascribed to an open reading frame (ORF) by a sequencing project [41]. pen reading frame (ORF) by a sequencing project [41]. Discussion While previous research mainly has reported the teratogenic and allelochemical effects of
PUAs as well as their role in cell to cell signaling (reviewed in [2–5]) and PUAs influence on Table 1. Confident target proteins found by 2D GE. 1
Protein
Gene name
Accession
No. Mass
(kDa)
Gel
1
Gel
2
Gel
3
1) Biological process
ATP biosynthetic process
ATP synthase subunit alpha, chloroplastic
AtpA
A0T0F1
54621.6
X
OO
O
ATP synthase subunit beta
AtpB
B7FS46
53619.2
X
OO
O
Photosynthesis
Fucoxanthin chlorophyll a/c protein
Lhcf10
B7G5B6
21352.7
X
OOO
O
Fucoxanthin chlorophyll a/c protein
Lhcf9
B7G955
22100.5
OOO
O
X
Fucoxanthin chlorophyll a/c protein
Lhcx2
B7FR60
21177.4
OO
O
X
Fucoxanthin chlorophyll a/c protein
Lhcf4
B7FRW2
21328.5
OO
O
X
2) Molecular function
Catalytic activity, isomerase activity
Ribulose-phosphate 3-epimerase
Rpe
B7FRG3
27812.0
OO
O
O
Catalytic activity, ligase activity
Predicted protein, family: Aspartate-ammonia ligase
PHATRDRAFT_44902a
B7FW24
43206.1
X
OO
O
Catalytic activity, oxidoreductase activity
Predicted protein, domains: Thioredoxin-like fold,
Thioredoxin domain
PHATRDRAFT_42566a
B7FNS4
24136.3
X
OOO
OOO
Predicted protein, domain: Rieske [2Fe-2S] iron-sulphur
domain
PHATRDRAFT_9046a
B7FPI8
17010.4
X
OO
O
Catalytic activity, transferase activity
Phosphoribulokinase
Prk
B5Y5F0
43325.4
X
OO
O
3) Predicted proteins without assignable function
Predicted protein
PHATRDRAFT_42612a
B7FNX7
24938.5
O
OOO
OOO
Predicted protein
PHATRDRAFT_45465a/
PHATRDRAFT_50215a
B7FXS8
37645.2
O
X
OOO
Predicted protein, family: SOUL haem-binding protein
PHATRDRAFT_37136a
B7G284
46049.4
O
OOO
OOO
Predicted protein
PHATRDRAFT_49286a
B7GA37
32141.6
OO
O
O
Predicted protein, domain: NAD(P)-binding domain
PHATRDRAFT_49287a
B7GA38
126885.6
X
OO
O
Predicted protein, family: Protein of unknown function
DUF1517
PHATRDRAFT_32071a
B7FQ47
33258.8
OO
X
O
1Proteins in this table were found in at least two of the three gels, a full list of labeled proteins can be found in S1 Information. OOO—only one protein per
excised gel spot was found, OO—identification of probe labeled protein besides other unlabeled proteins in a gel spot, O—more than one labeled protein
per excised gel spot, X—no hit. aNames are temporarily ascribed to an open reading frame (ORF) by a sequencing project [41]. doi:10 1371/journal pone 0140927 t001 Table 1. Confident target proteins found by 2D GE. 1 Gene name 1Proteins in this table were found in at least two of the three gels, a full list of labeled proteins can be found in S1 Information. Discussion OOO—only one protein per
excised gel spot was found, OO—identification of probe labeled protein besides other unlabeled proteins in a gel spot, O—more than one labeled protein
per excised gel spot, X—no hit. PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 9 / 17 Uptake and Protein Targets of Polyunsaturated Aldehydes in Diatoms gene regulation [30,31], almost nothing is known about underlying mechanistic aspects regard-
ing covalent protein interactions. Therefore, we applied a PUA-derived as well as control probe
to P. tricornutum. Probe design and labeling strategy PUAs are known to have diverse effects on planktonic organisms but defined molecular targets
are hitherto almost unidentified. Especially their function in cell to cell communication of dia-
toms has attracted much attention [7,8,10,14]. We undertook a labeling study to obtain a
deeper insight into the mechanism of action of these metabolites to reveal potential PUA-
uptake of phytoplankton and to identify protein targets of the compounds. Following previous
structure activity studies we addressed the specific activity of PUAs by comparison of probes
derived from the active unsaturated aldehyde (TAMRA-PUA) and the inactive saturated alde-
hyde (TAMRA-SA) [16,35]. The design of the probes allowed to employ them in two different
modes. For uptake studies we could use the TAMRA coupled molecules as described earlier for
the monitoring of PUA-targeting in copepods [35]. Interestingly, the different effects of
unmodified saturated aldehydes and PUAs observed in previous studies [16] were also mir-
rored in the effect of our different TAMRA-constructs. This indicates that the TAMRA substi-
tution has no significant influence on the action of the aldehydes. However we cannot exclude
that permeability is altered by the substitution. Probe concentration was set to 10μM, a value for which different algae showed response to
DD regarding cell membrane permeability of SYTOX Green [7], but P. tricornutum did not
[8]. For identification of protein targets we developed a two-step protocol involving incubation
with unmodified SA or DDY and, after work-up, coupling with the TAMRA-N3. This
approach allows to minimize the influence of bulky groups during in vivo interaction with tar-
get proteins. The well-established CuAAC coupling allowed to covalently link the dye to DDY-
labeled proteins [42,43]. As fluorescent reporter we selected the tetramethylrhodamine fluoro-
phore as it is relatively cheap, pH insensitive, photostable, cell permeable and easily excitable
with common lasers and filter sets [44]. Compared to experimental design of fluorescence
microscopy where physiological conclusions were relevant, probe concentration in the mecha-
nistic gel electrophoretic experiments was increased to 250μM DDY ensuring an adequate
detection of labeled proteins. TAMRA-PUA accumulates in P. tricornutum Fluorescence microscopy clearly shows an uptake and accumulation of the DD derived probe
by P. tricornutum (Fig 3). In contrast, TAMRA-SA, the probe derived from an almost inactive
aldehyde with otherwise similar physicochemical properties, compared to TAMRA-PUA, did
not substantially accumulate in the cells. Apparently the α,β,γ,δ-unsaturated structure element
found in PUAs is responsible for uptake and/or accumulation within diatom cells. A potential
mechanism explaining the accumulation could be an inhibited exfiltration due to covalent
adduct formation with cellular components such as proteins [17,18] as verified below or DNA
[19–21]. In contrast, the weaker fluorescence signal of TAMRA-SA is consistent with the much
lower reactivity of the underlying structure hexanal for which only few covalent reactions with
proteins have been reported [45,46]. By applying the hexanal derived TAMRA-SA to a P. tri-
cornutum culture we did not observe any covalently modified proteins in the corresponding
1D gel (S2 Fig). The intracellular accumulation can explain the specific elicitation of effects by PUAs [8]. We can exclude that the dye itself accumulates unspecifically in cells since TAMRA-N3 treated
P. tricornutum showed no different fluorescence signals compared to untreated controls PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 10 / 17 Uptake and Protein Targets of Polyunsaturated Aldehydes in Diatoms (Tukey’s HSD test p>0.05 between TAMRA-N3 and no probe), confirming the effective wash-
ing procedure to remove TAMRA [47]. 3D SIM images (Fig 4) reveal that fluorescence after
application of the TAMRA-PUA probe is distributed over almost the entire cell. The lack of
intracellular compartmentation can be explained with the high reactivity of such types of elec-
trophilic Michael acceptors [16,48,49]. Apparently no preferred shuttling of the probe into spe-
cific compartments occurs but also the cell walls and membranes do not represent a barrier for
this compound class. PUA-uptake might thus be a way to facilitate diatoms´ perception of this
compound class. Efficient uptake of essential metabolites has been earlier observed in diatoms
but specific uptake mechanisms of primary and secondary metabolites involving transporters
are not yet identified [50]. However, transporters of glucose that can support mixotrophic
growth of P. tricornutum are known, supporting the note of the capability of the alga to actively
take up organic metabolites from its environment [51]. This further supports the notion of a
possible cell to cell communication mechanism based on PUAs that requires cellular uptake. To unravel potential molecular targets within the proteome of P. DDY covalently modifies proteins of P. tricornutum We performed an in vivo labeling of P. tricornutum with a PUA-derived probe to identify tar-
get proteins and to deduce affected molecular functions and biochemical pathways. We
hypothesize that modified proteins may lose their function and that PUAs thereby interrupt or
disturb metabolic pathways. These changes on a molecular level probably lead to observed
effects of PUAs like growth inhibition and cell death [4,8]. Whereas Vardi et al. used a transcriptome analysis to search for DD affected genes and gene
products by screening for up- and downregulated transcripts [29], we performed a direct inves-
tigation on the covalent modification of the proteome by DDY. Thus, we discover interactions
with proteins, which do not necessarily have an influence on the transcript level but a direct
influence on the functionality of these proteins. Although the unsaturated aldehyde group of PUAs is universally reactive against nucleo-
philic amino acid side chains, we received moderate specific labeling of proteins (Table 1). This
agrees with previous findings that DD preferentially attacks distinct proteins and specific
nucleophilic sites if incubated with isolated purified proteins [17]. Underneath the confident
target proteins we found four fucoxanthin chlorophyll a/c proteins, which are part of the light
harvesting complex (LHC), responsible for the delivery of excitation energy between photosys-
tem I and II [52]. Compared to higher plants, LHCs of diatoms named fucoxanthin-chloro-
phyll-proteins bind chlorophyll c instead of b and fucoxanthin instead of lutein [53]. Three
groups of LHCs regarding their sequence and function are known, the found target proteins
(see Table 1) belong to two groups of them: Lhcx gene products are needed for protection
against surplus light and thus photoprotection and Lhcf gene products, the main antenna pro-
teins, function in light harvesting (reviewed in [54]). Effects of DD on photosystem efficiency
have already been shown for the diatoms Thalassiosira weissflogii [14] and a transgenic P. tri-
cornutum [29] and our findings now provide a mechanistic explanation for this action of
PUAs. The energy harvested during light reaction is mainly stored by forming adenosine triphos-
phate (ATP). We identified two probe labeled ATP synthase subunits (see Table 1) belonging
to the extrinsic catalytic sector, CF1 [55] of the chloroplastic ATP synthase. TAMRA-PUA accumulates in P. tricornutum tricornutum we undertook
further labeling studies. PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 DDY covalently modifies proteins of P. tricornutum ATP synthase,
located either in the mitochondria inner membrane or chloroplast thylakoid membrane, are
responsible for cellular ATP production from adenosine diphosphate and inorganic phosphor
in the presence of a proton gradient across the membrane [56]. The two PUA-targets ATP PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 11 / 17 Uptake and Protein Targets of Polyunsaturated Aldehydes in Diatoms synthase subunit alpha (AtpA) and ATP synthase subunit beta (AtpB) are located in the water
soluble CF1 complex of the chloroplastic ATP synthase. In the green algae Chlamydomonas
reinhardtii in the absence of the mitochondrial beta-subunit (gene name Atp2), ATP synthase
could not be assembled into an enzyme complex leading to decreased mitochondrial respira-
tion [57]. In conjunction with the finding of impairment of enzymes involved in light harvest-
ing the identified ATP synthase targets support the notion of a profound modulation of the
energy household under the influence of PUAs. Enzymes from the Calvin cycle are also PUA-targets connected to photosynthetic activity. Two PUA-targets, the kinase PRK and the epimerase RPE were found to be labeled after DDY
incubation. These enzymes are involved in carbon dioxide assimilation during the dark reac-
tion. RPE reversibly catalyzes the reaction of D-xylulose 5-phosphate to D-ribulose 5-phos-
phate in the Calvin cycle and pentose phosphate pathway [58]. The product D-ribulose-
5-phosphate is under ATP consumption further converted by PRK to D-ribulose-1,5-bispho-
sphate, which acts as acceptor for CO2 in photosynthetic carbon assimilation [59]. In the Cal-
vin cycle glyceraldehyde-3-phosphate dehydrogenase, the small protein CP12 and PRK form a
multi-enzyme complex, the redox state of PRK is regulated by thioredoxin-mediated thiol-
disulfide exchange in a light-dependent manner [59,60]. PRK is not active in the oxidized form
where cysteine residues at positions 16 and 55 in land plants and green algae form an intramo-
lecular disulfide bridge [61]. By reaction of those thiols with a PUA inactivation of PRK due to
spatial changes and loss of redox behavior is conceivable. Also labeling on other sites, such as
Lys may lead to loss of activity. Examples for alkylations were shown for PRK of different ori-
gin [62,63] and accordingly, alkylation of thiols and other nucleophilic residues by PUAs
might change activity of enzymes. It is striking that metabolic pathways such as the pentose phosphate pathway, photosynthe-
sis including photophosphorylation and Calvin cycle that are involved in the energy household
are specifically affected by PUA-treatment. Conclusion In this study we investigate the structure specificity of the uptake of PUA-derived probes and
analogues in P. tricornutum. We could also reveal PUA probe targets within the proteome of
the alga. Uptake experiments show a clear enrichment of TAMRA-PUA within the cells com-
pared to TAMRA-SA. Chemoproteomics allowed the identification of target proteins of TAM-
RA-PUA. Interestingly, preferential targets have important roles in biological processes
covering photosynthesis including ATP generation, conversion in Calvin cycle or the pentose
phosphate pathway. Besides three Lhcf- and one Lhcx-coding proteins important for light har-
vesting and photoprotection we found two ATP synthases. Generation of ATP is of major
importance since it supports nearly all cellular activities that require energy and its synthesis is
the most frequent chemical reaction in the biological word [56]. PRK, another PUA target cata-
lyzes the only reaction by which intermediates in the Calvin cycle can be contributed for fur-
ther CO2 assimilation [64]. RPE is important for both, the Calvin cycle and the reverse pentose
phosphate pathway. Loss of molecular functions of these proteins as it might occur through
covalent reactions of the nucleophilic protein residues with a PUA would immediately interfere
with the homeostasis of algae cells, explaining the fast adverse effect of PUAs. DDY covalently modifies proteins of P. tricornutum The response is more immediate than transcrip-
tomic regulation since proteins are the direct target of a covalent modification. Supporting Information A
561 nm laser was used for excitation, and fluorescence was filtered by a band pass filter (BP
570–620 nm) which opens up above 750 nm. WF—wide field. (ZIP) Supporting Information S1 Fig. Relative fluorescence intensity of P. tricornutum cells treated under different condi-
tions in two independent experiments. Cells were either incubated with TAMRA-PUA, 12 / 17 PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 Uptake and Protein Targets of Polyunsaturated Aldehydes in Diatoms TAMRA-N3, TAMRA-SA (only experiment 2) for one hour or kept under identical conditions
without probe. For each experiment one microscope slide per treatment with five cells (experi-
ment 1) or seven cells (experiment 2) was measured. For experiment 1, all microscope slides
were embedded in 2,2’-thiodiethanol as described in the materials and methods section, for
experiment 2 a poly (vinyl alcohol)/ n-propyl gallate antifade embedding medium [See Lu-
Walther H-W, Kielhorn M, Förster R, Jost A, Wicker K, Heintzmann R. fastSIM: a practical
implementation of fast structured illumination microscopy. Methods Appl Fluoresc. 2015;3
(1):014001] was used. Fluorescence intensities were recorded as mean gray value per pixel after
data treatment as described in the main text. To compare both experiments results were nor-
malized to “no probe”. Normalized averaged mean gray values per pixel of cells of each treat-
ment are presented as bars ±SD. Kruskal-Wallis one way analysis of variance on ranks revealed
differences in the median values among the treatment groups of each experiment (No. 1
H = 12.500, No. 2 H = 22.902; p<0.05). Tukey’s HSD test (p<0.05) attested significant differ-
ences between TAMRA-PUA and all other treatments within each experiment. (TIF) TAMRA-N3, TAMRA-SA (only experiment 2) for one hour or kept under identical conditions
without probe. For each experiment one microscope slide per treatment with five cells (experi-
ment 1) or seven cells (experiment 2) was measured. For experiment 1, all microscope slides
were embedded in 2,2’-thiodiethanol as described in the materials and methods section, for
experiment 2 a poly (vinyl alcohol)/ n-propyl gallate antifade embedding medium [See Lu-
Walther H-W, Kielhorn M, Förster R, Jost A, Wicker K, Heintzmann R. fastSIM: a practical
implementation of fast structured illumination microscopy. Methods Appl Fluoresc. 2015;3
(1):014001] was used. Fluorescence intensities were recorded as mean gray value per pixel after
data treatment as described in the main text. To compare both experiments results were nor-
malized to “no probe”. Normalized averaged mean gray values per pixel of cells of each treat-
ment are presented as bars ±SD. Supporting Information Kruskal-Wallis one way analysis of variance on ranks revealed
differences in the median values among the treatment groups of each experiment (No. 1
H = 12.500, No. 2 H = 22.902; p<0.05). Tukey’s HSD test (p<0.05) attested significant differ-
ences between TAMRA-PUA and all other treatments within each experiment. (TIF) S2 Fig. SDS-PAGE of in vivo treated samples of P. tricornutum. P. tricornutum was incu-
bated with 100μM of the reactive group (RG) DDY or SA or DMSO as control. After one hour
incubation cells were lysed, CuAAC with TAMRA-N3 was applied and SDS-PAGE and in-gel
fluorescence detection were accomplished (see also Fig 2). Only the DDY treated sample shows
specific fluorescent bands. S3 Fig. 2D GE images. Position of excised spots with identified proteins in the three 2D gels
(1, 2 and 3) presented in the Coomassie stained gels (A) and fluorescence images excited at
532nm for TAMRA-PUA detection (B). The positions of the spots were computed by Delta 2D
for each image by considering the Coomassie stained gel image as well as TAMRA-PUA and
Cy5 fluorescence images (for raw data of each image see S3 Folder). Slightly shifted positions
of spots between Coomassie and fluorescence images of each gel are due to change of gel
dimensions during Coomassie staining. (TIF) S1 Folder. Unmodified wide field fluorescence images of P. tricornutum treated with TAM-
RA-PUA, TAMRA-SA, TAMRA-N3 or without addition of a substance as control for
uptake experiments. Cells were measured with a Zeiss Elyra S1 system in wide field mode. A
561 nm laser was used for excitation, and fluorescence was filtered by a band pass filter (BP
570–620 nm) which opens up above 750 nm. WF—wide field. (ZIP) S1 Folder. Unmodified wide field fluorescence images of P. tricornutum treated with TAM-
RA-PUA, TAMRA-SA, TAMRA-N3 or without addition of a substance as control for
uptake experiments. Cells were measured with a Zeiss Elyra S1 system in wide field mode. A
561 nm laser was used for excitation, and fluorescence was filtered by a band pass filter (BP
570–620 nm) which opens up above 750 nm. WF—wide field. (ZIP) S1 Folder. Unmodified wide field fluorescence images of P. tricornutum treated with TAM-
RA-PUA, TAMRA-SA, TAMRA-N3 or without addition of a substance as control for
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21731545 12. Ribalet F, Wichard T, Pohnert G, Ianora A, Miralto A, Casotti R. S2 Folder. Unmodified bright field images of P. tricornutum treated with TAMRA-PUA, TAMRA-SA, TAMRA-N3 or without addition of a substance as control for uptake experi-
ments. Cells were measured with a Zeiss Elyra S1 system in bright field mode. Before data anal-
ysis tonal correction was optimized. BF—bright field. (ZIP) S3 Folder. Unmodified 2D GE images. Fluorescence images of TAMRA-PUA and Cy5
labeled protein gels and images of Coomassie stained gels. (ZIP) S1 Information. LC-MS/MS analysis and data processing. (DOCX) S1 Information. LC-MS/MS analysis and data processing. (DOCX) PLOS ONE | DOI:10.1371/journal.pone.0140927
October 23, 2015 13 / 17 Uptake and Protein Targets of Polyunsaturated Aldehydes in Diatoms S1 Table. Target proteins found by 2D GE. Proteins were classified into confident, labeled
and putative proteins subject to guidelines described in S1 Information and separated accord-
ing to their biological processes and molecular functions. (XLSX) S1 Table. Target proteins found by 2D GE. Proteins were classified into confident, labeled
and putative proteins subject to guidelines described in S1 Information and separated accord-
ing to their biological processes and molecular functions. (XLSX) Author Contributions Conceived and designed the experiments: SW GP BM OW. Performed the experiments: SW
NW YH BM HWLW. Analyzed the data: SW NW GP. Contributed reagents/materials/analysis
tools: SW NW RH OW AS. Wrote the paper: SW GP. Acknowledgments Kathrin Klehs, Ivana Šumanovac-Šestak and Martin Reifarth are acknowledged for helpful
advice on experimental design of fluorescence microscopy and choice of embedding medium. PLOS ONE | DOI:10.1371/journal.pone.0140927
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Supplementary Table 4 from Roles for MicroRNAs, miR-93 and miR-130b, and Tumor Protein 53–Induced Nuclear Protein 1 Tumor Suppressor in Cell Growth Dysregulation by Human T-Cell Lymphotrophic Virus 1
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Supplementary Table 4. Status of Tax expression in various
HTLV-1 cells
Cell line
Origin
tax gene transcript
Tax protein
MT-1
Leukemic
Expressed
Faintly
expressed
TLOM1
Leukemic
Not expressed
Not
detected
ED
Leukemic
Expressed,
but
contain
non-sense
mutation
Not
detected
ATL-43T
Leukemic
Not expressed
Not
detected
ATL-55T
Leukemic
Expressed,
but
contain deletion
Not
detected
ATL-48T
Leukemic
Expressed
Detected
ATL-2
Leukemic
Expressed
Detected
MT-4
Leukemic
Expressed
Detected
C81
Leukemic
Expressed
Detected Supplementary Table 4. Status of Tax expression in various
HTLV-1 cells
Cell line
Origin
tax gene transcript
Tax protein
MT-1
Leukemic
Expressed
Faintly
expressed
TLOM1
Leukemic
Not expressed
Not
detected
ED
Leukemic
Expressed,
but
contain
non-sense
mutation
Not
detected
ATL-43T
Leukemic
Not expressed
Not
detected
ATL-55T
Leukemic
Expressed,
but
contain deletion
Not
detected
ATL-48T
Leukemic
Expressed
Detected
ATL-2
Leukemic
Expressed
Detected
MT-4
Leukemic
Expressed
Detected
C81
Leukemic
Expressed
Detected Supplementary Table 4. Status of Tax expression in various
HTLV-1 cells
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https://scindeks-clanci.ceon.rs/data/pdf/0350-8501/2017/0350-85011777075T.pdf
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Khmer
| null |
Domestic violence in Serbia
|
Zbornik radova Pravnog fakulteta u Nišu
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cc-by-sa
| 4,376
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ȋæ©Ƭ©©ǡʹͲͲͷǣ͵ͲȌǤ10Psychological
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ϐ
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ȋ©ǡʹͲͳͳǣʹͲȌǤ 79 ʖ˹̆̈̅̀̂̈˸˼̆˺˸ʞ̈˸˺̅̆˻̌˸̂̋̃̊˽̊˸̋ʜ̀̐̋ȁʐ̡̈̆ȁʒ̆˼̀̅˸ ȁʹͲͳ ͳͳԘ ǡǡ
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de
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Ȍ
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ā
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ǡ
violence against women in marital and extramarital relations) was
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ruthless behavior
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Ǥ17 ͺ ʖ˹̆̈̅̀̂̈˸˼̆˺˸ʞ̈˸˺̅̆˻̌˸̂̋̃̊˽̊˸̋ʜ̀̐̋ȁʐ̡̈̆ȁʒ̆˼̀̅˸ ȁʹͲͳ
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Ǥ19
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ˋ˰ˈˆˑ˅ˋ˘ˋˊ˅˓˛ˋˎ˃˙˃Ǥ ʙ˯˖˚ːˈ˓ˈ˚ˋǣː˃˔ˋ˯ˈ˖˒ˑ˓ˑˇˋ˙ˋǡ˖ˊ˓ˑ˙ˋː˃˔ˋ˯˃ǡˆ˓˃˥˃ː˔ˍˑ˒˓˃˅ː˃ˊ˃˛˕ˋ˕˃
ˑˇː˃˔ˋ˯˃˖˒ˑ˓ˑˇˋ˙ˋǡˍ˓ˋ˅ˋ˚ːˑ˒˓˃˅ː˃ˊ˃˛˕ˋ˕˃ˑˇː˃˔ˋ˯˃˖˒ˑ˓ˑˇˋ˙ˋǡ˔˖ˇ˔ˍ˃
˒˓˃ˍ˔˃ˊ˃ˍ˓ˋ˅ˋ˚ːˑˇˈˎˑː˃˔ˋ˯ˈ˖˒ˑ˓ˑˇˋ˙ˋǤ 101 101
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https://openalex.org/W1528693124
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https://breast-cancer-research.biomedcentral.com/track/pdf/10.1186/bcr365
|
English
| null |
A novel phenylacetate-dextran derivative (NaPaC) inhibits breast cancer cell proliferation and modifies their interactions with endothelial cells
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Breast cancer research
| 2,001
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cc-by
| 24,675
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R Agresti, E Tagliabue, C Ghirelli, D Morelli, R Giovanazzi,
G Somenzi, M Campiglio, M Greco, A Balsari, S Menard Molecular Targeting Unit and General Surgery, Breast Unit, National
Cancer Institute, Milan, Italy Molecular Targeting Unit and General Surgery, Breast Unit, National
Cancer Institute, Milan, Italy Background: Disruption of the balance between apoptosis and pro-
liferation is considered to be an important factor in the development
and progression of tumor. In this study we determined the in vivo cell
kinetics along the spectrum of apparently normal epithelium, hyper-
plasia, preinvasive lesions and invasive carcinoma, in breast tissues
affected by fibrocystic changes in which preinvasive and/or invasive
lesions developed, as a model of breast carcinogenesis. Background: Clinical and experimental data have raised the possi-
bility that surgical removal of the primary tumor promotes the
growth of metastatic lesions. Purpose: This study was undertaken to determine the effect of
wound healing drainages and postsurgical sera obtained from
breast carcinoma (BC) patients on proliferation of dormant BC
cells and to assess the role of HER2 oncoprotein in this
proliferation. Materials and method: A total of 32 areas of apparently normal
epithelium and 135 ductal proliferative and neoplastic lesions were
studied. More than one epithelial lesion per case was analyzed. The apoptotic index (AI) and the proliferative index (PI) were
expressed as the percentage of TUNEL (TdT-mediated dUTP-nick
end-labelling) and Ki-67 positive cells, respectively. The prolifera-
tive/apoptotic index (P/A) was calculated for each case. Method: Proliferation of dormant BC cells was evaluated in vitro by
SRB colorimetric assay. Growth factors were identified by inhibi-
tion with specific antibodies and displacement of 125I-EGF from its
receptor. Cellular damage was measured by creatine phospho-
kinase level. The role of HER2 was analyzed by removal of HER2
from the membrane and inhibition by the anti-HER2 monoclonal
antibody herceptin. Results: Statistical analysis demonstrated significant differences
among the tissue groups for both indices (P<0.0001). The Als
and PIs were significantly higher in hyperplasia than in apparently
normal epithelium (P=0.04 and P=0.0005, respectively), in atypi-
cal hyperplasia than in hyperplasia (P=0.01 and P=0.04, respec-
tively) and in invasive carcinoma than in in situ
carcinoma
(P=0.0001 and P<0.0001, respectively). The two indices were
similar in atypical hyperplasia and in in situ carcinoma. The P/A
index increased significantly from normal epithelium to hyperplasia
(P=0.01) and from preinvasive lesions to invasive carcinoma
(P=0.04), whereas it was decreased (NS) from hyperplasia to
preinvasive lesions. A strong positive correlation between the Als
and the Pls was found (r=0.83; P<0.0001). Available online http://breast-cancer-research.com/content/3/S1 Available online http://breast-cancer-research.com/content/3/S1 The in vivo cell kinetics in breast carcinogenesis
NJ Agnantis, SA Kamina, PS Zagorianakou, A Demou,
A Katsaraki, P Kanavaros*, M Bai
Department of Pathology, Medical School, University of Ioannina,
Ioannina, Greece; *Department of Histology, Medical School,
University of Thessalia, Larisa, Greece R Agresti, E Tagliabue, C Ghirelli, D Morelli, R Giovanazzi,
G Somenzi, M Campiglio, M Greco, A Balsari, S Menard Meeting abstracts
23rd Congress of the International Association for Breast Cancer
Research
Düsseldorf, Germany
13–16 June 2001 Breast Cancer Res 2001, 3 (suppl 1):S1–S24
© 2001 BioMed Central Ltd
(Print ISSN 1465-5411; Online ISSN 1465-542X) Breast Cancer Res 2001, 3 (suppl 1):S1–S24
© 2001 BioMed Central Ltd
(Print ISSN 1465-5411; Online ISSN 1465-542X) Received: 10 May 2001
Published: 29 May 2001 A2 A5 The CC chemokine RANTES as a potential
contributor to breast cancer progression
E Azenshtein, G Luboshits, S Shina, E Neumark, N Vigler*,
S Chaitchik†, I Keydar, A Ben-Baruch
Department of Cell Research and Immunology, George S Wise
Faculty of Life Sciences, Tel-Aviv University; *Department of Oncology,
Tel-Aviv Sourasky Medical Center; †Department of Oncology, Sackler
Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel We collected 88 biopsy specimens, originally diagnosed benign,
from the patients who subsequently developed IDC in the ipsilat-
eral breast. Seven asynchronous lesions of initial biopsy (re-evalua-
tion was DCIS) and the respective IDC were subjected to LOH
analysis in this study. Thirteen microsatellite markers, which were
mapped to and/or very close to the tumor suppressor genes or
regions with frequent LOH in breast cancer, were used. In breast carcinoma, high levels of tumor-associated macrophages
are correlated with lymph node metastases and clinical aggressive-
ness. Potential candidates that may support the recruitment of
monocytes from the circulation into breast tumors are the members
of the CC subfamily of chemokines. In the present study we evalu-
ated the expression of the CC chemokine RANTES in sections of
breast cancer patients diagnosed in different stages of disease. Our results indicate that high incidence and intensity of RANTES
expression were directly correlated with a more advanced disease,
suggesting that the chemokine may be involved in breast cancer
progression. LOHs were observed in parallel with the tumor progression from
DCIS to IDC in all cases except for one that developed IDC in
another quadrant. The six patients developed IDC near the initial
biopsy, and presented similar or identical histopathologic features. LOH analysis of biopsy specimens from patients who subsequently
developed IDC demonstrated acquisition of genetic change at an
earlier stage, as the same allele at the same genomic locus was
lost in DCIS. Our results suggest that genetic alternations accumulate during
cancer progression from DCIS to IDC, and DCIS presents a high
risk of developing invasive transformation. Analyses performed by using the T47D and MCF-7 human breast
adenocarcinoma cells indicated that RANTES expression is tightly
regulated by cytokines. Furthermore, the results of our study indi-
cate that T47D-derived RANTES partially contributes to monocyte
migration, and suggest that in vivo this chemokine may be involved
in inducing monocyte infiltration to breast tumor sites. In the
present study we further characterized the paracrine and autocrine
mechanisms by which RANTES may support breast cancer pro-
gression. A5 The results suggest that RANTES may be involved in a
complex process, in which a crosstalk between infiltrating mono-
cytes and the tumor cells may affect tumor progression. Phagocytic activity of monocytes in patients with
breast cancer at different clinical stages
NN Arsenijevic, D Baskic, LD Acimovic* Institute of Microbiology and Immunology, and *Clinic for Surgery,
Faculty of Medicine, University of Kragujevac, Yugoslavia Asynchronous LOH analysis of ductal carcinoma
in situ from patients who subsequently developed
invasive ductal carcinoma M Amari, T Moriya*, Y Harada, T Ishida, K Ohnuki, N Ohuchi
Division of Surgical Oncology, Tohoku University School of Medicine,
Sendai, Japan; *Department of Pathology, Tohoku University Hospital,
Sendai, Japan Management of women with ductal carcinoma in situ (DCIS) is
currently a major concern. Biological characteristics in the light of
progression from DCIS to invasive ductal carcinoma (IDC) remain
unknown. Our previous study [1] investigating synchronous lesions
demonstrated higher LOH frequencies in parallel with the tumor
progression from atypical ductal hyperplasia (ADH) to DCIS and
IDC [1]. We report here an asynchronous LOH analysis of DCIS
from patients who subsequently developed IDC. A3 cancer group, related to the clinical stage. Thus, we noted a sixfold
decrease in the capacity of phagocytosis, fourfold decrease in per-
centage of phagocytosis and twofold decrease in phagocytic index
in patients with advanced stage (group C). The alterations in
number and function of PBMo in patients with benign and malig-
nant breast tumor were observed in close association with clinical
stage of disease, and thus they could be considered as indicators
of tumor progression. However, further studies are required to
determine whether monocyte dysfunction could provide additional
prognostic information in the case of breast cancer diagnosis and
therapy. R Agresti, E Tagliabue, C Ghirelli, D Morelli, R Giovanazzi,
G Somenzi, M Campiglio, M Greco, A Balsari, S Menard Results: Healing wound drainages and postsurgical sera from BC
patients stimulated the in vitro growth of BC cells. Removal of the
HER2 oncoprotein from BC cell membrane led to a dramatic
decrease in the induced proliferation. Drainage-induced prolifera-
tion was around 50% inhibited by antibodies directed against
EGF-like factors, including HB-EGF and TGF-α. Levels of these
growth factors in postsurgical sera, as well as the level of drainage-
induced proliferation, were directly correlated with the entity of
surgery (r=0.8, P=0.0007 and r=0.64, P=0.009, respectively). Treatment of the tumor cells with herceptin, abolished the patients’
drainage-induced proliferation when added to cultures before the
growth stimulus. Conclusion: These findings suggest accelerating cell turnover
along the continuum of breast carcinogenesis. Atypical hyper-
plasias and in situ carcinomas might be kinetically similar lesions. In the transition from normal epithelium to hyperplasia and from
preinvasive lesions to invasive carcinoma, the net growth of epithe-
lial cells results from a growth imbalance in favour of proliferation. In the transition from hyperplasia to preinvasive lesions there is an
imbalance in favour of apoptosis. Conclusion: HER2 overexpression by BC cells plays a major role
in the postsurgery rescue of metastatic BC cells from dormancy. Herceptin appears to inhibit this growth induction. A prospective
randomized clinical trial of perioperative treatment with herceptin of
BC patients is starting. reast Cancer Research Vol 3 Suppl 1
23rd Congress of the International Association for Breast Cancer Researc A6 The investigation was designed to evaluate numerical and func-
tional properties of peripheral blood monocytes (PBMo) in patients
with breast cancer at different clinical stages. Monocyte phagocy-
tosis test was performed in 19 patients with benign breast tumor,
29 patients with breast cancer and 10 healthy subjects. Cancer
patients were divided into three groups on the basis of the clinical
stage of disease: group A, patients with localized disease;
group B, patients with regional lymph node metastasis; and
group C, patients with distant metastasis. Patients with advanced
disease (group C) showed an increase in neutrophils, but no differ-
ences in the total count of leukocytes and absolute number of
lymphocytes as compared with healthy individuals, patients with
benign breast tumor or patients with lower stage. However, the
mean number of monocytes decreased in patients with benign
disease and further decreased in cancer patients, reaching the sig-
nificantly lowest value in patients with distant metastasis. Phago-
cytic activity of PBMo was found to be significantly lower in
patients with benign tumor, and it became further reduced in the Available online http://breast-cancer-research.com/content/3/S1 Despite these achievements, we are not able to select enough
patients with the need for radiotherapy, and the required dose in
this patient population. New techniques, such as comparative
genomic hybridization assay, DNA microarrays, functional DNA
screens and functional yeast assays, may guide us more pre-
cisely toward the optimal treatment strategy in individual patients. Furthermore, these research lines offer the possibility to investi-
gate the mechanism of action, and therefore lead to the develop-
ment of new drugs that will potentiate the cell-killing effect of
radiotherapy. This lecture focuses on the integration of these new
techniques in relation to the obtained results from the above-
mentioned clinical trials. (PRL) has been reported to inhibit apoptosis in various cell
types, including a Nb2 rat lymphoma cell line. In addition, there
is evidence that the human PRL-antagonist hPRL-G129R
induces apoptosis in breast cancer cell lines. We investigated a
possible relationship between prolactin receptor (PRL-R)
expression and apoptosis of CD3+ T lymphocytes, as well as
PRL plasma levels, in patients with breast cancer. Peripheral
blood mononuclear cells of patients (n = 11) and sex-matched
normal controls (n = 12) were stained with Annexin V, anti-Fas
mAb (CD95), mouse antihuman PRL-R mAb B6.2, anti-CD3
mAb and respective isotype control mAbs. Multicolor flow
cytometry was used to compare expression of these markers on
T cell. In patients, 37 ± 19% (median ± SD) of CD3+ cells bound
Annexin V, marking early apoptosis of T lymphocytes compared
with 17 ± 10% in controls (P < 0.004). Furthermore, 82 ± 15%
of the CD3+ T cells were Fas+ in patients, compared with
51 ± 9% in controls (P < 0.0001). All CD3+ T lymphocytes were
positive for PRL-R expression in breast cancer patients, as well
as in normal control individuals. The mean fluorescence intensity
of PRL-R on T lymphocytes of breast cancer patients was
106–172 (median 119) compared with 87–176 (median 123),
suggesting no difference in PRL-R expression on T lymphocytes
in patients versus controls. PRL plasma levels were comparable
in patients and normal controls (4.8 ± 3.4 ng/ml versus
9.8 ± 4.6 ng/ml). In concordance with these findings, PRL was
not able to inhibit the onset of apoptosis of Jurkat cells, a thymic
lymphoma cell line, incubated with Fas cross-linking CH-11
mAb. Monocyte phagocytic function in patients with
breast cancer during therapy
D Baskic, NN Arsenijevic, LD Acimovic*
Institute of Microbiology and Immunology; and *Clinic for Surgery,
Faculty of Medicine, University of Kragujevac, Yugoslavia The present study was designed to elucidate phagocytic function
of peripheral blood monocytes in patients with breast cancer
during surgery and chemotherapy. Absolute and relative number
of peripheral blood leukocytes and monocyte phagocytic function
(percentage of phagocytosis [PP], phagocytic index [PI] and
capacity of phagocytosis [CP]) were determined in 29 patients
with breast cancer and 10 healthy individuals. These parameters
were determined at the time of diagnosis, following surgery and
after chemotherapy. The total count of circulating leukocytes, and
absolute and relative counts of polymorphonuclears and lympho-
cytes were not significantly different between investigated groups,
before and after therapy. The mean number of monocytes was sig-
nificantly lower in cancer patients at diagnosis, but increased fol-
lowing surgery reaching the control value. There were no
significant postchemotherapy changes in the number of mono-
cytes. PP, PI and CP were decreased at the time of diagnosis. PP
and CP recovered to normal values following surgery, but PI
remained decreased. Following chemotherapy PP and CP
remained stable, whereas PI further decreased reaching the
values significantly lower than those found before the start of
chemotherapy. However, 3 months after last cycle of chemother-
apy, all tested parameters returned to normal values. These results
showed that phagocytic activity of cancer patients’ monocytes,
decreased at diagnosis, returned within the normal range after
surgical therapy. However, we need time to determine whether
the alteration in PBMo phagocytic activity may provide additional
prognostic information when monitoring surgically treated breast
cancer patients. B Betz, D Larbig, TO Goecke, C Nestle-Krämling, HG Bender,
D Niederacher Department of Obstetrics & Gynecology, Heinrich-Heine-University,
Düsseldorf, Germany Objective: Denaturing high-performance liquid chromatography
(DHPLC) is a recently developed method for detection of mutation
that is gaining importance as a screening method for analyzing
familial breast cancers, as well as heterogeneous tumor material. Method: DHPLC was established for mutation detection in
BRCA1/2 diagnostic, using more than 200 different positive con-
trols. Up until now, 64 DNA samples from patients with familial
background for breast cancer (BC) were analyzed by DHPLC for
BRCA1/2 mutations. An additional 136 sporadic BC were exam-
ined for p53 mutations, analyzing exons 5–8by DHPLC. Positive
results were confirmed by direct DNA sequencing. Objective: Denaturing high-performance liquid chromatography
(DHPLC) is a recently developed method for detection of mutation
that is gaining importance as a screening method for analyzing
familial breast cancers, as well as heterogeneous tumor material. Method: DHPLC was established for mutation detection in
BRCA1/2 diagnostic, using more than 200 different positive con-
trols. Up until now, 64 DNA samples from patients with familial
background for breast cancer (BC) were analyzed by DHPLC for
BRCA1/2 mutations. An additional 136 sporadic BC were exam-
ined for p53 mutations, analyzing exons 5–8by DHPLC. Positive
results were confirmed by direct DNA sequencing. The relevance of translational research for
radiotherapy in breast cancer
H Bartelink Antoni van Leeuwenhoek Ziekenhuis, The Netherlands Cancer
Institute, Amsterdam, The Netherlands In several EORTC trials the role of radiotherapy in breast cancer
has been examined. It has been shown that patients with DCIS
radiotherapy have a reduced risk of both invasive and noninvasive
ductal cancer recurrences. For patients with early breast cancer
we demonstrated that a boost of 16 Gy reduces the risk of recur-
rence in the breast by nearly a factor of 2, and is especially
clinically relevant for patients younger than 50 years. In locally
advanced breast cancer patients, a similar reduction in the local
recurrence rate was seen when chemotherapy or hormontherapy
was added to radiotherapy. In several EORTC trials the role of radiotherapy in breast cancer
has been examined. It has been shown that patients with DCIS
radiotherapy have a reduced risk of both invasive and noninvasive
ductal cancer recurrences. For patients with early breast cancer
we demonstrated that a boost of 16 Gy reduces the risk of recur-
rence in the breast by nearly a factor of 2, and is especially
clinically relevant for patients younger than 50 years. In locally
advanced breast cancer patients, a similar reduction in the local
recurrence rate was seen when chemotherapy or hormontherapy
was added to radiotherapy. Available online http://breast-cancer-research.com/content/3/S1 These results indicate that PRL/PRL-R might not be
involved in modulating Fas/Fas ligand interactions, which are, in
part, responsible for apoptosis of T lymphocytes, leading to
excessive turnover of T cells in the circulation of patients with
breast cancer. A8 Spontaneous apoptosis of circulating
T-lymphocytes and its correlation to their
prolactin receptor expression and prolactin
plasma levels in patients with breast cancer
T Bauernhofer, U Friebe-Hoffmann, T Hoffmann, G Dworacki,
B Vonderhaar, TL Whiteside
University of Pittsburgh Cancer Institute, Pittsburgh, Philadelphia; and
National Cancer Institute, NHI, Bethesda, Maryland, USA MP Boland, EA Kritikou, RS Chapman*, JL Heeley,
RWE Clarkson, CJ Watson Current therapies for most types of cancer focus on either surgical
or radiotherapeutic eradication of the primary tumor as the best
opportunity for cure. Therapy of disseminated disease has focused
on chemotherapy, but, with the exception of certain rarer types of
tumors, few patients are cured by chemotherapy, and even improve-
ments in survival have been difficult to demonstrate. TRANSGENE’s
current approaches to oncology focus on the stimulation of the
body’s own immune system to induce rejection of tumors. One of
these approaches is antigen-specific therapy. The first product can-
didate for antigen-specific therapy expresses the tumor-associated
MUC1 antigen, stimulating a cellular immune response that may be
useful in treating breast cancer and various epithelial cancers, such
as lung, pancreatic and ovarian cancers. The product developed is
a recombinant vaccinia virus containing sequences that code for
human MUC-1 antigen and interleukin-2. A phase I trial in nine
women with breast cancer was performed, in which the potential
product was well tolerated without serious side effects, and MUC1-
specific immune responses were observed. Phase II trials in breast
and in prostate cancer began during the second quarter of 1998. A
phase I trial in lung cancer patients is also in progress. During the
same period, a second-generation product was developed. The
new construct has been put into a highly attenuated vaccinia virus
(modified virus Ankara), the safety of which was tested in a clinical
trial in MUC1-positive cancer patients. Based on these results,
phase II studies are in preparation to assess the clinical efficacy of
this product in different populations of patients whose tumors
express the MUC-1 tumor antigen. Department of Pathology, University of Cambridge, Cambridge, UK;
*CRC Institute for Cancer Studies, University of Birmingham,
Birmingham, UK Postlactational regression of the mammary gland is characterized by
extensive apoptosis of the epithelial compartment. Involution occurs
in two phases: an early reversible phase and a later phase accom-
panied by breakdown of the extracellular matrix and remodeling of
the gland. We have used both knockout mice and a cell-culture
model to identify the transcription factors that regulate the early
phase of involution. Conditional deletion of Stat3 results in dimin-
ished apoptosis and delayed involution, whereas, in contrast, loss of
IRF-1, a downstream target of Stat1, accelerates the first phase of
involution. We have begun to analyze in more detail the molecular
events associated with the activation of these transcription factors. Association of the Epstein–Barr virus with breast
cancer: in vivo and in vitro studies
M Bonnet-Duqeynoy, H Arbach, K Takada*, I Joab
INSERM 99-32, Hôpital Saint Louis, Paris, France; *Institute for
Genetic Medicine, Hokkaido University, Sapporo, Japan Association of the Epstein–Barr virus with breast
cancer: in vivo and in vitro studies
M Bonnet-Duqeynoy, H Arbach, K Takada*, I Joab
INSERM 99-32, Hôpital Saint Louis, Paris, France; *Institute for
Genetic Medicine, Hokkaido University, Sapporo, Japan p
M Blazar, V Cirulli*, F Prins, SV Litvinov
Department of Pathology and Division of Laboratory Animals, Leiden
University Medical Center, Leiden, The Netherlands; *The Whittier
Institute for Diabetes, University of California San Diego, La Jolla,
California, USA M Blazar, V Cirulli*, F Prins, SV Litvinov
Department of Pathology and Division of Laboratory Animals, Leiden
University Medical Center, Leiden, The Netherlands; *The Whittier
Institute for Diabetes, University of California San Diego, La Jolla,
California, USA Epstein–Barr virus (EBV) may be a cofactor in the development of
different malignancies, including several types of carcinomas. We
demonstrated the presence of EBV in human breast cancers. We
detected the EBV genome by PCR in 51% of the tumor biopsies. In
90% of the cases studied, the virus was not detected in healthy
tissue. The presence of the EBV genome in breast tumors was con-
firmed by Southern-blot analysis. The EBV latent protein EBNA-1
was observed in a fraction (5–30%) of tumor epithelial cells. Expression of the EBV genes BNLF1 and BARF0 will be reported. A statistical relationship was established between the presence of
EBV and several poor prognostic factors. EBV may be a cofactor
in the development of a subset of breast cancers. Ep-CAM, an epithelial cell–cell adhesion receptor, is often over-
expressed in association with proliferation and remodeling in epithe-
lial tissues. Development of the mouse mammary gland during
pregnancy is associated with a progressive upregulation of Ep-
CAM expression, eventually reaching very high levels at day 16 of
pregnancy. This phenomenon is paralleled by a concomitant
branching of the mammary ductal tree and a sustained epithelial cell
proliferation. Using a MMTV-LTR/Ep-CAM transgenic mouse model,
we demonstrate that forced expression of Ep-CAM in the mammary
epithelium leads to an induction of budding and secondary branch-
ing of the glandular tree in virgin females. Interestingly, a complete
cycle of gestation in the Ep-CAM transgenic mice results in extreme
ductal hyperplasia/ductectasia and lobular hypoplasia, in combina-
tion with partially decreased differentiation of both ductal and alveo-
lar (lobular) epithelial cells. MP Boland, EA Kritikou, RS Chapman*, JL Heeley,
RWE Clarkson, CJ Watson Downstream targets have not been identified, although IGFBP-5
may be an indirect target of both Stat3 and IRF-1. In the absence of
Stat3, elevated levels of p21, p53 and Stat1 are observed. Using a
mammary epithelial cell culture model, KIM-2, and inducible activa-
tion of Stat3 and Stat5, we have shown that dimerization of Stat3
alone is sufficient to induce apoptosis of KIM-2 cells. Furthermore,
apoptosis can be significantly increased by blocking a survival
pathway. In contrast, dimerization of Stat5 provides a differentiation
signal and, subsequently, a survival signal for differentiated KIM-2
cells. Interplay between Stat3 and Stat5, identification of down-
stream targets, and crosstalk with other pathways is now being
investigated. Spontaneous apoptosis of circulating
T-lymphocytes and its correlation to their
prolactin receptor expression and prolactin
plasma levels in patients with breast cancer
T Bauernhofer, U Friebe-Hoffmann, T Hoffmann, G Dworacki,
B Vonderhaar, TL Whiteside
University of Pittsburgh Cancer Institute, Pittsburgh, Philadelphia; and
National Cancer Institute, NHI, Bethesda, Maryland, USA Results: The analysis of 64 DNA samples from patients with famil-
ial background for BC revealed several mutations and unclassified
variants (UVs). Twenty-three different p53 mutations could be
detected in 138 sporadic BC. Dilution of mutant DNA by wild-type
DNA revealed the high sensitivity of this method: 5% mutant DNA
is sufficient to achieve a positive DHPLC result. However, confirm-
ing a positive DHPLC result by DNA sequencing is difficult in het-
erogeneous tumor material. We have previously shown that a higher percentage of circulat-
ing CD3+ T lymphocytes undergo spontaneous apoptosis in
cancer patients as compared with normal controls. Prolactin Conclusion: DHPLC is a reliable, high-throughput technique for
detection of mutation in familial breast cancers, as well as in het-
erogeneous tumor material. Breast Cancer Research Vol 3 Suppl 1
23rd Congress of the International Association for Breast Cancer Research reast Cancer Research Vol 3 Suppl 1
23rd Congress of the International Association for Breast Cancer Researc A10
Specific immunotherapy of MUC1-positive
adenocarcinomas with a recombinant vaccinia
virus expressing MUC1 and IL-2
N Bizouarne, P Squiban, B Acres, JM Balloul, MA Ohresser,
R Figlin*, A Belldegrun*, R Herrman†, C Rochlitz†
TRANSGENE SA, Strasbourg, France; *UCLA School of Medicine,
USA; †Kantonspital, Basel, Switzerland A10 and increased rate of cell proliferation. These results support novel
morphoregulatory functions for the adhesion receptor Ep-CAM in
epithelial tissue development and homeostasis. A13 Novel morphoregulatory functions for the
adhesion receptor Ep-CAM in the mammary
epithelium
M Blazar, V Cirulli*, F Prins, SV Litvinov
Department of Pathology and Division of Laboratory Animals, Leiden
University Medical Center, Leiden, The Netherlands; *The Whittier
Institute for Diabetes, University of California San Diego, La Jolla,
California, USA Identification of HER2-positive breast carcinomas
as a particular subset with peculiar clinical
behaviours Bovine leukemia virus (BLV) is an oncogenic retrovirus that com-
monly infects cattle and causes a B cell leukemia/lymphoma in ‰
of 1% of infected cattle. BLV is present in much of marketed beef
and dairy products, and breast cancer incidence is greatest in
countries with high consumption of bovine foodstuffs. We were
therefore interested in determining whether humans were infected
with BLV, and whether it might play a role in breast cancer. In pre-
vious studies we found that many humans had antibodies to BLV
envelope glycoprotein (gp51) and capsid protein (p24), suggest-
ing humans might possibly be infected with BLV. We used
immunohistochemistry (IHC) and in situ PCR (IS-PCR) to detect
viral protein and proviral DNA, respectively, as signs of infection in
surgically excised human breast tissue sections. IHC utilized a
monoclonal antibody to the BLV p24 capsid protein. IS-PCR uti-
lized primers from the tax region of the BLV genome to amplify a
product with directly incorporated digoxigenin-11dUTP tags, which
were then detected with a peroxidase-conjugated antibody to
digoxigenin. The majority of the breast tissues had evidence of BLV
proviral genome and four out of 27 were positive for BLV capsid
protein. We are working to accumulate data on enough samples to
determine whether infection of breast tissue is associated with the
pathologic classification of the tissue. This research was sup-
ported by funds from the California Breast Cancer Research
Program. A large series of 2000 primary breast carcinomas was analyzed for
HER2 overexpression, and its prognostic potential. A subset analy-
sis, considering HER2-positive tumors as an independent subset
of breast carcinomas, was conducted. In our series, HER2 positiv-
ity was not associated with nodal status, unless the number of infil-
trated nodes was considered, whereas it was strongly associated
with large tumors (P>10–4), grade III tumors (P>10–4), lymphoid
infiltration (P>10–4) and absence of hormone receptor expression
(P>10–4). HER2 overexpression was a strong prognostic indicator
in N+ patients (P<10–7), whereas its prognostic impact was weak
and not statistically significant in the N– patients. Analysis of the
hazard ratio of relapse in relation to time from surgery indicate that
the poor prognosis associated with HER2-positivity in N+ patients
was found to be due to a peak of relapses in the first 3–4 years
from surgery. Association of the Epstein–Barr virus with breast
cancer: in vivo and in vitro studies
M Bonnet-Duqeynoy, H Arbach, K Takada*, I Joab
INSERM 99-32, Hôpital Saint Louis, Paris, France; *Institute for
Genetic Medicine, Hokkaido University, Sapporo, Japan Surprisingly, mammary gland involution
is affected because of a decreased frequency of apoptotic figures Latently EBV infected breast undifferentiated human epithelial cell
line, MDA-MB-231, was obtained and injected into nude mice. Tumors were obtained in which EBV persists. The persistence of EBV
in nude mice tumors, in the absence of any selection, suggests that
mammary epithelial cells could be a natural host for EBV. These
models will be used for the elaboration of specific therapeutic targets. Available online http://breast-cancer-research.com/content/3/S1 Available online http://breast-cancer-research.com/content/3/S1 Normal breast mammary epithelial cells are now being infected by
EBV in order to investigate the oncogenic potential of EBV in those
epithelial cells. clones are able to develop tumors in vivo despite high stable Fhit
expression, prompting us to investigate the different mechanisms
between the two types of Fhit-expressing clones. Supported by AIRC. A15 Restored expression of Fhit protein in Fhit-minus
breast cancer cells
M Campiglio, C Olgiati, P Aiello, CM Croce*, S Ménard
Molecular Targeting Unit, Department of Experimental Oncology,
Istituto Nazionale Tumori, Milan, Italy; *Kimmel Cancer Institute
Thomas Jefferson University, Philadelphia, USA Identification of HER2-positive breast carcinomas
as a particular subset with peculiar clinical
behaviours Multivariate analysis of different prognostic factors in
HER2+ and HER2– subsets indicated that grade is the most impor-
tant factor, followed by nodal status, lymphoid infiltration and tumor
size in HER2-negative breast carcinomas, whereas nodal status
was the most important prognostic factor, with tumor size showing
only borderline significance, in the HER2-positive group. Together,
the results indicate that HER2-positive breast carcinomas repre-
sent a particular subset of tumors with peculiar clinical and patho-
logical behaviours. Thus, conclusions drawn from clinical trials,
which serve as the basis for clinical management of breast carcino-
mas, might not always be valid for this low-frequency subset. Supported by AIRC. Bovine leukemia virus in human breast tissues
GC Buehring, KY Choi, HM Jensen*
University of California, Berkeley; *University of California, Davis,
California, USA Bovine leukemia virus in human breast tissues
GC Buehring, KY Choi, HM Jensen*
University of California, Berkeley; *University of California, Davis,
California, USA A17 Prevention of thymic atrophy in mammary tumor
bearers by IFN-γ
V Charyulu, B Adkins*, D Lobo*, DM Lopez*
Florida Atlantic University, Department of Health Sciences, Boca
Raton; *Department of Microbiology and Immunology, University of
Miami School of Medicine, Miami, Florida, USA Genetic modifiers of cancer risks conferred by
BRCA1 and BRCA2
P Devilee
Leiden University Medical Center, Leiden, The Netherlands Department of Obstetrics & Gynecology, Düsseldorf University
Medical Center, Düsseldorf; *Georg-Speyer-Haus
(Chemotherapeutical Research Institute), Frankfurt, Germany Department of Obstetrics & Gynecology, Düsseldorf University
Medical Center, Düsseldorf; *Georg-Speyer-Haus
(Chemotherapeutical Research Institute), Frankfurt, Germany One of the major goals of tumor immunotherapy is to overcome
immune escape and tumor anergy mechanisms. The identification of
(relatively) tumor-specific epitopes is more important for adoptive
immunotherapy strategies than their immunogenicity. In the immuno-
cellular approach, D44v-epitope-specific T-cells were cloned intro-
ducing a fusion gene encoding the single chain Fv-fragment of
CD44v-specific mAb and the zeta-chain of the TCR complex. MHC-
independent retargeted cytotoxicity could be shown toward antigen-
expressing tumor cells in vitro and in vivo. In a humoral approach, the
fusion gene for a Her2neu-specific scFv and a bacterial toxin was
expressed in E coli. After purification the fusion toxin showed signifi-
cant activity in animal experiments using Her2-neu-expressing tumors. Meanwhile, the first six patients suffering from Her2-neu-expressing
cancers have been treated topically so far. No significant systemic or
local side effects could be detected. Four out of six patients had a
local PR/CR. Further clinical studies are warranted and ongoing. Approximately 15% of all breast cancer patients have a positive
family history of the disease. BRCA1 and BRCA2 are two genes
that explain major proportions of families with multiple cases of early-
onset breast and/or ovarian cancer. Despite the high risks of breast
and ovarian cancer conferred by deleterious BRCA1 and BRCA2
mutations, a strong variability in phenotype has been observed
among families segregating the same mutation. This can range from
early-onset breast cancer and ovarian cancer, to late-onset breast
cancer without ovarian cancer. Even within a single pedigree, ages
of onset of cancer can vary substantially. These observations support
the idea that disease outcome in carriers is codetermined by other
factors. Different risk estimates for BRCA mutations, depending on
the type of population studied, also attest to this point. Risk esti-
mates derived from families with multiple cases of early-onset breast
cancer, used for linkage analysis to detect BRCA1 and BRCA2,
came out substantially higher than those from population-based
studies, and risks also appear to differ between populations. Both genetic and environmental factors are thought to interact with
BRCA1 and BRCA2. The influence of nongenetic factors is demon-
strated by the finding that even identical carrier twins may differ in
disease history. Prevention of thymic atrophy in mammary tumor
bearers by IFN-γ The gene FHIT, encompassing the FRA3B fragile site, is located in
a region of chromosome 3p14.2 that is often deleted in several
types of epithelial cancers and, therefore, it has been investigated
as a candidate tumor suppressor. In breast cancer inactivation of
FHIT occurred in 70% of the patients, and it is caused by both
alterations in the regulation of Fhit expression and by deletions of
the gene. Moreover, analysis of 500 cases of breast carcinomas
with 20 years of follow up demonstrated that loss of Fhit protein is
associated with high proliferative, large and undifferentiated
tumors, even though Fhit is not a prognostic factor. In order to elu-
cidate the possible role of FHIT as a tumor suppressor in breast
cancer and to identify its mechanisms, Fhit protein-negative breast
cancer cell lines lacking endogenous protein expression were
stable transfected with FHIT cDNA. Stable transfectant clones
showed no alteration in cell morphology and in in vitro anchorage-
dependent and independent proliferation. A significant delay in the
tumor growth in nude mice was observed for some Fhit-positive
clones; in an additional case the outgrowing of the tumor was due
to loss of Fhit expression in vivo. Interestingly, some Fhit-positive V Charyulu, B Adkins*, D Lobo*, DM Lopez*
Florida Atlantic University, Department of Health Sciences, Boca
Raton; *Department of Microbiology and Immunology, University of
Miami School of Medicine, Miami, Florida, USA Development of the in vivo transplantable D1-DMBA-3 mammary
tumors results in an alteration of several cytokines in the host, and
IFN-γ is one of the most severely downregulated. Notably, the thy-
muses of these mice display a profound atrophy that is associated
with a severe depletion of CD4+8+ thymocytes. Investigations into
the possible mechanisms that lead to this thymic atrophy revealed
that the levels of proliferation assessed by in vivo labeling with
5′-bromo-2′-deoxyuridine (BrdU) were similar in control and tumor-
bearing mice. However, our studies implicated a modest increase in
apoptosis, coupled with an arrest at the triple negative stage of dif-
ferentiation in the thymic hypocellularity in tumor bearers. Three-dimensional ultrasound-guided biopsy of
breast lesion: a new diagnostic support in the
preoperative diagnosis
L Delle Chiaie, S Schindelmann, I Heinich, V Heilmann,
G Helms, R Terinde
University Clinic, Ulm, Germany University Clinic, Ulm, Germany University Clinic, Ulm, Germany A safe and precise preoperative histologic diagnosis is the goal in
the modern treatment of breast cancer, to optimize the surgical
radicality and to reduce unnecessary mutilation without increasing
the risk of residual cancer and later recidives and to optimize the
degree of surgical radicality. Ultrasound-guided procedures are
useful in biopsying US-detectable breast lesions. In recent years
many bioptic procedures have been developed; each shows
advantages and disadvantages, but until now none has been
defined as the optimal one. Prevention of thymic atrophy in mammary tumor
bearers by IFN-γ We have
transfected the DA-3 mammary tumor cell line, derived in vitro from
the in vivo D1-DMBA tumors, with the IFN-γ gene and showed the Breast Cancer Research Vol 3 Suppl 1
23rd Congress of the International Association for Breast Cancer Research reast Cancer Research Vol 3 Suppl 1
23rd Congress of the International Association for Breast Cancer Researc production of high levels of IFN-γ protein by the transfected cells. Inoculation of hosts with IFN-γ transfected cells 4 days prior to chal-
lenge with the D1-DMBA-3 tumor resulted in a blockage of the
thymus involution in these mice. In contrast, using in the same pro-
tocol untransfected DA-3 cells, the progressive atrophy observed in
animals with D1-DMBA-3 tumors was observed. These results
suggest that the lack of IFN-γ may be an important factor in the
thymic atrophy that occurs during mammary tumorigenesis. and approximately 10% did not have malignant lesions). The diag-
nosis of malignancy or benignancy was confirmed in 97% of cases
(55/57); two false-negative bioptic results indicated hyperplasy
and suspected adenosis, but the successive postoperative diagno-
sis showed clear malignancy. With 3D US support we were able to reduce the number of biop-
sies for each lesion (two to three) without reduction of the histologic
results, also reducing the costs and the possible complications
(haematomas, infections and malignant cell spreading). Genetic modifiers of cancer risks conferred by
BRCA1 and BRCA2
P Devilee
Leiden University Medical Center, Leiden, The Netherlands Simple chance may determine age of onset, because
multiple genetic mutations are required for full tumorigenesis. Thus far
we are not even sure whether modifiers of BRCA-conferred risk actu-
ally exist, but some suggestive associations have been reported,
which will require independent confirmation. Rare alleles at HRAS1
were found to increase risk of ovarian cancer in BRCA1 carriers,
whereas breast cancer risk has been found to be modified by rare
alleles at the androgen receptor. Among Ashkenazi Jewish women, a
polymorphism in the 5′UTR of the RAD51 gene increased risk of
breast cancer fourfold, but only in carriers of the BRCA2-6174delT. Telomerase activity and bcl2 expression in human
breast cancer The Breast Cancer Centre, St George’s Hospital, London, UK Background: Telomerase is a ribonucleoprotein that synthesizes
telomers and plays an important role in cellular immortalization. Bcl2 gene encodes for a mitochondrial protein that is thought to
prevent apoptosis of normal cells. We previously reported telo-
merase activity in 74% of human invasive breast cancers, and
detected a significant association between telomerase activity and
prognostic parameters such as nodal status, tumour size and cellu-
lar proliferation. We hypothesized that telomerase reactivation in
human breast cancer was associated with reduced immunohisto-
chemical expression of bcl2. Materials and method: Bcl2 immunohistochemical expression was
determined in 25 infiltrating breast carcinomas with known telo-
merase activity (17 telomerase-positive and 8 telomerase-negative). The percentage of strongly and moderately stained tumour cells for
bcl2 was determined by a breast pathologist who was blinded to
telomerase data. Fisher’s exact test was used to examine the asso-
ciation between telomerase activity and bcl2 expression. We report preliminary results showing the functional existence of
such receptors in the human breast carcinoma cell line MCF-7. Using spectrofluorometry to measure the intracellular calcium con-
centration, evidence has been collected that the addition of E to
these cells causes a rapid rise in the intracellular calcium concen-
tration. This mechanism may prove to be an important initial signal-
ing pathway, leading to the activation of specific protein kinases
and subsequent proliferation. Results: The median percentage of strongly stained tumour cells
was 50% for telomerase-positive tumours (range 0–100%) and
45% for telomerase-negative tumours (range 0–100%). Twelve
(70%) out of 17 telomerase-positive tumours expressed strong or
moderate bcl2 staining in more than 50% of tumour cells, compared
with six (75%) out of eight telomerase-negative tumours (P=1.0). Conclusion: Telomerase reactivation appears to be independent A22 Local recurrence is often associated with subsequent occurrence
of distant metastases. Prophylactic (‘adjuvant’) systemic treatment
is theoretically justified, but its impact on prognosis is unclear. Hor-
monal treatment is recommended if the recurrence is positive for
ER/PR receptors. The use of chemotherapy is currently being
investigated in several multicentre studies. Available online http://breast-cancer-research.com/content/3/S1 Available online http://breast-cancer-research.com/content/3/S1 Available online http://breast-cancer-research.com/content/3/S1 Available online http://breast-cancer-research.com/content/3/S1 esterified by phenylacetic acid (NaPaC). In vitro, NaPaC can inhibit
threefold to fourfold more MCF-7ras proliferation than NaPa alone. Furthermore, we showed that the antiproliferative activity of NaPaC
was dependent on phenylacetate substitution. In vivo studies
showed that a very low dose of NaPaC (15mg/kg) inhibited the
MCF-7ras tumor growth of 60% without animal toxicity. The inhibi-
tion of tumor growth was concomitant with a reduction in angiogene-
sis and an increase in necrosis. Moreover, we demonstrated that
NaPaC inhibited the paracrine mitogenic effect of MCF-7ras condi-
tioned medium (CM) on fibroblasts and endothelial cells proliferation. esterified by phenylacetic acid (NaPaC). In vitro, NaPaC can inhibit
threefold to fourfold more MCF-7ras proliferation than NaPa alone. Furthermore, we showed that the antiproliferative activity of NaPaC
was dependent on phenylacetate substitution. In vivo studies
showed that a very low dose of NaPaC (15mg/kg) inhibited the
MCF-7ras tumor growth of 60% without animal toxicity. The inhibi-
tion of tumor growth was concomitant with a reduction in angiogene-
sis and an increase in necrosis. Moreover, we demonstrated that
NaPaC inhibited the paracrine mitogenic effect of MCF-7ras condi-
tioned medium (CM) on fibroblasts and endothelial cells proliferation. not definitively clear; the better survival after IBTR might be due to
a selection bias for breast preservation. A major goal of treatment for locoregionally recurrent breast cancer
is to achieve local control at the recurrent site. This includes
surgery and/or radiotherapy. For local control, the patterns of local
spread (scar or outside scar, multifocality, size, site) are important. In irresectable lesions, the addition of hyperthermia to radiotherapy
yields improved local control. Encouraging local control rates have
also been reported from some phase II studies with concurrent
radiochemotherapy. In general, patients with local control at the
recurrent site have a significantly better long-term prognosis as
compared with patients with re-recurrence. Telomerase activity and bcl2 expression in human
breast cancer
AE Elkak, K Kirkpatrick, L Mears, C Wells, M Ghilchick,
K Mokbel
The Breast Cancer Centre, St George’s Hospital, London, UK Research thus far has concentrated mainly on the classical steroid
hormone (SH) receptors and on the underlying mechanisms of
antihormone interactions with these receptors. This is mainly
because, in the conventional view, estrogen (E) and progesterone
produce
most
of
their
effects
through
interaction
with
cellular/nuclear receptors with subsequent alteration of the gene-
regulating machinery. However, emerging data suggest that these
lipophilic hormones are also able to produce rapid effects within
several seconds, which cannot be adequately explained through
the classical mechanism. Further investigation has recently led to
the discovery of membrane-bound forms of E-receptors, which are
coupled to cytosolic signal transduction proteins. These rapid
responses have been observed in several tissues such as myome-
trial cells, neurons, endothelium, osteoblasts, granulosa cells and
some breast cancer cell lines. The binding of E to these cell-
surface forms of E-receptors is thought to activate several second
messenger systems via the activation of G-proteins, resulting in the
activation of different protein kinases. One such kinase is the
mitogen-activated protein (MAP) kinase, which may serve as a
stimulus for cell proliferation. Protection against growth of MUC1/sec transfected
mammary tumor cells is mediated by an effector
cell with perforin-dependent cytotoxicity
T Fu, L Herbert, I Keydar*, DM Lopez
Department of Microbiology and Immunology, University of Miami School
of Medicine, Miami, Florida, USA; *Tel-Aviv University, Tel-Aviv, Israel Martin-Luther-University, Department of Radiotherapy, Halle, Germany Martin-Luther-University, Department of Radiotherapy, Halle, Germany Even in case of adequate curative treatment of the primary tumor,
approximately 10–20% of all patients will develop a locoregional
recurrence in the course of disease. At the time of diagnosis of the
recurrence, one-third of patients already has distant metastases. The 5-year survival for patients without metastases is approximately
40%. Prognostic factors are the initial lymph-node status and the
disease-free interval. Whether in-breast recurrences (IBTR) carry a
better prognosis than chest-wall recurrences after mastectomy is A23 Conclusion: Telomerase reactivation appears to be independent
of bcl2 protein expression in human breast cancer. A novel sodium phenylacetate-dextran derivative
ester inhibits the growth and angiogenesis of
MCF-7ras breast cancer xenografts
M Di Benedetto, D Briane, O Oudar, O Sainte Catherine,
J Jozefonvicz*, M Kraemer, M Crépin
UPRES 2360, Equipe d’Oncologie cellulaire et moléculaire des
tumeurs SMBH, Université Paris, Bobigny, France; *Laboratoire de
Recherches sur les Macromolécules (LRM), Institut Galilée,
Université Paris, Villetaneuse, France A novel sodium phenylacetate-dextran derivative
ester inhibits the growth and angiogenesis of
MCF-7ras breast cancer xenografts
M Di Benedetto, D Briane, O Oudar, O Sainte Catherine,
J Jozefonvicz*, M Kraemer, M Crépin
UPRES 2360, Equipe d’Oncologie cellulaire et moléculaire des
tumeurs SMBH, Université Paris, Bobigny, France; *Laboratoire de
Recherches sur les Macromolécules (LRM), Institut Galilée,
Université Paris, Villetaneuse, France The aim of our study was to improve and to optimize the reliability
of the high-speed breast core biopsy, using three-dimensional
ultrasound guidance. From September 2000 to March 2001, we performed 57 high-
speed breast core biopsies (Bard Instrument) under 3D US guid-
ance (three dimensional representation of the needle in the lesion):
13 lesions had a diameter >2cm and 44 had a diameter ≤2cm; in
13 of the latter the diameter was ≤1cm. From each tumor we
obtained only 2 to 3 bioptic cores (in at least one core we demon-
strated the presence of the needle central or marginally in the lesion). All of the patients underwent breast operation after the bioptic his-
tologic diagnosis (at biopsy approximately 90% had breast cancer, We previously showed that sodium phenylacetate (NaPa) and car-
boxymethyl benzylamide dextran (CMDB) are both able to block the
tumor growth of the breast cancer cell line MCF-7ras in athymic
mice. In this study, we studied the effect of a new molecule: a CMDB Regulation of episialin/MUC1 expression in
breast carcinomas: a complex interplay between
stimulatory and inhibitory factors
I Gaemers, H Volders, J Hilkens Since the discovery of the mouse mammary tumor virus (MMTV),
which was shown to be involved in mouse mammary carcinoma,
there has been an attempt to discover similar viruses associated
with human breast cancer. We have already shown that the human
mammary carcinoma cell line T47D releases retrovirus-like parti-
cles in response to steroid treatment. Division of Tumor Biology, Netherlands Cancer Institute, Amsterdam,
The Netherlands Division of Tumor Biology, Netherlands Cancer Institute, Amsterdam,
The Netherlands Episialin/MUC-1 is an epithelial mucin-like transmembrane glyco-
protein, which is highly overexpressed in a majority of human carci-
nomas, in particular breast and ovarian carcinomas. We and others
have shown that this overexpression results in reduced adhesion
and a higher metastatic potential of the tumor cells [1]. The overex-
pression of episialin originates mainly at the transcriptional level
(10-fold or more increased levels of episialin mRNA are found in
breast tumor specimens and breast carcinoma cell lines [2,3]). Consequently, information on the regulation of the episialin pro-
moter may provide a clue to the regulation of metastasis. Examina-
tion of the episialin promoter revealed several putative regulatory
elements. p
An RT transcript from T47D cells was isolated, using RT-PCR with
primers based on the published T47D endogenous retroviral pol
sequences. The PCR product encodes a 372-amino-acid long
protein. The new T47D RT is almost identical to both previously
described RTs from T47D cells, as well as to the enzymatically
active RT from human bone marrow cells (95 and 97% identity,
respectively). The DNA product was cloned into a bacterial expres-
sion system. A 42-kDa RT-related fragment was expressed, puri-
fied, and used to immunize rabbits. The antibodies recognize a
60–70kDa hormonally induced protein specifically in T47D cells. Moreover, steroid hormones induce the apperance of RT protein
foci in the cell cytoplasm, as demonstrated by confocal laser
microscopy. A parallel hormonal induction of the RT activity in cell
supernatants was observed. Expression of the RT-related protein
was also detected in tumor cells of breast cancer biopsies sec-
tions. These results support the idea that retroviruses may be asso-
ciated with human breast carcinoma. (1) We have shown that the episialin promoter is positively regulated
by STATs (signal transducers and activators of transcription) in
T47D breast and other carcinoma cell lines, using several estab-
lished inducers of episialin expression (eg IFN-γ and IL-6) as STAT-
activating ligands [3]. T Fu, L Herbert, I Keydar*, DM Lopez T Fu, L Herbert, I Keydar*, DM Lopez Department of Microbiology and Immunology, University of Miami School
of Medicine, Miami, Florida, USA; *Tel-Aviv University, Tel-Aviv, Israel Department of Microbiology and Immunology, University of Miami School
of Medicine, Miami, Florida, USA; *Tel-Aviv University, Tel-Aviv, Israel We have previously found that DA-3 mammary tumor cells trans-
fected with the secreted form of the MUC1-gene (DA-3/sec)
resulted in no tumor growth, whereas transfection with the reast Cancer Research Vol 3 Suppl 1
23rd Congress of the International Association for Breast Cancer Researc We conclude that the expression of episialin/MUC1 is determined
by a balance between positive and negative regulators, which is
distorted in tumors, leading to a higher episialin expression and
thus a more aggressive tumor type. neomycin vector alone (DA-3/neo) or with the transmembrane form
of MUC-1 (DA-3/TM) did not change the growth characteristics of
the DA-3 cells. Implantation of the DA-3/sec in nude mice resulted
in tumor development, indicating that the immune response is a
major cause of the lack of growth of these cells in immunologically
intact mice. In vitro activated spleen cells from DA-3/sec-injected
mice showed strong cytotoxicity against DA-3/sec, but not to
DA-3, DA-3/neo or DA-3/TM cells. This cytotoxicity was clearly
neutralized by in vivo administration of anti-CD3 monoclonal anti-
body, but not by anti-CD4 or anti-CD8 antibodies. Analysis of the
mechanism of killing of the effector cells revealed that anti-Fas anti-
body did not affect the reaction. Furthermore, FasL-transfected
EL-4 cells were not able to kill the DA-3 cells. In contrast, con-
canomycin A, a perforin-specific inhibitor, greatly reduced the cyto-
toxicity of spleen cells from DA-3/sec-injected mice. These data
suggest that a cell with a phenotype compatible to that of a NK
T cell, may be responsible, at least in part, for the protection
against the growth of DA-3/sec cells in immunocompetent mice. neomycin vector alone (DA-3/neo) or with the transmembrane form
of MUC-1 (DA-3/TM) did not change the growth characteristics of
the DA-3 cells. Implantation of the DA-3/sec in nude mice resulted
in tumor development, indicating that the immune response is a
major cause of the lack of growth of these cells in immunologically
intact mice. In vitro activated spleen cells from DA-3/sec-injected
mice showed strong cytotoxicity against DA-3/sec, but not to
DA-3, DA-3/neo or DA-3/TM cells. A27 Characterization of a hormonally induced reverse
transcriptase (RT) from the human breast cancer
cell line T47D: a possible involvement in human
breast cancer
M Golan, A Hizi*, I Keydar†, I Tsarfaty
Department of Human Microbiology and *Department of Cell Biology
and Histology, Sackler School of Medicine; †Department of Cell
Research and Immunology, George S Weis Faculty of Life Science,
Tel Aviv University, Tel Aviv, Israel Characterization of a hormonally induced reverse
transcriptase (RT) from the human breast cancer
cell line T47D: a possible involvement in human
breast cancer
M Golan, A Hizi*, I Keydar†, I Tsarfaty
Department of Human Microbiology and *Department of Cell Biology
and Histology, Sackler School of Medicine; †Department of Cell
Research and Immunology, George S Weis Faculty of Life Science,
Tel Aviv University, Tel Aviv, Israel Regulation of episialin/MUC1 expression in
breast carcinomas: a complex interplay between
stimulatory and inhibitory factors
I Gaemers, H Volders, J Hilkens IL-6 (an activator of STAT3) can stimulate the
episialin promoter, and binding of STAT3 to the STAT element in the
episialin promoter is observed in bandshift assays [3]. The possible
involvement of STATs in tumor progression (eg via episialin expres-
sion) is also indicated by the increased levels of activated STAT3
that are found in breast carcinoma cell lines with a high episialin
expression [4]. Similar results are found in vivo, where constitutive
activation of STAT1 and/or STAT3 is found in breast tumors [5]. References References
1. Hilkens et al: Cancer Lett, 1995. 2. Ligtenberg et al: J Biol Chem, 1990. 3. Gaemers et al: J Biol Chem, 2001. 4. Gaemers et al: (in preparation). 5. Watson, Miller: Br J Cancer, 1995. T Fu, L Herbert, I Keydar*, DM Lopez This cytotoxicity was clearly
neutralized by in vivo administration of anti-CD3 monoclonal anti-
body, but not by anti-CD4 or anti-CD8 antibodies. Analysis of the
mechanism of killing of the effector cells revealed that anti-Fas anti-
body did not affect the reaction. Furthermore, FasL-transfected
EL-4 cells were not able to kill the DA-3 cells. In contrast, con-
canomycin A, a perforin-specific inhibitor, greatly reduced the cyto-
toxicity of spleen cells from DA-3/sec-injected mice. These data
suggest that a cell with a phenotype compatible to that of a NK
T cell, may be responsible, at least in part, for the protection
against the growth of DA-3/sec cells in immunocompetent mice. Sentinel lymph node biopsies in breast cancer
A Hess, MO Flüß*, C Nestle-Krämling, HG Bender, P Dall
Institut für Frauenheilkunde und Geburtshilfe and *Institut für
Nuklearmedizin, Universität Düsseldorf, Germany The aim of this study was to analyze the transcriptional regulation of
different ERs via alternative cis-elements. For this purpose we per-
formed transient transfections with a luciferase reporter plasmid for
estrogen-responsive elements (ERE) and AP-1 elements, and
expression plasmids for ER-α, ER-β1 and ER-β2. Cells were left
unstimulated or stimulated with E2, tamoxifen or raloxifen. We found
that, in the breast cancer cell line SKBR3, ER-β1 lead to a signifi-
cant inhibition of AP-1 activity by E2, whereas through ER-β2 E2
lead to a stimulation of transcriptional activity. Antiestrogens inhib-
ited transcription through ER-β1 but did not exhibit an effect
through ER-β2. When transfecting ER-β1 in SKBR3 cells the basal
transcriptional activity increased, in contrast to the results obtained
when transfecting the human osteosarcoma cell line U2OS with the
same receptor. E2 in SKBR3 cells leads to a significant transcrip-
tional inhibition, whereas this effect is not seen in U2OS cells. Also
the antiestrogens tamoxifen and raloxifen exhibit in SKBR3 cells via
the same receptor a transcriptional inhibition, but in contrast in
U2OS cells they lead to a stimulation of transcription. In summary,
splice variants of ER-β are able to regulate the actvity of the AP-1
complex differentially, indicating that the relative expression of these
variants in a tumor could modulate its hormonal sensitvity. Sponsored b DFG Ha 2404/2 1 Introduction: Lymph node biopsy is not only important as a prog-
nostic factor, but also influences therapy. However, axillary lym-
phadenectomy is often accompanied by high morbidity. The
sentinel lymph node biopsy (SLN) should reduce the morbidity, but
give the same prognosic value. In 97.5% cases metastasis occurs
in lymph nodes (LN) of level I first, and only less than 3% directly in
LN level II. The first draining LN can be identified either by radioac-
tive material or colouring technique. Method: Patients with primary breast cancer (41 cases, age
30–80 years), 1 day before surgery, received peritumoral 1–3ml
nanocolloid containing 99m-technetium, with scintigraphy per-
formed 30–120 min later. Alternatively 2ml Patentblau (2.5% Byk
Gulden) was applied during the surgery 15 min before the axillary
lymphadenectomy. The marked LN was separated and sent to
pathology together with the other LN. p
gy
g
Results: Seventeen patients received Tc-nanocolloid, 21 Patent-
blue. Three patients were treated with both identification methods. Out of 41 patients, 14 had an axillary metastasis. Sentinel lymph node biopsies in breast cancer
A Hess, MO Flüß*, C Nestle-Krämling, HG Bender, P Dall
Institut für Frauenheilkunde und Geburtshilfe and *Institut für
Nuklearmedizin, Universität Düsseldorf, Germany Comparison of
radioactive labelling showed no false-negative results, but 7.3%
false negativity was obtained with the colour method. Sponsored by DFG Ha 2404/2-1. Antitumor potential of bisphosphonates
JR Green Novartis Pharma AG, Basel, Switzerland Novartis Pharma AG, Basel, Switzerland (2) Glucocorticoids also can stimulate episialin expression in T47D
cells through binding of the glucocorticoid receptor (GR) to GRE
half sites present in the episialin promoter [4]. Bisphosphonates (BPs), especially those with a nitrogen-contain-
ing substituent, are potent inhibitors of osteoclast-mediated bone
resorption. They have found extensive clinical use for the treatment
of both benign and malignant bone disease. BPs bind to hydroxy-
apatite and rapidly accumulate in bone where they inhibit the
mevalonate biosynthetic pathway, thereby preventing the post-
translational prenylation of small GTP-binding proteins and induc-
ing apoptosis in osteoclasts. Recent in vitro studies indicate that
BPs also inhibit proliferation, reduce viability and induce apoptosis
in several human tumor cell lines. In addition, BPs reduce the inva-
sion of tumor cells through extracellular matrix and impair the (3) In addition, we report that glucocorticoids can attenuate the
effect of IL-6/STAT3, using reporter, bandshift and FACS assays
[4]. The effect of glucocorticoids on STAT3-mediated episialin
expression occurs both through direct interactions between
STAT3 and GR, as well as via indirect pathways. Conversely, addi-
tion of IL-6 can augment GR-mediated episialin expression [4]. (4) We have also identified a 200-bp sequence fragment far
upstream in the episialin promoter that may bind a negative regula-
tor of episialin expression. The identity and exact binding sequence
of this putative inhibitor has not yet been determined. (3) In addition, we report that glucocorticoids can attenuate the
effect of IL-6/STAT3, using reporter, bandshift and FACS assays
[4]. The effect of glucocorticoids on STAT3-mediated episialin
expression occurs both through direct interactions between
STAT3 and GR, as well as via indirect pathways. Conversely, addi-
tion of IL-6 can augment GR-mediated episialin expression [4]. (4) We have also identified a 200-bp sequence fragment far
upstream in the episialin promoter that may bind a negative regula-
tor of episialin expression. The identity and exact binding sequence
of this putative inhibitor has not yet been determined. Available online http://breast-cancer-research.com/content/3/S1 Available online http://breast-cancer-research.com/content/3/S1 binding of tumor cells to bone in vitro. This growing body of evi-
dence suggests that BPs may have the potential to exert direct
antitumor effects in vivo, particularly in bone metastases where the
local BP concentration is elevated by the enhanced osteoclastic
resorption of BP-loaded bone. A30 Late radiation sequelae in women after breast-
conserving cancer therapy: effects of hyperbaric
oxygen therapy
KA Hartmann, JJ Feldmeier*, G Schmitt, UM Carl
Department of Radiation Oncology, University Düsseldorf, Düsseldorf,
Germany; *Department of Radiation Oncology, Medical College of
Ohio, Toledo, Ohio, USA Antitumor potential of bisphosphonates
JR Green Several experiments with zoledronic
acid (a highly potent BP with an imidazole substituent) adminis-
tered to mice injected with mammary, prostate or myeloma cancer
cells indicate not only inhibition of the tumor-induced osteolysis,
but also a reduction in the growth of bone metastases, accompa-
nied by the induction of tumor cell apoptosis. Moreover, zoledronic
acid has recently been shown to potently inhibit endothelial cell
proliferation in vitro and angiogenesis in mice bearing subcuta-
neous implants loaded with growth factors. Overall, these findings
provide a rationale for testing the antitumor potential of the more
potent nitrogen-containing BPs in pilot clinical trials. binding of tumor cells to bone in vitro. This growing body of evi-
dence suggests that BPs may have the potential to exert direct
antitumor effects in vivo, particularly in bone metastases where the
local BP concentration is elevated by the enhanced osteoclastic
resorption of BP-loaded bone. Several experiments with zoledronic
acid (a highly potent BP with an imidazole substituent) adminis-
tered to mice injected with mammary, prostate or myeloma cancer
cells indicate not only inhibition of the tumor-induced osteolysis,
but also a reduction in the growth of bone metastases, accompa-
nied by the induction of tumor cell apoptosis. Moreover, zoledronic
acid has recently been shown to potently inhibit endothelial cell
proliferation in vitro and angiogenesis in mice bearing subcuta-
neous implants loaded with growth factors. Overall, these findings
provide a rationale for testing the antitumor potential of the more
potent nitrogen-containing BPs in pilot clinical trials. radiation sequelae. The objective of this study was to assess the
efficacy of hyperbaric oxygen therapy in symptomatic patients after
breast cancer treatment. Patients and method: Forty-four patients with persisting symptoma-
tology after breast-conservation therapy were prospectively observed. Thirty-two women received hyperbaric oxygen therapy in a multiplace
chamber for a median of 25 sessions (7–60). One hundred per cent
oxygen was delivered at 240kPa for 90-min sessions, five times per
week. Twelve control patients received no further treatment. Changes
throughout the irradiated breast tissue were scored before and after
hyperbaric oxygen therapy, using modified LENT-SOMA criteria. Results: Hyperbaric oxygen therapy patients showed a significant
reduction in pain, edema and erythema scores as compared with
untreated controls (P<0.001). Fibrosis and teleangiectasia,
however, were not significantly affected by hyperbaric oxygen
therapy. Seven out of 32 women were free of symptoms after
hyperbaric oxygen therapy, whereas all 12 patients in the control
group had persisting complaints. A31 B Hanstein, T Flötotto, D Luke, D Niederacher, HG Bender
Universitäts-Frauenklinik Düsseldorf, Germany Sentinel lymph node biopsies in breast cancer
A Hess, MO Flüß*, C Nestle-Krämling, HG Bender, P Dall
Institut für Frauenheilkunde und Geburtshilfe and *Institut für
Nuklearmedizin, Universität Düsseldorf, Germany A29 Conclusion: Hyperbaric oxygen therapy should be considered as a
treatment option for patients with persisting symptomatology fol-
lowing breast-conserving therapy. Effect of anthracyclin-based neoadjuvant
chemotherapy on disseminated tumor cells in
breast cancer patients: an immunocytologic and
molecular approach
CM Jäger, A Müller, V Heilmann, R Grundmann, R Kreienberg
Universitäts-Frauenklinik, Ulm, Germany Effect of anthracyclin-based neoadjuvant
chemotherapy on disseminated tumor cells in
breast cancer patients: an immunocytologic and
molecular approach
CM Jäger, A Müller, V Heilmann, R Grundmann, R Kreienberg
Universitäts-Frauenklinik, Ulm, Germany The FHIT gene at 3p14.2 encompasses the common fragile site,
FRA3B, and is involved in frequent chromosome rearrangements in
human cancers. Fhit protein expression is reduced or lost in the
majority of esophageal, lung, gastric, cervical, pancreatic, kidney
and bladder cancers, and a large fraction of other cancers. Fhit
expression in sporadic breast cancers has been studied by several
groups, and reported to show alteration in expression of Fhit in
30–50%. Because familial breast cancers were reported to show a
higher frequency of LOH at 3p14.2 than sporadic breast cancers,
we were interested in whether common fragile regions might be
targets for repair by the Brca1 and Brca2 proteins, and might thus
be a downstream target in BRCA1- and BRCA2-induced familial
breast tumors. We studied a panel of Brca2-deficient breast tumors
and showed that only 18% expressed Fhit strongly, compared with
48% of sporadic tumors (P=0.002). Very recently we completed a
similar study of BRCA1 familial tumors, and observed that only 9%
of these tumors showed strong expression versus more than 40%
of sporadic breast tumors (P<0.001, odds ratio 0.09). We con-
clude that loss of BRCA1 and BRCA2 functions affect stability of
the FHIT/FRA3B locus and possibly other fragile loci. Objective: The leading cause of death from epithelial cancer is
metastatic tumor relapse due to early dissemination of tumor cells. Cytokeratins are specific markers of epithelial cancer cells in bone
marrow. As previously shown, these epithelial cells in bone marrow
seem to be resting ‘in dormancy’. This biological behaviour might
be an explanation for the resistance to cytotoxic agents. In the
present study, we evaluated whether primary chemotherapy in
locally advanced, nonmetastatic breast cancer can eliminate cyto-
keratin-positive cells in bone marrow. Furthermore, we investigated
the influence of primary chemotherapy on the tumor-associated
gene expression. Method: Twenty-one breast cancer patients underwent bone
marrow aspiration before and after neodjuvant chemotherapy. For
immunocytologic tumor cell detection we used the monoclonal
antibody 5D3 (Biogenex), which is directed against common
epitope on cytokeratin polypeptides, including cytokeratin 8/18/19. Organochlorines and breast cancer: effect of
exposure to dieldrin on risk and survival
AP Høyer, T Jørgensen*, AP Grandjean† KA Hartmann, JJ Feldmeier*, G Schmitt, UM Carl
Department of Radiation Oncology, University Düsseldorf, Düsseldorf,
Germany; *Department of Radiation Oncology, Medical College of
Ohio, Toledo, Ohio, USA Copenhagen Center for Prospective Population Studies, Denmark;
*Center of Preventive Medicine, KAS Glostrup, Denmark; †Institute of
Community Health, Odense, Denmark Copenhagen Center for Prospective Population Studies, Denmark;
*Center of Preventive Medicine, KAS Glostrup, Denmark; †Institute of
Community Health, Odense, Denmark Background: Persisting symptomatology after breast-conserving
surgery and radiation is frequently reported. In most cases symp-
toms in the breast resolve without further treatment. In some
instances, however, pain, erythema and edema can persist for
years and can impact on the patient’s quality of life. Hyperbaric
oxygen therapy was shown to be effective as treatment for late Some organochlorines have weak estrogenic activity, and may there-
fore interfere with breast cancer risk and survival. We assessed
prospectively risk and prognosis of breast cancer in relation to serum
concentrations of several compounds, which have shown to be
estrogenic in vitro and in vivo. Study participants (7712 women) Breast Cancer Research Vol 3 Suppl 1
23rd Congress of the International Association for Breast Cancer Research reast Cancer Research Vol 3 Suppl 1
23rd Congress of the International Association for Breast Cancer Researc ErbB2 is considered, therefore, as a target for cancer therapy. In
this respect, a growth inhibitory antibody (4D5) directed against
the extracellular domain of ErbB2 has been raised. Furthermore,
the humanized version (HerceptonTM) is now being used in the
clinic to treat metastatic breast cancer patients whose tumors
overxpress ErbB2. In our work, 4D5 has been applied as a selec-
tive inhibitor of ErbB2 function. Treatment with 4D5 blocks G1/S
phase progression in breast carcinoma cells that overexpress
ErbB2. This block correlates with a rapid reduction in ErbB2 phos-
photyrosine content, downregulation of signal transduction path-
ways, a reduction in the expression of proteins involved in the
sequestration of the cyclin-dependent kinase inhibitor p27, and
relocalization of p27 onto Cdk2 complexes. Strikingly, the 4D5-
induced G1 block can be rescued by treatment with various ErbB
ligands. The degree of rescue is ligand-related and is associated
with activation of specific signaling pathways. Additionally, through
comparison with an ErbB2-overexpressing gastric carcinoma cell
line (MKN7) that proliferates normally in the presence of 4D5, we
have demonstrated that decreased ErbB2 phosphotyrosine levels
do not necessarily lead to growth inhibition in response to 4D5. Effect of anthracyclin-based neoadjuvant
chemotherapy on disseminated tumor cells in
breast cancer patients: an immunocytologic and
molecular approach
CM Jäger, A Müller, V Heilmann, R Grundmann, R Kreienberg
Universitäts-Frauenklinik, Ulm, Germany For the molecular approach, after isolation of RNA, the reverse
transcription
with
Superscript
II
Reverse
Transkriptase
(Gibco/BRL) and Oligo (dT)12–18 Primers (Gibco/BRL) was per-
formed, followed by the amplification procedure with nested-RT-
PCR for β2-microglobulin, muc-1, CK-20 and carcinoembryogenic
antigen (CEA). ErbB family of receptors in breast cancer
NE Hynes, A Motoyama, HA Lane
Friedrich Miescher Institute, Basel, Switzerland Results:
Fifteen patients met the inclusion criteria. Before
chemotherapy, five out of 15 (30%) had cytokeratin-positive cells in
bone marrow. Four out of five were still tested positive after finishing
chemotherapy. With the RT-PCR procedure, all bone marrow aspi-
rates showed a signal for β2-microglobulin (positive control). Expression of cytokeratin-20 was absent, whereas muc-1 was
expressed in all aspirates. Two out of five showed expression of the
CEA before chemotherapy, which was absent in one and only slight Organochlorines and breast cancer: effect of
exposure to dieldrin on risk and survival
AP Høyer, T Jørgensen*, AP Grandjean† These data imply that ErbB2 overexpression alone is insufficient to
predict cellular response to ErbB2-directed therapies. The possi-
ble contribution of other ErbB receptors to the process of malig-
nant transformation will be discussed in relation to the evolution of
ErbB-directed treatment strategies. donated blood twice (1976–1978 and 1981–1983) and were fol-
lowed for 17 years with regard to development of breast cancer. Information on potential breast cancer risk factors and prognostics
were obtained through standardized questionnaires and by linkage
to the Danish Breast Cancer Cooperative Group. Breast cancer risk associated with baseline exposure in
1976–1978, and repeated measurements of organochlorines
(average concentration of the two measurements) was examined in
two cohort-nested case–control studies, including 240 cases and
477 controls, and 155 cases and 274 controls, respectively. The
cases served as a cohort in the survival analysis, in which the
average duration of follow up to death was 86 and 79 months after
the first and second sampling. The most consistent finding observed was dieldrin’s adverse effect
on breast cancer risk as well as prognosis. More than a twofold
increased risk was found among women with the highest baseline
concentration compared with those with the lowest, and a signifi-
cant trend was apparent. A high serum dieldrin concentration was
also significantly associated with an increased overall mortality,
being threefold for baseline measurements and almost sixfold
when repeated measurements was assessed. Similar results were
obtained when using breast cancer recurrence and/or death
caused by breast cancer as end-point. A33 A33
Fhit loss in familial breast cancer: is loss of DNA
repair function linked to alterations at
chromosome fragile sites? K Huebner, B Turner, WW Hauck*, N Popescu
Kimmel Cancer Institute, Jefferson Medical College, Philadelphia,
USA; *Laboratory of Experimental Carcinogenesis, National Cancer
Institute, Bethesda, Maryland, USA M Kimm, M Boer, I Gaemers, J Hilkens Crucial to the prognosis of cancer patients is not growth of the
primary tumor, but rather dissemination of neoplastic cells to other
organs As the process of the activation and inactivation of genes
that are involved in tumorigenesis and metastasis is still poorly
understood, the aim of this study is to identify novel mammary
cancer progression and metastasis genes in vivo. Proviral insertions
of mouse mammary tumor virus (MMTV) in mammary epithelial cells
are able to activate flanking oncogenes, leading to mammary tumor
induction. Classical examples of MMTV-induced oncogenes are
Wnt1 and Fgf3. Full neoplastic transformation to an invasive and
metastasizing
tumor
requires
activation
of
collaborating
onco/metastasis genes. Thus, additional proviral insertions may lead
to metastasis-inducing genes. In this study we used a BALB/c+
mouse strain (ie a BALB/c substrain that aquired C3H-MMTV by
forster-nursing), and compared extra proviral integrations in a series
of sets of independent primary tumors and metastases. As a first
step, the isolated tumor sets (primary tumor and metastases) were
analyzed by Southern blotting using a MMTV-LTR specific probe. A
number of the lung metastases indeed carried additional MMTV
integrations, which were not found in the primary tumor. These addi-
tional integrations might activate genes being responsible for the
lung metastases. To analyze the flanking sequences more efficiently,
an adaptor ligation-mediated PCR (Splinkerette-PCR) was modified
for the metastasis-related proviral MMTV integrations. To this end,
genomic DNA was digested and ligated to a suitable splinkerette
linker. The subsequent PCR gets its specificity by using a unique
MMTV-LTR-related oligonucleotide and a splinkerette-specific
oligonucleotide, which is only able to bind to the DNA if extension of
the MMTV oligonucleotide occurs. BLAST/NIX (DNA analysis soft-
ware) analysis of the derived additional sequences from 23 tumor
sets resulted in the discovery of a novel common integration site. The effect of this putative metastasis gene on the metastic potential
of mammary tumor cells is presently being investigated. Conclusion: COX-2 expression is significantly associated with
increased cellular proliferation and angiogenesis in invasive breast
cancer. The upregulation of COX-2 in ANCT suggests that COX-2
in the host is relevant to mammary carcinogenesis. ErbB family of receptors in breast cancer
NE Hynes, A Motoyama, HA Lane Friedrich Miescher Institute, Basel, Switzerland Friedrich Miescher Institute, Basel, Switzerland The ErbB2 receptor tyrosine kinase is overexpressed in many
human breast tumors, a phenomenon correlating with more
aggressive tumor characterisitcs and a worse patient prognosis. Available online http://breast-cancer-research.com/content/3/S1 Available online http://breast-cancer-research.com/content/3/S1 in the other case after chemotherapy. Before chemotherapy
immunocytologic tumor cell detection and RT-PCR procedure for
CEA was concurrently negative in 10 out of 15 patients. Conclusion: (1) A negative result in immunocytochemistry of bone
marrow of breast cancer patients seems to agree on the RT-PCR
for CEA. (2) Tumor cells could develop a different pattern of gene
expression under primary chemotherapy. (3) RT-PCR procedure
for muc-1 and CK-20 seems not to be useful for investigating dis-
seminated tumor cells in bone marrow. in the other case after chemotherapy. Before chemotherapy
immunocytologic tumor cell detection and RT-PCR procedure for
CEA was concurrently negative in 10 out of 15 patients. forms of COX: COX-1, which is expressed in many tissues; and
COX-2, which is the inducible form. COX-2 has been reported to
be involved in carcinogenesis and tumour angiogenesis. Therefore,
we hypothesized that COX-2 expression was associated with that
of vascular endothelial growth factor (VEGF) and proliferating cell
nuclear antigen (PCNA) in human breast cancer. Conclusion: (1) A negative result in immunocytochemistry of bone
marrow of breast cancer patients seems to agree on the RT-PCR
for CEA. (2) Tumor cells could develop a different pattern of gene
expression under primary chemotherapy. (3) RT-PCR procedure
for muc-1 and CK-20 seems not to be useful for investigating dis-
seminated tumor cells in bone marrow. Materials and method: RNA was extracted from 15 human breast
carcinomas and adjacent noncancerous tissue (ANCT). COX-2,
VEGF 189 and PCNA expressions were estimated by reverse tran-
scriptase-PCR (RT-PCR) and Taqman methodology in the RNA
samples. The results were analyzed using Spearman’s correlation
with Student’s t-test. Acidification-induced sensitization to
thermoradiotherapy in breast cancer
DB Leeper, LT Komarnicky Department of Radiation Oncology, Thomas Jefferson University,
Philadelphia, USA Department of Radiation Oncology, Thomas Jefferson University,
Philadelphia, USA Hyperthermia is an extensively studied cytotoxic agent, with strong
radio- and chemosensitizing potential. Recent positive clinical trials
combining superficial or deep heating techniques with radiation
therapy strongly support a role for hyperthermia as an adjuvant to
radiation. Many in vitro and in vivo studies have shown that acute
extracellular acidification will compromise fundamental protective
cellular responses and enhance tumor response to hyperthermia
and chemotherapy. Breast cancers, like most other tumors, exhibit elevated levels of
lactate production that provides a basis for selective acidification. A phase I/II clinical trial is underway to test the hypothesis that
hyperglycemia-induced acute acidification will sensitize carcinoma
of the breast to thermoradiotherapy. Six patients consented to fast
for at least 4h and ingest oral glucose (2g/kg, 0.44g/ml) 1.5h
before each hyperthermia treatment (HT) during a course of ther-
moradiotherapy. Hyperglycemia reduced tumor pHe before the first
hyperthermia session by 0.10±0.04pH unit (–0.29 to +0.08) from
7.12±0.11 (6.65–7.52); and during the third week of treatment
hyperglycemia
reduced
tumor
pHe
in
five
patients
by
0.01±0.04pH unit (–0.06 to +0.1). The three patients with a CR
(60%) exhibited tumor acidification during both sessions, in con-
trast to the two patients with a PR (40%) who exhibited tumor
acidification only during one session. Tumor acidification may indi-
cate tumor response. Cloning of novel mammary tumor progression and
metastasis genes Results: Median mRNA copy number for PCNA mRNA in
tumours was 1.65 × 106 (range 3.79 × 105–1.46 × 107). For
COX-2, the median mRNA copy number was 4.56 × 105 (range
2.48 × 103–5.10 × 106) in tumours and 2.26 × 106
(range
1.37E+05–4.79E+07) in ANCT. Copy numbers of VEGF mRNA
in tumours had a median value of 2.13 × 106
(range
3.42 × 101–3.37 × 107). There was a highly significant correlation
between COX-2 and PCNA levels in tumours (rs = 0.7896;
P = 0.000001) and VEGF in tumour samples (rs = 0.4610;
2P = 0.0320). K Kirkpatrick, W Ogunkolade, AE Elkak, S Bustin, P Jenkins,
M Ghilchick, K Mokbel K Kirkpatrick, W Ogunkolade, AE Elkak, S Bustin, P Jenkins,
M Ghilchick, K Mokbel Human tumor cells adapted to growth at pHe 6.7 do not show
thermosensitization until pHe is below 6.3 (pHi <6.45). Combin-
ing an inhibitor of respiration such as MIBG with hyperglycemia
blocks mitochondrial respiration and increases lactate produc-
tion. Thus, tumor oxygenation occurs coincidentally with acute
acidification. The Breast Cancer Centre, St George’s Hospital, Blackshaw Road,
London, UK The Breast Cancer Centre, St George’s Hospital, Blackshaw Road,
London, UK Background: Cyclo-oxygenase (COX) is the rate-limiting enzyme
in converting arachidonic acid to prostaglandins. There are two iso- reast Cancer Research Vol 3 Suppl 1
23rd Congress of the International Association for Breast Cancer Researc of a particular allele of class I molecules; for example, for H-2Kb,
the high-affinity binding of ovalbumin peptide SIINFEKL requires
P5 to be occupied F/Y, and P8 to have L/I, and other peptides
that lack these anchoring amino acids would bind with such a
low affinity that would be unable to induce, or be effective targets
for CTLs. Using MUC1 8 or 9mers as model peptides, we were
able to demonstrate the following: (1) these bind with low affinity;
(2) the binding is unusual and the peptides loop out of the
groove – indeed, the 9mer loops out so much it can be detected
by monoclonal anti-MUC1 peptide antibody; and (3) crystalliza-
tion structures of Kb with the 8mer (SAPDTRPA) demonstrates
that MUC1 and SIINFEKL have an identical shape within the
groove, the only difference being imposed by the side chains,
which are selectively recognized by T-cell receptor. The amino
acids occupied in the specific anchoring pockets are small
hydrophobic, rather than long/large hydrophobic F/Y or L/I
residues, thus the low affinity of the MUC1 peptide. The implica-
tion of the studies is that the rules for high-affinity binding in gen-
eration of CTL hold, but that these are not fixed rules: low-affinity
peptides bind, and indeed with the same general conformation
and shape as high-affinity peptides in one dimension; in another
they are clearly more ‘flexible’. Rats bearing the R3230 Ac rat mammary adenocarcinoma were
administered 1g/kg glucose ip, and/or 20mg/kg MIBG ip. The
median pO2 for glucose plus MIBG was increased from 5.3 to
13.8mmHg. A novel phenylacetate-dextran derivative (NaPaC)
inhibits breast cancer cell proliferation and
modifies their interactions with endothelial cells
S Malherbe, M Bérard, M Di Benedetto, N Peyri, M Crépin,
C Legrand, MX Wei
Inserm U553 Hôpital Saint-Louis, Paris, France Inserm U553 Hôpital Saint-Louis, Paris, France Breast cancer treatments are limited by secondary effects and
chemoresistances or hormone resistances. Sodium phenylacetate
(NaPa), a nontoxic metabolite, has been shown to induce in vivo and
in vitro antiproliferative effects on various cell types in our laboratory. We have previously shown that NaPa treatment induced breast
tumor cell apoptosis without acquired drug resistance. On the other
hand, we have demonstrated that a dextran derivative (CMDB) was
not only antitumoral but also antiangiogenic in a MCF-7ras tumor
model in animals. Recently, we have synthesized a novel hybrid
molecule, CMDB-NaPa ester, called NaPaC, and tested its effect
on the proliferation of MDA-MB-231 and MCF-7 breast tumor cells. NaPaC inhibits, dose dependently, the proliferation of MDA-MB-231
cell (IC50 0.5mmol/l) and of MCF-7 cell (IC50 1.5mmol/l). Primary
cultured endothelial cells (HUVEC) are weakly affected by NaPaC
treatment. Cytostatic effect of NaPaC was evidenced from the accu-
mulation of tumor cells in G0/G1 phase after 96h of treatment. The standardized mistletoe preparation Lektinol
has antitumoral potencies
U Mengs, A Burger*, D Wetzel, K Weber†, HH Fiebig*
Research and Development, Madaus AG, Cologne, Germany;
*University of Freiburg, Freiburg, Germany; †RCC, Itingen,
Switzerland Extracts of Viscum album L have been used for decades for non-
specific stimulation of the immune system in cancer therapy. Mistletoe lectins have been identified as the active components,
with cytotoxic and immunomodulatory activities. New experimental
data demonstrate that the special extract preparation Lektinol®
(Madaus AG, Cologne, Germany), standardized for bioactive
mistletoe lectin (ML), has antitumoral potencies in vitro and in
animal tumor models. As compared with the NaPa parent molecule, this new molecule
was 10-fold more efficient on these two tumor cell lines. Moreover,
NaPaC induces a strong apoptotic effect, as measured with
Annexin V-positive cells, on these tumor cells. In order to under-
stand the interactions between tumor cells and endothelial cells,
the conditioned media were prepared and added to HUVEC cells. Our results showed a clear killing effect on HUVEC cells. However,
this killing effect can be rescued by adding NaPaC. In vitro studies on human tumor cell lines and xenografts showed
Lektinol to be highly cytotoxic (ie toward breast, lung, prostate and
renal cell cancers). Taken together, our results showed that NaPaC is a powerful anti-
tumoral molecule, with cytostatic and proapoptotic effects on
MDA-MB-231 and MCF-7 tumor cells. Further studies should be
conducted to better understand the mechanism of these mutual
interactions between tumor cells and endothelial cells, especially
the killing effect on HUVEC cells. The in vivo antitumoral effects of Lektinol were examined in differ-
ent subcutaneously growing murine neoplasms following
repeated intraperitoneal treatment of 0.3–3–30–300 ng ML/kg. Marked tumor growth inhibition was observed with Renca renal
carcinoma, C8 colon 38, and F9 testicular teratoma. The
antimetastatic effects of Lektinol were investigated in the B16
melanoma model in mice. Following a single intravenous injection
of the melanoma cells, the daily treatment with 3–30–150 ng
ML/kg significantly reduced the formation of lung metastases. In
parallel, Lektinol enhanced several immune parameters (ie the
number of MAC-1+ mononuclear cells and CD4+8+ thymocytes
in the tumor-bearing animals). In a further study, the effects of
locally administered Lektinol were evaluated in the MB49 urinary
bladder carcinoma model in mice. After a single instillation of the
tumor cells, Lektinol was given repeatedly by intravesical adminis-
tration of 3–30 ng ML/0.1 ml/animal. Lektinol showed a distinct
effect on survival ratio, growth of primary bladder tumors and the
formation of multiple metastases. K Kirkpatrick, W Ogunkolade, AE Elkak, S Bustin, P Jenkins,
M Ghilchick, K Mokbel A single ip injection of glucose or MIBG in rats fasted
for 24h before irradiation did not show an increase in tumor
growth delay compared with 5Gy radiation alone. However, com-
bined treatment with glucose plus MIBG significantly inhibited
tumor growth delay. Radiation therapy and glucose plus MIBG was
more than additive. These results support our hypothesis that
hyperglycemia plus an inhibitor of respiration will sensitize tumors
to radiation by oxygenation, in addition to enhanced hyperthermia
sensitization by acute acidification. S
d i
b USPHS
CA59960 A41 The standardized mistletoe preparation Lektinol
has antitumoral potencies
U Mengs, A Burger*, D Wetzel, K Weber†, HH Fiebig*
Research and Development, Madaus AG, Cologne, Germany;
*University of Freiburg, Freiburg, Germany; †RCC, Itingen,
Switzerland Table 1 Table 1
AM 01
MM01
Patients
Adjuvant N>9
M1, PR or CR after conventional
induction, ER
Arm A
Tandem-E,C,TT
STAMP V
Arm B
EC ×4 →CMF ×3
Tandem STAMP V
q2w + G-CSF
Actually randomized
368
180/480
Status
Open
Open
Treatment related
0%
1.5%
mortality (%) The role of subareolar methylene blue in
identifying the sentinel node in patients with
invasive breast cancer The first trial tests high-dose Tandem E90C3000T400 + PBPC
versus dose-dense conventional chemotherapy with G-CSF
support. All patients received irradiation of the chest wall and the
supraclavicular lymph nodes, and tamoxifen in case of ER+ tumors. The trial will probably be closed by the end of the year. The first
interim analysis was done in 1/99, and the second one is planned in
1/02. Interim data will be presented. The trial in MBC randomizes
chemosensitive, ER-negative patients to 1× versus 2× STAMPV. The first interim analysis was done in 12/00. Data will be presented. Background: Recent studies have demonstrated that the sentinel
node biopsy (SNB) is a reliable and minimally invasive method for
determining the axillary node status in patients with breast cancer. However, the methods used for identifying the sentinel node (SN)
are heterogenous with variable success rates. Some studies have
reported low success rates with methylene blue (MB) dye for the
identification of the SN. The present study aims to examine the
accuracy of a simple method using subdermal injection of MB in
the subareolar region. High-risk breast cancer patients: comparison of
lymphocyte phenotypes and function in vitro Department of Gynecology and Obstetrics, HH-University Düsseldorf,
Germany Conclusion: Subareolar MB for identifying the SN in patients with
operable invasive breast cancer provides a simple and reliable
technique that can be used widely. The design of a phase III trial in high-risk breast cancer patients is
characterized by a dose-dense sequential control (arm B), and a
high-dose chemotherapy (HDC) arm with short induction phase and
tandem HDC (arm A). It is known that following such a treatment
there are profound changes in lymphocyte phenotypes and lympho-
cyte function. Here we show data on the changes in vitro during the
different treatment cycles, beginning with the tests 1995. High-dose chemotherapy with peripheral blood
progenitor cell support in breast cancer:
WSG AM01 and MM01 U Nitz, S Mohrmann, G Schütt, A Zander, N Kröger, M Frick,
HG Bender, on behalf of the West German Study Group (WSG),
Düsseldorf, Germany y
g
g
The lymphocyte membranes were tested using commercially avail-
able antibodies (Ortho) and the CytoronAbsolute cytofluorograf. The
lymphocyte functions were investigated in a H-3-thymidin incorpora-
tion test after stimulation with various stimuli (IL-2, IFN-γ, CD3, ConA,
Pokeweed, PHA and Candida antigen). In some of the patients, in
the supernatant of the lymphocyte proliferation test several cytokines
were determined (IL-2, IL-5, IL-10, IL-12, IL-13, IL-16, IFN-γ,
GM-CSF and TNF-α). From 80 examined patients, 16 died and eight
recurred. In arm A there were 57 patients (11 died and six got a
recurrence). Arm B contained 23 patients (five died and two
recurred). CD3+ cells were relatively equal in arm A and B patients,
whereas there was an enormous difference in the CD4+ and CD8+
cells after end of therapy. In the HDC patients CD4+ cells declined
during the follow up and CD8+ cells increased. In arm B there was a
normal decline during the therapy and a recovery after 24 months. In
a few patients of arm A we observed an elevation in B-cells (CD19),
shortly after the end of therapy. The functions of the cells will be pre-
sented in tables. All patients had sustained changes of their lympho-
cyte situation at least for 24 months. The WSG as a German interdisciplinary group initiated 05/94 a
large multicentre phase III trial to evaluate adjuvant high-dose
chemotherapy in high-risk breast cancer. About 100 centres all
over Germany participated. The second trial in metastatic breast
cancer (MBC) was initiated in 4/97 in co-operation with the
German Intergroup. The main characteristics of the two trials are
listed in Table 1. Structural features for peptides binding the class I
molecules Structural features for peptides binding the class I
molecules
I McKenzie, V Apostolopoulos, Y Mu*, IA Wilson*
The Austin Research Institute, Heidelberg, Victoria, Australia; *Scripps
Research Institute, La Jolla, California, USA I McKenzie, V Apostolopoulos, Y Mu*, IA Wilson*
The Austin Research Institute, Heidelberg, Victoria, Australia; *Scripps
Research Institute, La Jolla, California, USA Examination of the MHC crystal structures indicate that key
peptide binding positions are defined pockets within the groove Available online http://breast-cancer-research.com/content/3/S1 A42 disease in 12 (92.3%) out of 13 cases. The negative predictive
value for SN was 96% (22/23). We have estimated that the use of
MB rather than isosulfan blue as the labelling agent would save
approximately £1.3 million/year in the UK, should the SNB become
the standard of care. High-risk breast cancer patients: comparison of
lymphocyte phenotypes and function in vitro
S Mohrmann, A Oletzki, A Karaoglu, U Nitz, U Koldovsky
Department of Gynecology and Obstetrics, HH-University Düsseldorf,
Germany Human estrogen receptor-α (ER-α) transactivation
by selective estrogen receptor modulators (SERMs)
on VIT regulatory region in ER-α-negative breast
cancer cell line Evsa-T transiently transfected by
ER-α g
Patients and method: A total of 35 women with operable invasive
breast cancer undergoing axillary lymphadenectomy were recruited
at our centre over a 4-month period (April–July 2000). The SN was
identified in the axilla after injecting 1ml of 1% MB in the subareo-
lar region. The technical success rate, sensitivity and negative pre-
dictive value of this simple method were calculated. Furthermore,
the cost-benefit of using MB rather than isosulfan blue as the
labelling agent was determined. IP Nyamagana Butera, S Hadiy, G Leclercq IP Nyamagana Butera, S Hadiy, G Leclercq Laboratoire J-C Heuson de Cancérologie Mammaire, Institut Jules
Bordet, Brussels, Belgium The action of 11 selective estrogen receptor modulators (SERMs)
was investigated in two breast cancer cell lines, the estrogen
receptor-α-positive (ER-α+) MCF-7 and the ERα– Evsa-T. Results: The SN was successfully identified in 34 (97%) out of 35
patients. Thirteen (37%) out of 35 patients had metastasis in the
axillary nodes. The SN correctly predicted the presence of axillary reast Cancer Research Vol 3 Suppl 1
23rd Congress of the International Association for Breast Cancer Researc Using PCR and specific primers, a 660-bp sequence homologous
to the env gene of MMTV was detected in 38% of the human
breast cancers. This sequence was absent in normal tissues and
other tumors [1]. Samples from several geographical locations
have higher or lower frequencies. Our experiments were conducted by transient transfection of these
cells by a reporter plasmid carrying the luciferase gene under the
transcriptional control of the minimal promoter tk and the regulatory
region of vitellogenin A1 gene (Vit-tk-Luc). This latter region is known
to include a perfect estrogen responsive element (ERE). Evsa-T cells
were cotransfected with an expression vector for the human ER-α. The MMTV-like sequence was expressed as RNA in most positive
specimens [2]. The complete 9.9-kb proviral sequence of an
MMTV-like agent has now been amplified and sequenced in two
breast cancers. Structural features of this provirus suggest that it is
replicative competent [3]. Estradiol (E2) always increased transcription of Vit-tk-Luc basal
activity in both cell lines. Pure antiestrogens repressed it in MCF-7
cells, and had no effect in Evsa-T cells. Interestingly, in Evsa-T cells
as compared with MCF-7 cells, SERMs for which the chemical
structure contain clusters that mimic hydrophobic substituents
linked to the 11β-position of estradiol conferred greater transcrip-
tion. Of note, deletion of one half of the ERE site did not affect
transcription in Evsa-T cells, but abrogated it in MCF-7 cells. More-
over, substitution of Vit by an AP-1 site failed to activate transcrip-
tion in each case. Primary cultures of env positive tumors show budding retroviral
particles, and the supernatant particulate fractions show RT activ-
ity, presence of MMTV-like genes by RT-PCR and viral particles by
electron microscopy [4]. Experiments to prove infectivity are in
progress. Whether this virus is MMTV or a related human mammary tumor
virus is not certain, or is it known how humans are infected. MMP-9 production by T cells from mammary tumor
bearers is upregulated by tumor-derived VEGF
JL Owen, Z Gunja-Smith, DM Lopez
Department of Microbiology and Immunology, University of Miami
School of Medicine, Miami, Florida, USA MMP-9 production by T cells from mammary tumor
bearers is upregulated by tumor-derived VEGF
JL Owen, Z Gunja-Smith, DM Lopez
Department of Microbiology and Immunology, University of Miami
School of Medicine, Miami, Florida, USA A46 4. Melana et al: Proc Ann Meeting AACR 2001, 42:115. 4. Melana et al: Proc Ann Meeting AACR 2001, 42:115. Effect of interferon-γ (IFN-γ) on transforming
growth factor-β (TGF-β) regulation of sialomucin
complex/Muc4 SA Price-Schiavi, V Ramsauer, X Zhu, KL Carraway
Department of Cell Biology and Anatomy, University of Miami School
of Medicine, Miami, Florida, USA Sialomucin complex (SMC, rat Muc4) is a heterodimeric glycopro-
tein complex consisting of a mucin subunit ASGP-1 (Ascites
sialoglycoprotein-1) and a transmembrane subunit ASGP-2,
which is highly overexpressed on the surface of ascites 13762 rat
mammary adenocarcinoma cells. The complex is produced from a
single gene and polypeptide precursor. SMC is developmentally
regulated in normal rat mammary gland by multiple and complex
mechanisms, with levels in the lactating gland being 100-fold
those in the virgin gland. SMC transcript levels are enhanced in
normal rat mammary epithelial cells by fetal bovine serum, insulin,
and IGF-1 by an ERK-1/2-dependent pathway. SMC is post-tran-
scriptionally regulated by Matrigel (extracellular matrix) by inhibi-
tion
of
SMC
precursor
synthesis. SMC
is
also
post-transcriptionally regulated by TGF-β by disruption of SMC
precursor processing into mature ASGP-1 and ASGP-2. The inhi-
bition of SMC levels by TGF-β occurs by an ERK1/2-independent
pathway, suggesting that the SMAD or another pathway may be
involved in this effect. Interestingly, the inhibition of SMC levels by
TGF-β can be blocked by treatment with IFN-γ, which has been
shown to block TGF-β effects via a Jak/Stat-dependent pathway. This effect is dose-responsive and is dependent on the order in
which the cytokines are added, suggesting that the balance of
signaling inputs is important in determining the expression level of
SMC. Thus, SMC is regulated by multiple mechanisms, and the
delicate interplay of the pathways involved serves to maintain
normal levels of the complex and repress potential deleterious
effects of overexpression. IP Nyamagana Butera, S Hadiy, G Leclercq However, if a retrovirus is indeed involved in human breast carcino-
genesis, then preventive strategies can be planned. References Our results show that some SERMs may act as strong agonists on
transcription mediated by transfected ER-α in ER-α– breast tumors
with poor prognosis for antihormone therapy. We speculate that
additional binding sites for transcription factors, as well as different
coactivators, would be involved in this enhancement of activity. 1. Wang et al: Cancer Res 1995, 55:5173. 2. Wang et al: Cancer Res 1998; 4:2565. 3. Liu et al: Cancer Res 2001, 61:1754. A48 Matrix metalloproteinase-9 (MMP-9) has been shown to be impor-
tant in tumor invasion and metastasis, and may be implicated in
lymphoreticular cell extravasation. T cells from D1-DMBA-3
mammary tumor-bearing mice exhibit an overproduction of MMP-9
compared with the levels expressed in T cells from normal mice,
both at the transcriptional and translational levels. This upregula-
tion is more pronounced in animals bearing large tumors. We have
previously characterized several tumor derived factors in our
system using the in vitro DA-3 tumor cells derived from the in vivo
D1-DMBA-3 tumors (ie PGE2, GM-CSF and phosphatidyl serine). Treatment of normal T lymphocytes with these factors yielded no
increased production of MMP-9. TNF-α and IL-6, although not
expressed by the tumor cells themselves, are greatly increased in
the tumor bearers’ lymphoreticular cells and in their sera. Exposure
of normal T cells to these two cytokines also failed to upregulate
MMP-9 production. Vascular endothelial growth factor (VEGF) has
been shown to be produced by many tumors. Using a VEGF-spe-
cific ELISA, we determined that the DA-3 tumor cells, as well as
the T lymphocytes from tumor bearers, express high levels of this
growth factor. Treatment of normal T cells with VEGF resulted in an
overproduction of MMP-9. These results indicate that VEGF may
be responsible for the elevated levels of MMP-9 observed in T cells
from tumor-bearing mice. Effect of interferon-γ (IFN-γ) on transforming
growth factor-β (TGF-β) regulation of sialomucin Effect of interferon-γ (IFN-γ) on transforming
growth factor-β (TGF-β) regulation of sialomucin E Rethfeldt, M Becker, P Koldovsky Results: Mammography revealed malignant microcalcifications in
42 out of 55 patients (76%) and opacity with undefined borders
(greater diameter <1cm) in 13 out of 55 patients (24%). In 16 out
of 41 carcinomas (41%) there was immunostain positivity for bcl-2. In seven out of 16 patients (17.5%) mammography showed micro-
calcifications, whereas opacity was observed in nine out of 16
patients (22.5%). Twenty-three out of 41 carcinomas (56%) were
positive for fas. In 14 out of 23 patients (60%) mammography
showed microcalcifications and opacity in 11 out of 23 (47%). Thirty out of 41 carcinomas (73%) were positive for bax and DNA
fragmentation. In 17 out of 30 (58%) carcinomas that were posi-
tive for DNA fragmentation, mammography showed microcalcifica-
tions, whereas opacity was revealed in 16 out of 30 (53%)
patients. In 18 out of 30 (60%) carcinomas that were positive for
bax, mammography showed microcalcifications, whereas opacity
was detected in 14 out of 30 (46%) carcinomas. Hyperthermia has two major effects on cancer. (1) Tumor cells can
be killed, because they are more sensitive to heat than normal
cells. Thereby, membrane components can also be released. Both
events can induce antitumoral immunity. (2) It can revert chemo-
resistance of tumors. The patients in this study were postsurgery
and treated with radiotherapy and/or chemotherapy. Additionally to
chemotherapy, nonspecific immune stimulation was applied. All
patients were studied more than 5 years after the primary diagno-
sis. A total of 105 patients received the above-mentioned therapy. The 35 patients of the ‘hyperthermia group’ received whole-body
hyperthermia treatment. The distribution of tumor at various staging
was practically identical in both groups, as was the median follow-
up period: 70 months. In the control group (105 patients) 12
patients died and 61 developed metastasis within a mean period of
36 months. On the contrary, in the hyperthermia group (35
patients) no patient died and only three developed metastasis
within 52 months. Conclusion: Our results suggest the existence of significant corre-
lation between mammographic appearance and expression of
apoptosis factors in nonpalpable breast cancers, although the
number of patients evaluated was relatively small. Correlation with the mammographic image We evaluated the impact of c-erbB-2
overexpression on clinical and histopathological parameters of
patients with breast cancer. Objectives:
Retrospective evaluation of the mammographic
appearance of nonpalpable breast cancers and correlation with
apoptosis-related factors. g g
p
Conclusion: (1) Overexpression of c-erbB-2 oncoprotein in
primary breast cancer tumors may be an indicator of the extent of
lymph node metastases in patients. (2) Lobular carcinomas repre-
sent a defined subtype of breast carcinomas. p p
Method: Patients with nonpalpable breast lesions (n=211) were
evaluated between 1989 and 1999. All patients underwent preop-
erative mammographically guided needle-excision biopsy. Speci-
men radiography was always followed. Histological examination
revealed 55 cancers (26%; 30 ductal invasive [54.5%], 18 ductal
in situ [32.7%], five lobular invasive [9%] and two lobular in situ
[3.8%]). In 41 out of 55 carcinomas, immunohistochemistry was
conducted, using monoclonal antibodies against bcl-2, fas and
DNA fragmentation, and polyclonal antibody for bax. A retrovirus similar to MMTV associated with
human breast cancer
BG-T Pogo Mount Sinai School of Medicine, New York, USA Mount Sinai School of Medicine, New York, USA We investigated whether a retrovirus similar to the mouse
mammary tumor virus (MMTV) is implicated in human breast
carcinogenesis. Available online http://breast-cancer-research.com/content/3/S1 Available online http://breast-cancer-research.com/content/3/S1 A49 Results: C-erbB-2 overexpression was seen in 72 out of 417
(17%) of primary breast tumors. Patients with positive immunohisto-
chemistry (IHC) for c-erbB-2 were significantly younger (P=0.015),
on average. The number of involved lymph nodes was higher in
patients with positive IHC (P=0.014). Nearly all IHC positive
tumors (98.6%) were invasive ductal carcinomas, whereas all but
one lobular carcinoma were negative. Tumors with negative IHC
more often demonstrated positive estrogen (P=0.001) and proges-
terone (P=0.001) expression than did patients with positive IHC. There was a significant relation of c-erbB-2 IHC and nuclear
grading, Ki67 and p53. No association was found with menopausal
status, tumor size, T-staging and presence of metastases. Apoptosis-related factors in nonpalpable breast
tumors: an immunohistochemical study. Correlation with the mammographic image
P Ravazoula, E Likaki-Karatza*, FA Mpadra*, MV Karamouzis†,
P Aroukatos, E Tzorakoeleptherakis‡, HP Kalofonos†
Department of Pathology, *Department of Radiology, †Division of
Oncology 8211; Department of Medicine, ‡Department of Surgery,
University Hospital of Patras, Rion, Greece A50 C-erbB-2 overexpression in primary breast cancer:
relationship to clinical and histopathological
parameters of patients with breast cancer
S Regele*, FD Vogel*†, IB Runnebaum‡, R Kreienberg*
*Department of Obstetrics and Gynecology, Ulm, Germany;
‡University of Freiburg, Germany; †International Agency for Research
on Cancer, Lyon, France K Rhiem, M Münch, R Kreutzfeld, P Decker†, OD Wiestler*,
T Bauknecht, RK Schmutzler
Department of Obstetrics and Gynecology, University of Bonn,
Medical Center, Bonn; *Department of Neuropathology, University of
Bonn, Medical Center, Bonn; †Department of Surgery, University of
Bonn, Medical Center, Bonn, Germany Correlation with the mammographic image P Ravazoula, E Likaki-Karatza*, FA Mpadra*, MV Karamouzis†,
P Aroukatos, E Tzorakoeleptherakis‡, HP Kalofonos† Department of Pathology, *Department of Radiology, †Division of
Oncology 8211; Department of Medicine, ‡Department of Surgery,
University Hospital of Patras, Rion, Greece Objectives:
Retrospective evaluation of the mammographic
appearance of nonpalpable breast cancers and correlation with
apoptosis-related factors. Method: Patients with nonpalpable breast lesions (n=211) were
evaluated between 1989 and 1999. All patients underwent preop-
erative mammographically guided needle-excision biopsy. Speci-
men radiography was always followed. Histological examination
revealed 55 cancers (26%; 30 ductal invasive [54.5%], 18 ductal
in situ [32.7%], five lobular invasive [9%] and two lobular in situ
[3.8%]). In 41 out of 55 carcinomas, immunohistochemistry was
conducted, using monoclonal antibodies against bcl-2, fas and
DNA fragmentation, and polyclonal antibody for bax. Results: Mammography revealed malignant microcalcifications in
42 out of 55 patients (76%) and opacity with undefined borders
(greater diameter <1cm) in 13 out of 55 patients (24%). In 16 out
of 41 carcinomas (41%) there was immunostain positivity for bcl-2. In seven out of 16 patients (17.5%) mammography showed micro-
calcifications, whereas opacity was observed in nine out of 16
patients (22.5%). Twenty-three out of 41 carcinomas (56%) were
positive for fas. In 14 out of 23 patients (60%) mammography
showed microcalcifications and opacity in 11 out of 23 (47%). Thirty out of 41 carcinomas (73%) were positive for bax and DNA
fragmentation. In 17 out of 30 (58%) carcinomas that were posi-
tive for DNA fragmentation, mammography showed microcalcifica-
tions, whereas opacity was revealed in 16 out of 30 (53%)
patients. In 18 out of 30 (60%) carcinomas that were positive for
bax, mammography showed microcalcifications, whereas opacity
was detected in 14 out of 30 (46%) carcinomas. Conclusion: Our results suggest the existence of significant corre-
lation between mammographic appearance and expression of
apoptosis factors in nonpalpable breast cancers, although the
number of patients evaluated was relatively small. A50
C-erbB-2 overexpression in primary breast cancer:
relationship to clinical and histopathological
parameters of patients with breast cancer
S Regele*, FD Vogel*†, IB Runnebaum‡, R Kreienberg*
*Department of Obstetrics and Gynecology, Ulm, Germany;
‡University of Freiburg, Germany; †International Agency for Research
on Cancer, Lyon, France
Objective:
Amplification of c-erbB-2 oncoprotein has been
described in 10–35% of primary breast cancers. Breast tumors
with immunohistochemical overexpression of c-erbB-2 protein
seem to be more aggressive. Novel liposomal vectors for an enhanced gene
transfer in vitro G Röder, JB Prisack*, O Keil*, D Niederacher, HG Bender, P Dall
Department of Obstetrics and Gynaecology, and *Oncological
Chemistry, Heinrich-Heine-University, Duesseldorf, Germany Numerous studies have provided evidence that, although the trans-
formation of epithelial cells is the sine qua non condition for the
development of carcinomas, the nature of the connective/stromal
tissue environment is crucial for tumor progression. Matrix metallo-
proteinases (MMPs) that interact with stromal components have
been shown to contribute to malignancy in both the early and late
stages of tumor progression in human and mouse. Therefore, these
studies are of interest to improve our understanding of malignant
processes. In this context, the 11th member of the MMP family
(MMP11), also named stromelysin-3 (ST3), fulfills this paradigm. It
was discovered in 1990 because of its overexpression in a cDNA
library established from a human breast cancer biopsy. Later, clini-
cal trials showed that high levels of ST3 expression correlated with
a lower survival rate among patients with breast, head and neck, or
colon cancer. Therefore, the possibility that ST3 might play a role
during tumor progression was promising for the diagnosis, progno-
sis and design of new treatment. During the past 10 years, numer-
ous experiments have been performed to enhance the knowledge
of the biological function of ST3, and to evaluate its clinical rele-
vance. From the data, ST3 appears to be a unique member of the
MMP family, exhibiting peculiar features and function. Highly efficient gene transfer methods are basic requirements for a
successful gene therapy. Liposomal vectors based on cationic
lipids have been proven to be an attractive alternative to viral vector
systems concerning production and safety. The major disadvan-
tage of liposomes is their distinct lower transduction rate. To
improve the transduction efficiency in comparison with commer-
cially available liposomal vector systems, we have synthesized two
novel cationic lipids. In in vitro experiments with these novel liposo-
mal vectors, we examined gene transfer efficiency and cytotoxicity. As controls we used the commercially available DC-Chol and
FuGeneTM. We analyzed the cytotoxicity of our new lipids with a
dual-reporter-gene-assay and gene transfer efficiency via FACS-
analysis in seven gynaecological cancer cell lines. With the new
lipids, in different cell lines we achieved equivalent or better trans-
duction rates compared with the results obtained with DC-Chol or
FuGeneTM. Apart from improved transduction rates, cytotoxicity
was very low in all cases. K Rhiem, M Münch, R Kreutzfeld, P Decker†, OD Wiestler*,
T Bauknecht, RK Schmutzler Loss of heterozygosity constitutes a major mechanism of genetic
abberations in breast cancer, and strongly indicates the involve-
ment of tumor-suppressor genes in the affected chromosomal
regions. Ascertainment and refinement of such deleted regions by
highly polymorphic microsatellite markers is a prerequisite for the
identification of candidate genes and the isolation of novel genes. Prelimary results from our group indicate the existence of genes
located on chromosomal arm 15q that may be involved in breast
cancer progression to metastatic stage (Wick et al, Oncogene
1996). In this study a panel of 210 primary breast carcinomas, 28 Objective:
Amplification of c-erbB-2 oncoprotein has been
described in 10–35% of primary breast cancers. Breast tumors
with immunohistochemical overexpression of c-erbB-2 protein
seem to be more aggressive. We evaluated the impact of c-erbB-2
overexpression on clinical and histopathological parameters of
patients with breast cancer. Method: Primary tumors from 417 patients, who were treated at
our Department for breast cancer, were studied. Immunostaining of
c-erbB-2 oncoprotein was carried out utilizing the monoclonal anti-
body CB11. reast Cancer Research Vol 3 Suppl 1
23rd Congress of the International Association for Breast Cancer Researc studies comparing conventional dose adjuvant chemotherapy with
high-dose chemotherapy have been reported in abstract form: the
American Intergroup study (ASCO 1999) and the Dutch National
Study (ASCO 2000). The American study shows fewer relapses in
the high-dose arm. The Dutch study suggests a modest disease-
free survival advantage for the high-dose arm, but further follow up
is required to ascertain statistical significance (P=0.057, two-
sided, at the early analysis). In 2002, a 24% reduction in hazard
rate will be detectable with 80% power. Both the efficacy and toxi-
city of high-dose therapy may depend on the drugs, dosage and
schedules selected. In the Dutch study, a regimen was employed
that is similar to the frequently used CTCb (STAMP-V) regimen. The carboplatin dose is, however, twice as high, and the agents
are administered as short-term infusions rather than as continuous
96-h infusions. This may have an impact on the activation of the
prodrug cyclophosphamide; the activation route is strongly inhib-
ited by the presence of even low concentrations of thiotepa. metastases and 17 local recurrencies from primary breast carcino-
mas were analyzed for loss of heterozygosity by the use of 16
highly polymorphic markers spanning the chromosomal region
15q11-21. After PCR amplification, microsatellite markers were
separated by PAGE. Novel liposomal vectors for an enhanced gene
transfer in vitro These promising in vitro results led to
further analysis of possible usability of our new lipids in in vivo
experiments. A54 Pathway pathology: the wnt and erbB mammary
tumors
A Rosner, RD Cardiff
Department of Medical Pathology and Center for Comparative
Medicine, University of California, Davis, USA High-dose chemotherapy in breast cancer: Dutch
randomized studies
S Rodenhuis, H van Tinteren, EGE de Vries*
The Netherlands Cancer Institute, Amsterdam, The Netherlands;
*Groningen University Hospital, Groningen, The Netherlands K Rhiem, M Münch, R Kreutzfeld, P Decker†, OD Wiestler*,
T Bauknecht, RK Schmutzler LOH15q was seen in 30 out of 45 (67%)
metastases and recurrences, but only in 50 out of 210 (24%)
primary tumors (P<0.01). We identified two subregions defined
by microsatellite markers D15S514 (15q15) and CYP19
(15q21.1). LOH15q21.1 was most frequently detected in progres-
sive tumor stages. Importantly, analysis of LOH in several other
chromosomal regions (ie BRCA1 and BRCA2, TP53, RB1, ATM)
did not demonstrate a general increase in LOH frequencies, indi-
cating that LOH15q is a specific event associated with tumor pro-
gression. We are currently analyzing candidate genes located in
the regions of interest. A55
Novel liposomal vectors for an enhanced gene
transfer in vitro
G Röder, JB Prisack*, O Keil*, D Niederacher, HG Bender, P Dall
Department of Obstetrics and Gynaecology, and *Oncological
Chemistry, Heinrich-Heine-University, Duesseldorf, Germany A55
Novel liposomal vectors for an enhanced gene
transfer in vitro
G Röder, JB Prisack*, O Keil*, D Niederacher, HG Bender, P Dall
Department of Obstetrics and Gynaecology, and *Oncological
Chemistry, Heinrich-Heine-University, Duesseldorf, Germany High-dose chemotherapy in breast cancer: Dutch
randomized studies A Rosner, RD Cardiff
Department of Medical Pathology and Center for Comparative
Medicine, University of California, Davis, USA A Rosner, RD Cardiff Constitutional genomic instability of 9p23-24 in
BRCA2 mutation carriers
L Savelyeva, M Schwab CD31 is a surface molecule mediating homo- and heterotypic inter-
actions that control leukocyte trafficking through the endothelial
layer. Monoclonal antibodies against CD31 are used as markers of
neovascularization. Assessment of angiogenesis in 270 breast car-
cinomas revealed expression of CD31 in a single case of large
(5.2cm in diameter) high nuclear grade ductal carcinoma, in both
in situ and invasive components. Expression was limited to the cell
membrane, suggesting an adhesion function of CD31 in epithelial
cells. At variance with invasive breast carcinomas, angiogenesis is
not considered as a prognostic parameter in DCIS, and conse-
quently anti-CD31 MoAb are not included in standard testing. Thus, a reasonable explanation for our finding was that CD31
expression might be underscored in DCIS cells. Therefore, we
focused on 32 ductal carcinomas in situ (DCIS) larger than 2 cm,
pure or associated with invasive ductal carcinoma (IDC). Cancer
cells of seven extensive, high nuclear grade DCIS associated with
IDC were CD31+. CD31 was expressed by cells of DCIS the were
able to colonize lobules and large ducts extending to the nipple
(Paget’s disease). It was also expressed by IDC, but only in associ-
ation with CD44. Normal epithelium and hyperplastic epithelial
lesions were consistently CD31–. We conclude that CD31 expres-
sion is a feature acquired by breast cancer cells in DCIS model. Secondly, CD31 expression mainly correlates with tumor cell
spreading within the ductal system; and, finally, the invasive pheno-
type requires the coexpression of CD31 and CD44. Cytogenetics, German Cancer Research Center, Heidelberg, Germany Cytogenetics, German Cancer Research Center, Heidelberg, Germany Germ-line mutations in the BRCA2 gene account for a large propor-
tion of familial breast cancer cases in females, and for the majority of
familial breast cancers in males. Recent studies provide evidence for
a role of the BRCA2 protein in the maintenance of genomic integrity
by involvement in DNA repair and recombination. In order to identify
genetic damage resulting from mutated BRCA2 in humans, we ana-
lyzed constitutional karyotypes of BRCA2 mutation carriers. FISH
anlysis from lymphocytes of patients of breast cancer families with
germ-line BRCA2 mutation revealed additional constitutional chro-
mosomal alterations on 9p23-24. The rearrangements observed
include inversions, duplications and amplifications. Additionally, a
high level of random somatic chromosomal abnormalities on
9p23-24 has been shown. The 9p rearrangements are complex in all
families analysed, showing that this chromosomal region has suf-
fered a number of intrachromosomal recombinations. A Rosner, RD Cardiff A Rosner, RD Cardiff Human mammary cancer is frequently associated with the erbB
pathway, whereas ‘spontaneous’ mouse mammary tumor virus
(MMTV)-induced mammary tumors are associated with the wnt-1
pathway. Many members of both pathways have been studied in
genetically engineered mice. Using examples from the UCD Mutant
Mouse Pathology Laboratory, we studied the characteristics of
both pathways and found that they have unique, identifiable pheno-
types. These observations are the foundation for pathway pathol-
ogy. Members of the wnt1 pathway tend to form variations of the
classical, MMTV-induced, type A, B and P tumors described by The role of high-dose chemotherapy in the adjuvant treatment of
breast cancer will eventually be defined by a range of randomized
trials that still require years for maturation. Two underpowered
single-institution studies (from the MD Anderson Cancer Center
and from the Netherlands Cancer Institute) failed to show an
advantage for high-dose therapy. A randomized Scandinavian
study compared prolonged and intensive chemotherapy without
stem-cell support with brief chemotherapy followed by the
STAMP-V regimen. The intensive conventional treatment arm was
shown to be superior in terms of relapse-free survival. Two large Available online http://breast-cancer-research.com/content/3/S1 Dunn. Wnt1 tumors are expansile, surrounded by dense stroma,
develop around central ducts, retain myoepithelial differentiation,
and frequently have squamous metaplasia. Examples include the
following: wnt1, wnt10b, APC1, GSK, CKII, B-Catenin, and FGF
mice. In contrast, members of the erbB pathway are more likely to
resemble human tumors, to be invasive, lose myoepithelial differen-
tiation, form solid nodular asymetrical masses budding from individ-
ual ducts, have less stroma, and be less metaplastic. Examples
include the following: erbB2, PyV-mT, mutants and bigenics of
erbB and PyV-mT, src, and ras. Interestingly, GEM tumors initiated
by nuclear factors do not tend to have the characteristics of either
of these pathways. Examples: myc and lef1. These observations
suggest that the principles of pathway pathology can be applied to
human tumors of the breast and other organs. This work was sup-
ported by the DAAD (AR, individual grant), the State of California,
BCRP JB-0014, and RO1CA89140 from NCI. breast carcinomas in elderly women can also express apocrine
immuno-phenotype, and analyze the histological and clinical
aspects of such differentiation. A selected series of 50 NE tumors
(positive for NE markers in >50% of the cells) was tested for the
immunocytochemical expression of gross cystic disease fluid
protein-15 (GCDFP-15). Constitutional genomic instability of 9p23-24 in
BRCA2 mutation carriers
L Savelyeva, M Schwab The topogra-
phy of the 9p rearrangements can differ among family members,
even within an individual that can have cell populations with different
9p rearrangements. Collectively, these results point to an association
of mutant BRCA2 with genomic instability and gene alteration in
9p23-24, in at least a subset of BRCA2 mutation carriers. A Rosner, RD Cardiff About 50% of moderately (G2) and well-
differentiated (G1) NE breast carcinomas coexpressed the apoc-
rine marker. In these cases specific mRNA for GCDFP-15 (PIP)
and for chromogranin A was demonstrated using in situ hybridiza-
tion (ISH). Carcinomas of the alveolar subtype (G2) and poorly dif-
ferentiated carcinomas (G3) were pure NE carcinomas, devoid of
apocrine differentiation. The steroid receptor status of these
lesions was evaluated to test a possible involvement of androgen
receptors (AR) in apocrine differentiation. The level of AR and the
mean age of patients at diagnosis were significantly higher in apoc-
rine than in nonapocrine differentiated tumors. The histological
grade and the expression of estrogen receptor significantly influ-
enced the prognosis of these NE carcinomas, either pure or NE-
apocrine differentiated. In conclusion, NE breast carcinomas may
exhibit divergent apocrine differentiation that might be regulated by
the activation of AR in elderly patients. In addition, the possibility to
use Chs or GCDFP-15 serum values in the follow up of these
patients, as demonstrated in two cases of the present series, can
justify the immunophenotyping of the tumors. CD31 expression by cells of extensive ductal
in situ and invasive carcinomas of the breast
A Sapino, L Righi, P Cassoni, M Bongiovanni, S Deaglio*,
F Malavasi*, G Bussolati
Department of Biomedical Sciences and Human Oncology and
*Department of Genetics, Biology and Biochemistry, University of
Torino Medical School, Torino, Italy CD31 expression by cells of extensive ductal
in situ and invasive carcinomas of the breast
A Sapino, L Righi, P Cassoni, M Bongiovanni, S Deaglio*,
F Malavasi*, G Bussolati
Department of Biomedical Sciences and Human Oncology and
*Department of Genetics, Biology and Biochemistry, University of
Torino Medical School, Torino, Italy R Sinha, EG Snyderwine Department of Biomedical Sciences and Human Oncology, University
of Torino, Torino, Italy Department of Biomedical Sciences and Human Oncology, University
of Torino, Torino, Italy HCAs are mutagenic and carcinogenic compounds formed in meat
and fish prepared by high-temperature cooking methods, such as
frying, grilling and barbecuing. The precursors are amino acids, Neuroendocrine (NE) carcinomas of the breast are a rare entity
that diffusely expresses NE markers. We here demonstrate that NE Breast Cancer Research Vol 3 Suppl 1
23rd Congress of the International Association for Breast Cancer Research reast Cancer Research Vol 3 Suppl 1
23rd Congress of the International Association for Breast Cancer Researc reducing sugars and creatine, found specifically in muscle meat. One of the HCAs, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyri-
dine (PhIP), the most abundant HCA in the Western diet, has been
found to be a mammary gland carcinogen in rats. Studies in
rodents have also shown that PhIP is distributed to the mammary
gland and excreted into breast milk. Several epidemiologic studies
have found a moderately increased risk of breast cancer with
higher intake of red meat. Zheng et al (JNCI 1998) conducted a
case–control study within the cohort of the Iowa Women’s Health
Study to investigate the potential role of meat and HCAs and the
risk of breast cancer. A questionnaire was mailed to women in the
cohort who had breast cancer diagnosed during the period from
1992 to 1994, and to a random sample of cancer-free cohort
members to obtain information on usual intake of meats and
cooking practices. Color photographs showing various levels of
doneness for hamburger, beefsteak, and bacon were included. Using a HCA database (Sinha et al: Food Chem Toxicol 1998),
dietary intake of 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline
(MeIQx), 2-amino-3, 4, 8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx)
and PhIP were estimated. Multivariate analysis was performed on
data from 273 cases and 657 control subjects who completed the
survey. Well-done red meat intake was associated with increased
risk of breast cancer (Zheng et al: JNCI 1998). The odds ratios
(95% confidence interval) for categorical analysis of PhIP, with first
quintile as the referent group, were as follows: second quintile 1.1
(0.6–1.8); third quintile 1.2 (0.7–1.9); fourth quintile 1.4 (0.8–2.3);
and fifth quintile 1.9 (1.1–3.4) – P value for trend 0.001 (Sinha
et al: JNCI 2000). There was no statistically significant increase in
risk with either MeIQx or DiMeIQx. Control of apoptosis in breast by growth factors
and extracellular matrix: targets for therapeutic
intervention C Streuli, A Gilmore, P Wang, K Green, A Valentijn
School of Biological Sciences, University of Manchester, Manchester, UK R Sinha, EG Snyderwine Both animal carcinogenicity
studies and epidemiologic evidence suggests that consumption of
PhIP may increase the risk of breast cancer, but this hypothesis
needs to be investigated further. Simple changes in cooking
methods could eliminate the presence of PhIP in foods, if it is con-
clusively found to increase the risk of breast cancer. Thus, different classes of potent survival regulators (ie adhesion
and soluble factors) determine apoptotic cell fate within the same
cells through independent control of different mitochondrial acting
proapoptotic proteins. Our results broaden the scope for future
strategies of cancer intervention. Normo- and hyperbaric oxygenation of tumors:
from bench to bedside
O Thews Institute of Physiology & Pathophysiology, University of Mainz, Mainz,
Germany Institute of Physiology & Pathophysiology, University of Mainz, Mainz,
Germany Tumor hypoxia is an important factor limiting the efficacy of
sparsely ionizing radiation and O2-dependent chemotherapy. Because the tumor pO2 is the result of a dynamic steady state
between oxygen supply and O2 consumption of the tumor cells,
hypoxia could be reduced by improving the O2 supply for instance
by breathing hyperoxic gas mixtures to increase the arterial oxygen
partial pressure. This technique seems to be the most effective
method to improve tumor oxygenation, and thus to enhance the
efficacy of standard radiotherapy and chemotherapy in experimen-
tal malignancies, as well as in human tumors. However, the role of
varying inspiratory pCO2 on tumor oxygenation has been dis-
cussed controversially. Although carbogen (95% O2 + 5% CO2) is
used in the clinical setting, it remains unclear whether the benefi-
cial therapeutic effects are more pronounced than with pure
oxygen. Because in some tumor entities oxygenation is inadequate
and anisotropic, normobaric hyperoxia is often not sufficient to
completely eradicate tumor hypoxia. In these cases, breathing of
hyperoxic gases under hyperbaric conditions (2–3 atm) may be
sufficient to lead to therapeutic results. However, studies on exper-
imental tumors in animals as well as clinical trials in patients
showed nonuniform results concerning the therapeutic benefit of
hyperbaric hyperoxia, depending on the tumor entity, site of
growth, or tumor vascularization. Especially, squamous cell carci-
nomas of the head and neck region seem to benefit from additional
HBO therapy during radiotherapy, although several technical prob-
lems of irradiation during hyperbaric conditions are presently not
satisfactorily resolved. A63 Cell survival is adhesion-dependent in normal breast epithelium. Survival requires the integrin class of extracellular matrix (ECM)
receptors. We have demonstrated that specific ECM such as base-
ment membrane promote cell survival, whereas others, including
collagen I, do not. Basement membrane proteins are largely absent
around invasive breast cancer cells. Thus, cancer cells have lost
their specific ECM-dependency, presumably due to inappropriate
activation of adhesion-regulated survival enzymes. Such enzymes
represent potential targets for cancer intervention, particularly
where there is sufficient redundancy of signalling on basement
membrane to provide reduced or no dependency in normal cells. Downregulation of macrophage IL-12 production
by tumor-derived IL-11
M Torroella, DM Lopez
Department of Microbiology and Immunology, University of Miami
School of Medicine, Miami, Florida, USA M Torroella, DM Lopez CL Vogel We have previously shown that macrophages from mammary
tumor-bearing mice have a profound downregulation of IL-12,
which has been implicated in the low levels of IFN-γ and generally
depressed lymphoreticular cells functions in this tumor model. The
tumor used in our studies constitutively produces several factors
that have immunosuppressive activity (ie granulocyte macrophage-
colony stimulating factor [GM-CSF], prostaglandin E2 [PGE2], and
phosphatidyl serine [PS]). Of these factors, PGE2 and PS have
been shown to exert effects on macrophage functions. Recently,
we found that these tumor cells express IL-11 at both the transcrip-
tional and translational levels, as evidenced by RT-PCR and by
Western blots. Treatment of normal macrophages with IL-11
resulted in a downregulation of IL-12. In further studies using a
murine IL-11 ELISA, we observed low constitutive levels of this
cytokine in the supernatants of macrophage cultures from normal
mice, which is upregulated upon stimulation with LPS. Importantly,
macrophages from tumor bearers have higher production of IL-11
than their normal counterparts. Our results suggest that tumor cell
derived IL-11, in conjunction with the elevated levels of this
cytokine in tumor-bearing animal macrophages, play a role in the
depressed IL-12 production, leading to the impaired immune func-
tions observed during mammary tumorigenesis. Herceptin is the humanized monoclonal antibody targeting the
Her-2-Neu oncogene, which, when amplified, connotes a poor
prognosis for the 25% of breast cancer patients with gene amplifi-
cation. Pivotal clinical trials in metastatic breast cancer have estab-
lished clinical benefit in approximately one-third of patients treated
with one or two prior chemotherapy regimens for metastatic
disease; almost half of patients when used as a first-line single
agent; and in even higher percentages when used in combination
with cytotoxic chemotherapy. Antitumor response rates can be
maximized by selecting patients for treatment based on gene
amplification tests (eg FISH) rather than on immunohistochemistry. Although Herceptin is very well tolerated subjectively, it was, unex-
pectedly, found to be cardiotoxic, especially when used in conjunc-
tion with anthracyclines. This finding has made the development of
adjuvant programs a challenge, but several such adjuvant proto-
cols in earlier stage disease are in progress or planned. In addition
to adjuvant protocols, newer Herceptin combinations with other
cytotoxics and hormonal therapy will also be discussed. Herceptin
is the first (hopefully of many) targeted therapies that could revolu-
tionize breast cancer therapy in the decade to come. Available online http://breast-cancer-research.com/content/3/S1 We find that mutations inactivating cell cycle
checkpoints that regulate assembly of the mitotic spindle reverse
proliferative arrest, and foster transformation, in BRCA2-deficient
cells. These findings have implications not only for the evolution of
tumours following BRCA2 loss, but also for the mechanisms by
which cells perceive and respond to chromosome breakage. Loss of BRCA2 induces chromosomal instability characterized by
spontaneous breakage, in appropriate mitotic exchanges and chro-
mosomal fusions. Evidence will be presented that the repair of DNA
breaks that arise spontaneously during DNA replication require
BRCA2 for their error-free resolution. Loss of this function may foster
carcinogenesis by increasing the rate of spontaneous mutation. Conclusion: Serological Her2/neu concentrations paralleled the
clinical course of a patient with metastatic breast cancer receiving
Herceptin therapy. Prospective studies should be designed in
order to demonstrate the prognostic/predictive value of serological
Her2/neu determination. Paradoxically, BRCA2-deficient cells undergo cell cycle arrest
rather than the unrestrained proliferation that is typical of neoplas-
tic transformation. We find that mutations inactivating cell cycle
checkpoints that regulate assembly of the mitotic spindle reverse
proliferative arrest, and foster transformation, in BRCA2-deficient
cells. These findings have implications not only for the evolution of
tumours following BRCA2 loss, but also for the mechanisms by
which cells perceive and respond to chromosome breakage. Available online http://breast-cancer-research.com/content/3/S1 Available online http://breast-cancer-research.com/content/3/S1 Method: The sera samples of 10 Herceptin-patients were col-
lected immediately after standard hematological investigation. Serological Her2/neu was quantified using the Her2/neu Kit (Bayer
Diagnostics, Munich, Germany). Automatical determination was
performed on the immunoanalyzer Bayer Immuno 1™. The values
were analyzed in terms of the clinical course of each patient. Each
assay was performed in duplicate. dividing cells. The biological functions of the protein and its role in
tumour suppression remain uncertain. We have identified an essential function for BRCA2 in DNA repair by
homologous recombination. In BRCA2-deficient cells, DNA breaks
introduced into chromosomal substrates are inefficiently repaired by
homology-directed mechanisms, although repair by nonhomologous
end-joining is unaffected. BRCA2 interacts with the RAD51 recom-
bination protein, a functional homolog of bacterial RecA. The correct
intracellular localization and function of RAD51 are dependent upon
BRCA2, suggesting a mechanistic basis for its role in repair. Results: The 10 patients were observed 15 months (median),
range 6–21. Two patients had visceral metastases, two patients
bone metastases and six patients developed multiple occult metas-
tases. Seven patients had suffered a relapse. In all these seven
patients the serological Her2/neu concentration increased strik-
ingly at time of progress. The Her2/neu levels of the three patients
with stable disease did not change during the observation period. Results: The 10 patients were observed 15 months (median),
range 6–21. Two patients had visceral metastases, two patients
bone metastases and six patients developed multiple occult metas-
tases. Seven patients had suffered a relapse. In all these seven
patients the serological Her2/neu concentration increased strik-
ingly at time of progress. The Her2/neu levels of the three patients
with stable disease did not change during the observation period. Conclusion: Serological Her2/neu concentrations paralleled the
clinical course of a patient with metastatic breast cancer receiving
Herceptin therapy. Prospective studies should be designed in
order to demonstrate the prognostic/predictive value of serological
Her2/neu determination. BRCA2, suggesting a mechanistic basis for its role in repair. Loss of BRCA2 induces chromosomal instability characterized by
spontaneous breakage, in appropriate mitotic exchanges and chro-
mosomal fusions. Evidence will be presented that the repair of DNA
breaks that arise spontaneously during DNA replication require
BRCA2 for their error-free resolution. Loss of this function may foster
carcinogenesis by increasing the rate of spontaneous mutation. Paradoxically, BRCA2-deficient cells undergo cell cycle arrest
rather than the unrestrained proliferation that is typical of neoplas-
tic transformation. A64 Downregulation of macrophage IL-12 production
by tumor-derived IL-11
M Torroella, DM Lopez
Department of Microbiology and Immunology, University of Miami
School of Medicine, Miami, Florida, USA A Thomas, M Hoopmann, T Schöndorf, R Neumann*,
P Mallmann, U-J Göhring, CM Kurbacher, C Eisberg,
T Tanasale, M Warm University of Cologne, Department of Gynecology and Obstetrics,
Cologne; *Bayer Vital GmbH, Leverkusen, Germany Objective: Treatment with Herceptin® is one of the most promis-
ing therapies for patients with metastatic breast cancer whose
tumors overexpress the HER2/neu protein. Recent studies
provide evidence that patients receiving herceptin have a signifi-
cant benefit. Taking into account this clinical success and, addi-
tionally, the favorable side-effect pattern, addition of Herceptin in
first-line treatment of metastatic breast cancer is considered the
most encouraging therapeutic option. However, prognostic and/or
predictive markers justifying the therapy have not been available
until now. Therefore, we designed a retrospective study in order to
evaluate the serological Her2/neu determination accompanying a
Herceptin therapy. We have shown that pp125FAK mediates integrin survival signals
in breast epithelia, and phosphatidylinositol 3-kinase overcomes
apoptosis induced by dominant negative pp125FAK. Signals
downstream of pp125FAK regulate apoptosis through a control on
the activity of the proapoptotic protein Bax. Signal transduction through growth factor receptors can be regulated
by adhesive interactions via integrins. We have discovered that phar-
macological inhibition of epidermal growth factor receptor signalling
strongly induces apoptosis in breast epithelia. The mechanism of
apoptosis induction appears not to be through Bax activation, but
rather through dephosphorylation of the proapoptotic protein Bad. A69 tissue injury, and, at the same time, to produce significant anti-
tumor activity. In rats with R3230 AC mammary adenocarcinoma
tumors, a significant inhibition of tumor growth was observed after
intraperitoneal administration of 6mg/kg of manganese(III) tetrakis
(N-ethylpyridinium-2-yl) porphyrin (MnTE-2PyP). Furthermore, rats
that received MnTE-2PyP had a significant inhibition of the postra-
diation tumor regrowth. Animals pretreated with MnTE-2PyP
(6mg/kg intraperitoneal) 24h before implantation of R3230
mammary adenocarcinoma show a significant inhibition of tumor
angiogenesis. MnTE-2-PyP significantly delayed development of
RT-induced lung injury in rats after 28Gy of right hemithoracic irra-
diation. The magnitude of the change in breathing rate is, on
average, reduced by 30%, indicating the ability of MnTE-2PyP to
significantly reduce the severity of RT-induced lung injury. Six
months after the treatment, a significant increase in hydroxyproline
content per gram of dry or wet lung was observed in animals
receiving radiation only. Administration of 6mg/kg of MnTE-2-PyP
before RT resulted in a significant reduction in hydroxyproline
content. Furthermore, we have found a significant association
between the radioprotective effect of MnTE-2-PyP and changes in
plasma levels of transforming growth factor-β. This association
suggests a possible role of SOD mimetics in activation/regulation
of cytokines that are involved in development of radiation-induced
lung injury. This new strategy of utilizing a single compound with
antitumor activity to simultaneously protect normal tissues could
allow a higher dose of radiation to be delivered to the tumor
without increasing the risk of complications, and could further
improve breast-conserving cancer therapy. Association analysis was performed for 19 different BRCA muta-
tions (BRCA1: 14; BRCA2: 5), which were detected at least three
times in the German population. The aim of this study is the identifi-
cation of founder mutations and hot-spot mutations that are spe-
cific for the German population. Patients were genotyped for three
intragenic markers (D17S855, D17S1322 and D17S1323) in the
BRCA1 gene and for closely flanking markers (D13S1698,
D13S171 and D13S267) in the BRCA2 gene. Statistical analysis
was performed with an exact test of goodness-of-fit (Müller et al:
1991). The genotype data for the three markers analyzed each in
the BRCA1 and BRCA2 genes are in concordance with the pres-
ence of probable common haplotypes. Therefore, most of the fre-
quent mutations detected are likely to be founder mutations. Suprisingly, four C→T transitions in the BRCA1 gene, which had
been expected to result from independent mutational events, are
probably also founder mutations. On-site audits of the two clinical trials reported
from South Africa involving high-dose
chemotherapy (HDC) therapy for breast cancer
RB Weiss, CA Hudis, RA Beveridge The significance of tumor hypoxia extends beyond conventional
radiation resistance. It has been found that tumor hypoxia affects
drug resistance, angiognesis, cytokine production, cell cycle
control, apoptosis and development of distant metastases. Recently, it has been reported that hyperthermia improves tumor
oxygenation in both canine as well as human soft tissue sarcoma. This study describes a new optimized technique for pO2 mea-
surement in breast cancer patients using ultrasound-guided
placement of Eppendorf polarographic oxygen probes. Locally
advanced breast cancer patients, participating in a phase I/II
study of neoadjuvant liposomal doxorubicin/paclitaxel/hyperther-
mia treatment, were the subjects of this study. Tumor oxygena-
tion was measured before and 24 h after hyperthermia treatment. Advantages of the ultrasound-guided pO2 probe placement are
the following: accuracy with visualization and verification of the
Eppendorf electrode placement in tumor tissue; monitoring of the
electrode movement through the tumor tissue during the mea-
surement; ability to avoid electrode placement near or in large
blood vessels by using color Doppler imaging; and spatial repro-
ducibility of the second measurement. Despite progress in the
technology that can be used to measure tumor hypoxia, accurate
and verifiable placement of the oxygen probes in tumor tissue is
of tremendous importance. Ultrasound-guided pO2 probe place-
ment should become standard technique to improve accuracy
and reliability in the assessment of tumor oxygenation for disease
sites in which it is appropriate. An investigator at the University of the Witwatersrand in Johannes-
burg has reported (J Clin Oncol 1995; 13:2483–2489, and Proc
Am Soc Clin Oncol 1999, 18:2a) two clinical trials purporting to
be randomized prospective evaluations of HDC in comparison with
treatment administered in conventional doses. One involved
metastatic breast cancer, and the other high-risk primary disease. In both trials two cycles of a regimen devised at this institution
combining cyclophosphamide, mitoxantrone, and etopside were
used for the HDC. A total of 90 patients were reported in the
metastatic disease study, and 154 (or 151) were said to have been
treated in the high-risk study. Two separate on-site audits of avail-
able patient records have been performed. Results: Of the 154 (or 151) patients allegedly entered into the
high-risk study, medical records for only 58 patients (all appearing
to have received HDC) were made available for review. Of the 90
patients allegedly entered on the metastatic study (based on infor-
mation provided by the investigator), records for only 61 could be
found. Radioprotective and tumor antiangiogenic effect
of the novel synthetic superoxide dismutase
(SOD) mimetic compounds University of Cambridge, CRC Department of Oncology; The
Wellcome Trust Centre for Molecular Mechanisms in Disease,
Cambridge, UK Inherited mutations in the breast cancer susceptibility gene
BRCA2 predispose to breast, ovarian and other cancers. BRCA2
encodes a 3418-amino-acid protein that localizes to the nucleus of We have developed and tested several synthetic superoxide dis-
mutase (SOD) mimetic metalloporphyrin compounds to determine
their ability to protect/ameliorate radiation-induced (RT) normal st Cancer Research Vol 3 Suppl 1
23rd Congress of the International Association for Breast Cancer Research A69
Haplotype analysis in German families with
recurrent BRCA1 and BRCA2 mutations
B Wappenschmidt, A Golla*, A Kempe, A Meindl*,
RK Schmutzler, and the German Breast Cancer Consortium
Department of Obstetrics and Gynecology, University of Bonn, Bonn,
Germany; *Department of Medical Genetics, University of Munich,
Munich, Gemany A69 In contrast, the 4-bp deletion in
the BRCA1 gene (4184del4bp) and the most frequent mutation
3034delA in BRCA2 are recurrent mutations, for which no signifi-
cant association with specific founder alleles could be shown. Testing further informative family members to define the specific
haplotype is the aim of our current investigations. A68
Ultrasound-guided pO2 measurement in breast
cancer patients before and after hyperthermia
treatment
Z Vujaskovic, E Rosen, K Blackwell, EL Jones, LP Prosnitz,
TW Samulski, MW Dewhirst
Duke University Medical Center, Durham, North Carolina, USA A70 On-site audits of the two clinical trials reported
from South Africa involving high-dose
chemotherapy (HDC) therapy for breast cancer
RB Weiss, CA Hudis, RA Beveridge
Lombardi Cancer Center, Georgetown University, Washington,
Memorial Sloan-Kettering Cancer Center, New York; US Oncology,
Annadale, Virginia, USA Design and profile of low-molecular-weight
receptor tyrosine kinase inhibitors for
antiangiogenic therapy Method: Transgenic FVB mice were developed expressing the
human episialin gene under the control of the glucocorticoid
inducible MMTV promoter. Two transgenic founder lines were
selected: one expressing relatively low levels (F64) and one
expressing high levels of episialin (F8), both in a variety of glandular
epithelia. Oncology Research, Novartis Pharma AG, Basel, Switzerland Angiogenesis occurs physiologically during embryogenesis, ovula-
tion and wound healing, and pathologically in inflammation, psoria-
sis and tumor growth. Vascular endothelial growth factor (VEGF),
also known as vascular permeability factor (VPF), appears to be a
key factor in pathological situations that involve neovascularization
as well as enhanced vascular permeability. Our aim was to design
synthetic low-molecular-weight molecules that, by blocking the
VEGF/VEGF receptor system after oral administration, can be
used
therapeutically. One
compound
we
developed
is
PTK787/ZK 222584, a potent inhibitor of VEGF receptor tyrosine
kinases that is active in the submicromolar range. It also inhibits
other class III kinases, like the PDGFR-β tyrosine kinase, c-Kit and
c-Fms, but at higher concentrations. It is not active against
kinases from other receptor families such as EGFR, FGFR-1,
c-Met and Tie-2, or intracellular kinases such as c-Src, c-Abl,
PKC-α. PTK787/ZK 222584 inhibits VEGF-induced autophos-
phorylation of KDR, and endothelial cell proliferation, migration
and survival in the nanomolar range in cell-based assays. In con-
centrations up to 1 µmol/l, PTK787/ZK 222584 does not have
any cytotoxic or antiproliferative effect on cells that do not express
VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma
concentrations of PTK787/ZK 222584 remain above 1µmol/l for
more than 8 h. PTK787/ZK 222584 induces dose-dependent inhi-
bition of VEGF- and PDGF-induced angiogenesis in a growth
factor implant model, as well as a tumor cell-driven angiogenesis
model after once daily oral dose (25–100 mg/kg). In the same
dose range, it also inhibits the growth of xenografted human carci-
nomas either solid or in ascites formation, as well as a murine
renal carcinoma and its metastasis in syngeneic, orthotopic
models. Histological examination of tumors reveals inhibition of
microvessel formation in the interior of the tumor. PTK787/ZK
222584 is very well tolerated and does not impair wound healing
or hematopoietic recovery after concomitant cytotoxic anticancer
agent challenge. Results: Juvenile mice, either transgenic or not, showed significant
growth retardation at day 13 of age if fostered by a F8 transgenic
mother. Design and profile of low-molecular-weight
receptor tyrosine kinase inhibitors for
antiangiogenic therapy In the F8 mammary gland, large intracellular fat droplets
were present just beneath the apical membrane of the luminal
epithelial cells. In addition, the fat content in milk of fostering F8
transgenic mice was significantly reduced. This suggests that the
accumulation of large intracellular fat droplets is the result of ham-
pered fat secretion machinery in the mammary glands of these
transgenic mice. Moreover, the mammary glands of the F8 trans-
genic mice already showed histological signs of premature involu-
tion after 13 days of lactation. Moreover, lactoferrin levels in milk of
mice lactating for 13 days were higher in F8 mice than in nontrans-
genic mice, confirming that episialin overexpression induces pre-
mature involution. Conclusion: Overexpression of episialin strongly inhibits fat secre-
tion, and critically affects timing of involution of the lactating
mammary gland. On-site audits of the two clinical trials reported
from South Africa involving high-dose
chemotherapy (HDC) therapy for breast cancer
RB Weiss, CA Hudis, RA Beveridge Only 25 of these appeared to have received protocol treat-
ment (22 receiving HDC). The remainder could not be verified to
have received the purported study therapy. Many of the patients
reviewed for both studies did not meet the stated eligibility criteria,
and there was no evidence of any acceptable randomization
process. The reported results of these two studies cannot be used pp
p
Supported by a grant from the NIH CA42745 . Available online http://breast-cancer-research.com/content/3/S1 Available online http://breast-cancer-research.com/content/3/S1 as a basis for evaluating HDC for either metastatic or high-risk
primary breast cancer. as a basis for evaluating HDC for either metastatic or high-risk
primary breast cancer. FACS analysis revealed a slight increase in T lymphocytes
(CD3+, P = 0,13) and B lymphocytes (CD19+, P = 0.01) during
treatment. The ability of patient blood cells to release cytokines
was tested by measuring cytokine concentrations in the super-
natants of cell cultures stimulated with two different mitogens
(PHA or PWM). A mean increase was seen for IL-2, IL-10 and
TNF-α (P < 0.05, PHA method after 12 weeks of treatment). For
IL-1-α, IL-6, IFN-γ and IL-12 no clear effects were observed. However, the PHA and PWM methods produced different
results for some cytokines. FACS analysis revealed a slight increase in T lymphocytes
(CD3+, P = 0,13) and B lymphocytes (CD19+, P = 0.01) during
treatment. The ability of patient blood cells to release cytokines
was tested by measuring cytokine concentrations in the super-
natants of cell cultures stimulated with two different mitogens
(PHA or PWM). A mean increase was seen for IL-2, IL-10 and
TNF-α (P < 0.05, PHA method after 12 weeks of treatment). For
IL-1-α, IL-6, IFN-γ and IL-12 no clear effects were observed. However, the PHA and PWM methods produced different
results for some cytokines. J Wesseling, A Maas*, P Hageman*, J Storm*, M van der Valk*,
J Hilkens* Department of Pathology, University Hospital Groningen, Groningen,
The Netherlands; *Department of Tumor Biology, The Netherlands
Cancer Institute and Antoni van Leeuwenhoekhuis, Amsterdam, The
Netherlands Background: Episialin (MUC1, EMA, CA15.3) is a membrane-
associated mucin and is frequently overexpressed in adenocarcino-
mas. If overexpressed, it inhibits cell adhesion, promotes
invasiveness, and protects against cytotoxic T-cells in vitro. To
study the effects of episialin in vivo, we developed an episialin
transgenic mouse model. Background: Episialin (MUC1, EMA, CA15.3) is a membrane-
associated mucin and is frequently overexpressed in adenocarcino-
mas. If overexpressed, it inhibits cell adhesion, promotes
invasiveness, and protects against cytotoxic T-cells in vitro. To
study the effects of episialin in vivo, we developed an episialin
transgenic mouse model. Diminished milk fat secretion and premature
mammary gland involution in episialin/MUC1
transgenic mice Conclusion: Mild immunomodulatory effects were observed in
patients with breast and prostate cancer during treatment with a
mistletoe extract preparation standardized to mistletoe lectin (Lekti-
nol®). Changes in the different cytokines and lymphocyte subsets
remained within the physiological range. J Wesseling, A Maas*, P Hageman*, J Storm*, M van der Valk*,
J Hilkens* References 1. Yu FL, et al: A hypothesis on breast cancer. In: The 22nd Con-
gress of International Association for Breast Cancer Research;
September 25–28 1998; Athens, Greece. Edited by Ioannidou-
Mouzaka L, Agnantis NJ, Lopez DM. pp. 19–24. 2. Phillips DH, et al: Carcinogenesis 1994, 15:793–795. 1. Yu FL, et al: A hypothesis on breast cancer. In: The 22nd Con-
gress of International Association for Breast Cancer Research;
September 25–28 1998; Athens, Greece. Edited by Ioannidou-
Mouzaka L, Agnantis NJ, Lopez DM. pp. 19–24. 2. Phillips DH, et al: Carcinogenesis 1994, 15:793–795. A74
The mechanism of tamoxifen in breast cancer
prevention
F Yu, W Bender
Department of Biomedical Sciences, University of Illinois College of
Medicine at Rockford, Rockford, Illinois, USA A74
The mechanism of tamoxifen in breast cancer
prevention
F Yu, W Bender
Department of Biomedical Sciences, University of Illinois College of
Medicine at Rockford, Rockford, Illinois, USA F Yu, W Bender Department of Biomedical Sciences, University of Illinois College of
Medicine at Rockford, Rockford, Illinois, USA Tamoxifen (TAM) is known to have a dual mechanism of action: (1)
to compete with 17β-estradiol (E2) at the receptor site and to block
the promotional role of E2 in breast cancer; and (2) to bind DNA
after metabolic activation and to initiate carcinogenesis. Recent
large clinical trials indicate that TAM is also an effective chemopre-
ventive agent against breast cancer. The mechanism is unknown. Because E2 requires activation by epoxidation to bind DNA forming
DNA adducts [1], and the same is true for TAM [2], the question is
whether this preventive effect of TAM against breast cancer is con-
tributory to the possibility that TAM, as an effective competitor for
epoxidation, prevents the formation of E2 epoxide and conse-
quently breast cancer. Evidence will be presented to show that,
indeed, when incubated together with E2 for epoxidation, TAM was
able to dramatically reduce the formation of E2 epoxide as mea-
sured by both the loss of the ability of E2 to inhibit nuclear RNA
synthesis, and the reduced binding of [3H]labeled E2 to nuclear
DNA. Identical results were obtained when TAM and estrone (E1)
were used. These results suggest that the breast cancer preven-
tive effect of TAM is through a competitive epoxidation mechanism
with E1/E2. R f A76 A76
CD95 ligand expression mediates immune escape
in breast cancer
C Moers, M Müschen*, MW Beckmann†, P Mallmann
Klinik für Geburtshilfe und Gynäkologie and *Klinik für Genetik,
Universität zu Köln, Köln; †Klinik für Geburtshilfe und Gynäkologie,
Universität Erlangen, Erlangen, Germany LATE SUBMISSIONS A75
CD95 ligand expression in dedifferentiated breast
cancer
C Moers, M Müschen*, MW Beckmann†, P Mallmann
Klinik für Geburtshilfe und Gynäkologie and *Klinik für Genetik,
Universität zu Köln, Köln; †Klinik für Geburtshilfe und Gynäkologie,
Universität Erlangen, Erlangen, Germany CD95 ligand expression mediates immune escape
in breast cancer C Moers, M Müschen*, MW Beckmann†, P Mallmann
Klinik für Geburtshilfe und Gynäkologie and *Klinik für Genetik,
Universität zu Köln, Köln; †Klinik für Geburtshilfe und Gynäkologie,
Universität Erlangen, Erlangen, Germany Interaction of CD95 and its ligand (CD95L) plays an important
role in the regulation of immune response, since CD95+ lym-
phocytes may be killed after engagement of the CD95 receptor. Studying the CD95/CD95L system in 40 cases of breast
cancer, the malignant cells expressed CD95L, but lost CD95
expression, when compared to non-malignant mammary epithe-
lia. In addition, four breast cancer lines expressed CD95L, which
was further enhanced, when the cells were treated with IFN-γ. This was functionally relevant, because Jurkat T cells incubated
on breast cancer cells underwent CD95L specific apoptosis
and the rate of apoptosis was demonstrated by inhibition of
matrix metalloproteinases, CD95L expressed on breast cancer
cells could also be shed from the cell membrane into the culture
supernatant and supernatants derived from breast cancer cell
cultures induced CD95L specific apoptosis in Jurkat T cells. Interestingly, in breast cancer patients depletion of CD4+ and
CD8+ peripheral blood lymphocytes was tightly correlated with
CD95 ligand expression in the tumours, which is suggestive for
a relationship between CD95 ligand expression by tumour and
systemic immunosuppression. Patterns of cytokines and lymphocyte subsets in
patients with breast and prostate cancer treated
with a standardized mistletoe extract preparation
D Wetzel, A Gromöller, U Elsässer-Beile*
Research and Development, Madaus AG, Cologne, Germany;
*Department of Urology, University of Freiburg, Germany In an open, multicentre clinical trial (GCP), 136 patients with
cancer were included to develop a quality of life instrument. After completion of primary cancer therapy, patients received
subcutaneous injections of a standardized mistletoe extract
preparation corresponding to a dose of approximately 5 ng
mistletoe lectin twice weekly for 3 months. In 19 patients with
breast or prostate cancer, immunological parameters were
determined at baseline, and after 6 and 12 weeks of treatment. Compounds that inhibit VEGF, such as PTK787/ZK 222584, have
the potential to provide a novel, effective and well-tolerated therapy
for the treatment of solid tumors, and may provide a new therapeu-
tic approach for the treatment of other diseases where angiogene-
sis plays an important role. reast Cancer Research Vol 3 Suppl 1
23rd Congress of the International Association for Breast Cancer Researc A74 tional test, CD95+ target cells were cultured on breast cancer sec-
tions. The target cells underwent apoptosis when cultured on
breast cancer sections, but could be rescued when CD95L was
specifically blocked by a CD95-Fc fusion molecule. In conclusion, the findings suggest an inverse regulation of CD95
and CD95L expression during dedifferentation of breast cancer
and that killing of bystander cells by the CD95L expressing tumour
could be involved in tissue invasion. Resistance to CD95-mediated apoptosis in breast
cancer is not due to somatic mutation of the CD95
gene cancer is not due to somatic mutation of the CD95
gene
C Moers, M Müschen*, MW Beckmann†, P Mallmann
Klinik für Geburtshilfe und Gynäkologie and *Klinik für Genetik,
Universität zu Köln, Köln; †Klinik für Geburtshilfe und Gynäkologie der
Universität Erlangen, Erlangen, Germany C Moers, M Müschen*, MW Beckmann†, P Mallmann
Klinik für Geburtshilfe und Gynäkologie and *Klinik für Genetik,
Universität zu Köln, Köln; †Klinik für Geburtshilfe und Gynäkologie der
Universität Erlangen, Erlangen, Germany C Moers, M Müschen*, MW Beckmann†, P Mallmann
Klinik für Geburtshilfe und Gynäkologie and *Klinik für Genetik,
Universität zu Köln, Köln; †Klinik für Geburtshilfe und Gynäkologie der
Universität Erlangen, Erlangen, Germany CD95 ligand expression has been observed in various malignan-
cies. Studying the CD95 ligand (CD95L) and receptor (C95)
system in benign and malignant breast tumours from 48 patients
mRNA and protein expression were determined by quantitative
RT-PCR and immunofluorescence. mRNA levels of CD95 corre-
lated inversely (r = 0.90; P < 0.01) and CD95L positively
(r = 0.89; P < 0.01) with histopathological grading of the breast
tumours. CD95 mRNA levels were low in high-grade carcino-
mas, but high in benign mammary tissues. In contrast, CD95
mRNA levels were low in adenomas, but increased 20-fold in
grade I, 120-fold in grade II and 310-fold in grad III breast
cancer. Resistance to CD95 (Apo-1/Fas)-mediated apoptosis is a typical
feature of breast cancer cells. Recent studies identified deleterious
mutations of the CD95 gene not only in a variety of B cell lym-
phomas but also in a number of solid tumour entities. Therefore, we amplified and sequenced selected regions (includ-
ing regulatory promoter regions and the last exon coding for the
death domain) of the CD95 gene from 48 breast cancer cases and
10 breast cancer cell lines but no mutation was found. In the pres-
ence of both polymorphic allele, loss of heterozygosity was
excluded in 27 informative cases. We conclude that relevant
somatic mutations of the CD95 gene occur, if at all, at a very low
frequency and are not the primary cause for resistance to CD95-
mediated apoptosis in breast cancer. Since CD95L acts as an efficient inducer of apoptosis in CD95+
cells, apoptotic cells were identified on the tissue sections. Tumour
infiltrating lymphocytes and stroma cells in close proximity to
CD95L expressing breast cancer underwent apoptosis. As a func-
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Poly(Ionic Liquid): A New Phase in a Thermoregulated Phase Separated Catalysis and Catalyst Recycling System of Transition Metal-Mediated ATRP
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Polymers
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polymers
Article
Poly(Ionic Liquid): A New Phase in a
Thermoregulated Phase Separated Catalysis and
Catalyst Recycling System of Transition
Metal-Mediated ATRP
Lan Yao 1 , Bingjie Zhang 1 , Hongjuan Jiang 1,2, *, Lifen Zhang 1, *
1
2
3
*
ID
and Xiulin Zhu 1,3
Suzhou Key Laboratory of Macromolecular Design and Precision Synthesis, Jiangsu Key Laboratory of
Advanced Functional Polymer Design and Application, State and Local Joint Engineering Laboratory for
Novel Functional Polymeric Materials, Department of Polymer Science and Engineering, College of
Chemistry, Chemical Engineering and Materials Science, Soochow University, 199 Ren-ai Road,
Suzhou 215123, China; 20174209022@stu.suda.edu.cn (L.Y.); zbj593971779@163.com (B.Z.);
xlzhu@suda.edu.cn (X.Z.)
Changzhou Huake Polymers Co., Ltd., 602 Yulong Road, Xinbei District, Changzhou 213022, China
Global Institute of Soft Technology, No. 5 Qingshan Road, Suzhou National Hi-Tech District,
Suzhou 215163, China
Correspondence: jianghongjuan@126.com (H.J.); zhanglifen@suda.edu.cn (L.Z.);
Tel.: +86-512-6588-2056 (L.Z.); Fax: +86-512-6588-2787 (L.Z.)
Received: 25 February 2018; Accepted: 16 March 2018; Published: 21 March 2018
Abstract: Poly(ionic liquid)s (PILs) have become the frontier domains in separation science because
of the special properties of ionic liquids as well as their corresponding polymers. Considering their
function in separation, we designed and synthesized a thermoregulated PIL. That is, this kind of
PIL could separate with an organic phase which dissolves the monomers at ambient temperature.
When heated to the reaction temperature, they become a homogeneous phase, and they separate
again when the temperature falls to the ambient temperature after polymerization. Based on this,
a thermoregulated phase separated catalysis (TPSC) system for Cu-based atom transfer radical
polymerization (ATRP) was constructed. The copper catalyst (CuBr2 ) used here is easily separated
and recycled in situ just by changing the temperature in this system. Moreover, even when the
catalyst had been recycled five times, the controllability over resultant polymers is still satisfying.
Finally, only 1~2 ppm metal catalyst was left in the polymer solution phase, which indicates the really
high recycling efficiency.
Keywords: poly(ionic liquid)s (PILs); atom transfer radical polymerization (ATRP); thermoregulated
phase separated catalysis (TPSC); catalyst recycle; living radical polymerization
1. Introduction
Atom transfer radical polymerization (ATRP) is one of the most widely used radical
polymerizations, and it is well-known for its numerous advantages, such as a wide range of suitable
monomers, basically commercially available initiators, ligands, catalysts, and mild polymerization
conditions. The resultant polymers can be synthesized with designed structures, including random,
block, graft, star, gradient, hyper-branched (co) polymers, end functional polymers, etc. [1–3].
Usually, ATRP is mediated by transition metal catalysts in the presence of heat [4,5], light [6–8],
electrochemistry [9–12], or microwave [13–15]. However, the transition metal residual in polymer
product will pollute/color the polymers, which will not only result in the waste of metal catalysts,
Polymers 2018, 10, 347; doi:10.3390/polym10040347
www.mdpi.com/journal/polymers
Polymers 2018, 10, 347
2 of 12
but also hinder ATRP industrialization and limit the application of polymer product especially in
electronic and biomedical materials field.
In order to deal with the transition metal residual issue in ATRP, scientists developed a series
of solutions to separate and recycle the transition metal catalysts [16]. Biphasic catalysis systems
are effective ways. In solid–liquid biphasic catalysis systems, solid-supported catalysis by physical
adsorption [17,18] and by covalent bonding [19–22], as well as immobilized/soluble hybrid [23,24]
systems are easily removed by differences in physical properties between the solid phase and the
liquid phase. However, these methods usually result in poor control over polymerization.
Especially, in liquid–liquid biphasic catalysis systems, the thermoregulated phase separation
catalysis (TPSC) system, which was reported by Jin’s group in an organic reaction system [25], has
unique advantages with the properties of “one phase reaction coupled with two-phase separation”.
In the TPSC biphasic system, the metal catalysts and ligands are less soluble in nonpolar organic
solvents at room temperature but dissolve in temperature-sensitive polar solvents. When heated
above the critical temperature, the two phases are infinitely miscible to perform the reaction
homogeneously, and the two phases are separated again when the reaction is cooled to room
temperature. The temperature-sensitive phase containing the catalyst complex can be separated
from the nonpolar organic phase containing production easily. Recently, we have extended the concept
of TPSC to an ATRP process [26–30]. Herein, the ATRP catalyst complexes dissolve in one phase
(usually in a polar solvent) while monomer and other substrates with excellent solubility in another
phase (usually a nonpolar solvent). The two solvents dissolve unlimitedly and react in a homogeneous
condition when heating the polymerization temperature; however, a thorough phase separation
happens easily and spontaneously when cooling down to ambient or lower temperature, and the
catalyst complexed can be separated from the polymer phase by an easy and efficient decantation.
Due to the good combination of homogeneous and heterogeneous separation, this system ensures
polymer control and prominence of catalyst selectivity while recycling the catalyst.
In traditional ATRP processes, a lower oxidation state transition metal (e.g., CuBr, FeCl2 ) was
used as the original catalyst, which can be easily oxidized to a higher oxidation state during operation.
Therefore, to obtain consistent results, special handling procedures are required, and the preformed
catalysts must be stored under an inert atmosphere. Oxygen or other oxidants should be removed
from the system prior to addition of the catalyst in the lower oxidation state. Therefore, the process of
catalyst complex handling can be challenging [16]. In order to overcome the drawbacks of traditional
ATRP, some improved ATRP methods, such as activators (re)generated by electron transfer (A(R)GET)
ATRP [31–33], supplemental activators and reducing agents (SARA) ATRP [34,35], and initiators
for continuous activator regeneration ATRP (ICAR ATRP) [36,37] have been reported. In these
methods, higher oxidation state transition metals (e.g., CuBr2 , FeCl3 ) are used, which facilitate
the operation of catalyst separation and recycling. In the ICAR ATRP system, a thermal initiator
(e.g., azobis(isobutyronitrile) (AIBN)) is used to generate radicals, which can react with higher oxidation
state transition metals to generate active catalysts (lower oxidation state transition metal) in situ.
Therefore, AIBN actually acts as a reducing agent in this case.
It is well known that ionic liquids (ILs) are salts that are liquids at low temperature (<100 ◦ C)
which present a new class of solvents [38]. They are famous for their plenty of unique strengths,
such as low melting point, no measurable vapor pressure, highly thermostable, adjustable density and
viscosity [39], satisfying coordination ability with organic/inorganic substances, easy to handle and get
raw materials, high design-ability, etc. [40]. Poly(ionic liquid)s (PILs) refer to a kind of ionic polymers
connected through a polymeric backbone to form a macromolecular architecture [30]. Therefore, it is a
kind of a unique material which has the special qualities both of polymers and ionic liquids. PILs can
be obtained just by the chain polymerizing of ionic liquids monomers [41]. Since Salamone et al. first
polymerized several vinylimidazolium based ILs in the 1970s [42] various PILs based on different ionic
liquid monomers have been polymerized. Most PILs gotten nowadays are solid with a relatively low
glass transition temperature, which means that they are liquids at ambient temperature. Due to their
as thermoregulated phase and organic phase, respectively. In this novel system, a transition metal
catalyst could be easily separated just by changing the polymerization temperature and this has
nearly no negative effect on catalytic efficiency, polymer molecular weight, or molecular weight
distribution.
Polymers 2018, 10, 347
3 of 12
2. Experimental Section
2.1.
Materials
charge
features, PILs can stabilize catalytically active metal and metal oxide nanoparticles [43]. As a
result,
PIL-based
polymerization
as supportsfrom
facilitate
the recovery,
recycling,
andCo.
further
Methyl methacrylate
(MMA,systems
+99%) purchased
Shanghai
Chemical
Reagents
Ltd.
use of the China)
transition
metal
catalysts
in comparison
molecular
ILs column
[44,45]. to
Compared
with ILs,
(Shanghai,
were
purified
by passing
through atoneutral
alumina
remove inhibitors.
the viscosities
PILs would
change
greatly
with the temperature
changes,China)
making
it aused
stable
Tris
((2-pyridyl)ofmethyl)
aminenot
(TPMA,
98%,
Sigma-Aldrich
Co. Ltd., Shanghai,
was
as
phase
in
polymerization
system
[46–50].
received. 2,2′-Azobis (isobutyronitrile) (AIBN, 97%, Shanghai Chemical Reagents Co. Ltd.) was
Combination
the advantages
ofroom
TPSC,temperature
ICAR ATRP,
and vacuum
PILs, in before
this work,
we designed a
recrystallized
from of
ethanol
and dried at
under
use. Monomethoxy
recycled
thermoregulated
PIL
which
are
used
in
the
ATRP
system
as
a
solvent
and
poly(ethylene glycol)-350 (MPEG350, number-average molecular weight 350 constructed
g/mol, J&Ka
PIL/organic biphasic TPSC system
for catalyst separation
and recycling.
used as
2 was sodium
Scientific,Shanghai,China),
ethyl-2-bromo-2-phenyl
acetate
(EBPA,Herein,
97%, CuBr
J&K),
the
original
catalyst,
tris
((2-pyridyl)
methyl)
amine
(TPMA)
as
the
ligand,
ethyl-2-bromo-2-phenyl
triacetoxyborohydride (97%, Saan Chemical Technology Co. Ltd., Shanghai, China), acryloyl chloride
acetateEnergy
(EBPA)Chemical),
as the ATRP
initiator,
and AIBN(+96%,
as the reducing
addition, methyl
methacrylate
(97%,
and
2-bromoethanol
Shanghaiagent.
Titan In
Technology
Co., Ltd.,
Shanghai,
(MMA) is
an important
industry,
which plays
role in manufacturing
of organic
glass,
China)
were
used as monomer
received. in
Copper
bromide
(CuBra2vital
, analytical
reagent), acetic
acid glacial
coatings,
adhesives,
and
medical
polymers
[51],
therefore
MMA
was
used
as
a
model
monomer
for
this
(analytical agent), tetraethylammonium bromide (+97%), triethylamine (N(Et)3, analytical reagent),
novel
polymerization
system.
In
addition,
we
chose
a
thermoregulated
IL
monomer,
MPEG350-MI-MA,
methanesulfonyl chloride (analytical reagent), saturated sodium bicarbonate, benzene (analytical
which was
first synthesized
our group
to polymerize
aim PIL
[30]. Thedichloride
synthetic pathway
is
reagent),
toluene
(analytical in
agent),
methanol
(analyticalthe
agent),
methylene
(analytical
shown
in
Scheme
1.
Furthermore,
the
resultant
PIL
and
benzene
were
selected
as
thermoregulated
agent), tetrahydrofuran (THF, analytical reagent), cyclohexane (analytical reagent), n-hexane
phase and organic
respectively.
In this
novel
system,from
a transition
metal
catalyst
could be
easily
(analytical
reagent),phase,
and all
other chemicals
were
obtained
Shanghai
Chemical
Reagents
Co.
Ltd.
separated
just
by
changing
the
polymerization
temperature
and
this
has
nearly
no
negative
effect
on
and used as received unless mentioned. The liquid ionic monomer, and thermoregulated poly (ionic
catalytic
efficiency,
polymer
molecular
weight,
or
molecular
weight
distribution.
liquid) were synthesized separately according to the procedures shown in Schemes 1 and 2.
NH
CH3SO 2Cl
N
CH 3SO2-PEG-OCH3
HO-PEG-OCH3
NaOH
1
Cl HO
N
N
PEG
OCH3
2
Br
O
O
Br
O
3
N
N
PEG
OCH3
2
O
+
O
3
O
Br
O
N
N PEG OCH3
Br
MPEG350-MI-MA
-MI-MA.
Scheme 1. Synthetic pathway of the thermoregulated ionic liquid monomer MPEG
MPEG350
350-MI-MA.
2. Experimental Section
2.1. Materials
Methyl methacrylate (MMA, +99%) purchased from Shanghai Chemical Reagents Co. Ltd.
(Shanghai, China) were purified by passing through a neutral alumina column to remove inhibitors.
Tris ((2-pyridyl) methyl) amine (TPMA, 98%, Sigma-Aldrich Co. Ltd., Shanghai, China) was used
as received. 2,20 -Azobis (isobutyronitrile) (AIBN, 97%, Shanghai Chemical Reagents Co. Ltd.) was
recrystallized from ethanol and dried at room temperature under vacuum before use. Monomethoxy
poly(ethylene glycol)-350 (MPEG350 , number-average molecular weight 350 g/mol, J&K Scientific,
Shanghai, China), ethyl-2-bromo-2-phenyl acetate (EBPA, 97%, J&K), sodium triacetoxyborohydride
(97%, Saan Chemical Technology Co. Ltd., Shanghai, China), acryloyl chloride (97%, Energy Chemical),
and 2-bromoethanol (+96%, Shanghai Titan Technology Co., Ltd., Shanghai, China) were used
as received. Copper bromide (CuBr2 , analytical reagent), acetic acid glacial (analytical agent),
tetraethylammonium bromide (+97%), triethylamine (N(Et)3 , analytical reagent), methanesulfonyl
chloride (analytical reagent), saturated sodium bicarbonate, benzene (analytical reagent), toluene
(analytical agent), methanol (analytical agent), methylene dichloride (analytical agent), tetrahydrofuran
(THF, analytical reagent), cyclohexane (analytical reagent), n-hexane (analytical reagent), and all other
Polymers 2018, 10, 347
4 of 12
chemicals were obtained from Shanghai Chemical Reagents Co. Ltd. and used as received unless
mentioned. The liquid ionic monomer, and thermoregulated poly (ionic liquid) were synthesized
separately according to the procedures shown in Schemes 1 and 2.
Polymers 2018, 10, x FOR AUTHORS PROOFREADING
4 of 12
O
O
radical polymerization
n
O
NH
Br
N
PEG
H3 CO
AIBN
MPEG350-MI-MA
O
NH
Br
N
PEG
H3CO
PIL
Scheme
Scheme 2.
2. Synthetic
Synthetic pathway
pathway of
of poly(ionic
poly(ionic liquid)
liquid) (PIL).
(PIL).
2.2.
2.2. Synthesis
Synthesis of
of the
the Thermoregulated
Thermoregulated Poly(Ionic
Poly(Ionic Liquid)
Liquid) (PIL)
(PIL)
The
ionic liquid
liquid monomer
monomer MPEG
MPEG350
350-MI-MA
The synthetic
synthetic pathway
pathway of
of the
the thermoregulated
thermoregulated ionic
-MI-MA is
is shown
shown
in
1 and
2 were
synthesized
according
to the reported
literatureliterature
[30]. Besides,
in Scheme
Scheme1.1.Intermediates
Intermediates
1 and
2 were
synthesized
according
to the reported
[30].
the
intermediate
3
was
synthesized
according
to
another
previous
work
[52].
15
mmol
of
MPEG
350Besides, the intermediate 3 was synthesized according to another previous work [52]. 15 mmol
MI-MA
and
0.246
g
AIBN
(1.5
mmol)
were
added
to
a
dried
Schelenk
tube
with
a
stir
bar,
and
then
of MPEG350 -MI-MA and 0.246 g AIBN (1.5 mmol) were added to a dried Schelenk tube with a stir bar,
10
mL
of DMSO
also added
to the
tube.toAfter
bubbled
with
argon with
for 15argon
min, the
tube
wasthe
sealed
and
then
10 mL was
of DMSO
was also
added
the tube.
After
bubbled
for 15
min,
tube
◦
and
settled
under
80
°C
for
three
days.
When
the
reaction
was
finished
and
the
mixture
was
cooled
was sealed and settled under 80 C for three days. When the reaction was finished and the mixture
to
room
temperature,
washed the washed
resultedthe
solution
with
methanol
3–5 times,
poly
(ionic
was
cooled
to room temperature,
resulted
solution
withfor
methanol
forthen
3–5 the
times,
then
the
liquid)
(PIL)
was
obtained.
The
PIL
was
determined
by
gel
permeation
chromatograph
(GPC)
with
poly (ionic liquid) (PIL) was obtained. The PIL was determined by gel permeation chromatograph
M
n,GPC =
26,300
g/mol= and
Mwg/mol
/Mn = 1.46.
(GPC)
with
Mn,GPC
26,300
and Mw /Mn = 1.46.
2.3. Typical
Typical Procedure
Procedure for the TPSC-Based ICAR ATRP of MMA and Catalyst Recycling
2.3.
Recycling
The typical
typical procedure
procedurefor
forthe
theTPSC-based
TPSC-basedICAR
ICARATRP
ATRPofofMMA
MMAisisdemonstrated
demonstrated
below
with
The
below
with
a
a molar
ratio
of [MMA]
200:1:1:3:1.
CuBr
mg,
molar
ratio
of [MMA]
0/[EBPA]
0/[CuBr
2]0/[TPMA]
0/[AIBN]00/[AIBN]
= 200:1:1:3:1.
2 (10.4 mg,
0.047
mmol),
0 /[EBPA]
0 /[CuBr
2 ]0 /[TPMA]
0 = CuBr
2 (10.4
0.047 mmol),
µL, MMA
0.047 mmol),
MMA
(1 mL,
mmol),
PIL (200
mg,AIBN
0.108 (7.8
mmol),
EBPA
(8.3 µL,EBPA
0.047 (8.3
mmol),
(1 mL, 9.4
mmol),
PIL9.4
(200
mg, 0.108
mmol),
mg, AIBN
0.047
(7.8 mg,and
0.047
mmol),(3.0
andmL)
benzene
(3.0 mL)
were
into a 5with
mL ampoule
with
clean
stir bar.
mmol),
benzene
were added
into
a 5 added
mL ampoule
a clean stir
bar.a The
ampoule
The bubbled
ampoulewith
was argon
bubbled
argon
for to
about
15 minthe
to dissolved
eliminate oxygen,
the dissolved
oxygen,
and then
was
forwith
about
15 min
eliminate
and then
flame-sealed.
flame-sealed.
The sealed
ampoule
was
to 70 ◦ C for polymerization.
desired time of
The
sealed ampoule
was heated
to 70
°C heated
for polymerization.
After the desiredAfter
time the
of polymerization,
polymerization,
ampoule
was
out and
ampoule
was cooled
by immersing
into iced
the
ampoule wasthe
taken
out and
thetaken
ampoule
wasthe
cooled
by immersing
it into
iced water.itAfter
the
water. After
system was
completely phase-separated,
ampoule
wasand
broken,
and transferred
the
system
was the
completely
phase-separated,
the ampoulethe
was
broken,
transferred
the upper
upper polymer
solution
intotoTHF
get a diluted
one,the
then
the mixture
was precipitated
in methanol.
polymer
solution
into THF
get atodiluted
one, then
mixture
was precipitated
in methanol.
The
The precipitated
product
was filtered
off suction
with suction
a vacuum
distillation
flask.
To obtain
precipitated
product
was filtered
off with
from afrom
vacuum
distillation
flask. To
obtain
a drieda
dried product,
the filtered
product
placed
a vacuum
oven
at 30
about3–4
3–4h.h.The
Theweight
weight of
of
product,
the filtered
product
was was
placed
in ain
vacuum
oven
at 30
°C◦ C
forfor
about
the dried
driedproduct
product was
wasmeasured
measured and
andthe
themonomer
monomerconversion
conversionwas
wascalculated
calculatedaccording
accordingto
toit.
it.Finally,
Finally,
the
The PIL
PIL phase
phase containing
containing the
the catalyst
catalyst was
was transferred
transferred to
to aanew
new ampoule
ampouleand
andfresh
freshEBPA
EBPA (8.3
(8.3 µL,
µL,
The
0.047 mmol), MMA
mmol),
AIBN
(7.8(7.8
mg,mg,
0.047
mmol)
and benzene
(3 mL)
0.047
MMA(1(1mL,
mL,9.49.4
mmol),
AIBN
0.047
mmol)
and benzene
(3were
mL) also
wereadded
also
for catalyst
recycling
experiments.
All remaining
polymerization
operations
were the
same
above.
added
for catalyst
recycling
experiments.
All remaining
polymerization
operations
were
theas
same
as
above.
2.4. Typical Procedure for Chain Extension of PMMA
2.4. Typical
Procedure
Chain Extension
of PMMA procedure of PMMA with the molar ratio of
A typical
chainforextension
polymerization
[MMA]
/[PMMA]
200:1:1:3:1of
wasPMMA
as follows:
0 /[CuBr
2 ]0 /[TPMA]
0 /[AIBN]0 =
A 0typical
chain
extension
polymerization
procedure
withThe
thepolymer
molar obtained
ratio of
was
filtered
with
neutral
alumina
and
reprecipitated,
then
it
was
dried
in
a
vacuum
oven
to a constant
[MMA]0/[PMMA]0/[CuBr2]0/[TPMA]0/[AIBN]0 = 200:1:1:3:1 was as follows: The polymer obtained
was
weight. with
A predetermined
quantity
PMMA (0.024then
mmol)
was dried
addedininaavacuum
dry 5 mLoven
ampule
a stir
filtered
neutral alumina
and of
reprecipitated,
it was
to awith
constant
bar as the
macro-initiator,quantity
then the of
corresponding
CuBr
PILampule
(0.108 mmol),
2 (0.047
weight.
A predetermined
PMMA (0.024amount
mmol) of
was
added
in a mmol),
dry 5 mL
with a
MMA
(9.4
mmol),
AIBN
(0.047
mmol),
and
benzene
(3.0
mL)
were
added.
The
rest
of
the
procedure
stir bar as the macro-initiator, then the corresponding amount of CuBr2 (0.047 mmol), PIL
(0.108
was the MMA
same as
themmol),
typicalAIBN
procedure
the TPSC-based
ICAR
ATRP
above.
mmol),
(9.4
(0.047formmol),
and benzene
(3.0
mL) described
were added.
The rest of the
procedure was the same as the typical procedure for the TPSC-based ICAR ATRP described above.
2.5. Characterizations
The number-average molecular weight (Mn,GPC) and molecular weight distribution (Mw/Mn)
Polymers 2018, 10, 347
5 of 12
Polymers 2018, 10, x FOR AUTHORS PROOFREADING
5 of 12
2.5. Characterizations
Thewith
number-average
molecular
weight
(Mn,GPC
) andTSKgel
molecular
weight distribution
(M(4.6
equipped
a refractive-index
detector
(TOSOH),
using
guardcolumn
SuperMP-N
×n )
w /M
values
of
PMMA
were
determined
using
a
TOSOH
HLC-8320
gel
permeation
chromatograph
20 mm) and two TSKgel SupermultiporeHZ-N (4.6 × 150 mm) with measurable molecular weights
−1 at
(GPC) equipped
a 6refractive-index
detector
(TOSOH),
using TSKgel
guardcolumn
SuperMP-N
ranging
from 103 towith
1 × 10
g·mol−1. THF was
employed
as the eluent
at a flow
rate of 0.35 mL·min
20 GPC
mm) samples
and twowere
TSKgel
SupermultiporeHZ-N
(4.6
× 150 mm)and
with
measurable
40(4.6
°C.×
The
injected
using a TOSOH plus
auto-sampler
calibrated
withmolecular
PMMA
3 to 1 × 10
6 g·mol−1 . THF was employed as the eluent at a flow rate of
1
weights
ranging
from
10
standards purchased from TOSOH. H NMR spectra were recorded on an INOVA 400 MHz nuclear
−1 at 40 ◦ C. The GPC samples were injected using a TOSOH plus auto-sampler and
0.35 mL·min
magnetic
resonance
(NMR) instrument using CDCl3 and DMSO-d6 as the solvents and
1 H NMR spectra were recorded on an
calibrated with PMMA
purchased
TOSOH.
tetramethylsilane
(TMS) asstandards
an internal
standard from
at ambient
temperature.
INOVA 400 MHz nuclear magnetic resonance (NMR) instrument using CDCl3 and DMSO-d6 as the
3.solvents
Results and
and tetramethylsilane
Discussion
(TMS) as an internal standard at ambient temperature.
3. Results
Discussion
3.1.
Selectionand
of Solvent
for TPSC-Based ICAR (Initiators for Continuous Activator Regeneration) ATRP
System
3.1. Selection of Solvent for TPSC-Based ICAR (Initiators for Continuous Activator Regeneration)
ATRP
TheSystem
PIL (Mn,GPC = 26,300 g/mol, Mw/Mn =1.46) was confirmed with 1H NMR spectroscopy. From
Figure 1, it can be seen that the monomer’s double bond disappeared at δ = 5.84–6.47 ppm and a new
The PIL (Mn,GPC = 26,300 g/mol, Mw /Mn =1.46) was confirmed with 1H NMR spectroscopy. From
group of chemical shifts were found at δ = 2.65–2.95 ppm (f in Figure 1) assigned to polymer backbone
Figure 1, it can be seen that the monomer’s double bond disappeared at δ = 5.84–6.47 ppm and a new
hydrogen bonds, which indicated that the PIL was obtained successfully. In order to construct the
group of chemical shifts were found at δ = 2.65–2.95 ppm (f in Figure 1) assigned to polymer backbone
TPSC-based ATRP system, we screened out the optimal solvent system firstly. The results are shown
hydrogen bonds, which indicated that the PIL was obtained successfully. In order to construct the
in Table 1. Six organic solvents including p-xylene, o-xylene, toluene, benzene, cyclohexane, and nTPSC-based ATRP system, we screened out the optimal solvent system firstly. The results are shown in
hexane were investigated to form different mixed solvents with the synthesized PIL. At room
Table 1. Six organic solvents including p-xylene, o-xylene, toluene, benzene, cyclohexane, and n-hexane
temperature, only cyclohexane/PIL solvent pair was miscible, which indicating that it did not meet
were investigated to form different mixed solvents with the synthesized PIL. At room temperature,
the biphasic solvent requirement of TPSC system. After increasing the temperature to 70–90 °C, it is
only cyclohexane/PIL solvent pair was miscible, which indicating that it did not meet the biphasic
found that only benzene/PIL solvent pair became miscible completely, namely
forming a
solvent requirement of TPSC system. After increasing the temperature to 70–90 ◦ C, it is found that
homogeneous solution. Therefore, benzene was selected as the optimal organic solvent applied in
only benzene/PIL solvent pair became miscible completely, namely forming a homogeneous solution.
this TPSC system. Actually, as shown in Figure 2, the homogeneous polymerization and
Therefore, benzene was selected as the optimal organic solvent applied in this TPSC system. Actually,
heterogeneous separation process could be easily realized just by changing the reaction temperature
as shown in Figure 2, the homogeneous polymerization and heterogeneous separation process could
with benzene/PIL solvent system.
be easily realized just by changing the reaction temperature with benzene/PIL solvent system.
f
n
O
DMSO
O
c
Br
b
d
N
a
NH
b
c
O
d
O
n
e
CDCl3
10
8
TMS
b c ef
a
6
4
2
0
Chemical shift(ppm)
Figure
NMR
spectrum
ofofpoly(ionic
3 and
DMSO-d
6 as
mixed
solvents
Figure1.1.1H1 H
NMR
spectrum
poly(ionicliquid)
liquid)(PIL)
(PIL)with
withCDCl
CDCl
DMSO-d
mixed
solvents
3 and
6 as
and
tetramethylsilane
and
tetramethylsilane(TMS)
(TMS)asasananinternal
internalstandard.
standard.
Figure 2. Photographs of thermoregulated phase separated catalysis (TPSC)-based ICAR-ATRP
(initiators for continuous activator regeneration-atom transfer radical polymerization) in PIL/benzene
biphasic solvent system.
10
TMS
b c ef
a
8
6
4
2
0
Chemical shift(ppm)
Polymers
2018, 1.
10,1H
347NMR spectrum of poly(ionic liquid) (PIL) with CDCl3 and DMSO-d6 as mixed solvents6 of 12
Figure
and tetramethylsilane (TMS) as an internal standard.
Figure 2.
2. Photographs
catalysis (TPSC)-based
(TPSC)-based ICAR-ATRP
ICAR-ATRP
Photographs of
of thermoregulated
thermoregulated phase separated catalysis
(initiators for
for continuous
continuous activator
activatorregeneration-atom
regeneration-atomtransfer
transferradical
radicalpolymerization)
polymerization)ininPIL/benzene
PIL/benzene
(initiators
biphasic solvent system.
system.
Table 1. Selection of organic solvents with PIL (174 mg) for the TPSC-based polymerization a .
Solvent (2.0 mL)
25 ◦ C
70–90 ◦ C
25 ◦ C
p-Xylene
o-Xylene
Toluene
Benzene
Cyclohexane
n-Hexane
I
I
I
I
M
I
S
S
S
M
M
S
I
I
I
I
M
I
a
I indicates that the two solvents are immiscible at investigated temperature; S indicates that the two
solvents are slightly miscible at investigated temperature; and M indicates that the two solvents are miscible
at investigated temperature.
Subsequently, we investigated the effect of types of ligands (Me6TRAN, PMDETA, TPMA, and
TDA-1), reducing agents (AsAc, AsAc-Na, glucose, and AIBN) on polymerization. As listed in Table 2,
all the polymerizations could be performed smoothly (Entries 1–8, Table 2). However, TPMA and
AIBN were selected as the ligand and reducing agent, respectively, by considering polymerization
rate and controllability over PMMA molecular weight and its distribution. Therefore, an optimal
ICAR ATRP system could be constructed by using MMA as the model monomer, EBPA as the ATRP
initiator, CuBr2 as the catalyst, TPMA as the ligand, AIBN as the reducing agent, and benzene/PIL as
the solvent pair system. In addition, we also investigated the effect of molar ratio of CuBr2 to AIBN,
and the results are shown in Table 3. It can be seen from Table 3 that the polymerization could be
successfully carried out with a wide range of [AIBN]0 :[CuBr2 ]0 ([AIBN]0 :[CuBr2 ]0 = 0.5–2:1). However,
the polymerization rate increased with the increase of the amount of AIBN since more active catalyst
CuBr could be generated correspondingly as expected by ICAR ATRP mechanism [37].
Table 2. Effect of kinds of ligand and reducing agent on polymerization.
Entry
Ligand
Reducing
Agent
Time (h)
Conv. (%)
M n,th a
(g/mol)
M n,GPC
(g/mol)
M w /M n
1
2
3
4
5
6
7
8
TPMA
PMDETA
Me6TRAN
TDA-1
TPMA
TPMA
TPMA
TPMA
AIBN
AIBN
AIBN
AIBN
AIBN
Glucose
AsAc
AsAc-Na
11.5
10
10
10
5.5
57
57
5.5
73.1
81.9
83.9
72.5
69.9
46.4
62.5
66.1
14,800
16,400
16,800
14,500
14,000
9500
12,700
13,400
14,600
10,300
8700
29,100
12,000
11,400
16,200
19,300
1.17
1.39
1.51
1.58
1.31
1.11
1.11
1.12
Polymerization conditions: [MMA]0 :[EBPA]0 :[CuBr2 ]0 :[ligand]0 :[reducing agent]0 = 200:1:1:3:1, V MMA = 1.0 mL,
V benzene = 3.0 mL and mPIL = 200 mg, temperature = 70 ◦ C. a Mn,th = ([M]0 /[EBPA]0 ) × Mw,MMA
× conv. %. TPMA: tris(2-pyridylmethyl)amine; PMDETA: pentamethyldiethylenetriamine; Me6 TRAN:
tris(2-dimethylaminoethyl)amine; TDA-1: tris(3,6-dixa-heptyl)amine; AsAc: ascorbic acid; AsAc-Na:
sodium ascorbate.
Polymers 2018, 10, 347
7 of 12
Table 3. Effect of molar ratio of [CuBr2 ]0 :[AIBN]0 on polymerization.
Entry
y/x
Time (h)
Conv. (%)
M n,th a (g/mol)
M n,GPC (g/mol)
M w /M n
1
2
3
4
5
0.5
0.8
1.0
1.5
2.0
5.5
5.5
5.5
5.5
5.5
19.8
30.2
39.4
43.6
46.3
4000
6000
7900
8700
9300
5600
8100
8900
10,200
10,300
1.10
1.11
1.11
1.13
1.14
Polymerization conditions: [MMA]0 :[EBPA]0 :[CuBr2 ]0 :[TPMA]0 :[AIBN]0 = 200:1:x:3:y, V MMA = 1.0 mL,
Polymers
2018, =10,
FORand
AUTHORS
PROOFREADING
7 of 12
V benzene
3.0x mL
mPIL = 200
mg, temperature = 70 ◦ C. a Mn,th = ([M]0 /[EBPA]0 ) × Mw,MMA × conv. %.
3.2.
3.2. Polymerization
Polymerization Kinetics
Kinetics of
of MMA
MMA
To
lowlow
viscosity
reaction
condition
and a fit
polymerization
rate as wellrate
as the
To make
makea arelatively
relatively
viscosity
reaction
condition
and
a fit polymerization
as
better
controllability
of
polymerization,
we
finally
chose
the
molar
ratio
well as the better controllability of polymerization, we finally chose the molar ratio of
of
[MMA]
0/[TPMA]
0/[AIBN]
0 = 200:1:1:3:0.8
and
3.0
mL
of
benzene,
200
mg
PIL
to
[MMA]00/[EBPA]
/[EBPA]00/[CuBr2]
/[CuBr2]
/[TPMA]
/[AIBN]
=
200:1:1:3:0.8
and
3.0
mL
of
benzene,
200
mg
PIL
to
0
0
0
further
investigate
the
polymerization
kinetics
of
MMA
in
this
polymerization
system.
The
first-order
further investigate the polymerization kinetics of MMA in this polymerization system. The first-order
kinetics
(Figure3a)3a)
indicated
the propagating
in theremained
system constant
remained
constant
kinetics (Figure
indicated
thatthat
the propagating
radicalsradicals
in the system
throughout
throughout
the polymerization
up to high
monomer (more
conversion
(moreFigure
than 95%).
Figure
3bM
shows
the polymerization
up to high monomer
conversion
than 95%).
3b shows
that
n,GPC
that
M
n,GPC
increased
linearly
with
monomer
conversion
and
the
molecular
weight
distributions
kept
increased linearly with monomer conversion and the molecular weight distributions kept narrow
narrow
(M
w
/M
n
<
1.20).
These
results
indicated
the
“living”/controlled
features
of
the
TPSC-based
(Mw /Mn < 1.20). These results indicated the “living”/controlled features of the TPSC-based ICAR
ICAR ATRP
of MMA
in PILL/benzene
biphasic
system.
ATRP
of MMA
in PILL/benzene
biphasic
system.
(b) 16000
1.50
3.5
14000
M
(kg/mol)
n,GPC
12000
Mw /Mn
2.5
Mn,GPC(kg/mol)
ln([M]0/[M])
3.0
2.0
1.5
1.0
0.5
1.45
1.40
1.35
10000
1.30
8000
1.25
6000
1.20
1.15
4000
1.10
2000
1.05
0.0
0
0
2
4
6
8
10
12
14
16
Time (h)
(a)
18
Mw/Mn
(a)
0
10
20
30
40
50
60
70
80
1.00
90 100
Conversion(%)
(b)
Figure 3. In([M]
function
ofof
(a)(a)
time
and
(b)(b)
number-average
molecular
weight
(Mn,GPC
) and)
In([M]00/[M])
/[M])asasa a
function
time
and
number-average
molecular
weight
(Mn,GPC
molecular
weight
distribution
(Mw/M(M
n) w
versus
conversion
for TPSC-based
ICAR ATRP
of
and molecular
weight
distribution
/Mn ) monomer
versus monomer
conversion
for TPSC-based
ICAR
ATRP
MMA.
[MMA]
/[EBPA]
/[CuBr
]
/[TPMA]
/[AIBN]
=
200:1:1:3:0.8,
V
=
1.0
mL,
MMA. of
[MMA]
0/[EBPA]
0/[CuBr
2
]
0
/[TPMA]
0
/[AIBN]
0
=
200:1:1:3:0.8,
V
MMA
=
1.0
mL,
V
benzene
=
3.0
mL,
0
0
2 0
0
0
MMA
V
=mg
3.0 and,
mL, m
mbenzene
PIL = 200
temperature
= and,
70 °C.temperature = 70 ◦ C.
PIL = 200 mg
3.3. Chain-End Analysis and Chain Extension
In order to verify the structure of the resultant polymer and the chain-end functionality, we
1 HNMR
made
analysis of
ofaaresultant
resultantPMMA
PMMA(M
(M
n,GPC =
= 4250
4250 g/mol,
g/mol, M
n ==
made analysis
Mww/M
/M
1.22)by
by1H
NMRspectroscopy.
spectroscopy.
n 1.22)
n,GPC
From the 11H
NMR
spectrum
of
the
polymer
(Figure
4),
it
can
be
seen
that
the
chemical
shifts
at δat
=
H NMR spectrum of the polymer (Figure 4), it can be seen that the chemical
shifts
4.0–4.1
ppm
(e in
4) and
δ =δ7.15–7.35
ppm
(as (as
in Figure
4) are
attributed
to the
methyl
of
δ = 4.0–4.1
ppm
(e Figure
in Figure
4) and
= 7.15–7.35
ppm
in Figure
4) are
attributed
to the
methyl
initiator
EBPA
and
hydrogen
of
aromatic
rings,
respectively.
This
indicated
that
the
initiator
EBPA
of initiator EBPA and hydrogen of aromatic rings, respectively. This indicated that the initiator
moieties
had successfully
attached
on the on
structure
of the polymers.
The chemical
shifts at shifts
δ = 3.78
EBPA moieties
had successfully
attached
the structure
of the polymers.
The chemical
at
ppm
(c in
Figure
are attributed
to the bromine-terminated
methyl ester
group
at group
the chain
endchain
[13].
δ = 3.78
ppm
(c in4)Figure
4) are attributed
to the bromine-terminated
methyl
ester
at the
In
to further
the “living”
featurefeature
of theofresulting
polymers,
we used
the
endaddition,
[13]. In addition,
to demonstrate
further demonstrate
the “living”
the resulting
polymers,
we used
resultant
PMMA
as aasmacroinitiator
forfor
thethe
chain
extension
the resultant
PMMA
a macroinitiator
chain
extensionvia
viaTPSC-based
TPSC-basedICAR
ICARATRP
ATRP method.
method.
The
molecular
weight
increased
to
M
n,GPC
=
39,000
g/mol
from
M
n,GPC
=
6600
g/mol
after
chain
The molecular weight increased to Mn,GPC = 39,000 g/mol from Mn,GPC = 6600 g/mol after chain
extension, while the molecular weight distribution kept relatively narrow (Mw/Mn = 1.36) (Figure 5).
The successful chain extension further demonstrated the “living” character of this novel ATRP
catalyst separation and recycling system.
Polymers 2018, 10, 347
8 of 12
extension, while the molecular weight distribution kept relatively narrow (Mw /Mn = 1.36) (Figure 5).
The successful chain extension further demonstrated the “living” character of this novel ATRP catalyst
separation
and recycling system.
Polymers 2018, 10, x FOR AUTHORS PROOFREADING
8 of 12
Polymers 2018, 10, x FOR AUTHORS PROOFREADING
ee
O
O
H22
aa H
C
C
C
C
H
O
H33C
C
O
dd
aa
aa CH
a
CH33 a
H
H
H22
H22
C
C nC
C
C C
n
O
C
O C
O
O b
b
CH
CH33
d
7.4
7.4
1.00
1.00
8 of 12
aa
CH
CH33
C
C Br
Br
C
C O
O
O
O c
c
CH
CH3
bb
H
H22O
O
3
e
4.2
4.2
7.2
7.2
4.0
4.0
DMSO
DMSO
aa
cc
A
A
A
A
90.88
90.88
8
7
6
5
4
3
2
Chemical shifts (ppm)
1
0
Figure
4. 111HHNMR
NMR spectrumofofthe
theresultant
resultant PMMA(M
(Mn,GPC
= 4250 g/mol,
Mw /Mn =via
1.22) via
Figure
Figure 4.
4. H NMR spectrum
spectrum of
resultant PMMA
PMMA (Mn,GPC
n,GPC =
= 4250
4250 g/mol,
g/mol, M
Mww/M
/Mnn == 1.22)
1.22) via TPSCTPSCTPSC-based ICAR ATRP of MMA using DMSO-d6 as the solvent and TMS as the internal standard.
based
based ICAR
ICAR ATRP
ATRP of
of MMA
MMA using
using DMSO-d
DMSO-d66 as
as the solvent
solvent and
and TMS
TMS as
as the
the internal
internal standard.
standard.
permeation
chromatograph
(GPC)
traces
of
and
chain
extension
using
Figure 5.
Gel
permeation
chromatograph
(GPC)
traces
of before
and after
extension
5. Gel
Gel
permeation
chromatograph
(GPC)
traces
of before
before
and after
after
chain chain
extension
using
PMMA
(obtained
by
TPSC-based
ICAR
ATRP
of
MMA)
as
the
macroinitiator.
Original
using
(obtained
by TPSC-based
ICAR ofATRP
of as
MMA)
as the macroinitiator.
PMMA PMMA
(obtained
by TPSC-based
ICAR ATRP
MMA)
the macroinitiator.
Original
PMMA:[MMA]
00:[EBPA]
22]
00 =
== 1.0
3.0
mL
Original
PMMA:[MMA]
]0 :[TPMA]
:[AIBN]0 = V
200:1:1:1:0.8,
Vbenzene
PMMA:[MMA]
:[EBPA]00:[CuBr
:[CuBr
]00:[TPMA]
:[TPMA]
:[AIBN]
= 0200:1:1:1:0.8,
200:1:1:1:0.8,
VMMA
MMA
1.0 mL,
mL, V
V
benzene
= 1.0
3.0 mL,
mL
0 :[EBPA]
0 :[CuBr002:[AIBN]
MMA =
◦
and
m
mg,
temperature
70
PMMA:[MMA]
22]
V
= 3.0
and m==PIL
= Chain-extended
200.0 mg, temperature
=00:[PMMA]
70 C.00:[CuBr
Chain-extended
and
mPIL
PIL =
= 200.0
200.0
mg,mL
temperature
70 °C.
°C.
Chain-extended
PMMA:[MMA]
:[PMMA]
:[CuBr
]00:[TPMA]
:[TPMA]00::
benzene
[AIBN]
MMA
mL,
3.0
PIL
PMMA:[MMA]
=m
V MMA === 70
1.0°C.
[AIBN]00 == 200:0.05:1:1:0.8,
200:0.05:1:1:0.8,
V
MMA =
= 1.0
1.0
mL, V
Vbenzene
benzene
= [AIBN]
3.0 mL
mL 0and
and
m200:0.05:1:1:0.8,
PIL =
= 200
200 mg,
mg, temperature
temperature
70
°C.mL,
0 :[PMMA]V
0 :[CuBr
2 ]0 :[TPMA]
0: =
V benzene = 3.0 mL and mPIL = 200 mg, temperature = 70 ◦ C.
3.4. Catalyst Recycling and Reuse
3.4. Catalyst
Recycling andTPSC-based
Reuse
In the constructed
ICAR ATRP, the recycling efficiency is the most important
parameter.
weTPSC-based
conducted the
following
the recovered
PIL phase.
The
In the Therefore,
constructed
ICAR
ATRP, experiments
the recyclingusing
efficiency
is the most
important
monomer (MMA),
ligand
reducing
agent (AIBN),
and organic
solvent
(benzene) needed
for
parameter.
Therefore,
we(TPMA),
conducted
the following
experiments
using
the recovered
PIL phase.
the
typical
MMA
polymerization
were
respectively
added
to
the
recovered
PIL
phase
and
carried
out
The monomer (MMA), ligand (TPMA), reducing agent (AIBN), and organic solvent (benzene) needed
the the
subsequent
recovered
polymerization.
When the polymerization
was completed,
partcarried
of the
for
typical MMA
polymerization
were respectively
added to the recovered
PIL phasea and
polymer
phase
was
taken
out
to
have
an
inductively
coupled
plasma
(ICP)
test
to
determine
out the subsequent recovered polymerization. When the polymerization was completed, a partthe
of
amount
of transition
metal
in an
theinductively
polymer solution.
recycling
experiments
and
the
polymer
phase was
takenremaining
out to have
coupled The
plasma
(ICP) test
to determine
corresponding
results aremetal
shown
in Table 4.
can
be seensolution.
that afterThe
fiverecycling
recovery experiments
experiments,and
the
the
amount of transition
remaining
in Itthe
polymer
catalysis efficiency of the catalyst was still maintained at a high level, and the resulting polymers kept
narrow molecular weight distributions. Importantly, the residual Cu catalyst in polymer solution
phase was less than 2.2 ppm in every recycling experiment. That is to say even after five recycling
polymerizations the catalyst recycling efficiency still remained high (more than 94%, as is shown in
Figure 6). These results was much better than that (less than 90%) reported by our previous similar
Polymers 2018, 10, 347
9 of 12
corresponding results are shown in Table 4. It can be seen that after five recovery experiments, the
catalysis efficiency of the catalyst was still maintained at a high level, and the resulting polymers kept
narrow molecular weight distributions. Importantly, the residual Cu catalyst in polymer solution
phase was less than 2.2 ppm in every recycling experiment. That is to say even after five recycling
polymerizations the catalyst recycling efficiency still remained high (more than 94%, as is shown in
Figure 6). These results was much better than that (less than 90%) reported by our previous similar
Polymers
2018, 10,
x FORATRP
AUTHORS
PROOFREADING biphasic system [27].
9 of 12
TPSC-based
ICAR
in p-xylene/PEG-200
Table
4. Polymerization
Table 4.
Polymerization results
results of PIL recycling
recycling via TPSC-based ICAR ATRP a..
R e c y c lin g E f f ic ie n c y ( % )
Recycling
Conv.
Mn,th b
Mn,GPC
CR c
b
EntryRecycling
Mw/Mn
M
M
n,GPC
n,th
Times Conv.(%)
(g/mol)
(g/mol)
(%)
Entry
M w /M n (ppm)
CR c (ppm)
Times
(g/mol)
(g/mol)
1
1
59.8
11,980
18,500
1.12
1.3
1
1
59.8
11,980
18,500
1.12
2
2
55.7
11,200
15,600
1.09
1.5 1.3
2
2
55.7
11,200
15,600
1.09
3
3
60.2
12,100
18,100
1.12
2.2 1.5
3
3
60.2
12,100
18,100
1.12
4
4
52.4
10,500
18,900
1.10
2.0 2.2
4
4
52.4
10,500
18,900
1.10
2.0
5
5
52.3
13,000
12,200
1.07
1.7
5
5
52.3
13,000
12,200
1.07
1.7
a Polymerization conditions: [MMA]0:[EBPA]0:[CuBr2]0:[TPMA]0:[AIBN]0 = 200:1:1:3:0.8, VMMA = 1.0
a Polymerization conditions: [MMA] :[EBPA] :[CuBr ] :[TPMA] :[AIBN] = 200:1:1:3:0.8, V
= 1.0 mL,
MMA
0
0
2 0
0
0
b Mn,th =
mL,
Vbenzene
3.0mmL,= m
PIL = 200 mg for the first run, t = 4.5 h, and temperature
70 °C;
V benzene
= 3.0 =mL,
200
mg for the first run, t = 4.5 h, and temperature = 70 ◦ C; b Mn,th =
= ([M]
PIL
0 /[initiator]0 )
c
c CRresidual
× M0w,MMA
× conv.%;
means
the catalyst
polymer
solution
phase
which
was
([M]
/[initiator]
0) × MCR
w,MMA
× conv.%;
meansinthe
catalyst
residual
in(benzene
polymerphase),
solution
phase
determined
by
inductively
coupled
plasma (ICP).
(benzene phase), which was
determined
by inductively coupled plasma (ICP).
100
80
60
1
2
3
4
5
R e c y c lin g T im e s
Figure 6. Recycling efficiency as a function of recycling times. Polymerization conditions:
Figure 6. Recycling efficiency as a function of recycling times. Polymerization conditions:
[MMA]0/[EBPA]0/[CuBr2]0/[TPMA]0/[AIBN]0 = 200:1:1:3:1, VMMA = 1.0 mL, Vbenzene = 3.0 mL, mPIL = 200
[MMA]0 /[EBPA]0 /[CuBr2 ]0 /[TPMA]0 /[AIBN]0 = 200:1:1:3:1, V MMA = 1.0 mL, V benzene = 3.0 mL,
mg, t = 4.5 h, and temperature = 70 °C. Recycling
efficiency was calculated by the percentage of
mPIL = 200 mg, t = 4.5 h, and temperature = 70 ◦ C. Recycling efficiency was calculated by the percentage
residual copper catalyst in poly(ionic liquid) phase to the original copper catalyst, which was
of residual copper catalyst in poly(ionic liquid) phase to the original copper catalyst, which was
determined by ICP.
determined by ICP.
4. Conclusions
4. Conclusions
A novel TPSC-based ICAR ATRP system with high catalyst recycling efficiency was successfully
A novel TPSC-based ICAR ATRP system with high catalyst recycling efficiency was successfully
constructed via a thermoregulated PIL/benzene as the solvent pair strategy. In this system, the Cu
constructed via a thermoregulated PIL/benzene as the solvent pair strategy. In this system, the Cu
catalyst is miscible with the monomer/polymer at the polymerization temperature (70 ◦°C); when
catalyst is miscible with the monomer/polymer at the polymerization temperature (70 C); when
cooled to room temperature, the ATRP catalyst dissolved in PIL was in situ separated from the
cooled to room temperature, the ATRP catalyst dissolved in PIL was in situ separated from the polymer
polymer organic solution (benzene solution) easily and recycled for the next polymerization facilely.
organic solution (benzene solution) easily and recycled for the next polymerization facilely. Therefore,
Therefore, this strategy can avoid the conventional tedious post-treatment steps of recycling catalyst,
this strategy can avoid the conventional tedious post-treatment steps of recycling catalyst, which will
which will be much beneficial for the industrial process of ATRP.
be much beneficial for the industrial process of ATRP.
Acknowledgments:
Acknowledgments: The
The financial
financial support
support from
from the
the National
National Natural
Natural Science
Science Foundation
Foundationof
of China
China(Grant
(GrantNos.
Nos.
21174096,
21774082),
the
Natural
Science
Foundation
of
Jiangsu
Province
for
Youth
(No.BK20170321),
21174096, 21774082), the Natural Science Foundation of Jiangsu Province for Youth (No.BK20170321), the
the Key
Key
Technology
SupportProgram
Programof of
Changzhou
Industry
(No.CE20160035)
andProject
the Project
by the
Technology Support
Changzhou
for for
Industry
(No.CE20160035)
and the
FundedFunded
by the Priority
Academic
Program Development
of Jiangsu Higher
Education
are gratefully
Priority
Academic
Program Development
of Jiangsu
HigherInstitutions
Education(PAPD)
Institutions
(PAPD) acknowledged.
are gratefully
acknowledged.
Author Contributions: Hongjuan Jiang, Lifen Zhang, and Xiulin Zhu designed the experiments. Lan Yao and
Bingjie Zhang performed the experiments and analyzed the data. Lan Yao, Hongjuan Jiang, and Lifen Zhang
wrote the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Polymers 2018, 10, 347
10 of 12
Author Contributions: Hongjuan Jiang, Lifen Zhang, and Xiulin Zhu designed the experiments. Lan Yao and
Bingjie Zhang performed the experiments and analyzed the data. Lan Yao, Hongjuan Jiang, and Lifen Zhang
wrote the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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© 2018 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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https://openalex.org/W2100004878
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https://europepmc.org/articles/pmc3602182?pdf=render
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English
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Airway epithelial cells initiate the allergen response through transglutaminase 2 by inducing IL-33 expression and a subsequent Th2 response
|
Respiratory research
| 2,013
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cc-by
| 6,407
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* Correspondence: dlee5522@snu.ac.kr
1Department of Biomedical Sciences, Laboratory of Immunology and Cancer
Biology, Seoul, Korea
2Interdisciplinary Program of Cancer Biology, Cancer Research Institute, Seoul,
Korea
Full list of author information is available at the end of the article Oh et al. Respiratory Research 2013, 14:35
http://respiratory-research.com/content/14/1/35 Oh et al. Respiratory Research 2013, 14:35
http://respiratory-research.com/content/14/1/35 Abstract Background: Transglutaminase 2 (TG2) is a post-translational protein-modifying enzyme that catalyzes the
transamidation reaction, producing crosslinked or polyaminated proteins. Increased TG2 expression and activity
have been reported in various inflammatory conditions, such as rheumatoid arthritis, inflammation-associated
pulmonary fibrosis, and autoimmune encephalitis. In particular, TG2 from epithelial cells is important during the
initial inflammatory response in the lung. In this study, we evaluated the role of TG2 in the pathogenesis of allergic
asthma, particularly whether TG2 affects initial activation signaling leading to Th2 differentiation against antigens. Methods: We induced allergic asthma by ovalbumin sensitization and intranasal challenge in wild-type (WT) BALB/c
and TG2-deficient mice. Broncheoalveolar lavage fluid cells and intracellular cytokine production were analyzed by
flow cytometry. Interleukin (IL)-33 and TG2 expression in lung epithelial cells was detected by confocal microscopy. Results: Airway responsiveness was attenuated in TG2-deficient mice compared to that in the WT control. In
addition, recruitment of eosinophils and Th2 and Th17 differentiation decreased in TG2-deficient mice. Treatment
with cysteamine, a transglutaminase inhibitor, also reduced airway hypersensitivity, inflammatory cell recruitment,
and T helper cell differentiation. TG2-deficient mice showed reduced IL-33 expression following induction of allergic
asthma compared to those in the WT control. Conclusions: We found that pulmonary epithelial cells damaged by allergens triggered TG2-mediated IL-33
expression leading to type 2 responses by recruiting both innate and adaptive arms of the immune system. Keywords: Epithelium, IL-33, Transglutaminase 2, Asthma, Animal models Keywords: Epithelium, IL-33, Transglutaminase 2, Asthma, Animal models chronic airway inflammatory disease [2]. T cells play an
important role during asthma pathogenesis, and T helper
type 2 (Th2) cell differentiation is important in initiating
and perpetuating events in asthma, particularly in experi-
mental models [1,2]. The role of innate inflammatory cells,
such as mast cells, basophils, and recently defined innate
lymphoid cells, has been suggested to provide a local cyto-
kine environment that induces Th2 differentiation [3]. In
addition, epithelial cells at the mucosal surfaces have been
accepted as integral components of innate and adaptive
immunity [4,5]. The important role of pulmonary epithe-
lial cells during lung infection has been documented [5-7],
and the critical role of epithelial cells in inflammatory Airway epithelial cells initiate the allergen
response through transglutaminase 2 by inducing
IL-33 expression and a subsequent Th2 response Keunhee Oh1,2,3, Myung Won Seo1, Ga Young Lee1, Ok-Jin Byoun1, Hye-Ryun Kang4, Sang-Heon Cho4
and Dong-Sup Lee1,2,3,5* RESEARCH Open Access © 2013 Oh 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. © 2013 Oh 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. Materials and methods
Mice BALB/c mice were obtained from the Jackson Laboratory
(Bar Harbor, ME). TG2−/−mice were backcrossed to BALB/c
mice for 12 generations (N12). Female, 8-12-week-old mice
were used for experiments. All animal experiments were
performed with the approval of the Institutional Animal
Care and Use Committee at Seoul National University
(authorization no. SNU05050203). Histopathology and immunofluorescence of lung tissue Histopathology and immunofluorescence of lung tissue
Lung tissues were fixed in 4% paraformaldehyde, pro-
cessed, and embedded in paraffin. Sections were stained
with H&E for histopathological analysis. To investigate
IL-33 expression in epithelial cells, lung tissue from
airway hypersensitivity-induced mice were stained with
anti-IL-33 (R&D Systems, Minneapolis, MN), anti-TG2
(Neomarker,
Fremont,
CA),
and
anti-pro-surfactant
protein-C (pro-SP-C, Millipore, Billerica, MA). Alexa 488-
conjugated donkey anti-goat IgG and Alexa 546-conjugated
anti-rabbit IgG antibodies (Invitrogen, Carlsbad, CA) were
used for visualization. Image acquisition and processing
was performing using a confocal fluorescence microscope
(Olympus, Center Valley, PA) and FV10-ASW 2.0 Viewer
(Olympus). In this study, we investigated the role of TG2 in the
pathogenesis of allergic asthma, particularly whether TG2
affects initial activation signaling by inducing IL-33 and
downstream molecules leading to Th2 differentiation
against antigens. Allergic asthma was induced by ovalbu-
min (OVA) sensitization and intranasal challenge. We
found that airway hypersensitivity was attenuated in TG2-
deficient mice compared to that in wild-type (WT) con-
trols and recruitment of eosinophils and Th2 and Th17
differentiation was decreased in TG2-deficient mice. Treat-
ment with cysteamine, a transglutaminase inhibitor, also
reduced airway hypersensitivity, inflammatory cell recruit-
ment, and T helper cell differentiation. TG2-deficient mice
revealed decreased IL-33 expression following the induc-
tion of allergic asthma compared to that in the WT Immunization Mice
were
intraperitoneally
administered
phosphate
buffered saline (PBS) containing OVA (grade V, Sigma-
Aldrich, St Louis, MO) and aluminum hydroxide (alum)
(Sigma-Aldrich) (20 μg OVA + 2 mg alum) two times at
a 7-day interval. Intranasal OVA challenge (50 μg) was
performed for 3 consecutive days starting on days 14
and 21 after the first immunization. Mice were injected
intraperitoneally with cysteamine (40 mg/kg/day, Sigma-
Aldrich) to inhibit TG2 activity. Background Allergic asthma is characterized by airway hyperreactivity,
mucus hypersecretion, eosinophilic infiltration, and ele-
vated serum IgE levels [1]. Although the early proposed
role of abnormal airway smooth muscles has not been
clearly defined, infiltration of inflammatory cells such as
eosinophils, macrophages, and lymphocytes in the airways
of patients with asthma and the efficacy of corticosteroids
in the majority of patients indicate that asthma is a Oh et al. Respiratory Research 2013, 14:35
http://respiratory-research.com/content/14/1/35 Page 2 of 9 amplification following non-infectious damage has re-
cently been reported by our group [8]. control. Thus, we provide evidence that TG2 in pulmonary
epithelial cells initiates allergic responses by inducing the
IL-33-Th2 signaling pathways. Transglutaminase 2 (TG2) is a post-translational pro-
tein-modifying enzyme that catalyzes the transamidation
reaction, producing crosslinked or polyaminated proteins. TG2 is expressed ubiquitously in various cellular compart-
ments and participates in many biological processes, in-
cluding extracellular matrix formation, wound healing,
apoptosis, and differentiation [9,10]. TG2 has also been
implicated in many disease processes. Increased TG2 ex-
pression and activity have been reported in various inflam-
matory conditions, such as rheumatoid arthritis, gouty
arthritis, and organ fibrosis [11-13]. Altered forms of
proteins modified by TG2 enzymatic activity have been
suggested in the pathogenesis of various diseases, such
as celiac disease, cataracts, and Huntington’s disease
[9,10,14-16]. Controversies exist regarding the pathological
and protective roles of TG2 during inflammation, TG2
sustains inflammation through the release of inflammatory
cytokines while minimizing inflammation by increasing the
clearance of apoptotic cells [12,17]. Recent disease animal
models using TG2-deficient mice have revealed the im-
portant role of TG2 during the pathogenesis of bacterial
sepsis, inflammation-associated pulmonary fibrosis, and
autoimmune encephalitis [8,18,19]. TG2 is important du-
ring the initial inflammatory response. Specifically, TG2 in-
duces
nuclear
factor-κB-dependent
interleukin
(IL)-6
secretion from lung epithelial cells, leading to Th17 diffe-
rentiation in the lung [8]. Epithelial cell-derived signaling
mediators, such as IL-33, thymic stromal lymphopoietin
(TSLP), and IL-25, initiate Th2 immune responses and
each can direct the Th2 response either alone or through
downstream mediators [4]. Among these, IL-33 has been
implicated as the most upstream mediator of epithelial
cytokines [4]. Since epithelial TG2 can initiate the in-
flammatory response of non-infectious tissue damage [8],
we assumed the TG2 may also play an important role in initi-
ating and perpetuating the epithelial inflammatory response
leading to Th2 differentiation. Airway responsiveness
Ai
i Airway responsiveness was assessed as a change in airway
function after challenge with aerosolized methacholine
(Sigma-Aldrich) via the airways. Mice progressively in-
haled 6.25–50 mg/ml methacholine for 5 min at 24 h
after the last OVA intranasal challenge. Airway respon-
siveness was measured using the OCP3000 instrument
(One Chamber Plethysmography for animals; Allmedicus,
Anyang, Gyeonggi-do, Korea). Analysis of broncheoalveolar lavage fluid (BALF) Broncheoalveolar lavage was performed with five 1.0-mL
aliquots of PBS through a tracheal cannula. Cytospin Oh et al. Respiratory Research 2013, 14:35
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http://respiratory-research.com/content/14/1/35 times at a 7-day interval, and an intranasal OVA challenge
(50 μg OVA) for 3 consecutive days starting on days 14 and
21 to evaluate the role of TG2 during the pathogenesis of
allergic asthma. Airway hyperresponsiveness (AHR) was
assessed by methacholine challenge 1 day following the
last intranasal OVA challenge, and the whole body
method was used to measure enhanced pause. Broncho-
alveolar lavage (BAL) fluid and lung tissue were sampled
the next day (Figure 1A). TG2 expression from paren-
chymal lung tissue, especially from type II alveolar cells
increased in OVA-immunized and -challenged mice com-
pared to that in unmanipulated control mice (Figure 1B). The histopathological analysis of lung tissue following
disease induction revealed that TG2−/−mice had reduced
airway inflammation with decreased inflammatory cell in-
filtration surrounding the airways as compared with WT
mice (Figure 1C). Airway hyperresponsiveness also de-
creased in TG2 mice compared to that in WT mice
(Figure 1D). specimen was obtained and the cells were stained with
Wright-Giemsa. BALF were analyzed for IL-13 and IL-17
and IL-4 and IFN-γ levels by sandwich ELISA. To evaluate
cytokine production, cells were restimulated with 50 ng/ml
PMA and 1 μg/ml ionomycin (Sigma-Aldrich) for 4 h. For
intracellular staining, Golgi plug (BD-Pharmingen, San
Diego, CA) was added during the final 2 h of stimulation. Cells were labeled with anti-CD4, anti-IL-4, anti-IL-13,
anti-IL-17, and anti-IFN-γ antibodies (eBioscience, San
Jose, CA). Intracellular cytokine levels were analyzed
using a FACSCalibur (BD Biosciences, San Jose, CA) and
FlowJo software (Tree Star, Ashland, OR). Statistical analysis Statistical significance was analyzed using the Student’s
t-test. A p value of < 0.05 was taken to indicate statis-
tical significance. Results Airway inflammation was attenuated in TG2-deficient mice
WT BALB/c and TG2-deficient mice with a BALB/c
background (N12) (TG2−/−) were intraperitoneally admi-
nistered OVA and alum (20 μg OVA + 2 mg alum) two Recruitment of eosinophils is reduced in TG2-deficient mice
OVA-sensitized and –challenged WT mice showed in-
creased numbers of inflammatory cells in BAL fluid Figure 1 Transglutaminase 2 (TG2) deficiency attenuates airway hypersensitivity. (A) Wild-type (WT) and TG2 deficient (TG2−/−) mice were
immunized with ovalbumin (OVA) plus aluminum hydroxide (alum) and intranasally challenged with OVA. (B) Immunohistochemistry of TG2
expression in the lungs from WT mice. Sections were prepared 24 h after intranasal OVA challenge (original magnification, ×400). (C)
Representative photographs of lungs from WT and TG2−/−mice after sensitization and challenge with PBS or OVA. Sections were stained with
hematoxylin and eosin (original magnification, ×100). (D) Airway responsiveness was measured in sensitized and challenged mice. Pause was
recorded for 3 min after aerosol methacholine treatment. The results for each group are expressed as means ± standard errors (n = 5). Figure 1 Transglutaminase 2 (TG2) deficiency attenuates airway hypersensitivity. (A) Wild-type (WT) and TG2 deficient (TG2−/−) mice were
immunized with ovalbumin (OVA) plus aluminum hydroxide (alum) and intranasally challenged with OVA. (B) Immunohistochemistry of TG2
expression in the lungs from WT mice. Sections were prepared 24 h after intranasal OVA challenge (original magnification, ×400). (C)
Representative photographs of lungs from WT and TG2−/−mice after sensitization and challenge with PBS or OVA. Sections were stained with
hematoxylin and eosin (original magnification, ×100). (D) Airway responsiveness was measured in sensitized and challenged mice. Pause was
recorded for 3 min after aerosol methacholine treatment. The results for each group are expressed as means ± standard errors (n = 5). Oh et al. Respiratory Research 2013, 14:35
http://respiratory-research.com/content/14/1/35 Page 4 of 9 whereas interferon (IFN)-γ increased, in TG2−/−mice
compared to WT mice (Figure 3B). compared to those in the PBS-treated control mice. In
addition, TG2−/−mice showed decreased inflammatory
cell infiltration compared to that in WT mice (Figure 2A). Cytospin analysis with Wright–Giemsa stain revealed that
TG2−/−mice showed a dramatic decrease in eosinophils,
but not macrophages or lymphocytes, in BAL fluid. Thus,
the reduction in infiltrating inflammatory cells in these
mice reflected selective reduction of eosinophils (Figure 2B
and 2C). Results We also confirmed a selective decrease of eosin-
ophils in the TG2−/−mice as compared with WT mice
using flow cytometric analysis of BAL fluid (Figure 2D). Cysteamine treatment reduced recruitment of airway
inflammatory cells We intraperitoneally injected cysteamine twice daily
(40 mg/kg) from the day of the first intraperitoneal OVA
sensitization to evaluate the effect of the transglutaminase
pharmacological inhibitor on the pathogenesis of allergic
asthma. Cysteamine treatment decreased airway inflam-
mation compared to that in the PBS-treated control
(Figure 4A). The BAL fluid analysis revealed that cyste-
amine greatly reduced the number of infiltrating inflam-
matory
cells
compared
to
the
PBS-treated
control
(Figure 4B). The decrease in inflammatory cells was not
confined to eosinophils, as cysteamine treatment reduced
all components of innate and adaptive cells recruited to Reduced Th2/Th17 differentiation in TG2-deficient mice Reduced Th2/Th17 differentiation in TG2-deficient mice
Intracellular cytokine analysis of BAL fluid cells from OVA
exposed mice revealed that TG2−/−mice showed decreased
amounts of IL-4- and/or IL-13-secreting CD4+ T cells
compared to WT mice (Figure 3A). IL-4, IL-13, IL-17, and
OVA-specific-IgE
levels
in
the
BAL
fluid
decreased, Figure 2 Transglutaminase 2 (TG2) deficiency reduces eosinophil recruitment. (A–C) Bronchoalveolar lavage (BAL) fluid from WT and TG2
−/−mice was obtained 48 h after the last OVA challenge. BAL cells were counted and analyzed by Wright-Giemsa staining. Total (A) and
differential cell counts (B) were performed. (C) Representative photographs of BAL cells. Cells were stained with Wright–Giemsa (original
magnification, ×1000). Arrows present eosinophils. (D) Inflammatory cells in BAL fluid from WT and TG2−/−mice were labeled with anti-CCR3 and
anti-Siglec-F antibodies and analyzed by flow cytometry. (A-B) Data are means ± standard deviation of three independent determinations with
BAL cells from n = 5 mice/group. Figure 2 Transglutaminase 2 (TG2) deficiency reduces eosinophil recruitment. (A–C) Bronchoalveolar lavage (BAL) fluid from WT and TG2
−/−mice was obtained 48 h after the last OVA challenge. BAL cells were counted and analyzed by Wright-Giemsa staining. Total (A) and
differential cell counts (B) were performed. (C) Representative photographs of BAL cells. Cells were stained with Wright–Giemsa (original
magnification, ×1000). Arrows present eosinophils. (D) Inflammatory cells in BAL fluid from WT and TG2−/−mice were labeled with anti-CCR3 and
anti-Siglec-F antibodies and analyzed by flow cytometry. (A-B) Data are means ± standard deviation of three independent determinations with
BAL cells from n = 5 mice/group. Oh et al. Respiratory Research 2013, 14:35
http://respiratory-research.com/content/14/1/35 Page 5 of 9 Figure 3 TG2 deficiency reduces Th2 and Th17 differentiation. (A) BAL cells were harvested 48 h after the last OVA challenge and
restimulated with phorbol myristic acid (PMA) and ionomycin for 4 h. Intracellular cytokine levels were analyzed by flow cytometry. (B) Levels of
interleukin (IL)-4, IL-13, IL-17, and interferon (IFN)-γ in BAL fluid were determined by ELISA. OVA-specific IgE in BAL fluid was also detected by
ELISA. Data are means ± standard deviations of three independent determinations with BAL cells from n = 5 mice/group. Figure 3 TG2 deficiency reduces Th2 and Th17 differentiation. (A) BAL cells were harvested 48 h after the last OVA challenge and
restimulated with phorbol myristic acid (PMA) and ionomycin for 4 h. Intracellular cytokine levels were analyzed by flow cytometry. Reduced Th2/Th17 differentiation in TG2-deficient mice (B) Levels of
interleukin (IL)-4, IL-13, IL-17, and interferon (IFN)-γ in BAL fluid were determined by ELISA. OVA-specific IgE in BAL fluid was also detected by
ELISA. Data are means ± standard deviations of three independent determinations with BAL cells from n = 5 mice/group. Figure 4 Cysteamine treatment reduces airway hypersensitivity. (A-C) WT mice were immunized with OVA plus alum and intranasally
challenged with OVA. Mice were injected intraperitoneally with cysteamine (40 mg/kg/day) to inhibit TG2 activity. (A) Representative photographs
of lungs from PBS- or cysteamine-treated mice. Sections are stained with hematoxylin and eosin (original magnification, ×200). (B, C) BAL fluid
was obtained 48 h after the last challenge with OVA. BAL cells were counted and analyzed by Wright–Giemsa staining. Total (B) and differential
cell counts (C) were performed. Data are means ± standard deviations of three independent determinations with BAL cells from
n = 5 mice/group. Figure 4 Cysteamine treatment reduces airway hypersensitivity. (A-C) WT mice were immunized with OVA plus alum and intranasally
challenged with OVA. Mice were injected intraperitoneally with cysteamine (40 mg/kg/day) to inhibit TG2 activity. (A) Representative photographs
of lungs from PBS- or cysteamine-treated mice. Sections are stained with hematoxylin and eosin (original magnification, ×200). (B, C) BAL fluid
was obtained 48 h after the last challenge with OVA. BAL cells were counted and analyzed by Wright–Giemsa staining. Total (B) and differential
cell counts (C) were performed. Data are means ± standard deviations of three independent determinations with BAL cells from
n = 5 mice/group. Page 6 of 9 Oh et al. Respiratory Research 2013, 14:35
http://respiratory-research.com/content/14/1/35 Oh et al. Respiratory Research 2013, 14:35
http://respiratory-research.com/content/14/1/35 the lung, including eosinophils, macrophages, and lym-
phocytes (Figure 4C). epithelial cytokine mediator leading to the Th2 pheno-
type [16]. PBS-treated WT mice revealed increased IL-33
expression in pulmonary epithelial cells following intra-
nasal OVA challenge, whereas CyM-treated mice showed
reduced IL-33 expression (Figure 5D). Cysteamine treatment reduced T helper cell
differentiation Cysteamine
(CyM)
treatment
reduced
all
cytokine-
secreting lymphocytes tested when we analyzed BAL
fluid infiltrating lymphocytes using intracellular cytokine
staining. Not only IL-4-, IL-13-, and IL-17-secreting
lymphocytes, but also IFN-γ-secreting lymphocytes de-
creased in percentage and number compared to those in
the PBS-treated control (Figure 5A and 5B). T helper
cell differentiation to all pathways tested also decreased
following cysteamine treatment when we gated CD4+
T cells (Figure 5A and 5C). We assessed IL-33 expression
from pulmonary epithelial cells following disease induc-
tion to evaluate the role of TG2 during the initiation of
Th2 differentiation and airway hyperresponsiveness, as
IL-33
has
been
implicated
as
the
most
upstream Reduced IL-33 expression in TG2-deficient mice
WT mice revealed increased IL-33 expression in pulmon-
ary epithelial cells, whereas TG2−/−mice showed reduced
IL-33 expression (Figure 6A). The kinetics of IL-33 ex-
pression also showed delayed and reduced IL-33 expres-
sion in TG2−/−mice compared to WT mice (Figure 6A). Reduced IL-33 expression in the TG2−/−mice was also re-
vealed by reverse transcription polymerase chain reaction
(RT-PCR) analysis of the lung. TG2−/−mice also showed re-
duced TSLP expression compared to WT mice (Figure 6B). Th2 responses occurred in the wild-type mice only fol-
lowing 4 times or more of intranasal OVA challenges, Figure 5 Cysteamine treatment reduces T helper cell differentiation. BAL cells were harvested 48 h after the last OVA challenge and
restimulated with phorbol myristic acid (PMA) and ionomycin for 4 h. Intracellular cytokine levels were analyzed by flow cytometry. (A) The
proportions of interleukin (IL)-4-, IL-13-, IL-17-, and interferon (IFN)-γ-producing cells are shown. (B) Number of cytokine-producing cells. (C)
Number of cytokine-producing CD4+ T cells. (D) Immunofluorescence staining of IL-33 (green) and pro-surfactant protein C (SPC) (red) in the
lungs from PBS or CyM-treated WT mice. Sections were prepared 24 h after the last ovalbumin (OVA) challenge (original magnification, ×1000). (A-C) Data are means ± standard deviations of three independent determinations with BAL cells from n = 5 mice/group. Figure 5 Cysteamine treatment reduces T helper cell differentiation. BAL cells were harvested 48 h after the last OVA challenge and
restimulated with phorbol myristic acid (PMA) and ionomycin for 4 h. Intracellular cytokine levels were analyzed by flow cytometry. (A) The
proportions of interleukin (IL)-4-, IL-13-, IL-17-, and interferon (IFN)-γ-producing cells are shown. (B) Number of cytokine-producing cells. (C)
Number of cytokine-producing CD4+ T cells. Cysteamine treatment reduced T helper cell
differentiation (D) Immunofluorescence staining of IL-33 (green) and pro-surfactant protein C (SPC) (red) in the
lungs from PBS or CyM-treated WT mice. Sections were prepared 24 h after the last ovalbumin (OVA) challenge (original magnification, ×1000). (A-C) Data are means ± standard deviations of three independent determinations with BAL cells from n = 5 mice/group. Oh et al. Respiratory Research 2013, 14:35
http://respiratory-research.com/content/14/1/35 Page 7 of 9 Figure 6 Reduced IL-33 in TG2−/−mice. (A) Immunofluorescence staining of IL-33 (green) and pro-SPC (red) in the lung from WT and TG2−/−
mice. Sections were prepared 24 h after OVA challenge. (original magnification, ×1000). (B) IL-33 mRNA expression in lung tissues of WT and
TG2−/−mice were determined by reverse transcription-polymerase chain reaction. (C) BAL cells were harvested 24 h after OVA challenge and
restimulated with PMA and ionomycin for 4 h. Intracellular cytokine levels were analyzed by flow cytometry. The proportions of IL-4- and
IL-13-producing cells are shown. Figure 6 Reduced IL-33 in TG2−/−mice. (A) Immunofluorescence staining of IL-33 (green) and pro-SPC (red) in the lung from WT and TG2−/−
mice. Sections were prepared 24 h after OVA challenge. (original magnification, ×1000). (B) IL-33 mRNA expression in lung tissues of WT and
TG2−/−mice were determined by reverse transcription-polymerase chain reaction. (C) BAL cells were harvested 24 h after OVA challenge and
restimulated with PMA and ionomycin for 4 h. Intracellular cytokine levels were analyzed by flow cytometry. The proportions of IL-4- and
IL-13-producing cells are shown. which indicated the Th2 responses developed when IL-
33 expression reached its peak level (Figure 6C). a broad-spectrum transglutaminase inhibitor, reduces pul-
monary inflammation and fibrosis following intratracheal
bleomycin instillation [8]. Cystamine, a dimeric form of
cysteamine, also ameliorates IgE-induced passive cutane-
ous anaphylaxis and phorbol myristic acid-induced atopic
dermatitis [20]. Sohn et al. [21] developed recombinant
peptides with dual inhibitory capacity against TG2 and
phospholipase
A2,
and
these
recombinant
peptides
reversed the inflammation of allergic conjunctivitis. These
authors also found that R2 peptide treatment ameliorates
inflammatory allergic asthma induced by OVA sensi-
tization and challenge [22]. A correlation between TG2
expression and disease progression has been suggested in
patients with toluene diisocyanate-induced occupational
asthma and exercise-induced bronchoconstriction [23,24]. Although an important role for TG2 in allergic asthma
pathogenesis has been suggested by these reports, all stud-
ies used rather nonspecific pharmacological inhibitors or
conducted relative expression studies. Cysteamine treatment reduced T helper cell
differentiation Therefore, the crit-
ical role of TG2 during asthma pathogenesis has not been
properly evaluated using genetically modified mice. In this Discussion In this study, we observed that TG2 plays an important
role in initiating the allergic Th2 response by inducing
IL-33 and downstream molecules leading to Th2 differ-
entiation following allergen sensitization and challenge. Airway hyperresponsiveness was attenuated in TG2-
deficient mice compared to WT control mice, and
recruitment of eosinophils and Th2 and Th17 differenti-
ation decreased in TG2-deficient mice. We confirmed
the role of TG2 in the pathogenesis of allergic asthma
using cysteamine, a transglutaminase inhibitor. TG2-
deficient mice revealed reduced IL-33 expression follow-
ing asthma induction. Thus, we provide evidence that
TG2 in pulmonary epithelial cells initiates allergic re-
sponses through the IL-33-Th2 signaling pathways. The roles of TG2 in the pathogenesis of organ-specific
and systemic inflammatory responses including hyper-
sensitivity reactions have been documented. Cysteamine, Oh et al. Respiratory Research 2013, 14:35
http://respiratory-research.com/content/14/1/35 Page 8 of 9 and pathological inflammation from both infectious and
non-infectious stimuli [34,35]. IL-33 and its ST2 recep-
tor are mainly expressed in bronchial epithelial cells in
the lung [25]. Thus, airway epithelial cells are also active
players in the pathogenesis of asthma through epithelial
cytokines including IL-33, TSLP, and IL-25, which are
produced and released by epithelial cells in response to
various exogenous stimuli or by cellular damage [36]. In
our previous report, epithelial TG2 played a critical role
in initiating the inflammatory response leading to the
type 17 response following bleomycin treatment [8]. respect, our results utilizing TG2-deficient mice show a
critical role for TG2 during the pathogenesis of experi-
mental allergic asthma and suggest that TG2 is a putative
novel disease target of allergic asthma. Asthma is a chronic inflammatory disease with charac-
teristics of type 2 cytokine production. IL-33 is a strong
inducer of the Th2 immune response, and its role in the
immunopathogenesis of allergic asthma had been docu-
mented. Higher IL-33 expression occurs in patients with
asthma and in murine models of asthma [25,26]. In-
creased airway hyper-responsiveness and inflammatory
cell infiltration is observed by either IL-33 administration
or
by
studies
with
IL-33
overexpressing
transgenic
[27,28]. IL-33-deficient mice reveal reduced inflammatory
cell recruitment to the lung and attenuated airway hyper-
responsiveness compared to that of a WT control [29]. In
addition, blocking ST2 signaling either by blocking anti-
bodies or by using a soluble receptor protein inhibits pul-
monary inflammation and airway hyperresponsiveness
[30]. Competing interests
h
h
h
d
l Competing interests
The authors have declared that no conflict of interest exists. p
g
The authors have declared that no conflict of interest exists. The authors have declared that no conflict of interest exists. Authors’ contributions KO designed the research, performed the experiments, interpreted the data
and wrote the manuscript; MWS, GYL, and OJB performed the experiments;
HRK and SHC interpreted the data and reviewed the manuscript; DSL
designed the research, interpreted the data, wrote and edited the
manuscript. All authors read and approved the final manuscript. Discussion IL-33 induces type 2 cytokine production through
the ST2 receptor expressed on multiple innate and adap-
tive immune cells, including type 2 innate lymphoid cells,
Th2 cells, mast cells, basophils, eosinophils, and natural
killer T cells [31]. Further, IL-33 stimulates the expres-
sion of TSLP and its receptor and, thus, indirectly in-
duces Th2 responses [4,32], which was also reduced in
TG2-deficient mice in our study. Data on the IL-33 in-
duction signaling pathway are incomplete, but a recent
report revealed a role for extracellular ATP in triggering
IL-33 release following airborne allergen exposure [33]. In our study, expression of IL-33 increased in the type
II pulmonary epithelial cells following intranasal OVA
treatment and increased further following additional in-
tranasal injection of OVA (Figure 6A). TG2 expression
on the pulmonary epithelial cells also increased by intra-
nasal OVA injection and further increased with additional
OVA challenge, which closely paralleled IL-33 expression
kinetics. Among the several suggested mechanisms of
TG2, transglutaminase enzyme activity may be involved in
the regulation of IL-33 expression in that inhibition of en-
zyme activity using CyM also decreased IL-33 expression
comparable to TG2 knockout mice (Figure 5D). In
addition, we found that Th2 responses occurred in the
wild-type mice only following 4 times or more of intrana-
sal OVA challenges in our experimental settings, which in-
dicated
the
Th2
responses
developed
when
IL-33
expression reached its peak level (Figure 6C). Thus we
suggest that TG2 and downstream IL-33 expression
seem to be important in the induction of Th2 response
and eosinophil recruitment. The important role of lung epithelial cells in initiating
pulmonary inflammation during allergic responses re- Author details
1 1Department of Biomedical Sciences, Laboratory of Immunology and Cancer
Biology, Seoul, Korea. 2Interdisciplinary Program of Cancer Biology, Cancer
Research Institute, Seoul, Korea. 3Transplantation Research Institute, Seoul,
Korea. 4Department of Internal Medicine, Seoul National University College of
Medicine, Seoul, Korea. 5Seoul National University College of Medicine, 103
Daehak-ro Jongno-gu, Seoul, Korea. Received: 4 December 2012 Accepted: 19 February 2013
Published: 13 March 2013 Received: 4 December 2012 Accepted: 19 February 2013
Published: 13 March 2013 Received: 4 December 2012 Accepted: 19 February 2013
Published: 13 March 2013 Acknowledgements This study was supported by grants from Korea Healthcare technology R&D
Project, Ministry for Health, Welfare & Family Affairs (D-S.L. No. A100190) and
Basic Science Research Program through the National Research Foundation
of Korea (NRF) funded by the Ministry of Education, Science and Technology
(K.O. No. 2012008122) Conclusion In this study, we found that pulmonary epithelial cells
damaged by allergens triggered TG2-mediated IL-33 ex-
pression leading to type 2 responses by recruiting both in-
nate and adaptive arms of the immune system. Thus,
epithelial TG2 was a common critical link transducing epi-
thelial tissue damage to initiate Th2 or Th17 inflammatory
responses depending on the context of the stimuli. Abbreviations Abbreviations
TG2: Transglutaminase; BALF: Bronchoalveolar lavage fluid. 3.
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Submit your manuscript at
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inflammatory effects of the R2 peptide, an inhibitor of transglutaminase
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and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
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Submit your manuscript at
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and take full advantage of: Submit your next manuscript to BioMed Central
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https://openalex.org/W4380741812
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https://ojs.wiserpub.com/index.php/RRCS/article/download/2637/1448
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English
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Enhancing Performance of Wide Area CIoT SDN by US-ML Based Optimum Controller Placement
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Research reports on computer science
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Received: 8 March 2023; Accepted: 28 March 2023 Abstract: It is a critical area of study for enhancing the effectiveness of wide-area Cellular Internet of Things (CIoT)
networks. One solution is to merge Software Defined Networking (SDN) with Internet of Things (IoT) network to boost
efficiency. The main challenge is determining the best location for the SDN controller and evaluating SDN clustering. This paper proposed an Un-Supervised Machine-Learning (US-ML) approach based on silhouette distance along with
gap statistic for finding the optimum number of controllers for network under consideration. In addition, the Partition
Around Medoids (PAM) approach is opted for allocation of controller locations. Apart from SDN, another approach
is to create efficient Low-Power Wide Area Networks (LPWAN). As a result, this research contributed to the study of
various LPWAN design approaches and offered a method of optimal controller location for IoT-SDN cellular networks
in industries. Several outstanding research challenges are noted, and prospective research objectives for LPWAN are
offered. For the case study of wide area networks (WAN), a graphical representation of the SDN controller positioning
method is presented. It is determined that effective placement can improve SDN performance in worst-case network
scenarios. Keywords: IoT, LPWAN, cellular network, SDN, clustering, positioning of controller, LoRaWAN Keywords: IoT, LPWAN, cellular network, SDN, clustering, positioning of controller, LoRaWAN Research Reports on Computer Science
https://ojs.wiserpub.com/index.php/RRCS/
Research Article in Special Issue: Selected Papers from the 4th International
Conference on Machine Learning, Image Processing, Network Security and Data
Sciences (MIND-2022) Research Reports on Computer Science
https://ojs.wiserpub.com/index.php/RRCS/
Research Article in Special Issue: Selected Papers from the 4th International
Conference on Machine Learning, Image Processing, Network Security and Data
Sciences (MIND-2022) Research Reports on Computer Science
https://ojs.wiserpub.com/index.php/RRCS/ Research Reports on Computer Science
https://ojs.wiserpub.com/index.php/RRCS/ Research Article in Special Issue: Selected Papers from the 4th International
Conference on Machine Learning, Image Processing, Network Security and Data
Sciences (MIND-2022) Research Article in Special Issue: Selected Papers from the 4th International
Conference on Machine Learning, Image Processing, Network Security and Data
Sciences (MIND-2022) Amrita Khera
*, Uma Shankar Kurmi Department of Electronics and Communication Engineering, LNCT University, Bhopal, India
E-mail: engg.ams@gmail.com Copyright ©2023 Amrita Khera, et al.
DOI: https://doi.org/10.37256/rrcs.2320232637
This is an open-access article distributed under a CC BY license
(Creative Commons Attribution 4.0 International License)
https://creativecommons.org/licenses/by/4.0/ 1. Introduction Sensing devices are deployed in the Internet of Things (IoT) network in a widespread range. The requirement
of improving the performance of cellular IoT-based wide area networks (WANs) had grown dramatically in recent
years. This challenge can be met by combining Low-Power WAN (LPWAN) and Cellular IoT (CIoT) networks [1]. Another strategy is to implement a hybrid combination of Software Defined Networking (SDN) and IoT networks
[2], for improving the system effectiveness. The SDN-IoT network has recently become popular in industrial uses
for control engineering and modeling of autonomous management. This paper proposed to test the Un-Supervised
Machine-Learning (US-ML) based SDN controller’s allocation problem. Figure 1 depicts the most common uses of
SDN-IoT. The SDN is increasingly being employed for sensors interface and constrained monitoring of IoT for WAN
implementations. These networks deployment has recently become popular in the industrial uses for control engineering 112 | Amrita Khera, et al. 112 | Amrita Khera, et al. Research Reports on Computer Science and modeling of autonomous management. The network applications as mentioned in Figure 1 include production
control, smart cities, automation of smart homes, inventory management, etc. Each of these applications needs to
communicate a huge amount of information. Applications of SDN-IoT
network
Smart grids
Production
Smart cities
Inventory
management
Manufacturing
Smart homes
Figure 1. Applications of SDN-IoT in industries Smart cities Applications of SDN-IoT
network Production Figure 1. Applications of SDN-IoT in industries 2. Classifications of WAN protocols The broad classification of the WAN performance enhancement methodologies is given in Figure 2. The LoRaWAN
as well as Narrowband IoT (NB-IoT) seem to be the most attractive long range communication techniques worldwide,
and they are generating a vast IoT networks. LPWAN
WAN designs
SDN-IoT
NB-IoT
LoRaWAN
SigFox
Figure 2. IoT-based WAN technology classification WAN designs Figure 2. IoT-based WAN technology classification The energy efficiency, longevity, quality of service (QoS) and long range coverage of these networks are all
important variables to consider when evaluating their WAN performance. The 3rd Generation Partnership Project (3GPP)
created the NB-IoT idea for cellular networks. NB-IoT offers the features of a fourth generation (4G) mobile network,
such as worldwide coverage and long range, also with energy efficiency using LPWANs to enhance energy savings. NB-IoT is also intended to improve interior coverage and enable a variety of low devices [3, 4]. It’s designed to cater
to the high-value IoT sector, which values low latency and great service quality [3-6]. LoRaWAN, on the other hand, is
aimed for lower-cost devices with long range (high coverage), intermittent communication needs, and long battery life. LoRaWAN and SigFox are examples of LPWAN technologies that use upgraded physical layer (PHY) technologies for
achieving long range. Narrowband (NB) cellular networks, such as NB-IoT and Long-Term Evolution in the Machines
(LTE-M) provide decreased capacity and simpler node and networking management measures. 1.1 Contributions The main design problems are appropriate SDN controller location and adoption of low-energy devices for
LPWAN applications. It has been discovered that better controller arrangement can reduce the communication distance
between sensor nodes, resulting in improved performance. This paper will offer a case study of these two difficulties. The effectiveness of several LPWAN technologies is initially compared and assessed. The validation results of the US-
ML based on method of silhouette and gap statistics optimal allocation of controller location for enhancing the WAN
performance consideration are then shown, depending on latencies and cluster selection. Special Issue|2023| 113 Research Reports on Computer Science Research Reports on Computer Science 4. Related works A great deal of effort has gone into improving cellular IoT networks. Adopting the SDN-IoT framework is one
option. The segregation of the data and control planes in the SDN paradigm has been the technical platform, where
network nodes in the data plane conduct forwarding activities under the administration and control of SDN controllers
within the control plane. As a result, many SDN-enabled devices used in modern IoT networks might provide variable
network architectures at a cheap capital cost. This section provides a comprehensive review for LPWAN as well as
SDN-based techniques. A summary of the review work is given in Table 1. Table 1. Summary of the clustering based routing protocols for WAN
Authors
Methodology
Approach
Onumanyi et. al. [1]
Based on an universal network
infrastructure as well as a PHY layer
concept. Cognitive radio (CR) solutions in an LPWAN network must be instal
the gateway (GW) rather than the LPWAN end nodes. Sebastian et al. [2]
The unlicensed LPWAN systems
performed well at distances of up to
10 km, with SigFox having the highest
coverage in the LPWAN section. A single testbed for measuring and comparing the performance of LP
as well as NB-IoT devices. Ballerini et al. [3]
Evolves the effectiveness of LoRaWAN
as well as NB-IoT accurate field
measurements in the same application
system for fair energy efficiency
evaluation. Based on experimental information gathered at a LoRa, a wireless po
sensor was created to assess fractures in reinforced concrete construc
Nair et al. [4]
It is dependent on the technology,
which was examined using several key
performance indicators (KPIs). Addresses the huge scale of IoT installations as well as other essentia
like low power, low cost, extensive coverage, and long battery life. Jiang [5]
Introduces the LoRa and NB-IoT testbeds
that were utilized in the studies, as
well as the technique for evaluating the
performance benefit of protocols. The purpose of this study is to compare the effectiveness of LoRa and
IoT in respect of connectivity quality. Ugwuanyi et al. [6]
The effectiveness of LoRaWAN as well
as NB-IoT employing one Evolved Node
B (eNB) and two user equipment (UEs)
was examined. Aims to provide long-term potential for IoT growth in underdevelope
nations by comparing LoRaWAN with NB-IoT in regard to power
consumption, privacy, latency, and throughput. Maurya et al. [7]
Have designed a multiple-input multiple-
output (MIMO)-cognitive relaying
network. 3. Comparison of LPWAN methods This section of the paper evaluates the NB-IoT, SigFox and LoRa approaches of LPWAN implementation [7-
10]. The comparative evaluation is based on a survey of the literature for coverage and energy. Figure 3 depicted an
overview of the performance evaluation of LPWAN techniques. The figure clearly illustrates that NB-IoT outperforms
overall in regard to coverage and energy performance. Over existing network topologies, NB-IoT offers a longer life of batteries and much more flexible energy
management, with an operational lifespan of far more than 15 years. This is beneficial for small-footprint gadgets such
as smart watches. Although LoRa has optimal maximum range up to 20 km, it is extremely challenging to adjust and
install. 114 | Amrita Khera, et al. Research Reports on Computer Science Coverage in
dB
Maximum
range in km
Frequency
up to in GHz
Battery life
in years
Implementation
cost in $
NB-IoT
Sigfox
LoRa
20
40
60
80
100
120
140
160
180
0
Figure 3. Performance summaries of LPWAN technologies Coverage in
dB
Maximum
range in km
Frequency
up to in GHz
Battery life
in years
Implementation
cost in $
NB-IoT
Sigfox
LoRa
20
40
60
80
100
120
140
160
180
0
Figure 3. Performance summaries of LPWAN technologies Figure 3. Performance summaries of LPWAN technologies 4.1 Review of SDN control Mobile devices are used to build WAN networks. As a result of deploying SDN in combination with an IoT
network, Song et al. [14] advocated software-defined (SD)-IoT networks. Their study concentrated on recognizing
spatial events and splitting challenges among several flow paths. Kobo et al. [15] created a novel SD wireless network
election and effective controller placements. It allows to quickly and easily spread-ing SD network communication
control over your IoT network. Rahman et al. [16] used SDN-IoT during the COVID-19 epidemic to offer robust data
connection on the surfaces and devices required for Industry 4.0. Li et al. [17], Tran et al. [11] and Hans et al. [12]
offered different techniques for SDN controller placement to optimize performance. Similar literature is presented in [13,
18, 19]. 4. Related works Designed an energy-efficient cognitive radio network with a relay pre Table 1. Summary of the clustering based routing protocols for WAN Table 1. Summary of the clustering based routing protocols for WAN A single testbed for measuring and comparing the performance of LPWAN
as well as NB-IoT devices. Based on experimental information gathered at a LoRa, a wireless power
sensor was created to assess fractures in reinforced concrete constructions. Addresses the huge scale of IoT installations as well as other essential needs
like low power, low cost, extensive coverage, and long battery life. The purpose of this study is to compare the effectiveness of LoRa and NB-
IoT in respect of connectivity quality. Aims to provide long-term potential for IoT growth in underdeveloped
nations by comparing LoRaWAN with NB-IoT in regard to power
consumption, privacy, latency, and throughput. Designed an energy-efficient cognitive radio network with a relay precoder. Special Issue|2023| 115 Research Reports on Computer Science Table 1. Continued Table 1. Continued
Authors
Methodology
Approach
Ismail et al. [8]
LPWANs are a cutting-edge technology
that allows applications. Identifies the important LPWAN prospects and suggests future research
topics for LPWAN. Chen et al. [9]
Wireless communication as well as
heterogeneous devices, such as smart
buildings and greener IoT artificial
intelligence (AI) applications for smart
homes, health monitoring, self-driving,
and emotional engagement. Concentrate on modern wireless communication technology, notably
cellular communications. Tran et al. [11]
SDN controller positioning. For controller placement, this paper used the optimized submodular
dependent approach. Hans et al. [12]
SDN controllers with optimization
algorithm placement. Developed strategy for improving SDN-IoT network applications. Jain et al. [13]
Presented deep learning based algorithm. This method is presented for wearable devices. Identifies the important LPWAN prospects and suggests future research
topics for LPWAN. Identifies the important LPWAN prospects and suggests future research
topics for LPWAN. Concentrate on modern wireless communication technology, notably
cellular communications. Developed strategy for improving SDN-IoT network applications. 5. Optimum allocation of controller in SDN network Perform clustering: Find the minimum sum for all clusters
7. If N medoids < K
Update medoids
repeat 5
end if
8. Calculate worst case and average latencies ← O, T, V, P, Lavg, Lworst1,2,3,4
9. Finally, find optimum controller location using the silhouette method. And gap statistic. Algorithm 1. The proposed PAM based clustering Algorithm 1. The proposed PAM based clustering
The proposed PAM based clustering
1. Input data set D and, the number of medoids K = 4
2. Determine the weight matrix based on ← distance of edges K m
3. Calculate the shortest distance matrix using Johnson’s algorithm
4. Find the minimum sum of distances and select initial medoids
5. If N medoids < K
repeat 2, 3
end if
6. Perform clustering: Find the minimum sum for all clusters
7. If N medoids < K
Update medoids
repeat 5
end if
8. Calculate worst case and average latencies ← O, T, V, P, Lavg, Lworst1,2,3,4
9. Finally, find optimum controller location using the silhouette method. And gap statistic. 5. If N medoids < K
repeat 2, 3
end if end if
6. Perform clustering: Find the minimum sum for all clusters
7
If N
d id < K 7. If N medoids < K
Update medoids
repeat 5
end if end if
8. Calculate worst case and average latencies ← O, T, V, P, Lavg, Lworst1,2,3,4
9. Finally, find optimum controller location using the silhouette method. And gap statistic. Research Reports on Computer Science 5. Optimum allocation of controller in SDN network The goal of this part is to test and asses the effectiveness of the existing map graph comprising nodes in WAN-SDN
under the modified worst case of latencies. Additionally, it is proposed to analyze the performance of controller smart
placement in SDN. The effectiveness of SDN-IoT is proposed or predicted to improve by adopting optimal allocation of
controller locations, since controllers serve as the system’s heart. Figure 4 depicts the major sequential processes of the
proposed controller placement technique utilized for WAN application. Start
Generate topology data from gml file
determine adjacency matrix
Get coordinates and level matrix from
node map
Find geographical distance
vector
End
Evaluate the statistics of network
parameters
Estimate the controller positions based
on cluster and distance
Using k-medoids for best controller
placement
Figure 4. Design steps of SDN controller optimum placement Generate topology data from gml file
determine adjacency matrix Evaluate the statistics of network
parameters Get coordinates and level matrix from
node map Find geographical distance
vector Figure 4. Design steps of SDN controller optimum placement 116 | Amrita Khera, et al. 116 | Amrita Khera, et al. Research Reports on Computer Science Research Reports on Computer Science The topological data or information is created utilizing the MATLAB environment from an input example of a
gml file. The network node coordinates and the level matrix generated from the node map. Estimate the controller
placements using cluster and distance information. Based on silhouette techniques, the paper analyzed the worst-
case latency and clustering counts. To find the best placements for all of these controllers, the proposed method uses a
location-allocation approach called the Partition About Medoids (PAM) algorithm clustering using propagation delay. The k-medoids are employed for the allocation of the controller locations. In the foundation of controller placement, we
use these techniques to detect the number of controllers that minimizes total network transmission latency. The algorithm of the proposed controller placement using PAM based clustering is given in Algorithm 1. Algorithm 1. The proposed PAM based clustering
The proposed PAM based clustering
1. Input data set D and, the number of medoids K = 4
2. Determine the weight matrix based on ← distance of edges K m
3. Calculate the shortest distance matrix using Johnson’s algorithm
4. Find the minimum sum of distances and select initial medoids
5. If N medoids < K
repeat 2, 3
end if
6. 6. Results and discussions Various proposed results are presented based on US-ML based modified latencies calculation. Table 2 displays
simulation settings used to assess the best SDN controller placement strategy. For the study, four controllers for 14
WAN application sites are considered. It can be seen that latency is evaluated for the worst-case network conditions,
which follow the same pattern as real latency. Table 2. SDN based on the study using approximate parameters
Parameters
Description, range or value
Nodes
14 cities’ default locations
Methodology
Cluster formations and latency improvement
Nc
The number of controllers is set to 4
S
The silhouette values range from -0.5 to 1
C
Cluster number Table 2. SDN based on the study using approximate parameters Figure 5 depicts the hierarchical data information extracted from the gml file, as well as the levels and coordinate
plot of the WAN network. The proposed approach is used to assess cluster quality by calculating the shortest distance
between data points. Research Reports on Computer Science Latitude (degrees)
Longitude (degrees)
50
45
40
35
30
25
-140
-120
-100
-80
-60
Figure 5. Representation of input collected node locations topological knowledge from the gml file Figure 5. Representation of input collected node locations topological knowledge from the gml file In the scope of controller placement, we use these techniques to determine the number of controllers which
minimize total network propagation delay (i.e., switch-to-switch latency)i The general latencies for the network are defined as The general latencies for the network are defined as 0
0
/ (
(
. ))
m
n
avg
L
dist
length graph node
= ∑∑
(1) (1) 0max
worst
toy
L
dist
=
(2) (2) In this paper, the modified calculation of the worst case of latencies for four controller locations are defined as In this paper, the modified calculation of the worst case of latencies for four controller locations are defined as 5
(
(
. 6. Results and discussions )) 2 10
avg
O
T
V
P
L
length graph node
+
+
+
= ∑
∗∗
(3) (3) where corresponding to four controller clusters where corresponding to four controller clusters 1
2
3
4
(
);
(
);
(
);
(
)
avg
avg
avg
avg
O
sum dist
T
sum dist
V
sum dist
P
sum dist
=
=
=
=
(4) (4) The modified worst-case latencies for four distances are defined as 1
2
3
4
1
2
3
4
5
5
5
5
max(
)
max(
)
max(
)
max(
)
;
;
;
2 10
2 10
2 10
2 10
avg
avg
avg
avg
worst
worst
worst
worst
dist
dist
dist
dist
L
L
L
L
=
=
=
=
∗
∗
∗
∗
(5) (5) Research Reports on Computer Science
118 | Amrita Khera, et al. cience
118 | Amrita Khera, et al. Research Reports on Computer Science Research Reports on Computer Science 118 | Amrita Khera, et al. Number of controllers
Overall latency (ms)
1
2
3
4
0
1
2
3
4
5
6
7
Average latency
Worst-case latency
× 10
-3
(a) Validated latencies
Number of controllers
Overall latency (ms)
1
2
3
4
0
0.002
0.004
0.006
0.008
0.01
Average latency
Worst-case latency
(b) Proposed modified distance based worst latencies
Figure 6. Results of the validation and proposed average and worst-case latency vs. number of controllers Number of controllers
Overall latency (ms)
1
2
3
4
0
0.002
0.004
0.006
0.008
0.01
Average latency
Worst-case latency (b) Proposed modified distance based worst latencies Figure 6. Results of the validation and proposed average and worst-case latency vs. number of controllers In Figure 6 (a) and (b), the experimental findings of computed average latency and worst-case latencies are shown. It is clear that the proposed modified distance based latency calculation (Equations (3) to (5)) performs better under
worst case and worst latencies are much closer to the average required latencies. Figure 7 presented a much better representation of the worst-case latencies with the proposed method and existing
approach. It can be observed that the modified latencies even for the worst case are less for around 50% of cases of
controllers. Special Issue|2023| 119 7. Conclusions and discussions The report presents a research on the performance of LPWAN systems. Furthermore, the US-ML based silhouette
PAM method-based optimal placement of the SDN-IoT controllers on WAN networks is investigated. The coverage
performance of WAN networks is studied and compared for the NB-IoT, LoRa, and SigFox technology specifications. It is found that, when compared to current network topologies, NB-IoT appears to have a longer battery life and more
effective energy management, with a functional lifetime of significantly more than 15 years. It is concluded that although the maximum recommended distance for LoRa to be utilized in WAN architecture
is roughly 20 km, implementing it is a difficult task at hand. SDN controller placement is tested and simulated, and
it is determined that effective positioning might better redistribute clusters in the network and lower the likelihood of
incorrect cluster formation. It is found that for the proposed approach the majority of silhouette values are positive, indicating a better
distribution of clusters in the network, with less than of around 10% of clusters having an incorrect distribution having
negative silhouette values with modified latencies calculation. The method uses random distance measures, thus
outcomes are probabilistic and expected to change a bit. Limitation can be eliminated by simulation using a fixed set of
distances measured for network formation. Overall, it is concluded that combining SDN with IoT networks can improve the overall effectiveness of the WAN
network by increasing data control. In future, it is expected to test and design the performance of the LoRaWAN for
the WAN performance enhancement. In the upcoming future, the effectiveness of a NB-IoT network at reduced power
consumption must be tested. 6. Results and discussions Number of controllers
Overall latency (ms)
× 10
-3
1
2
3
4
5
6
7
8
9
1
1.5
2
2.5
3
3.5
4
Existing worst-case latency
Modified worst-case latency
Figure 7. Results comparison of modified worst-case latency performance Existing worst-case latency
Modified worst-case latency Figure 7. Results comparison of modified worst-case latency performance Using the unsupervised silhouette distances based method to obtain the highest k percent of the values of the
measures for silhouette, bend, and gap. Figure 8(a) depicts the cluster levels vs. the silhouette values. The silhouette
values range from -0.5 to 1. It is required to minimize negative values for better network performance. Using the unsupervised silhouette distances based method to obtain the highest k percent of the values of the
measures for silhouette, bend, and gap. Figure 8(a) depicts the cluster levels vs. the silhouette values. The silhouette
values range from -0.5 to 1. It is required to minimize negative values for better network performance. It is obvious from Figure 8(b) that for the proposed approach the majority of silhouette values are positive,
indicating a better distribution of clusters in the network, with less than of around 10% of clusters having an incorrect
distribution having negative silhouette values (shown by red color) with modified latencies calculation. Special Issue|2023| 119 Research Reports on Computer Science Cluster level
4
3
2
1
Silhouette value
0.2
-0.6
-0.4
-0.2
0.4
0.6
0.8
0
1
(a) Validation of silhouette values
Cluster
4
3
2
1
Silhouette value
-0.2
0
0.2
0.6
0.8
0.4
1
(b) Silhouette values with our proposed worst-case latency
Figure 8. Comparison of cluster count effectiveness for silhouette value for proposed four controller system Cluster
4
3
2
1
Silhouette value
-0.2
0
0.2
0.6
0.8
0.4
1 Cluster level
4
3
2
1
Silhouette value
0.2
-0.6
-0.4
-0.2
0.4
0.6
0.8
0
1 Cluster level Cluster Silhouette value (a) Validation of silhouette values (a) Validation of silhouette values (b) Silhouette values with our proposed worst-case latency ure 8. Comparison of cluster count effectiveness for silhouette value for proposed four controller system Figure 8. Comparison of cluster count effectiveness for silhouette value for proposed four controller system Conflict of interest Authors declare that the current work does not have any conflict of interest. [1] Onumanyi AJ, Abu-Mahfouz AM, Hancke GP. Towards cognitive radio in low power wide area network for
industrial IoT applications. In: 2019 IEEE 17th International Conference on Industrial Informatics (INDIN).
Helsinki, Finland: IEEE; 2019. p.947-950. https://doi.org/10.1109/INDIN41052.2019.8972333 Research Reports on Computer Science p
p
g
[2] Sebastian JE, Sikora A, Schappacher M, Amjad Z. Test and measurement of LPWAN and cellula References [1] Onumanyi AJ, Abu-Mahfouz AM, Hancke GP. Towards cognitive radio in low power wide area network for
industrial IoT applications. In: 2019 IEEE 17th International Conference on Industrial Informatics (INDIN). Helsinki, Finland: IEEE; 2019. p.947-950. https://doi.org/10.1109/INDIN41052.2019.8972333 Research Reports on Computer Science 120 | Amrita Khera, et al. in a unified testbed. In: 2019 IEEE 17th International Conference on Industrial Informatics (INDIN). Helsinki,
Finland: IEEE; 2019. p.1521-1527. https://doi.org/10.1109/INDIN41052.2019.8972256 in a unified testbed. In: 2019 IEEE 17th International Conference on Industrial Informatics (INDIN). Helsinki,
Finland: IEEE; 2019. p.1521-1527. https://doi.org/10.1109/INDIN41052.2019.8972256 [3] Ballerini M, Polonelli T, Brunelli D, Magno M, Benini L. NB-IoT versus LoRaWAN: An experimental evaluation
for industrial applications. IEEE Transactions on Industrial Informatics. 2020; 16(12): 7802-7811. https://doi. org/10.1109/TII.2020.2987423 [4] Nair KK, Abu-Mahfouz AM, Lefophane S. Analysis of the narrow band internet of things (NB-IoT) technology. In:
2019 Conference on Information Communications Technology and Society (ICTAS). Durban, South Africa: IEEE;
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[5] Jiang L. Comparision of LoRa and NB-IoT in terms of connectivity. Degree project. KTH Royal Institute of
Technology; 2020. https://www.diva-portal.org/smash/get/diva2:1453316/FULLTEXT01.pdf gy
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[6] Ugwuanyi S, Paul G, Irvine J. Survey of IoT for developing countries: Performance analysis of LoRaWAN and
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International Conference on Industrial and Information Systems (ICIIS). Roorkee, India: IEEE; 2016. p.749-752. https://doi.org/10.1109/ICIINFS.2016.8263038 [8] Ismail D, Rahman M, Saifullah A. Low-power wide-area networks: opportunities, challenges, and directions. In: Proceedings of the Workshop Program of the 19th International Conference on Distributed Computing and
Networking. New York, United States: ACM; 2018. p.1-6. https://doi.org/10.1145/3170521.3170529 [9] Chen M, Miao Y, Jian X, Wang X, Humar I. Cognitive-LPWAN: Towards intelligent wireless services in hybrid
low power wide area networks. IEEE Transactions on Green Communications and Networking. 2018; 3(2): 409-
417. https://doi.org/10.1109/TGCN.2018.2873783 [10] Mane SY. LPWAN’s–Overview, market scenario and performance analysis of Lora, Sigfox using NB-
Fi range calculator. In: 2021 International Conference on Smart Generation Computing, Communication
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SMARTGENCON51891.2021.9645902 [11] Tran AK, Piran MJ, Pham C. SDN controller placement in IoT networks: An optimized subm
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Communications Letters. 2017; 6(5): 646-649. https://doi.org/10.1109/LWC.2017.2726533 Special Issue|2023| 121 Research Reports on Computer Science Research Reports on Computer Science
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English
| null |
Tetrakis[μ-1,3-bis(4-methyl-2-pyridylimino)isoindolinato]trimercury(II) dinitrate methanol tetrasolvate
|
Acta crystallographica. Section E
| 2,006
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Download date: 24. Oct. 2024 University of Southern Denmark Tetrakis[µ-1,3-bis(4-methyl-2-pyridylimino)isoindolinato]trimercury(II) dinitrate m
tetrasolvate Citation for pulished version (APA):
Wicholas, M., Dietrich, B. L., Anderson, O., & Cour, A. L. (2006). Tetrakis[µ-1,3-bis(4-methyl-2-
pyridylimino)isoindolinato]trimercury(II) dinitrate methanol tetrasolvate. Acta Crystallographica Section E:
Crystallographic Communications, (E62), m1689-m1690. https://doi.org/10.1107/S1600536806023944 Citation for pulished version (APA):
Wicholas, M., Dietrich, B. L., Anderson, O., & Cour, A. L. (2006). Tetrakis[µ-1,3-bis(4-methyl-2-
pyridylimino)isoindolinato]trimercury(II) dinitrate methanol tetrasolvate. Acta Crystallographica Section E:
Crystallographic Communications, (E62), m1689-m1690. https://doi.org/10.1107/S1600536806023944 Go to publication entry in University of Southern Denmark's Research Portal Terms of use
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If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk metal-organic papers Tetrakis[l-1,3-bis(4-methyl-2-pyridylimino)isoindolin-
ato]trimercury(II) dinitrate methanol tetrasolvate Acta Crystallographica Section E
Structure Reports
Online Acta Crystallographica Section E
Structure Reports Acta Crystallographica Section
Structure Reports
Online
ISSN 1600-5368 Structure Reports
Online Mark Wicholas,a* Brandon L.
Dietrich,a Oren P. Andersonb
and Agnete la Courb The
title
compound,
[Hg3(C20H16N5)4](NO3)24CH3OH,
consists of a trinuclear HgII dipositive cation, [Hg3(40-
MeL)4]2+, two nitrate anions and four occluded methanol
molecules. The Hg atoms of the cation lie on a crystallographic
twofold axis and are tetrahedrally coordinated by nitrogen-
donor atoms of the bridging 1,3-bis(4-methyl-2-pyridylimino)-
isoindoline ligands. The central Hg atom is coordinated by
four pyridine N atoms, one from each of the 40-MeL ligands,
while the two outer Hg atoms are each coordinated by two
pyridine and two pyrrole N atoms. Received 30 May 2006
Accepted 22 June 2006 aDepartment of Chemistry, Western Washington
University, Bellingham, Washington 98225,
USA, and bDepartment of Chemistry, Colorado
State University, Fort Collins, Colorado 80523,
USA aDepartment of Chemistry, Western Washington
University, Bellingham, Washington 98225,
USA, and bDepartment of Chemistry, Colorado
State University, Fort Collins, Colorado 80523,
USA Correspondence e-mail:
wicholas@chem.wwu.edu Correspondence e-mail:
wicholas@chem.wwu.edu Received 30 May 2006
Accepted 22 June 2006 doi:10.1107/S1600536806023944
Wicholas et al.
[Hg3(C20H16N5)4](NO3)24CH4O
m1689 Comment Tridentate nitrogen-donor ligands have played an important
role in inorganic chemistry in recent years as templates for
models of metalloproteins and as templates for catalytic
processes (Chaudhuri & Wieghardt, 1987; Trofimenko, 1999). The pyridine-arm isoindoline 40-MeLH [1,3-bis(4-methyl-2-
pyridylimino)isoindoline] when deprotonated normally coor-
dinates meridionally to metal ions via a pyrrole and two
pyridine N atoms and forms a series of bis-tridentate octa-
hedral complexes M(40-MeL)2 with the divalent first row
transition metal ions, M = Mn–Zn (Gagne´ et al., 1981). We
have shown through serendipitous discovery that 40-MeLH
when deprotonated reacts with zinc perchlorate in an equi-
molar ratio to form the trinuclear complex [Zn3(C20H16N5)4]-
(ClO4)25H2O, which contains tetrahedrally coordinated Zn
atoms (Anderson et al., 2003). In an effort to determine
whether other d10 metal ions form similar tetrahedrally coor-
dinated trinuclear complexes, we have examined the reactions
of 40-MeLH with CdII and HgII. Key indicators
Single-crystal X-ray study
T = 165 K
Mean (C–C) = 0.007 A˚
H-atom completeness 81%
R factor = 0.033
wR factor = 0.066
Data-to-parameter ratio = 15.3 For details of how these key indicators were
automatically derived from the article, see
http://journals.iucr.org/e. Figure 1 g
The structure of the trinuclear complex cation (50% probability
displacement ellipsoids). Unlabeled atoms of the cation are related to
the labeled atoms by operation of the twofold crystallographic symmetry
axis through the three Hg atoms (x, y, 1
2 – z); a symmetry-related nitrate
anion and two symmetry-related occluded methanol molecules are not
shown. Experimental scans
Absorption correction: multi-scan
(SADABS; Sheldrick, 2000)
Tmin = 0.145, Tmax = 0.253
(expected range = 0.086–0.151)
33142 measured reflections
8318 independent reflections
6461 reflections with I > 2(I)
Rint = 0.052
max = 26.4
Refinement
Refinement on F 2
R[F 2 > 2(F 2)] = 0.033
wR(F 2) = 0.066
S = 0.97
8318 reflections
542 parameters
H-atom parameters constrained
w = 1/[2(Fo
2) + (0.0264P)2]
where P = (Fo
2 + 2Fc
2)/3
(/)max = 0.006
max = 1.09 e A˚ 3
min = 1.75 e A˚ 3 Crystal data
[Hg3(C20H16N5)4](NO3)24CH4O
Mr = 2143.34
Monoclinic, C2=c
a = 26.285 (5) A˚
b = 19.755 (4) A˚
c = 19.376 (4) A˚
= 126.24 (3)
V = 8115 (3) A˚ 3
Z = 4
Dx = 1.754 Mg m3
Mo K radiation
= 5.74 mm1
T = 165 (2) K
Prism, yellow
0.55 0.50 0.33 mm Data collection: SMART (Bruker, 2001); cell refinement: SAINT
(Bruker, 2003; data reduction: SAINT; program(s) used to solve
structure: SHELXTL (Sheldrick, 2000); program(s) used to refine
structure: SHELXTL; molecular graphics: SHELXTL; software used
to prepare material for publication: SHELXTL. MW acknowledges the support of Research Corporation. The diffractometer at Colorado State University was provided
by a grant from the National Institutes of Health Shared
Instrumentation Grant Program. Data collection
Bruker SMART 1000
diffractometer
! scans
Absorption correction: multi-scan
(SADABS; Sheldrick, 2000)
Tmin = 0.145, Tmax = 0.253
(expected range = 0.086–0.151)
33142 measured reflections
8318 independent reflections
6461 reflections with I > 2(I)
Rint = 0.052
max = 26.4 Bruker SMART 1000
diffractometer
! scans
Absorption correction: multi-scan
(SADABS; Sheldrick, 2000)
Tmin = 0.145, Tmax = 0.253
(expected range = 0.086–0.151)
33142 measured reflections
8318 independent reflections
6461 reflections with I > 2(I)
Rint = 0.052
max = 26.4 metal-organic papers Figure 1
The structure of the trinuclear complex cation (50% probability
displacement ellipsoids). Unlabeled atoms of the cation are related to
the labeled atoms by operation of the twofold crystallographic symmetry
axis through the three Hg atoms (x, y, 1
2 – z); a symmetry-related nitrate
anion and two symmetry-related occluded methanol molecules are not
shown. of CdII for octahedral coordination (Sigel & Martin, 1994), the
title trinuclear HgII complex, (I), is formed readily from the
reaction of mercuric nitrate and deprotonated 40-MeLH. The cation in the title compound is composed of two short
helices of opposite hand, one coiling around Hg1 and one
coiling around Hg3, that meet at Hg2. The three Hg atoms are
located on a crystallographic twofold axis, and thus form a
linear backbone for the complex cation. As a result, the
trinuclear cation has crystallographic twofold symmetry about
that Hg3 axis. The crystallographically unique nitrate anion
exhibits no sign of disorder, presumably because of multiple
weak [O O = 2.966 (8)–3.137 (7) A˚ ] hydrogen bonding
between its O atoms and the hydroxyl groups of the occluded
methanol molecules. Key indicators For details of how these key indicators were
automatically derived from the article, see
http://journals.iucr.org/e. For details of how these key indicators were
automatically derived from the article, see
http://journals.iucr.org/e. doi:10 1107/S1600536806023944
Wicholas et al
[Hg3(C20H16N5)4](NO3)24CH4O
m1689
While we have been unsuccessful in preparing the CdII
trinuclear congener, perhaps because of the known preference Acta Cryst. (2006). E62, m1689–m1690
doi:10.1107/S1600536806023944
Wicholas et al. [Hg3(C20H16N5)4](NO3)24CH4O
m1689
# 2006 International Union of Crystallography
All rights reserved
While we have been unsuccessful in preparing the CdII
trinuclear congener, perhaps because of the known preference While we have been unsuccessful in preparing the CdII
trinuclear congener, perhaps because of the known preference While we have been unsuccessful in preparing the CdII
trinuclear congener, perhaps because of the known preference # 2006 International Union of Crystallography
All rights reserved Acta Cryst. (2006). E62, m1689–m1690 metal-organic papers References Addison, A. W. & Burke, P. J. (1981). J. Heterocycl. Chem. 18, 803–805. Addison, A. W. & Burke, P. J. (1981). J. Heterocycl. Chem. 18, 803–805. Anderson, O. P., la Cour, A., Dodd, A., Garrett, A. D. & Wicholas, M. (2003). Inorg. Chem. 42, 122–127. Anderson, O. P., la Cour, A., Dodd, A., Garrett, A. D. & Wicholas, M. (2003). Inorg. Chem. 42, 122–127. Bruker (2001). SMART. Version 5.625. Bruker AXS Inc., Madison, Wisconsin,
USA. Gagne´, R. R., Marritt, W. A., Marks, D. N. & Siegl, W. O. (1981). Inorg. Chem.
20, 3260–3267. Experimental g
The structure of the trinuclear complex cation (50% probability
displacement ellipsoids). Unlabeled atoms of the cation are related to
the labeled atoms by operation of the twofold crystallographic symmetry
axis through the three Hg atoms (x, y, 1
2 – z); a symmetry-related nitrate
anion and two symmetry-related occluded methanol molecules are not
shown. The isoindoline ligand was prepared by a solid state melt reaction
(Addison
&
Burke,
1981). Solid
Hg(NO3)23H2O
(346 mg,
0.914 mmol) was added to 25 ml of a warm methanol solution (25 ml)
of 40-MeLH (327 mg, 1.0 mmol), which had been deprotonated by the
addition of 1.0 M Bu4NOH (1.0 ml, 1.0 mmol) in methanol. The
orange–yellow solution was refluxed vigorously for 2 h, and the
resulting orange solution was filtered while warm to remove cloudi-
ness. Evaporation of the solvent from the filtrate over a two-day
period produced large yellow crystals (45% yield) of (I), which were
washed with ice-cold methanol and air-dried. Uiso(H) = 1.2Ueq(aromatic C) and 1.5.Ueq(methyl C). H atoms were
not included for the occluded methanol molecules, due to the fact
that they could not be located in the difference map. While the
highest peak in the difference map was 1.26 A˚ from C41, the next 19
peaks were due to residual density from the Hg atoms. The deepest
hole in the map was 0.90 A˚ from Hg3. Crystal data
[Hg3(C20H16N5)4](NO3)24CH4O
Mr = 2143.34
Monoclinic, C2=c
a = 26.285 (5) A˚
b = 19.755 (4) A˚
c = 19.376 (4) A˚
= 126.24 (3)
V = 8115 (3) A˚ 3
Z = 4
Dx = 1.754 Mg m3
Mo K radiation
= 5.74 mm1
T = 165 (2) K
Prism, yellow
0.55 0.50 0.33 mm
Data collection
Bruker SMART 1000
diffractometer
! Chaudhuri, P. & Wieghardt, K. (1987). Prog. Inorg. Chem. 35, 329–436. S2. Experimental The isoindoline ligand was prepared by a solid state melt reaction (Addison & Burke, 1981). Solid Hg(NO3)2·3H2O (346
mg, 0.914 mmol) was added to 25 ml of a warm methanol solution of 4′-MeLH (327 mg, 1.0 mmol), which had been
deprotonated by the addition of 1.0 M Bu4NOH (1.0 ml, 1.0 mmol) in methanol. The resulting orange–yellow solution
was refluxed vigorously for 2 h, and the resulting orange solution was filtered while warm to remove cloudiness. Evaporation of the solvent from the filtrate over a two-day period produced large yellow crystals (45% yield) of (I),
which were washed with ice-cold methanol and air-dried. S1. Comment Tridentate nitrogen-donor ligands have played an important role in inorganic chemistry in recent years as templates for
models of metalloproteins and as templates for catalytic processes (Chaudhuri & Wieghardt, 1987; Trofimenko, 1999). The pyridine-arm isoindoline 4′-MeLH [1,3-bis(4-methyl-2-pyridylimino)isoindoline] when deprotonated normally
coordinates meridionally to metal ions via a pyrrole and two pyridine N atoms and forms a series of bis-tridentate
octahedral complexes M(4′-MeL)2 with the divalent first row transition metal ions, M = Mn–Zn (Gagné et al., 1981). We
have shown through serendipitous discovery that 4′-MeLH when deprotonated reacts with zinc perchlorate in an
equimolar ratio to form the trinuclear complex [Zn3(C20H16N5)4](ClO4)2·5H2O, which contains tetrahedrally coordinated
Zn atoms (Anderson et al., 2003). In an effort to determine whether other d10 metal ions form similar tetrahedrally
coordinated trinuclear complexes, we have examined the reactions of 4′-MeLH with CdII and HgII. While we have been unsuccessful in preparing the CdII trinuclear congener, perhaps because of the known preference of
CdII for octahedral coordination (Sigel & Martin, 1994), the title trinuclear HgII complex, (I), is formed readily from the
reaction of mercuric nitrate and deprotonated 4′-MeLH. While we have been unsuccessful in preparing the CdII trinuclear congener, perhaps because of the known preference of
CdII for octahedral coordination (Sigel & Martin, 1994), the title trinuclear HgII complex, (I), is formed readily from the
reaction of mercuric nitrate and deprotonated 4′-MeLH. The cation in the title compound is composed of two short helices of opposite hand, one coiling around Hg1 and one
coiling around Hg3, that meet at Hg2. The three Hg atoms are located on a crystallographic twofold axis, and thus form a
linear backbone for the complex cation. As a result, the trinuclear cation has crystallographic twofold symmetry about
that Hg3 axis. The crystallographically unique nitrate anion exhibits no sign of disorder, presumably because of multiple
weak [O···O = 2.966 (8)–3.137 (7) Å] hydrogen bonding between the its O atoms and the hydroxyl groups of the
occluded methanol molecules. Refinement Refinement
Refinement on F 2
R[F 2 > 2(F 2)] = 0.033
wR(F 2) = 0.066
S = 0.97
8318 reflections
542 parameters
H-atom parameters constrained
w = 1/[2(Fo
2) + (0.0264P)2]
where P = (Fo
2 + 2Fc
2)/3
(/)max = 0.006
max = 1.09 e A˚ 3
min = 1.75 e A˚ 3 H-atom parameters constrained
w = 1/[2(Fo
2) + (0.0264P)2]
where P = (Fo
2 + 2Fc
2)/3
(/)max = 0.006
max = 1.09 e A˚ 3
min = 1.75 e A˚ 3 Refinement on F 2
R[F 2 > 2(F 2)] = 0.033
wR(F 2) = 0.066
S = 0.97
8318 reflections
542 parameters Bruker (2003). SAINT. Version 6.45. Bruker AXS Inc., Madison, Wisconsin,
USA. Chaudhuri, P. & Wieghardt, K. (1987). Prog. Inorg. Chem. 35, 329–436. Gagne´, R. R., Marritt, W. A., Marks, D. N. & Siegl, W. O. (1981). Inorg. Chem. 20, 3260–3267. Sheldrick, G. M. (2000). SHELXTL (Version 6.14) and SADABS (Version
2.10). Bruker AXS Inc., Madison, Wisconsin, USA. )
,
,
,
Sigel, H. & Martin, R. B. (1994). Chem. Soc. Rev. 28, 83–91. H atoms of the cation were placed in idealized positions and
refined using a riding model with C—H = 0.93 and 0.96 A˚ , and with (
)
Trofimenko, S. (1999). Scorpionates – The Coordination Chemistry of
Polypyrazolylborate Ligands. London: Imperial College Press. Acta Cryst. (2006). E62, m1689–m1690 m1690
Wicholas et al. [Hg3(C20H16N5)4](NO3)24CH4O supporting information supporting information Acta Cryst. (2006). E62, m1689–m1690 [doi:10.1107/S1600536806023944] Acta Cryst. (2006). E62, m1689–m1690 [doi:10.1107/S1600536806023944] Acta Cryst. (2006). E62, m1689–m1690 [doi:10.1107/S1600536806023944] S3. Refinement H atoms of the cation were placed in idealized positions and refined by using a riding model with C—H = 0.93 and 0.96
Å, and with Uiso(H) = 1.2Ueq(aromatic C) and 1.5.Ueq(methyl C) [please check changes]. H atoms were not included on
the occluded methanol molecules, due to the fact that they could not be located in the difference map, the highest peaks Acta Cryst. (2006). E62, m1689–m1690 sup-1 supporting information of which were due to residual electron density from the Hg atoms. Please specify locations of highest peak and deepest
hole. 1 Figure 1 g
The structure of the trinuclear complex cation (50% probability displacement ellipsoids). Unlabeled atoms of the cation
are related to the labeled atoms by operation of the twofold crystallographic symmetry axis through the three Hg atoms; a
symmetry-related nitrate anion and two symmetry-related occluded methanol molecules are not shown. Tetrakis[µ-1,3-bis(4-methyl-2-pyridylimino)isoindolinato]trimercury(II) dinitrate methanol tetrasolvate Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and
goodness of fit S are based on F2, conventional R-factors R are based
on F, with F set to zero for negative F2. The threshold expression of
F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is
not relevant to the choice of reflections for refinement. R-factors based
on F2 are statistically about twice as large as those based on F, and R-
factors based on ALL data will be even larger. Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and
goodness of fit S are based on F2, conventional R-factors R are based
on F, with F set to zero for negative F2. The threshold expression of
F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is
not relevant to the choice of reflections for refinement. R-factors based
on F2 are statistically about twice as large as those based on F, and R-
factors based on ALL data will be even larger. supporting information Refinement
Refinement on F2
Least-squares matrix: full
R[F2 > 2σ(F2)] = 0.033
wR(F2) = 0.066
S = 0.97
8318 reflections
542 parameters
0 restraints
Primary atom site location: structure-invariant
direct methods
Secondary atom site location: difference Fourier
map
Hydrogen site location: inferred from
neighbouring sites
H-atom parameters constrained
w = 1/[σ2(Fo2) + (0.0264P)2]
where P = (Fo2 + 2Fc2)/3
(Δ/σ)max = 0.006
Δρmax = 1.09 e Å−3
Δρmin = −1.75 e Å−3 Refinement
Refinement on F2
Least-squares matrix: full
R[F2 > 2σ(F2)] = 0.033
wR(F2) = 0.066
S = 0.97
8318 reflections
542 parameters
0 restraints
Primary atom site location: structure-invariant
direct methods
Secondary atom site location: difference Fourier
map
Hydrogen site location: inferred from
neighbouring sites
H-atom parameters constrained
w = 1/[σ2(Fo2) + (0.0264P)2]
where P = (Fo2 + 2Fc2)/3
(Δ/σ)max = 0.006
Δρmax = 1.09 e Å−3
Δρmin = −1.75 e Å−3 Secondary atom site location: difference Four
map
Hydrogen site location: inferred from
neighbouring sites
H-atom parameters constrained
w = 1/[σ2(Fo2) + (0.0264P)2]
where P = (Fo2 + 2Fc2)/3
(Δ/σ)max = 0.006
Δρmax = 1.09 e Å−3
Δρmin = −1.75 e Å−3 Tetrakis[µ-1,3-bis(4-methyl-2-pyridylimino)isoindolinato]trimercury(II) dinitrate methanol tetrasolvate Crystal data
[Hg3(C20H16N5)4](NO3)2·4CH4O
Mr = 2143.34
Monoclinic, C2/c
Hall symbol: -C 2yc
a = 26.285 (5) Å
b = 19.755 (4) Å
c = 19.376 (4) Å
β = 126.24 (3)°
V = 8115 (3) Å3
Z = 4
F(000) = 4168
Dx = 1.754 Mg m−3
Mo Kα radiation, λ = 0.71073 Å
Cell parameters from 999 reflections
θ = 3.2–26.4°
µ = 5.74 mm−1
T = 165 K
Prism, yellow
0.55 × 0.50 × 0.33 mm
Data collection
Bruker SMART 1000
diffractometer
Radiation source: fine-focus sealed tube
Graphite monochromator
ω scans
Absorption correction: multi-scan
(SADABS; Sheldrick, 2000)
Tmin = 0.145, Tmax = 0.253
33142 measured reflections
8318 independent reflections
6461 reflections with I > 2σ(I)
Rint = 0.052
θmax = 26.4°, θmin = 3.2°
h = −32→32
k = −24→24
l = −24→24 sup-2 Acta Cryst. (2006). E62, m1689–m1690 supporting information Acta Cryst. (2006). E62, m1689–m1690 Special details Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x
y
z
Uiso*/Ueq
Hg1
0.0000
−0.177441 (12)
0.2500
0.02208 (7)
Hg2
0.0000
0.055905 (12)
0.2500
0.02167 (7)
Hg3
0.0000
0.275941 (12)
0.2500
0.01788 (7)
N1
0.0759 (2)
−0.2662 (2)
0.2992 (3)
0.0374 (11)
N1A
−0.01204 (17)
0.36481 (19)
0.1579 (2)
0.0269 (9)
N2
0.12162 (18)
−0.22458 (19)
0.2312 (2)
0.0244 (9)
N2A
−0.11536 (17)
0.33158 (18)
0.0384 (2)
0.0218 (9)
N3
0.03385 (17)
−0.14694 (18)
0.1794 (2)
0.0193 (8)
N3A
−0.09376 (16)
0.25344 (17)
0.1508 (2)
0.0171 (8)
N4
−0.02641 (16)
−0.04366 (17)
0.1163 (2)
0.0199 (8)
N4A
−0.10706 (17)
0.15451 (17)
0.2112 (2)
0.0192 (9)
N5
−0.08361 (16)
−0.01277 (17)
0.1652 (2)
0.0174 (8)
N5A
−0.00916 (17)
0.12594 (16)
0.3331 (2)
0.0191 (9)
C1
0.0753 (3)
−0.3157 (3)
0.3460 (5)
0.062 (2)
H1
0.0517
−0.3092
0.3670
0.075*
C1A
0.0343 (2)
0.4111 (3)
0.1914 (3)
0.0385 (14)
H1A
0.0731
0.4008
0.2423
0.046*
C2
0.1079 (3)
−0.3759 (3)
0.3647 (4)
0.0555 (18)
H2
0.1066
−0.4085
0.3981
0.067*
C2A
0.0273 (3)
0.4727 (3)
0.1544 (3)
0.0384 (14)
H2A
0.0605
0.5034
0.1802
0.046* Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x
y
z
Uiso*/Ueq Acta Cryst. (2006). Acta Cryst. (2006). E62, m1689–m1690 Special details E62, m1689–m1690 sup-3 supporting information pp
g
C3
0.1423 (2)
−0.3869 (2)
0.3332 (4)
0.0371 (14)
C3A
−0.0293 (2)
0.4886 (2)
0.0787 (3)
0.0318 (12)
C4
0.1426 (2)
−0.3364 (2)
0.2855 (3)
0.0326 (13)
H4
0.1650
−0.3426
0.2627
0.039*
C4A
−0.0766 (2)
0.4409 (2)
0.0428 (3)
0.0287 (12)
H4A
−0.1152
0.4503
−0.0089
0.034*
C5
0.1102 (2)
−0.2755 (2)
0.2698 (3)
0.0253 (11)
C5A
−0.0675 (2)
0.3790 (2)
0.0825 (3)
0.0223 (11)
C6
0.1792 (3)
−0.4515 (3)
0.3535 (4)
0.0574 (18)
H6A
0.1587
−0.4875
0.3618
0.086*
H6B
0.2211
−0.4455
0.4048
0.086*
H6C
0.1812
−0.4628
0.3069
0.086*
C6A
−0.0369 (3)
0.5563 (2)
0.0367 (4)
0.0501 (16)
H6A1
−0.0323
0.5503
−0.0085
0.075*
H6A2
−0.0052
0.5871
0.0785
0.075*
H6A3
−0.0779
0.5745
0.0136
0.075*
C7
0.0902 (2)
−0.1699 (2)
0.1970 (3)
0.0217 (11)
C7A
−0.1249 (2)
0.2784 (2)
0.0677 (3)
0.0189 (10)
C8
0.1132 (2)
−0.1178 (2)
0.1663 (3)
0.0205 (10)
C8A
−0.1769 (2)
0.2313 (2)
0.0105 (3)
0.0213 (10)
C9
0.1673 (2)
−0.1151 (2)
0.1710 (3)
0.0284 (12)
H9
0.1967
−0.1501
0.1953
0.034*
C9A
−0.2218 (2)
0.2328 (3)
−0.0771 (3)
0.0290 (11)
H9A
−0.2224
0.2671
−0.1104
0.035*
C10
0.1762 (2)
−0.0582 (3)
0.1378 (3)
0.0329 (13)
H10
0.2125
−0.0547
0.1401
0.039*
C10A
−0.2663 (2)
0.1807 (2)
−0.1138 (3)
0.0328 (13)
H10A
−0.2966
0.1798
−0.1728
0.039*
C11
0.1321 (2)
−0.0061 (3)
0.1012 (3)
0.0322 (12)
H11
0.1391
0.0312
0.0786
0.039*
C11A
−0.2662 (2)
0.1311 (2)
−0.0643 (3)
0.0340 (13)
H11A
−0.2968
0.0975
−0.0905
0.041*
C12
0.0781 (2)
−0.0086 (2)
0.0976 (3)
0.0262 (11)
H12
0.0488
0.0264
0.0739
0.031*
C12A
−0.2217 (2)
0.1297 (2)
0.0234 (3)
0.0264 (11)
H12A
−0.2219
0.0961
0.0569
0.032*
C13
0.0697 (2)
−0.0659 (2)
0.1310 (3)
0.0184 (10)
C13A
−0.1766 (2)
0.1809 (2)
0.0594 (3)
0.0225 (11)
C14
0.0193 (2)
−0.0837 (2)
0.1395 (3)
0.0183 (10)
C14A
−0.1229 (2)
0.1940 (2)
0.1489 (3)
0.0182 (10)
C15
−0.13338 (19)
−0.0180 (2)
0.1677 (3)
0.0184 (10)
H15
−0.1388
0.0146
0.1975
0.022*
C15A
0.0377 (2)
0.1337 (2)
0.4162 (3)
0.0247 (11)
H15A
0.0717
0.1040
0.4417
0.030*
C16
−0.1766 (2)
−0.0695 (2)
0.1279 (3)
0.0226 (11)
H16
−0.2108
−0.0714
0.1304
0.027*
C16A
0.0377 (2)
0.1836 (2)
0.4656 (3)
0.0255 (11)
H16A
0.0714
0.1876
0.5231
0.031* sup-4 Acta Cryst. (2006). Acta Cryst. (2006). E62, m1689–m1690 Acta Cryst. (2006). E62, m1689–m1690 Acta Cryst. (2006). E62, m1689–m1690 Special details E62, m1689–m1690 supporting information supporting information
sup 5
A
C
(
) E
1
1
C17
−0.1686 (2)
−0.1185 (2)
0.0841 (3)
0.0245 (11)
C17A
−0.0128 (2)
0.2280 (2)
0.4289 (3)
0.0259 (11)
C18
−0.1179 (2)
−0.1124 (2)
0.0804 (3)
0.0213 (11)
H18
−0.1119
−0.1441
0.0503
0.026*
C18A
−0.0615 (2)
0.2197 (2)
0.3426 (3)
0.0233 (11)
H18A
−0.0962
0.2485
0.3159
0.028*
C19
−0.0755 (2)
−0.0587 (2)
0.1221 (3)
0.0184 (10)
C19A
−0.0583 (2)
0.1686 (2)
0.2962 (3)
0.0183 (10)
C20
−0.2138 (2)
−0.1764 (2)
0.0414 (3)
0.0348 (13)
H20A
−0.2198
−0.1970
0.0810
0.052*
H20B
−0.1970
−0.2093
0.0234
0.052*
H20C
−0.2535
−0.1601
−0.0074
0.052*
C20A
−0.0140 (3)
0.2827 (3)
0.4815 (3)
0.0409 (15)
H20D
−0.0292
0.2644
0.5121
0.061*
H20E
−0.0414
0.3185
0.4447
0.061*
H20F
0.0279
0.3001
0.5215
0.061*
N6
0.3285 (2)
−0.0977 (3)
0.3344 (3)
0.0530 (14)
O1
0.3477 (2)
−0.1471 (3)
0.3191 (3)
0.0901 (18)
O2
0.3121 (2)
−0.1032 (3)
0.3827 (3)
0.0888 (17)
O3
0.3275 (2)
−0.0426 (3)
0.3054 (4)
0.105 (2)
C40
0.3059 (4)
−0.2905 (4)
0.3660 (5)
0.085 (3)
O40
0.3407 (2)
−0.2470 (3)
0.4391 (3)
0.0823 (15)
C41
0.2550 (4)
0.0936 (3)
0.3046 (5)
0.085 (3)
O41
0.2585 (3)
0.0400 (3)
0.3531 (4)
0.0954 (18)
Atomic displacement parameters (Å2)
U11
U22
U33
U12
U13
U23
Hg1
0.02581 (15)
0.01864 (13)
0.03105 (17)
0.000
0.02190 (14)
0.000
Hg2
0.01963 (14)
0.01255 (12)
0.02745 (15)
0.000
0.01095 (12)
0.000
Hg3
0.01165 (13)
0.01850 (12)
0.01461 (14)
0.000
0.00288 (11)
0.000
N1
0.043 (3)
0.024 (2)
0.061 (3)
0.013 (2)
0.039 (3)
0.015 (2)
N1A
0.017 (2)
0.030 (2)
0.021 (2)
−0.0076 (18)
0.0040 (19)
0.0025 (19)
N2
0.027 (2)
0.022 (2)
0.030 (2)
0.0029 (18)
0.020 (2)
−0.0019 (19)
N2A
0.016 (2)
0.027 (2)
0.018 (2)
0.0019 (16)
0.0074 (18)
0.0026 (17)
N3
0.025 (2)
0.0192 (19)
0.020 (2)
−0.0007 (17)
0.0161 (19)
−0.0031 (17)
N3A
0.014 (2)
0.0181 (19)
0.014 (2)
−0.0005 (15)
0.0053 (17)
−0.0005 (15)
N4
0.019 (2)
0.0185 (19)
0.023 (2)
−0.0027 (17)
0.0129 (18)
0.0002 (17)
N4A
0.014 (2)
0.0159 (19)
0.022 (2)
0.0003 (16)
0.0076 (19)
−0.0013 (17)
N5
0.016 (2)
0.0163 (18)
0.018 (2)
0.0029 (16)
0.0090 (18)
0.0038 (16)
N5A
0.019 (2)
0.0145 (19)
0.024 (2)
0.0002 (16)
0.013 (2)
0.0012 (17)
C1
0.070 (5)
0.046 (4)
0.111 (6)
0.031 (3)
0.076 (5)
0.042 (4)
C1A
0.026 (3)
0.040 (3)
0.030 (3)
−0.007 (3)
0.006 (3)
0.008 (3)
C2
0.066 (5)
0.034 (3)
0.087 (5)
0.018 (3)
0.056 (4)
0.029 (3)
C2A
0.042 (3)
0.032 (3)
0.037 (3)
−0.015 (3)
0.020 (3)
0.000 (3)
C3
0.031 (3)
0.019 (3)
0.047 (4)
0.007 (2)
0.015 (3)
0.000 (2)
C3A
0.044 (3)
0.026 (3)
0.035 (3)
0.004 (2)
0.029 (3)
0.002 (2)
C4
0.033 (3)
0.026 (3)
0.037 (3)
0.007 (2)
0.020 (3)
−0.004 (2) Atomic displacement parameters (Å2) sup-5 supporting information C4A
0.029 (3)
0.030 (3)
0.024 (3)
0.006 (2)
0.014 (2)
0.010 (2)
C5
0.019 (3)
0.022 (2)
0.033 (3)
0.001 (2)
0.015 (2)
−0.006 (2)
C5A
0.023 (3)
0.023 (2)
0.022 (3)
0.005 (2)
0.014 (2)
0.004 (2)
C6
0.055 (4)
0.033 (3)
0.064 (5)
0.022 (3)
0.025 (4)
0.004 (3)
C6A
0.071 (5)
0.031 (3)
0.058 (4)
0.005 (3)
0.043 (4)
0.012 (3)
C7
0.019 (3)
0.027 (3)
0.020 (3)
−0.005 (2)
0.012 (2)
−0.009 (2)
C7A
0.012 (2)
0.025 (2)
0.015 (2)
0.0034 (19)
0.006 (2)
−0.003 (2)
C8
0.020 (3)
0.027 (2)
0.016 (3)
−0.004 (2)
0.011 (2)
−0.007 (2)
C8A
0.015 (2)
0.026 (2)
0.016 (2)
0.003 (2)
0.005 (2)
−0.005 (2)
C9
0.022 (3)
0.038 (3)
0.032 (3)
−0.004 (2)
0.019 (3)
−0.010 (2)
C9A
0.024 (3)
0.034 (3)
0.023 (3)
0.007 (2)
0.011 (2)
−0.002 (2)
C10
0.027 (3)
0.047 (3)
0.033 (3)
−0.012 (3)
0.022 (3)
−0.005 (3)
C10A
0.018 (3)
0.040 (3)
0.022 (3)
0.006 (2)
0.001 (2)
−0.013 (2)
C11
0.033 (3)
0.040 (3)
0.027 (3)
−0.012 (2)
0.020 (3)
−0.001 (2)
C11A
0.020 (3)
0.027 (3)
0.036 (3)
−0.001 (2)
0.006 (3)
−0.015 (3)
C12
0.025 (3)
0.033 (3)
0.021 (3)
−0.006 (2)
0.014 (2)
0.000 (2)
C12A
0.020 (3)
0.021 (2)
0.030 (3)
0.000 (2)
0.010 (2)
−0.009 (2)
C13
0.019 (2)
0.025 (2)
0.012 (2)
−0.0074 (19)
0.009 (2)
−0.0056 (18)
C13A
0.014 (2)
0.021 (2)
0.025 (3)
0.0032 (19)
0.007 (2)
−0.009 (2)
C14
0.020 (3)
0.019 (2)
0.017 (3)
−0.008 (2)
0.011 (2)
−0.009 (2)
C14A
0.012 (2)
0.017 (2)
0.021 (3)
−0.0005 (19)
0.007 (2)
−0.006 (2)
C15
0.016 (2)
0.018 (2)
0.021 (3)
0.0047 (19)
0.011 (2)
0.003 (2)
C15A
0.021 (3)
0.022 (2)
0.026 (3)
0.000 (2)
0.012 (2)
0.007 (2)
C16
0.015 (2)
0.027 (3)
0.027 (3)
0.0016 (19)
0.013 (2)
0.005 (2)
C16A
0.029 (3)
0.029 (3)
0.017 (3)
0.000 (2)
0.012 (2)
0.004 (2)
C17
0.015 (3)
0.025 (2)
0.021 (3)
−0.005 (2)
0.004 (2)
0.004 (2)
C17A
0.028 (3)
0.029 (2)
0.025 (3)
−0.002 (2)
0.018 (2)
−0.001 (2)
C18
0.020 (3)
0.019 (2)
0.021 (3)
−0.0031 (19)
0.010 (2)
−0.007 (2)
C18A
0.020 (3)
0.022 (2)
0.032 (3)
0.005 (2)
0.017 (2)
0.001 (2)
C19
0.015 (2)
0.016 (2)
0.018 (2)
0.0029 (19)
0.007 (2)
0.0039 (19)
C19A
0.017 (2)
0.020 (2)
0.018 (3)
−0.0014 (19)
0.011 (2)
0.0028 (19)
C20
0.033 (3)
0.029 (3)
0.039 (3)
−0.013 (2)
0.019 (3)
−0.008 (2)
C20A
0.049 (4)
0.045 (4)
0.031 (3)
0.004 (3)
0.025 (3)
−0.008 (3)
N6
0.025 (3)
0.067 (4)
0.041 (3)
0.010 (3)
0.005 (3)
−0.005 (3)
O1
0.074 (4)
0.103 (4)
0.067 (4)
0.029 (3)
0.027 (3)
−0.022 (3)
O2
0.058 (3)
0.156 (5)
0.070 (4)
−0.004 (3)
0.048 (3)
−0.017 (4)
O3
0.062 (4)
0.103 (5)
0.128 (5)
0.022 (3)
0.044 (4)
0.057 (4)
C40
0.086 (6)
0.103 (7)
0.072 (6)
−0.037 (5)
0.049 (5)
−0.022 (5)
O40
0.077 (4)
0.100 (4)
0.067 (3)
−0.009 (3)
0.041 (3)
0.017 (3)
C41
0.118 (7)
0.039 (4)
0.070 (5)
−0.025 (4)
0.041 (5)
0.008 (4)
O41
0.096 (4)
0.093 (4)
0.123 (5)
−0.014 (3)
0.079 (4)
−0.027 (4) Geometric parameters (Å, º)
Hg1—N3i
2.114 (4)
C7A—C8A
1.472 (6)
Hg1—N3
2.114 (4)
C8—C9
1.371 (6)
Hg1—N1i
2.391 (4)
C8—C13
1.379 (6)
Hg1—N1
2.391 (4)
C8A—C13A
1.371 (6) sup-6 supporting information supporting information 2.241 (4)
C8A—C9A
1.381 (6)
2.241 (4)
C9—C10
1.381 (6)
2.258 (4)
C9—H9
0.9300
2.258 (4)
C9A—C10A
1.395 (6)
2.089 (3)
C9A—H9A
0.9300
2.089 (4)
C10—C11
1.391 (7)
2.388 (4)
C10—H10
0.9300
2.388 (4)
C10A—C11A
1.370 (7)
1.333 (6)
C10A—H10A
0.9300
1.340 (6)
C11—C12
1.380 (6)
1.344 (6)
C11—H11
0.9300
1.348 (6)
C11A—C12A
1.381 (7)
1.281 (5)
C11A—H11A
0.9300
1.388 (6)
C12—C13
1.386 (6)
1.287 (5)
C12—H12
0.9300
1.386 (6)
C12A—C13A
1.392 (6)
1.384 (5)
C12A—H12A
0.9300
1.398 (5)
C13—C14
1.471 (6)
1.389 (5)
C13A—C14A
1.475 (6)
1.395 (5)
C15—C16
1.373 (6)
1.277 (5)
C15—H15
0.9300
1.392 (5)
C15A—C16A
1.373 (6)
1.283 (5)
C15A—H15A
0.9300
1.391 (6)
C16—C17
1.383 (6)
1.333 (5)
C16—H16
0.9300
1.341 (5)
C16A—C17A
1.386 (6)
1.338 (6)
C16A—H16A
0.9300
1.341 (5)
C17—C18
1.384 (6)
1.383 (7)
C17—C20
1.496 (6)
0.9300
C17A—C18A
1.390 (6)
1.368 (7)
C17A—C20A
1.498 (6)
0.9300
C18—C19
1.398 (6)
1.375 (7)
C18—H18
0.9300
0.9300
C18A—C19A
1.387 (6)
1.372 (7)
C18A—H18A
0.9300
0.9300
C20—H20A
0.9600
1.362 (7)
C20—H20B
0.9600
1.508 (6)
C20—H20C
0.9600
1.376 (7)
C20A—H20D
0.9600
1.518 (6)
C20A—H20E
0.9600
1.399 (6)
C20A—H20F
0.9600
0.9300
N6—O1
1.211 (6)
1.388 (6)
N6—O3
1.218 (7)
0.9300
N6—O2
1.245 (6)
0.9600
O1—O40
3.137 (7)
0.9600
O2—O40
2.976 (8)
0.9600
O2—O41
3.060 (8)
0.9600
O3—O41
2.967 (8) Hg2—N5A
2.241 (4)
C8A—C9A
1.381 (6)
Hg2—N5Ai
2.241 (4)
C9—C10
1.381 (6)
Hg2—N5
2.258 (4)
C9—H9
0.9300
Hg2—N5i
2.258 (4)
C9A—C10A
1.395 (6)
Hg3—N3A
2.089 (3)
C9A—H9A
0.9300
Hg3—N3Ai
2.089 (4)
C10—C11
1.391 (7)
Hg3—N1Ai
2.388 (4)
C10—H10
0.9300
Hg3—N1A
2.388 (4)
C10A—C11A
1.370 (7)
N1—C5
1.333 (6)
C10A—H10A
0.9300
N1—C1
1.340 (6)
C11—C12
1.380 (6)
N1A—C1A
1.344 (6)
C11—H11
0.9300
N1A—C5A
1.348 (6)
C11A—C12A
1.381 (7)
N2—C7
1.281 (5)
C11A—H11A
0.9300
N2—C5
1.388 (6)
C12—C13
1.386 (6)
N2A—C7A
1.287 (5)
C12—H12
0.9300
N2A—C5A
1.386 (6)
C12A—C13A
1.392 (6)
N3—C7
1.384 (5)
C12A—H12A
0.9300
N3—C14
1.398 (5)
C13—C14
1.471 (6)
N3A—C14A
1.389 (5)
C13A—C14A
1.475 (6)
N3A—C7A
1.395 (5)
C15—C16
1.373 (6)
N4—C14
1.277 (5)
C15—H15
0.9300
N4—C19
1.392 (5)
C15A—C16A
1.373 (6)
N4A—C14A
1.283 (5)
C15A—H15A
0.9300
N4A—C19A
1.391 (6)
C16—C17
1.383 (6)
N5—C19
1.333 (5)
C16—H16
0.9300
N5—C15
1.341 (5)
C16A—C17A
1.386 (6)
N5A—C15A
1.338 (6)
C16A—H16A
0.9300
N5A—C19A
1.341 (5)
C17—C18
1.384 (6)
C1—C2
1.383 (7)
C17—C20
1.496 (6)
C1—H1
0.9300
C17A—C18A
1.390 (6)
C1A—C2A
1.368 (7)
C17A—C20A
1.498 (6)
C1A—H1A
0.9300
C18—C19
1.398 (6)
C2—C3
1.375 (7)
C18—H18
0.9300
C2—H2
0.9300
C18A—C19A
1.387 (6)
C2A—C3A
1.372 (7)
C18A—H18A
0.9300
C2A—H2A
0.9300
C20—H20A
0.9600
C3—C4
1.362 (7)
C20—H20B
0.9600
C3—C6
1.508 (6)
C20—H20C
0.9600
C3A—C4A
1.376 (7)
C20A—H20D
0.9600
C3A—C6A
1.518 (6)
C20A—H20E
0.9600
C4—C5
1.399 (6)
C20A—H20F
0.9600
C4—H4
0.9300
N6—O1
1.211 (6)
C4A—C5A
1.388 (6)
N6—O3
1.218 (7)
C4A—H4A
0.9300
N6—O2
1.245 (6)
C6—H6A
0.9600
O1—O40
3.137 (7)
C6—H6B
0.9600
O2—O40
2.976 (8)
C6—H6C
0.9600
O2—O41
3.060 (8)
C6A—H6A1
0.9600
O3—O41
2.967 (8) sup-7 Acta Cryst. Acta Cryst. (2006). E62, m1689–m1690 Acta Cryst. (2006). E62, m1689–m1690 supporting information (2006). E62, m1689–m1690 supporting information pp
g
C6A—H6A2
0.9600
C40—O40
1.432 (8)
C6A—H6A3
0.9600
C41—O41
1.384 (8)
C7—C8
1.485 (6)
N3i—Hg1—N3
146.87 (19)
C9A—C8A—C7A
130.6 (5)
N3i—Hg1—N1i
83.99 (14)
C8—C9—C10
117.0 (5)
N3—Hg1—N1i
121.52 (14)
C8—C9—H9
121.5
N3i—Hg1—N1
121.52 (14)
C10—C9—H9
121.5
N3—Hg1—N1
83.99 (14)
C8A—C9A—C10A
117.5 (5)
N1i—Hg1—N1
85.7 (2)
C8A—C9A—H9A
121.2
N5A—Hg2—N5Ai
103.75 (17)
C10A—C9A—H9A
121.2
N5A—Hg2—N5
114.95 (13)
C9—C10—C11
121.4 (5)
N5Ai—Hg2—N5
108.66 (13)
C9—C10—H10
119.3
N5A—Hg2—N5i
108.66 (13)
C11—C10—H10
119.3
N5Ai—Hg2—N5i
114.95 (13)
C11A—C10A—C9A
121.0 (5)
N5—Hg2—N5i
106.16 (17)
C11A—C10A—H10A
119.5
N3A—Hg3—N3Ai
155.43 (18)
C9A—C10A—H10A
119.5
N3A—Hg3—N1Ai
114.04 (13)
C12—C11—C10
121.4 (5)
N3Ai—Hg3—N1Ai
84.58 (13)
C12—C11—H11
119.3
N3A—Hg3—N1A
84.58 (13)
C10—C11—H11
119.3
N3Ai—Hg3—N1A
114.04 (13)
C10A—C11A—C12A
121.7 (5)
N1Ai—Hg3—N1A
85.35 (19)
C10A—C11A—H11A
119.1
C5—N1—C1
118.0 (4)
C12A—C11A—H11A
119.1
C5—N1—Hg1
123.2 (3)
C11—C12—C13
116.7 (4)
C1—N1—Hg1
117.6 (3)
C11—C12—H12
121.6
C1A—N1A—C5A
118.2 (4)
C13—C12—H12
121.6
C1A—N1A—Hg3
117.2 (3)
C11A—C12A—C13A
117.0 (5)
C5A—N1A—Hg3
122.9 (3)
C11A—C12A—H12A
121.5
C7—N2—C5
127.7 (4)
C13A—C12A—H12A
121.5
C7A—N2A—C5A
128.5 (4)
C8—C13—C12
121.6 (4)
C7—N3—C14
109.5 (4)
C8—C13—C14
107.8 (4)
C7—N3—Hg1
123.0 (3)
C12—C13—C14
130.5 (4)
C14—N3—Hg1
122.0 (3)
C8A—C13A—C12A
121.8 (5)
C14A—N3A—C7A
109.3 (4)
C8A—C13A—C14A
107.1 (4)
C14A—N3A—Hg3
122.4 (3)
C12A—C13A—C14A
131.1 (5)
C7A—N3A—Hg3
123.7 (3)
N4—C14—N3
129.9 (4)
C14—N4—C19
124.3 (4)
N4—C14—C13
122.4 (4)
C14A—N4A—C19A
123.5 (4)
N3—C14—C13
107.7 (4)
C19—N5—C15
119.4 (4)
N4A—C14A—N3A
128.5 (4)
C19—N5—Hg2
114.7 (3)
N4A—C14A—C13A
123.5 (4)
C15—N5—Hg2
124.8 (3)
N3A—C14A—C13A
108.0 (4)
C15A—N5A—C19A
118.7 (4)
N5—C15—C16
122.5 (4)
C15A—N5A—Hg2
121.5 (3)
N5—C15—H15
118.8
C19A—N5A—Hg2
118.9 (3)
C16—C15—H15
118.8
N1—C1—C2
123.5 (5)
N5A—C15A—C16A
122.9 (4)
N1—C1—H1
118.3
N5A—C15A—H15A
118.6
C2—C1—H1
118.3
C16A—C15A—H15A
118.6
N1A—C1A—C2A
123.2 (5)
C15—C16—C17
119.2 (4) sup-8 Acta Cryst. (2006). E62, m1689–m1690 supporting information Acta Cryst. (2006). E62, m1689–m1690 supporting information N1A—C1A—H1A
118.4
C15—C16—H16
120.4
C2A—C1A—H1A
118.4
C17—C16—H16
120.4
C3—C2—C1
119.2 (5)
C15A—C16A—C17A
119.4 (5)
C3—C2—H2
120.4
C15A—C16A—H16A
120.3
C1—C2—H2
120.4
C17A—C16A—H16A
120.3
C1A—C2A—C3A
119.3 (5)
C16—C17—C18
118.1 (4)
C1A—C2A—H2A
120.4
C16—C17—C20
121.1 (4)
C3A—C2A—H2A
120.4
C18—C17—C20
120.8 (4)
C4—C3—C2
117.0 (5)
C16A—C17A—C18A
117.6 (4)
C4—C3—C6
122.5 (5)
C16A—C17A—C20A
120.4 (4)
C2—C3—C6
120.5 (5)
C18A—C17A—C20A
122.0 (4)
C2A—C3A—C4A
118.0 (5)
C17—C18—C19
119.9 (4)
C2A—C3A—C6A
119.3 (5)
C17—C18—H18
120.0
C4A—C3A—C6A
122.7 (5)
C19—C18—H18
120.0
C3—C4—C5
122.0 (5)
C19A—C18A—C17A
120.1 (4)
C3—C4—H4
119.0
C19A—C18A—H18A
120.0
C5—C4—H4
119.0
C17A—C18A—H18A
120.0
C3A—C4A—C5A
120.9 (5)
N5—C19—N4
113.8 (4)
C3A—C4A—H4A
119.5
N5—C19—C18
120.8 (4)
C5A—C4A—H4A
119.5
N4—C19—C18
125.1 (4)
N1—C5—N2
123.0 (4)
N5A—C19A—C18A
121.3 (4)
N1—C5—C4
120.3 (5)
N5A—C19A—N4A
115.4 (4)
N2—C5—C4
116.3 (4)
C18A—C19A—N4A
123.1 (4)
N1A—C5A—N2A
122.2 (4)
C17—C20—H20A
109.5
N1A—C5A—C4A
120.4 (4)
C17—C20—H20B
109.5
N2A—C5A—C4A
117.2 (4)
H20A—C20—H20B
109.5
C3—C6—H6A
109.5
C17—C20—H20C
109.5
C3—C6—H6B
109.5
H20A—C20—H20C
109.5
H6A—C6—H6B
109.5
H20B—C20—H20C
109.5
C3—C6—H6C
109.5
C17A—C20A—H20D
109.5
H6A—C6—H6C
109.5
C17A—C20A—H20E
109.5
H6B—C6—H6C
109.5
H20D—C20A—H20E
109.5
C3A—C6A—H6A1
109.5
C17A—C20A—H20F
109.5
C3A—C6A—H6A2
109.5
H20D—C20A—H20F
109.5
H6A1—C6A—H6A2
109.5
H20E—C20A—H20F
109.5
C3A—C6A—H6A3
109.5
O1—N6—O3
120.3 (7)
H6A1—C6A—H6A3
109.5
O1—N6—O2
119.5 (7)
H6A2—C6A—H6A3
109.5
O3—N6—O2
120.1 (7)
N2—C7—N3
132.3 (4)
N6—O1—O40
96.3 (5)
N2—C7—C8
120.0 (4)
N6—O2—O40
103.6 (4)
N3—C7—C8
107.7 (4)
N6—O2—O41
96.1 (4)
N2A—C7A—N3A
131.5 (4)
O40—O2—O41
159.9 (2)
N2A—C7A—C8A
121.3 (4)
N6—O3—O41
101.4 (5)
N3A—C7A—C8A
107.2 (4)
C40—O40—O2
109.6 (4)
C9—C8—C13
121.9 (4)
C40—O40—O1
85.9 (4)
C9—C8—C7
130.7 (4)
O2—O40—O1
40.52 (13)
C13—C8—C7
107.3 (4)
C41—O41—O3
90.9 (4) sup-9 Acta Cryst. (2006). E62, m1689–m1690 supporting information
C13A—C8A—C9A
121.0 (4)
C41—O41—O2
132.3 (5)
C13A—C8A—C7A
108.4 (4)
O3—O41—O2
41.43 (15)
Symmetry code: (i) −x, y, −z+1/2. C13A—C8A—C9A
121.0 (4)
C41—O41—O2
132.3 (5)
C13A—C8A—C7A
108.4 (4)
O3—O41—O2
41.43 (15) Symmetry code: (i) −x, y, −z+1/2. Symmetry code: (i) −x, y, −z+1/2. sup-10
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https://openalex.org/W2157176660
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https://ogst.ifpenergiesnouvelles.fr/articles/ogst/pdf/2006/01/colliou_vol61n1.pdf
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English
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Adapting the NADI (TM) Concept to Heavy Duty Engines
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Oil & gas science and technology
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cc-by
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Adapting the NADITM Concept
to Heavy Duty Engines
T. Colliou1, R. Tilagone1 and B. Martin1
1 IFP-Lyon, BP 3, 69390 Vernaison - France
e-mail: thierry.colliou@ifp.fr - richard.tilagone@ifp.fr - brigitte.martin@ifp.fr Résumé — Adaptation du concept NADI™aux moteurs de poids lourd — Le moteur diesel est
particulièrement bien adapté aux motorisations de véhicules poids lourds mais doit encore être amélioré. Le compromis sur les émissions NOx/particules doit encore être amélioré tout en maintenant un
rendement élevé malgré l’augmentation des puissances spécifiques. Si la technologie de post traitement
SCR est prête pour atteindre les objectifs fixés par les normes Euro V et les suivantes, les combustions
de type HCCI (Homogeneous Charge Compression Ignition) ou HPC (Highly Premixed Combustion)
offrent également un fort potentiel pour atteindre les futurs niveaux d’émission de NOx et de particules. En réponse à ces nouveaux challenges, l’IFP a développé un système de combustion capable d’atteindre
de très bas niveaux de particules et de NOx tout en maintenant un niveau de performance comparable à
un moteur diesel traditionnel en combustion standard. Ce moteur bi mode appelé NADITM (pour
Narrow Angle Direct Injection) autorise des combustions HCCI à charge partielle et commute sur des
combustions conventionnelles pour atteindre les exigences des fortes charges. Ce papier présente les derniers développements de ce concept sur moteur monocylindre et les
améliorations obtenues en terme de plage d’utilisation des combustions HPC, d’émissions de CO et de
HC, de consommation de carburant et du bruit de combustion. À pleine charge, la chambre de
combustion NADITM est comparable aux chambres actuelles avec cependant une légère augmentation
de la consommation et des émissions de fumée. En combustion HPC une PME de 13 bar a été atteinte à mi-régime du cycle ESC avec un niveau
de NOx inférieur à 0,3 g/kWh ce qui est environ 25 fois plus faible qu’un diesel classique. Les
émissions de particules sont comparables avec le niveau du moteur standard. Les émissions de CO et de
HC sont sensiblement supérieures mais restent compatibles avec les performances des catalyseurs
d’oxydation actuels. La pénalité interne de consommation due aux débits d’admission et aux taux d’EGR supérieurs
nécessaires pour le contrôle des combustions HPC peut être estimée par un code 0D de la boucle de
suralimentation. La puissance de refroidissement de l’air et de l’EGR est également calculée et montre
une augmentation importante par rapport au moteur de série pour maintenir la température admission à
un niveau acceptable. La voie de la réduction du rapport volumétrique a également été explorée et permet d’augmenter
significativement la plage de fonctionnement HCCI. Oil & Gas Science and Technology – Rev. IFP, Vol. 61 (2006), No. 1, pp. 73-84
Copyright © 2006, Institut français du pétrole Oil & Gas Science and Technology – Rev. IFP, Vol. 61 (2006), No. 1, pp. 73-84
Copyright © 2006, Institut français du pétrole Oil & Gas Science and Technology – Rev. IFP, Vol. 61 (2006), No. 1, pp. 73-84
Copyright © 2006, Institut français du pétrole Development and Control of Combustion Systems
Évolutions et contrôle des systèmes de combustion
D o s s i e r Adapting the NADITM Concept
to Heavy Duty Engines
T. Colliou1, R. Tilagone1 and B. Martin1
1 IFP-Lyon, BP 3, 69390 Vernaison - France
e-mail: thierry.colliou@ifp.fr - richard.tilagone@ifp.fr - brigitte.martin@ifp.fr Les principaux inconvénients proviennent de la
difficulté de démarrage à froid et de la chute du rendement de thermodynamique. Le challenge est maintenant de définir des stratégies d’injection multiple permettant d’étendre la
plage HCCI vers les fortes charges et de rendre les conditions de fonctionnement pleinement 74 Oil & Gas Science and Technology – Rev. IFP, Vol. 61 (2006), No. 1 compatibles avec les performances de la boucle de suralimentation et de la puissance des échangeurs
à mettre en œuvre. Abstract — Adapting the NADITM Concept to Heavy Duty Engines — Diesel combustion is well
adapted for heavy duty engines but the challenge is twofold. On the one hand, the trade off between
NOx and particulate emissions has to be improved. On the other hand, efficiency needs to be
maintained and the trend is to increase specific power and torque output. If SCR technology is ready to
meet Euro IV and beyond, homogeneous charge compression ignition (HCCI) or highly premixed
combustion (HPC) are new technologies that also offer great potential for meeting future NOx and
particulate emissions regulation targets. In response to the challenges that the Diesel engine faces, IFP has developed a combustion system
which is able to achieve near zero particulates and NOx emissions while maintaining the performance
standards of the D.I Diesel engine. This “dual mode” engine application called NADITM (Narrow Angle
Direct Injection) applies Homogeneous Charge Compression Ignition at part load and switches to
conventional Diesel combustion to reach high and full load requirements. Thanks to the financial support from European Commission, the “Hy-SPACE” project leaded by IFP,
was launched in partnership with Daimler Chrysler, Iveco, Delphi, Volvo, Chalmers University
(Contract G3RD-CT-2002-00 787) with the aim to adapt the NADITM concept to heavy duty engines. This paper presents the latest developments of the NADITM concept on a single cylinder heavy duty
engine, and the associated improvements regarding HPC (Highly Premixed Combustion) operating
range, CO and HC emissions, fuel consumption and combustion noise. At full load, the NADITM system
is consistent with present Diesel performances with a slight increase in fuel consumption and smoke
level. In HPC combustion mode, above mid speed of the ESC cycle, a BMEP of 13 bar was achieved with
NOx emissions below 0.3 g/kWh which is 25 times lower than a conventional Diesel engine. The
particulate emissions are comparable to the standard engine level. Adapting the NADITM Concept
to Heavy Duty Engines
T. Colliou1, R. Tilagone1 and B. Martin1
1 IFP-Lyon, BP 3, 69390 Vernaison - France
e-mail: thierry.colliou@ifp.fr - richard.tilagone@ifp.fr - brigitte.martin@ifp.fr CO and HC emissions were limited
and compatible with an oxidation after-treatment catalyst performance. The penalty resulting from the higher intake flow and EGR rate needed to control the HPC combustion
can be predicted by air-loop 0D calculations. The cooling power for air and EGR is also important to
maintain acceptable intake temperature. The way to reduce compression ratio was explored and led to a significantly increase in HCCI range. The mains drawbacks are the cold starting conditions and the increase in fuel consumption. The challenge is now to find appropriate multiple staged injection strategies to extend the HCCI range
up to higher load and to be fully compatible with the turbocharger performances and cooler
efficiencies. The challenge is now to find appropriate multiple staged injection strategies to extend the HCCI range
up to higher load and to be fully compatible with the turbocharger performances and cooler
efficiencies. IVECO C8 single cylinder engine specifications IVECO C8 single cylinder engine specifications Area of HPC/HCCI combustion in an equivalent fuel/air ratio
and combustion temperature map. combustion at low and medium loads and conventional
combustion at high load. IFP has developed a combustion chamber which is called
NADITM (Narrow Angle Direct Injection), allowing for good
performances with the two combustion modes [1], [6-9]. The general architecture of the engine is very close to a
traditionnal diesel engine with some differences listed below: – narrow spray cone angle to limit fuel liner impingement;
– adapted specific bowl design for fuel wall guiding effect;
– reduced geometric compression ratio;
l i l
i j
i
(C
il f
l i j
i – narrow spray cone angle to limit fuel liner impingement;
d
d
ifi b
l d i
f
f
l
ll
idi
ff – narrow spray cone angle to limit fuel liner impingement;
– adapted specific bowl design for fuel wall guiding effect;
– reduced geometric compression ratio; – narrow spray cone angle to limit fuel liner impingement;
– adapted specific bowl design for fuel wall guiding effect; p y
g
p g
– adapted specific bowl design for fuel wall guiding effect; – reduced geometric compression ratio; Heat release analysis was done using a standard 0D in-
house code. Exhaust gas emissions of CO, HC, NOx, O2 and
CO2 were recorded by Pierburg analysers. An AVL415S
smoke meter was used for soot measurement. – multiple stage injection (Common rail fuel injection
system). Figure 2
Overview of the NADITM combustion system concept. INTRODUCTION far from thermal NOx and soot formation areas identifying
for conventional Diesel combustion with premixed and
diffusion flame. For many reasons, Diesel combustion is a suitable approach
for heavy duty engines but still faces two main drawbacks. The principle of HCCI combustion consists of: The principle of HCCI combustion consists of: On the one hand, the trade-off between NOx and particulate
emissions has to be improved to meet the most stringent
regulations. On the other hand, specific power and torque
must be increased without any loss of thermal efficiency. Selective Catalytic Reduction technology is ready to cope
with Euro IV regulations and the following ones, but
Homogeneous Charge Compression Ignition (HCCI) or
highly premixed combustion (HPC) offer great potential for
meeting future NOx and particulate emissions regulation
target [1 to 20]. On the one hand, the trade-off between NOx and particulate
emissions has to be improved to meet the most stringent
regulations. On the other hand, specific power and torque
must be increased without any loss of thermal efficiency. Selective Catalytic Reduction technology is ready to cope
with Euro IV regulations and the following ones, but
Homogeneous Charge Compression Ignition (HCCI) or
highly premixed combustion (HPC) offer great potential for
meeting future NOx and particulate emissions regulation
target [1 to 20]. – preparing a highly diluted air/fuel mixture by burned gases
recirculation; – promoting simultaneous ignition in the whole space of the
combustion chamber; – controlling precisely the combustion heat release to
achieve best the compromise in terms of efficiency and
pollutant emissions. The main difficulties are linked to the ability to control
combustion speed , to lower pollutant emissions and to avoid
oil dilution. One additional aspect is the reduced HCCI range
leading to a “dual mode” approach, using the HCCI or HPC As shown in Figure 1, HCCI or HPC, which concern more
or less bulk homogeneous mixture, are located in the area 75 T Colliou et al. / Adapting the NADITM Concept to Heavy Duty Engines emissions and combustion noise while maintaining very low
NOx and particulate emissions. Figure 1
Area of HPC/HCCI combustion in an equivalent fuel/air ratio
and combustion temperature map. Temperature
Equivalent ratio
HCCI
Combustion
Soot
Conventional
Diesel
Combustion
NOx 1 EXPERIMENTAL SET-UP The engine is a single cylinder IVECO Cursor 8 engine with
specifications given in Table 1. No oxidation catalyst was used, so the HC and CO
emissions reported in this paper are engine out values. A cylinder pressure transducer was fitted into the cylinder
head to record high-speed data at 0.1 crank angle resolution. TABLE 1 TABLE 1
IVECO C8 single cylinder engine specifications
Displacement
1.3 l
Bore
115 mm
Stroke
125 mm
Connecting Rod Length
200 mm
Valves per cylinder
4
Injection system
DELPHI common-rail
Compression ratio
Adjustable from 8 to 20
Max. in cyl peak pressure
220 bar
Injection
Up to 8 per cycle
Max. BMEP
20 bar
ESC speeds
1300, 1580 and 1870 rpm Adaptation of the NADI™ Concept
to Heavy Duty Engines Developed for passenger car applications, the NADITM
concept was conceived as a universal concept applicable to
any Diesel engine. Its potential is so appealing that the Hy-
SPACE project intended at developing clean heavy duty
engines decided to try it out. The dimensions of the heavy duty engines (bore) are well
in accordance with narrow cone angle approach expectation
in terms of wall wetting. CFD was used in order to accelerate the adaptation of
passenger car know-how to heavy-duty engines. Such an
adaptation was carried out by a widening of piston bowl and
injector spray cone angle. Several bowls and corresponding
injection systems were tested. Overview of the NADITM combustion system concept. Overview of the NADITM combustion system concept. At full load, configuration selection was based on power
requirements, the NADITM chamber with a compression ratio
of 14:1 having to provide as much power as the original
chamber (compression ratio of 17:1) without any fuel
consumption loss and with a peak pressure limit of 220 bar. This paper summarises the results obtained with this
concept on a heavy duty application and presents the
development of this concept on a single cylinder engine
regarding HCCI or HPC operating range, CO and HC Oil & Gas Science and Technology – Rev. IFP, Vol. 61 (2006), No. 1 76 Figure 4
Validation of HCCI investigations. 90
80
70
60
50
40
30
20
10
0
-100 -80
-60
-40
-20
0
Crank angle (°)
20
40
60
80
100
Pressure (bar)
Test bench motored
Test bench fired: 1370 ml/min
@ 100 bar
IFP-C3D: 1200 ml/min
@ 100 bar
IFP-C3D: 1370 ml/min
@ 100 bar After a few CFD iterations, a bowl design was selected
leading to the best compromise between power performances
and HPC running. The injector characteristics were also
adapted to the new combustion chamber geometry: cone
angle, flow, number of holes, as illustrated in Figure 3. The
next selection criterion was emissions levels at part load. An
operating point was chosen and injection strategies tested on
the selected bowls. Results showed that HCCI combustion
was favoured by the configuration with the narrowest spray
cone angle, indicating that an ideal optimum would need to
be found between full load (wider) and part load (narrower)
configurations. The narrow configuration was manufactured
for test bench investigations. Figure 3
NADITM bowls tested and configurations selected after full
load investigations. Adaptation of the NADI™ Concept
to Heavy Duty Engines 90° dome angle
90° spray angle
110° dome angle
100° spray angle Validation of HCCI investigations. 2.1 Conventional Combustion As the standard engine is tuned to reach the EURO IV target
with SCR and without any particulate filter, the results with
the NADITM chamber are presented in comparison to the
serial chamber with the same NOx emissions level (about
8 g/kWh), Figure 5. If the difference of fuel consumption is
limited below 2%, the smoke emissions are slightly increased
at B speed (1580 rpm) as presented below. NADITM bowls tested and configurations selected after full
load investigations. The higher smoke level obtained at part load condition
with the NADITM chamber are not detrimental as these points
will be run in HCCI combustion, which is characterised
by very low smoke level. At higher load, conventional All previous HCCI combustion CFD work was performed
without any test bench data. Once the selected configuration
was manufactured and test bench available a validation of the
previous CFD work was performed by computing a test
bench HCCI operating point. Figure 5
Smoke emissions comparison at B speed (1580 rpm). 0
0.1
0.2
0.3
0.4
0.5
25
50
75
100
Smoke (FSN)
Load (%)
Serial Chamber
NADI chamber Results are illustrated in Figure 4 (in cylinder pressure
curve) corresponding to the running condition described in
Table 2. The trust put in the CFD tools for piston bowl pre-
design for part load HCCI operating conditions is confirmed. Two CFD cases are present: one for the theoretical injector
static mass flow rate and one with the effective injector static
mass flow (that measured on the injector test bed after
manufacturing: 1370ml/min@100bar). 2.2 Homogeneous Combustion Several tests in homogeneous combustion were performed on
steady-state conditions to improve the performance of the
NADITM combustion chamber on the B speed (ESC cycle). Engine parameter settings were optimised looking at EURO V
limits without any NOx after-treatment: rail pressure,
injection timing, number of injection, EGR ratio, injector
mass flow rate and number of hole. Figure 6
Effect of injection pressure on smoke level on B50. Figure 7
Effect of injection pressure on HC and CO emissions on B50. Figure 8
Effect of injection timing on smoke level at different injection
pressure. 0
1
2
3
4
5
6
7
8
9
10
-60
0
Injection timing (°CA)
Smoke (FSN)
1600 bar
1200 bar
800 bar
-50
-40
-30
-20
-10
0
500
1000
1500
2000
2500
3000
3500
4000
4500
Injection pressure (bar)
HC (ppm)
0.0
0.5
1.0
1.5
2.0
2.5
CO (%)
HC
CO
500
750
1000
1250
1500
1750
0
1
500
1750
1500
1250
1000
750
Injection pressure (bar) p
(
y
)
Engine parameter settings were optimised looking at EURO V
limits without any NOx after-treatment: rail pressure,
injection timing, number of injection, EGR ratio, injector
mass flow rate and number of hole. Figure 6 Effect of injection pressure on smoke level on B50. In order to take into account running conditions, limiting
factors have been selected: 0
500
1000
1500
2000
2500
3000
3500
4000
4500
Injection pressure (bar)
HC (ppm)
0.0
0.5
1.0
1.5
2.0
2.5
CO (%)
HC
CO
500
750
1000
1250
1500
1750 Figure 7
Effect of injection pressure on HC and CO emissions on B50. 0
500
1000
1500
2000
2500
3000
3500
4000
4500
Injection pressure (bar)
HC (ppm)
0.0
0.5
1.0
1.5
2.0
2.5
CO (%)
HC
CO
500
750
1000
1250
1500
1750 – Noise: < 95 dB; – NOx: < 0.3 g/kWh; – Particulates: < 0.025 g/kWh; – HC: < 2 g/kWh (compatible with a EURO V after-
treatment performances); – CO: < 5 g/kWh (compatible with a EURO V after-
treatment performances. No oil dilution (very important); – Fuel consumption < + 4% (to be competitive with SCR
approach); – Compatible with standard air loop; – Maximum peak pressure: 220 bar; – Minimum compression ratio: 14:1 (starting condition). High EGR rate operating conditions for a single cylinder engine High EGR rate operating conditions for a single cylinder engine
Rpm
Air flow
Qinj
λ
EGR
Prail
rate (kg/h)
(mg)
rate
(MPa)
1580
35.6
41.4
1.25
55.4%
140 Smoke emissions comparison at B speed (1580 rpm). T Colliou et al. / Adapting the NADITM Concept to Heavy Duty Engines 77 Figure 6
Effect of injection pressure on smoke level on B50. 0
1
2
3
4
5
500
1750
Smoke (FSN)
1500
1250
1000
750
Injection pressure (bar) combustion leads to a slight increase of smoke level. In order
to reduce the gap in terms of particulate emissions, post-
injection is efficient way without any severe damage for fuel
consumption. Unburned hydrocarbon and carbon monoxide
emissions are slightly higher than the conventional chamber
but significantly lower than the Euro V limitation. 0
1
2
3
4
5
500
1750
Smoke (FSN)
1500
1250
1000
750
Injection pressure (bar) 2.2.1 Injection Pressure Several tests have been performed with an early single-
injection approach from 600 bar to the maximum pressure
allowed by the fuel injection system (1600 bar). The engine
setting concerning the EGR rate and the equivalence ratio is
always the same. In these conditions, the injection timing is
adjusted to obtain the lower smoke emission. The best result
in terms of smoke emission with fixed EGR rate and
equivalence ratio is obtained with the maximum injection
fuel pressure. As we can see on Figure 6, the smoke level
decreases from 4.5 to 0.2 when the fuel pressure increases
from 600 to 1600 bar. g
Effect of injection pressure on HC and CO emissions on B50. Figure 8
Effect of injection timing on smoke level at different injection
pressure. 0
1
2
3
4
5
6
7
8
9
10
-60
0
Injection timing (°CA)
Smoke (FSN)
1600 bar
1200 bar
800 bar
-50
-40
-30
-20
-10 0
1
2
3
4
5
6
7
8
9
10
-60
0
Injection timing (°CA)
Smoke (FSN)
1600 bar
1200 bar
800 bar
-50
-40
-30
-20
-10 As seen on Figure 7, higher injection pressure leads to an
effective reduction of unburned hydrocarbons and carbon
monoxide divided by 7. The smoke level curve also depends on the injection
pressure but for early injection timing, no difference can be
noticed. In general, at very early injection timings (50°CA
before top dead center), low in-cylinder density and spray
angle effects lead to fuel over penetration and probably
impingement on cylinder liner surfaces which cause exces-
sive smoke level (Fig. 8). Figure 8 Effect of injection timing on smoke level at different injection
pressure. Oil & Gas Science and Technology – Rev. IFP, Vol. 61 (2006), No. 1 Oil & Gas Science and Technology – Rev. IFP, Vol. 61 (2006), No. 1 78 level give the limit. The results obtained in term of emissions
and noise for the B speed are plotted on Figure 10. In fact, for medium injection timing, the low smoke level
is a result of the flow rate balancing the fuel introduced in
combustion chamber during the auto-ignition delay, and the
spray atomisation. For retarded injection timing, the smoke
level decreases because of the increase of the auto-ignition
delay. It is also possible to obtain a very low particulate
emission level but with an important fuel penalty. 2.2.1 Injection Pressure In order to
highlight the interest of the injection pressure, tests have been
performed with two injectors with different fuel flow rates. Figure 9 presents the results obtained for optimised injection
timing with lowest smoke level. The HC level increase in comparison to the standard
engine but is still compatible with a common after-treatment
system. At low load, CO level is lower than the Cursor 8
engine. Nevertheless, equivalence ratio is increased close to
the maximum HCCI load in order to keep the noise level
below the objective. The consequence of this setting is an
increase both in the carbon monoxide emission level at part
load and in fuel consumption. As expected, the NOx level is
below the target, more than 10 times lower than Cursor 8
engine. The particulate level is very low but slightly higher
than the reference. Figure 9
Effect of injection pressure on B50 for Z injection flows. 0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Smoke (FSN)
1.37 ml/min
1.08 ml/min
Equivalent flow (l/min)
High pressure
Low pressure 0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Smoke (FSN)
1.37 ml/min
1.08 ml/min
Equivalent flow (l/min)
High pressure
Low pressure The fuel penalty for HCCI combustion in comparison to
the results obtained with the standard engine is plotted on
Figure 11. The results do not include turbocharger behavior:
on single cylinder, exhaust back-pressure is higher than the
intake pressure level to promote necessary condition for
EGR. Taking into account these particular conditions, the
HPC combustion has a positive impact at low load. Due to
equivalent ratio setting and as a result of EGR rate and
injection timing, the benefit is reduced so that fuel
consumption is comparable up to 50% load (10 bar BMEP). Equivalent flow l
InjP
CylP
Inj fl
( / min)
(
) /
=
−
⋅
100
ow – InjP: injection pressure (MPa); Thanks to the better homogeneous mixing due to the early
injections, the CO emissions are divided by two. The better
control of the combustion speed leads to a 4dB-noise
reduction. Nevertheless, HC emissions are higher than with
single injection and have to be treated to meet EURO V
limitation. Notice that the intake pressure is lower (about
0.4 bar) and is more compatible with the turbocharger
performances. The new settings allow for an extension of the
HCCI range from 10 to 14 bar BMEP (50 to 70% load). – CylP: cylinder pressure at the beginning of injection
(MPa); – Inj flow: injector flow (l/min @ 10MPa). – Inj flow: injector flow (l/min @ 10MPa). With the same equivalent flow, the best results are
obtained with the higher pressure. 2.2.3 HPC Results with Multiple Stage Injection One possibility offered by the NADITM concept based on the
narrow cone angle is to manage very early injections. This
possibility have been tested under several conditions. If the
interest is limited at low load (heat release controled with
single injection), multiple injection opens new possibilities at
part load and upper load. As shown on Figure12 for 50%
load on B speed condition, the multiple early injection allows
for a better combustion control. The maximum heat release
rate is 50% less and allows for a reduction of EGR and
equivalence ratio setting without exeeding the maximum
noise level allowed. Effect of injection pressure on B50 for Z injection flows. The X-axis represents the equivalent flow in l/min which
is calculated as following: Equivalent flow l
InjP
CylP
Inj fl
( / min)
(
) /
=
−
⋅
100
ow Equivalent flow l
InjP
CylP
Inj fl
( / min)
(
) /
=
−
⋅
100
ow 2.2.2 HPC Results with Single Injection The objective was to reach the highest load in accordance
with the defined targets and with a maximum injection
pressure of 160 MPa. The injection timing was adjusted to
obtain the minimum smoke level with the best fuel
consumption. The EGR temperature was close to 100°C
compatible with a standard cooler efficiency. In these
conditions the maximum load is 10 bar (50% load) BMEP
(Brake Mean Effective Pressure). The noise and the smoke Multiple injection extends the HCCI Range without any
penalty for fuel consumption (Fig. 13). One of the severe constraints is the maximum in-cylinder
pressure that limits the intake pressure improvement. Moreover, the EGR rate is at the minimum to reach the NOx
objective. So, the way to increase the HPC range is the use of
new injection strategies in odder to delay the combustion T Colliou et al. / Adapting the NADITM Concept to Heavy Duty Engines T Colliou et al. / Adapting the NADITM Concept to Heavy Duty Engines
79
0
0.5
1.0
1.5
2.0
HC (g/kWh)
STD
HPC
0
25
50
100
75
Load (%)
0
0.5
1.0
1.5
2.0
2.5
CO (g/kWh)
STD
HPC
0
25
50
100
75
Load (%)
82
84
86
88
90
92
94
96
Noise (dBA)
STD
HPC
0
25
50
100
75
Load (%)
0
0.005
0.010
0.015
0.020
Part. (g/kWh)
STD
HPC
0
25
50
100
75
Load (%)
STD
HPC
0
2
4
6
8
10
12
NOx (g/kWh)
0
25
50
100
75
Load (%)
Figure 10
Results at B speed compared to the standard chamber. 79 0
0.5
1.0
1.5
2.0
HC (g/kWh)
STD
HPC
0
25
50
100
75
Load (%)
0
0.5
1.0
1.5
2.0
2.5
CO (g/kWh)
STD
HPC
0
25
50
100
75
Load (%)
96
0
0.005
0.010
0.015
0.020
Part. 2.2.2 HPC Results with Single Injection (g/kWh)
STD
HPC
0
25
50
100
75
Load (%)
STD
HPC
0
2
4
6
8
10
12
NOx (g/kWh)
0
25
50
100
75
Load (%) STD
HPC
0
2
4
6
8
10
12
NOx (g/kWh)
0
25
50
100
75
Load (%) 0
0.5
1.0
1.5
2.0
HC (g/kWh)
STD
HPC
0
25
50
100
75
Load (%) 0
0.5
1.0
1.5
2.0
2.5
CO (g/kWh)
STD
HPC
0
25
50
100
75
Load (%) 82
84
86
88
90
92
94
96
Noise (dBA)
STD
HPC
0
25
50
100
75
Load (%) Results at B speed compared to the standard chamber. Results at B speed compared to the standard chamber. Oil & Gas Science and Technology – Rev. IFP, Vol. 61 (2006), No. 1 80 -6
-4
-2
0
2
4
6
8
10
12
0
20
40
60
80
100
Fuel penalty (%)
Load (%)
0
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
Heat release rate (1/°CA)
320
330
340
350
360
370
380
390
400 410
Crank angle (°)
Single injection
Multiple injection
Figure 11
Fuel consumption with single injection compared with the
standard chamber (without turbocharger effect). Figure 12
HRR with single and multiple injection . -8
-6
-4
-2
0
2
4
6
8
10
12
0
20
40
60
80
100
Fuel penalty (%)
Load (%)
Single injection
Multiple injection
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1.0
1.2
Pintake - Pexhaust (bar)
0
25
50
75
100
Load (%)
Standard engine
Single injection
Multiple injection
Figure 13
Fuel consumption with single and multiple injection compared
with the standard chamber (without turbocharger effect). Figure 14
Turbocharger effect on low pressure loop efficiency. 0
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
Heat release rate (1/°CA)
320
330
340
350
360
370
380
390
400 410
Crank angle (°)
Single injection
Multiple injection -6
-4
-2
0
2
4
6
8
10
12
0
20
40
60
80
100
Fuel penalty (%)
Load (%) 350
360
370
Crank angle (°) Figure 11 Figure 12
HRR with single and multiple injection . Fuel consumption with single injection compared with the
standard chamber (without turbocharger effect). Fuel consumption with single injection compared with the
standard chamber (without turbocharger effect). HRR with single and multiple injection . 2.2.2 HPC Results with Single Injection g
p
j
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1.0
1.2
Pintake - Pexhaust (bar)
0
25
50
75
100
Load (%)
Standard engine
Single injection
Multiple injection
Figure 14
T
b
h
ff
t
l
l
ffi i -8
-6
-4
-2
0
2
4
6
8
10
12
0
20
40
60
80
100
Fuel penalty (%)
Load (%)
Single injection
Multiple injection
Figure 13
Fuel consumption with single and multiple injection compared
with the standard chamber (without turbocharger effect). -8
-6
-4
-2
0
2
4
6
8
10
12
0
20
40
60
80
100
Fuel penalty (%)
Load (%)
Single injection
Multiple injection Fuel penalty (%) Pintake - Pexhaust (bar) Fuel penalty (%) Figure 13 Turbocharger effect on low pressure loop efficiency. Fuel consumption with single and multiple injection compared
with the standard chamber (without turbocharger effect). timing. The other way based on reducing the compression
ratio will be discussed later. At low load, the turbocharger effect is the same. When the
BMEP increases, the power absorbed by the compressor to
produce high-pressure charge is attainable with a closed
turbine nozzle position. With a multiple injection approach,
the limited EGR rate and the slight increase of exhaust
temperature needed allow for more VNT opening and so
limits the exhaust back pressure. 2.2.4 Air and EGR Loop Calculations 0D calculations for a single stage turbocharger have been
performed with IFP’s in-house code. The results obtained
quantify the impact of the air-loop on the feasibility of the
HPC running conditions for a multicylinder engine. The corrected fuel consumption compared to the standard
engine is presented on Figure 15. Calculations have been performed with a variable
geometry turbine. Its position is adjusted to obtain enough
energy for the compressor driving. The results obtained in
term of pressure difference between intake and exhaust is
presented Figure 14. The fuel penalty is limited up to a 40% load using single
injection. For a higher load, the EGR needed to limit
combustion noise is so great that the exhaust back pressure
leads us to increase fuel penalty by more than 10%. T Colliou et al. / Adapting the NADITM Concept to Heavy Duty Engines 81 Figure 15
Fuel consumption with single and multiple injection compared
with the standard chamber. -8
-6
-4
-2
0
2
4
6
8
10
12
Fuel penalty (%)
0
20
40
60
80
100
Load (%)
Single injection
Multiple injection The intake temperature increases from 40°C to 80°C when
the load increases from 25 to 70%. Those values have to be
compared to the standard variation from 25°C to 50°C at full
load conditions. In this case the cooling power is about
4 times higher than a common Diesel engine. This aspect
should be taken into account for further application as a
potential limitation to extend HCCI range. Fuel penalty (%) Fuel penalty (%) The 0D calculations also give also information on exhaust
temperature downstream from the turbine as presented on the
Figure 17. If the HPC exhaust temperature is comparable to the serial
engine up to 25% load, the difference between the two
configurations reaches 100°C at 70% load. The maximum
temperature for HPC combustion is about 250°C. which
assumes good oxidation catalyst efficiency to be able to treat
the higher level of carbon monoxide and unburned hydro-
carbons. The oxidation catalyst efficiency required to reach
the EURO V target is about 70%. Fuel consumption with single and multiple injection compared
with the standard chamber. 2.2.5 Compression Ratio Effect As we can see above, one limitation of the HCCI range is the
smoke level or the maximum in-cylinder pressure. The
objective is also to introduce all the fuel during the auto
ignition delay. So, one way for an HCCI range extension is to
reduce the compression ratio. Two additional ratios have
been tested, 12:1 and 10:1. A compression ratio of 10:1 was
achieved with a new piston bowl chamber with the same
dome angle and geometry as a compression ratio of 14:1. Because of the oil gallery proximity, it was not possible to
attain the same bowl design with a compression ratio of 10:1. The combustion chamber volume was adjusted by increasing
the squish volume. For the same load, the multiple injection allows us to limit
fuel penalty by less than 4%. With this setting and the single
stage turbocharger, the maximum range for limited fuel
penalty is about 13 bar (65% load) of BMEP. For a higher
load, the intake pressure needs to be reduced to limit the
turbine power necessary, otherwise a double stage turbo-
charger adaptation is necessary. One other aspect for HPC running condition is the power
needed to cool the intake air and EGR mass flow. The
calculations performed for a multicylinder engine in HCCI
mode are compared to the standard engine on Figure 16. Figure 16
Air + EGR power for HCCI running conditions. Figure 17
Exhaust temperature downstream from the turbine for
different combustion modes. 0
10
20
30
40
50
60
70
80
Cooling power (kW)
0
25
50
75
100
Load (%)
Standard engine
Single injection
Multiple injection
0
50
100
150
200
250
300
350
400
450
500
Exhaust temp. (°C)
0
25
50
75
100
Load (%)
Standard engine
Single injection
Multiple injection 0
10
20
30
40
50
60
70
80
Cooling power (kW)
0
25
50
75
100
Load (%)
Standard engine
Single injection
Multiple injection Figure 17
Exhaust temperature downstream from the turbine for
different combustion modes. 0
50
100
150
200
250
300
350
400
450
500
Exhaust temp. (°C)
0
25
50
75
100
Load (%)
Standard engine
Single injection
Multiple injection 0
50
100
150
200
250
300
350
400
450
500
Exhaust temp. 2.2.5 Compression Ratio Effect (°C)
0
25
50
75
100
Load (%)
Standard engine
Single injection
Multiple injection Cooling power (kW) Figure 16 Figure 17 Air + EGR power for HCCI running conditions. Exhaust temperature downstream from the turbine for
different combustion modes. 82 Oil & Gas Science and Technology – Rev. IFP, Vol. 61 (2006), No. 1 With the compression ratio of 14:1, the injection timing
range in accordance with smoke level below the target is
small: between 41 CA and 47 CA. With a compression ratio of
10:1, the smoke target is attainable with an extended injection
timing range. Of course the fuel penalty increases but can be
limited to 5%. Concerning the air loop, the reduced compres-
sion ratio allows to decrease the EGR rate without any
combustion control difficulty. In these conditions, the intake
pressure could be reduced and be more compatible with the
turbocharger performances. Of course fuel consumption is
higher (as discussed before) due to the reduced compression
ratio but this drawback is minimised with the limited effect of
the turbine on the exhaust back-pressure. At a first time, tests were performed in 50% load running
conditions with the same EGR, equivalence ratio and
injection setting. The heat release rate is presented on the
Figure 18. As we can see, the lower compression ratio allows for
smooth combustion with better HRR phasing. With compres-
sion ratio reduction of 4 points, the noise level can be
reduced by 8 dBA The unburned hydrocarbons increase probably due to the
lower in-cylinder density (wall impingement). These results
are presented in Figure 19. Figure 20 presents the smoke level versus injection timing. The smoke level is also significantly lower. Figure 18
Heat release rate depending on compression ratio. Figure 19
HC and smoke level compared to STD engine. Figure 20
Smoke level depending on injection timing. Figure 21
HCCI range with different compression ratio. 2.2.5 Compression Ratio Effect 0
0.02
0.04
0.06
0.08
0.10
0.12
0.14
320
330
340
350
360
370
380
390
400 410
Heat Release Rate (1/°CA)
C.R = 14:1
C.R = 12:1
C.R = 10:1
CA50 = 359.6 °CA
CA50 = 363.6 °CA
CA50 = 355.5 °CA
Crank angle (°)
0
100
200
300
400
500
600
700
800
HC (ppm)
0
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
Smoke (FSN)
HC
Smoke
STD
HCCI/14:1 HCCI/12:1 HCCI/10:1
Compression ratio
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Smoke (FSN)
Injection timing (°CA)
-60
-55
-50
-45
-40
-35
-30
Comp. Ratio = 14:1
Comp. Ratio = 12:1
Comp. Ratio = 10:1
0
5
10
15
20
25
C.R. = 10:1
C.R. = 12:1
C.R. = 14:1
HCCI range (bar)
Single injection
Multiple injection 0
0.02
0.04
0.06
0.08
0.10
0.12
0.14
320
330
340
350
360
370
380
390
400 410
Heat Release Rate (1/°CA)
C.R = 14:1
C.R = 12:1
C.R = 10:1
CA50 = 359.6 °CA
CA50 = 363.6 °CA
CA50 = 355.5 °CA
Crank angle (°) Figure 19
HC and smoke level compared to STD engine. 0
100
200
300
400
500
600
700
800
HC (ppm)
0
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
Smoke (FSN)
HC
Smoke
STD
HCCI/14:1 HCCI/12:1 HCCI/10:1
Compression ratio Heat release rate depending on compression ratio. Heat release rate depending on compression ratio. HC and smoke level compared to STD engine. 0
5
10
15
20
25
C.R. = 10:1
C.R. = 12:1
C.R. = 14:1
HCCI
(b
)
Single injection
Multiple injection 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Smoke (FSN)
Injection timing (°CA)
-60
-55
-50
-45
-40
-35
-30
Comp. Ratio = 14:1
Comp. Ratio = 12:1
Comp. Ratio = 10:1 Smoke (FSN) Figure 21 Smoke level depending on injection timing. HCCI range with different compression ratio. T Colliou et al. / Adapting the NADITM Concept to Heavy Duty Engines 83 the HC and CO emissions are compatible with exhaust
temperatures and oxidation catalyst performances. The main advantage of reducing the compression ratio is
the possible extension of the HCCI range. The limit for each
value is presented on the Figure 21. REFERENCES 1
Walter, B., Monteiro, L., Miche, M. and Gatellier, B. (2004)
Improvement of Exhaust and Noise Emissions of the
NADITM Concept Using PreMixed Type Combustion with
Multiple Stages Injection. SIA Paper. When the maximum range is reached with single injection,
multiple injection opens new settings. It is possible to reduce
noise and smoke emission with a fuel consumption reduction. It is also possible to reduce the EGR rate and increase
equivalent ratio running conditions. These new settings create
the possibility of extending the maximum HPC range up to a
70% load with the compression ratio of 14. 2
Duffy, K., Fluga, E., Faulkner, S., Heaton, D., Schleyer, C. and Sobotowski, R. (2004) Latest Development in Heavy
Duty Diesel HCCI. Which Fuels For Low CO2 Engine? Editions Technip. 3
Duffy, K., Fluga, E., Faulkner, S., Gutmann, P and Herzog,
P.L. (2004) Passenger Vehicle Diesel Engines for the U.S. SAE paper 20004-01-1452. 4
Cowland, C., Gutmann, P. and Herzog, P.L. (2004) Passenger
Vehicle Diesel Engines for the U.S. SAE paper 20004-01-
1452. No-dimensional calculations for a single stage turbo-
charger have been performed with IFP’s in-house code. The
results obtained showed that the high EGR rates needed to
reach HCCI combustion have an important impact on the
turbo-charger running conditions. The higher energy needed
to drive the compressor goes to close the turbine. However,
the consequence on exhaust back pressure is serious and has a
negative effect on fuel consumption. Running with multi-
injection settings, by the reduced EGR rate, allows a
limitation of this drawback. The calculations show the high
cooling (air + EGR) power needed to keep the intake
temperature compatible with the HPC combustion. Therefore,
the power and the exhaust back pressure limitations allow for
an HCCI range up to 13 bar BMEP. The calculations also that 5
Kahrstedt, J., Behnk, K., Sommer, A. and Wormbs, T. (2003)
Combustion Processes to Meet Future Emission Standards. MTZ 10/2003. 6
Weissbäck, M., Csato, J., Glensvig, M., Sams, T. and
Herzog, P. (2003) An Approach for Future HSDI Diesel
Engines. MTZ 9/2003. 7
Umwelt Bundes Amt. (2003) Future Diesel - Exhaust Gas
Legislation for Passenger Cars, Light-Duty Commercial
Vehicles, and Heavy Duty Vehicles - Updating of Limit
Values for Diesel Vehicles. www.umweltdaten.de/uba-info-
presse/ hintergrund/FutureDiesel_e.pdf 8
Walter, B. and Gatellier, B. (2003) Near Zero NOx Emissions
and High Fuel Efficiency Diesel Engine: the NADITM
Concept Using Dual Mode Diesel Combustion. 2.2.5 Compression Ratio Effect Reducing the compression ratio could be an alternative to
extending the HCCI range up to a 75% load with single
injection strategy (compared to 50% with compression ratio
of 14) but the cold starting difficulties and the limited
efficiency could be serious drawbacks. A variable compres-
sion ratio engine is needed to consider using lower values
than 14:1. Multiple injection hasn’t been tested with the lower
compression ratio. It could be possible that the HCCI range
should be extended by 3 or 4 bar more. However, the cold starting difficulties and the lower
efficiency should be major drawbacks for a reduction of
compression ratio below 14:1. The major challenge is now to find new settings which
should allow for an increase in HCCI range with reduction in
EGR and intake mass flow. A two stage turbo-charger loop
seems essential to limit the fuel penalty due to the exhaust
back-pressure. New multi-injection settings and high injec-
tion pressure should be the solution to these problems. SUMMARY AND CONCLUSIONS The work carried out at IFP has allowed a first vision of the
potential of adapting the NADITM chamber to an heavy duty
engine. CFD calculations have allowed us to determine an adapted
chamber which gives good results in conventional combus-
tion. These NADITM chamber performances compared to a
standard engine with a higher compression ratio are not
exactly similar but the difference is limited. For high BMEP
(> 50% load ) the fuel penalty is 1 or 2% higher with a slight
increase in smoke level. The exhaust temperature is compa-
rable to the serial engine. The carbon monoxide and unburned
hydrocarbons emissions are slightly higher be still bellow
EURO V target. ACKNOWLEDGEMENT The authors would like to thank Wilfrid Robert who
conducted the engine tests and Benjamin Reveille who
performed the 3D calculations. The authors are grateful to Delphi who prepared and
delivered the fuel injection system and to Iveco who assist us
for the engine. Many thanks to European Commission for HySpace
project funding. In HPC conditions, the maximum fuel injection system
pressure is used to minimise the smoke level. The limitation
was about 1600 bar and it seems that higher pressure should
improve the results. High pressure is also beneficial for HC
and CO emissions. It also make it possible to reduce smoke
level by a increasing the fuel introduction speed and thus
allowing for better combustion timing. Copyright © 2006 Institut français du pétrole
Permission to make digital or hard copies of part or all of this work for personal or classroom use is granted without fee provided that copies are not made
or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page. Copyrights for components of this
work owned by others than IFP must be honored. Abstracting with credit is permitted. To copy otherwise, to republish, to post on servers, or to redistribute
to lists, requires prior specific permission and/or a fee: Request permission from Documentation, Institut français du pétrole, fax. +33 1 47 52 70 78,
or revueogst@ifp.fr. REFERENCES Oil and Gas
Science and Technology, 58, 1, pp. 101-114. Oil & Gas Science and Technology – Rev. IFP, Vol. 61 (2006), No. 1 84 15 Kimura, S., Aoki, O., Ogawa, H. and Muranaka, S. (1999)
New Combustion Concept for Ultra-Clean and High-
Efficiency Small DI Diesel Engines. SAE Paper 1999-01-
3681. 09 Gatellier, B. and Walter, B (2002) Development of the High
Power NADITM
Concept Using Dual Mode Diesel
Combustion to Achieve Zero NOx and Particulate Emissions. Thiesel 2002 Conference on thermo- and fluid Dynamic
Processes in Diesel Engines. 16 Christensen, M., Hultqvist, A. and Johansson, B. (1999)
Demonstrating the Multi Fuel Capability of a Homogeneous
Charge Compression Ignition Engine with Variable Com-
pression Ratio. SAE Paper 1999-01-3679. 10 Walter, B. and Gatellier, B. (2002) Development of the High
Power NADITM Concept Using Dual Mode Diesel Combus-
tion to Achieve Zero NOx and Particulate Emissions. SAE
paper 2002-01-1744. 17 Richter, M., Franke, A., Aldén, M., Hultqvist, A. and
Johansson, B. (1999) Optical Diagnostics Applied to a
Naturally Aspired Homogeneous Charge Compression
Ignition Engine. SAE Paper 1999-01-3649. 11 Gatellier, B., Walter, B. and Miche, M. (2001) New Diesel
Combustion Process to Achieve near Zero NOx and
Particulates Emissions. IFP International Congress - A new
generation of engine combustion processes for the future? 18 Iwabuchi, Y., Kawai, K., Shoji, T. and Takeda, Y. (1999)
Trial of New Concept Diesel Combustion System – Premixed
Compression – Ignited Combustion. SAE Paper 1999-01-
0185. 12 Hultqvist, A., Engdar, U., Johansson, B. and Klingmann, J. (2001) Reacting Boundary Layers in a Homogeneous Charge
Compression Ignition (HCCI) Engine. SAE Paper 2001-01-
1032. 19 Odaka, M., Suzuki, H., Koike, N. and Ishii, H. (1999) Search
for Optimizing Method of Homogeneous Charge Diesel
Combustion. SAE Paper 1999-01-0184. 13 Kimura, S., Aoki, O., Kitahara, Y. and Aiyoshizawa, E. (2001) Ultra-Clean Combustion Technology Combining a
Low – Temperature and Premixed Combustion Concept for
Meeting Future Emission Standards. SAE Paper 2001-01-
0200. 20 Akagawa, H., Miyamoto, T., Harada, A., Sasaki, S.,
Shimazaki, N., Hashizume, T. and Tsujimura, K. (1999)
Approaches to Solve Problems of the Premixed Lean Diesel
Combustion. SAE Paper 1999-01-0183. 14 Christensen, M. and Johansson, B. (2000) Supercharged
Homogeneous Charge Compression Ignition (HCCI) with
Exhaust Gas Recirculation and Pilot Fuel. SAE Paper 2000-
01-1835. Final manuscript received in November 2005
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Simplified Generation of Biomedical 3D Surface Model Data for Embedding into 3D Portable Document Format (PDF) Files for Publication and Education
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PloS one
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cc-by
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Abstract The usefulness of the 3D Portable Document Format (PDF) for clinical, educational, and research purposes has recently been
shown. However, the lack of a simple tool for converting biomedical data into the model data in the necessary Universal 3D
(U3D) file format is a drawback for the broad acceptance of this new technology. A new module for the image processing
and rapid prototyping framework MeVisLab does not only provide a platform-independent possibility to create surface
meshes out of biomedical/DICOM and other data and to export them into U3D – it also lets the user add meta data to these
meshes to predefine colors and names that can be processed by a PDF authoring software while generating 3D PDF files. Furthermore, the source code of the respective module is available and well documented so that it can easily be modified
for own purposes. Citation: Newe A, Ganslandt T (2013) Simplified Generation of Biomedical 3D Surface Model Data for Embedding into 3D Portable Document Format (PDF) Files
for Publication and Education. PLoS ONE 8(11): e79004. doi:10.1371/journal.pone.0079004 Editor: Peter M.A. van Ooijen, University of Groningen, University Medical Center Groningen, Netherlands Received July 23, 2013; Accepted September 25, 2013; Published November 15, 2013 Copyright: 2013 Newe, Ganslandt. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors acknowledge support by Deutsche Forschungsgemeinschaft (DFG) and Friedrich-Alexander-University Erlangen-Nuremberg within the
funding programme Open Access Publishing. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: axel.newe@fau.de Competing Interests: The authors have declared that no competing interests exist. * E-mail: axel.newe@fau.de * E-mail: axel.newe@fau.de Several authors have proven these 3D models embedded into
PDF documents to be useful for electronic publication in biology
[2,3], (bio-)chemistry [4,5,6,7] and medicine [8,9,10,11,12] and
superior over alternative solutions. Spatial relationships (like the
vessel systems in the liver or neuronal fibers in the central nervous
system) can easily be differentiated and perceived much better
than by textual description [2,13]. Abstract The consumer of a document is
not dependent on the one single view the author has selected for a
3D scene, but can freely decide which view(s) shall be used for a
printout, based on his own preferences or interests. Furthermore,
the interaction aspect might be a trigger for a detailed exploration
driven by the reader’s curiosity [2]. Even journals start requesting
their authors to embed multimedia content directly into their
publications [14], because the former concept of supplemental
external resources undermines the concept of a completely self-
contained document with all necessary information [15]. Simplified Generation of Biomedical 3D Surface Model
Data for Embedding into 3D Portable Document Format
(PDF) Files for Publication and Education Axel Newe1*, Thomas Ganslandt2
1 Chair of Medical Informatics, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany, 2 Medical Centre for Information and Communication
Technology, University Hospital Erlangen, Erlangen, Germany 1 Chair of Medical Informatics, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany, 2 Medical Centre for Information and Communication
Technology, University Hospital Erlangen, Erlangen, Germany Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany, 2 Medical Centre for Information and Communicatio
Erlangen, Erlangen, Germany The Portable Document Format with Embedded 3D
Models The Portable Document Format with Embedded 3D
Models The Portable Document Format (PDF) is a comprehensive
document description language used to define electronic docu-
ments independently of its creating, displaying and printing
software, hardware and operating system. A PDF file encapsulates
all resources to completely describe the content and layout of an
electronic document, including texts, fonts, images and multime-
dia elements without the need of external resources. Starting with version 1.6 of the PDF Specification [1],
implemented and published first in 2007 with Adobe Acrobat
3D Version 8 and Adobe Reader 8.1, three-dimensional mesh
models can be embedded into this widely known and used file
format (more than 500 million users worldwide, according to
Adobe: http://www.adobe.com/uk/pdf/), which has been the de-
facto standard for the exchange of electronic documents for years
now. An alternative is not in sight. Besides that, it is a simple fact, that much of raw data in science
is 3D by its nature: molecules, microscopic and macroscopic
anatomy, propagation of radiation – traditional ways of presenting
this kind of data in 2D come with an inherent loss of information. No 2D image, illustration, stereograph or descriptive text will ever
describe 3D data as precisely and in full extent as a 3D
representation can do and therefore should. The
Adobe
Reader
(http://get.adobe.com/reader/
otherversions/) offers many options to display these mesh models
(solid surface, transparent surface, wireframe, point cloud, contour
lines, illumination) and to let the user interact with them (zooming,
panning, rotating, selection of components). Using embedded
scripting, even complex animations and interaction with other
components (e.g. form elements) of the respective PDF document
are possible. Background and Related Work A general major issue regarding the exchange of medical data is
privacy and security. PDF provides the possibility to encrypt
documents (with AES or RC4) and to sign them digitally. Although [17] has proved that PDF security is not waterproof in
all respects, the contents of PDF documents themselves could not
be disclosed. This makes PDF documents suitable for the exchange
of medical data. In 2008, the Association for Information and
Image Management (AIIM) has released a Best Practice Guide for
the implementation of PDF in healthcare (AIIM BP02–2008), also
known
as
PDF/H
(http://www.aiim.org/Research-and-
Publications/Standards/Articles/PDF-Healthcare, [18]), that is
officially accepted by Adobe [19]. The Universal 3D (U3D) File Format (
)
PDF allows importing two different 3D model file formats: the
Product Representation Compact (PRC) format and the Universal
3D (U3D) format. Although PRC is the older format (first
appearance around 2002) and published as ISO 14739-1, U3D
seems to have become more accepted and is nowadays available as
export format for many software applications dealing with 3D
models. It was initially defined as an exchange format for 3D
model data in Computer Aided Construction (CAD) by a
consortium of companies related to this industry (including e.g. Intel, Siemens and Boeing). In December 2004, the Ecma
International (formerly known as European Computer Manufac-
turers Association, ECMA) published the first edition of its
standard ECMA-363 (Universal 3D File Format); the latest version
is the 4th edition from June 2007 [21]. The replacement of the last tool in this chain is not reasonable. Some kind of PDF authoring tool will always be needed since it
cannot be expected that an application that generates 3D scene
data also provides the ability to set text layout, process screenshots
etc. Therefore a one-click-solution as discussed by [6] is not really
feasible, but the number of tools should be reduced to a maximum
of two applications: one for generating the 3D scene data and one
for generating the final PDF. In this paper, we present a novel way
to create this scene data. Background and Related Work PDF Features and Suitability for Biomedical Documents
The PDF specification (latest version 1.7, extension level 5) is
very well documented and available to the full extent from its
developer
Adobe
(http://www.adobe.com/devnet/pdf/
pdf_reference.edu.html). The usage is free of charge, as well as
the Adobe Reader that is available for all major operating systems
(MS Windows, Mac OS, Linux) and currently the only software
for displaying and printing PDF documents that fully supports all
features of PDF (including multimedia and 3D). Adobe Acrobat is
the Reader’s commercial counterpart for creating and editing PDF
documents. Although there are many commercial and free tools
available for creating PDF files or for converting other documents
into PDF, Adobe Acrobat is the only off-the-shelf software that
fully supports all PDF features (especially regarding 3D models:
http://convert-pdf-software-review.toptenreviews.com/). Besides
that, it is also available for Windows, Mac OS and Linux. Universal 3D is a binary file format that contains all necessary
information to describe a 3D scene graph. This includes the
geometry data, palette definitions, lighting, cameras (‘‘views’’),
texturing and pre-defined animations (‘‘motions’’). A U3D scene consists of an arbitrary number of objects that can
be sorted in a monohierarchic object tree. The geometry of each
object can be defined as a triangulated surface mesh, a set of lines
or a set of points (‘‘point cloud’’). For smooth rendering, the level
of detail can be defined depending on the distance to the viewpoint
(CLOD – Continuous Level of Detail). A proprietary bit encoding
algorithm allows for a highly compressed storage of the geometry
data. The possibility to re-use resources once defined (e.g. objects
of the same geometry with different colors) further contributes to
the reduction of the final file size [21]. U3D files are sequences of ‘‘blocks’’, always starting with a ‘‘File
Header Block’’ (block type 0x00443355, which reads as ‘‘U3D’’ in
ASCII). The File Header Block is followed by ‘‘Declaration
Blocks’’ and ‘‘Continuation Blocks’’. Declaration Blocks contain
information about the objects (e.g. mesh definitions or texture
resources) in the file and Continuation Blocks can provide
additional information for objects declared in a Declaration Block
(e.g. the vertex coordinates of a mesh) [21]. PDF specification 1.7 is also published by the International
Organization for Standardization as ISO 32000–1:2008 [16] and
fulfills all requirements for an interactive publication document as
postulated by Thoma et. al. [15]. Simplifying the Generation of U3D Model Data The generation of the necessary mesh model data is still
cumbersome. Previous authors needed a tool chain of at least three
[6,8] or even four [11] different software applications and up to 22 November 2013 | Volume 8 | Issue 11 | e79004 PLOS ONE | www.plosone.org 1 Generation of Biomedical 3D Model Data for PDF only hardware that is currently not capable of rendering 3D scenes
is the growing field of tablet computers. single steps until the final PDF was created. Furthermore, some of
these tools are not available for all platforms (OsiriX only for
MacOS, used by [11]), are commercial software with closed source
and license costs (Amira and Adobe 3D Toolkit, used by [2]) or
need intermediate file formats and processing steps (MeshLab,
used by [6] & [8]). The WEMSaveAsU3D module and an auxiliary
ComposeWEMDescriptionForU3D module The WEMSaveAsU3D module and an auxiliary
ComposeWEMDescriptionForU3D module The new WEMSaveAsU3D module that has recently been
integrated into the standard distribution of MeVisLab saves
WEMs that consist of triangle faces into U3D files as defined in
Standard ECMA-363. If a WEM contains more than one patch,
each patch is converted to a U3D object. Therefore, each patch
should have a unique name, specified by its ‘‘Label’’ property. If
the names of the patches in a WEM are not unique (or not
specified at all), the module creates new (unique) names for the
U3D file. Within the U3D file, each U3D object carries the name
(label) of the WEM patch it was created from. In MeVisLab, surface meshes are internally represented as
Winged Edge Meshes (WEM) as proposed by Baumgart [26,27]. Each WEM in MeVisLab can consist of a number of WEM
‘‘patches’’, whereat each patch represents a closed set of ‘‘faces’’
that in total form the surface of a 3D model. These faces can be
polygonal, but triangles are preferred and recommended. The
standard distribution of MeVisLab contains about 4 dozens of pre-
defined modules for creating, rendering, loading, saving and
manipulating WEMs, including the ‘‘WEMIsoSurface’’ module
that can be directly used to create a surface mesh out of a DICOM
image (e.g. a segmentation mask). More U3D object properties can be specified using the
‘‘Description’’ property of a WEM patch: the color (including
transparency) of a single object and of an object group, the
reflective color of an object, the name of an object group and the
name of the overall model. These additional U3D properties need
to be composed to a single string and thereafter written to the
‘‘Description’’ property of a WEM patch to be parsed by the
module. The helper module ComposeWEMDescriptionForU3D
facilitates the generation of valid string encoded U3D properties. The standard ‘‘WEMSave’’ module of MeVisLab provides the
possibility to store WEM meshes in different formats, i.a. the
popular STL format (STereoLithography format [28], also known
as Standard Tessellation Language) into a file, but meta data
besides the pure surface geometry is exported only for the
proprietary binary Winged Edge Mesh format. The current version of the WEMSaveAsU3D module does not
implement all U3D features of the ECMA-363 standard. It is
limited to triangle meshes, coloring, lighting and grouping of
objects into a tree hierarchy. The missing features are discussed
below. Generation of Biomedical 3D Model Data for PDF Tutorial’’ is recommended to be perused by newcomers. It is
available
for
direct
download
(http://www.mevislab.de/
fileadmin/docs/current/MeVisLab/Resources/Documentation/
Publish/SDK/GettingStarted.pdf) as well as with the MeVisLab
installation (Menu ‘‘Help’’ R ‘‘Show Help Overview’’ R ‘‘Getting
Started’’). Tutorial’’ is recommended to be perused by newcomers. It is
available
for
direct
download
(http://www.mevislab.de/
fileadmin/docs/current/MeVisLab/Resources/Documentation/
Publish/SDK/GettingStarted.pdf) as well as with the MeVisLab
installation (Menu ‘‘Help’’ R ‘‘Show Help Overview’’ R ‘‘Getting
Started’’). An additional module named ‘‘ComposeWEMDescription-
ForU3D’’ was created to facilitate the user-friendly generation of
meta data necessary for coloring and naming U3D objects. This
module was implemented as a MeVisLab Macro Module using
Python as programming language by reason that it is not a time-
critical module and way easier to modify and extend this way. The modular design of MeVisLab allows for simple assembling
of image processing ‘‘networks’’ [24] and comes with more than
800 pre-defined standard components (‘‘modules’’). About 1800
additional modules completely wrap ITK and VTK, which makes
the total module base very comprehensive. MeVisLab has been
evaluated as a very good platform for creating application
prototypes using visual data-flow programming [25], is very well
documented and supported by an active online community. The WEMSaveAsU3D module and an auxiliary
ComposeWEMDescriptionForU3D module To
overcome
this
lack,
a
new
export
module
named
‘‘WEMSaveAsU3D’’ was created. Since U3D files can contain
very detailed information about objects and the whole scene, a
functional extension of the existing WEMSave module that
predominantly only stores geometry data was not reasonable. As
all modules for MeVisLab, the WEMSaveAsU3D was written in
C++. Microsoft Visual Studio 2008 was used for editing and
compiling the source code, as well as for debugging. The module
class inherits from the ‘‘WEMInspector’’ base class since it serves
as final module in a WEM processing chain. A New Module for MeVisLab To achieve the goal of simplifying the creation of U3D files by
reducing the number of necessary tools to only one application in
(but not limited to) the field of biomedical image processing, a new
module for MeVisLab (http://www.mevislab.de/) was created. MeVisLab
is
an
image
processing
framework
and
visual
development environment, developed by MeVis Medical Solutions
AG and Fraunhofer MEVIS (formerly MeVis Research GmbH) in
Bremen, Germany. It is available for all major platforms (MS
Windows,
Mac
OS
and
Linux:
http://www.mevislab.de/
download/) and offers a variety of licensing options, including a
‘‘MeVisLab SDK Unregistered’’ license which is free for use in
non-commercial
organizations
and
research
(http://www. mevislab.de/mevislab/versions-and-licensing/). MeVisLab
can
not only be used as a toolbox for simple image processing, but
also as a framework for creating sophisticated applications with
graphical user interfaces that hide the underlying platform and do
not require substantial programming knowledge [22,23,24]. The
general usage of MeVisLab is explained in its comprehensive and
easy-to-understand
documentation
(http://www.mevislab.de/
developer/documentation/). Especially
the
‘‘Getting
Started In addition to that, DICOM Supplement 104: ‘‘DICOM
Encapsulation of PDF Documents’’ [20] defines a SOP Class to
encapsulate PDF documents into a Composite DICOM SOP
Instance using the Secondary Capture object, so that PDF files can
be exchanged using the appropriate DICOM Service Classes. Caveats regarding the PDF format with embedded 3D models
discussed by other authors (e.g. [2]) are almost obsolete. Long-time
compatibility and readability should be solved with the transfer of
the PDF specification to ISO 32000. Even simple desktop
hardware is nowadays capable of displaying interactive 3D scenes. In the case that processing power is not sufficient for a smooth
rendering, Adobe Reader dynamically reduces details during the
interaction and renders again with full details right after the
interactive manipulation of the respective scene has ended. The November 2013 | Volume 8 | Issue 11 | e79004 PLOS ONE | www.plosone.org 2 Generation of Biomedical 3D Model Data for PDF Usage of the Modules A detailed description of both modules and their usage is
available with the MeVisLab documentation as well as online
(http://www.mevislab.de/docs/current/MeVisLab/Standard/
Documentation/Publish/ModuleReference/WEMSaveAsU3D. html). To simplify the adding of new features, a set of tool methods was
implemented and the complete set of constant definitions (e.g. material attributes and block type codes) of the ECMA-363
Standard were made available in a dedicated C++ Header file
(WEMSaveAsU3D_Definitions.h, Figure 1). )
Figure 2 shows the basic usage of the two modules; the
MeVisLab network in the upper part (A) is the standard example
network for the WEMSaveAsU3D module and implements the
simplest processing chain: loading of a mesh, modifying the U3D
properties and saving the U3D file. For MeVisLab novices, we
strongly recommend reading the ‘‘Getting Started’’ tutorial
mentioned above to understand how to create and work with a
MeVisLab network. For a quick assessment of our modules, follow
the instructions in Figure 3 to reproduce and use this example
network. The
source
code
was
verbosely
annotated
to
facilitate
programmers to understand and expand the implementation. Almost every line of code that is directly related to the U3D
standard has a comment pointing to the respective chapter of the
ECMA-363 document (Figure 1). Figure 1. Code snippet of pre-defined constants. This code snippet from WEMSaveAsU3D_Definitions.h shows comments pointing to the
chapters of the ECMA-363 standard where the respective block type constants are defined. doi:10.1371/journal.pone.0079004.g001 Figure 1. Code snippet of pre-defined constants. This code snippet from WEMSaveAsU3D_Definitions.h shows comments pointing to the
chapters of the ECMA-363 standard where the respective block type constants are defined. doi:10.1371/journal.pone.0079004.g001 November 2013 | Volume 8 | Issue 11 | e79004 November 2013 | Volume 8 | Issue 11 | e79004 PLOS ONE | www.plosone.org 3 Generation of Biomedical 3D Model Data for PDF Figure 2. Example Network for the new MeVisLab modules. (A) This example network illustrates the basic usage of the WEMSaveAsU3D
module and the ComposeWEMDescriptionForU3D module. The network is available with the standard distribution of MeVisLab (right-click on the
instance of a WEMSaveAsU3D module and select ‘‘Show Example Network’’). The LocalWEMLoad module loads a 3D model defined in Object File
Format (‘‘venus. off’’, part of the MeVisLab demo data) and the WEMSaveAsU3D modules writes it into a U3D file. The ComposeWEMDesription-
ForU3D module sets the color of the model as well as object and group names. The result is displayed on the bottom (B). Usage of the Modules doi:10 1371/journal pone 0079004 g002 Figure 2. Example Network for the new MeVisLab modules. (A) This example network illustrates the basic usage of the WEMSaveAsU3D
module and the ComposeWEMDescriptionForU3D module. The network is available with the standard distribution of MeVisLab (right-click on the
instance of a WEMSaveAsU3D module and select ‘‘Show Example Network’’). The LocalWEMLoad module loads a 3D model defined in Object File
Format (‘‘venus. off’’, part of the MeVisLab demo data) and the WEMSaveAsU3D modules writes it into a U3D file. The ComposeWEMDesription-
ForU3D module sets the color of the model as well as object and group names. The result is displayed on the bottom (B). doi:10.1371/journal.pone.0079004.g002 Figure 2. Example Network for the new MeVisLab modules. (A) This example network illustrates the basic usage of the WEMSaveAsU3D
module and the ComposeWEMDescriptionForU3D module. The network is available with the standard distribution of MeVisLab (right-click on the
instance of a WEMSaveAsU3D module and select ‘‘Show Example Network’’). The LocalWEMLoad module loads a 3D model defined in Object File
Format (‘‘venus. off’’, part of the MeVisLab demo data) and the WEMSaveAsU3D modules writes it into a U3D file. The ComposeWEMDesription-
ForU3D module sets the color of the model as well as object and group names. The result is displayed on the bottom (B). doi:10.1371/journal.pone.0079004.g002 Figure 2. Example Network for the new MeVisLab modules. (A) This example network illustrates the basic usage of the WEMSaveAsU3D
module and the ComposeWEMDescriptionForU3D module. The network is available with the standard distribution of MeVisLab (right-click on the
instance of a WEMSaveAsU3D module and select ‘‘Show Example Network’’). The LocalWEMLoad module loads a 3D model defined in Object File
Format (‘‘venus. off’’, part of the MeVisLab demo data) and the WEMSaveAsU3D modules writes it into a U3D file. The ComposeWEMDesription-
ForU3D module sets the color of the model as well as object and group names. The result is displayed on the bottom (B). doi:10.1371/journal.pone.0079004.g002 November 2013 | Volume 8 | Issue 11 | e79004 PLOS ONE | www.plosone.org 4 Generation of Biomedical 3D Model Data for PDF Figure 3. Quick reference to creating and using the modules. These screenshots illustrate how to create and use the modules for U3D export. 1. (A) Create a new network (Menu ‘‘File’’ R‘‘New’’). 2. (A) Create an instance of the WemSaveAsU3D module (type the name into the ‘‘Search
Modules’’ field (1) and hit Enter). Usage of the Modules The module icon (2) should appear in the workspace. 3. (A) Open the example network of the module (right-click the
module icon (2) and select ‘‘Show Example Network’’ (3) from the context menu). 4. (B) A new network tab and two module panels should open
automatically. (If not, open the panels manually by double-clicking the module icons of WemSaveAsU3D and ComposeWEMDescriptionForU3D.) 5. (B) Modify the U3D model properties using the ComposeWEMDescriptionForU3D panel (4). 6. (B) To save the U3D file, go to the WemSaveAsU3D
panel, specify a file name (5) and click ‘‘Save’’ (6). Other surface models (e.g. in the popular STL format) can be loaded by means of the LocalWEMLoad
module (double-click the respective module icon and select the ‘‘Browse’’ button from the module panel). doi:10.1371/journal.pone.0079004.g003 Figure 3. Quick reference to creating and using the modules. These screenshots illustrate how to create and use the modules for U3D export. 1. (A) Create a new network (Menu ‘‘File’’ R‘‘New’’). 2. (A) Create an instance of the WemSaveAsU3D module (type the name into the ‘‘Search
Modules’’ field (1) and hit Enter). The module icon (2) should appear in the workspace. 3. (A) Open the example network of the module (right-click the
module icon (2) and select ‘‘Show Example Network’’ (3) from the context menu). 4. (B) A new network tab and two module panels should open
automatically. (If not, open the panels manually by double-clicking the module icons of WemSaveAsU3D and ComposeWEMDescriptionForU3D.) 5. (B) Modify the U3D model properties using the ComposeWEMDescriptionForU3D panel (4). 6. (B) To save the U3D file, go to the WemSaveAsU3D
panel, specify a file name (5) and click ‘‘Save’’ (6). Other surface models (e.g. in the popular STL format) can be loaded by means of the LocalWEMLoad
module (double-click the respective module icon and select the ‘‘Browse’’ button from the module panel). doi:10.1371/journal.pone.0079004.g003 Figure 4 shows a more complex processing chain. The
corresponding network is provided as File S1. Figure 5 gives an
impression of a human femur, that has been segmented with
MeVisLab and exported to U3D using various names and colors. formats (MCS, STL, VRML) and workflow discontinuity (see
Figure 5 for a segmentation result example). A Simple and Straight Forward Way to Create 3D Model
Data for Embedding in PDF g
The WEMSaveAsU3D module for MeVisLab offers a simple
way to create U3D files from surface meshes of biomedical data. It
works ‘‘out-of-the-box’’ and comes with the standard MeVisLab
distribution. The creation of the surface mesh itself can be
completely handled within MeVisLab; the result can be exported
directly into a U3D file. MeVisLab is available for free and for all
three major platforms (Windows, Mac OS and Linux). By using
MeVisLab for the generation of U3D model data, the direct
export function of 3D content into PDF as demanded by
conclusion #3 in [8] is almost fulfilled. The WEMSaveAsU3D
module does not create a PDF file, but a U3D file with all
necessary meta data for a direct import using PDF authoring
software. The replacement of this authoring step seems not
reasonable since 3D content will probably never be the only
content of a PDF. By combining free PDF authoring tools like
LaTeX (http://www.latex-project.org/) or the iText library
(http://itextpdf.com/) with U3D models coming from MeVisLab,
the complete PDF authoring process can be performed without
any commercial software and on any of the major platforms. Usage of the Modules And even if the problem itself has already been solved by
another software, MeVisLab and the WEMSaveAsU3D module
can still be used to convert existing model data into U3D if the
model surface is available in a popular alternative format
(STereoLithography/Standard Tessellation Language, Object File
Format, Wavefront or Polygon File Format). MeVisLab also offers
the possibility to convert Open Inventor Scenes into WEMs which
then can be exported into U3D as shown in Figure 4. November 2013 | Volume 8 | Issue 11 | e79004 Further Development Example of a more complex application network. (A) This example network simulates a complex image processing chain (read from
bottom to top). The network generates an Open Inventor Scene with a cube and a sphere as ‘‘segmentation results’’ (B). The two objects are then
converted into WEM patches (SoWEMConvertInventor modules) and the properties (names and colors) are set (WEMModify modules). Finally the two
WEM patches are merged into one WEM and afterwards written into a U3D file. The result is displayed on the bottom right (C). A file containing this
network is provided as File S1. doi:10.1371/journal.pone.0079004.g004 Figure 4. Example of a more complex application network. (A) This example network simulates a complex image processing chain (read from
bottom to top). The network generates an Open Inventor Scene with a cube and a sphere as ‘‘segmentation results’’ (B). The two objects are then
converted into WEM patches (SoWEMConvertInventor modules) and the properties (names and colors) are set (WEMModify modules). Finally the two
WEM patches are merged into one WEM and afterwards written into a U3D file. The result is displayed on the bottom right (C). A file containing this
network is provided as File S1. doi:10.1371/journal.pone.0079004.g004 The DICOM Supplement 132 [31] defines a Surface Segmen-
tation Storage SOP Class based on triangle meshes. Although
MeVisLab currently does not comprise an import module for
DICOM Supplement 132 files, it is desirable to add one as heir to
the ‘‘WEMGenerator’’ base class. Once implemented, such
a module would close the gap between generic DICOM
segmentation results stored as surface meshes and their conversion
into U3D files for embedding into PDF. constricted by representation in a 2D figure that should ideally be
presented as a 3D model to reveal the full information content. The last missing feature of U3D is pre-defined animation
(‘‘motion’’ in U3D terms) which is limited to rotation and
translation - a model deformation is not possible. This makes it
impossible to display e.g. the dynamics of a beating heart whereas
the animation of moving joints and their adjacent bones is
conceivable, e.g. for educational purposes. Although the currently available version of the WEMSaveA-
sU3D module cannot utilize any of the previously discussed U3D
features, their implementation is planned for future releases. Further Development There are four U3D features of minor importance for
biomedical imaging still missing as regards WEM export from
MeVisLab: labeling, textures, alternative geometry definition
(point clouds & line set) and pre-defined animations. The possibility to embed 3D labels (‘‘2D Glyphs’’ in U3D
terminology, demonstrated in fig. 2 and fig. 7 of [29]) makes it easy
to clearly identify objects within the space of an interactive 3D
scene, independently from the view selected by the user. Especially
for PDFs with educational purpose as discussed in [12], e.g. for
teaching anatomy to medical students, an undoubtful labeling of
structures
with
complex
spatial
relationships
can
be
very
serviceable. Application of textures to 3D models (e.g. a human face as
demonstrated in Additional File #1 of [10]), is of limited utility,
except for a simulated volume rendering as shown in fig. 5 of [29]. The main disadvantages of this simulated volume rendering are
fixed windowing and file size. The rendering can be embedded
with only one pre-defined window setting that must match the
preference and intent of the viewer. In Addition to that, a
complete set of textured slices for each of the three Cartesian axes
must be embedded, which inflates the file size. On the other hand,
simulated volume rendering within PDF documents offers a new
way of publishing biomedical 3D images. The generation of the model surface still has to be done, but
that is also a step MeVisLab can be used for. More than 2600
image processing modules (including ITK and VTK) provide a
vast potential of finding a solution for many biomedical image
processing and analysis challenges. To give an example: all four software applications used for
segmentation & surface mesh generation (Mimics by Materialise),
scene assembling (Maya by Alias), object categorization (Deep
Exploration Standard by Right Hemisphere) and coloring (3D
Reviewer by Adobe) for the Visible Korean Project [12] could
probably have been replaced by a single MeVisLab image
processing network, thus avoiding the use of intermediate file U3D models can be defined as point clouds (fig. 1 of [29]) or
line sets. The latter could be used for visualization of vessel
centerlines, nervous fiber tracking or 3D ECG diagrams. Fig. 2(B)
of [30] is a good example of 3D ECG data visualization November 2013 | Volume 8 | Issue 11 | e79004 PLOS ONE | www.plosone.org 5 Generation of Biomedical 3D Model Data for PDF Figure 4. Further Development Since
the source code of the module is verbosely commented and
available with the MeVisLab distribution since version 2.4, the
implementation can also be done by any user with sufficient C++
programming skills. All necessary tool methods and constants for
writing the respective U3D Modifier Blocks and Resource Blocks
(chapter 9.7 and 9.8 of [21]) are already implemented and used by
the current version of the module. The source code can be found
after the complete installation of MeVisLab in [Install Path]/
Packages/MeVisLab/Standard/Sources/ML/MLWEMModules/
WEMSaveAsU3D. File Size Considerations The last and probably most important issue regarding U3D
data and the respecting PDF files incorporating them is the overall
file size. 3D model data can be very large: one of the results of the
Visible Korean Project [12], a highly detailed 3D PDF of a male
head has a size of almost 100 MiBytes even though the raw data
has been reduced reasonably. To achieve the smallest possible file
size while preserving the most of the comprising information, an
intelligent reduction of the number of surface triangles is
inevitable. The ‘‘WEMReducePolygons’’ module of MeVisLab
allows for reducing the number triangles by collapsing edges using
a Quadric Error Metric [32]. Each of these collapse operations PLOS ONE | www.plosone.org November 2013 | Volume 8 | Issue 11 | e79004 6 Generation of Biomedical 3D Model Data for PDF introduces an error in the resulting mesh. Edges that cause as little
error as possible are collapsed first thus preserving as much of the
original shape as possible. Using this method, triangles defining
plane surfaces have highest priority to be replaced by a more
coarse mesh. A good e
triangle reduction strat
fig. 1 of [8]: the top fac
triangles that could be
Figure 5. Model of a segmented femur. Model of a left human femur segmented with MeVisLab
surface between compact bone and spongy bone (green) and the surface of the bone marrow (blu
doi:10.1371/journal.pone.0079004.g005 i
d
i
h
l i
h Ed
h
li l
h A
d
l f
bl
li
i
f hi
Figure 5. Model of a segmented femur. Model of a left human femur segmented with MeVisLab. The model shows the outer surface (red), the
surface between compact bone and spongy bone (green) and the surface of the bone marrow (blue). doi:10.1371/journal.pone.0079004.g005 Figure 5. Model of a segmented femur. Model of a left human femur segmented with MeVisLab. The model shows the outer surface (red), the
surface between compact bone and spongy bone (green) and the surface of the bone marrow (blue). doi:10.1371/journal.pone.0079004.g005 coarse mesh. A good example for a reasonable application of this
triangle reduction strategy is the orthodontic model embedded in
fig. 1 of [8]: the top face of the model is composed of hundreds of
triangles that could be reduced to a number of only 7 without introduces an error in the resulting mesh. References (2011)
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June 2013. 6. Kumar P, Ziegler A, Grahn A, Hee CS, Ziegler A (2010) Leaving the structural
ivory tower, assisted by interactive 3D PDF. Trends Biochem Sci 35: 419–422. 22. Koenig M, Spindler W, Rexilius J, Jomier J, Link F, et al. (2006) Embedding
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Supplementary version of this article with
embedded 3-d figures. (PDF) Modern science produces data with three-dimensional nature in
many disciplines. PDF technology offers the possibility to publish
this data in all its dimensions and should therefore be used
accordingly. With MeVisLab and only one additional PDF
authoring tool, the complete process of generating 3D PDF
documents for biomedical publications can be handled in a
consolidated working environment, free of license costs and with File Size Considerations Edges that cause as little
error as possible are collapsed first thus preserving as much of the
original shape as possible. Using this method, triangles defining
plane surfaces have highest priority to be replaced by a more November 2013 | Volume 8 | Issue 11 | e79004 November 2013 | Volume 8 | Issue 11 | e79004 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 7 Generation of Biomedical 3D Model Data for PDF all major operating systems. The new WEMSaveAsU3D module
does not feature all capabilities of the U3D standard, but covers
most of the current use cases for 3D visualization in the biomedical
domain. Due to the availability of the well documented source
code, additional features can be added with low effort if needed. losing any information. The creator of the final model has to make
a tradeoff between model details and file size, but in most cases the
number of triangles can be reduced by a large percentage without
losing substantial information while greatly reducing file size. From our experience, a reduction rate of 95% (based on a voxel-
precise mesh) is acceptable for most illustrational purposes and was
applied for Figure 5. Regarding modern broadband internet
connections and network speed, file sizes of around 10 MiBytes
should not be a problem. Supporting Information File S1
MeVisLab network file of the image processing
chain shown in Figure 4 (A). (MLAB) Author Contributions Conceived and designed the experiments: AN. Wrote the paper: AN TG. Implemented the software: AN. References Accessed 10 June 2013. 32. Garland M, Heckbert PS (1997) Surface simplification using quadric error
metrics. Proc. 24th Annual Conference on Computer Graphics and Interactive
Techniques: 209–216. 17. Castiglione A, De Santis A, Soriente C (2010) Security and privacy issues in the
Portable Document Format. J Syst Software 83: 1813–1822. November 2013 | Volume 8 | Issue 11 | e79004 PLOS ONE | www.plosone.org 8
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An Empirical Approach to Phishing Countermeasures Through Smart Glasses and Validation Agents
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To cite this version: Jema David Ndibwile, Edith Talina Luhanga, Doudou Fall, Daisuke Miyamoto, Gregory Blanc, et
al.. An empirical approach to phishing countermeasures through smart glasses and validation agents. IEEE Access, 2019, 7, pp.130758-130771. 10.1109/access.2019.2940669. hal-02438600 An empirical approach to phishing countermeasures
through smart glasses and validation agents
Jema David Ndibwile, Edith Talina Luhanga, Doudou Fall, Daisuke
Miyamoto, Gregory Blanc, Youki Kadobayashi HAL Id: hal-02438600
https://hal.science/hal-02438600v1
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abroad, or from public or private research centers. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access Date of publication xxxx 00, 0000, date of current version xxxx 00, 0000. Digital Object Identifier 10.1109/ACCESS.2017.DOI JEMA DAVID NDIBWILE1, EDITH TALINA LUHANGA2, DOUDOU FALL1, DAISUKE
MIYAMOTO4, GREGORY BLANC3, AND YOUKI KADOBAYASHI.1, (IEEE, Member) ,
,
, (
1Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, Japan, 630-0192
2Nelson Mandela African Institution of Science and Technology, P.O.Box 447, Arusha, Tanzania
3Samovar, CNRS, Télécom SudParis, Institut Polytechnique de Paris, 9 rue Charles Fourier, 91011 Evry Cedex, France
4Tokyo University, 7 Chome-3-1 Hongo, Bunkyo City, Tokyo, Japan 113-8654 “This work was supported by JSPS KAKENHI Grant Number JP17K00180.” “This work was supported by JSPS KAKENHI Grant Number JP17K00180.” ABSTRACT Phishing attacks have been persistent for more than two decades despite mitigation efforts
from academia and industry. We believe that users fall victim to attacks not only because of lack of
knowledge and awareness, but also because they are not attentive enough to security indicators and visual
abnormalities on the webpages they visit. This is also probably why smart device users, who have more
limited screen size and device capabilities compared to desktop users, are three times more likely to fall
victim to phishing attacks. To assert our claim, we first investigated general phishing awareness among
different groups of smartphone users. We then used smart eyeglasses (electro-oculographic) to experimen-
tally measure the mental effort and vigilance exhibited by users while surfing a website and while playing
an Android phishing game that we developed. The results showed that knowledge and awareness about
phishing do not seem to have a significant impact on security behaviours, as knowledgeable participants
exhibited insecure behaviours such as opening email attachments from unfamiliar senders. However,
attentiveness was important as even participants with low cybersecurity knowledge could effectively identify
attacks if they were reasonably attentive. Based on these results, we asserted that users are more likely to
continue falling victim to phishing attacks due to insecure behaviours, unless tools to lessen the identification
burden are provided. We thus recommended implementing a lightweight algorithm into a custom Android
browser for detecting phishing sites deceptively without a user interaction. We used fake login credentials
as validation agents and monitor the destination server HTTP responses to determine the authenticity of a
webpage. We also presented initial evaluation results of this algorithm. INDEX TERMS Social engineering, Phishing, Smart glasses, Mobile devices, Deceptive login, Android
browser, Electro-oculographic, Cybersecurity psychology. INDEX TERMS Social engineering, Phishing, Smart glasses, Mobile devices, Deceptive login, Android
browser, Electro-oculographic, Cybersecurity psychology. VOLUME 4, 2016 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. JEMA DAVID NDIBWILE1, EDITH TALINA LUHANGA2, DOUDOU FALL1, DAISUKE
MIYAMOTO4, GREGORY BLANC3, AND YOUKI KADOBAYASHI.1, (IEEE, Member)
1Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, Japan, 630-0192
2Nelson Mandela African Institution of Science and Technology, P.O.Box 447, Arusha, Tanzania
3Samovar, CNRS, Télécom SudParis, Institut Polytechnique de Paris, 9 rue Charles Fourier, 91011 Evry Cedex, France
4Tokyo University, 7 Chome-3-1 Hongo, Bunkyo City, Tokyo, Japan 113-8654 JEMA DAVID NDIBWILE1, EDITH TALINA LUHANGA2, DOUDOU FALL1, DAISUKE
MIYAMOTO4, GREGORY BLANC3, AND YOUKI KADOBAYASHI.1, (IEEE, Member)
1Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, Japan, 630-0192
2Nelson Mandela African Institution of Science and Technology, P.O.Box 447, Arusha, Tanzania
3Samovar, CNRS, Télécom SudParis, Institut Polytechnique de Paris, 9 rue Charles Fourier, 91011 Evry Cedex, France
4Tokyo University, 7 Chome-3-1 Hongo, Bunkyo City, Tokyo, Japan 113-8654 I. INTRODUCTION emails and instant messages purporting to be from a trusted
contact, and the victims are deceived to click on a malicious
link. Clicking the link can result in installation of malicious
software (malware), locking of the user’s computing devices
(ransomware) or redirection to a fake website. T T
HE increase in the use of smart devices such as smart-
phones and the increasing amount of important infor-
mation they store make them a prime target by attackers in-
terested in exploiting them [1]. The exploitation can either be
through the device itself or a human. The most common and
classic human exploitation is phishing, which mostly relies
on the naivety of users. Phishing is an art of deception where
a user is fraudulently convinced by an attacker to divulge
sensitive information such as credit card or login credentials. The attacks are launched via electronic communications e.g., The number of phishing attacks is increasing every year
[2]. Research shows that despite the persistence of cyber-
attacks that rely on the naivety of users e.g., phishing, cy-
bersecurity knowledge among users is globally still low [3]. Several mitigation strategies have been proposed [4] [5] [7]
[8], but preventing attacks that target user-application inter- 1 VOLUME 4, 2016 This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access p Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS actions is still a major challenge as the deception techniques
used and the fake websites deployed are increasingly sophis-
ticated. Falling victim to attacks when using mobile devices
is also three times more likely than on desktops [6] due
to the devices’ limited screen size and capabilities. Limited
computational power, for instance, limited the number of PC
solutions that can be implemented on smart devices. A simple
example is the omission of mobile phone browser plugins. Likewise, web features such as URL bar are not spacious
enough to accommodate a lengthy URL address which is
normally truncated and, sometimes, hidden. Regardless of
these additional challenges, little has been done to address
this. I. INTRODUCTION As smartphone penetration and internet usage via mobile
devices continue to rise, it is important to understand users’
cybersecurity behaviours and what factors are most responsi-
ble for cybersafety behaviours. were more consistent and positive than non-tech-savvy par-
ticipants. The tech-savvy participants who exhibited higher
mental efforts had a higher phishing identification rate than
those with lower or average efforts. However, despite the
correlations among non-tech-savvy participants not being as
strong as the tech-savvy participants, it was still positive and
linear. Based on these findings, we believe that users in general
are more likely to continually fall victim to phishing attacks
due to insecure behavior and negligence regardless of being
educated or aware of the attack. From a usable security view-
point, we believe that the burden to identify phishing attacks
should not entirely be borne by the users. Thus, to help
smart device users to browse safely online, we recommend
an Android application prototype that can be incorporated
inside a native Android browser. The prototype uses dummy
login credentials to thwart phishing sites, as phishing sites
almost always allow users to proceed to the next page even
if the credentials are wrong [44]. Incorporating the prototype
inside a native browser means the user does not need to install
third-party security plugins or download another standalone
app. It also removes the necessity of the user to manually
check whether a website is a phishing one. In this study, we aimed to investigate whether the role of
users’ cybersecurity knowledge level, attention and vigilance
to security indicators displayed on smartphone web browsers
have an impact on their abilities to identify phishing (fake)
websites from legitimate ones. We first conducted a survey
with 206 participants to determine their self-reported cy-
bersecurity knowledge, attitudes and behaviours, and to ob-
jectively determine how accurate their self-reported knowl-
edge levels were. We then sampled 40 participants (50%
computer scientists (tech-savvy) and 50% non-computer sci-
entists (non-tech-savvy) from different demographics) and
assessed the mental effort they used when viewing a mixture
of fake and legitimate websites via an Android game that
we developed. Mental effort was estimated through smart
eyewear (JINS MEME glasses) which can detect eye and
body movement and horizontal and vertical electrooculogram
(EOG) signals which are amplified by metal dry electrodes
near the nasion and rhinion parts of the nose and eye [9]. II. BACKGROUND AND RELATED WORK This section highlights some works that analyze human cog-
nitive ability through eye movements on performing certain
tasks. It also describes some previous works that show the
usefulness of validation agents such as dummy login creden-
tials and how a user can avoid entering web-login credentials
manually. 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 In
the game, we kept count of the number of correctly identified
phishing websites. We hypothesized that while cybersecurity
professionals would easily spot phishing sites or emails, av-
erage users would have varying degrees of success depending
on their vigilance. II. BACKGROUND AND RELATED WORK B. MENTAL EFFORT ON PHISHING IDENTIFICATION The idea of analyzing eye movements for correlating them
with some cognitive tasks has recently been gaining pace,
influenced by smart-eyewear technologies. "Eye can tell"
by Miyamoto et al. [10] is one of the classic work of eye-
movement analysis for estimating the attentiveness of users
on phishing webpages, however it was carried out on a
PC environment equipped with an eye-tracking device, and
not using an eyewear. Thus, it is neither suitable for small
devices nor capable of revealing a user response to changes
in her mind and body. The experiment was solely limited
to the use of a display device (computer) for verifying what
kind of information a user checks on a website for phishing
identification. Similarly, a strong correlation between eye movements
and activities such as reading has been observed in several
research where the number of words a user reads is estimated
[11]. However, the estimation is solely based on a powerful
computer and a display device which highly limits its im-
plementation in several environments. In one of the recent
works, Kunze et al. implemented EOG-based technique with
JINS MEME smart glasses to estimate the word counts read
by a user [12]. EOG-based technique through devices like
JINS MEME smart glasses has also been used in a range of
unobtrusive activity tracking. Ishimaru et al. used an early
prototype of JINS MEME smart glasses, and demonstrated
how simple eye-movement visualization, body posture, and
eye blink can be used to recognize and analyze certain human
activity patterns such as talking, reading, and walking [13]. Shahriar et al. proposed and implemented a desktop-based
testing tool named Phishtester [7]. Phishtester works by test-
ing the trustworthiness of a number of suspicious websites
through a provision of unknown random inputs to the login
page. The tester checks the login page response against the
pre-established known symptoms for a malicious site. This
solution uses Finite State Machine (FSM) logic and only
works based on a trigger that depends on certain conditions,
signifying phishing attempt, to be true as pre-specified. Like
any other rule-based system, if all the antecedent(s) of a
rule are true, then the system is triggered. This might not
be suitable to all problem domains, it is only suitable when
a system behavior can be decomposed into separate states
with well-defined conditions for state transitions [16]. Wu
et al. III. PHISHING AND PRIVACY KNOWLEDGE OF USERS
This section describes how we conducted our first study, from
data collection to analysis and presents the findings. Briefly, the discussed related approaches were mainly im-
plemented in PC in which they require huge amount of com-
putational power that might completely not work in small-
sized devices or cause undesirable user experiences. Some
features that might be necessary for security functionality for
PCs are removed in mobile devices in order to accommodate
their limited computational resources. For instance, browser
plugins for mobile devices are often omitted or ignored due
to their resource drainage. Browser developers point of view
for not including plugins and extensions is the unmanageable A. WEB LOGIN AUTOMATION Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access
thor et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS consumption of resources (CPU/GPU and RAM). When
these plugins are active on mobile devices they may lead to an
uncontrollable browsing experience and power consumption. Furthermore, these works require intensive analysis of the
features to identify malicious sites including the dependence
on certain rules (rule based) to be true or certain amount of
traffic for them to work. automatically submitted, without user intervention, to the
web service based on the login endpoint and the expiration
date of the login token. The challenge with these solutions
is the requirement of an extra Bluetooth-enabled device and
browser plugins which are computationally expensive for a
small device. Another important approach to address phishing attacks
is by testing the trustworthiness of the login webpage by
injecting random login inputs into the webpage form fields. However, most of these works are browser-based plugins, and
particularly designed to Mozilla Firefox browser. Yue et al. proposed Firefox browser-based solution to protect against
phishing attacks with bogus bites [5]. A Firefox browser
extension transparently inputs a relatively large number of
bogus credentials into a suspicious phishing website, rather
than attempts to prevent vulnerable users from browsing it. These bogus bites conceal victims’ real credentials among
bogus credentials and enable legitimate websites to identify
stolen credentials in a timely manner. However, the installa-
tion must be done at both client and server side. Users need to
install BogusBiter and a legitimate server needs to deploy the
defensive line enabled by BogusBiter. Moreover, the other
concern regarding a massive deployment of BogusBiter is
that if the login page of a legitimate website is wrongly
flagged as a phishing page, the load on the authentication
web server will increase significantly due to a large number
of bogus bites. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. B. MENTAL EFFORT ON PHISHING IDENTIFICATION proposed MobiFish [4] mobile application, which is
implemented through Optical Character Recognition (OCR)
for checking visual similarities between malicious and legiti-
mate websites. However, visual similarity approaches have a
tendency of missing well-presented phishing webpages [17]. From this perspective, the cognitive effort can be estimated
through the eye movements when a user is trying to identify
a phishing website. The information obtained can be used
by the solution developers to gain an insight on what proper
security indicators to add or omit into the existing anti-
phishing tools. III. PHISHING AND PRIVACY KNOWLEDGE OF USERS
This section describes how we conducted our first study, from
data collection to analysis and presents the findings. A. WEB LOGIN AUTOMATION To avoid entering web-login credentials, a user of a smart
device such as smartphone can authenticate herself to another
device such as a laptop or another mobile device through a
close-range Bluetooth communication. This strategy prevents
phishing attacks as it uses a multi-factor authentication. Han et al. introduced a Bluetooth-enabled smart device
as a platform to store the login information of a user such
as ID and password [14]. A smart device pre-stores infor-
mation features of a login user interface. Then before the
user enters the authentication information in another device,
a plugin on her web browser communicates with the smart
device through Bluetooth to verify the login credentials. Af-
ter passing the login credential verification, the smart device
automatically fills the login information to the login page
on behalf of the user. However, for performance issues most
smartphone browsers are not equipped with plugins. To summarize, our research objectives were: 1) To examine security behaviours of users pertaining to
phishing. 2) To examine the impact of mental effort in identifying
phishing attacks on websites and emails. 3) To use the results of objectives number 1 and 2 to
improve our previous solution for phishing detection
and intervention. Our findings for the first study revealed that a majority of
the participants are aware of security issues but it does not
translate into security-conscious behaviours. For instance, we
noticed that education level does not significantly influence
the behaviours of users in phishing attacks. In our second
study, we observed some positive correlations between the
estimated mental effort of the users and phishing attack iden-
tification. The correlations among tech-savvy participants Similarly, Bridge et al. introduced a method for automati-
cally submitting login credentials, seamlessly, for a user of
a web service [15]. The login information and credentials
corresponding to the login form of a web service are stored
and then used to authenticate the user for a session of the web
service. A login token, generated by the web service, and its
expiration date are tracked. The login credentials are then 2 2 VOLUME 4, 2016 This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. VOLUME 4, 2016 TABLE 1. PARTICIPANTS’ DEMOGRAPHIC FOR A USER STUDY Privacy Concerns and Preferences: The majority of par-
ticipants confirmed that protecting personal privacy was very
important (n=155, 75%) and that they would benefit more if
they received security advice and tips on how to protect their
devices. A. STUDY TOOLS The study used the following tools: The study used the following tools: • JINS MEME smart glasses • Android smartphone (Nexus 7) with a phishing game
• Smartphone for recording JINS MEME readings • Android smartphone (Nexus 7) with a phishing game • Smartphone for recording JINS MEME readings JINS MEME is an eyewear that can detect head and eye
movements [9]. The prototype looks just like normal eye
glasses and it is made of 3 electrodes (electrooculography)
and motion sensors (accelerometer and gyroscope) around
the nose to detect eye and head movements respectively,
as well as a Bluetooth LE module to stream the data to a
computer, smartphone or tablet. JINS MEME smart glasses
measure EOG signals of roughly over 100 Hz generated by
human brain non-invasively and over 50 Hz of the motion
sensor. Additionally, JINS MEME provides raw data that
could easily be visualized and retrieved in CSV. The smart
glasses battery can operate up to 8 hours [13]. B. USER STUDY RESULTS Cybersecurity Knowledge and Behaviours: Participants aged
between 45-54 were most confident with their knowledge
level (38.46% reported high level and 61% reported low)
whereas participants aged between 18-24 were least confi-
dent (11.9% reported high and 88.1% reported low). Female
and male participants had about the same level of confidence
with 19.15% and 20.50% respectively reporting high. Overall
22.8% of participants felt they had low cybersecurity knowl-
edge while 59.22% felt they had just enough knowledge to
protect themselves online and 18% had reported themselves
as highly knowledgeable. We developed a simple Android quiz game for participants
with three main interfaces. The start screen shows a start
button, a score line and the page number. The second screen
contains 20 different pages which are a mix of emails and
webpages. The pages include both legitimate and phishing
samples and the user has to select which sample (phishing
or legitimate) is being shown on each page. The last screen
shows the player’s score at the end of the quiz. We twisted
the webpage and email features such as URL, logo, graphic,
structure to look as close and authentic to legitimate ones so
that it is difficult to tell the difference. Thus, to get a good
score, a user needs a certain level of vigilance to identify a
phishing web page. Fig. 1 shows the sample interfaces from
the game. We sampled the phishing emails and websites from
https://www.phishing.org/ and https://www.phishtank.com/. Their contents are already verified as phishing by the re-
spective organizations. For the legitimate ones, we collected
random legitimate emails and websites. The objective assessment of actual knowledge revealed
that these assessments were highly inaccurate (Table 2). An alarming number of the participants did have very
little cybersecurity knowledge as evidenced by the fact that
59.71% (n=123) being likely or very likely to open links from
unknown emails, which is the common means of launching a
phishing attack. This means a vast majority of the participants
are susceptible to phishing attacks (p<0.001, Student’s t-test)
[40]. In a one-way multivariate analysis of variance with
both Welch’s [41] and Hotelling Lawley t-test [42], we found
that education levels have no significant impact on security-
conscious behaviours. IV. ESTIMATION OF USER MENTAL EFFORT ON A
PHISHING PAGE for their time and they had to meet the following criteria to
be eligible: for their time and they had to meet the following criteria to
be eligible: This section outlines the second part of the study - estimation
of mental effort displayed when viewing websites on smart
devices, and the implications of mental effort level on phish-
ing website identification. We present the tools used for the
experiment, the procedure and the results. • Own either an Android- or an iOS-based smartphone. • Have experience with basic smartphone operations The survey was administered online and consisted of sev-
eral questions, both multiple-choice and open-ended. The
first part of the survey collected participants’ demographic
details including education level, age and gender, and the
second part asked questions on cybersecurity knowledge (e.g. "how much do you know about information security?", "how
and when did you learn about it?", "are software updates
important for your device?"), cybersecurity behaviours (e.g. "do you update your device when prompted?", "do you
open link from an unknown email sender?", "how often
do you read internet policies, terms and conditions?") and
privacy concerns and preferences (e.g. "do you lock your
smartphone?", "what are the attributes of a good password?",
"would you like to receive security tips and advice for your
device?"). Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS TABLE 1. PARTICIPANTS’ DEMOGRAPHIC FOR A USER STUDY
Education Level
Percentage
Non-graduate
24.75%
Undergraduate
48.5%
Postgraduate
26.75% TABLE 1. PARTICIPANTS’ DEMOGRAPHIC FOR A USER STUDY This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. p 10.1109/ACCESS.2019.2940669, IEEE Access
Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS A. STUDY METHOD We conducted a survey with 206 people (62.35% male,
37.65% female) of varying education levels. Table 1 shows
the proportion of participants without university degrees,
with undergraduate degrees, and with postgraduate degrees. The average age was 30 years (σ = 7.15) and 33 years (σ =
13) for male and female participants, respectively. Recruit-
ment was conducted online. Participants were compensated VOLUME 4, 2016 VOLUME 4, 2016 3 This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access p This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. B. EXPERIMENT SETUP Despite the low cybersecurity knowledge levels and inse-
cure behaviours, participants were strongly motivated to pro-
tect their private data and acted to do so. The most prevalent
security behaviour was the use of screen lock authentication
on smartphones (n=165, 80%). We sampled 40 participants to participate in the study, 50%
computer science students and 50% non-computer science
students. Table 3 shows the education level and IT expertise
of the participants. 4 VOLUME 4, 2016 VOLUME 4, 2016 This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS FIGURE 1. Screen shots of the game showing the starting screen, sample webpage/email and a user’s score. FIGURE 1. Screen shots of the game showing the starting screen, sample webpage/email and a user’s score. TABLE 2. PHISHING SUSCEPTIBILITY BASED ON OUR ASSESSMENTS
Demographic
Category
Susceptibility by (%)
Age
18 - 24
57.14%, n=40/70
25 - 34
63.77%, n=44/69
35 - 44
68.42%, n=26/38
45 - 54
44.83%, n=13/29
Average
59.70%, n=123/206
Education
Non graduate
66.67%, n=34/51
Undergraduate
57.00%, n=57/100
Postgraduate
65.45%, n=36/55
FIGURE 2. JINS MEME smart glasses worn by one of the participants. FIGURE 1. Screen shots of the game showing the starting screen, sample webpage/email and a user’s score. FIGURE 1. Screen shots of the game showing the starting screen, sample webpage/email and a user’s score. FIGURE 1. Screen shots of the game showing the starting screen, sample webpage/email and a user’s score. FIGURE 2. JINS MEME smart glasses worn by one of the participants. TABLE 2. PHISHING SUSCEPTIBILITY BASED ON OUR ASSESSMENTS Demographic
Category
Susceptibility by (%)
Age
18 - 24
57.14%, n=40/70
25 - 34
63.77%, n=44/69
35 - 44
68.42%, n=26/38
45 - 54
44.83%, n=13/29
Average
59.70%, n=123/206
Education
Non graduate
66.67%, n=34/51
Undergraduate
57.00%, n=57/100
Postgraduate
65.45%, n=36/55
Average
66.65%, n=127/206
Gender
Female
58.97%, n=46/78
Male
60.15%, n=77/128
Total Average
59.71%, n=123/206 FIGURE 2. JINS MEME smart glasses worn by one of the participants. FIGURE 3. Sample signal recordings by the JINS MEME official App showing
a participant’s focus rate while taking up a quiz. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. C. JINS MEME EXPERIMENT RESULTS We observed a strong positive correlation between the mental
effort measured and quiz scores among tech-savvy users
and inconsistent correlation among non-tech-savvy users. Therefore, tech-savvy users with higher mental effort had
a higher phishing identification rate than those with lower
or average effort. We calculated the correlation between the
mental effort and quiz score for the tech-savvy users by
using Pearson’s product correlation [43] as shown in Table
4 and Fig. 4. The calculated correlation coefficient (r) is
given by the Equation 1 and Equation 2. Whereas, the rxy
is the measure of linear dependence or association between
x (mental effort) and y (phishing quiz score), and it ranges
between -1 and 1. r =
Pn
i=1(xi −¯x)(yi −¯y)
pPn
i=1(xi −¯x)2pPn
i=1(yi −¯y)2
(1)
r = rxy =
n P xiyi−P xi
P yi
p
n P x2
i −(P xi)2p
n P y2
i −(P yi)2 (2) (1) FIGURE 5. Correlation between mental focus and quiz score for
non-tech-savvy participants. (2) coefficient (r) is 0.4882285 (about average) and data signifi-
cance with p-value = 0.001393. FIGURE 4. Correlation between mental focus and quiz score for tech-savvy
participants. TABLE 3. PARTICIPANTS’ EDUCATION AND EXPERTISE FOR THE JINS
MEME EXPERIMENT Demographic
Category
Education
Undergraduate: 27.5%
Postgraduate: 72.5%
IT Expertise
Tech-savvy: 50%
Non-tech-savvy: 50% Demographic
Category
Education
Undergraduate: 27.5%
Postgraduate: 72.5%
IT Expertise
Tech-savvy: 50%
Non-tech-savvy: 50% The procedure was as follows: JINS MEME is turned on
and connected via Bluetooth to a smartphone that has the offi-
cial application for recording the signals. A user puts on JINS
MEME glasses with the sensors intact to the nose pad and
bridge. When the user is ready, she gives a signal and start
the quiz. The starting of the quiz is synchronized with the
start of the smart glasses recording by its application. Once
a user completes the quiz, the recording also synchronously
stops. The smart glasses use its own designated algorithm to
calculate and estimate the mental effort of a user by checking
frequently gazed points, starred points, deep focuses, normal
focuses, eye movements, blink power and when a user is not
focusing at all. Fig. 2 shows the JINS MEME smart glasses FIGURE 3. Sample signal recordings by the JINS MEME official App showing
a participant’s focus rate while taking up a quiz. worn by one of the participants and Fig. 3 shows the varying
levels of focus exhibited during the experiment. 5 VOLUME 4, 2016 This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access p Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS TABLE 4. A SUMMARY STATISTICAL TABLE FOR FIGURE 4
t
df
p-value
confidence interval
correlation
7.0281
18
1.472e-06
0.6658
0.9418
0.8561035
FIGURE 5. Correlation between mental focus and quiz score for
non-tech-savvy participants. D. POST-EXPERIMENT INTERVIEW After every game session, we held a brief interview about the
participants’ experience. We also played the game with each
participant in order to explain the answers. All participants
agreed that the game and the scores they archived were fair. However, we noticed that a good number (about 38%) of par-
ticipants, even among the tech savvy, did not know some of
the phishing indicators. Some of the missed indicators were
just due to lack of attention and some were due to lack of
knowledge or experience with phishing attacks. Most notably
was the https://www.amazonn.co.jp/ where most participants
missed the additional ‘n’ because naturally, the text was not
big enough due to the screen size, and roughly, it was easy
to think that the URL was legit. Additionally, some of the
participants had no idea that if you are addressed as "Dear
user, Dear friend, Dear customer etc." could be one of the
biggest hints that you are on a phishing email. Legitimate
businesses address their customers with their names but
phishers do not often know the identity of their targets. FIGURE 4. Correlation between mental focus and quiz score for tech-savvy
participants. For the non-tech-savvy participants, we noticed two
things: some participants with extremely higher mental effort
did not necessarily get higher quiz scores in the game and
some participants with lower mental effort did not necessarily
got lower quiz scores. We believe that the participants who
had lower quiz scores despite higher mental effort did not
have even a slight idea of how the phishing sites look. On the other hand, the participants who got higher scores
despite exhibiting less mental effort may have just guessed
the correct answers or had a great knowledge of the subject. However, on average, non-tech-savvy participants who ex-
hibited reasonable mental effort performed as well as much
as most of the tech-savvy participants. Fig. 5 shows the
mental effort of non-tech-savvy users against phishing quiz
scores and its statistical summary on Table 5. Red dots denote
the odd outcomes i.e. high mental efforts with lower scores
and vice versa. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. B. RECOMMENDED SYSTEM STRATEGY FOR
PHISHING DETECTION ON SMARTPHONES Based on our conducted studies and experiments on smart-
phone behaviours of users and the estimation of their mental
efforts on identifying phishing attacks, we reckon that users
require automated assistance for phishing attacks. Users are
responsible for understanding how phishing attacks work and
make efforts to learn about it through various awareness
programs. However, leaving the entire burden of phishing
identification to users may not be a plausible idea as we have
seen that awareness alone is not enough. For instance, when
a user is tired or exhausted she can make irrational decision
despite being aware of the phishing attacks. Thus, we recom-
mend an anti-phishing strategy that can independently and
automatically detect phishing attacks and intervenes during
the user login process. The key focus of the strategy is low-
computational power requirement. As we have already dis-
cussed in Section II.A, most existing anti-phishing strategies
are not suitable for small-sized devices such as smartphones. Our study, like few others [17] [18], indicates that being
aware of phishing attacks alone is not enough for a user
to stay safe from phishing attacks. The awareness of risks
has to be linked to a perceived vulnerability or a mitiga-
tion strategy. Downs et al. found that prior experience and
knowledge of phishing attacks architecture help to predict
behavioural responses to phishing attacks [19], but this is not
contrary to our findings or those from the studies in [17] [18]
because experience with phishing attacks and architecture
is very different from mere awareness about existence of
the attacks. The study by Downs et al. further suggests
that deeper understanding of the web architecture, such as
being able to precisely interpret URLs, page redirection and
understanding other cues such as what a lock signifies, is
associated with less vulnerability to phishing attacks. It also
posits that perceived severity of the consequences does not
predict secure behaviour. Thus, efforts to educate users on the
architectures and security features on browsers, rather than
merely informing them on what phishing is and warning them
about the risks is necessary. Many universities now require
students to read policies related to IT facility usage before
being granted access to use them, and these policies usually
have sections on cybersecurity [20] [21]. In spite of this, our
study showed that even people with higher education level
do not necessarily have higher cybersecurity knowledge. users should be employed. FIGURE 6. Correlation between mental focus and quiz score for all
participants. Additionally, Chen et al. posit that behavioral characteris-
tics such as intolerance of risk, curiosity, and trust can be used
to predict individual ability to identify phishing interfaces. In "Real or Bogus", they investigated characteristics of users
and how they influence their ability to predict phishing at-
tacks. They found that participants who are intolerant of risk
were more likely to regard legitimate interfaces as phishing. In contrast, participants who were more trusting and less
curious performed better on a phishing security quiz [24]. In our study, we found that women, who are usually more
risk-averse in cyber and other behaviours than men [25] [26],
were only less susceptible if they had higher education levels. This might be because higher education level means they are
generally more computer-literate, and therefore understand
or have had experiences with cyber-attacks such as computer
viruses or spam messages. Statistically, our results show that
the females whose education levels are at least bachelor
degrees are less susceptible to phishing than men of the
same education levels (21% vs. 38% respectively exhibited
insecure behaviours). On the other hand, females with no
bachelor degrees are more susceptible than men in the similar
category (31% vs. 12.70%, respectively). FIGURE 6. Correlation between mental focus and quiz score for all
participants. A. FACTORS THAT CAN SUPPORT PHISHING SITE
IDENTIFICATION The results derived from our study with JINS MEME eye-
wear suggest that the combination of good computer literacy
and reasonable attention to a webpage has a positive impact
in identifying phishing attacks. However, little focus can
easily lead a user into skipping important cues for phishing
attacks regardless of phishing knowledge and awareness as
shown in Fig. 4. The performance of non-tech-savvy partici-
pants (Fig. 5) and for all users (Fig. 6) also suggests that a lot
of effort does not guarantee a higher chance of identifying
phishing attacks either. Moderate and reasonable effort can
yield better phishing identification chance in both cases. B. RECOMMENDED SYSTEM STRATEGY FOR
PHISHING DETECTION ON SMARTPHONES Previous studies have shown that policy statements are rarely
read [22] [23], and therefore more engaging means to educate In our previous work [34], we developed a prototype of an
Android application (UnPhishMe) that simulates a user login
procedure by using dummy login credentials to thwart phish-
ing attacks. We used the automation framework (Selendroid
[27]) which has the ability to manipulate the User Interface
(UI) of Android native and web apps. In this section, we
outline the system design, performance analysis and the
weakness of the prototype and how it can be improved and
implemented as a native Android browser. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. V. DISCUSSION In this section, we discuss the role of attention and prior
phishing knowledge on the ability of a user to identify phish-
ing attacks. We also discuss UnPhishMe application proto-
type and its improvements for assisting users with phishing
attacks identification. TABLE 5. A SUMMARY STATISTICAL TABLE FOR FIGURE 5 TABLE 5. A SUMMARY STATISTICAL TABLE FOR FIGURE 5 Fig. 6 shows the overall correlation among tech-savvy and
non-tech-savvy participants combined where the correlation t
df
p-value
confidence interval
correlation
0.9811
18
0.3846
-0.2607
0.5940
0.2055 6 6 VOLUME 4, 2016 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Algorithm 1 IsClientValidate(userID, password, Flag) Algorithm 1 IsClientValidate(userID, password, Flag) Algorithm 1 IsClientValidate(userID, password, Flag) Normally, when a user successfully authenticates herself to
a legitimate webpage, she expects to see a new webpage
with some requested resources. Technically, the current URL
string changes to a new or extended URL string. Result: true or false
initialization if Flag == 0 then
if serverConnection==0 userID.contain(@) then
return true
else
return false
end
else
if serverConnection==0 then
return true
else
return false
end
end This is also true for a phishing webpage, however unlike
a legitimate webpage, the login inputs correctness do not
matter. Contrary to that, on a legitimate website if a user pro-
vides incorrect login inputs there will be various outcomes. Certainly, the login inputs will be rejected and the webpage
remains the same with its contents slightly altered e.g., a
display of an error message. As a result, the URL string
remains unaltered and the page does not shift. However, some
websites such as Facebook [28] provide more than one alter-
native login pages. Once a first login attempt fails, a user is
presented with another similar page, which is modified with
some additional input requirements such as reCAPTCHA. A page shift automatically alters the URL string into a new
one. Therefore, its new computed hashcode will be different
from its original hashcode even if the authentication fails. Thus, the login automation should be iterative in such case
as summarized in the system overview in Fig. 8. In brief, we developed algorithms 1 and 2 (reference
to pseudocode). Algorithm 1 handles client-side validation
issues on webpages e.g., checking whether the username is
an email or has the minimum length of characters, while
Algorithm 2 monitors the authentication response of the
destination server. In our experiments with Alexa websites,
we found that most websites, 198/200 (98%), did not have
hard rules for usernames e.g., types of characters used. They
only specified the minimum length. We therefore created
a database of usernames that satisfy the different lengths
specified and email addresses using these usernames. By actively monitoring the URL changes after the authen-
tication attempt, the application can compare the original
URL hashcode h(URL) and the subsequent URL hashcode
h(URL’). In principal, the URL exhibited before a user logs
into a website remains the same even after the authentication
attempt fails on a legitimate webpage. Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS FIGURE 7. Checking HTTP response and URL consistency. authentication procedure through lightweight Java classes
and methods. It intercepts a login page opened by a user
and simulates the login procedure with fake credentials. Technically, an authentication attempt to a login webpage
with incorrect login details examines the trustworthiness of
that page. However, a user needs to have prior knowledge and
remembers to do so every time she encounters a suspicious
page. In small size devices, this procedure could be very
tedious when done manually. FIGURE 7. Checking HTTP response and URL consistency. Our proposed approach uses Java lightweight library class
(HTTP request client) that incurs a very small amount of
CPU and memory to analyze a destination server HTTP
header information [45]. In addition, instead of analyzing the
URL as a string of characters for determining its changing
dynamic, we analyzed it as numeric characters by program-
matically computing its hashcode every time a page was
loaded. Comparing numerals is much faster and easier than
comparing strings. The login automation module sends login credentials to
a resource server and computes a current URL hashcode. Later the resource server response about the authentication is
given and again the new URL hashcode h(URL’) is computed
for comparison with the original one. Thereafter, the server
response and the URL string status are intercepted by the
application to generate a proper warning message for the
user. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. 1) OVERVIEW UnPhishMe’s logic is to automatically intervene in the login
page in the background process of a device. It simulates user 7 VOLUME 4, 2016 This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access p Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS 3) EXPERIMENT AND RESULTS the webpage will not allow these details to be sent. In this
case, Algorithm 1 return true where there is a zero connection
to the destination server (serverConnection=0) and sets the
Flag to 0 in Algorithm 2 to denote an email address is needed. Setting the Flag to 0 allows the algorithm to enter and send
an email address from our database along with a password. In both cases, if Algorithm 1 returns false (serverConnec-
tion=1), the entered login details have successfully been sent
for authentication checking and therefore Algorithm 2 takes
over to monitor the appropriate response. We show the experiment procedure and results of our ap-
plication in this section. We depict its detection accuracy,
significance and computational cost. We tested about 700 websites, 500 suspicious ones from
Phishtank [14] and a supplementary test with top 200 le-
gitimate ones from Alexa.com [35]. Most of the suspicious
websites (96%) responded positively to the authentication
automation with completely dummy login credentials. A pos-
itive response, a successful authentication and inconsistent
URL string, raised an alert every time a malicious site was
opened. In the supplementary test with 200 legitimate web-
sites, we obtained an overall accuracy of 91%. We explain
these results in the V.D.(2) section of the Discussion. Algorithm 2 TestPhishingSite() Algorithm 2 TestPhishingSite()
Result: Alert User
initialization
URL or Link
Count=10
start loginPage()
Flag=1
if !(IsClientValidate(userID, password, Flag)) then
if (!IsAuthenticated(userID, password)) then
if hashcode1=hashcode2 then
if http_status_code is in {400,499} then
Legitimate Site
else
other error
end
else
while Count!=0 do
TestPhishingSite()
Count-=1
Flag=0
end
end
else
Suspicious site
end
else
end
return Legitimate site Result: Alert User
initialization We evaluated the performance of UnPhishMe through an
Android device with the following specifications: • Type: Samsung Galaxy SIII • Android version: 4.2.2 • Mode: GT-I9300 • CPU: ARMv7 Processor, 1400 MHz, 4 Cores • Internal memory: 853 Mb • Internal storage: 11,000 Mb Memory usage and performance differs from one device to
the other. The lower limit for a low and medium density
screen device is 16 Megabytes (Mb). That is a baseline for
Android memory usage by an application [29]. We have
optimized UnPhishMe CPU performance and memory usage
to a minimum by implementing lightweight Java classes and
methods. The measurements of CPU and memory usage
while using UnPhishMe were less than 5% and 10Mb respec-
tively, which is relatively low for a mobile device. 3) EXPERIMENT AND RESULTS We show
the performance test results of UnPhishMe from an Android
Profiler tool of the Android Studio [30] in Fig. 9. Algorithm 1 IsClientValidate(userID, password, Flag) If a user is successfully
authenticated, it is certainly expected that the URL will
change into a new one. For a malicious site, the URL almost
always changes with a successful authentication even when a
user provides incorrect login credentials [44]. When a webpage loads, Algorithm 2 invokes Algorithm
1. Initially, Flag is set to 1 in Algorithm 2. This means
the algorithm enters a username, not an email address. The
algorithm then enters and attempts to send a username from
our database and a password. If the destination server re-
quires an email address, the client-side validation script on UnPhishMe consists of two engines, one is for an authen-
tication automation and the other one is for server response
interpretation and alert generation to a mobile device user as
shown in Fig. 7. 8 8 VOLUME 4, 2016 This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. 4) SCOPE AND ASSUMPTIONS RECOMMENDED SYSTEM IMPROVEMENTS
Despite the UnPhishMe prototype being suitable for a real-
time phishing detection and timely feedback, it is not a
replacement for a browser. However, the entire logic of
the prototype can be implemented inside a native Android
browser which by default does not offer any form of phishing
protection [32] and can freely be customized. Moreover, this
kind of implementation waives off the need of a browser to
FIGURE 10. Implementation of the standard RFC. 10
VOLUME FIGURE 8. The work flow of UnPhishMe. FIGURE 9. UnPhishMe application prototype CPU and memory usage. FIGURE 10. Implementation of the standard RFC. FIGURE 9. UnPhishMe application prototype CPU and memory usage. 4) SCOPE AND ASSUMPTIONS This experiment had several assumptions and a scope that is
described in this section. We discuss some implementation
and performance issues and provide some recommendations
on how to improve this work. Our work focuses on mobile devices that use an Android
operating system. However, it is not limited to Android-
based devices only, it can be re-developed for other de-
vice operating systems such as iOS (formerly iPhone OS). However, currently, due to iOS restrictions to manipulate its
drivers, we could not implement our approach on it. Further-
more, since our solution depends on RFC 2616 [31] indus-
trial standard response for client requests, we are limited to
legitimate websites that conform to that standard. Some web
servers, such as http://www.nike.com, that implement such
server standards for an authentication failure, fit well into our
solution scope as indicated in Fig. 10. Together with other
criteria, it is easier to deduce a phishing site when it does not
behave like a legitimate one. The standard information can
be a certain response code equivalent to a client request such
as HTTP 401 Unauthorized or 403 Forbidden. The type of operations for the algorithms is ele-
mentary, because all the operations are checking and
affectation. Thus
the
time
complexity
is
linear
i.e., T(TestPhishingSite()) ∈θ(n)
(3) (3) As for the space complexity, an array for error codes needs
99 units (400-499) and all other remaining variables require
just 1 unit of space. That means, the space complexity is also
linear such that. (4) 9 VOLUME 4, 2016 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access This article has been accepted fo Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS FIGURE 8. The work flow of UnPhishMe. FIGURE 8. The work flow of UnPhishMe. FIGURE 9. UnPhishMe application prototype CPU and memory usage. C. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. C. RECOMMENDED SYSTEM IMPROVEMENTS Despite the UnPhishMe prototype being suitable for a real-
time phishing detection and timely feedback, it is not a
replacement for a browser. However, the entire logic of
the prototype can be implemented inside a native Android
browser which by default does not offer any form of phishing
protection [32] and can freely be customized. Moreover, this
kind of implementation waives off the need of a browser to FIGURE 10. Implementation of the standard RFC. 10 1) IMPLEMENTATION ON A BROWSER Without changing the logic of UnPhishMe prototype, we ini-
tiate and customize an Android browser by modifying a Web
View (WV) through the Android WebKit and invoke the input
data in the background process. For setting up the custom
Web View browser, the WV object has to be created whereas,
the WebViewClient handles onPageFinished(), shouldOver-
rideUrlLoading() methods, etc., which are responsible for
controlling the web format and structure when loaded while
the WebChromeClient handles Javascript’s alert() and other
functions. VOLUME 4, 2016 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS FIGURE 11. Implementation of UnPhishMe algorithm with an Android WebView browser. FIGURE 11. Implementation of UnPhishMe algorithm with an Android WebView browser. have third party anti-phishing plugins which are computa-
tionally heavy. the browser is installed. The getElementById() method is
then used to inject the credentials to the appropriate login
fields and the onClick() method submit the form. Meanwhile
the boolean onDestroy() function is set to FALSE so as the
process could be finalized and an alert generated to the user. We have summarized this procedure with a diagram in Fig. 11. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. p p Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS FIGURE 12. Performance evaluation of UnPhishMe on a Webview browser. database through an Android dbhelper. database through an Android dbhelper. Listing 1. Password Generator Function
function pass_gen (len) {
var length = (len)?(len):(10);
var string=’abcdefghijklmnopqrstuvwxyz’;
var numeric = ’0123456789’;
var pct=’!@#%^&*()_+~‘|}{[]\:;?><,./-=’;
var password = "";
var character = ""; var crunch = true;
while(password.length<length) {
unphish1 = Math.ceil(string.length*Math. ,→random()*Math.random());
unphish2 = Math.ceil(numeric.length*Math. ,→random()*Math.random());
unphish3 = Math.ceil(pct.length*Math.random
,→()*Math.random());
up = string.charAt(unphish1);
up = (password.length\%2==0)?(up.toUpperCase
,→()):(up);
character += up;
character += numeric.charAt(unphish2);
character += pct.charAt(unphish3);
password = character; }
password=password.split(’’).sort(function(){
return 0.5-Math.random()}).join(’’);
return password.substr(0,len); }
console.log( pass_gen () ); FIGURE 12. Performance evaluation of UnPhishMe on a Webview browser. restrictions. The iOS architecture is closed-source thus its kernel
restricts the developers to have a full control over the hardware,
timing, file system, interrupts, drivers and power management. As a
result, in iOS, implementing detective techniques like ours is very
difficult unless a developer illegally manipulates the jail-broken
iOS device to get a root access. Thus, we could not find this
technique implemented in iOS. However, there a few solutions that
work on iOS. Again the main challenge is that they have similar
disadvantages that motivated us into developing our solution. For
instance, Anti-Phishing - Identity Guard [36], Metacert anti-phish
app [37], Avira Mobile Security for iOS [38], and Defense against
mobile phishing attack are some of the anti-phishing techniques
that are available in iOS [39]. However, they work by collecting
information about known phishing sites from many sources and
automatically update their list of phishing sites. Thus, they are
not different from any other blacklist-based techniques which are
not able to efficiently perform in a real-time and on zero-day or
zero-hour phishing attack. Moreover, applications such as Avira
mobile solution for iOS, for instance, necessitate the use of cloud or
proxy server to separately analyze the heuristic features for phishing
detection. For example, we tested some of these applications with
malicious websites from Phishtank, they took at least a couple
of seconds up to two minutes to determine whether a simple and
obvious phishing site is malicious. In that case, a user might divulge
her sensitive information even before an alert is generated. password = character; } return 0.5-Math.random()}).join(’’); return password.substr(0,len); } console.log( pass_gen () ); 3) BENEFITS OF THE RECOMMENDED IMPROVED
IMPLEMENTATION An implementation of UnPhishMe’s logic on a web browser reduces
the risk of a user exposing her login information to an attacker. Most
importantly, there is no need for a web browser to have third party
plugins which normally slow down mobile devices with limited
computational power. Additionally, this implementation reduces
even the risks that are not associated with an action of a user. For
instance, phishing attacks that are associated with DNS and Web
cache poisoning or typo squatting. Thus, if an attacker compromises
a resource, proxy or DNS server, and redirects the user’s traffic
to a rogue server, the user can still be safe with the assistance of
UnPhishMe. We obtained the performance data dump from the Android Studio
3.1.3 Profiler. With the same testing environment, UnPhishMe im-
plementation on a browser had about the same amount of memory
usage as the prototype, which ranges between (3% - 4%) of unused
memory (500 Mb) which equates to a range of 15 - 20 Mb. However
the CPU usage was about 25% which is 20% higher than the
prototype as indicated in Fig. 12. Additionally, the average network
usage was 3.5 Mbps. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. 2) LOGIN CREDENTIALS GENERATION AND
MANAGEMENT The fake passwords used by UnPhishMe were generated
using a simple script (Listing 1). In our experiment with
Alexa sites, we found that apart from one website, the top
200 sites follow the same password requirements. The excep-
tional website did not accept ‘@’ or ‘-’ as special characters. Listing 1 generated a random list of characters that satisfy
the password requirements of the top 200 websites. We used
this approach to ensure that if a Phisher implements a client-
side validation for a password, the fake password would
be viewed as legitimate since it meets the required criteria. However, in our testing of phishing sites, we only found
rare cases of input validation on userID (either an email or
a username is acceptable) but not on entered passwords. The
fake passwords were stored in HashMap tables of an SQLite The fake passwords used by UnPhishMe were generated
using a simple script (Listing 1). In our experiment with
Alexa sites, we found that apart from one website, the top
200 sites follow the same password requirements. The excep-
tional website did not accept ‘@’ or ‘-’ as special characters. When a webpage is opened by a user, the shouldOver-
rideUrlLoading() method is called and the browser checks
for the existence of the HTML login form fields by scanning
the HTML tags. If there are no login form fields available, the
process is killed by setting the Boolean onDestroy() function
to TRUE. To detect if the page contains a login form, our
approach looks for the HTML form action attribute, <form
action = " " method = " " >. If the login form fields exist,
the browser invokes the retrieval of dummy credentials from
an SQLite database which is automatically installed when 11 VOLUME 4, 2016 VOLUME 4, 2016 This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access 2) DETECTION ACCURACY Despite attaining an overall detection accuracy of 96% for phishing
sites from Phishtank, our approach missed about 4% of the suspi-
cious entities. The failure was due to the non-standard HTML-based
pages such as Flash- and PDF-based. Our approach depends heavily
on the availability of HTML tags (input ID, name, type, form action)
for login fields such as username, password and submit button. However, webpages are made of the HTML technology, often in
conjunction with other technologies such as CSS, Javascript, JSON
etc., thus non-HTML sites would render so poorly on smartphones
to the point that they can easily be recognized effortlessly even
without the techniques such as UnPhishMe. [4] L. Wu, X. Du, and J. Wu, "Effective Defense Schemes for Phishing At-
tacks on Mobile Computing Platforms," IEEE Transactions on Vehicular
Technology, vol. 65, no. 8, pp. 6678-6691, 2016. [5] C. Yue and H. Wang., 2008, December. Anti-phishing in offense and
defense. In Computer Security Applications Conference, 2008. ACSAC
2008. Annual (pp. 345-354). IEEE. [6] M. Jakobbson, Understanding social engineering based scams. New York:
Springer, 2016. [7] H. Shahriar and M. Zulkernine, "PhishTester: Automatic Testing of Phish-
ing Attacks," 2010 Fourth International Conference on Secure Software
Integration and Reliability Improvement, 2010. On the other hand, another significant issue worth mentioning is
the performance of our approach on legitimate webpages. We tested
the approach with additional top 200 webpages from Alexa.com
and achieved a 91% detection accuracy. The unsuccessful 9% were
due to the webpages that require different authentication techniques
such as QR code scanners, One Time Password (OTP) sent to a
user mobile phone through SMS, and graphical passwords which
work by having a user selects a matching image, in a specific order,
presented in a graphical user interface (GUI). Since our approach is
basically a text-based login automation bot, it cannot automate such
complex authentication. However, technically speaking, Phishers
may find it difficult or nearly impossible to replicate these kinds
of authentications. Moreover, these kinds of websites can be white-
listed in a module within UnPhishMe. [8] L. Wu, X. Du, and J. Wu, "MobiFish: A lightweight anti-phishing scheme
for mobile phones," 2014 23rd International Conference on Computer
Communication and Networks (ICCCN), 2014. [9] S. Kanoh, S. Ichi-Nohe, S. Shioya, K. Inoue, and R. VI. CONCLUSION In this paper, we investigated the awareness of phishing attacks
and cybersecurity behaviours of 206 users through a survey. We
then used smart eyeglasses (electro-oculographic) to measure the
mental effort and vigilance of 40 participants when browsing web-
sites and when playing an Android phishing sites identification
game that we developed. We found that knowledge and awareness
of phishing attacks were insufficient for users’ cyber protection
because even knowledgeable participants had insecure behaviours
like opening email attachments from unfamiliar senders. However,
paying attention when browsing websites enabled participants to
effectively identify phishing sites. Based on these results, we assert
that browsers should automatically help users detect phishing web-
sites as it is difficult to maintain high mental focus for long periods
of time. This is particularly true for mobile device browsers, as the
limited screens means it is difficult to see web browser security
indicators and the limited capabilities mean many existing solutions
are difficult to implement. We recommend automatic login with fake
credentials to be deployed on browsers for phishing detection, as
phishing sites tend to grant access even with wrong login credentials
and this solution requires very low computational resources and
would not require any user effort, making it sustainable and usable
by both cybersecurity-aware and non-aware users. [15] H. Bridge, B. Goodger, G. Murphy, and J. N. Jitkoff , "Background auto-
submit of login credentials." U.S. Patent 8,607,306. [16] V. S. Bagad., and S. P. Kawachale. VLSI design. Technical Publications,
2008. [17] R. Dhamija, J. D. Tygar, and M. Hearst, "Why phishing works," Proceed-
ings of the SIGCHI conference on Human Factors in computing systems -
CHI 06, 2006. [18] D. Harley and A. Lee, "Phish Phodder: is User Education Helping or
Hindering?," in Virus Bulletin Conference Proceedings, 2007. [19] J. S. Downs, M. Holbrook, and L. F. Cranor, "Behavioral response to
phishing risk," Proceedings of the anti-phishing working groups 2nd
annual eCrime researchers summit on - eCrime 07, 2007. [20] N. F. Doherty, L. Anastasakis, and H. Fulford, "The information security
policy unpacked: A critical study of the content of university policies,"
International Journal of Information Management, vol. 29, no. 6, pp. 449-
457, 2009. [21] A. Ghazvini, Z. Shukur, and Z. Hood, "Review of Information Security
Policy based on Content Coverage and Online Presentation in Higher
Education," International Journal of Advanced Computer Science and
Applications, vol. 9, no. 8, 2018. [22] A. D. D. LIMITATIONS AND FUTURE WORK
1) ARCHITECTURAL ISSUES Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS an attacker always attempts to exploit the naivety of some website
users rather than exploiting weaknesses of a system [33]. an attacker always attempts to exploit the naivety of some website
users rather than exploiting weaknesses of a system [33]. [2] M. Jensen, A. Durcikova, and R. Wright, "Combating Phishing Attacks:
A Knowledge Management Approach," Proceedings of the 50th Hawaii
International Conference on System Sciences (2017), 2017. [3] E. Kritzinger and S. V. Solms. 2013. A Framework for Cyber Security in
Africa. Journal of Information Assurance & Cybersecurity (2013), 1-10. DOI:http://dx.doi.org/10.5171/2012.322399 2) DETECTION ACCURACY Kawashima, "Devel-
opment of an eyewear to measure eye and body movements," 2015 37th
Annual International Conference of the IEEE Engineering in Medicine and
Biology Society (EMBC), 2015. [10] D. Miyamoto, G. Blanc, and Y. Kadobayashi, "Eye Can Tell: On the
Correlation Between Eye Movement and Phishing Identification," Neural
Information Processing Lecture Notes in Computer Science, pp. 223-232,
2015. [11] D. E. Irwin, "Fixation Location and Fixation Duration as Indices of
Cognitive Processing ," J. M. Henderson & F. Ferreira (Eds.), The interface
of language, vision, and action: Eye movements and the visual world. New
York, NY, US: Psychology Press., pp. 105-133, 2004. Lastly, only 7% of the tested legitimate webpages implemented
client-side validation, they check whether the format of the input
data is valid. However, this causes a small delay of 9-32 millisec-
onds. We believe this could be one of the reasons most legitimate
websites (84%) did not implement client-side validation, rather they
just check whether the provided credentials match their records in
the back-end server. [12] K. Kunze, M. Katsutoshi, Y. Uema, and M. Inami, "How much do you
read?," Proceedings of the 6th Augmented Human International Confer-
ence on - AH 15, 2015. [13] S. Ishimaru, K. Kunze, K. Tanaka, Y. Uema, K. Kise, and M. Inami, "Smart
Eyewear for Interaction and Activity Recognition," Proceedings of the
33rd Annual ACM Conference Extended Abstracts on Human Factors in
Computing Systems - CHI EA 15, 2015. [14] W. Han, Y. Cao, and C. Lei, "Using a Smart Phone to Strengthen Password-
Based Authentication," 2011 International Conference on Internet of
Things and 4th International Conference on Cyber, Physical and Social
Computing, 2011. D. LIMITATIONS AND FUTURE WORK
1) ARCHITECTURAL ISSUES The authentication automation in mobile devices is more challeng-
ing than in desktop computers. The necessary automation drivers
that are available on desktops are normally not found on mobile
devices. In this work, we automated the user authentication proce-
dure through Selendroid with JavaScript Object Notation (JSON). However, Selendroid is only compatible with Android devices and
may not work on other types of devices. g
In future work, this automatic functionality can be fully in-
tegrated into commercial smartphone browsers with full browser
functionalities. Furthermore, since the method used in our appli-
cation is constantly scanning the HTML tags for login fields, we
intend to improve it so that it can detect the presence of login fields
without scanning the entire list of HTML tags. Another significant challenge for implementing our solution is
the requirement of specific websites that implement or follow in-
dustrial standards for authentication such as RFC 2616. Following
such standards makes it difficult for an attacker to simulate fake
legitimate responses. However, there is a possibility for an attacker
to try to authenticate the UnPhishMe request traffic to an associated
legitimate website on the fly in order to fool a user into providing
true credentials. UnPhishMe has several numbers of iterations for
addressing that problem, as we have demonstrated in the system
model in section V.B.2. Additionally, a client-server connection
time can be shortened on a client side to give an attacker less space
to subvert a user request to a legitimate site. However, semantically, In comparison with similar approaches in iOS [36], [37], [38],
[39] and PC, our approach fares much better as it eliminates zero-
day or zero-hour phishing attacks, with limited resources possible,
in a real-time environment. As we mentioned earlier, our design is
simple thus it favors small-sized devices computationally but does
not favor large-sized devices. The complexities of the algorithms are
demonstrated in Equation (3) and Equation (4). In iOS, however, the restrictions necessitate most researchers
to test their algorithms in Android-based devices, which have less 12 VOLUME 4, 2016 This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Her re-
search interests include health behavior change,
understanding users and human-computer interactions. [29] https://developer.android.com/reference/android/app/ActivityManager.html
getLargeMemoryClass (Accessed in May 2017). [30] "Measure
app
performance
with
Android
Profiler
|
Android
Developers,"
Android
Developers. [Online]. Available:
https://developer.android.com/studio/profile/android-profiler. [Accessed:
07-March-2017]. [31] IETF. [Online]. Available: https://www.ietf.org/rfc/rfc2616.txt. [Accessed:
06-January-2017]. [32] N. Virvilis, N. Tsalis, A. Mylonas, and D. Gritzalis, "Mobile Devices: A
Phisher’s Paradise," Proceedings of the 11th International Conference on
Security and Cryptography, 2014. [33] B. Schneier, "Semantic attacks: The third wave of network attacks,"
Crypto-Gram Newsletter 14, 2000. DOUDOU FALL received his M.E. degree in
Data Transmission and Information Security from
University Cheikh Anta Diop, Senegal in 2009. He received another M.E. & Ph.D. degrees in
Information Science from Nara Institute of Sci-
ence and Technology (NAIST), Japan in 2012 and
2015, respectively. He is currently an Assistant
Professor in the Graduate School of Information
Science, NAIST. His research interests include
cloud computing security, vulnerability & security risk analysis. [34] J. D. Ndibwile, Y. Kadobayashi, and D. Fall, "UnPhishMe: Phishing
Attack Detection by Deceptive Login Simulation through an Android
Mobile App," 2017 12th Asia Joint Conference on Information Security
(AsiaJCIS), 2017. [35] Alexa top 1000. [Online]. Available: https://www.alexa.com. [Accessed:
26-June-2019]. [36] Anti
Phishing
-
Identity
Guard. [Online]. Avail-
able:
https://apps.apple.com/us/app/anti-phishing-identity-
guard/id1136376302. [Accessed: 26-June-2019]. [37] Innovative anti-phishing app comes to iPhones. [Online]. Available:
https://www.computerworld.com/article/3327240/innovative-anti-
phishing-app-comes-toiphones.html. [Accessed: 26-June-2019]. risk analysis. DAISUKE MIYAMOTO is an Associate Professor
in the Graduate School of Information Science
and Technology, the University of Tokyo, Japan
(UTokyo) since 2018. He received the Bachelor
of Commerce from Kwansei Gakuin University,
Japan, in 2000, the Master and Doctorate degree
of Engineering from Nara Institute of Science and
Technology, Japan (NAIST), in 2002 and 2009
respectively. DAISUKE MIYAMOTO is an Associate Professor
in the Graduate School of Information Science
and Technology, the University of Tokyo, Japan
(UTokyo) since 2018. He received the Bachelor
of Commerce from Kwansei Gakuin University,
Japan, in 2000, the Master and Doctorate degree
of Engineering from Nara Institute of Science and
Technology, Japan (NAIST), in 2002 and 2009
respectively. [38] Avira Mobile Security for iOS: Powerful protection against phishing
attacks and identity theft. [Online]. Available: https://blog.avira.com/avira-
mobile-security-for-ios/. [Accessed: 26-June-2019]. [39] J. Hou and Q. Yang, "Defense against mobile phishing attack,"
in Computer Security Course Project, http://wwwpersonal. umich. edu/yangqi/pivot/mobile phishing defense.pdf, 2012. [40] Student, "The Probable Error of a Mean," Biometrika, vol. 6, no. 1, pp. 1-25, 1908. [41] B. L. Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS [25] S. Kleitman, M. K. H. Law, and J. Kay, "It’s the deceiver and the receiver:
Individual differences in phishing susceptibility and false positives with
item profiling," Plos One, vol. 13, no. 10, 2018. JEMA DAVID NDIBWILE received a Master
of Technology (M.Tech) in Information Security
from Jawaharlal Nehru Technological University
(JNTU), Hyderabad, India in 2015. He is currently
a Ph.D candidate at the Nara Institute of Science
and Technology (NAIST) in Japan. His research
interests include phishing countermeasures, the
psychology of cybersecurity, and ethical hacking. JEMA DAVID NDIBWILE received a Master
of Technology (M.Tech) in Information Security
from Jawaharlal Nehru Technological University
(JNTU), Hyderabad, India in 2015. He is currently
a Ph.D candidate at the Nara Institute of Science
and Technology (NAIST) in Japan. His research
interests include phishing countermeasures, the
psychology of cybersecurity, and ethical hacking. JEMA DAVID NDIBWILE received a Master
of Technology (M.Tech) in Information Security
from Jawaharlal Nehru Technological University
(JNTU), Hyderabad, India in 2015. He is currently
a Ph.D candidate at the Nara Institute of Science
and Technology (NAIST) in Japan. His research
interests include phishing countermeasures, the
psychology of cybersecurity, and ethical hacking. [26] S. Sheng, M. Holbrook, P. Kumaraguru, L. F. Cranor, and J. Downs,
"Who falls for phish?," Proceedings of the 28th international conference
on Human factors in computing systems - CHI 10, 2010. [27] M. Tan and P. Cheng, "Research and implementation of automated testing
framework based on Android," Information Technology, p. 035, May
2016. [28] Facebook. [Online]. Available: https://www.facebook.com/. [Accessed:
06-May-2019]. EDITH TALINA LUHANGA received a BEng in
Electronic and Computer Engineering and an MSc
in Advanced Computing Science from the Univer-
sity of Nottingham in 2010 and 2011 respectively
and a Ph.D. in Information Science at the Nara
Institute of Science and Technology (NAIST) in
Japan. She is currently working as a Lecturer at
the Nelson Mandela African Institution of Science
and Technology (NM-AIST) in Tanzania. Her re-
search interests include health behavior change,
human-computer interactions. EDITH TALINA LUHANGA received a BEng in
Electronic and Computer Engineering and an MSc
in Advanced Computing Science from the Univer-
sity of Nottingham in 2010 and 2011 respectively
and a Ph.D. in Information Science at the Nara
Institute of Science and Technology (NAIST) in
Japan. She is currently working as a Lecturer at
the Nelson Mandela African Institution of Science
and Technology (NM-AIST) in Tanzania. p p Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS VI. CONCLUSION Veiga, "Comparing the information security culture of employees
who had read the information security policy and those who had not,"
Information and Computer Security, vol. 24, no. 2, pp. 139-151, 2016. [23] "Cyber security - why you’re doing it all wrong," ComputerWeekly.com. [Online]. Available:
https://www.computerweekly.com/opinion/Cyber-
security-why-youre-doing-it-all-wrong. [Accessed: 04-May-2019]. REFERENCES [1] R. Bitton, A. Finkelshtein, L. Sidi, R. Puzis, L. Rokach, and A. Shabtai,
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cols.io.n74dhqw)," protocols.io, 2018. 13 VOLUME 4, 2016 VOLUME 4, 2016 VOLUME 4, 2016 This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI
10.1109/ACCESS.2019.2940669, IEEE Access Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Welch, "The Generalization of "Students" Problem when Several
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of Science and Technology, Japan. His research
interests include cybersecurity, web security, and
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of ACM and IEEE Communications Society. 14 14 VOLUME 4, 2016 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/.
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https://openalex.org/W4223539357
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English
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Structure and ensemble refinement against SAXS data: combining MD simulations with Bayesian inference or with the maximum entropy principle
|
bioRxiv (Cold Spring Harbor Laboratory)
| 2,022
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cc-by
| 13,822
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Abstract Small-angle X-ray scattering (SAXS) is a powerful method for tracking conformational tran-
sitions of proteins or soft-matter complexes in solution. However, the interpretation of the
experimental data is challenged by the low spatial resolution and the low information con-
tent of the data, which lead to a high risk of overinterpreting the data. Here, we illustrate
how SAXS data can be integrated into all-atom molecular dynamics (MD) simulation to
derive atomic structures or heterogeneous ensembles that are compatible with the data. Besides providing atomistic insight, the MD simulation adds physico-chemical information,
as encoded in the MD force fields, which greatly reduces the risk of overinterpretation. We
present an introduction into the theory of SAXS-driven MD simulations as implemented
in GROMACS-SWAXS, a modified version of the GROMACS simulation software. We dis-
cuss SAXS-driven parallel-replica ensemble refinement with commitment to the maximum
entropy principle as well as a Bayesian formulation of SAXS-driven structure refinement. Practical considerations for running and interpreting the simulations are presented. The
methods are freely available via GitLab at https://gitlab.com/cbjh/gromacs-swaxs. Keywords Small-angle scattering, SAXS, SANS, molecular dynamics simulation, SAXS-driven MD,
explicit solvent Affiliations 1Theoretical Physics and Center for Biophysics, Saarland University, Saarbrücken 66123,
Germany Authors Leonie Chatzimagas1 and Jochen S. Hub1 Running title Structure and ensemble refinement against SAXS data Title Structure and ensemble refinement against SAXS data: combining
MD simulations with Bayesian inference or with the maximum en-
tropy principle .
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
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doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
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doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It
The copyright holder for this prepri
this version posted April 10, 2022. ;
https://doi.org/10.1101/2022.04.05.487171
doi:
bioRxiv preprint . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
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doi:
bioRxiv preprint .
CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
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https://doi.org/10.1101/2022.04.05.487171
doi:
bioRxiv preprint 1
Introduction Understanding the function of biomolecules requires understanding of their conformational
dynamics. An increasingly popular method for tracking conformational transition of biomolecules
is small-angle X-ray scattering (SAXS), which provides structural information that is not
accessible by other techniques. Unlike NMR spectroscopy, which probes local distances and 1 1 . CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
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bioRxiv preprint angles of smaller biomolecules, SAXS provides information on the overall shape and is ap-
plicable to both small and large biomolecules. Unlike crystallography or cryo electron mi-
croscopy, SAXS probes molecules at ambient temperatures in solution, enabling experiments
that track conformational transition after application of external stimuli. The accuracy of
SAXS data has greatly improved over recent years thanks to sample delivery coupled with
size exclusion chromatography (SEC-SAXS), thereby reducing sample aggregation artifacts,
single-photon counting detractors, and standards for sample preparation (Berthaud et al.,
2012; Jeffries et al., 2016). Software and algorithms for data analysis and for SAXS-based
structural modeling has greatly developed (Gräwert and Svergun, 2020). These properties
and developments establish SAXS as an indispensable tool for integrative structural biology
(Rout and Sali, 2019; Brosey and Tainer, 2019). The interpretation of the SAXS data is challenged by the low information content of
the experimental signals. Because the biomolecules are randomly oriented during solution
scattering, SAXS curves I(q) represent an orientational average and, consequently, are only
a one-dimensional smooth function of momentum transfer q = 4π sin(θ)/λ. Here, λ is the
X-ray wavelength and 2θ is the scattering angle. The number of data points in I(q) that
provide independent structural information is estimated by the number of Shannon-Nyquist
channels (Moore, 1980; Rambo and Tainer, 2013) (1) NShan = (qmax −qmin)D/π,
(1) NShan = (qmax −qmin)D/π, where qmax and qmin denote the maximum and minimum momentum transfer in I(q), re-
spectively, and D is the maximum diameter of the solute. 1
Introduction CC-BY 4.0 International license
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bioRxiv preprint between two conformational states. This study neglected effects from the hydration layer
on the SAXS curve, rationalized by the fact that hydration layer effects may approximately
cancel upon taking SAXS curve differences. Our group developed SAXS-driven MD focused
on absolute SAXS curves and implemented the method into an extension of the GROMACS
software, coined GROMACS-SWAXS (https://gitlab.com/cbjh/gromacs-swaxs). The imple-
mentation uses explicit-solvent SAXS curve calculations using atomistic representations for
the hydration layer and excluded solvent (Chen and Hub, 2015, 2014). Kimanius et al. (2015)
suggested refining protein structures against SAXS data using metadynamics. However, since
the SAXS curve calculations neglected the buffer subtraction, the implementation was not
yet ready for refinement against experimental data. Paissoni et al. (2020) provided a method
for SAXS-driven simulations implemented in PLUMED, which was primarily motivated with
the aim of accelerating SAXS-driven simulations. This method maps the atomistic model to
a coarse-grained representation for computing a SAXS curve. The method neglects effects
from the hydration layer, and the coarse-grained approximation is limited to smaller scatter-
ing angles (Bernetti and Bussi, 2021). However, as discussed in this chapter and previously,
the SAXS-driven MD simulations implemented in GROMACS-SWAXS are subject to only
a small computational overhead between 5% and 20%, suggesting that SAXS-driven MD
simulations based on all-atom SAXS predictions are likewise computationally feasible. Hsu
et al. (2020) presented SAXS-driven MD simulations utilizing the Debye equation, similar
to Björling et al. (2015); however, these authors included effects from the hydration layer
by increasing atomic form factors of solvent-exposed atoms, similar to the FoXS method
(Schneidman-Duhovny et al., 2010). This chapter describes the refinement of protein and soft-matter complexes by cou-
pling all-atom MD simulations to experimental SAXS data, as implemented in GROMACS-
SWAXS. We first describe SAXS-driven MD simulations coupled with a harmonic restraint
to the data (Chen and Hub, 2015). The method was extended for simultaneous refinement
against SAXS and several small-angle neutron scattering (SANS) data sets collected at
different D2O concentrations (Chen et al., 2019). 1
Introduction NShan is in the range of 5 to 30 for
many SAXS experiments. For comparison, even a small protein with 100 residues contains
approx. 200 flexible backbone angles, demonstrating that SAXS data is by far insufficient for
defining all degrees of freedom of a biomolecule. Consequently, structure refinement against
SAXS data is highly ambiguous, that is, many different structures fit the data equally well. Challenges due to the low information content are enhanced by the presence of heteroge-
neous ensembles and by uncertainties in the experimental and predicted SAXS curves. The
low information content of the data together with larger number of degrees of freedom leads
to a significant risk of overinterpretation upon fitting structural models against experimental
data (Putnam et al., 2007; Hub, 2018). To mitigate the risk of overinterpretation during structure refinement, two main strate-
gies have been devised. First, nearly all degrees of freedom of the biomolecule have been
constrained, leading to methods such as rigid-body or normal mode refinement (Petoukhov
and Svergun, 2005; Pelikan et al., 2009; Gorba et al., 2008). Such methods use only sim-
ple energy terms to discriminate plausible from prohibited conformations, such as volume
exclusion terms between protein domains or a multidimensional harmonic potential along
normal modes. Second, physico-chemical information has been added to the SAXS data with
the help of atomistic molecular dynamics (MD) simulations (Björling et al., 2015; Chen and
Hub, 2015; Kimanius et al., 2015; Paissoni et al., 2020). During MD simulations, all degrees
of freedom are kept flexible, but the force field restrains the biomolecule to realistic con-
formations with acceptable free energy. Simulations with a coupling to experimental SAXS
curves have been coined SAXS-driven or SAXS-guided MD simulations. Several previous studies described SAXS-driven simulations, in which the MD simula-
tions were coupled to experimental SAXS data with an energetic restraint. Björling et al. (2015) presented a method with a focus on the interpretation of SAXS curve differences 2 . CC-BY 4.0 International license
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bioRxiv preprint . 2.1
Experiment-supported energetic bias Similar to refinement against other experimental data (Jack and Levitt, 1978), SAXS-
guided refinement is implemented by augmenting the MD force field energy VFF(R) with an
experiment-derived energy Eexp(R, D) that drives the simulation into conformations R that
are compatible with the data D: Ehybrid = VFF(R) + Eexp(R, D)
(2) (2) Here, the data is given by the experimental SAXS intensities Iexp(qi) and errors σ(qi), where
i = 1, . . . , Nexp and Nexp is the number of experimental data points. The MD simulation is
coupled to the data using a harmonic restraint on the SAXS intensities following Here, the data is given by the experimental SAXS intensities Iexp(qi) and errors σ(qi), where
i = 1, . . . , Nexp and Nexp is the number of experimental data points. The MD simulation is
coupled to the data using a harmonic restraint on the SAXS intensities following Eexp(R(t), D) = 1
2fc kBT
Nexp
X
i=1
[Ic(qi; R(t)) −(fIexp(qi) + c)]2
[fσ(qi)]2
. (3) (3) Here, Ic is the SAXS curve calculated on-the-fly from the MD simulation, fc is an overall
force constant, whereas kB and T denote the Boltzmann constant and the temperature. The
factor 1/2 is introduced to enable a Bayesian interpretation of the refined ensemble in the
special case fc = 1 as described below. The symbols f and c denote fitting parameter that
adjust the overall scale and an offset of the experimental curve relative to the calculated
curve; f and c are adjusted throughout the simulation by minimizing Eexp. Hence, only
those differences between Ic and Iexp that cannot be explained by f and c contribute to
Eexp and, thereby, drive the simulation. The offset c absorbs uncertainties due to the buffer
subtraction and may be omitted when coupling to high-precision SAXS data. As an example, Figure 1 presents structure refinement of the nuclear exportin CRM1,
which transports protein cargos across the nuclear pore (Chen and Hub, 2015). Free simula-
tions of CRM1 are highly force field-dependent, evident from the fact that simulations with
the Amber99sb or with the Charmm22* force fields lead to overly open or overly closed con-
formations, respectively (Fig. 1A, B, D top). Simulations with each force field exhibit poor
agreement with experimental SAXS data (Fig. 1C, black and solid yellow or green curves). 2.1
Experiment-supported energetic bias Upon restraining the simulations to SAXS data, excellent agreement with the data is ob-
tained (Fig. 1C, dashed yellow or green curves), and both two force fields lead to a consensus
partly open conformation (Fig. 1D bottom). Hence, the SAXS data overrules imperfections
in the two force fields. 1
Introduction We discuss how SAXS-guided structure
refinement is embedded into rigorous probability theory, as the obtained MD ensemble may
be interpreted as the posterior distribution of a Bayesian inference problem (Shevchuk and
Hub, 2017). Such viewpoint enables statements of confidence intervals, which are still under-
developed in SAXS-guided modeling. Finally, we describe the refinement of heterogeneous
ensembles against SAXS-data using a minimal bias, that is, with commitment to Jaynes’
principle of maximum entropy (Hermann and Hub, 2019; Ivanović et al., 2020). The methods described here are freely available via an extension of the GROMACS simu-
lation software (Abraham et al., 2015) developed at https://gitlab.com/cbjh/gromacs-swaxs. We present the theoretical basis of SAXS-driven MD simulations. We discuss conceptual
considerations as well as practical guidelines for running and interpreting the calculations. Additional tutorials and documentation is available at https://cbjh.gitlab.io/gromacs-swaxs-
docs. Although we here refer mostly to SAXS, the methods described here are likewise ap-
plicable for refinement against small-angle neutron scattering (SANS) data or for refinement
against combined SAXS/SANS data. SAXS-driven MD simulations require a forward model for predicting SAXS curves from
given MD simulations. The methods implemented in GROMACS-SWAXS utilize explicit-
solvent calculations, thereby taking explicit representations of the hydration layer and ex- 3 . CC-BY 4.0 International license
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bioRxiv preprint cluded solvent into account (Merzel and Smith, 2002; Park et al., 2009; Chen and Hub,
2014). For more details on explicit-solvent SAXS predictions we refer to a chapter “Predict-
ing solution scattering patterns with explicit-solvent molecular simulations” in Part A of
this monograph. 2.2
Increasing the computational efficiency by smoothing and re-
binning the experimental curve Owing to the large number of pixels on modern X-ray detectors, Iexp(qi) is heavily over-
sampled. Iexp(qi) contains typically 800–2500 noisy estimates of the true underlying smooth 4 . CC-BY 4.0 International license
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bioRxiv preprint Figure 1: Structure refinement of the nuclear exportin CRM1 against SAXS data. Free MD
simulations with the Amber99sb (yellow) or Charmm22* force fields lead to different confor-
mations (A/B; D, top), which both disagree with experimental SAXS data (C, see legend). During SAXS-driven simulations, the different force fields lead to similar conformations (D,
bottom), in excellent agreement with the data. Adapted and reused with permission from
Chen and Hub (2015). Figure 1: Structure refinement of the nuclear exportin CRM1 against SAXS data. Free MD
simulations with the Amber99sb (yellow) or Charmm22* force fields lead to different confor-
mations (A/B; D, top), which both disagree with experimental SAXS data (C, see legend). During SAXS-driven simulations, the different force fields lead to similar conformations (D,
bottom), in excellent agreement with the data. Adapted and reused with permission from
Chen and Hub (2015). SAXS curve, which contains only few independent data points (Nexp ≫NShan). Conse-
quently, the formulation in Eq. 3 is inefficient, since it requires the calculation of Ic(qi; R)
for a large number Nexp of q-points. The computational cost is reduced by smoothing the raw experimental data, thereby
merging neighboring Iexp(qi) pffoints within the same Shannon bin into a smoothed curve
¯Iexp(q). A shell script for smoothing the curve based on the ATSAS module DATGNOM
(Manalastas-Cantos et al., 2021) is available at https://cbjh.gitlab.io/gromacs-swaxs-docs. Upon smoothing the curve, Nexp raw experimental data points are merged into a smooth
curve with only NShan independent features, suggesting that the uncertainties of the smoothed
curve follow ¯σ2(q) ≈σ2(q)/ns, where ns = Nexp/NShan is the number of experimental points
per Shannon bin. 2.2
Increasing the computational efficiency by smoothing and re-
binning the experimental curve Let ∆I(qi) := Ic(qi; R) −(fIexp(qi) + c) denote the residuals between ex-
perimental and calculated curve and χ2 =
Nexp
X
i=1
∆I(qi)
fσ(qi)
2
,
(4) (4) such that Eexp = fc kBTχ2/2. Let us further decompose the residuals ∆Ii(qi) = ∆¯Ii + δIi
into contributions (i) relative to the smoothed curve ∆¯Ii, which can be fitted by adjusting
the biomolecular structure R, and (ii) owing to statistical noise in the data δIi, which cannot 5 . CC-BY 4.0 International license
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bioRxiv preprint 0
1
2
3
q (nm
−1)
10
-1
10
0
10
1
10
2
10
3
SAXS curve
Oversampled data
Smoohted
Rebinned
0
1
2
3
q (nm
−1)
-10
-5
0
5
10
15
SAXS curve
Figure 2: Smoothing and rebinning the experimental curve to increase the computational
efficiency of SAXS-driven MD simulations. Black dots: Oversampled experimental SAXS
data for bovine serum albumin dimers taken from Jeffries et al. (2016). Curve smoothed
with DATGNOM (pink) and resampled with DATREGRID, here using 1.5 q-points per
Shannon bin (Manalastas-Cantos et al., 2021). 0
1
2
3
q (nm
−1)
10
-1
10
0
10
1
10
2
10
3
SAXS curve
Oversampled data
Smoohted
Rebinned
0
1
2
3
q (nm
−1)
-10
-5
0
5
10
15
SAXS curve Figure 2: Smoothing and rebinning the experimental curve to increase the computational
efficiency of SAXS-driven MD simulations. Black dots: Oversampled experimental SAXS
data for bovine serum albumin dimers taken from Jeffries et al. (2016). Curve smoothed
with DATGNOM (pink) and resampled with DATREGRID, here using 1.5 q-points per
Shannon bin (Manalastas-Cantos et al., 2021). Figure 2: Smoothing and rebinning the experimental curve to increase the computational
efficiency of SAXS-driven MD simulations. Black dots: Oversampled experimental SAXS
data for bovine serum albumin dimers taken from Jeffries et al. (2016). 2.2
Increasing the computational efficiency by smoothing and re-
binning the experimental curve Curve smoothed
with DATGNOM (pink) and resampled with DATREGRID, here using 1.5 q-points per
Shannon bin (Manalastas-Cantos et al., 2021). be fitted. Then, χ2 can be rewritten as be fitted. Then, χ2 can be rewritten as χ2 =
Nexp
X
i=1
∆¯I(qi)
fσ(qi)
2
+
Nexp
X
i=1
δI(qi)
fσ(qi)
2
+ 2
Nexp
X
i=1
∆¯I(qi) δI(qi)
[fσ(qi)]2
(5) (5) The third term in Eq. 5 vanishes approximately because the noise δI(qi) is symmetrically
distributed around zero. The second term adds a constant offset of ≈1 per experimental
data point, i.e., a total offset of Nexp to χ2 that cannot be fitted. The first term quantifies
the deviation with respect to the smoothed curve and contains all the relevant structural
information. Using that ∆¯I(qi) and σ(qi) are approximately constant within each Shannon
bin, the first term of Eq. 5 simplifies to a sum over the Shannon bins, χ2 ≈
NShan
X
b=1
∆¯I(qb)2
f 2¯σ(qb)2 + Nexp,
(6) (6) where we used ¯σ2(q) ≈σ2(q)/ns. Hence, up to a structurally irrelevant constant offset Nexp
owing to experimental noise, χ2 and Eexp can be evaluated using only the NShan intensities
and errors of the smoothed curve. In case that more than NShan q-points are used to evaluate
Eexp, a correcting prefactor is needed and we arrive at E′
exp ≈1
2fc kBT NShan
Nused
Nused
X
i=1
[Ic(qi; R) −(f ¯Iexp(qi) + c)]2
[f ¯σ(qi)]2
,
(7) (7) (7) where Nused may be chosen in the range of 1–2 NShan. Evidently, using the formulation in
Eq. 7 greatly improves the computational efficiency of SAXS-driven MD simulations as it
requires ∼30 to 300 times fewer SAXS intensity calculations as compared to the formulation
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Increasing the computational efficiency by smoothing and re-
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bioRxiv preprint 0
1
2
3
4
5
6
7
q (nm
−1)
10
3
10
4
10
5
10
6
σ (e
2)
σtot
σbuf
σexp
σc
Figure 3: Typical errors applied during SAXS-driven MD. At small angles, systematic errors
σbuf (blue) dominate the total uncertainty σtot (green circles), here modeled by a buffer
density uncertainty of 1%. At wide angles, experimental errors dominate (orange). Calculated
statistical errors are typically insignificant (black). 0
1
2
3
4
5
6
7
q (nm
−1)
10
3
10
4
10
5
10
6
σ (e
2)
σtot
σbuf
σexp
σc Figure 3: Typical errors applied during SAXS-driven MD. At small angles, systematic errors
σbuf (blue) dominate the total uncertainty σtot (green circles), here modeled by a buffer
density uncertainty of 1%. At wide angles, experimental errors dominate (orange). Calculated
statistical errors are typically insignificant (black). 2.3
Accounting for systematic and calculated errors SAXS data collected with single-photon counting detectors may be subject to tiny statistical
errors σexp(q) at small scattering angles. Consequently, the overall uncertainty of the data
may be dominated by unknown systematic errors, for instance owing to imprecise buffer
subtraction or minor undetected radiation damage. When applying only the tiny statistical
errors, the SAXS-derived energies take spuriously large values at small angles due to the
1/¯σ(qi)2 term, which would further propagate into large SAXS-derived forces. Such problems are avoided by modeling of systematic errors. We previously modeled
systematic errors via an uncertainty δρbuf of 0.1% to 1% of the buffer density (Chen and
Hub, 2015). The uncertainty δρbuf propagates into an uncertainty σbuf(q) of the SAXS curve,
which is large at low angles and small at wide angles. Consequently, σbuf(q) dominates the
overall uncertainty at low angles and avoids spuriously large SAXS-derived forces, whereas
¯σ(q) dominates at wide angles. Apart from the systematic errors, GROMACS-SWAXS takes
statistical errors of the calculated curve σc(qi) into account (Fig. 3). This is implemented by
replacing the errors ¯σ in Eq. 7 with σ2
tot = f 2¯σ2+σ2
buf +σ2
c, leading to the final SAXS-derived
energy applied by GROMACS-SWAXS: Ef
exp ≈1
2fc kBT NShan
Nused
Nused
X
i=1
[Ic(qi; R) −(f ¯Iexp(qi) + c)]2
f 2¯σ2(qi) + σ2
buf(qi) + σ2c(qi),
(8) (8) 2.4
On-the-fly averaging of the calculated SAXS curve Explicit-solvent SAXS curve predictions require averaging over multiple MD frames for com-
puting a SAXS curve. The number of frames required for obtaining a converged SAXS curve
depends on the contrast between the biomolecule and the pure buffer; the smaller the elec-
tron density contrast, the more frames are required to converge the SAXS curve. During
SAXS-driven MD simulations discussed here, the SAXS curve is therefore computed as a
running temporal average with a memory kernel that decays exponentially into the past. 7 . CC-BY 4.0 International license
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bioRxiv preprint The on-the-fly average of a quantity X at simulation time t is then given by ⟨X⟩(t) = N −1
Z t
0
X(t′) e−(t−t′)/τdt′,
(9) (9) where τ is the memory time, typically chosen between 50 and 200 ps, and N is a normaliza-
tion constant. Likewise, on-the-fly averaging of the scattering amplitudes of the biomolecular
and the pure-solvent system using Eq. 9 provide an on-the-fly averaged SAXS curve (Chen
and Hub, 2015). Implementing the coupling to the experimental SAXS curve with an on-the-fly averaged
calculated curve has two key advantages: first, thermal fluctuations on the time scale 1–2 τ
such as solvent, side chain, or loop fluctuations are taken into account before comparing the
calculated with the experimental curve in Eq. 8. Such thermal fluctuations may significantly
influence the SAXS curve of proteins at moderate scattering angles (q > 0.25 Å−1) (Tiede
et al., 2002; Chen and Hub, 2014; Moore, 2014). Second, there is no need to compute
SAXS intensities every MD step, but instead one SAXS update every ∼0.5 ps is sufficient. The update interval together with the memory time must be chosen such that the SAXS
curves converge within τ. The longer update interval renders SAXS-driven MD simulation
computationally efficient with an overhead of only 5–20% relative to unbiased simulations. 2.4
On-the-fly averaging of the calculated SAXS curve However, owing to the on-the-fly average, the dynamics are not conservative because the
energy Ef
exp depends not only on the current but also on previous conformations. Hence, a
reasonably tight temperature coupling scheme is required to avoid energy drifts. 2.5
SAXS-derived forces applied during MD CC-BY 4.0 International license
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bioRxiv preprint When coupling to SANS data measured at a large D2O concentration, the contrast and
the factor fcontrast may even become negative because a D2O buffer exhibits a larger neutron
scattering length density than proteins. Consequently, upon moving a protein domain to the
left, the contrast may move to the right; such effect is correctly taken into account by a
negative factor fcontrast. tlab.io/gromacs- 2.5
SAXS-derived forces applied during MD SAXS-derived forces applied in the MD simulations are given by the negative gradients of
the SAXS-derived energy, Fk
=
−∇kEf
exp(R, D)
(10)
=
−fc kBT NShan
Nused
Nused
X
i=1
Ic(qi; R(t)) −(f ¯Iexp(qi) + c)
σtot(qi)2
∇kIc(qi; R(t)),
(11) (10) (11) where ∇kIc(qi; R(t)) denotes the on-the-fly averaged gradients of the SAXS intensities with
respect to the coordinates of atom k. For large biomolecular systems, storing these gradients
may require several Gigabytes of memory (Chen and Hub, 2015). where ∇kIc(qi; R(t)) denotes the on-the-fly averaged gradients of the SAXS intensities with
respect to the coordinates of atom k. For large biomolecular systems, storing these gradients
may require several Gigabytes of memory (Chen and Hub, 2015). GROMACS-SWAXS computes the forces Fk and the gradients ∇kIc(qi; R(t)) only for
the solute atoms but not for the solvent atoms. Therefore, in addition to the derivatives of
the Ic with respect to the solute coordinates, a correction factor fcontrast = ρsolu −ρsolv
ρsolu
(12) (12) is applied, where ρsolu and ρsolv denote the average solute and solvent densities, respectively. Here, density refers to the scattering length density, that is, to the electron density in SAXS
or to the neutron scattering length density in SANS. As a numerical example, the factor
fcontrast accounts for the fact that, upon moving a protein domain with density ρsolu =
440 e nm−3 inside solvent with density ρsolv = 334 e nm−3, only the contrast of 106 e nm−3 is
moved. Consequently, change of Ic(qi) upon a domain movement in mobile water is reduced
by the factor fcontrast = 0.24 as compared to a domain movement at fixed water positions. is applied, where ρsolu and ρsolv denote the average solute and solvent densities, respectively. Here, density refers to the scattering length density, that is, to the electron density in SAXS
or to the neutron scattering length density in SANS. As a numerical example, the factor
fcontrast accounts for the fact that, upon moving a protein domain with density ρsolu =
440 e nm−3 inside solvent with density ρsolv = 334 e nm−3, only the contrast of 106 e nm−3 is
moved. Consequently, change of Ic(qi) upon a domain movement in mobile water is reduced
by the factor fcontrast = 0.24 as compared to a domain movement at fixed water positions. 8 . 2.6
Protocol A CC-BY 4.0 International license
made available under a
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bioRxiv preprint ; fit scale f and offset c of experimental curve
waxs-Iexp-fit
= scale-and-offset
; update on-the-fly averaged SAXS curve every 0.5ps with dt=0.002
waxs-nstcalc
= 250
; nr of q-values, N_used. Use approx. 1.5 N_Shan
waxs-nq
= 15
; qmin and qmax in nm^(-1)
waxs-startq
= 0
waxs-endq
= 5
; nr of q-vectors for orientational average, use ~0.2*(D*qmax)^2
waxs-nsphere
= 500
; density of solvent (e/nm3), used for a precise buffer subtraction
waxs-solvdens
= 334
; relative uncertainty of the solvent density, used to
; estimate systematic errors. Use 0.1% to 0.5%
waxs-solvdens-uncert = 0.005
; D2O concentration (e.g. 0% and 100%) for each SANS calc. waxs-deuter-conc = [0 1] ; fit scale f and offset c of experimental curve ; fit scale f and offset c of experimental curve
waxs-Iexp-fit
= scale-and-offset p
Iexp-fit
= scale-and-offset p
; update on-the-fly averaged SAXS curve every 0.5ps with dt=0.002
waxs-nstcalc
= 250 ; nr of q-values, N_used. Use approx. 1.5 N_Shan
15 ; nr of q-values, N_used. Use approx. 1.5 N_Shan
waxs-nq
= 15 q
; nr of q-vectors for orientational average, use ~0.2*(D*qmax)^2
waxs-nsphere
= 500 p
; density of solvent (e/nm3), used for a precise buffer subtraction
waxs-solvdens
= 334 ; relative uncertainty of the solvent density, used to
%
% waxs solvdens uncert
0.005
; D2O concentration (e.g. 0% and 100%) for each SANS calc. waxs-deuter-conc = [0 1] ; D2O concentration (e.g. 0% and 100%) for each SANS calc. 5. Smooth the experimental target curve. Upon smoothing the curve, the errors are re-
duced by a factor of n1/2
s
, where ns is the number of experimental data points per Shan-
non bin. A Shell script for this purpose is available at https://cbjh.gitlab.io/gromacs-
swaxs-docs. The shell script requires installation of the ATSAS software (Manalastas-
Cantos et al., 2021). 5. Smooth the experimental target curve. Upon smoothing the curve, the errors are re-
duced by a factor of n1/2
s
, where ns is the number of experimental data points per Shan-
non bin. A Shell script for this purpose is available at https://cbjh.gitlab.io/gromacs-
swaxs-docs. 2.6
Protocol A To carry out SWAXS-driven MD simulations with the GROMACS-SWAXS implementa-
tion published at https://gitlab.com/cbjh/gromacs-swaxs, the following protocol is recom-
mended: 1. Download and compile GROMACS-SWAXS, following the installation instructions of
official GROMACS. Alternatively, GROMACS-SWAXS can be easily installed using
Spack, which is available at many high-performance computing centers. 2. Setup the MD simulation system following freely available GROMACS tutorials. Make
sure to use an approximately 1 nm larger simulation box compared to common simu-
lations via the GROMACS module gmx editconf -d. 3. First compute a SAXS curve from an equilibrium simulation using the rerun functional-
ity of the GROMACS-SWAXS gmx mdrun module, following tutorials at https://cbjh.git
swaxs-docs and as described in a chapter in Part A of this monograph. Computing a
SAXS curve involves the setup of a pure-solvent simulation systems, definition of the
atomic form factors with the gmx genscatt module, and building of the envelope with
gmx genenv, which defines the solvent region contributing to the SAXS curve. From
visual inspection of the SAXS curve, is it plausible that a conformational transition of
the biomolecule explains the deviation between the calculated and the experimental
curve? 4. Generate the run-input (tpr) file with the gmx grompp module. For a typical SAXS-
driven MD simulation, the MD parameter (mdp) file may look as follows: 4. Generate the run-input (tpr) file with the gmx grompp module. For a typical SAXS-
driven MD simulation, the MD parameter (mdp) file may look as follows: = xray [neutron neutron ...] ; solute group
waxs-solute
= Protein ; or Protein-Masses g
p
te
= Protein ; or Protein-Masses ;
g
p
waxs-solvent
= Water_and_ions ; rotational fit group as defined with gmx genenv
waxs-rotfit
= C-alpha p
; memory time for on-the-fly average
waxs-tau
= 200 ; turn on SAXS energy E_exp slowly, e.g. over 5ns
waxs-t-target
= 5000 g
; define coupling potential type. Alternative: log waxs-potential
= linear p
; use experiental, calculated and systematic errors ; use experiental, calculated and syst waxs-weights
= exp+calc+solvdens ; force constant, use 1-5. fc=1 enables Bayesian interpretation
waxs-fc
= 1 9 . CC-BY 4.0 International license
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Protocol A It is
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bioRxiv preprint 3.1
Posterior, likelihood, and prior distributions Following pioneering work by Rieping, Habeck, and coworkers on refinement against NMR
data (Rieping et al., 2005; Habeck et al., 2006), SAXS-driven MD simulations have been
reformulated as tool for Bayesian inference of biomolecular structures from experimental
data. Accordingly, the goal of structure refinement is to find the conditional probability
P(R, θ|D, K) that quantifies the plausibility for the biomolecular structure R in the light of
the experimental data D and prior physical knowledge K. In the context of SAXS-guided
modeling, the data D is given by the experimental curve Iexp(q) and its errors, whereas the
prior knowledge K represents the MD force field, the starting conformation of the simulation,
and other prior experience. The symbol θ summarizes so-called nuisance parameters such
as the unknown fitting parameters or unknown systematic errors, which must be estimated
simultaneously with the structure. In Bayesian inference, P(R, θ|D, K) should not be interpreted as probabilities of random
events, as common in the “frequentist interpretation” of probability theory, but instead as
a quantification of our state of knowledge and ignorance. A wide distribution P(R, θ|D, K)
implies that many different conformations R are compatible with D and K, implying a
high degree of ignorance and large confidence intervals on R. By the same token, a narrow
P(R, θ|D, K) implies that only few structures are plausible in the light of D and K, implying
precise knowledge of R and small confidence intervals on R. Hence, computing P(R, θ|D, K)
provides rigorous confidence intervals for the refined structure founded in probability theory. The conditional probability is evaluated using Bayes’ theorem, P(R, θ|D, K) ∝L(D|R, θ, K) π(R|K) π(θ|K),
(13) (13) where π(R|K) and π(θ|K) denote the prior distributions of conformations and of the nui-
sance parameters, respectively. L(D|R, θ, K) is the likelihood that the data D was measured
given that the structure R was present in the experiment and given a specific set of nuisance
parameters θ. The distribution P(R, θ|D, K) is called posterior distribution. When running
MD simulations, the prior π(R|K) is naturally given by the Boltzmann distribution where π(R|K) and π(θ|K) denote the prior distributions of conformations and of the nui-
sance parameters, respectively. L(D|R, θ, K) is the likelihood that the data D was measured
given that the structure R was present in the experiment and given a specific set of nuisance
parameters θ. 2.6
Protocol A The shell script requires installation of the ATSAS software (Manalastas-
Cantos et al., 2021). 6. Finally, specify the envelope files with environment variables and run the SAXS-driven
MD: 6. Finally, specify the envelope files with environment variables and run the SAXS-driven
MD: export GMX_ENVELOPE_FILE=envelope.dat export GMX_WAXS_FIT_REFFILE=envelope-ref.gro sw=/path/to/pure-solvent.tpr p
p
p
fw=/path/to/pure-solvent.xtc p
p
mdrun -s topol.tpr -fw $fw -sw $sw -is t gmx mdrun -s topol.tpr -fw $fw -sw $sw -is target.xvg ... Here, pure-solvent.tpr and pure-solvent.xtc are the run-input and trajectory
files of the pure-solvent systems used for computing the buffer subtraction. These have
already been set up for computing the SAXS curve from an equilibrium simulation in
step 3. 7. To analyze the simulation, visualize it in a molecular viewer and validate that the
conformation is reasonable. Inspect in waxs_final.xvg whether the simulation was
capable of finding a conformation that is compatible with the target SAXS curve. Inspect the SAXS-derived energy Ef
exp, which is stored in the energy (edr) file and
available via with the gmx energy command. Validate that the energy is in the range
of several kBT after the structure has been refined. The contributions of individual
q-points to Ef
exp, available in waxs_pot.xvg, may reveal q-regions that could not be
explained. 8. Since waxs_final.xvg represents the final on-the-fly average of the calculated curve,
it represents only the final 1–2 τ (∼200 ps) of the simulation. Use the rerun func-
tionality of mdrun (gmx mdrun -rerun traj.xtc) to compute the SAXS curve that
is uniformly averaged over a longer part of the SAXS-driven simulation, for instance
over the final 10 ns. A uniform average is enabled with the mdp option waxs-tau = 0. Use more q-points, such as waxs-nq = 100. 8. Since waxs_final.xvg represents the final on-the-fly average of the calculated curve,
it represents only the final 1–2 τ (∼200 ps) of the simulation. Use the rerun func-
tionality of mdrun (gmx mdrun -rerun traj.xtc) to compute the SAXS curve that
is uniformly averaged over a longer part of the SAXS-driven simulation, for instance
over the final 10 ns. A uniform average is enabled with the mdp option waxs-tau = 0. Use more q-points, such as waxs-nq = 100. 10 . CC-BY 4.0 International license
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Posterior, likelihood, and prior distributions The distribution P(R, θ|D, K) is called posterior distribution. When running
MD simulations, the prior π(R|K) is naturally given by the Boltzmann distribution π(R|K) ∝e−VFF(R)/kBT
(14) (14) with the MD force field VFF(R), i.e., by the ensemble obtained before incorporating the data
D. A reasonable choice for π(θ|K) is less obvious; if no prior information on the nuisance
parameters is available, a non-informative prior such as a flat or a Jefferys’ prior may be
used. It is advisable to test multiple priors in order to exclude that the conclusions depend
on the choice of the priors. For instance, testing multiple choices of π(R|K) corresponds to
running simulations with difference force fields VFF. Assuming Gaussian independent errors σtot, the likelihood function is L(D|R, θ, K)
∝
Nexp
Y
i=1
exp
−[Ic(qi; R) −I′
exp(qi; θ)]2
2[fσ(qi; θ)]2
! =
exp
−1
2
Nexp
X
i=1
[Ic(qi; R, θ) −I′
exp(qi; θ)]2
[fσ(qi; θ)]2
,
(15) (15) 11 . CC-BY 4.0 International license
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bioRxiv preprint Here, the I′
exp(qi; θ) = f Iexp(qi) + c is the experimental data adjusted by the scale f and
the offset c, which appear as nuisance parameters (cf. Eq. 3). In GROMACS-SWAXS, the
likelihood function is modified twofold relative to Eq. 15. First, as described above, the raw
experimental intensities (Iexp, σ) and errors are replaced with the smoothed curve (¯Iexp, ¯σ),
thereby replacing the sum over Nexp values with a sum over NShan values. Second, the errors
are augmented with the calculated and systematic errors, leading to the final likelihood
function: Lf(D|R, θ, K)
∝
exp
−1
2
NShan
Nused
Nused
X
j=1
(Ic(qj; R) −¯I′
exp(qj; θ))2
f 2¯σ2(qi) + σ2
buf(qi; θ) + σ2c(qi)
(16) (16) Hence, Lf contains three nuisance parameters θ = {f, c, δρbuf}. 3.1
Posterior, likelihood, and prior distributions The posterior distribution P(R, θ|D, K) cannot be computed analytically but is instead
obtained by importance sampling. Here, Newtonian dynamics as implemented by MD sim-
ulations are used for sampling the conformations. By taking the negative logarithm of the
posterior, we turn the probability functions into energy terms, E
=
−kBT ln P(R, θ|D, K)
=
VFF(R) + Ef
exp(R, D, θ)
fc=1 −kBT ln π(θ|K),
(17) (17) where we used Eqs. 8, 13, 14, and 16. Several aspects of this results are notable: • The original SAXS-derived energy (Eq. 8) required the choice of a force constant fc that
weights the experimental data relative to the force field VFF. Using Bayesian inference,
in contrast, no force constant is needed because the weight of the experimental data
is fully determined by probability theory. In practice, it may be useful to first drive a
conformational transition with a larger fc and, in a follow-up simulation, sample the
posterior with fc = 1. • Sampling the posterior P(R, θ|D, K) has been implemented using Gibbs sampling. Accordingly, nuisance parameters θ are sampled with Metropolis Monte-Carlo at fixed
conformations R, followed by Newtonian dynamics of R at fixed θ, and so on. • Sampling the fitting parameters f and c is not required because they can be marginal-
ized out analytically at the level of the likelihood when assuming flat priors for f
and c, i.e., π(f|K) = π(c|K) = const. Then, the likelihood in Eq. 16 is replaced with
˜L(D|R, δρbuf, K) =
R
L(D|R, θ, K) df dc, thereby taking “all possible” values of f and
c into account. Evaluating this integral shows that ˜L takes the same form as L, ex-
cept that f and c are replaced with their maximum-likelihood estimates fml and cml
(Shevchuk and Hub, 2017). 3.2
Bayesian treatment of systematic errors at small angles GROMACS-SWAXS models systematic errors at small angles via an uncertainty of the buffer
density δρbuf (see above and Fig. 3). In Bayesian SAXS-driven MD, δρbuf can be treated
as one of the nuisance parameters θ (Shevchuk and Hub, 2017). Accordingly, the relative
uncertainty δρbuf is sampled simultaneously with the structure R to obtain a joint posterior 12 . CC-BY 4.0 International license
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bioRxiv preprint 0
2
4
6
8
δρbuf (e nm
−3)
0
0.2
0.4
0.6
0.8
Posterior Pbuf(δρbuf|D,K)
Leucine binding protein, calculated Iexp(q)
HSP90+ATP, experimental Iexp(q)
Figure 4: Example of posterior distributions over the uncertainty of the buffer density δρbuf
taken from SAXS-driven MD simulations of leucine binding protein (LBP, solid curve) or
HSP90 bound to ATP (dashed curve). For LBP, the posterior peaks at δρbuf = 0; hence,
systematic errors are not required to explain the data, but they cannot be excluded either. This finding is expected since the SAXS-driven MD simulation were carried out against
a calculated target curve without any uncertainties. In contrast, the posterior for HSP90
peaks at larger δρbuf = 4 e nm−3, suggesting that significant systematic errors are strictly
required to explain the data. This finding reflects that the SAXS-driven MD was carried out
against experimental data with substantial systematic errors at small angles. Data taken
from Shevchuk and Hub (2017). 0
2
4
6
8
δρbuf (e nm
−3)
0
0.2
0.4
0.6
0.8
Posterior Pbuf(δρbuf|D,K)
Leucine binding protein, calculated Iexp(q)
HSP90+ATP, experimental Iexp(q) Figure 4: Example of posterior distributions over the uncertainty of the buffer density δρbuf Figure 4: Example of posterior distributions over the uncertainty of the buffer density δρbuf
taken from SAXS-driven MD simulations of leucine binding protein (LBP, solid curve) or
HSP90 bound to ATP (dashed curve). 3.2
Bayesian treatment of systematic errors at small angles For LBP, the posterior peaks at δρbuf = 0; hence,
systematic errors are not required to explain the data, but they cannot be excluded either. This finding is expected since the SAXS-driven MD simulation were carried out against
a calculated target curve without any uncertainties. In contrast, the posterior for HSP90
peaks at larger δρbuf = 4 e nm−3, suggesting that significant systematic errors are strictly
required to explain the data. This finding reflects that the SAXS-driven MD was carried out
against experimental data with substantial systematic errors at small angles. Data taken
from Shevchuk and Hub (2017). distribution over structures and buffer density uncertainties P(R, δρbuf|D, K). GROMACS-
SWAXS applies a Gaussian prior distribution for δρbuf. Let rbuf := δρbuf/ρbuf, then the
prior is π(rbuf|K) ∝exp(−r2
buf/2ϵ2
buf)
(18) (18) where ρbuf is the solvent density and ϵbuf is given with the mdp parameter waxs-solvdens-uncer
typically set between 0.1% and 1%. This algorithm detects automatically whether the ex-
perimental data is biased by systematic errors at small angles. Namely, if the MD force field
permits a conformational transition that explains the experimental data Iexp(q), the poste-
rior of δρbuf would peak at small values, indicating that systematic errors are not required
to explain the experimental data (Fig. 4, blue solid). In contrast, if the MD simulations does
not find a conformation that explains Iexp at small angles, the posterior of δρbuf would peak
at larger values, indicating that significant systematic errors are plausible in the light of the
data and the force field (Fig. 4, orange dashed). Clearly, treatment of systematic errors in SAXS-based modeling is still underdeveloped. To harvest increasingly finer details in SAXS data, it will be useful to explicitly model other
sources of errors, such as a small fraction of aggregated samples. We believe that Bayesian
inference will provide a rigorous framework to learn systematic errors simultaneously with
the biomolecular structures. 3.3
Protocol B The Bayesian interpretation of SAXS-driven MD simulations are enabled by using a force
constant of unity, and optionally, by sampling δρbuf. The mdp file for a Bayesian SAXS-
driven MD simulation should contain, apart from the options described above: 13 . CC-BY 4.0 International license
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bioRxiv preprint waxs-fc
= 1
waxs-solvdens-uncert
= 0.005
waxs-solvdens-uncert-bayesian
= yes waxs-fc
= 1
waxs-solvdens-uncert
= 0.005
waxs-solvdens-uncert-bayesian
= yes The simulation frames in the trajectory of the SAXS-driven MD may be interpreted as
samples from a the high-dimensional posterior distribution over conformations P(R|D, K). Obtaining posteriors over intuitive properties, such as the center-of-mass distance dcom be-
tween two domains, is straight-forward: dcom values may be extracted from the trajectory
frames and plotted as a histogram. The histograms is the posterior Pcom(dcom|D, K) over
the center-of-mass distances, suggesting that the peak position and the width of Pcom pro-
vide the most plausible dcom and its uncertainty in the light of the data and the force field. Mathematically, obtaining Pcom(dcom|D, K) from P(R|D, K) would involve a marginaliza-
tion, that is the integration over all other degrees of freedom except dcom; however, since
the trajectory of the Bayesian SAXS-driven MD contains samples of dcom, there is no need
carry out a marginalization in practice. Samples from the posterior over the relative uncertainty of the buffer density, δρbuf/ρbuf,
are written into separate output file waxs_solvDensUncert.xvg allowing the calculation of
a histogram and, hence, the posterior over the uncertainty of the buffer density. 4.1
Theoretical background N replicas are simulated si-
multaneously, each providing a calculated curve I1, . . . , IN. Coupling the replica-averaged
SAXS curve to the experiment with a harmonic restraint leads to the maximum entropy
ensemble. Figure 5: Illustration of parallel-replica ensemble refinement. N replicas are simulated si-
multaneously, each providing a calculated curve I1, . . . , IN. Coupling the replica-averaged
SAXS curve to the experiment with a harmonic restraint leads to the maximum entropy
ensemble. that maximizes the Shannon entropy that maximizes the Shannon entropy S(p) = −
X
i
p(Ri) ln p(Ri)
(19) (19) under the given constrains. under the given constrains. In ensemble refinement, however, we are typically interested in refining a prior ensemble
from a free MD simulation against experimental data, suggesting that it is useful to maximize
the relative entropy between the unbiased and the refined ensemble (Caticha, 2004). Because
the relative entropy is the negative of the Kullback-Leibler divergence DKL(p1|p0) (Kullback
and Leibler, 1951), maximizing the relative entropy implies that we should find a refined
ensemble distribution p1(Ri) that minimizes DKL(p1|p0) =
X
i
p1(Ri) ln p1(Ri)
p0(Ri)
(20) (20) under the given constrains, where p0(Ri) is the prior ensemble distribution. Taking the
prior from an unbiased MD ensemble, the aim is to find an updated ensemble that is (i)
in agreement with the data and (ii) updated as minimally as possible with respect to the
unbiased prior ensemble. In other words, the ensemble is only updated as strictly needed to
explain the data, while any bias that is not supported by the data is avoided. Specifically,
the formulation assures that the ensemble is not updated if the prior ensemble is already in
agreement with the data. The minimization problem can be solved with the help of Lagrangian multipliers, where
one multiplier is required for each experimental constraint (Pitera and Chodera, 2012). However, the Lagrangian multipliers must be optimized in a iterative manner, which may
be tedious in presence of a larger number of experimental constraints (Boomsma et al., 2014). An alternative for implementing a minimal bias is the parallel-replica approach. Here, several
copies of the system (replicas) are simulated in parallel, and only the calculated data averaged
over the replias is restrained to the data with a harmonic restraint (Fig. 5). Roux and Weare
(2013) and Cavalli et al. 4.1
Theoretical background Since SAXS is a solution method, experimental SAXS intensities represent the average over
a structural ensemble. For structurally stable proteins, the ensemble may be approximated
by a single, most prominent conformation, enabling the use of structure refinement methods
described above. In contrast, for solutes that adopt a heterogeneous ensemble, the ensemble
is adequately represented by a large number of conformations or by conformational distribu-
tions. Typical examples are intrinsically disordered proteins (IDPs) and proteins with dis-
ordered regions, domains connected with flexible linkers, or dynamic soft-matter complexes. Upon refining such heterogeneous ensembles against experimental SAXS data Iexp(q), the
SAXS curves computed from the individual conformations may differ from Iexp(q), and only
the ensemble-averaged computed curve should agree with the data. However, ensemble refinement against SAXS data is an ill-posed problem because many
different ensembles would agree with Iexp(q), even if the conformational space is restrained
with an all-atom force field. Two strategies have been put forward for choosing a justified
refined ensemble from all the ensembles that satisfy the data (Ravera et al., 2016). Following
the strategy of maximum parsimony, the aim is to explain the data with as few conforma-
tions as possible. Such approach is most justified if the biomolecule adopts a few well-defined
conformational states. The second approach is founded in statistical physics and is based on
Jaynes’ maximum-entropy principle (Jaynes, 1957; Boomsma et al., 2014; Cesari et al., 2018;
Hermann and Hub, 2019). According to Jaynes, we should choose the ensemble distribution
with the greatest uncertainty (or with the least information) that satisfies a given set of con-
straints. In the context of structure refinement, the constraints are our requested agreement
with the experimental data. The principle is satisfied by finding an ensemble distribution 14 . CC-BY 4.0 International license
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bioRxiv preprint Figure 5: Illustration of parallel-replica ensemble refinement. 4.1
Theoretical background (2013) showed that the replica-averaged harmonic restraint imposed
a minimal bias in the limit of a large number of replicas. 15 . CC-BY 4.0 International license
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Parallel-replica ensemble refinement against SAXS data Parallel-replica refinement against SAXS data is illustrated for an IDP in Fig.5. First, the
SAXS intensity is averaged over the replicas intensities Ic(qi, Rα), ¯Ic(qi; R1, ..., RN) =
1
Nrepl
Nrepl
X
α=1
Ic(qi, Rα) ,
(21) (21) where α is the replica index and Nrepl the number of replicas. Then, the systems are coupled
to the data with a harmonic restraint similar to Eq. 8, except that the SAXS curve from a
single simulation is substituted by the replica-averaged curve: where α is the replica index and Nrepl the number of replicas. Then, the systems are coupled
to the data with a harmonic restraint similar to Eq. 8, except that the SAXS curve from a
single simulation is substituted by the replica-averaged curve: Eexp(R1, ..., RN; Iexp) = 1
2NreplfckBT NShan
Nused
Nused
X
j
¯Ic(qj; R1, ..., RN) −¯I′
exp(qj)
2
f 2 ¯σ2(qj)
(22) (22) Here, ¯I′
exp denotes the smoothed experimental curve adjusted by the fitting parameters f
and c, as used above. The biasing energy is multiplied with Nrepl, such that the factor cancels
with 1/Nrepl in Eq. 21 when taking the derivatives with respect to atomic coordinates, as
done for computing the SAXS-derived forces (Hummer and Köfinger, 2015). Here, ¯I′
exp denotes the smoothed experimental curve adjusted by the fitting parameters f
and c, as used above. The biasing energy is multiplied with Nrepl, such that the factor cancels
with 1/Nrepl in Eq. 21 when taking the derivatives with respect to atomic coordinates, as
done for computing the SAXS-derived forces (Hummer and Köfinger, 2015). 4.4
Protocol C Multi-replica SAXS-driven MD simulations are set up similar to the single-replica refinement
described above. 1. Compile GROMACS-SWAXS with MPI support using cmake -DGMX_MPI=ON .. 1. Compile GROMACS-SWAXS with MPI support using cmake -DGMX_MPI=ON ... 2. When using four replicas, for instance, use the following mdp options: 2. When using four replicas, for instance, use the following mdp options: waxs-ensemble-type
= maxent-ensemble
waxs-ensemble-nstates
= 4
waxs-scale-i0
= no ; yes with small contrast, e.g. with IDP
gen-vel
= yes waxs-ensemble-type
= maxent-ensemble
waxs-ensemble-nstates
= 4
waxs-scale-i0
= no ; yes with small contrast, e.g. with IDP
gen-vel
= yes waxs-ensemble-type With the gen-vel = yes option, the replicas start with different initial velocities, pro-
viding independent simulations. For solutes with a small contrast, such as an IDP, the
forward scattering intensity Ic(q = 0) may not converge within the memory time τ. To
greatly accelerate the convergence, Ic(q = 0) may be fixed to the forward intensity of the
target curve by adding a small constant density to the solvent, turned on with the mdp
option waxs-scale-i0 = yes. Prepare one tpr file for each replica, and place the tpr files
into different sub-directories 000, 001, etc: gmx grompp -f maxent.mdp -o 000/topol.tpr
gmx grompp -f maxent.mdp -o 001/topol.tpr e 3. Run the multi-replica simulation with the -multidir functionality of mdrun: sw=/path/to/pure-solvent.tpr
fw=/path/to/pure-solvent.xtc
target=$(realpath Itarget_trans.xvg)
export GMX_ENVELOPE_FILE=$(realpath envelope.dat)
export GMX_WAXS_FIT_REFFILE=$(realpath envelope-ref.gro)
mpiexec -np 4 gmx_mpi mdrun \
-s topol.tpr -sw $sw -fw $fw -is $target -multidir 000 001 sw=/path/to/pure-solvent.tpr fw=/path/to/pure-solvent.xtc target=$(realpath Itarget_trans.xvg) p
p
p
export GMX_WAXS_FIT_REFFILE=$(realpath envelope-ref.gro) p
p
g
p
-s topol.tpr -sw $sw -fw $fw -is $target -multidir 000 001 4. Carry out the same analysis as described above for regular SAXS-driven MD. Now, the file
waxs_final.xvg contains the final on-the-fly average of the replica-averaged SAXS curve. Carry out a rerun with the trajectories of the four replicas to compute a uniformly averaged
SAXS curve for each replica, and henceforth average the calculated curve. Inspect whether
the replica-averaged curve agrees with the experimental target curve. 5. Compute distributions h1(ξ) of interesting observables ξ, combined from all replica trajec-
tories, such as the distribution of the radius of gyration or the secondary structure content
of an IDP. Such distributions quantify the heterogeneity of the ensemble. 6. With a new simulation system, redo the simulation with increasing numbers of replicas
Nrepl. 4.4
Protocol C Re-compute distributions over observables using the same aggregated simulation time
(e.g., 1×400 ns, 2×200 ns, 4×100 ns, etc.). Inspect convergence of the entropy (or the width)
of the distributions h1(ξ) as function of Nrepl. 4.3
Choosing the number of replicas CC-BY 4.0 International license
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Choosing the number of replicas The number of replicas that are required to follow the maximum entropy principle depends
on the system (Boomsma et al., 2014; Hermann and Hub, 2019). A possible strategy for
finding a good value for Nrepl is to investigate distributions h(ξ) of a few important degrees
of freedom ξ, such as distribution of the radius of gyration of an IDP or of the moments of
inertia of a soft-matter complex. Accordingly, the Kullback-Leibler divergence DKL(h1|h0)
between the biased distribution h1(ξ) and unbiased distribution h0(ξ) may be plotted versus
the number of replicas. A sufficient value of Nrepl would be indicated by a plateau region of
such plot. A disadvantage of DKL is its numerical instability; namely, since p0 appears in the de-
nominator of Eq. 20, DKL is unstable if some conformations of the biased ensemble p1
were hardly sampled in the unbiased distribution p0. A numerically more stable alternative
is given by the Jensen-Shannon divergence, which may be considered as a smoothed and
symmetrized version of DKL, DJS(h1|h0) = [DKL(h0|hM) + DKL(h1|hM)]/2,
(23) (23) where hM = (h0 + h1)/2 is the average of h0 and h1 (Hermann and Hub, 2019). Another
useful measure is given by the entropy of biased distributions h1, S[h1] =
R
h1(ξ) ln h1(ξ)dξ. If the number of replias is too low, the ensemble is still overly biased, which may lead to
overly narrow ensemble and overly narrow distributions h1(ξ). Hence, it is useful to plot the
entropy (or even simply the width) of h1(ξ) versus the number of replicas. For the refinement
of an IDP ensemble we found previously that using only 4–8 replicas were sufficient. This
finding is likely explained by the fact that, when using explicit-solvent SAXS predictions,
Ic(qi, Rα) represent on-the-fly averages with a memory time of 50–200 ps. Hence, even the
SAXS curves from individual replicas represent some conformation heterogeneity, explaining
why only few replicas are sufficient to represent the heterogeneous overall shape of the IDP
ensemble that is encoded by the data. 16 . CC-BY 4.0 International license
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Example: ensemble refinement of a detergent micelle Figure 6 presents a multi-replica-average ensemble refinement of a DDM detergent micelle
using an increasing number of 1–10 replicas. The distributions of moments of inertia as com-
puted from the aggregated simulations strongly depend on the number of replicas. Namely, 17 . CC-BY 4.0 International license
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bioRxiv preprint Figure 6: (A) Parallel-replica ensemble refinement of a DDM detergent micelle. (B) Excellent
agreement is found with the data, irrespective of the number of replicas between 1 and 10. (C) However, different numbers of replicas lead to different conformation ensembles, here
quantified by the distributions of the moments of inertia along the three principal axes,
demonstrating that agreement with the data does by far not guarantee that the ensemble
is correct. Reprinted with permission from Ivanovic et al., J. Phys. Chem. Lett. 2020, 11,
945–951, Copyright 2020 American Chemical Society. Figure 6: (A) Parallel-replica ensemble refinement of a DDM detergent micelle. (B) Excellent
agreement is found with the data, irrespective of the number of replicas between 1 and 10. (C) However, different numbers of replicas lead to different conformation ensembles, here
quantified by the distributions of the moments of inertia along the three principal axes,
demonstrating that agreement with the data does by far not guarantee that the ensemble
is correct. Reprinted with permission from Ivanovic et al., J. Phys. Chem. Lett. 2020, 11,
945–951, Copyright 2020 American Chemical Society. the distributions from single-copy refinement are overly narrow, indicative of an overly re-
strained ensemble in violation of the maximum entropy principle (Fig. 6C, black). Using a
larger number of replicas, the distributions are wider, reflecting a larger degree of hetero-
geneity. Critically, all simulations reveal quantitative agreement with the data, even when
using only a single replica (Fig. 6B). This demonstrates that agreement with the data does
by far not guarantee that the ensemble is correct. 4.5
Example: ensemble refinement of a detergent micelle the distributions from single-copy refinement are overly narrow, indicative of an overly re-
strained ensemble in violation of the maximum entropy principle (Fig. 6C, black). Using a
larger number of replicas, the distributions are wider, reflecting a larger degree of hetero-
geneity. Critically, all simulations reveal quantitative agreement with the data, even when
using only a single replica (Fig. 6B). This demonstrates that agreement with the data does
by far not guarantee that the ensemble is correct. 5
Discussion: conceptual considerations and recommen-
dations 5.1
SAXS-driven MD simulations (should) feel only a weak bias by
the SAXS data 5.1
SAXS-driven MD simulations (should) feel only a weak bias by
the SAXS data Structural data is typically insufficient for defining all degrees of freedom of a biomolecule. This is true not only for SAXS data, but also for data from X-ray crystallography, NMR
spectroscopy, or cryo electron microscopy. To avoid overfitting during structure refinement, 18 . CC-BY 4.0 International license
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bioRxiv preprint structural data is complemented with addition physico-chemical information that restraints
the biomolecule into realistic conformations. The required amount of additional information
critically depends on the information content of the data; the lower information content of the
data, the more predictive additional information is needed. For instance, excluding atomic
overlaps and restraining chemical bond geometries is often sufficient for the refinement of
atomic models against crystallographic data but would be by far insufficient for refinement
against SAXS data. During SAXS-driven MD, the structure is largely imposed by the all-atom force field,
which restraints not only chemical geometries but also maintains a proper hydrogen bond
network and favorable electrostatic interactions. Overfitting is avoided by using only a small
force constant fc for the SAXS-derived restraints in the order of unity, such that biomolecular
dynamics are largely controlled by the force field VFF(R) whereas the energy Eexp only mildly
pushes the biomolecule into agreement with the data (see Eqs. 8 and 17). Indeed, Ef
exp takes
values of few up to tens of kilojoules per mole, whereas the potential energy contributions
from Lennard-Jones or Coulomb interactions are typically in the range of hundred thousands
to millions of kilojoules per mole. 5.2
Accelerating transitions with SAXS data and sampling limita-
tions SAXS-driven MD simulations have been used to accelerate large-scale conformational tran-
sitions of biomolecules, which would require prohibitively long simulation times during unbi-
ased simulations. For instance, we showed that SAXS data may be used to drive large-scale
opening transition of the large proteins Hsp90 or ATCase in MD simulations (Shevchuk and
Hub, 2017; Chen and Hub, 2015). However, SAXS-driven MD works as an enhanced sam-
pling technique only if the SAXS-derived forces point into the direction of the sought-after
conformation transition. This is typically true in the case of large-scale domain motions, in
particular if these motions modulate the radius of gyration. In contrast, achieving a complex,
nonlinear rearrangements in the simulation such as the folding of an unstructured tail into
an α-helix is far more challenging. In such cases, the correct final state might be detectable
via a low χ2 in Eq. 5, but it is unlikely that SAXS-derived forces would accelerate the folding
transition. Hence, for guiding complex transitions with SAXS data, SAXS-driven MD may
be combined with established enhanced sampling techniques such as simulated tempering
or Hamiltonian replica exchange. 6
Summary (iii) The parallel-replica approach allows one to refine heterogeneous ensembles with com-
mitment to the maximum-entropy principle. Here, the SAXS curve averaged over sev-
eral replicas is coupled to the experimental data using harmonic restrains, such that
the updated ensemble is compatible with the data but biased as minimally as possible
with respect to the unbiased ensemble. All approaches require a forward model for calculating the SAXS intensities from the
atomistic structures. We discussed structure and ensemble refinement based on explicit-
solvent SAXS curve calculations, as used in the WAXSiS method, thereby accurately rep-
resenting the hydration layer and the excluded solvent Chen and Hub (2014); Knight and
Hub (2015). An implementation of the methods described here employing the explicit-solvent
SAXS curve calculations is freely available in GROMACS-SWAXS (https://gitlab.com/cbjh/gromac
swaxs). 7
Acknowledgements We thank Milos Ivanovic for preparing Figure 6A. This study was supported by the Deutsche
Forschungsgemeinschaft (HU 1971/3-1). Björling, A., Niebling, S., Marcellini, M., van der Spoel, D., and Westenhoff, S. (2015).
Deciphering solution scattering data with experimentally guided molecular dynamics sim-
ulations. J. Chem. Theory Comput., 11:780–787. 6
Summary SAXS is an increasingly valuable tool of integrative structural biology thanks to the accuracy
of data collected at modern SEC-SAXS beamlines and thanks to its structural information
content, which is not accessible by other techniques. However, the interpretation of the data
is challenged by the low information content of the experimental signals, leading to the
risk of overinterpreting the data. This risk is mitigated by using MD simulations that add
physico-chemical information to the data. In this chapter, we presented three approaches for refining structural ensembles against
SAXS data by integrating the experimental data on-the-fly into all-atom MD simulations: 19 . CC-BY 4.0 International license
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bioRxiv preprint (i) During SAXS-driven MD, an MD simulation is coupled to experimental SAXS data
using harmonic restrains on the data, thereby refining an ensemble that may be ap-
proximated by a single, most prominent conformation. The SAXS-driven simulations
promote conformational transitions compatible with the data. They are capable of
overcoming force field imperfections and sampling limitations of unbiased simulations,
given that the transitions are geometrically simple. (ii) Using SAXS-driven MD as tool for Bayesian inference of biomolecular structures, a
posterior distribution is sampled by the MD simulation, quantifying the plausibility of
a biomolecular structure in the light the experimental data and prior physico-chemical
information, as encoded in the MD force fields. This Bayesian framework may estimate
systematic errors at small angles simultaneously with the structure, which enables one
to assess whether systematic errors are required to explain the experimental data. (iii) The parallel-replica approach allows one to refine heterogeneous ensembles with com-
mitment to the maximum-entropy principle. Here, the SAXS curve averaged over sev-
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the updated ensemble is compatible with the data but biased as minimally as possible
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https://doi.org/10.1101/2022.04.05.487171
doi:
bioRxiv preprint Putnam, C. D., Hammel, M., Hura, G. L., and Tainer, J. A. (2007). X-ray solution scatter-
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CC-BY 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted April 10, 2022.
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Estágio em psicologia escolar e educacional: ruptura com a medicalização da educação
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Psicologia Escolar e Educacional
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| 1,756
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Pasantía en psicología escolar y educacional: ruptura con la
medicalización de la educación Vânia Aparecida Calado
Universidade Potiguar – RN Todos esses elementos citados precarizam as rela
ções construídas na escola, provocam sofrimento em todos
os seus atores, prejudicam a qualidade do processo de en
sino e aprendizagem e revelam uma complexidade que será
enfrentada pelo psicólogo escolar e educacional. A Psicologia Escolar e Educacional é uma área de
estudos e de atuação da Psicologia que compreende o
fenômeno educacional como resultado das relações que
se estabelecem no interior da escola. A escola deve ser
compreendida a partir de elementos como as políticas edu
cacionais, pela história local de sua constituição enquanto
instituição, como referência educacional pelos sujeitos que a
constituem e nela se constituem, além de aspectos sociais e
ideológicos (Patto, 2008; Souza 2009).l Souza (2009, 2010) traz alguns desafios para a for
mação e atuação do psicólogo escolar educacional: superar
a concepção e atuação profissional a partir de uma perspec
tiva adaptacionista; compromisso social com a construção
de uma escola democrática e de qualidade; a construção de
uma práxis psicológica frente a queixa escolar; romper com
práticas medicalizantes na instituição educacional. A escola reflete as desigualdades sociais, econômi
cas e culturais e também as reproduz. Historicamente, os
alunos que apresentam dificuldades são encaminhados
para trajetórias caracterizadas pelo fracasso escolar, perme
adas por sofrimento e exclusão. Atualmente, muitos desses
alunos, com base na concepção de que o fracasso escolar
se deve às disfunções neurológicas, são diagnosticados
com pretensas doenças do não aprender, incluindo-se aqui
a hiperatividade, a disfunção cerebral mínima, os distúrbios
de aprendizagem, a dislexia, doenças que nunca foram
comprovadas. O objetivo desse texto é descrever um relato de expe
riência que se refere ao acompanhamento de uma turma do
curso de Psicologia de uma instituição de Ensino Superior
Privada do Rio Grande do Norte na disciplina de estágio bá
sico em Psicologia Escolar e Educacional, ministrada no pri
meiro semestre de 2013; relatar a trajetória dos estagiários
a partir da imersão em instituições educacionais públicas e
sua contribuição para a formação dos estudantes, tendo em
vistas os desafios colocados por Souza (2009, 2010). Para Moysés e Collares (2010), esse fenômeno se
refere à medicalização da educação que busca transformar
problemas do sistema educacional em doenças que a me
dicina poderia resolver. Para as autoras, esse movimento
transfere para o campo médico, questões coletivas, de or
dem social e política, reduzindo-as a aspectos biológicos,
isentando de responsabilidade outras instâncias de poder. Revista Quadrimestral da Associação Brasileira de Psicologia Escolar e Educacional, SP. Volume 18, Número 3, Setembro/Dezembro de 2014: 567-569. http://dx.doi.org/10.1590/ 2175-3539/2014/0183828 http://dx.doi.org/10.1590/ 2175-3539/2014/0183828 Brasileira de Psicologia Escolar e Educacional, SP. Volume 18, Número 3, Setembro/Dezembro de 2014: 567-569. Pasantía en psicología escolar y educacional: ruptura con la
medicalización de la educación O
resultado é a individualização e a culpabilização das crian
ças e adolescentes. A proposta do estágio foi apresentada às equipes
gestoras, de escolas públicas de ensino fundamental e mé
dio, que relataram algumas queixas escolares relacionadas
a algumas turmas, como por exemplo: indisciplina, violência,
desinteresse pela escolarização, distanciamento da família,
desestrutura familiar, transtornos de aprendizagem como
dislexia e transtorno de déficit de atenção (TDAH). Revista Quadrimestral da Associação Brasileira de Psicologia Escolar e Educacional, SP. Volume 18, Número 3, Setembro/Dez com que desacreditassem em sua capacidade de aprender
e desistissem de pensar no futuro.i Os estagiários foram divididos em grupos de 3 a 4
pessoas, acompanharam 15 turmas de alunos distribuídas
em 8 escolas, realizaram 6 a 7 visitas a campo, quando
desenvolveram as seguintes atividades: investigação da his
tória da escola, entrevistas com equipe gestora, professores
e demais funcionários, 4 a 5 encontros com as turmas indi
cadas, observação de diversos momentos da rotina escolar
(entrada e saída dos alunos, intervalo, aula vaga, uso da
biblioteca, sala de multimídia e de informática, reunião de
pais) leitura do projeto político pedagógico, discussão e pla
nejamento das atividades com os professores das turmas. As supervisões aconteciam antes de cada visita a campo
para reflexão, problematização, discussão de textos e pla
nejamento da próxima visita à instituição. Para os profissionais e para as turmas que participa
ram do estágio, ter vivenciado um espaço em que podiam
falar o que pensavam e sentiam, possibilitou o fortaleci
mento de seus potenciais. A relação entre equipe e alunos
ficou mais horizontal, flexível e aberta. Aquelas crianças e
adolescentes que inicialmente não paravam quietas, que
não eram capazes de refletir, dar opinião e realizar uma
produção escrita, ao final dos encontros, faziam tudo isso. Os encontros tinham contribuído para a problematização da
queixa vivenciada e sua ressignificação (Checchia, 2010). As supervisões possibilitaram que os estagiários
pouco a pouco realizassem uma ruptura epistemológica de
uma visão adaptacionista da Psicologia, que percebessem a
complexidade do fenômeno escolar e o quanto explicações
reducionistas como a desestrutura familiar, a violências das
comunidades, as supostas doenças do não aprender eram
superficiais e preconceituosas, buscavam reduzir e culpabi
lizar ora o aluno, ora a família ora os professores e gesto
res (Asbahr, Martins, & Mazzolini, 2011). Pasantía en psicología escolar y educacional: ruptura con la
medicalización de la educación Compreendiam a
necessidade da construção de uma práxis frente à queixa
escolar que possibilitasse o trabalho participativo com todos
os setores do processo educativo, o fortalecimento do traba
lho do educador, a análise coletiva dos diferentes discursos
para o enfrentamento dos desafios e ruptura de práticas
medicalizantes. Apresentavam outro posicionamento ético e
político, o compromisso com a luta por uma escola democrá
tica e de qualidade (Souza, 2010). Durante as primeiras supervisões os estagiários rela
taram experiências de intenso sofrimento devido à compre
ensão da realidade das instituições educacionais públicas,
caracterizada por: infraestrutura precária; falta de ventilação,
iluminação, material de limpeza; sem manutenção das insta
lações impedindo o uso de espaços como quadra; ausência
de refeitório; bibliotecas não utilizadas; falta de merenda e
de transporte escolar que causava cancelamento das aulas;
muitas aulas vagas; ausência de espaços de reflexão e troca
de experiências; utilização de estratégias de homogeneiza
ção na formação de turmas; discurso da instituição em rela
ção aos alunos e seus familiares com preconceitos ligados
à raça, gênero e classe social. Para Souza (2007), esses
problemas caracterizam ambientes escolares que produzem
fracasso escolar e sofrimento em muitas instituições educa
cionais públicas. O contato com a equipe gestora, principalmente com
a coordenação pedagógica, revelou centralização de poder
de decisão, resolução de conflitos de sala de aula a partir
de ações autoritárias, como ameaças e punições, restando
pouca disponibilidade de tempo para o trabalho de orienta
ção e apoio pedagógico à equipe. A ação em conjunto com
os professores possibilitou compreender que trabalhavam
isoladamente, com precário material didático, baixos salários
e desvalorização profissional. No geral, a equipe encontrava
-se desanimada, frustrada, apresentando estresse e descré
dito em relação aos alunos e às políticas educacionais. Referências Interinstitucional Queixa Escolar (Orgs.), Medicalização de
crianças e adolescentes: conflitos silenciados pela redução de
questões sociais a doença de indivíduos (pp. 57-67). São Paulo:
Casa do Psicólogo. Recebido em: 29/10/2013
Aprovado em: 07/06/2014 Recebido em: 29/10/2013
Aprovado em: 07/06/2014 Souza, M. P. R. (2010). Retornando à patologia para justificar a
não aprendizagem escolar: a medicalização e o diagnóstico de
transtornos de aprendizagem em tempos de neoliberalismo.
Em Conselho Regional de Psicologia de São Paulo & Grupo p
(
@y
)
Professora do Curso de Psicologia da Universidade Potiguar – Laureate International Universities, Campus Roberto Freire, Natal, RN. Mestre em
Psicologia Escolar e do Desenvolvimento Humano pela Universidade de São Paulo. Referências Asbahr, F. S. F., Martins, E., & Mazzolini, B. P. M. (2011). Psicologia,
formação de psicólogos e a escola: desafios contemporâneos. Psicologia em Estudo, 16(1), 157-163. Checchia, A. K. A. (2010). Adolescência e escolarização numa
perspectiva crítica em Psicologia Escolar. Campinas: Alínea. Moysés, M.A., & Collares, C. A. L. (2010). Dislexia e TDAH: uma
análise a partir da ciência médica. Em Conselho Regional de
Psicologia de São Paulo & Grupo Interinstitucional Queixa Escolar
(Orgs.), Medicalização de crianças e adolescentes: conflitos
silenciados pela redução de questões sociais a doença de
indivíduos (pp. 71-110). São Paulo: Casa do Psicólogo. Os pais que participaram valorizavam os professores
e a escola. Percebiam a grande quantidade de aulas vagas
provocadas pelas faltas dos docentes e por disciplinas sem
docentes. Não reclamaram da qualidade da aula dos pro
fessores, apenas da relação com seus filhos, muitas vezes
desrespeitosa e autoritária. Todas as escolas estabeleciam
relação hierárquica com os pais, que por sua vez, não en
contravam espaço de escuta e participação e eram culpabili
zados pelos problemas vivenciados pela instituição. Patto, M. H. S. (2008). A produção do fracasso escolar: histórias de
submissão e rebeldia. São Paulo: Casa do Psicólogo. Souza, B. P. (2007). Funcionamentos escolares e produção do
fracasso escolar e sofrimento. Em Souza, B. P. (Org.), Orientação
à queixa escolar (pp. 241-278). São Paulo: Casa do Psicólogo. As turmas de alunos trouxeram muitas queixas rela
cionadas aos professores, gestores, infraestrutura da escola,
aos colegas e familiares. Queixaram-se de não serem vistos
como crianças e adolescentes, de não terem espaço de es
cuta e acolhimento de suas manifestações. A percepção de
aulas desmotivantes, monótonas, professores impacientes e
autoritários, relatos de experiências de humilhação, faziam Souza, M. P. R. (2009). Psicologia Escolar e Educacional em busca
de novas perspectivas. Psicologia Escolar e Educacional, 13(1),
179-182. 568 Relato de Prática Profissional. Souza, M. P. R. (2010). Retornando à patologia para justificar a
não aprendizagem escolar: a medicalização e o diagnóstico de
transtornos de aprendizagem em tempos de neoliberalismo. Em Conselho Regional de Psicologia de São Paulo & Grupo
Interinstitucional Queixa Escolar (Orgs.), Medicalização de
crianças e adolescentes: conflitos silenciados pela redução de
questões sociais a doença de indivíduos (pp. 57-67). São Paulo:
Casa do Psicólogo. Interinstitucional Queixa Escolar (Orgs.), Medicalização de
crianças e adolescentes: conflitos silenciados pela redução de
questões sociais a doença de indivíduos (pp. 57-67). São Paulo:
Casa do Psicólogo. Sobre a autora Vânia Aparecida Calado (vaniacalado1@yahoo.com.br) Vânia Aparecida Calado (vaniacalado1@yahoo.com.br)
Professora do Curso de Psicologia da Universidade Potiguar – Laureate International Universities, Campus Roberto Freire, Natal, RN. Mestre em
Psicologia Escolar e do Desenvolvimento Humano pela Universidade de São Paulo. Professora do Curso de Psicologia da Universidade Potiguar – Laureate International Universities, Campus Roberto Freire, Natal, RN. Mestre em
Psicologia Escolar e do Desenvolvimento Humano pela Universidade de São Paulo. O presente artigo foi parcialmente apresentado no III Seminário Internacional “A Educação Medicalizada – reconhecer e acolher as diferenças”,
realizado de 10 a 13 de Julho de 2013 em São Paulo. Estágio em psicologia escolar e educacional * Vânia Aparecida Calado 569
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https://ejurnal.itenas.ac.id/index.php/elkomika/article/download/8947/3360
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Rancang Bangun Purwarupa Manipulator Lengan Robot Dengan Tiga Derajat Kebebasan
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Elkomika
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cc-by-sa
| 5,474
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Rancang Bangun Purwarupa Manipulator Lengan
Robot Dengan Tiga Derajat Kebebasan
IDHAM HANIF AYEGA, TUA AGUSTINUS TAMBA, BAGUS MADE ARTHAYA
Program Studi Teknik Elektro, Universitas Katolik Parahyangan, Indonesia
Email: ttamba@unpar.ac.id Received 31 Mei 2023 | Revised 26 Juni 2021 | Accepted 3 Juli 2023 Received 31 Mei 2023 | Revised 26 Juni 2021 | Accepted 3 Juli 2023 ABSTRAK Makalah ini melaporkan hasil penelitian terkait rancang bangun suatu purwarupa
manipulator lengan robot dengan tiga derajat kebebasan. Proses perancangan
dilakukan dalam tiga tahap yang mencakup pemodelan matematis kinematika
maju dan balik pada robot, desain dan simulasi purwarupa robot menggunakan
pemrograman MATLAB dan SOLIDWORKS terintegrasi, dan konstruksi perangkat
keras robot menggunakan teknolog 3D printing.Proses simulasi dan eksperimen
selanjutnya dilakukan untuk membandingkan kesesuaian operasional dan kinerja
perangkat keras dan model simulasi komputer yang telah dirancang. Berdasarkan
hasil simulasi dan eksperimen, disimpulkan bahwa desain perangkat keras robot
memiliki tingkat kepresisian yang sangat baik dengan batas kesalahan maksimum
untuk solusi kinematika maju yang dihasilkan adalah sebesar 2.745% serta batas
kesalahan maksimum untuk solusi kinematika balik adalah sebesar 0.06%. Kata kunci: lengan robot, kinematika, robotic toolbox, 3D printing 1. PENDAHULUAN Lengan robot merupakan instrumen elektro-mekanik yang umumnya dapat diprogram untuk
memiliki fungsi menyerupai lengan manusia (Craig, 2022). Permasalahan yang sering
ditemukan pada perancangan lengan robot adalah penentuan model matematis baik untuk
peninjauan/ analisis kinematika maupun dinamika yang kemudian akan digunakan sebagai
basis perancangan pengontrol lengan robot pada tahapan berikutnya. Keberhasilan dari
pengontrolan lengan robot ditentukan berdasarkan keakuratan serta kepresisian pergerakan
lengan robot dari suatu konfigurasi/titik ke konfigurasi/titik lainnya. Manipulator lengan robot memiliki tiga komponen utama, yaitu link, sambungan (joint) dan
end effector (Craig, 2022). Joint pada lengan robot adalah sambungan penghubung antar
benda tegar (link) pada robot. Sementara itu, link merupakan benda tegar yang terhubung
dengan joint. End effector adalah bagian paling ujung dari lengan robot. Lengan robot dapat
digunakan untuk melakukan berbagai fungsi yang diantaranya memilih dan memindahkan
(pick and place) objek tertentu hingga proses pengelasan (welding) (Lynch, dkk,
2017)(Dzedzickis, dkk, 2021). Dengan perkembangan teknologi yang pesat saat ini,
lengan robot diprediksi akan semakin banyak digunakan untuk membantu pekerjaan
manusia (Dave, dkk, 2022). Dalam bidang pendidikan, lengan robot merupakan salah satu sistem yang paling banyak
digunakan untuk mengilustrasikan berbagai teknik desain dan analisis robot mulai dari
penentuan model, analisis karakteristik respons, dan implementasi pengontrol (Dewi,
2020)(Qian, dkk, 2022)(Lobbezoo, dkk, 2023). Penggunaan perangkat keras
purwarupa lengan robot dalam kegiatan pembelajaran khususnya terkait topik kinematika,
dinamika, dan teori kontrol dapat mengoptimalkan proses studi dan pengalaman mahasiswa
(Bai, 2021)(Manjula, 2018)(Baby, dkk, 2017). Meskipun saat ini telah terdapat
beragam jenis manipulator lengan robot, pemahaman mengenai sistematika proses
perancangan dan konstruksi perangkat keras lengan robot terbilang masih terbatas (Lee,
2020)(Long, dkk, 2020). Beberapa penelitian sebelumnya cenderung difokuskan pada
salah satu aspek tertentu, misalnya pemodelan kinematika (Qian, dkk, 2022) (Bai, 2021)
atau desain pengontrol (Dewi, 2020) pada manipulator saja. Desain purwarupa atau alat
peraga sistem lengan robot yang lebih sistematis dan terstruktur yang dapat digunakan
untuk mendemonstrasikan tahapan penentuan model matematis, analisis respons, desain
komponen, proses assembly, serta pengujian dan kontrol sistem lengan robot tentu sangat
diperlukan untuk mendukung proses pembelajaran/penelitian robotika (Zeng, dkk, 2022). Makalah ini melaporkan hasil studi dan penelitian terkait desain, konstruksi, dan analisis
secara terstruktur suatu purwarupa manipulator lengan robot dengan tiga (3) derajat
kebebasan. Sebagai kontribusi terhadap penelitian sebelumnya, makalah ini memaparkan
tahapan yang lebih sistematis terkait desain dan perancangan purwarupa lengan robot yang
mencakup tahapan pemodelan, desain dan konstruksi purwarupa lengan robot. Rancang Bangun Purwarupa Manipulator Lengan Robot Dengan Tiga Derajat Kebebasan Rancang Bangun Purwarupa Manipulator Lengan Robot Dengan Tiga Derajat Kebebasan ABSTRACT The research reported in this paper was aimed at developing a prototype of a
robotic arm/manipulator with three degrees of freedom. The prototype was
developed in three main stages, namely forward and inverse kinematics
modeling of the robot, simulation modeling of the prototype in MATLAB-
SOLIDWORKS integrated environment, and finally the hardware development of
the robot using 3D printing techniques. The operational performance of the
constructed robotic hardware was then analyzed and compared with that of the
developed simulation model. The experimental results of the robot performance
evaluations suggested that the robot prototype has good operational precision
performance in which the resulting maximum error for forward kinematics task is
only about 2.745% whereas the resulting maximum error for inverse kinematics
task is only about 0.06%. Keywords: robotic arm, forward kinematics, robotic toolbox, 3D printing Keywords: robotic arm, forward kinematics, robotic toolbox, 3D printing ELKOMIKA – 796 Ayega, dkk Ayega, dkk Ayega, dkk 1. PENDAHULUAN Proses
perancangan yang dipaparkan mencakup desain model matematis kinematika gerak lengan
robot, desain simulator model kinematika (forward dan inverse) dengan mengintegrasikan
perangkat lunak MATLAB/SIMULINK dan SOLIDWORKS (Kundu, dkk, 2022)(Al Tahtawi,
dkk, 2021), konstruksi perangkat keras purwarupa lengan robot dengan menggunakan 3D
printer, serta analisis dan validasi hasil purwarupa robot yang dibangun. Hasil percobaan
terkait perbandingan pergerakan antara perangkat keras purwarupa lengan robot dan model
simulasi lengan robot menunjukkan tingkat akurasi yang cukup baik di mana eror maksimum
pada penentuan solusi permasalahan forward kinematics adalah 2.745% dan eror maksimum
pada penentuan solusi permasalahan inverse kinematics adalah 0,06%. ELKOMIKA – 797 2. METODOLOGI PERANCANGAN Pemodelan awal manipulator lengan robot dilakukan untuk menggambarkan rancangan
komponen utama yang terdiri dari link, joint, dan end effector. Gambar 1 menunjukkan
rancangan komponen utama robot serta penentuan sistem koordinat lokal pada setiap joint
sebagai dasar pemodelan kinematika pergerakannya. Pada gambar ini, 𝒙𝟎−𝒚𝟎−𝒛𝟎 adalah
kerangka koordinat inersial statis pada robot sebagai referensi global, dan 𝒙𝒊−𝒚𝒊−𝒛𝒊
untuk i = 1,2,3 adalah kerangka koordinat lokal untuk setiap link pada robot. Arah sumbu 𝒛𝟎
dan 𝒛𝒊 adalah keluar dari kertas dan tidak ditunjukkan secara eksplisit pada Gambar 1. Pemodelan di Gambar 1 dilakukan dengan analisis kinematika yang mencakup kinematika
maju (forward kinematics, FK) dan kinematika balik (inverse kinematics, IK). Analisis FK
pada dasarnya berkaitan dengan penentuan posisi dan orientasi end effector berdasarkan
informasi nilai sudut joint θ୧ yang diberikan. Sebaliknya, analisis IK berkaitan dengan
penentuan kombinasi nilai sudut θ୧ yang sesuai dengan informasi posisi dan orientasi end
effector tertentu yang diinginkan (Corke, 2022)(Corke, 2017). Gambar 1. Rancangan Model Manipulator Lengan Robot Gambar 1. Rancangan Model Manipulator Lengan Robot 2.1 Pemodelan dan Analisis FK Pemodelan dan analisis FK dilakukan berdasarkan aturan dan parameter Denavit-Hartenberg
(DH) (Corke, 2022). Pada ilustrasi di Gambar 1, tabel parameter DH model robot
ditunjukkan di Tabel 1 dengan parameter 𝛼𝒊, 𝑎𝒊, 𝜃 dan d୧ masing-masing menyatakan link
twist, link length, joint angle, dan joint offset. Kemudian, konstanta lଵ=110 mm, lଶ=85 mm,
dan lଷ=60 mm menyatakan panjang link 1, link 2, dan link 3. Berdasarkan Tabel 1, maka matriks transformasi homogeneous yang menyatakan pose link i
relatif terhadap link (i-1) untuk i = 1,2,3 dapat ditentukan seperti pada Persamaan (1). 𝑇
ିଵ
= ൦
cos 𝜃
−sin 𝜃
0
𝑙cos 𝜃
sin 𝜃
cos 𝜃
0
𝑙sin 𝜃
0
0
1
0
0
0
0
1
൪
(1) 𝑇
ିଵ
= ൦
cos 𝜃
−sin 𝜃
0
𝑙cos 𝜃
sin 𝜃
cos 𝜃
0
𝑙sin 𝜃
0
0
1
0
0
0
0
1
൪
(1) (1) Matriks transformasi homogeneous yang menyatakan pose end effector terhadap posisi base
ditentukan dengan Persamaan (2) di mana 𝑥∗= 𝑙ଵcos 𝜃ଵ+ 𝑙ଶcos(Σୀଵ
ଶ
𝜃) + 𝑙ଷcos(Σୀଵ
ଷ
𝜃)
dan 𝑥∗= 𝑙ଵsin 𝜃ଵ+ 𝑙ଶsin(Σୀଵ
ଶ
𝜃) + 𝑙ଷsin(Σୀଵ
ଷ
𝜃) adalah pose robot relatif terhadap base. Rancang Bangun Purwarupa Manipulator Lengan Robot Dengan Tiga Derajat Kebebasan Rancang Bangun Purwarupa Manipulator Lengan Robot Dengan Tiga Derajat Kebebasan 𝑇
ଷ
= 𝑇
ଵ
𝑇
ଶ
ଵ
𝑇
ଷ
ଶ൦
cos(Σ𝑖=1
3 𝜃𝑖)
−sin(Σ𝑖=1
3 𝜃𝑖)
0
𝑥∗
sin(Σ𝑖=1
3 𝜃𝑖)
cos(Σ𝑖=1
3 𝜃𝑖)
0
𝑦∗
0
0
1
0
0
0
0
1
൪
(2) (2) Tabel 1. Parameter dan Tabel DH Model Robot di Gambar 1
Link i
𝜶𝒊
𝒂𝒊
𝒅𝒊
𝜽𝒊
i=1
0
1l
0
𝜃ଵ
i=2
0
2l
0
𝜃ଶ
i=3
0
3l
0
𝜃ଷ Validasi parameter DH di Tabel 1 dan matriks transformasi homogeneous di Persamaan (2),
simulasi FK dilakukan menggunakan Robotics Toolbox pada MATLAB (Corke, 2022)(Corke,
2017). Nilai parameter 𝜃= 90° dipilih untuk i = 1,2,3. Berdasarkan Persamaan (2), matriks
transformasi homogeneous model robot ditunjukkan pada Persamaan (3). Validasi parameter DH di Tabel 1 dan matriks transformasi homogeneous di Persamaan (2),
simulasi FK dilakukan menggunakan Robotics Toolbox pada MATLAB (Corke, 2022)(Corke,
2017). Nilai parameter 𝜃= 90° dipilih untuk i = 1,2,3. Berdasarkan Persamaan (2), matriks
transformasi homogeneous model robot ditunjukkan pada Persamaan (3). 𝑇
ଷ
= ൦
0
1
0
−85
−1
0
0
50
0
0
1
0
0
0
0
1
൪
(3) (3) yang mengindikasikan posisi end effector adalah 𝜃= 85 dan y = 50 . Hasil simulasi pada
Robotics Toolbox ditunjukkan di Gambar 2 dengan nilai matriks transformasi homogeneous
yang sama dengan hasil pada Persamaan (3). Dapat disimpulkan bahwa model FK yang
ditentukan sesuai dengan desain model robot yang dirancang. Gambar 2. Hasil simulasi untuk 𝜽𝒊 dengan 𝒊= 𝟏, 𝟐, 𝟑 Gambar 2. Hasil simulasi untuk 𝜽𝒊 dengan 𝒊= 𝟏, 𝟐, 𝟑 2. METODOLOGI PERANCANGAN Matriks transformasi homogeneous yang menyatakan pose end effector terhadap posisi base
ditentukan dengan Persamaan (2) di mana 𝑥∗= 𝑙ଵcos 𝜃ଵ+ 𝑙ଶcos(Σୀଵ
ଶ
𝜃) + 𝑙ଷcos(Σୀଵ
ଷ
𝜃)
dan 𝑥∗= 𝑙ଵsin 𝜃ଵ+ 𝑙ଶsin(Σୀଵ
ଶ
𝜃) + 𝑙ଷsin(Σୀଵ
ଷ
𝜃) adalah pose robot relatif terhadap base. ELKOMIKA – 798 ELKOMIKA – 798 ELKOMIKA – 798 2.2 Pemodelan dan Analisis IK Solusi permasalahan IK ditentukan menggunakan geometri model robot seperti ditunjukkan
pada Gambar 3. Berdasarkan gambar ini, sudut orientasi 𝜙 antara end effector lengan robot
terhadap koordinat base adalah: 𝜙= 𝜃1 + 𝜃2 + 𝜃3. Maka dapat dituliskan: 𝑥= 𝑙ଵcos 𝜃1 + 𝑙2 cos(Σ𝑖=1
2 𝜃𝑖) + 𝑙3 cos 𝜙= 𝑥̅ + 𝑙3 cos 𝜙
(4a)
𝑦= 𝑙ଵsin 𝜃1 + 𝑙2 sin(Σ𝑖=1
2 𝜃𝑖) + 𝑙3 sin 𝜙= 𝑦ത+ 𝑙3 sin 𝜙
(4b) (4a)
(4b) (4b) di mana variabel 𝑥̅ dan 𝑦ത diilustrasikan seperti di Gambar 3. di mana variabel 𝑥̅ dan 𝑦ത diilustrasikan seperti di Gambar 3. di mana variabel 𝑥̅ dan 𝑦ത diilustrasikan seperti di Gambar 3. ELKOMIKA – 799 ELKOMIKA – 799 Ayega, dkk Ayega, dkk Ayega, dkk
Gambar 3. Geometri robot untuk penentuan solusi IK Gambar 3. Geometri robot untuk pene Gambar 3. Geometri robot untuk penentuan solusi IK 2.3 Simulasi Desain Model Berbasis MATLAB-SOLIDWORKS 2.3 Simulasi Desain Model Berbasis MATLAB SOLIDWORKS
Berdasarkan model kinematika yang dibangun pada MATLAB, selanjutnya dirancang
simulator desain purwarupa lengan robot pada SOLIDWORKS untuk memperoleh gambaran
nyata purwarupa yang angan dibangun. Secara khusus, rancangan purwarupa robot pada
SOLIDWORKS ini sangat penting karena setiap komponen penyusun robot yang
dikembangkan dicetak langsung berdasarkan desain komponen tiga dimensi (3D) pada
SOLIDWORKS. Dalam hal ini, desain simulator harus dibangun dengan mengintegrasikan
model 3D pada SOLIDWORKS dengan model kinematika yang telah dirancang pada MATLAB
(Kundu, dkk, 2022)(Al Tahtawi, dkk, 2021). Proses integrasi dilakukan menggunakan
fitur SIMSCAPE pada MATLAB yang memungkinkan rancangan model pada SOLIDWORKS
untuk diakses melalui SIMULINK. Dalam hal ini, setiap perintah (command) dan data pada
MATLAB dan SOLIDWORKS dapat saling dipertukarkan. Rancangan model perangkat keras
robot pada SOLIDWORKS ditunjukkan di Gambar 4 sedangkan diagram blok model
SIMULINK/SIMSCAPE antara MATLAB dan SOLIDWORKS ditunjukkan di Gambar 5. Setelah diperoleh integrasi model MATLAB-SOLIDWORKS, selanjutnya dilakukan simulasi
untuk menguji karakteristik nilai torsi yang dibutuhkan pada desain purwarupa robot untuk
menghasilkan nilai sudut tertentu yang diinginkan pada setiap joint robot. Pada proses
simulasi ini, nilai sudut joint yang diinginkan merupakan input pada MATLAB sedangkan nilai
torsi yang dibutuhkan adalah output pada SOLIDWORKS. Pada proses simulasi, sudut rotasi
pada link 1 diputar terlebih dahulu hingga mencapai nilai 𝜃ଵ= 90° pada detik ke-60,
kemudian sudut pada link 2 diputar hingga mencapai nilai θଶ= 45° pada detik ke-120, dan
akhirnya sudut pada link 3 diputar untuk mencapai nilai θଷ= 45° pada detik ke-200. Contoh
nilai torsi yang dibutuhkan oleh link 1 untuk mencapai rotasi ditunjukkan pada Gambar 6. Gambar 4. Desain perangkat keras purwarupa robot pada SOLIDWORKS Gambar 4. Desain perangkat keras purwarupa robot pada SOLIDWORKS ELKOMIKA – 800 ELKOMIKA – 800 Rancang Bangun Purwarupa Manipulator Lengan Robot Dengan Tiga Derajat Kebebasan Gambar 5. Diagram blok simulator pada MATLAB SIMSCAPE
Gambar 6. Karakteristik torsi motor (kiri) dan nilai sudut (kanan) pada link 1 Gambar 5. Diagram blok simulator pada MATLAB SIMSCAPE Gambar 5. Diagram blok simulator pada MATLAB SIMSCAPE Gambar 6. Karakteristik torsi motor (kiri) dan nilai sudut (kanan) pada link 1 Gambar 6. Karakteristik torsi motor (kiri) dan nilai sudut (kanan) pada link 1 3. HASIL DAN PEMBAHASAN 3.1 Hasil Rancangan Perangkat Keras 3.1 Hasil Rancangan Perangkat Keras 3.1.1 Rancangan Struktur Base 3.1.1 Rancangan Struktur Base Komponen struktur base merupakan tumpuan utama bagi purwarupa lengan robot yang
dirancang menggunakan kombinasi material polylactic acid (PLA) dan acrylic (Landers, dkk,
2020). Penggerak joint pada base terdiri dari tiga komponen utama, yaitu timing belt,
timing pulley, dan gear (Rahul, dkk, 2020). Struktur base pada dasarnya dirancang
sebagai sebuah joint tipe revolute, namun pada penelitian ini tidak difungsikan dan hanya
bersifat statis/diam. Revolute joint pada base dibangun dengan aktuator berupa motor
stepper NEMA 23 yang mampu menghasilkan torsi maksimal sebesar 1,8 Nm. Gambar 8
menunjukkan hasil rancang bangun komponen base dengan detail komponen utama serta
fungsi yang ditunjukkan pada Tabel 3. Tabel 2. Komponen mekanikal-elektrikal robot
Komponen mekanikal-elektrikal
Keterangan
Mikrokontroler AM 2560
Pemrograman robot
Motor driver (TB 6560/6600)
Penggerak aktuator
Motor stepper NEMA 23/17/11
Aktuator pada joint robot
Catu daya 12 volt, 5 Ampere
Sumber daya listrik
Breadboard dan kabel jumper
Rangkaian elektronik robot
Link dan joint lengan robot
Komponen gerak pada robot
Komponen pencapit (gripper)
Komponen end effector robot
Struktur base kerangka inersial
Rangka statis penyangga robot
Timing belt, timing pulley, gear
Penggerak motor stepper
Fastener
Konektor komponen robot Tabel 2. Komponen mekanikal-elektrikal robot Tabel 2. Komponen mekanikal-elektrikal robot
Komponen mekanikal-elektrikal
Keterangan
Mikrokontroler AM 2560
Pemrograman robot
Motor driver (TB 6560/6600)
Penggerak aktuator
Motor stepper NEMA 23/17/11
Aktuator pada joint robot
Catu daya 12 volt, 5 Ampere
Sumber daya listrik
Breadboard dan kabel jumper
Rangkaian elektronik robot
Link dan joint lengan robot
Komponen gerak pada robot
Komponen pencapit (gripper)
Komponen end effector robot
Struktur base kerangka inersial
Rangka statis penyangga robot
Timing belt, timing pulley, gear
Penggerak motor stepper
Fastener
Konektor komponen robot
Gambar 8. Struktur base: tampak samping (kiri), isometris (tengah), assembly (kanan)
Tabel 3. Komponen mekanikal-elektrikal pada base
Komponen struktur base
Keterangan
Base motor holder
Tempat peletakan motor stepper pada base
Motor shaft
Meneruskan putaran motor stepper ke base
Motor stepper NEMA 23
Aktuator joint struktur base
Ball bearing (28 x 12 x 8 mm)
Mengurangi gesekan antara shaft dan base
Thrust bearing (28x12x9 mm)
Menahan gaya aksial dari lengan robot Gambar 8. Struktur base: tampak samping (kiri), isometris (tengah), assembly (kanan) Tabel 3. Komponen mekanikal-elektrikal pada base Tabel 3. 3.1 Hasil Rancangan Perangkat Keras Gambar 7 menunjukkan hasil rancangan perangkat keras purwarupa manipulator lengan
robot dengan komponen mekanik dan elektrikal utama sebagaimana ditunjukkan di Tabel 2. Perangkat keras purwarupa robot dibangun melalui penggabungan beberapa struktur utama
yang terdiri dari struktur base, struktur link 1-3, dan struktur end effector (Covaciu, 2019). ELKOMIKA – 801
Gambar 7. Rancangan perangkat keras purwarupa lengan robot ELKOMIKA – 801
Gambar 7. Rancangan perangkat keras purwarupa lengan robot Ayega, dkk Ayega, dkk 3.1.1 Rancangan Struktur Base Komponen mekanikal-elektrikal pada base
Komponen struktur base
Keterangan
Base motor holder
Tempat peletakan motor stepper pada base
Motor shaft
Meneruskan putaran motor stepper ke base
Motor stepper NEMA 23
Aktuator joint struktur base
Ball bearing (28 x 12 x 8 mm)
Mengurangi gesekan antara shaft dan base
Thrust bearing (28x12x9 mm)
Menahan gaya aksial dari lengan robot
Bearing/motor holder
Pengikat bearing base dan motor pada base
Gear base
Komponen penggerak base
Struktur kaki robot
Stabilisasi postur robot 3.1.2 Rancangan Struktur Link 1 3.1.2 Rancangan Struktur Link 1 3.1.2 Rancangan Struktur Link 1
Link 1 sebagai lengan pertama pada rancangan robot memiliki pergerakan yang diatur oleh
joint tipe revolute. Aktuator pada revolute joint di link 1 adalah motor stepper NEMA 17. Gambar 9 menunjukkan hasil rancang bangun komponen link 1 dengan detail komponen
utama serta fungsi yang ditunjukkan pada Tabel 4. ELKOMIKA – 802 ELKOMIKA – 802 Rancang Bangun Purwarupa Manipulator Lengan Robot Dengan Tiga Derajat Kebebasan Gambar 9. Struktur link 1: tampak samping (kiri), isometris (tengah), assembly (kanan) mbar 9. Struktur link 1: tampak samping (kiri), isometris (tengah), assembly (kanan) Gambar 9. Struktur link 1: tampak samping (kiri), isometris (tengah), assembly (kanan) mbar 9. Struktur link 1: tampak samping (kiri), isometris (tengah), assembly (kanan) Tabel 4. Komponen mekanikal-elektrikal pada link 1 Tabel 4. Komponen mekanikal-elektrikal pada link 1
Komponen link 1
Keterangan
Struktur utama link 1
Tempat terhubungnya semua komponen link 1
Motor stepper NEMA 17
Aktuator untuk pada link 1
Gear link 1
Komponen penggerak link 1
Cover 2 link 1
Tempat penyambungan gear link 1
Spacer link 1
Tempat melekatnya bearing holder pada link 1
Cover 1 link 1
Tempat menempelkan gear link 1 dan penutup bagian link 1
Shaft link 1
Poros pergerakan joint link 1 3.1.3 Rancangan struktur link 2 dan link 3 3.1.3 Rancangan struktur link 2 dan link 3 g
Link 2 dan link 3 dirancang secara bersamaan dan simultan dengan mekanisme pergerakan
joint tipe revolute. Pergerakan link 2 dan link 3 diatur oleh tiga komponen utama yaitu timing
belt, timing pulley, dan gear. Gambar 10 menunjukkan hasil rancang bangun link 2 dan link
3 setelah melalui tahap assembly. Link 3 pada gambar ini merupakan link terakhir yang
terhubung dengan end effector sebagai gripper pada robot. Hasil rancang bangun link 2 dan
link 3 juga terdiri dari beberapa komponen seperti diringkaskan pada Tabel 5. Gambar 10. 3.1.1 Rancangan Struktur Base Desain link 2-3: tampak atas (kiri), isometris (tengah), assembly (kanan) ambar 10. Desain link 2-3: tampak atas (kiri), isometris (tengah), assembly (kanan) Tabel 5. Komponen mekanikal-elektrikal pada link 2-3
Komponen link 2-3
Keterangan
Struktur utama link 2-3
Tempat terhubungnya semua komponen link 2 dan link 3
Motor stepper NEMA 11
Aktuator untuk joint pada link 3
Belt guide
Penghubung timing belt dengan gear link 3
Link 3
Link akhir pada robot yang terhubung dengan end effector
Gear link 3
Komponen penggerak link 3
Shaft link 3
Meneruskan putaran yang dihasilkan oleh motor stepper ELKOMIKA – 803
3.1.4 Rancangan struktur end effector
End effector merupakan bagian paling ujung lengan robot dengan bentuk dan mekanisme
gerak yang disesuaikan dengan fungsi robot. Pada penelitian ini, jenis end effector yang 3.1.4 Rancangan struktur end effector Ayega, dkk Ayega, dkk digunakan adalah gripper untuk pencengkram objek target robot. Gambar 11 menunjukkan
desain komponen end effector yang dibangun pada penelitian ini dengan komponen utama
seperti diringkaskan di Tabel 6. digunakan adalah gripper untuk pencengkram objek target robot. Gambar 11 menunjukkan
desain komponen end effector yang dibangun pada penelitian ini dengan komponen utama
seperti diringkaskan di Tabel 6. Gambar 11. Desain end effector: tampak atas (kiri) dan isometris (kanan) Gambar 11. Desain end effector: tampak atas (kiri) dan isometris (kanan) Tabel 6. Komponen mekanikal-elektrikal pada end effector Tabel 6. Komponen mekanikal-elektrikal pada end effector
Komponen end effector
Keterangan
Struktur utama end effector
Tempat terhubungnya semua komponen end effector
Motor servo SG90
Aktuator untuk joint pada link 3
Gear 1 end effector
Pencengkram sisi kiri
Gear 2 end effector
Pencengkram sisi kanan 3.2 Analisis Cakupan Kerja (Working Envelope) 3.2 Analisis Cakupan Kerja (Working Envelope)
Cakupan kerja atau working envelope merupakan bidang/ruang jangkauan yang dapat
diraih/dicapai oleh lengan robot dengan minimal satu orientasi. Suatu manipulator lengan
robot hanya dapat beroperasi atau melakukan fungsinya di dalam working envelope (Corke,
2022)(Craig, 2022). Umumnya, derajat kebebasan suatu manipulator lengan robot akan
berbanding secara proporsional dengan besar working envelope yang dihasilkan. Working
envelope suatu robot juga dipengaruhi oleh beberapa hal lain seperti panjang setiap (link),
tinggi struktur base, dan arah sumbu putar (Kojima, dkk, 2022). Working envelope purwarupa lengan robot yang dirancang mencakup bidang horizontal dan
vertikal seperti ditunjukkan pada Gambar 12. Sebagaimana ditunjukkan pada gambar ini,
penambahan joint pada struktur base robot menghasilkan working envelope maksimum di
bidang horizontal berbentuk area setengah lingkaran dengan radius maksimum 50 mm. Selain itu, pergerakan link 1 hingga end effector menghasilkan working envelope pada
bidang horizontal berbentuk tiga per empat lingkaran dengan radius maksimum 95 mm. Hasil identifikasi working envelope ini dapat menjadi basis dalam penentuan jenis fungsi
operasi dan bidang kerja yang dapat dilakukan oleh robot 3.3 Pengujian Kepresisian Pergerakan Robot
Salah satu hal penting dalam desain dan pengontrolan robot adalah terkait ketepatan
perubahan variabel gerak (dalam hal ini sudut rotasi setiap joint) aktual pada perangkat
keras purwarupa robot sesuai dengan nilai perubahan variabel yang ditentukan pada
program/perangkat lunak mikrokontroller robot. Untuk menguji ketepatan gerak robot yang
dirancang, dilakukan percobaan untuk membandingkan nilai sudut yang diberikan pada
modul mikrokontroller dengan pengukuran nilai sudut aktual yang dihasilkan pada perangkat
keras purwarupa robot (Bi, dkk, 2020). ELKOMIKA – 804 3.4 Pengujian Implementasi Analisis FK g j
p
Pengujian selanjutnya adalah terkait implementasi FK untuk penentuan posisi dan orientasi
end effector relatif terhadap kerangka koordinat base berdasarkan input nilai sudut rotasi
yang ditentukan pada setiap link robot. Untuk kombinasi nilai sudut setiap link yang
ditentukan, pengujian dilakukan untuk membandingkan posisi dan orientasi end effector
yang dihasilkan pada perangkat keras purwarupa robot dengan hasil simulasi berdasarkan
model FK robot pada Tabel 1 dan Persamaan (2). Percobaan implementasi FK pada
purwarupa robot dilakukan untuk beberapa kombinasi sudut rotasi pada setiap link robot. Pengujian I dilakukan untuk menguji konfigurasi inersial dari kerangka koordinat base robot. Pada pengujian ini, nilai parameter sudut link pada simulator MATLAB dan pada input
program mikrokontroller diprogram untuk menentukan nilai sudut 𝜃= 0° untuk 𝑖= 1,2,3. Percobaan dilakukan sebanyak tiga (3) kali untuk mengamati konsistensi nilai posisi dan
orientasi model simulasi maupun perangkat keras purwarupa robot. Tabel 7 meringkaskan
perbandingan posisi end effector robot yang dihasilkan pada model simulasi MATLAB serta
pada purwarupa lengan robot. Berdasarkan Tabel 7, dapat disimpulkan bahwa rerata eror
implementasi di sumbu X adalah 1.33% dengan nilai maksimal 2.745%, sedangkan rerata
eror pada sumbu 𝑍 adalah 2.06% dengan nilai tertinggi 2.654%. Tabel 7. Hasil implementasi FK pada pengujian I (kerangka base)
Percobaan ke-
Solusi FK pada MATLAB
Solusi FK pada Purwarupa
x
y
z
x
y
z
1
255
0
113
250
0
110
2
255
0
113
248
0
110
3
255
0
113
253
0
112 Tabel 7. Hasil implementasi FK pada pengujian I (kerangka base) Pengujian II dilakukan dengan terlebih dahulu merotasikan base pada konfigurasi sudut
𝜃= −90° dan selanjutnya mengatur nilai sudut rotasi untuk setiap link masing-masing
𝜃ଵ= 90°, 𝜃ଶ= 𝜃ଷ= −90°. Untuk kombinasi sudut tersebut, hasil simulasi model pada
MATLAB dan hasil pengujian pada perangkat keras lengan robot ditunjukkan pada Gambar
14. Perbandingan posisi end effector yang dihasilkan model simulasi MATLAB dan pada
perangkat keras lengan robot ditunjukkan di Tabel 8. Untuk percobaan ini, nilai eror
implementasi di sumbu Y adalah 2.446% sedangkan eror di sumbu Z adalah 0,743%. Percobaan selanjutnya dilakukan untuk kombinasi sudut berbeda di mana base dirotasikan
pada sudut 𝜃= 90° dan sudut rotasi setiap link ditentukan sebesar 𝜃ଵ= 60°, 𝜃ଶ= −30°,
dan 𝜃ଷ= −90°. Hasil simulasi MATLAB dan pengujian pada perangkat keras lengan robot
untuk percobaan ini ditunjukkan pada Gambar 15 dengan perbandingan konfigurasi end
effector diringkaskan pada Tabel 8. Ayega, dkk Ayega, dkk Ayega, dkk Rancang Bangun Purwarupa Manipulator Lengan Robot Dengan Tiga Derajat Kebebasa Rancang Bangun Purwarupa Manipulator Lengan Robot Dengan Tiga Derajat Kebebasan Gambar 12. Working envelope di bidang horizontal (kiri) dan vertikal (kanan) Gambar 12. Working envelope di bidang horizontal (kiri) dan vertikal (kanan) Pengukuran nilai perubahan sudut aktual joint pada setiap struktur link robot dilakukan
menggunakan sensor rotary encoder yang memiliki karakteristik statis 1600 pulse-per-
revolution (PPR) dan batas kesalahan ±0,225°. Data direkam dengan menghubungkan
rotary encoder yang terpasang pada perangkat keras purwarupa robot dengan perangkat
lunak MATLAB sehingga diperoleh data pengukuran berupa nilai sudut dan jumlah sinyal
pulsa (Oguntosin, dkk, 2019). Pengukuran dilakukan dengan memvariasikan input sudut
pada mikrokontroller dan membandingkannya dengan pengukuran sensor rotary encoder. Hasil pengukuran nilai sudut oleh rotary encoder untuk input variasi nilai sudut yang
diberikan pada mikrokontroller untuk link 1, link 2, dan link 3 ditunjukkan masing-masing
pada Gambar 13. Dari hasil pengukuran tersebut, diperoleh informasi bahwa nilai eror
maksimum antara nilai sudut yang diinginkan dan sudut aktual yang dihasilkan adalah ±0,5°
untuk link 1 (pada pengukuran 5°), ±0,5° untuk link 2 (pada pengukuran 10°), dan ±0,925°
untuk link 3 (pada pengukuran 10°), ). Karena diperoleh bahwa nilai eror sudut maksimum
pergerakan setiap joint pada robot yang dirancang adalah ±0,925°, maka disimpulkan
pergerakan setiap link pada robot memiliki tingkat ketelitian yang cukup baik. ELKOMIKA – 805
(a) (b)
(c)
Gambar 13. Hasil pengukuran sudut rotasi link 1 (a), link 2 (b), dan link 3 (c) (b) (a) (b) ELKOMIKA – 805
(c)
Gambar 13. Hasil pengukuran sudut rotasi link 1 (a), link 2 (b), dan link 3 (c) Ayega, dkk Ayega, dkk Solusi permasalahan IK pada purwarupa robot adalah 𝜃ଵ= 8,11°, 𝜃ଶ= −39,23°, dan 𝜃ଵ=
61,12° seperti ditunjukkan pada Gambar 16 (kanan). Karena hasil solusi IK yang diperoleh
dari simulasi dan eksperimen cukup berbeda, kemudian dilakukan pengujian FK pada kedua
solusi tersebut untuk membandingkan posisi end effector yang dihasilkan masing-masing
kombinasi sudut. Hasil perbandingan tersebut ditunjukkan di Tabel 9 yang mengindikasikan
kedua kombinasi sudut menghasilkan posisi end effector yang sesuai. Hasil percobaan ini
mengindikasikan kemungkinan didapatkan beberapa solusi kombinasi parameter (sudut
rotasi atau panjang translasai) untuk posisi end effector yang sama pada permasalahan IK
pada sistem manipulator lengan robot. 4. KESIMPULAN Penelitian ini telah melakukan studi dan penelitian terkait desain, konstruksi, dan analisis
purwarupa manipulator lengan robot dengan tiga derajat kebebasan. Proses rancang bangun
yang dilakukan mencakup desain model matematis kinematika (maju/forward dan balik/
inverse) pergerakan lengan robot, desain simulator model kinematika lengan robot dengan
mengintegrasikan perangkat lunak MATLAB dan SOLIDWORKS, serta konstruksi/integrasi
komponen perangkat keras purwarupa lengan robot menggunakan 3D printer. Untuk tujuan
pengujian, analisis perbandingan dilakukan antara pergerakan purwarupa robot yang
dibangun dengan simulasi pergerakan model robot pada perangkat lunak MATLAB. Hasil
percobaan terkait perbandingan pergerakan antara perangkat keras dan model simulasi
lengan robot yang dilakukan menunjukkan tingkat akurasi yang cukup baik di mana eror
maksimum pada penentuan solusi permasalahan forward kinematics adalah 2.745%
sementara eror maksimum pada penentuan solusi permasalahan inverse kinematics adalah
0,06%. Penelitian selanjutnya akan diarahkan pada upaya peningkatan tingkat presisi
pergerakan pada pengoperasian purwarupa manipulator robot yang saat ini telah dirancang. 3.4 Pengujian Implementasi Analisis FK Diperoleh nilai eror implementasi di sumbu 𝑌 adalah
0,285% sedangkan pada sumbu 𝑍 adalah 0,609%. ELKOMIKA – 806
Gambar 14. Hasil Simulasi (kiri) dan Eksperimen (kanan) FK untuk Pengujian II ELKOMIKA – 806
Gambar 14. Hasil Simulasi (kiri) dan Eksperimen (kanan) FK untuk Pengujian II Rancang Bangun Purwarupa Manipulator Lengan Robot Dengan Tiga Derajat Kebebasan Tabel 8. Hasil implementasi FK pada pengujian II (kombinasi sudut link)
Percobaan ke-
Solusi FK Pada MATLAB
Solusi FK Pada Purwarupa
x
y
z
x
y
z
1
0
-85
163
-0,025
-82.92
161,78
2
0
158,6
198,8
0,1583
158,15
197,58
Gambar 15. Hasil simulasi (kiri) dan eksperimen (kanan) FK untuk Percobaan II Gambar 15. Hasil simulasi (kiri) dan eksperimen (kanan) FK untuk Percobaan II Gambar 16. Hasil simulasi IK pada MATLAB (kiri) dan pada Arduino (kanan). Gambar 16. Hasil simulasi IK pada MATLAB (kiri) dan pada Arduino (kanan). Tabel 9. Hasil implementasi iK pada pengujian II (kombinasi sudut link) Solusi IK Pada MATLAB
Solusi FK Pada Purwarupa
Parameter
𝜽𝟏
𝜽𝟐
𝜽𝟑
𝜽𝟏
𝜽𝟐
𝜽𝟑
60°
-60°
30°
8,11°
61,12°
-39,23°
Koordinat
x
y
z
x
y
z
191
125
0
191.004
124,994
0 3.5 Pengujian Implementasi Analisis IK 3.5 Pengujian Implementasi Analisis IK Pengujian dilakukan untuk membandingkan solusi IK pada model simulasi dan purwarupa
lengan robot. Posisi end effector yang ditinjau adalah 𝑥= 191, 𝑦= 125, 𝑧= 0 relatif terhadap
kerangka koordinat link 1, dan objektif pengujian adalah menentukan kombinasi sudut rotasi
yang sesuai dengan posisi end effector yang diketahui. Pengujian terlebih dahulu dilakukan
pada MATLAB dengan solusi IK 𝜃ଵ= 60°, 𝜃ଶ= −60°, 𝜃ଵ= 30° dan konfigurasi robot
seperti ditunjukkan pada Gambar 16 (kiri). Pengujian perangkat keras purwarupa robot
kemudian dilakukan dengan solusi permasalahan IK ditentukan berdasarkan nilai sudut rotasi
sebagaimana tercatat pada modul pengontrol ARDUINO pada robot (Rahul, dkk, 2019). ELKOMIKA – 807 Ayega, dkk DAFTAR RUJUKAN Al Tahtawi, A. R., Agni, M., & Hendrawati, T. D. (2021). Small-scale Robot Arm Design with
Pick and Place Mission Based on Inverse Kinematics. Journal of Robotics and Control
(JRC), 2(6), 469–475. https://doi.org/10.18196/jrc.26124 Baby, A., Augustine, C., Thampi, C., George, M., A P, A., & C Jose, P. (2017). Pick and Place
Robotic Arm Implementation Using Arduino. IOSR Journal of Electrical and Electronics
Engineering, 12(02), 38–41. https://doi.org/10.9790/1676-1202033841 Baby, A., Augustine, C., Thampi, C., George, M., A P, A., & C Jose, P. (2017). Pick and Place
Robotic Arm Implementation Using Arduino. IOSR Journal of Electrical and Electronics
Engineering, 12(02), 38–41. https://doi.org/10.9790/1676-1202033841 Bai, Y., Luo, M., & Pang, F. (2021). An Algorithm for Solving Robot Inverse Kinematics Based
on FOA Optimized BP Neural Network. Applied Sciences, 11(15), 7129. https://doi.org/10.3390/app11157129 Bai, Y., Luo, M., & Pang, F. (2021). An Algorithm for Solving Robot Inverse Kinematics Based
on FOA Optimized BP Neural Network. Applied Sciences, 11(15), 7129. https://doi.org/10.3390/app11157129 Bi, Z. M., Zhonghua, M., Bin, Z., & Chris, Z. (2020). The State of the Art of Testing Standards
for Integrated Robotic Systems. Robotics and Computer-Integrated Manufacturing,
63(1), 101893. https://doi.org/10.1016/j.rcim.2019.101893 Bi, Z. M., Zhonghua, M., Bin, Z., & Chris, Z. (2020). The State of the Art of Testing Standards
for Integrated Robotic Systems. Robotics and Computer-Integrated Manufacturing,
63(1), 101893. https://doi.org/10.1016/j.rcim.2019.101893 Corke, P. (2017). Robot Manipulator Capability in MATLAB: A Tutorial on Using the Robotics
System Toolbox [Tutorial]. IEEE Robotics & Automation Magazine, 24(3), 165–166. ELKOMIKA – 808 ELKOMIKA – 808 Rancang Bangun Purwarupa Manipulator Lengan Robot Dengan Tiga Derajat Kebebasan Rancang Bangun Purwarupa Manipulator Lengan Robot Dengan Tiga Derajat Kebebasan Rancang Bangun Purwarupa Manipulator Lengan Robot Dengan Tiga Derajat Kebebasan https://doi.org/10.1109/MRA.2017.2718418 Corke, P. (2022). Robotics and Control (1st ed., Vol. 141). Springer International Publishing. https://doi.org/10.1007/978-3-030-79179-7 Covaciu, F. (2019). Control and Actuation System of a Six Degrees of Freedom Robotic Arm. Acta Technica Napocensis-Series: Applied Mathematics, Mechanics, and Engineering,
62(1), 99–106. https://atna-mam.utcluj.ro/index.php/Acta/article/view/1150 Craig, J. J. (2022). Introduction to Robotics: Mechanics and Control (4th ed.). Pearson
Education Limited. https://www.pearson.com/en-us/subject-catalog/p/introduction-
to-robotics-mechanics-and-control/P200000003304/9780137848744 Dave, H. K., Chanpura, M. D., Kathrotiya, S. J., Patolia, D. D., Dodiya, D. D., & Kharva, P. S. (2022). Design, Development and Control of SCARA for Manufacturing Processes. In
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Analysis of 4 DOF Pick and Place Arm Robot Manipulator using Fuzzy Logic Controller. International Journal of Electrical & Computer Engineering, 10(2), 2088–8708. https://doi.org/h10.11591/ijece.v10i2.pp1376-1386 Dzedzickis, A., Subačiūtė-Žemaitienė, J., Šutinys, E., Samukaitė-Bubnienė, U., & Bučinskas,
V. (2021). Advanced Applications of Industrial Robotics: New Trends and Possibilities. Applied Sciences, 12(1), 135. https://doi.org/10.3390/app12010135 Kojima, A., Tran, D. T., & Lee, J.-H. (2022). Investigation of the Mounting Position of a
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MATLAB/SimMechanics. In R. Kumar, V. S. Chauhan, M. Talha, & H. Pathak (Eds.),
Machines, Mechanism and Robotics (Lecture Notes in Mechanical Engineering) (1st
ed., Vol. 1, pp. 661–671). Springer. https://doi.org/10.1007/978-981-16-0550-5_65 Landers, R. G., Barton, K., Devasia, S., Kurfess, T., Pagilla, P., & Tomizuka, M. (2020). A
Review of Manufacturing Process Control. Journal of Manufacturing Science and
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119624–119634. https://doi.org/10.1109/ACCESS.2020.3003807 Lobbezoo, A., & Kwon, H. J. (2023). Simulated and Real Robotic Reach, Grasp, and Pick-and- ELKOMIKA – 809 Ayega, dkk Ayega, dkk Place Using Combined Reinforcement Learning and Traditional Controls. Robotics,
12(1), 12. https://doi.org/10.3390/robotics12010012 Place Using Combined Reinforcement Learning and Traditional Controls. Robotics,
12(1), 12. https://doi.org/10.3390/robotics12010012 Long, D. T., Binh, T. V., Hoa, R. V., Anh, L. V., & Toan, N. V. (2020). Robotic Arm Simulation
by using Matlab and Robotics Toolbox for Industry Application. International Journal
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Press. Manjula, V. S., & Karamagi, R. I. (2018). Automatic Pick and Place Robot Manipulation Using
a Microcontroller. Journal of Applied & Computational Mathematics, 7(3), 1–8. https://doi.org/10.4172/2168-9679.1000408 Oguntosin, V., Olasina, J., & Akindele, A. (2019). Control Design and Analysis of Cruise
Control System. Journal of Physics: Conference Series, 2437(1), 020170. https://doi.org/10.1088/1742-6596/1299/1/012108 Qian, H., Chen, B., Xia, X., Deng, S., & Wang, Y. (2022). D-H Parameter Method-based
Wearable Motion Tracking. Journal of Physics: Conference Series, 2216(1), 012027. https://doi.org/10.1088/1742-6596/2216/1/012027 Rahul, K., Raheman, H., & Paradkar, V. (2020). Design of 4 DOF Parallel Robot Arm and
Firmware Implementation on Embedded System to Transplant Pot Seedlings. Artificial
Intelligence in Agriculture, 4(1), 172–183. https://doi.org/10.1016/j.aiia.2020.09.003 Rahul, T. N. M., Reddy, K. T., & Mary, A. V. A. (2019). Cost Effective Robotic System Using
Arduino. Journal of Computational and Theoretical Nanoscience, 16(8), 3222–3227. https://doi.org/10.1166/jctn.2019.8166 Zeng, C., Zhou, H., Ye, W., & Gu, X. (2022). iArm: Design an Educational Robotic Arm Kit for
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Social Media Terms and Conditions and Informed Consent From Children: Ethical Analysis
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JMIR pediatrics and parenting
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cc-by
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Abstract Background: Terms and conditions define the relationship between social media companies and users. However, these legal
agreements are long and written in a complex language. It remains questionable whether users understand the terms and conditions
and are aware of the consequences of joining such a network. With children from a young age interacting with social media,
companies are acquiring large amounts of data, resulting in longitudinal data sets that most researchers can only dream of. The
use of social media by children is highly relevant to their mental and physical health for 2 reasons: their health can be adversely
affected by social media and their data can be used to conduct health research. Objective: The aim of this paper is to offer an ethical analysis of how the most common social media apps and services inform
users and obtain their consent regarding privacy and other issues and to discuss how lessons from research ethics can lead to
trusted partnerships between users and social media companies. Our paper focuses on children, who represent a sensitive group
among users of social media platforms. Methods: A thematic analysis of the terms and conditions of the 20 most popular social media platforms and the 2 predominant
mobile phone ecosystems (Android and iOS) was conducted. The results of this analysis served as the basis for scoring these
platforms. Results: The analysis showed that most platforms comply with the age requirements issued by legislators. However, the consent
process during sign-up was not taken seriously. Terms and conditions are often too long and difficult to understand, especially
for younger users. The same applies to age verification, which is not realized proactively but instead relies on other users who
report underaged users. Conclusions: This study reveals that social media networks are still lacking in many respects regarding the adequate protection
of children. Consent procedures are flawed because they are too complex, and in some cases, children can create social media
accounts without sufficient age verification or parental oversight. Adopting measures based on key ethical principles will safeguard
the health and well-being of children. This could mean standardizing the registration process in accordance with modern research
ethics procedures: give users the key facts that they need in a format that can be read easily and quickly, rather than forcing them
to wade through chapters of legal language that they cannot understand. Social Media Terms and Conditions and Informed Consent From
Children: Ethical Analysis Christophe Olivier Schneble, MSc; Maddalena Favaretto, MSc; Bernice Simonne Elger, Prof Dr; David Martin Shaw,
PhD hneble, MSc; Maddalena Favaretto, MSc; Bernice Simonne Elger, Prof Dr; David Martin Shaw, Christophe Olivier Schneble, MSc; Maddalena Favaretto, MSc; Bernice Simonne Elger, Prof Dr; David Martin Shaw,
PhD Institute of Biomedical Ethics, University of Basel, Basel, Switzerland Institute of Biomedical Ethics, University of Basel, Basel, Switzerland Corresponding Author:
Christophe Olivier Schneble, MSc
Institute of Biomedical Ethics
University of Basel
Bernoullistrasse 28
Basel, 4056
Switzerland
Phone: 41 61 207 02 03
Email: christophe.schneble@unibas.ch Corresponding Author:
Christophe Olivier Schneble, MSc
Institute of Biomedical Ethics
University of Basel
Bernoullistrasse 28
Basel, 4056
Switzerland
Phone: 41 61 207 02 03
Email: christophe.schneble@unibas.ch Abstract Improving these processes would help safeguard the
mental health of children and other social media users. (JMIR Pediatr Parent 2021;4(2):e22281) doi: 10.2196/22281 (JMIR Pediatr Parent 2021;4(2):e22281) doi: 10.2196/22281 (JMIR Pediatr Parent 2021;4(2):e22281) doi: 10.2196/22281 JMIR Pediatr Parent 2021 | vol. 4 | iss. 2 | e22281 | p. 1
(page number not for citation purposes) JMIR PEDIATRICS AND PARENTING JMIR PEDIATRICS AND PARENTING Schneble et al Original Paper Background Social media companies have experienced tremendous growth
during the last decade; however, they have largely neglected
the issues of privacy and confidentiality. In addition to
connecting people, social media apps (the companies) are also
tremendous data collectors, gathering a wide range of
information that spans from nonsensitive to highly sensitive
data. Although many data might be nonsensitive in isolation,
the combination of various types of data might subsequently
allow insights into sensitive health issues [1]. In fact, many
studies have used social media data to gain insights into the
mental state of users [2,3]. Moreover, with children and young
adults using social media apps from a young age, companies
have acquired data over long time spans, which is similar to
longitudinal data used in research. Keeping this in mind and
knowing that predictive algorithms will become more accurate,
it is of major importance to build governance and inform users
about the use of their data to foster data protection. This is all
the more important given the latest scandal surrounding
Cambridge Analytica [4,5] and the sharing of data between
Facebook and device manufacturers such as Apple and top-rated
apps such as Spotify and Netflix [6]. These are prominent
examples of misbehavior that illustrate the urgent need for a
trusted partnership between users and social media companies. Letting children use parents’ accounts also bypasses the age
requirements imposed by social media companies. In their terms
of service, social media apps and services defined the minimum
age at which adolescents or children can use the app or service
without obtaining parental consent. With regard to age
requirements, the law plays an important role by setting
boundaries for protecting children’s privacy, data sharing, and
profiling. In the United States, COPPA defines 13 years as the
minimum age to join such communities. Before that age, explicit
parental consent is needed to sign up. The EU has recently
introduced the GDPR, in which Article 8 defines the necessity
of parental consent for all youths aged below 16 years in
situations where information society services are offered directly
to them. However, the member states are free to choose and
adopt their own particular regulation within the age range of
13-16 years. Some countries, such as the United Kingdom, have
opted for an age of 13 years, whereas others such as Germany
have set the boundary at 16 years [10]. KEYWORDS g data; ethics; children; health data; terms and conditions; trusted partnership; medical ethics; mobile phon social media; big data; ethics; children; health data; terms and conditions; trusted partnership; medical et social media; big data; ethics; children; health data; terms and conditions; trusted partnership; medical ethics; mobile phone JMIR Pediatr Parent 2021 | vol. 4 | iss. 2 | e22281 | p. 1
(page number not for citation purposes) https://pediatrics.jmir.org/2021/2/e22281 XSL•FO
RenderX JMIR PEDIATRICS AND PARENTING Schneble et al [12]. When children access social media through their parents’
accounts, parents might feel that they have control over their
children’s media use. This is problematic for 2 reasons: first,
parents will not be able to control every click, and second, as
the UK Children’s Commissioner points out, children might be
presented with explicit adult content of which their parents
remain unaware. Objectives When signing up for such a service, users consent by reading
or at least scrolling through the terms of service and by clicking
the agree button. However, these terms and conditions are often
long and written in a complex legal language. Thus, it remains
questionable whether users—particularly children and young
adults—truly understand the terms and conditions and are aware
of the consequences of joining a network. Most of the platforms
offer their service for free but require users to accept the preset
package of conditions with limited privacy choices to permit
access to their services. This paper provides an ethical analysis of the most popular
social media platforms and services used by children and
adolescents (in the EU and the United States). It focuses on age
requirements, how information about the platform is presented,
how consent is obtained, how (and if) age verification is
implemented, whether resources are provided to educate parents
or children, and if there are community guidelines. It then
discusses the emerging issues and the predominant regulations
of our target countries and illustrates how experiences from
research ethics could be used to develop a trusted relationship
between users and companies, facilitating the ethical functioning
of social media networks. Social media apps are ubiquitous in today’s world and have
changed the way we communicate, share, and interact with each
other daily. They are also omnipresent in the lives of young
people, and it is estimated that 1 in 3 of all internet users is
under the age of 18 years [10,11]. A recent study by the UK
Children’s Commissioner has shown that a significant number
of children access social media through their parents’accounts,
whereas most adolescents (71% in the United States and 85%
in Europe) have one or more social media accounts or identities Background The GDPR would thus
not prohibit the use of such services before the minimum age
requiring children’s self-consent but would instead require
parental consent to access these services and process the
personal data of children, as defined in the GDPR. Most of the
companies however set their minimum age requirements at the
age imposed by national law, as shown in our results. Contractual law in the terms and conditions (also known as
terms of services) and privacy policies define how privacy,
confidentiality, and data sharing are handled. They are the
predominant legal and contractual mechanisms that define the
relationship between users and social media companies. These
mechanisms are subject to various national and international
regulations. The General Data Protection Regulation (GDPR)
of the European Union (EU) [7] sets boundaries concerning the
processing of data. In the United States, the Children’s Online
Privacy Protection Act (COPPA) [8] and the fair information
principles issued by the Federal Trade Commission [9] are the
2 predominant regulations. However, the efficacy of such age regulations remains to be
questionable as the primary research strands in children’s digital
rights show that children and parents feel social pressure to join
such communities [12] and thus might lie about their age when
joining social media services [13]. Doing so is easy because
normally, signing up relies only on the honesty of the user. https://pediatrics.jmir.org/2021/2/e22281 JMIR Pediatr Parent 2021 | vol. 4 | iss. 2 | e22281 | p. 2
(page number not for citation purposes) JMIR Pediatr Parent 2021 | vol. 4 | iss. 2 | e22281 | p. 3
(page number not for citation purposes) Methods We conducted a thematic analysis [14] of the terms and
conditions of the 20 most popular social media platforms in
2019 [15] and the 2 predominant mobile phone ecosystems,
Android and iOS. Within this sample of 20 platforms, we JMIR Pediatr Parent 2021 | vol. 4 | iss. 2 | e22281 | p. 2
(page number not for citation purposes) JMIR Pediatr Parent 2021 | vol. 4 | iss. 2 | e22281 | p. 2
(page number not for citation purposes) XSL•FO
RenderX JMIR PEDIATRICS AND PARENTING Schneble et al excluded all apps and social networks targeting only
Chinese-speaking users (because of a lack of terms and
conditions in English; WeChat, QQ, QZone, and Sina Weibo),
discussion websites (Reddit), and those targeting only adults
(LinkedIn or Viber), resulting in 10 platforms relevant to
children. The terms and conditions were read in depth, emerging
topics of ethical interest were identified, and categories for
further in-depth analysis were created. The categories identified
were the minimum age to join, how the consent process was
handled, the age verification process, the presence of parental portals (educating parents on the use of the respective
platforms), and the possibility of requesting account deletion
in the cases of underaged users. Note that most of the platforms
are available either as web apps or as smartphone apps. The
results of this in-depth analysis are summarized in Table 1, and
the apps are scored according to the criteria in Table 2. As most
of the apps are available on smartphones, we also decided to
include the quasi-standard platforms such as Android and
Google, as they have a gatekeeping function (in terms of age)
to allow children to access those networks. https://pediatrics.jmir.org/2021/2/e22281 https://pediatrics.jmir.org/2021/2/e22281 XSL•FO
RenderX JMIR PEDIATRICS AND PARENTING JMIR PEDIATRICS AND PARENTING Schneble et al Table 1. Overview of the most popular social media apps. JMIR Pediatr Parent 2021 | vol. 4 | iss. 2 | e22281 | p. 4
(page number not for citation purposes) Methods Parent porta
or communi
ty guideline
Parental con-
sent
Possibility
to request
deletion of
the account
Age verifica-
tion
Minimum
age (years)
Viewable
without
signing in
Predominant
content
Provider
Active
users (in
millions)
Platform or app
Social media
Yes
Consent by
user
Yes (form)
Verification
of official
document
when ac-
count is
locked
13
Yes
Video or text
or images or
social messag-
ing
Facebook
Inc
2234
Facebook
Yes
Consent by
user or par-
ents if below
13 years
Yes
Background
check or ver-
ification of
official docu-
ment or cred-
it card verifi-
cation when
locked
13
(≥14/≥16)a
Yes
Video creation
Google
1900
YouTube
No
Consent by
user
No
By SMS
messaging
13
No
Social messag-
ing (video or
text or music)
WhatsApp
Inc (Face-
book Inc)
1500
WhatsApp
Yes
Consent by
user
Yes (form)
Verification
of ID when
locked
13 (16)a
Yes
Images or
video
Facebook
Inc
1000
Instagram
No
Yes (for cer-
tain coun-
tries)
Yes (mail)
No
13 (14)a
No
Music or im-
ages
Beijing
Bytedance
Technology
500
TikTok
No
Consent by
user
Yes
Yes for sensi-
tive posts
No
Yes
Text
Twitter Inc
335
Twitter
No
Consent by
user
No
No
No
No
Social messag-
ing
Microsoft
Corporation
300
Skype
Yes
Consent by
user
Yes (mail)
By peer or
birthday can
be changed
only a limit-
ed number
of times
13
No
Video or pho-
to posting
Snap Inc
291
Snapchat
Yes
Consent by
parents if un-
deraged use
Yes (form)
By peer
13
Yes
Images
13
250
Pinterest
No
No
No
No
No
No
Social messag-
ing
LINE Corpo-
ration
203
LINE
Ecosystems
Yes
Consent by
parents if un-
deraged
users
Yes
Yes (Credit
card or
SMS)
13
N/A
Apps
Apple
N/Ab
iOS (Apple
ID)
Yes
Consent by
parents if un-
deraged
users
Yes
Back check
or verifica-
tion of offi-
cial docu-
ment or cred-
it card verifi-
cation
13
(≥14/≥16)a
N/A
Apps
Google
N/A
Android
Play Store Table 1. Overview of the most popular social media apps. aOn the basis of the country, the companies have adopted a different minimum age. bN/A: not applicable. aOn the basis of the country, the companies have adopted a different minimum age. bN/A: not applicable. JMIR Pediatr Parent 2021 | vol. 4 | iss. 2 | e22281 | p. Age Requirements and Age Verification Table 1 shows that all companies except LINE have adopted a
minimum age of 13 years for the use of their services. However,
the Apple and Google (Android) ecosystems offer the possibility
of using their various services at a younger age with parental
consent. Google achieves this by integrating the child’s account
into the so-called Family Link [17], a platform to group and
administrate family member accounts; the same applies to
Apple, which has also set up an infrastructure to manage family
accounts. Most service providers rely on other users reporting
underage use and offer either a mailing address or a form as the
only way of contact when requesting the deletion of an account
created by underage children. A more sophisticated method has
been adopted by Google, where a background check is Scoring System On the basis of the data in Table 1, our scoring system (Table
2) awards each platform a possible score of 1 (+) or 0 (none) Table 3. Constraints of the scoring system. Criteria for no point
Criteria for point
Topic
No age restriction or no age verification present
Age restriction and implemented age verification present
Minimum age or age verification
No
Yes
Possibility to request deletion
Consent by user
Consent by parents
(Parental) consent process
No parent portal
Parent portal present
Parent portal Table 3. Constraints of the scoring system. performed by verifying the age entered in any one of its services
whenever the user uses another service that is part of its
ecosystem. Once an account is locked, Instagram and Facebook
request a copy of an official document (ID card or passport) to
unlock it. Android, iOS, and YouTube adopt another way of
handling this issue, where the check is performed against a valid
credit card, resulting in a parent giving de facto consent. In
contrast, Snapchat allows users to change their date of birth
only a certain number of times [18]. Methods 4
(page number not for citation purposes) https://pediatrics.jmir.org/2021/2/e22281 XSL•FO
RenderX JMIR PEDIATRICS AND PARENTING Schneble et al Table 2. Scoring the most popular social media apps. Total score
Parent portal or communi-
ty guidelines
Possibility to request
deletion of the account
Parental consent
Minimum age or age verification
Platform or app
3
Parent portal present
Yes
Consent by user
Age restriction and implemented age
verification present
Facebook
4
Parent portal present
Yes
Consent by parents
Age restriction and implemented age
verification present
YouTube
2
No parent portal
No
Consent by parents
Age restriction and implemented age
verification present
WhatsApp
3
Parent portal present
Yes
Consent by user
Age restriction and implemented age
verification present
Instagram
1
No parent portal
No
Consent by parents
No age restriction or no age verifica-
tion present
TikTok
1
No parent portal
Yes
Consent by user
No age restriction or no age verifica-
tion present
Twitter
2
No parent portal
No
Consent by parents
Age restriction and implemented age
verification present
Skype
3
Parent portal present
Yes
Consent by user
Age restriction and implemented age
verification present
Snapchat
4
Parent portal present
Yes
Consent by parents
Age restriction and implemented age
verification present
Pinterest
1
No parent portal
No
Consent by user
Age restriction and implemented age
verification present
LINE
Results
across the 5 different categories used in our analysis. The criteria
are presented in Table 3 The category for minimum age and Table 2. Scoring the most popular social media apps. across the 5 different categories used in our analysis. The criteria
are presented in Table 3. The category for minimum age and
age verification is cumulative. One point will be awarded only
if both criteria are met, because we believe this fulfills the
gatekeeper function. Studies suggest that children are often
happy to lie about their age and that parents even encourage
their children to sign up [13,16]; thus, the efficacy of a minimum
age requirement in the absence of verification remains ethically
questionable. https://pediatrics.jmir.org/2021/2/e22281 Results The results of our analysis will be discussed thematically, in
turn, after presenting the results of our scoring mechanism. Consent Process Full text
You grant Pinterest and our users a non-exclusive, royalty-free, transferable, sublicensable, worldwide license to
use, store, display, reproduce, save, modify, create derivative works, perform, and distribute your User Content on
Pinterest solely for the purposes of operating, developing, providing, and using Pinterest. Nothing in these Terms
restricts other legal rights Pinterest may have to User Content, for example under other licenses. We reserve the right
to remove or modify User Content or change the way it’s used in Pinterest, for any reason. This includes User Content
that we believe violates these Terms, our Community Guidelines, or any other policies. Simplified version
If you post your content on Pinterest, we can show it to people and others can save it. Don't post porn or spam or be
a jerk to other people on Pinterest. You grant Pinterest and our users a non-exclusive, royalty-free, transferable, sublicensable, worldwide license to
use, store, display, reproduce, save, modify, create derivative works, perform, and distribute your User Content on
Pinterest solely for the purposes of operating, developing, providing, and using Pinterest. Nothing in these Terms
restricts other legal rights Pinterest may have to User Content, for example under other licenses. We reserve the right
to remove or modify User Content or change the way it’s used in Pinterest, for any reason. This includes User Content
that we believe violates these Terms, our Community Guidelines, or any other policies. Simplified version Discussions Currently, some providers request verification by email or phone
by sending the user a short message during the registration
process (the standard procedure for setting up a WhatsApp
account). The latter provides an additional security layer as cell
phone companies have a minimum age for issuing a contract;
when a child has a cell phone, the parents have at least agreed
to the use of such a device and thus are aware that the child
might sign up for such a service, even if they are potentially
unaware of the services that the child subsequently signs up for. However, this might be a problem in countries where
pay-as-you-go phones require no identification, either by age
or by verification with an official ID card or social security card. Furthermore, implementing an age verification process by
requesting verification through a text message could be seen as
discriminating against children who do not possess a cell phone
at all and, thus, solely have to rely on a parent to register. Parent Portals or Community Guidelines Almost every platform (except social messaging platforms)
offers a parent’s portal or community guidelines. This ranges
from simply linking to interesting articles (Snapchat) to
providing an information center (Instagram and Facebook) to
video sequences (Facebook) on problematic behavior along
with short sequences showing a safe way to use the service. Principal Findings On the basis of our scoring system (Table 2), most providers
scored 3 out of 4 points. However, one-third of the service
providers achieved poor results. This shows that the regulations
that service providers comply with, either by themselves or by
law, offer at least some protection for users. However, TikTok,
Twitter, and LINE only scored 1 point and only 2 companies
achieved the maximum score (Pinterest and YouTube). In the following section, we will therefore discuss the categories
presented in Table 1 and suggest possible improvements within
the framework of the 4 guiding ethical principles. Simplified version If you post your content on Pinterest, we can show it to people and others can save it. Don't post porn or spam or be
a jerk to other people on Pinterest. If you post your content on Pinterest, we can show it to people and others can save it. Don't post porn or spam or be
a jerk to other people on Pinterest. an age requirement is largely useless in the absence of
verification. Therefore, we argue that a robust age verification
process needs to be adopted by service providers in the coming
years. However, establishing such mechanisms needs to be
implemented in a way that complies with privacy and the
principles of data minimization [19]. The survey mentioned
earlier [13] has shown that some children lie about their age
and the ease of registering for a social media service (requiring
only a few minutes) does not constitute a barrier. https://pediatrics.jmir.org/2021/2/e22281 JMIR Pediatr Parent 2021 | vol. 4 | iss. 2 | e22281 | p. 6
(page number not for citation purposes) Consent Process Upon registration, the user was asked to accept the terms and
conditions. In most cases, the user agrees to the terms and
conditions by checking a checkbox and subsequently clicking JMIR Pediatr Parent 2021 | vol. 4 | iss. 2 | e22281 | p. 5
(page number not for citation purposes) https://pediatrics.jmir.org/2021/2/e22281 XSL•FO
RenderX JMIR PEDIATRICS AND PARENTING Schneble et al recommendations on the consent process [19], we were not able
to identify a standard presentation form or standard procedure
in presenting terms and conditions. Most forms show their terms
and conditions only in continuous text, whereas others have
adopted a question and answer form (eg, Facebook, Instagram,
and Pinterest). Pinterest is the only platform that provides a
simplified version in addition to the full version of its terms
(Textbox 1). the register button or even by only clicking the register button
(Facebook and Instagram). the register button or even by only clicking the register button
(Facebook and Instagram). Sometimes, the link to the terms and conditions is in a smaller
font (see Table S2 in Multimedia Appendix 1 for an overview)
so that it is hardly identifiable (Snapchat). On Instagram and
Facebook, it is highlighted in bold font. Although the Article
29 Working Party (an independent European advisory body on
data protection and privacy created by the EU) offers some Textbox 1. Full text versus simplified terms and conditions (Pinterest). Textbox 1. Full text versus simplified terms and conditions (Pinterest). Full text
You grant Pinterest and our users a non-exclusive, royalty-free, transferable, sublicensable, worldwide license to
use, store, display, reproduce, save, modify, create derivative works, perform, and distribute your User Content on
Pinterest solely for the purposes of operating, developing, providing, and using Pinterest. Nothing in these Terms
restricts other legal rights Pinterest may have to User Content, for example under other licenses. We reserve the right
to remove or modify User Content or change the way it’s used in Pinterest, for any reason. This includes User Content
that we believe violates these Terms, our Community Guidelines, or any other policies. Simplified version
If you post your content on Pinterest, we can show it to people and others can save it. Don't post porn or spam or be
a jerk to other people on Pinterest. Obtaining Consent underage children by analyzing their physical face properties
(such as the Amazon recognition application programming
interface [20]) or using written language with neurolinguistic
programming for processing natural language. We are fully
aware that the use of such technologies can lead to other ethical
and legal concerns. Although these concerns are too complex
to address in depth in this paper, we discuss them briefly in the
following section. underage children by analyzing their physical face properties
(such as the Amazon recognition application programming
interface [20]) or using written language with neurolinguistic
programming for processing natural language. We are fully
aware that the use of such technologies can lead to other ethical
and legal concerns. Although these concerns are too complex
to address in depth in this paper, we discuss them briefly in the
following section. Obtaining valid user consent (and in the case of children,
parental consent) is one of the 6 lawful bases to process personal
data, as listed in Article 6 of the GDPR. Generally, as consent
is a tool that gives users data subjects control over whether
personal data concerning them will be processed [19], to do so,
valid consent has to meet certain criteria; it must be freely given,
be specific, and be informed and include an unambiguous
indication of the data subject’s wishes. How consent is presented
to the user, whether it is written or presented pictorially or in
short video sequences, is up to the controller (company). This
means that harmonization is not currently envisaged. However,
the Article 29 Working Party (an advisory board of the EU on
data protection issues) does lay out how data subjects (users)
should provide consent. Obtaining consent by simply scrolling
down and ticking a checkbox is not seen as appropriate from
an ethical standpoint, although it might be sufficient from a
policy perspective. Thus, the Working Party provides 2
examples of how a valid mechanism could look (outlined in
Textbox 2), which is not currently met by any of the services
that are subject to our investigation. As shown in our analysis,
users are presented with written information on their rights and
rights of companies on topics such as data protection,
community rules, and minimum age. Minimum Age to Sign Up for a Service Our analysis reveals that most apps have adopted the minimum
age of 13 years for children to sign up to use their services. This
complies with the US COPPA and GDPR. In contrast with the
COPPA, the GDPR provides a minimum age requirement
ranging from 13 to 16 years for children to register for a service. Owing to the GDPR’s extraterritorial force (as mentioned in
Article 3 of the GDPR), other states and companies outside the
EU have to comply with EU standards when targeting users
(and children) in an EU member state. Other providers delegate age verification to their users by setting
up forms where one can report underage use. However, this
method does not guarantee age verification and, in the absence
of other measures, it suggests that the service provider is neither
serious nor proactively interested in complying with the
minimum age requirement. Today's technologies could make it possible to approach the
minimum age to check more proactively. For example, artificial
intelligence could enable the use of techniques such as image
classification algorithms or natural language processing to detect Strongly intertwined with the definition of the minimum age is
the issue of age verification. As Table 1 shows, the issue of age
verification is currently not taken seriously by companies, and XSL•FO
RenderX JMIR PEDIATRICS AND PARENTING Schneble et al Schneble et al https://pediatrics.jmir.org/2021/2/e22281 Appropriate way Swiping a bar on a screen, waiving in front of a smart camera, turning a smartphone around clockwise, or in a
figure-eight motion may be options to indicate agreement, as long as clear information is provided, and it is clear
that the motion in question signifies agreement to a specific request (e.g., if you swipe this bar to the left, you agree
to the use of information X for purpose Y. Repeat the motion to confirm). The controller must be able to demonstrate
that consent was obtained this way, and data subjects must be able to withdraw consent as easily as it was given. I
i JMIR Pediatr Parent 2021 | vol. 4 | iss. 2 | e22281 | p. 7
(page number not for citation purposes) Obtaining Consent Appropriate way
Swiping a bar on a screen, waiving in front of a smart camera, turning a smartphone around clockwise, or in a
figure-eight motion may be options to indicate agreement, as long as clear information is provided, and it is clear
that the motion in question signifies agreement to a specific request (e.g., if you swipe this bar to the left, you agree
to the use of information X for purpose Y. Repeat the motion to confirm). The controller must be able to demonstrate
that consent was obtained this way, and data subjects must be able to withdraw consent as easily as it was given. Inappropriate way
Scrolling down or swiping through a website will not satisfy the requirement of a clear and affirmative action. This
is because the alert that continuing to scroll will constitute consent may be difficult to distinguish and/or maybe missed
when a data subject is quickly scrolling through large amounts of text and such an action is not sufficiently
unambiguous. A special category for obtaining consent is imposed for children
below the age of legal maturity in their respective countries. In
such cases, the GDPR and COPPA require approval from the
parent or guardian. This has several positive and negative
aspects. On the one hand, this regulation places the burden on
the parents to protect children from potential harm, which could,
in turn, be built by safeguarding mechanisms of the platforms. On the other hand, overrestrictive consent processes could be
a driver of inequality, as strict parents could hinder beneficial
A
l
t
(
h
i
th
t ’
credit card or facial recognition) is always associated with mor
data being collected not only from the child but also from th
parent. Thus, balancing data minimization against sufficien
safeguards plays an important role in designing an ethica
consent process. Emphasizing consent is important; however, other scholars hav
argued that solely focusing on this aspect and implying parenta
consent is not enough. By making data protection impac
assessment mandatory (as required by the GDPR), risks can b Obtaining Consent A further issue is that some
of the services only provide a checkbox to tick or, in the worst
case, only a button to register where the terms and conditions
are not displayed during the account’s creation unless the user
clicks the link. This fosters a click and forget mentality and is
far from providing a sustainable and respectful partnership
between service providers and users. Often, the link to the terms
and conditions is presented in smaller fonts and stands in
contrast with the large textboxes filled during the registration
process, as shown in the examples in Table S2 in Multimedia
Appendix 1. Article 9 of the GDPR places biometric data in a special
category: processing is prohibited unless special circumstances
are met. However, notably Article 9 [7] of the GDPR permits
each EU member country to introduce certain derogations with
respect to restrictions on processing biometric data (member
states may maintain or introduce further conditions, including
limitations). For instance, the Netherlands has provided an
opt-out option for biometric data if necessary, for authentication
or security purposes, and Croatia’s new data protection law
exempts surveillance security systems [21]. In the United States,
no federal law regulates the collection of biometric data. However, 3 states—Illinois, Washington, and Texas—have
implemented regulations on biometric data [21]. On the ethical
side, the introduction of such technologies to tackle the issue
of age verification is also potentially problematic, as appropriate
consent must be obtained from the user, who should also have
a full overview where the biometric data are being used, as these
types of data represent special categories that are harmful when
misused. Thus, the use of such technologies should follow clear
ethical guidelines. For example, such technologies should not
be used to collect more information about users and data than
is necessary, and they should always be used for a specific
purpose. This is also because an increasing number of predictive
analyses are possible [2,22] from simple social media data. Textbox 2. Example of how to obtain consent (examples of the Article 29 Working Party). Textbox 2. Example of how to obtain consent (examples of the Article 29 Working Party). Inappropriate way Scrolling down or swiping through a website will not satisfy the requirement of a clear and affirmative action. This
is because the alert that continuing to scroll will constitute consent may be difficult to distinguish and/or maybe missed
when a data subject is quickly scrolling through large amounts of text and such an action is not sufficiently
unambiguous. Scrolling down or swiping through a website will not satisfy the requirement of a clear and affirmative action. This
is because the alert that continuing to scroll will constitute consent may be difficult to distinguish and/or maybe missed
when a data subject is quickly scrolling through large amounts of text and such an action is not sufficiently
unambiguous. credit card or facial recognition) is always associated with more
data being collected not only from the child but also from the
parent. Thus, balancing data minimization against sufficient
safeguards plays an important role in designing an ethical
consent process. A special category for obtaining consent is imposed for children
below the age of legal maturity in their respective countries. In
such cases, the GDPR and COPPA require approval from the
parent or guardian. This has several positive and negative
aspects. On the one hand, this regulation places the burden on
the parents to protect children from potential harm, which could,
in turn, be built by safeguarding mechanisms of the platforms. On the other hand, overrestrictive consent processes could be
a driver of inequality, as strict parents could hinder beneficial
usage. A complex consent process (such as using the parents’ Emphasizing consent is important; however, other scholars have
argued that solely focusing on this aspect and implying parental
consent is not enough. By making data protection impact
assessment mandatory (as required by the GDPR), risks can be JMIR Pediatr Parent 2021 | vol. 4 | iss. 2 | e22281 | p. 7
(page number not for citation purposes) XSL•FO
RenderX JMIR PEDIATRICS AND PARENTING Schneble et al Children's Emergency Fund) warned of the formation of a
significant digital divide [23], highlighting the gap between
children who can connect and subsequently sign up for social
media networks. This divide could be the result of either having
more permissive parents who agree to the use of such services
or because the child is wealthy enough to purchase a
pay-as-you-go phone with data to access social media services
secretly. Inappropriate way Conversely, children who are left out of social media
because their parents are more law-abiding or controlling or
because their socioeconomically disadvantaged background
makes personal phones unaffordable or are forced to share their
parents’ devices. Children in the latter group feel left out of
their friends’ social lives and end up being ostracized by their
peers or even bullied. already identified at an earlier stage [22]. Combining these 2
approaches for making the terms and conditions more readable
and fostering data protection impact assessments would help to
protect children’s rights. already identified at an earlier stage [22]. Combining these 2
approaches for making the terms and conditions more readable
and fostering data protection impact assessments would help to
protect children’s rights. Educating Users and Parents As the report of the UK Children’s Commissioner [12] has
shown, the safe use of social media depends on building
awareness and educating children about its use and fostering
digital literacy. Parents and teachers play an important role. Most of the apps we analyzed offered parents websites where
the companies either provided links to useful literature (the
simplest way to deal with that issue) or by providing short
YouTube sequences to inform children and parents about
potential harm and the security measures to take when using
social media. With the introduction of the GDPR and the adjustment of the
minimum age to 16 years in certain countries, it is expected that
the topic of social pressure will defuse itself at least on an
institutional level because institutions must adhere to this
requirement. However, social media companies’ adhesion to
the GDPR age requirement could, on the other hand, worsen
social pressure for children as the gap between the legal age at
which it is possible to join social media and children’s actual
social practices differs [24]. In medical care, children can give
consent for themselves below the legal age of maturity; however,
this exception does not apply in the case of compliance with
GDPR. Given the importance of educating parents and teens [12], we
suggest
that
future
legislation
should
mandate
the
implementation of such parental portals. From an ethical point
of view, it would be good to encourage companies to spend a
reasonable amount of their revenue in educating parents and
children about the potential harm resulting from the use of their
services. A good example is provided in the Facebook Help
Center, which offers short YouTube sequences and quizzes on
the topics of data protection and possible harm. JMIR Pediatr Parent 2021 | vol. 4 | iss. 2 | e22281 | p. 8
(page number not for citation purposes) JMIR PEDIATRICS AND PARENTING JMIR PEDIATRICS AND PARENTING Figure 1. Mapping the four ethical biomedical principles of the use of social media to issues arising from the use of social media and links them to
possible fields of actions. (Enlarged age verification: Using sophisticated mechanisms such as credit card charges could foster digital divide; Parental
consent: Parents might prevent kids joining resulting in negative consequences for them). media use. This debate is not new in the legal context;
Brunschwig [26] was one of the first to show how contractual
law can be exemplified with comics fostering a better
understanding of otherwise complex matters. Several scholars
have been working on this topic, proposing nutrition label–like
terms and conditions [27] and grid-based terms and conditions
[28]. Such pictorial forms of consent are best practices in
research ethics settings, especially with sensitive study
participants or those with low literacy levels. There might be
some implementation issues with such solutions. Nevertheless,
when we are speaking about children—a sensitive group—such
terms and conditions are a much better means of informing
users about potential harm. This is not a purely theoretical
discussion and approach, as Apple recently presented nutrition
labels for their App Store [29]. In research ethics, the informed consent process plays a crucial
role and contributes to a trusted partnership between subjects
and researchers. When approached about the possibility of
involvement in a clinical study (and increasingly for interviews
or survey participation), potential participants are given all
relevant information and time to digest and consider it before
signing an informed consent form. In the past, the information
provided to participants often ran to over 100 pages, thus raising
the same concerns about accessibility and comprehensibility as
social media terms and conditions. In recent years, however,
there has been a move toward making such information much
more patient- and participant-friendly, with, for example, the
UK Human Research Authority supporting the use of simple
information sheets in a question and answer format running to
a maximum of 5-10 pages. This practice focus on
communicating relevant information about risks and harms in
a concise and comprehensible format could also serve as a model
for building trusted relationships between social media users
and companies. The problem with using terms and conditions
as an information sheet is that such policies are essentially legal
documents and written in dense legal language. JMIR Pediatr Parent 2021 | vol. 4 | iss. 2 | e22281 | p. 9
(page number not for citation purposes) https://pediatrics.jmir.org/2021/2/e22281 Research Ethics as a Model for a Trust-Based
Partnership Social media apps have become ubiquitous among children and
adolescents. It has become difficult to refuse to be part of such
networks, because of both social pressure and an increasing
number of institutions (such as schools) requiring such channels,
resulting in social pressure to use these services for
communication, regardless of whether parents regard the use
of these services to be appropriate for their children. This could
also be seen as a loss of autonomy concerning the freedom to
decide whether and when to join. We can imagine a scenario
in which children who want to participate in social media life
are pressured to lie about their age on the internet by fellow
schoolmates or friends because this peer group’s main vehicle
of social interaction is heavily mediated by online- messaging
and social media, for example, children need to be on WhatsApp
to be able to meet with others because all of the peer meetings
are communicated that way. It is also possible that parents could
incentivize their offspring to engage in online misconduct as
they want their children to use online messaging services (eg,
WhatsApp) out of convenience or for monitoring purposes. These phenomena can create new social inequalities. In fact, in
its 2017 report, UNICEF (United Nations International Similar to social media today, biomedical research used to have
a bad reputation in terms of involving participants. People were
included in medical studies without their consent, and their data
were shared without their knowledge. To prevent such unethical
practices, 4 main ethical principles have become fundamental
to research ethics and biomedical ethics more widely: respect
for autonomy, nonmaleficence, beneficence, and justice. In the
context of social media, all of these principles are relevant;
however, this is particularly true of respect for autonomy and
nonmaleficence. Figure 1 illustrates how social media can
innovate to ensure age verification, valid consent, and other
aspects to make sure that these key ethical principles are
respected. Fundamentally, it is an ethical imperative to ensure
that children are of suitable age and understand the risks of
social media to reduce the risk of harm to their emotional
well-being and mental health: evidence suggests that social
media can have substantial impacts in the areas of self-esteem
and well-being, with issues related to cyberbullying and
Facebook Depression [25]. https://pediatrics.jmir.org/2021/2/e22281 https://pediatrics.jmir.org/2021/2/e22281 XSL•FO
RenderX JMIR PEDIATRICS AND PARENTING Schneble et al JMIR PEDIATRICS AND PARENTING Disentangling
lengthy legal texts from the salient information required to
provide informed consent is essential for social media
companies. However, today’s relationships are still unbalanced
from the very beginning, with users required to sign up with a
simple click after having to read information that is only
presented in written form and complex language. This means
that many users remain to be unaware of exactly what they are
signing up for. Moving toward some sort of pictorial consent
system would be a much more appropriate approach to
informing both children and adults about the risks of social Another possible solution, and a step in the right direction, is
the simplified text-based rules for several social media apps
developed by the UK Children's Commissioner [30]. Research
ethics also requires that data can typically only be shared and
processed with the consent of the persons concerned. However,
recent social media scandals [4,31] have shown that some social
media companies have neglected this issue, which must also be
addressed more clearly in terms and conditions. Another
essential aspect of research ethics is the right to withdraw
consent and the possibility of deleting data (or an account if
research takes place via the internet) by the user. However, for
underaged users (with respect to the minimum age required by
the companies), it should also be possible for parents to delete
an account without going through a complicated process. This
could be done, for example, by specifying a parental contact XSL•FO
RenderX JMIR PEDIATRICS AND PARENTING Schneble et al information that users need from the complex legal language
would also have the benefit of facilitating standardization;
regardless of the jurisdictions, the language for consent
documents should be simple and straightforward. In addition,
in some cases, using pictorial versions of the terms and
conditions would surely leverage the efficacy of today’s mostly
unread versions. The vast majority of social media users have
given only uninformed consent; however, the click, consent,
and forget at your peril model must be relegated to history in
favor of a more transparent and ethical system. The
standardization of terms and conditions is only possible if an
effective political intervention is implemented. Recent
developments and discussions about monopolistic large social
media companies in the US Congress are a step toward
harmonization. Furthermore, the role model function of the
GDPR as a quasi-standard for new data protection regulations
will eventually simplify standardization. JMIR PEDIATRICS AND PARENTING Adopting measures
based on key ethical principles will safeguard children’s health
and well-being and those of other social media users. when registering the account. Finally, research ethics also
address the potential risks in participating in a study. Most
companies in our sample address possible harms of using their
services in their parent portals and community guidelines. Conflicts of Interest None declared. Conclusions Our analysis reveals that social media networks are still lacking
in many respects with regard to adequate protection for children. Consent procedures are flawed because they are too complex,
and in some cases, children can create social media accounts
without sufficient age verification or parental oversight. Given
the high risks of inappropriate content being shared and the
targeting of children with specific advertisements, social media
companies must improve their procedures to protect not only
children but also all users. This can be achieved by standardizing
the registration process in accordance with modern research
ethics procedures described earlier: give users the key facts that
they need in a format that can be read easily and quickly, rather
than forcing them to wade through chapters of legal language
that they cannot understand. Disentangling the practical Multimedia Appendix 1 Links and presentation of terms and conditions. Links and presentation of terms and conditions. [DOCX File , 619 KB-Multimedia Appendix 1] [DOCX File , 619 KB-Multimedia Appendix 1] https://pediatrics.jmir.org/2021/2/e22281 Acknowledgments g
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i
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31. Schneble CO, Elger BS, Shaw DM. Google's Project Nightingale highlights the necessity of data science ethics review. EMBO Mol Med 2020 Mar 06;12(3):e12053 [FREE Full text] [doi: 10.15252/emmm.202012053] [Medline: 32064790] p
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31. Schneble CO, Elger BS, Shaw DM. Google's Project Nightingale highlights the necessity of data science ethics review. JMIR PEDIATRICS AND PARENTING EMBO Mol Med 2020 Mar 06;12(3):e12053 [FREE Full text] [doi: 10.15252/emmm.202012053] [Medline: 32064790] Abbreviations
COPPA: Children's Online Privacy Protection Act
EU: European Union
GDPR: General Data Protection Regulation https://pediatrics.jmir.org/2021/2/e22281 XSL•FO
RenderX Schneble et al JMIR PEDIATRICS AND PARENTING Edited by S Badawy; submitted 10.07.20; peer-reviewed by K Devon; comments to author 25.07.20; revised version received 04.09.20;
accepted 07.03.21; published 22.04.21
Please cite as:
Schneble CO, Favaretto M, Elger BS, Shaw DM
Social Media Terms and Conditions and Informed Consent From Children: Ethical Analysis
JMIR Pediatr Parent 2021;4(2):e22281
URL: https://pediatrics.jmir.org/2021/2/e22281
doi: 10.2196/22281
PMID: Please cite as:
Schneble CO, Favaretto M, Elger BS, Shaw DM
Social Media Terms and Conditions and Informed Consent From Children: Ethical Analysis
JMIR Pediatr Parent 2021;4(2):e22281
URL: https://pediatrics.jmir.org/2021/2/e22281
doi: 10.2196/22281
PMID: ©Christophe Olivier Schneble, Maddalena Favaretto, Bernice Simonne Elger, David Martin Shaw. Originally published in JMIR
Pediatrics and Parenting (https://pediatrics.jmir.org), 22.04.2021. This is an open-access article distributed under the terms of the
Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work, first published in JMIR Pediatrics and Parenting, is properly cited. The complete bibliographic information, a link to the original publication on http://pediatrics.jmir.org, as well as this copyright
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Determining end-of-life policy for recoverable products
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International journal of production research
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Appendix A. Examples In this appendix we provide illustrative examples to show how the methods in the main text are
applied. Daniel W. Steeneck
and Subhash C. Sarin
aGrado Department of Industrial and Systems Engineering, Virginia Tech, Blacksburg, VA; b Center for
Transportation and Logistics, Massachusetts Institute of Technology, Cambridge, MA aGrado Department of Industrial and Systems Engineering, Virginia Tech, Blacksburg, VA; b Center for
Transportation and Logistics, Massachusetts Institute of Technology, Cambridge, MA (Received 00 Month 20XX; accepted 00 Month 20XX) (Received 00 Month 20XX; accepted 00 Month 20XX) A.1
Consideration of other EOL options To illustrate how to consider other EOL options in the proposed analysis framework, consider
the example Πmax and Πj
EOL functions shown in Figure C1 and the corresponding updated Πmax
depicted in Figure C2. Since we are always choosing the next most pro table option when generating Πupdated
max
, the slopes
are decreasing, and therefore Πupdated
max
will be concave as well. In Figure C1, we see that Π1
max has
the greatest slope, and therefore, it is the rst segment of Πupdated
max
in Figure C2. In this case, the
rst C1 cores collected are disassembled and their parts used for remanufacturing. However, the
next best segment is Π1
EOL, in which case the next d1 cores are used for EOL Option 1. Core supply
is not yet exhausted and the next best EOL option is Π2
max, in which case the remaining cores are
collected in order to obtain additional parts for remanufacturing and salvage. ∗Corresponding author. Email: steeneck@gmail.com. Author's current a liation is The Air Force Insitute of Technology,
Department of Operational Sciences, Wright-Patterson AFB, OH. May 2, 2017 May 2, 2017 May 2, 2017 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research SteeneckSarinEOLMapping_IJPR_appendix_v3.0 To appear in the International Journal of Production Research
Vol. 00, No. 00, 00 Month 20XX, 112 To appear in the International Journal of Production Research
Vol. 00, No. 00, 00 Month 20XX, 112 g
g
Department of Operational Sciences, Wright-Patterson AFB, OH. Department of Operational Sciences, Wright-Patterson AFB, OH. Daniel W. Steeneck a,b∗and Subhash C. Sarin a Daniel W. Steeneck a,b∗and Subhash C. Sarin a
aGrado Department of Industrial and Systems Engineering, Virginia Tech, Blacksburg, VA; b Center for
Transportation and Logistics, Massachusetts Institute of Technology, Cambridge, MA ∗Corresponding author. Email: steeneck@gmail.com. Author's current a liation is The Air Force Insitute of nline appendix: Determining end-of-life policy for recoverable products Daniel W. Steeneck a,b∗and Subhash C. Sarin a SteeneckSarinEOLMapping_IJPR_appendix_v3.0 Determination of Pricing and optimal EOL policy Determination of Pricing and optimal EOL policy We illustrate the procedure for jointly determining pricing and optimal EOL policy (given
by Proposition 8) for two cases of δ with M = 10, 000, and report the optimal solution for various
other values of δ. Assume that δ = 0.5. Since PK
k=1 skψk −θ ≥0, there exists no limiting part. In Table A3, the
iterations are shown for the method in Proposition 8 to nd p∗. The columns give various important
values obtained while implementing Algorithm EOL-CCPP. For convenience, when checking con-
straint violation, we report µz(p
′
R), de ned to be the value of pN when pR = p
′
R and (pN, pR) ∈µ=
z . To begin, since K = 7, z = 7 and since there is no limiting part, R
′ = R7
4 and i = 4 (by Steps
0 and 1 of Algorithm EOL-CCPP). Also, in Step 1, we nd the unconstrained optimal solution of
EOL-CCPP-R7
4 to be p
′ = {105, 843, 45, 005}, in which case p
′
N < µ8(p
′
R) = 150, 016 (and thus
µ=
8 is violated). The conditions of Step 2.3 are met and by solving EOL-CCPP-R4-µ8 we nd the
optimal solution to make 2,931 new trucks and to sell them for $106,030 each. The optimal policy
is to collect all cores possible and salvage parts. pm
Iteration
z
p
′
N
p
′
R
µz+1(p
′
R)
µz(p
′
R)
µ
′
v
h
′
v
µv
m ∪µv
Result
New µv
1
7
105, 843
45, 005
150, 016
150, 013
µ=
8
∅
∅
p∗by solving
EOL-CCPP-R4-µ=
8
{µ=
8 } However, in case δ = 0.8, a dierent solution is found. In Table A4, the iterations are shown for
the method in Proposition 8 to nd p∗. To begin, since K = 7, z = 7, and there is no limiting part so
R
′ = R7
4 and i = 4 (by Steps 0 and 1 of Algorithm CCPP). Also, in Step 1, we nd the unconstrained
optimal of EOL-CCPP-R7
4 to be p
′ = {106, 030, 82, 801}, in which case the p
′
N > µ7(p
′
R) = 105, 336
(and thus µ=
7 is violated), and by Step 2.4, z = 6. optimal policy hold. The major barrier to remanufacturing is the remanufactured product price. Table A1. Part replacement cost, salvage value, good part recovery yield, and
disposal cost for a Class 8 truck. T Table A1. Part replacement cost, salvage value, good part recovery yield, and
disposal cost for a Class 8 truck. k
Part
cp
k
sk
ψk
cdisp
k
1
Cab
20,306
13,537
0.97
90.00
2
Engine
14,480
9,653
0.84
78.75
3
Transmission
5,405
3,604
0.83
18.00
4
Radiator
1,931
1,288
0.82
6.75
5
Hood
4,049
2,699
0.74
20.00
6
Front Axle Assembly
2,257
1,505
0.60
33.75
7
Rear Dierential
2,725
1,816
0.59
112.5
Table
A2. Additional
pa-
rameters and their values
pR
35,000
cR
10,000
cN
70,000
cc
16,000
cd
2,000
λ
0.7 A.2
Application of the proposed model to Class 8 trucking industry We illustrate use of the model described in the main text with an example from the Class 8 trucking
industry. The major components of a Class 8 truck are: cab, engine, transmission, radiator, hood,
front axle assembly, and rear dierential. The part replacement costs, salvage values, part yields,
and disposal costs for these parts are given in Table A1. The remanufactured product price, cost to
recover a truck, cost to disassemble a truck, demands of new and remanufactured trucks, and the
proportion of new trucks produced that can be recovered, are given in Table A2. Determination of optimal EOL policy Assume that
pR
=
40, 000. For this product, salvage is the optimal EOL policy as
ρR ≤PK
k=1 sk and PK
k=1 ψk −θ ≥0 (by de nition of the Salvage EOL policy type). Note
that ρR = 40, 000 −10, 000 = 30, 000 and PK
k=1 sk = 34, 102, and indeed, ρR ≤PK
k=1 sk. It can
also be veri ed that PK
k=1 skψk −θ ≥0, and thus, the optimality conditions for salvage as the SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research optimal policy hold. The major barrier to remanufacturing is the remanufactured product price. increment in δ. Both the consumer and producer gain with increment in δ. increment in δ. Both the consumer and producer gain with increment in δ. Table A5. Optimal Solutions for various values of δ for example with parameters given in Table A1 and M = 10, 000
Cost per
δ
dN
dR
dN+R
pN
pR
Π
¯
UN
¯
UR
Truck
Optimal Policy
0.5
2,931
0
2,931
106,030
N/A
1.29×108
21,985
-
62,018
S2
0.6
2,931
0
2,931
106,030
N/A
1.29×108
21,985
-
62,018
S2
0.7
2,931
0
2,931
106,030
N/A
1.29×108
21,985
-
62,018
S2
0.75
2,931
0
2,931
106,030
N/A
1.29×108
21,985
-
62,018
S2
0.8
2,212
914
3,126
105,850
82,487
1.32×108
27,558
5,483
57,752
S7
4 , λdN = dR
ψ7
0.85
2,002
1,149
3,151
105,323
87,326
1.36×108
29,667
7,328
55,600
S4
4 , λdN = dR
ψ4
0.9
1,983
1,166
3,149
104,507
92,482
1.40×108
30,618
7,872
55,913
S2
4 , λdN = dR
ψ2
0.95
1,875
1,272
3,147
103,745
97,651
1.44×108
32,195
9,067
55,524
S1
4 , λdN = dR
ψ1 Determination of Pricing and optimal EOL policy Returning to Step 1, R
′ = R6
4 and i = 4, and
we nd the unconstrained optimal of EOL-CCPP-R6
4 to be p
′ = {105, 843, 82, 504}, in which case
p
′
N < µ7(p
′
R) = 105, 843 (and thus µ=
6 is violated). By Step 2.4, z = 5. Returning to Step 1, R
′ = R5
4
and i = 4, and we nd the unconstrained optimal of EOL-CCPP-R6
4 to be p
′ = {105, 685, 83, 631},
in which case p
′
N < µ7(p
′
R) = 105, 875. Since µ=
6 is doubly violated, by Step 2.1 we solve EOL-
CCPP-R4-µ6 and nd the optimal solution is to make 2,2212 new trucks and 913 remanufactured
trucks and to sell them at $105,850 and $82,487, respectively. Table A5 gives optimal solutions for various values of δ. In this case, the values of δ less than
about 0.75 result in the salvage EOL policy. For the values of δ greater than 0.8, the price of new
products falls, as does demand, while the price and demand for remanufactured products increases
and pro t increases as well. Additionally, the number of customers purchasing trucks increases with
increment in δ and the average utility for both new and remanufactured trucks increases as well. Finally, we notice that the average cost per truck (both new and remanufactured) decreases with 2 May 2, 2017 International Journal of Production Research International Journal of Production Research Table A4. Application of Proposition 8 for example with parameters given in Table A1 and M = 10, 000, δ = 0.8
pm
Iteration
z
p
′
N
p
′
R
µz+1(p
′
R)
µz(p
′
R)
µ
′
v
h
′
v
µv
m ∪µv
Result
New µv
1
7
106,030
82,051
102,563
105,336
µ7
∅
∅
Iterate
{µ=
7 }
2
6
105,843
82,505
105,871
105,904
µ7
∅
µ7
µ7
doubly
violated:
solve EOL-CCPP-R4-
µ7
{µ=
7 } Appendix B. Proofs In this appendix we provide proofs of the propositions in the body of this paper. Some properties
needed for some of the proofs are presented and proven in this appendix as well. The following
notation is used to simplify the presentation of the proofs. σi,j =
i
X
k=1
skψk +
K
X
k=j+1
cp
kψk,
(B1)
κi,j =
i
X
k=1
sk +
K
X
k=j+1
cp
k,
(B2)
σc
i,j =
j
X
k=i+1
cp
kψk,
(B3)
κc
i,j =
j
X
k=i+1
cp
k,
(B4)
σs
i,j =
j
X
k=i+1
skψk, and
(B5)
κs
i,j =
j
X
k=i+1
sk. (B6) σi,j =
i
X
k=1
skψk +
K
X
k=j+1
cp
kψk,
(B1)
κi,j =
i
X
k=1
sk +
K
X
k=j+1
cp
k,
(B2)
σc
i,j =
j
X
k=i+1
cp
kψk,
(B3)
κc
i,j =
j
X
k=i+1
cp
k,
(B4) (B1) (B2) (B3) (B4) (B5) (B6) 3 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research Taking the derivatives of (13) and (14) with respect to C results in Taking the derivatives of (13) and (14) with respect to C results in Π
′
0,z =ρRψz +
z−1
X
k=1
sk(ψk −ψz) −
K
X
k=z+1
cp
k(ψz −ψk) −θ
for C ∈[0, dR
ψz
) and z = 1, . . . , K + 1,
(B7) (B7) and Π
′
z,z+1 =
z
X
k=1
skψk +
K
X
k=z+1
cp
kψk −θ
for C ∈[dR
ψz
, dR
ψz+1
) and z = 1, . . . , K. (B8) (B8) Proof of Proposition 1. The expression for Π (see (1)) can be grouped by its linear (L) terms and
piecewise linear (PWL) terms. Let Proof of Proposition 1. The expression for Π (see (1)) can be grouped by its linear (L) terms and
piecewise linear (PWL) terms. Let L = ρRQR −θC,
(B9) (B9) and PWL =
K
X
k=1
sk
max
ψkC −QR, 0
−
K
X
k=1
cp
k
max
QR −ψkC, 0
. (B10) (B10) e PWL terms can be stated as The PWL terms can be stated as The PWL terms can be stated as z
X
k=1
sk(ψkC −QR) −
K
X
k=z+1
cp
k(QR −ψkC) for QR
ψz
≤C ≤QR
ψz+1
, z = 0, . . . Appendix B. Proofs , K,
(B11)
hat z
X
k=1
sk(ψkC −QR) −
K
X
k=z+1
cp
k(QR −ψkC) for QR
ψz
≤C ≤QR
ψz+1
, z = 0, . . . , K,
(B11) (B11) Note that Note that max{ψkC −QR, 0} =
(
ψkC −QR for
C ≥QR
ψk ,
0
otherwise. (B12) (B12) and and max{QR −ψkC, 0} =
(
QR −ψkC for
C ≤QR
ψk ,
0
otherwise. (B13) (B13) Since the value of ψk, k = 1, . . . , K decreases with increment in k, in case
QR
ψz
≤C
≤
QR
ψz+1 , PK
k=1 sk
max
ψkC −QR, 0
= Pz
k=1 sk(ψkC −QR) and PK
k=1 cp
k
max
QR −ψkC, 0
=
PK
k=z+1 cp
k(QR −ψkC). Since the value of ψk, k = 1, . . . , K decreases with increment in k, in case
QR
ψz
≤C
≤
QR
ψz+1 , PK
k=1 sk
max
ψkC −QR, 0
= Pz
k=1 sk(ψkC −QR) and PK
k=1 cp
k
max
QR −ψkC, 0
=
PK
k=z+1 cp
k(QR −ψkC). R
R
R
R k=z+1
k(Q
ψk )
Additionally, let C1 : QR
1
ψz1 ≤C1 ≤
QR
1
ψz1+1 and let C2 : QR
2
ψz2 ≤C2 ≤
QR
2
ψz2+1 where z1 < z2. Let A(C, QR) =
z
X
k=1
sk(ψkC −QR) −
K
X
k=z+1
cp
k(QR −ψkC). (B14) (B14) 4 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research Let A(C1, QR
1 ) and A(C2, QR
2 ) represent A(C, QR) for C∞and C∈. We have, Let A(C1, QR
1 ) and A(C2, QR
2 ) represent A(C, QR) for C∞and C∈. We have, Let A(C1, QR
1 ) and A(C2, QR
2 ) represent A(C, QR) for C∞and C∈. We have, A(C1, QR
1 ) =(σz1,z2 + σc
z1,z2)C1 −(κz1,z2 + κc
z1,z2)QR
1 , and
(B15)
A(C2, QR
2 ) =(σz1,z2 + σs
z1,z2)C2 −(κz1,z2 + κs
z1,z2)QR
2 . (B16)
C1 + (1 −γ) C2 and QR
γ = γQR
1 + (1 −γ) QR
2
R
QR A(C1, QR
1 ) =(σz1,z2 + σc
z1,z2)C1 −(κz1,z2 + κc
z1,z2)QR
1 , and
(B15)
A(C2, QR
2 ) =(σz1,z2 + σs
z1,z2)C2 −(κz1,z2 + κs
z1,z2)QR
2 . Appendix B. Proofs (B16) (B15)
(B16) Let Cγ = γC1 + (1 −γ) C2 and QR
γ = γQR
1 + (1 −γ) QR
2
Assume QR
γ
ψz1 ≤Cγ ≤
QR
γ
ψz1+1 and γA(C1, QR
1 ) + (1 −γ)A(C2, QR
2 ) ≤A
Cγ, QR
γ
. (B17) (B17) Note that the expression for A
Cγ, QR
γ
is given by (B15). Substituting the expressions for
A(C1, QR
1 ), A(C2, QR
2 ) and A
Cγ, QR
γ
into (B17), rearranging terms and simplifying yields Note that the expression for A
Cγ, QR
γ
is given by (B15). Substituting the expressions for
A(C1, QR
1 ), A(C2, QR
2 ) and A
Cγ, QR
γ
into (B17), rearranging terms and simplifying yields σc
z1,z2 −σs
z1,z2
C2 −
κc
z1,z2 −κs
z1,z2
QR
2 ≥0. (B18) (B18) W.l.o.g. let QR
2 = ψ
′C2
(B19) QR
2 = ψ
′C2 (B19) where ψ
′ ≤ψz2 (since by assumption QR
2 ≤ψz2C2). Substituting (B19) into (B18) gives σc
z1,z2 −σs
z1,z2
−
κc
z1,z2 −κs
z1,z2
ψ
′ ≥0. (B20) (B20) Note that σc
z1,z2 −σs
z1,z2 = Pz2
k=z1+1(cp
k−sk)ψk. Since ψ
′ ≤ψz2, (κc
z1,z2 +κs
z1,z2)ψ
′ = Pz2
k=z1+1(cp
k−
sk)ψ
′ ≤Pz2
k=z1+1(cp
k −sk)ψk and so (B20) is true. Note that σc
z1,z2 −σs
z1,z2 = Pz2
k=z1+1(cp
k−sk)ψk. Since ψ
′ ≤ψz2, (κc
z1,z2 +κs
z1,z2)ψ
′ = Pz2
k=z1+1(cp
k−
sk)ψ
′ ≤Pz2
k=z1+1(cp
k −sk)ψk and so (B20) is true. Now assume
QR
γ
ψz2 ≤Cγ ≤
QR
γ
ψz2+1 and that (B17) still holds. However, now the expression for
A
Cγ, QR
γ
is now given by (B16). As a result, substituting the expressions for A(C1, QR
1 ), A(C2, QR
2 )
and A
Cγ, QR
γ
into (B17) and rearranging terms and simplifying yields σc
z1,z2 −σs
z1,z2
C1 −
κc
z1,z2 −κs
z1,z2
QR
1 ≤0. (B21) (B21) W.l.o.g. let W.l.o.g. let QR
1 = ψ
′C1
(B22) QR
1 = ψ
′C1 (B22) where ψ
′ ≥ψz1+1 (since by assumption QR
1 ≥ψz1+1C1). Substituting (B22) into (B21) gives where ψ
′ ≥ψz1+1 (since by assumption QR
1 ≥ψz1+1C1). Substituting (B22) into (B21) gives σc
z1,z2 −σs
z1,z2
−
κc
z1,z2 −κs
z1,z2
ψ
′ ≤0. Property 2. (Dominance Properties) Π0,n ≤Πn−1,n for C ∈[ dR
ψn−1 , dR
ψn ), n = z + 1, . . . , K + 1
(see Figure C4) which is true, by assumption. which is true, by assumption. Appendix B. Proofs (B23) (B23) Note that σc
z1,z2 −σs
z1,z2 = Pz2
k=z1+1(cp
k −sk)ψk. Since ψ
′ ≥ψz1+1, (κc
z1,z2 + κs
z1,z2)ψ
′ =
Pz2
k=z1+1(cp
k −sk)ψ
′ ≥Pz2
k=z1+1(cp
k −sk)ψk and so (B23) is true. Note that σc
z1,z2 −σs
z1,z2 = Pz2
k=z1+1(cp
k −sk)ψk. Since ψ
′ ≥ψz1+1, (κc
z1,z2 + κs
z1,z2)ψ
′ =
Pz2
k=z1+1(cp
k −sk)ψ
′ ≥Pz2
k=z1+1(cp
k −sk)ψk and so (B23) is true. P
k
z1+1( k
)
P
k
z1+1( k
)
That these results hold for any z1 and z2 where z1 < z2 and z1, z2 ∈{1, . . . , K} the PWL term
is a concave function of C and QR. Since L is a linear function of these variables, L + PWL is a
piecewise linear concave function. k
z1+1
k
k
z1+1
k
That these results hold for any z1 and z2 where z1 < z2 and z1, z2 ∈{1, . . . , K} the PWL term
is a concave function of C and QR. Since L is a linear function of these variables, L + PWL is a
piecewise linear concave function. That these results hold for any z1 and z2 where z1 < z2 and z1, z2 ∈{1, . . . , K} the PWL term
is a concave function of C and QR. Since L is a linear function of these variables, L + PWL is a
piecewise linear concave function. 5 May 2, 2017 International Journal of Production Research SteeneckSarinEOLMapping_IJPR_appendix_v3.0 To prove Proposition 2 we introduce the following properties of Π. Let ΠdR be the function given
by the union of Πk,k+1 for all k = 1, . . . , K. Property 1. ΠdR is a concave function of C. Proof of Property 1. We prove the result by showing that Π
′
z−1,z ≥Π
′
z,z+1 for all z = 1, . . . , K. By
(B8), Π
′
z−1,z ≥Π
′
z,z+1, implies z−1
X
k=1
skψk +
K
X
k=z
cp
kψk −θ ≥
z
X
k=1
skψk +
K
X
k=z+1
cp
kψk −θ. (B24) (B24) By simplifying (B24), we have By simplifying (B24), we have cp
k ≥sk,
(B25) (B25) which is true, by assumption. Property 2. (Dominance Properties) I. For some z, z = 1, . . . , K, the following statements are equivalent:
a. the slopes of the segments of Π are such that Π
′
0,z ≤Π
′
z−1,z, I. For some z, z = 1, . . . , K, the following statements are equivalent:
a. the slopes of the segments of Π are such that Π
′
0,z ≤Π
′
z−1,z, 0,z
b. Π0,z ≥Π0,n for C ∈[0, dR
ψn ), n = 1, . . . , z −1 and ,
b. Π0,z ≥Π0,n for C ∈[0, dR
ψn ), n = 1, . . . , z −1 and ψ
c. Π0,z ≥Πn−1,n for C ∈[ dR
ψn−1 , dR
ψn ), n = 1, . . . , z
(see Figure C3). c. Π0,z ≥Πn−1,n for C ∈[ dR
ψn−1 , dR
ψn ), n = 1, . . . , z
(see Figure C3). (
g
)
II. For some z, z = 1, . . . , K, the following statements are equivalent:
a. the slopes of the segments of Π are such that Π
′
0,z ≥Π
′
z,z+1,
b. Π0,z ≥Π0,n for C ∈[0, dR
ψz ) , n = z + 1, . . . , K + 1, and
c. Π0,n ≤Πn−1,n for C ∈[ dR
ψn−1 , dR
ψn ), n = z + 1, . . . , K + 1
(see Figure C4). II. For some z, z = 1, . . . , K, the following statements are equivalent:
a. the slopes of the segments of Π are such that Π
′
0,z ≥Π
′
z,z+1, II. For some z, z = 1, . . . , K, the following statements are equivalent:
a. the slopes of the segments of Π are such that Π
′
0,z ≥Π
′
z,z+1, p
f
g
f
0,z
z,z+1
b. Π0,z ≥Π0,n for C ∈[0, dR
ψz ) , n = z + 1, . . . , K + 1, and
R
R b. Π0,z ≥Π0,n for C ∈[0, ψz ) , n
z + 1, . . . , K + 1, and
c. Π0,n ≤Πn−1,n for C ∈[ dR
ψn−1 , dR
ψn ), n = z + 1, . . . , K + 1
see Figure C4). ψ
c. Proof of Property 2. Case I. 1. We want to show that statements a and b are equivalent. We begin by showing Π
′
0,z ≤Π
′
z−1,z for some z, z = 1, . . . , K i Π0,z ≥Π0,z−1 for C ∈[0, dR
ψz−1
). (B26) Π
′
0,z ≤Π
′
z−1,z for some z, z = 1, . . . , K i Π0,z ≥Π0,z−1 for C ∈[0, dR
ψz−1
). (B26) (B26) To show the only if part of (B26) assume To show the only if part of (B26) assume Π
′
0,z ≤Π
′
z−1,z. (B27) Π
′
0,z ≤Π
′
z−1,z. (B27) Note that
d Note that
d Note that
i. Π0,z( dR
ψz ) = Π
′
0,z · dR
ψz (origin of Π0,z is (0, 0)),
ii. Π0,z−1( dR
ψz−1 ) = Π
′
0,z−1 ·
dR
ψz−1 (origin of Π0,z−1 is (0, 0)), and
iii. Π
′
z−1,z =
Π0,z( dR
ψz )−Π0,z−1(
dR
ψz−1 )
dR
ψz −
dR
ψz−1
(by the piece-wise structure of Π). Substituting (i.) and (ii.) into (iii.), we can rewrite Π
′
z−1,z in terms of Π
′
0,z and Π0,z−1: Note that
i. Π0,z( dR
ψz ) = Π
′
0,z · dR
ψz (origin of Π0,z is (0, 0)), Note that
i. Π0,z( dR
ψz ) = Π
′
0,z · dR
ψz (origin of Π0,z is (0, 0)),
ii Π0
1( dR ) = Π
′
·
dR
(origin of Π0
1 is (0 0)) and i. Π0,z( dR
ψz ) = Π
′
0,z · dR
ψz (origin of Π0,z is (0, 0)), ψz
ψz
ii. Π0,z−1( dR
ψz−1 ) = Π
′
0,z−1 · (origin of Π0,z−1 is (0, 0)), and iii. Π
′
z−1,z =
Π0,z( dR
ψz )−Π0,z−1(
dR
ψz−1 )
dR
ψz −
dR
ψz−1
(by the piece-wise structure of Π). ψz
ψz−1
Substituting (i.) and (ii.) into (iii.), we can rewrite Π
′
z−1,z in terms of Π
′
0,z and Π0,z−1 Π
′
z−1,z =
Π
′
0,z · dR
ψz −Π
′
0,z−1 ·
dR
ψz−1
dR
ψz −
dR
ψz−1
. Proof of Property 2. Case I. (B28) (B28) 6 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research Substituting (B28) into (B27), we have Substituting (B28) into (B27), we have Substituting (B28) into (B27), we have Substituting (B28) into (B27), we have Π
′
0,z ≤
Π
′
0,z · dR
ψz −Π
′
0,z−1 ·
dR
ψz−1
dR
ψz −
dR
ψz−1
,
(B29) (B29) which simpli es to which simpli es to Π
′
0,z ≥Π
′
0,z−1. (B30) Π
′
0,z ≥Π
′
0,z−1. (B30) Since Π0,z and Π0,z−1 share the same origin, (B30) implies that Since Π0,z and Π0,z−1 share the same origin, (B30) implies that Π0,z ≥Π0,z−1. (B31) Π0,z ≥Π0,z−1. (B31) Thus, we have shown that if Π
′
0,z ≤Π
′
z−1,z, then Π0,z ≥Π0,n for n = z −1. To show the if
part of B26, we may simply work backwards from the above logic by assuming Π0,z ≥Π0,z−1. Next, we show that part of B26, we may simply work backwards from the above logic by assuming Π0,z ≥Π0,z−1. Next, we show that Next, we show that Π
′
0,z ≤Π
′
z−1,z =⇒Π
′
0,z−1 ≤Π
′
z−2,z−1. (B32) (B32) Again, assume the relationship in (B27). Note that Again, assume the relationship in (B27). Note that g
,
p
(
)
i. Π0,z−1( dR
ψz−1 ) = Π
′
0,z−1 ·
dR
ψz−1 (origin of Π0,z is (0, 0)), g
(
)
i. Π0,z−1( dR
ψz−1 ) = Π
′
0,z−1 ·
dR
ψz−1 (origin of Π0,z is (0, 0)),
ii. Πz−1,z( dR
ψz ) = Π
′
z−1,z · ( dR
ψz −
dR
ψz−1 ) + Π0,z−1( dR
ψz−1 ) (by the piece-wise structure of Π), and
iii. Π
′
0,z =
Πz−1,z( dR
ψz )
dR
ψz
(by the piece-wise structure of Π). Substituting (i.) into (ii.) and (ii.) into (iii.) yields i. Π0,z−1( d
ψz−1 ) = Π0,z−1 ·
d
ψz−1 (origin of Π0,z is (0, 0)),
ii. Πz−1,z( dR
ψz ) = Π
′
z−1,z · ( dR
ψz −
dR
ψz−1 ) + Π0,z−1( dR
ψz−1 ) (by the piece-wise structure of Π), and
iii. Π
′
0,z =
Πz−1,z( dR
ψz )
dR
ψz
(by the piece-wise structure of Π). Substituting (i.) into (ii.) and (ii.) into (iii.) yields 0,z
1( ψz−1 )
0,z
1
ψz−1 (
g
0,z
( , )),
. Proof of Property 2. Case I. Case II. Case II. The proof for this case follows along the same lines as for Case I. Case II. The proof for this case follows along the same lines as for Case I. Case II. The proof for this case follows along the same lines as for Case I. Case
The proof for this case follows along the same lines as for Case I. Property 3. A Part m, m = 1, . . . , K + 1 is a key part if Property 3. A Part m, m = 1, . . . , K + 1 is a key part if Π
′
m,m+1 ≤Π
′
0,m ≤Π
′
m−1,m. (B38) (B38) Proof of Property 3. By De nition 2, Part m is key if Condition (15) holds. Note that this condition
holds only if statement b of Property 2, Case I and statement b of Property 2, Case II hold, which
are equivalent to statement a of Property 2 (i.e., Π
′
0,m ≤Π
′
m−1,m), Case I and statement a of
Property 2, Case II (i.e., Π
′
m,z+1 ≤Π
′
0,m), respectively. Thus, we have the result. Proof of Proposition 2. By Property 3, if a Part, m, is the key part, then Π
′
0,m ≤Π
′
m−1,m for
C ∈[0, dR
ψm ). In case m = 1, substituting (B7) for Π
′
0,m and (B8) for Π
′
m−1,m, yields Π
′
0,1 = Π
′
0,1,
which is always true. y
In case m = 2, . . . , K, substituting (B7) for Π
′
0,m and (B8) for Π
′
m−1,m, we have that ρRψm +
m−1
X
k=1
sk(ψk −ψm) −
K
X
k=m+1
cp
k(ψm −ψk) −θ ≤
m−1
X
k=1
skψk +
K
X
k=m
cp
kψk −θ,
(B39) (B39) which simpli es to ρR ≤Pm−1
k=1 sk + PK
k=m cp
k, or ρR ≤κm−1 (since κm−1 = Pm−1
k=1 sk + PK
k=m cp
k
by (6)) which simpli es to ρR ≤Pm−1
k=1 sk + PK
k=m cp
k, or ρR ≤κm−1 (since κm−1 = Pm−1
k=1 sk + PK
k=m cp
k
by (6)). Proof of Property 2. Case I. Πz−1,z( dR
ψz ) = Π
′
z−1,z · ( dR
ψz −
dR
ψz−1 ) + Π0,z−1( dR
ψz−1 ) (by the piece-wise structure of Π), and
Π
( dR ) ψz
Substituting (i.) into (ii.) and (ii.) into (iii.) yields ψz
Substituting (i.) into (ii.) and (ii.) into (iii.) yields Π
′
0,z =
Π
′
z−1,z · ( dR
ψz −
dR
ψz−1 ) + Π
′
0,z−1 ·
dR
ψz−1
dR
ψz
. (B33) (B33) Substituting (B33) into (B27), we have Substituting (B33) into (B27), we have Π
′
z−1,z · ( dR
ψz −
dR
ψz−1 ) + Π
′
0,z−1 ·
dR
ψz−1
dR
ψz
≤Π
′
z−1,z,
(B34) (B34) which simpli es to 0,z−1 ≤Π
′
z−1,z. (B35) Π
′
0,z−1 ≤Π
′
z−1,z. (B35) By Property 1, ΠdR is a concave function of C, and thus, Π
′
z−1,z ≤Π
′
z−2,z−1,
(B36) Π
′
z−1,z ≤Π
′
z−2,z−1, (B36) and by the transitivity property Π
′
0,z−1 ≤Π
′
z−2,z−1,
(B37) Π
′
0,z−1 ≤Π
′
z−2,z−1, (B37) which is the desired result to show (B32). 7 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research Note that Π
′
0,z ≤Π
′
z−1,z implies Π0,z ≥Π0,z−1 and Π
′
0,z−1 ≤Π
′
z−2,z−1 by (B26) and (B32),
respectively. By these same relationships Π
′
0,z−1 ≤Π
′
z−2,z−1 implies Π0,z−1 ≥Π0,z−2 and
Π
′
0,z−2 ≤Π
′
z−3,z−2. The iterative re-application of (B26) and (B32) proves the only if part
of the proposition. dR The if part of the proposition is shown by the fact that if Π0,z ≥Π0,n for C ∈[0, dR
ψn ),
n = 1, . . . , z −1, then Π
′
0,z ≥Π
′
0,z−1 by (B26) The proof of the equivalence of statements a and c follows along the same lines as above. p
q
g
3. The equivalence of statements b and c follows by the transitivity property, i.e., statement b's
is equivalence to statement a, and statement a's is equivalence to statement c imply that
statements b and c are equivalent. 3. The equivalence of statements b and c follows by the transitivity property, i.e., statement b's
is equivalence to statement a, and statement a's is equivalence to statement c imply that
statements b and c are equivalent. Case II. The proof for this case follows along the same lines as for Case I. Proof of Property 2. Case I. y ( ))
In case m = K + 1, substituting (B7) for Π
′
0,m and (B8) for Π
′
m−1,m, we have that y ( ))
In case m = K + 1, substituting (B7) for Π
′
0,m and (B8) for Π
′
m−1,m, we have that ρRψK+1 +
K
X
k=2
sk(ψk −ψK+1) −θ ≤
K
X
k=1
skψk −θ,
(B40) (B40) which simpli es to ρR ≤PK
k=1 sk, or ρR ≤κK (since κK = PK
k=1 sk by (6)). Also by Property 3 if Part m is the key part then Π
′
≤Π
′ Also, by Property 3, if Part m is the key part, then Π
′
m,m+1 ≤Π
′
0,m. In case m = 1, . . . , K, substituting (B7) for Π
′
0,m and (B8) for Π
′
m,m+1, we have that ,
+
,
In case m = 1, . . . , K, substituting (B7) for Π
′
0,m and (B8) for Π
′
m,m+1, we have that ,
,
In case m = 1, . . . , K, substituting (B7) for Π
′
0,m and (B8) for Π
′
m,m+ ,
,
n case m = 1, . . . , K, substituting (B7) for Π
′
0,m and (B8) for Π
′
m,m+1, we have that m
X
k=1
skψk +
K
X
k=m+1
cp
kψk −θ ≤ρRψm +
m−1
X
k=1
sk(ψk −ψm) −
K
X
k=m+1
cp
k(ψm −ψk) −θ,
(B41) (B41) which simpli es to ρR ≥Pm
k=1 sk + PK
k=m+1 cp
k, or ρR ≥κm (since κm = Pm
k=1 sk + PK
k=m+1 cp
k by
(6)). 8 International Journal of Production Research
SteeneckSarinEOLMapping_IJPR_appendix_v3.0 May 2, 2017 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research In case m = K + 1, Π
′
m,m+1 is unde ned and so ρR ≤κK is su cient for Part K + 1 to be
key. Proof of Property 4. We now prove each case of Property 4.
C
I Proof of Property 5. We need to consider two cases. Case I. In this case, Πmax is given by (17). By Property 2, Case II, Π
′
0,z ≥Π
′
z,z+1. By Property 1, ΠdR is
a concave function of C and Πz,z+1 ∈ΠdR. Thus, the slope of Πmax is non-increasing and is itself a
concave function of C. Case II. In this case, Πmax = ΠS, which is a linear function. Proof of Property 5. We need to consider two cases. Case I. Proof of Property 5. We need to consider two cases. Case I. Property 4. (Generation of Πmax). I. If for some m, m = 1, . . . , K, the slopes of the segments of Π are such that Π
′
m,m+1 ≤Π
′
0,m ≤
Π
′
m−1,m, i.e., Part m is the key part, then Πmax is given by (17) (see Figure C5). II. If there does not exist an m, m = 1, . . . , K, for which the slopes of the segments of Π satisfy
Π
′
m,m+1 ≤Π
′
0,m ≤Π
′
m−1,m, then: Πmax = ΠS for C ∈[0, ∞),
(B42) (B42) Πmax = ΠS for C ∈[0, ∞), i.e., Part K + 1 is the key part. This outcome is incurred i Π
′
K,K+1 ≥Π
′
0,K (see Figure C6). Proof of Property 4. We now prove each case of Property 4.
C
I C
To show the only if case, assume that Parts z = 1, . . . , K are not key. Therefore, both cases of
Property 2 do not hold for Parts z = 1, . . . , K. First, we show that if Part z = 1, . . . , K is not key,
the conditions for one of the cases of Property 2 will hold. We denote Property 2, Case I as simply
Case I (i.e., Π
′
0,z ≤Π
′
z−1,) and Property 2, Case II as simply Case II (i.e, Π
′
0,z ≥Π
′
z,z+1). When
Part 1 is not a key part, note that Case I is always true, i.e. Π
′
0,1 ≤Π
′
0,1, thus one of the cases is
true. However, by assumption, Case II does not hold and thus Π
′
0,1 ≤Π
′
1,2. By the application of
(B26), we have Π
′
0,1 ≤Π
′
1,2 =⇒Π
′
0,2 ≤Π
′
0,1. (B43) (B43) However, this means that Case I holds for Part 2. Thus, Case II must not hold, and therefore,
Π
′
0,2 ≤Π
′
2,3. By re-applying this logic, we have that Π
′
0,K ≤Π
′
K,K+1. Since Π
′
0,K+1 = Π
′
K,K+1 and
Π
′
0,K+1 ≥Π
′
0,K, Π0,K+1 = Π0,S ≥Π0,K, thereby implying Part K + 1 to be key. Note that, if
Π
′
0,K ≤Π
′
K,K+1 the same logic holds, implying that Parts z = 1, . . . , K are not key. However, this means that Case I holds for Part 2. Thus, Case II must not hold, and therefore,
Π
′
0,2 ≤Π
′
2,3. By re-applying this logic, we have that Π
′
0,K ≤Π
′
K,K+1. Since Π
′
0,K+1 = Π
′
K,K+1 and
Π
′
0,K+1 ≥Π
′
0,K, Π0,K+1 = Π0,S ≥Π0,K, thereby implying Part K + 1 to be key. Note that, if
Π
′
0,K ≤Π
′
K,K+1 the same logic holds, implying that Parts z = 1, . . . , K are not key. ,
,
+
The if case directly follows by assuming Π
′
0,K ≤Π
′
K,K+1 and working backwards using similar
logic given in the only if case. Property 5. Πmax is a concave function of C. Property 5. Πmax is a concave function of C. Proof of Property 5. We need to consider two cases.
Case I. In this case, Πmax is given by (17). By Property 2, Case II, Π
′
0,z ≥Π
′
z,z+1. By Property 1, ΠdR is
a concave function of C and Πz,z+1 ∈ΠdR. Thus, the slope of Πmax is non-increasing and is itself a
concave function of C. In this case, Πmax is given by (17). By Property 2, Case II, Π
′
0,z ≥Π
′
z,z+1. By Property 1, ΠdR is
a concave function of C and Πz,z+1 ∈ΠdR. Thus, the slope of Πmax is non-increasing and is itself a
concave function of C. In this case, Πmax = ΠS, which is a linear function. Proof of Proposition 3. It is a direct consequence of Property 4 and Property 5. Proof of Proposition 4. By Property 4, we know that Πmax may only take certain policy types (see
Section 4). Each policy type, along with the speci cation of a key part, limiting part, and domain
for A, imply a speci c optimal policy, i.e., choice of C and QR, since Πmax is a concave function of
C (by Property 5). Let C∗and QR∗be the optimal solutions for C and QR, respectively. Proof of Proposition 4. By Property 4, we know that Πmax may only take certain policy types (see
Section 4). Each policy type, along with the speci cation of a key part, limiting part, and domain
for A, imply a speci c optimal policy, i.e., choice of C and QR, since Πmax is a concave function of
C (by Property 5). Let C∗and QR∗be the optimal solutions for C and QR, respectively. 9 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 May 2, 2017 International Journal of Production Research 1. No Collection
In this case, Πmax ≤0 for all C ∈[0, A], and thus C∗= 0 and QR∗= 0. 1. No Collection
In this case, Πmax ≤0 for all C ∈[0, A], and thus C∗= 0 and QR∗= 0. 2. Salvage
By the de nition of the Salvage type, QR = 0 and Πmax = ΠS, which is linear with positive
slope. Thus, C∗= A and QR∗= 0. R 3. Remanufacture and Salvage or Remanufacturing with Part m key and A ∈[0, dR
ψm )
In this case, Πmax = Π0,m (by Property 4), which is a non-decreasing function of C in the
domain A ∈[0, dR
ψm ). Thus, C∗= A and QR∗= ψmA. ψ
4. Proof of Property 5. We need to consider two cases.
Case I. Remanufacture and Salvage with Key Part m, no limiting part and A ∈[ dR
ψm , ∞)
In this case, Πmax = ΠdR (by Property 4), and it is non-decreasing since there is no limiting
part. Thus, C∗= A and QR∗= dR. R
R 5. Remanufacturewhere Part m key, Part l is limiting, and A ∈[ dR
ψ1 , dR
ψl ]
In this case, Πmax = ΠdR (by Property 4), and it is non-decreasing for C ∈[0, dR
ψl ]. Thus,
C∗= A and QR∗= dR. 5. Remanufacturewhere Part m key, Part l is limiting, and A ∈[ dR
ψ1 , dR
ψl ]
In this case, Πmax = ΠdR (by Property 4), and it is non-decreasing for C ∈[0, dR
ψl ]. Thus,
C∗= A and QR∗= dR. ψ
ψ
In this case, Πmax = ΠdR (by Property 4), and it is non-decreasing for C ∈[0, dR
ψl ]. Thus,
C∗= A and QR∗= dR. C
A and Q
d . 6. Remanufacture Part l limiting, and A ∈( dR
ψl , ∞)
In this case, Πmax = ΠdR (by Property 4). It is a non-decreasing function of C ∈[0, dR
ψl ], and a
non-increasing function of C ∈( dR
ψl , ∞) since there is a limiting part, l. Thus, for A ∈( dR
ψl , ∞),
C∗= dR
ψl and QR∗= dR. Q
6. Remanufacture Part l limiting, and A ∈( dR
ψl , ∞)
In this case, Πmax = ΠdR (by Property 4). It is a non-decreasing function of C ∈[0, dR
ψl ], and a
non-increasing function of C ∈( dR
ψl , ∞) since there is a limiting part, l. Thus, for A ∈( dR
ψl , ∞),
C∗= dR
ψl and QR∗= dR. Proof of Proposition 5. Assume Proof of Proposition 5. Assume Proof of Proposition 5. Assume Proof of Proposition 5. Assume Π
′
0,l = (pR −cR)ψl +
l−1
X
k=1
sk(ψk −ψl) −
K
X
k=l+1
cp
k(ψl −ψk) −θ ≤0. (B44) (B44) k=1
k=l+1
his may be rewritten as
l
1
K
!! l
1
K
! This may be rewritten as his may be rewritten as This may be rewritten as This may be rewritten as pR −cR
−
l−1
X
k=1
sk +
K
X
k=l+1
cp
k
!! ψl +
l−1
X
k=1
skψk +
K
X
k=l+1
cp
kψk −θ
! ≤0. Proof of Property 6. Let xi = {pN
i , pR
i , dN
i , dR
i }. Let Πm
i (xi) denote Πm
3 as a function of the variables
in xi (Πm
3 is given by (20) in Table 1 with the (pN −cN) term included). Let m1 < m2. The terms
in Πm1
3
and Πm2
3
may be grouped using the expressions for σm1,m2, κm1,m2, σc
m1,m2, κc
m1,m2, σs
m1,m2, SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research Proof of Property 5. We need to consider two cases.
Case I. (B45) (B45) or equivalently or equivalently (pR −cR) −κl
ψl +
l−1
X
k=1
skψk +
K
X
k=l+1
cp
kψk −θ
! ≤0. (B46) (B46) Since Part l is assumed to be limiting, Pl−1
k=1 skψk +PK
k=l+1 cp
kψk −θ ≤Pl
k=1 skψk +PK
k=l+1 cp
kψk −
θ ≤0. Also, by assumption, pR −cR ≤κl, and so Equation (B46) is true. By the concave nature of
ΠdR, its maximum occurs at Πl−1,l( dR
ψl ) = Π0,l( dR
ψl ). Since Π
′
0,l < 0, we have Π0,l( dR
ψl ) < 0. Property 6. Πmax is a convex function over the domain of R3 for a given pN. Proof of Property 6. Let xi = {pN
i , pR
i , dN
i , dR
i }. Let Πm
i (xi) denote Πm
3 as a function of the variables
in xi (Πm
3 is given by (20) in Table 1 with the (pN −cN) term included). Let m1 < m2. The terms
in Πm1
3
and Πm2
3
may be grouped using the expressions for σm1,m2, κm1,m2, σc
m1,m2, κc
m1,m2, σs
m1,m2, 10 10 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research κs
m1,m2, and therefore κs
m1,m2, and therefore Πm1
3 (x) = (pN −cN)dN + (pR −cR)ψmλdN + (σm1,m2 + σc
z −θ)λdN −(κm1,m2 + κc
z)ψmλdN
(B47)
Πm2
3 (x) = (pN −cN)dN + (pR −cR)ψmλdN + (σm1,m2 + σs
z −θ)λdN −(κm1,m2 + κs
z)ψmλdN
(B48) Also, let x1 = {pN, pR
1 , dN
1 , dR
1 } and x2 = {pN, pR
2 , dN
1 , dR
1 }. By Proposition 2, pR
1 ≥pR
2 . Also, let x1 = {pN, pR
1 , dN
1 , dR
1 } and x2 = {pN, pR
2 , dN
1 , dR
1 }. By Proposition 2, pR
1 ≥pR
2 . Assume that γΠm1
3 (x1) + (1 −γ)Πm2
3 (x2) ≥Πm1
3 (γx1 + (1 −γ)x2),
(B49) (B49) for all γ ∈[0, 1] such that γx1 + (1 −γ)x2 ∈Rm1
3 . Substituting the expressions for Πm1
3
and Πm2
3
into (B49), rearranging terms and simplifying yields κm1,m2 −(pR
2 −cR)
(ψm1 −ψm2) + κc
m1,m2ψm1 −κs
m1,m2)ψm2
λdN
2
≥γ(pR
1 −pR
2 )(dN
2 −dN
1 ))ψm1)λdN
2 −(σc
m1,m2 −σs
m1,m2)λdN
2 . Proof of Property 5. We need to consider two cases.
Case I. (B50) (B50) Subtracting (κc
m1,m2−κs
m1,m2)ψm1 and adding (Cm2−Sm2)(ψm1−ψm2) to both sides and simplifying
yields
(κm1,m2 + κs
m1,m2 + (cp
m2 −sm2) −(pR
2 −cR))(ψm1 −ψm2)
dN
2
R
R
N
N
(κm1,m2 + κs
m1,m2 + (cp
m2 −sm2) −(pR
2 −cR))(ψm1 −ψm2)
dN
2
≥γ(pR
1 −pR
2 )(dN
2 −dN
1 ))ψm1
+
(σc
m1,m2 −σs
m1,m2) −(C2
m1,m2 −S2
m1,m2)ψm1 + (cp
m2 −sm2)(ψm1 −ψm2)
λdN
2
(B51) ≥γ(pR
1 −pR
2 )(dN
2 −dN
1 ))ψm1 (B51) +
(σc
m1,m2 −σs
m1,m2) −(C2
m1,m2 −S2
m1,m2)ψm1 + (cp
m2 −sm2)(ψm1 −ψm2)
λdN
2 Recognizing that κm1,m2+κs
m1,m2+(cp
m2−sm2) = κm2−1 ≥(pR
2 −cR), the left-hand side of (B51) is
positive. On the right-hand side of (B51), (pR
2 −pR
1 )(dN
2 −dN
1 ) < 0 since pR
1 ≥pR
2 , and it can easily be
veri ed that dN
2 −dN
1 ≤0. Also, on the right-hand side of (B51), σc
m1,m2 −σs
m1,m2 −(cp
m2 −sm2)ψm2 =
Pm2−1
k=m1+1(cp
k −sk)ψk is less than (κc
m1,m2 −κs
m1,m2)ψm1 −(cp
m2 −sm2)ψm1 = Pm2−1
k=m1+1(cp
k −sk)ψm1
since ψm1 ≥ψm2 and cp
k ≥sk for all k = 1, . . . , K. It follows that the right-hand side of (B51) is
negative and thus (B51) is true. Now assume that γΠm1
3 (x1) + (1 −γ)Πm2
3 (x2) ≥Πm2
3 (γx1 + (1 −γ)x2),
(B52) (B52) for all γ ∈[0, 1] such that γx1 + (1 −γ)x2 ∈Rm1
3 . m
m for all γ ∈[0, 1] such that γx1 + (1 −γ)x2 ∈Rm1
3 . m
m Substituting the expressions for Πm1
3
and Πm2
3
into (B52), rearranging terms and simplifying
yields (σc
m1,m2 −σs
m1,m2)dN
1 ≥(1 −γ)(pR
2 −pR
1 )(dN
1 −dN
2 )ψm2
+ [(κm1,m2 −(pR
1 −cR))(ψm1 −ψm2)
+ κc
m1,m2ψm1 −κs
m1,m2ψm2)]dN
1 . (B53) (B53) 11 11 May 2, 2017 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research Subtracting (κc
m1,m2 −κs
m1,m2)ψm2λdN
1 from both sides yields Subtracting (κc
m1,m2 −κs
m1,m2)ψm2λdN
1 from both sides yields [(σc
m1,m2 −σs
m1,m2) −(κc
m1,m2−κs
m1,m2)ψm2]dN
1 ≥
(1 −γ)(pR
2 −pR
1 )(dN
1 −dN
2 )ψm2
+ (κm1,m2 + κc
m1,m2 −(pR
1 −cR))(ψm1 −ψm2)dN
1 . Proof of Property 5. We need to consider two cases.
Case I. (B54) −κs
m1,m2)ψm2]dN
1 ≥
(1 −γ)(pR
2 −pR
1 )(dN
1 −dN
2 )ψm2
+ (κm1,m2 + κc
m1,m2 −(pR
1 −cR))(ψm1 −ψm2)dN
1 . (B54) [(σc
m1,m2 −σs
m1,m2) −(κc
m1,m2−κs
m1,m2)ψm2]dN
1 ≥ (B54) On the left-hand side of (B54), σc
m1,m2 −σs
m1,m2 = Pm2
k=m1+1(cp
k −sk)ψk is greater than (κc
m1,m2 −
κs
m1,m2)ψm2 = Pm2
k=m1+1(cp
k −sk)ψm2 since ψm2 ≤ψm1 and cp
k ≥sk for all k = 1, . . . , K. Therefore,
the left-hand side of (B54) is positive. Recognizing that κm1,m2 + κc
m1,m2 = ρm1 ≤(pR
1 −cR) (by
Proposition 2) and (pR
2 −pR
1 )(dN
2 −dN
1 ) < 0 since pR
1 ≥pR
2 , and it can easily be veri ed that
dN
2 −dN
1 ≤0, it follows that the right-hand side of (B54) is negative and thus (B51) is true. A similar logic holds for any m1 < m2 and value of pN Proof of Proposition 6. Since EOL-CCPP is a maximization problem and by Property 6 for any pN,
Π3 is convex within the region of Π3, the optimal values of pN and pR will be at the boundary of R3
shared by R1 or R2, if pR = κK+1+cR, or R4 or R5, if pR = ρm+cR . The solutions on the boundary
of R3 shared by R1 or R2 are also in (pN, pR) ∈µ=
K+1. Thus, the optimal solution within R3 will
also be in (pN, pR) ∈µ=
K+1 or at a boundary with R4 ∪R5. It follows that p∗∈R4 ∪R5 ∪µ=
K+1. Property 7. Πmax is concave function over the regions Rz
4 and R5. Proof of Property 7. Let Πρ = (pN −cN)dN + (pR −cR)dR
(B55) (B55) Referring to Table 3, regions Rz
4 and R5, imply EOL polices Sz
4 and S5 and referring to Table
1, these policies correspond to pro ts Πz
4, z = 1, . . . , min{k, l} and Π5 with the (pN −cN) term
included, respectively. These expressions have the form Πρ +L where L is linear in dN and dR (and
thereby linear in pN and pR). Referring to Table 3, regions Rz
4 and R5, imply EOL polices Sz
4 and S5 and referring to Table
1, these policies correspond to pro ts Πz
4, z = 1, . . . Proof of Property 5. We need to consider two cases.
Case I. , min{k, l} and Π5 with the (pN −cN)
term included, respectively. Πz
4, z = 1, . . . , min{k, l} and Π5 may be grouped by expressions for
σz,l, κz,l, σc
z,l, κc
z,l, σs
z,l, κs
z,l and therefore Proof of Proposition 7. : Let Part m be the key part and Part l be the limiting part. Assume that
pN
4 and pR
4 are such that dR
4
ψz ≤λdN
4 ≤
dR
4
ψz+1 where m ≤z ≤min{l, k}, and pN
5 and pR
5 are such that
λdN
5 ≥dR
5
ψl . Referring to Table 3, regions Rz
4 and R5 imply EOL polices Sz
4 and S5 and referring to
Table 1, these policies correspond to pro ts Πz
4, z = 1, . . . , min{k, l} and Π5 with the (pN −cN)
term included, respectively. Πz
4, z = 1, . . . , min{k, l} and Π5 may be grouped by expressions for
σz,l, κz,l, σc
z,l, κc
z,l, σs
z,l, κs
z,l and therefore Πz
4 = (pN −cN)dN + (pR −cR)dR + (σz,l + σc
z,l −θ)λdN −(κl + κc
z,l)dR
(B59)
Π5 = (pN −cN)dN + (pR −cR)dR + (σz,l + σs
z,l −θ)dR
ψl
−(κl + κs
z,l)dR
(B60) (B59) (B60) Note that Πz
4 and Π5 are of the form Πρ + L where Πρ is given by (B55) and L represent
the terms linear in pN and pR. Let Πz,L
4
and ΠL
5 be linear terms of Πz
4 and Π5, respectively. Let
xi = {pN
i , pR
i , dN
i , dR
i }, i = 4, 5. Also, let dN
γ = γdN
4 + (1 −γ)dN
5 and dR
γ = γdR
4 + (1 −γ)dR
5 . Assume
that γΠz,L
4 (x4) + (1 −γ)ΠL
5 (x5) ≤ΠL
4 (γx4 + (1 −γ)x5),
(B61) (B61) for all γ such that dR
γ
ψz ≤λdN
γ ≤
dR
γ
ψz+1 . L for all γ such that dR
γ
ψz ≤λdN
γ ≤
dR
γ
ψz+1 . L Substituting the expressions for Πz,L
4
and ΠL
5 into (B61), and expanding and rearranging terms
yield (σz,l + σc
z,l −θ)λdN
5 −(σz,l + σs
z,l −θ)dR
5
ψl
≥(κc
z,l −κs
z,l)dR
5 . (B62) (B62) W.l.o.g. Proof of Property 5. We need to consider two cases.
Case I. , min{k, l} and Π5 with the (pN −cN) term
included, respectively. These expressions have the form Πρ +L where L is linear in dN and dR (and
thereby linear in pN and pR). Substituting the expressions for dN and dR given by (24) and (25), respectively, into (B55) and
rearranging terms yields Πρ = −
1
δ(1 −δ)
pR2 + δpR(2pN −cN) −δ(pN −cN) +
pN −(1 −δ)
+ cR(δpN −pR)
. (B56) (B56) The Hessian of Πρ is negative de nite if and only if The Hessian of Πρ is negative de nite if and only if −
2
1 −δv2
1 +
4
1 −δv1v24 +
2
1 −δ −2
δ
v2
2 < 0,
(B57) (B57) for all v1, v2 ̸= 0, which is true since for all v1, v2 ̸= 0, which is true since (v1 −v2)2 + 1 −δ
δ
> 0,
(B58) (B58) for δ ∈[0, 1]. It follows that Πρ is a concave function of pN and pR. Since Πz
4 and Π5 dier from Πρ
by terms linear in pN and pR, they are concave functions of pN and pR by similar argument. for δ ∈[0, 1]. It follows that Πρ is a concave function of pN and pR. Since Πz
4 and Π5 dier from Πρ
by terms linear in pN and pR, they are concave functions of pN and pR by similar argument. Property 8. Πmax concave over domain of R4. Property 8. Πmax concave over domain of R4. Proof of Property 8. It follows directly by the logic similar to that for the proof of Proposition 1
and Property 7. Proof of Property 8. It follows directly by the logic similar to that for the proof of Proposition 1
and Property 7. 12 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research Proof of Proposition 7. : Let Part m be the key part and Part l be the limiting part. Assume that
pN
4 and pR
4 are such that dR
4
ψz ≤λdN
4 ≤
dR
4
ψz+1 where m ≤z ≤min{l, k}, and pN
5 and pR
5 are such that
λdN
5 ≥dR
5
ψl . Referring to Table 3, regions Rz
4 and R5 imply EOL polices Sz
4 and S5 and referring to
Table 1, these policies correspond to pro ts Πz
4, z = 1, . . . Proof of Property 5. We need to consider two cases.
Case I. Substituting the expressions for Πz,L
4
and ΠL
5 into (B66) and expanding and rearranging terms
yield (σz,l + σc
z,l −θ)λdN
4 −(σz,l + σs
z,l −θ)dR
4
ψl
≤(κc
z,l + κs
z,l)dR
4 . (B67) (B67) W.l.o.g. let dR
4 = ψ
′λdN
4
(B68) (B68) ψz+1 (since by assumption dR
1 ≥ψz+1λdN
4 ). Substituting (B68) into (B67) gives (σz,l + σc
z,l −θ)λdN
4 −(σz,l + σs
z,l −θ)ψ
′λdN
4
ψl
≤(κc
z,l + κs
z,l)ψ
′λdN
4 . (B69) (B69) Replacing (σz,l + σs
z,l −θ) ψ
′
ψl with (σz,l + σs
z,l −θ) + (σz,l + σs
z,l −θ)( ψ
′
ψl −1) in (B64) and simplifying
yields Replacing (σz,l + σs
z,l −θ) ψ
′
ψl with (σz,l + σs
z,l −θ) + (σz,l + σs
z,l −θ)( ψ
′
ψl −1) in (B64) and simplifying
yields Replacing (σz,l + σs
z,l −θ) ψ
′
ψl with (σz,l + σs
z,l −θ) + (σz,l + σs
z,l −θ)( ψ
′
ψl −1) in (B64) and simplifying
yields (σc
z,l −σs
z,l) −(κc
z,l + κs
z,l)ψ
′ ≤(σz,l + σs
z,l −θ)(ψ
′
ψl
−1)
(B70) (B70) Since by assumption Part l is limiting and cp
k ≥sk for all k = 1, . . . , K, (σz,l + σc
z,l −θ) ≥
(σz,l + σs
z,l −θ) ≥0 and (σz,l + σc
z,l −θ) −(σz,l + σs
z,l −θ) = Pl
k=z+1(cp
k −sk)ψk. Since ψ
′ ≥ψz+1,
(κc
z,l + κs
z,l)ψ
′ ≥Pl
k=z+1(cp
k −sk)ψk. Finally, since ψ
′
ψl ≥1,(σz,l + σc
z,l −θ) −(σz,l + σs
z,l −θ) ψ
′
ψl ≤
(σz,l + σc
z,l −θ) −(σz,l + σs
z,l −θ). Thus (B70) is true. Since by assumption Part l is limiting and cp
k ≥sk for all k = 1, . . . , K, (σz,l + σc
z,l −θ) ≥
(σz,l + σs
z,l −θ) ≥0 and (σz,l + σc
z,l −θ) −(σz,l + σs
z,l −θ) = Pl
k=z+1(cp
k −sk)ψk. Since ψ
′ ≥ψz+1,
(κc
z,l + κs
z,l)ψ
′ ≥Pl
k=z+1(cp
k −sk)ψk. Finally, since ψ
′
ψl ≥1,(σz,l + σc
z,l −θ) −(σz,l + σs
z,l −θ) ψ
′
ψl ≤
(σz,l + σc
z,l −θ) −(σz,l + σs
z,l −θ). Thus (B70) is true. Proof of Property 5. We need to consider two cases.
Case I. let dR
5 = ψ
′λdN
5
(B63) (B63) where ψ
′ < ψl (since by assumption dR
5 ≤ψlλdN
5 ). Substituting (B63) into (B62) and simplifying
gives where ψ
′ < ψl (since by assumption dR
5 ≤ψlλdN
5 ). Substituting (B63) into (B62) and simplifying
gives (σz,l + σc
z,l −θ) −(σz,l + σs
z,l −θ)ψ
′
ψl
≥(κc
z,l −κs
z,l)ψ
′. (B64) (B64) Replacing (σz,l + σs
z,l −θ) ψ
′
ψl with (σz,l + σs
z,l −θ) −(σz,l + σs
z,l −θ)(1 −ψ
′
ψl ) in (B64) and simplifying
yields (σz,l + σs
z,l −θ)(1 −ψ
′
ψl
) ≥(κc
z,l −κs
z,l)ψ
′ −(σc
z,l −σs
z,l). (B65) (B65) Also, (σz,l + σs
z,l −θ)(1 −ψ
′
ψl ) ≥0 since ψ
′ ≤ψl, and since Part l is limiting, σz,l + σs
z,l −θ > 0. It
follows that the left-hand side of (B65) is positive. Note that (κc
z,l −κs
z,l)ψ
′ = Pl
k=z+1(cp
k −sk)ψ
′
is less than (κc
z,l −κs
z,l) = Pl
k=z+1(cp
k −sk)ψk since by assumption cp
k ≥sk for all k = 1, . . . , K and
ψ
′ ≤ψk for k = z+1, . . . l. It follows that the right-hand side of (B65) is negative. Thus (B65) is true. Also, (σz,l + σs
z,l −θ)(1 −ψ
′
ψl ) ≥0 since ψ
′ ≤ψl, and since Part l is limiting, σz,l + σs
z,l −θ > 0. It
follows that the left-hand side of (B65) is positive. Note that (κc
z,l −κs
z,l)ψ
′ = Pl
k=z+1(cp
k −sk)ψ
′
is less than (κc
z,l −κs
z,l) = Pl
k=z+1(cp
k −sk)ψk since by assumption cp
k ≥sk for all k = 1, . . . , K and
ψ
′ ≤ψk for k = z+1, . . . l. It follows that the right-hand side of (B65) is negative. Thus (B65) is true. 13 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research Now assume that γΠz,L
4 (x4) + (1 −γ)ΠL
5 (x5) ≤ΠL
5 (γx4 + (1 −γ)x5),
(B66) (B66) for all γ such that dR
γ
ψl ≤λdN
γ ≤
dR
γ
ψl+1 . for all γ such that dR
γ
ψl ≤λdN
γ ≤
dR
γ
ψl+1 . Proof of Property 10. The proof of the if part follows by contrapositive argument: If the optimal
solution is not on µm, m = 2, . . . , K, then µm is not a doubly violated constraint. Consider two
adjacent regions Rm and Rm+1 with common boundary µ=
m. By assumption, p∗/∈µ=
m. Let µv be
the set of boundaries violated by pm. There are two cases: (i) µ=
m ∈µv and (ii) µ=
m /∈µv. SteeneckSarinEOLMapping_IJPR_appendix_v3.0 Proof of Property 5. We need to consider two cases.
Case I. Also, by the contrapositive argument of the only if part of
Proposition 9, we know that p∗∈µ≥
m since pm+1 does not violate µ=
m. p
∈µ≥
m since pm violates µm. Also, by the contrapositive argument of the only if part of
Proposition 9, we know that p∗∈µ≥
m since pm+1 does not violate µ=
m. Proposition 9, we know that p∗∈µ≥
m since pm+1 does not violate µ=
m. Property 11. p∗satis es one of the following conditions: Property 11. p∗satis es one of the following conditions: (i) p∗= pm if pm does not violate any boundaries of Rm, (ii) p∗∈µ=
m if µ=
m is a doubly violated constraint, or
(
) ( )
µm
µm
(iii) p∗
∈
hv ∪µv, otherwise, where hv constitute the violated constraints of h=
z , z
=
1, . . . , min{l, K}, and µv are the violated constraints of µ=
z , z = 1, . . . , min{l, K}. (iii) p∗
∈
hv ∪µv, otherwise, where hv constitute the violated constraints of h=
z , z
=
1, . . . , min{l, K}, and µv are the violated constraints of µ=
z , z = 1, . . . , min{l, K}. Proof of Property 11. Case (i) holds clearly since Πmax is a concave function of (pN, pR). Case (ii)
is true by Proposition 10. Fore Case (iii), there are no doubly violated boundaries or unconstrained
optimal solutions that satisfy a region's constraints, and so the solution must be on some violated
constraint. Proof of Property 11. Case (i) holds clearly since Πmax is a concave function of (pN, pR). Case (ii)
is true by Proposition 10. Fore Case (iii), there are no doubly violated boundaries or unconstrained
optimal solutions that satisfy a region's constraints, and so the solution must be on some violated
constraint. Proof of Proposition 8. Follows directly from Propositions 2 and 6 and Properties 9, 10. Proof of Property 5. We need to consider two cases.
Case I. ,
z,l
,
z,l
By the above results and Properties 7 and 8, it follows that Πmax is a concave function over the
region R4 ∪R5. Property 9. Let pm be the unconstrained optimal solution to an optimization problem with feasible
region where (pN, pR) ∈µ≤
m then pm violates µm if and only if p∗∈µ≥
m. Proof of Property 9. The proof of the if part follows by contrapositive argument: If p∗∈µ<
m, then
pm does not violate µm. Since p∗∈µ<
m and Πmax is a concave function over R4 ∪R5 (by Proposition
7), the improving directions for points on µ=
m are towards the region (pN, pR) ∈µ<
m. Thus, pm does
not violate µ=
m. µm
The proof of the only if part follows by contrapositive argument: If pm does not violate µ=
m, then
p∗∈µ<
m. If pm does not violate µ=
m, the improving directions of points on µ=
m for Πmax are toward
points in (pN, pR) ∈µ<
m and since Πmax is concave over R4 ∪R5 we have p∗∈µ<
m. Property 10. µm, m = 2, . . . , K is a doubly violated boundary, if and only if p∗∈µ=
m. Property 10. µm, m = 2, . . . , K is a doubly violated boundary, if and only if p∗∈µ=
m. Proof of Property 10. The proof of the if part follows by contrapositive argument: If the optimal
solution is not on µm, m = 2, . . . , K, then µm is not a doubly violated constraint. Consider two
adjacent regions Rm and Rm+1 with common boundary µ=
m. By assumption, p∗/∈µ=
m. Let µv be
the set of boundaries violated by pm. There are two cases: (i) µ=
m ∈µv and (ii) µ=
m /∈µv. 14 May 2, 2017 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research (i) µ=
m ∈µv
The points on µ=
m are also shared between Rm and Rm+1 and are known to be suboptimal. For these points, by Property 9 we know that moving in the direction of Rm is suboptimal. Thus, improving directions must be in the direction of (pN, pR) ∈µ>
m and so pm+1 will not
violate µ=
m. (i) µ=
m ∈µv
The points on µ=
m are also shared between Rm and Rm+1 and are known to be suboptimal. Proof of Property 5. We need to consider two cases.
Case I. For these points, by Property 9 we know that moving in the direction of Rm is suboptimal. Thus, improving directions must be in the direction of (pN, pR) ∈µ>
m and so pm+1 will not
violate µ=
m. (ii) µm /∈µv
In this case, µ=
m is not violated by pm and thus, cannot be violated twice. ( ) µ
/ µ
In this case, µ=
m is not violated by pm and thus, cannot be violated twice. The proof of the only if part follows by contrapositive argument: If µm is not doubly violated,
then p∗/∈µ=
m. There are two cases: (i) µ=
m is not violated, and (ii) µ=
m is violated once. The proof of the only if part follows by contrapositive argument: If µm is not doubly violated,
then p∗/∈µ=
m. There are two cases: (i) µ=
m is not violated, and (ii) µ=
m is violated once. (i.) µ=
m is not violated
In this case the contrapositive argument of the if part of Property 9 applies. (i.) µm is not violated
In this case the contrapositive argument of the if part of Property 9 applies. (ii ) µ= is violated only once (ii.) µ=
m is violated only once
Let pm violate µ=
m, but pm+1 does not violate µ=
m. By the if part of Property 9, we know
p∗∈µ≥
m since pm violates µ=
m. Also, by the contrapositive argument of the only if part of
Proposition 9, we know that p∗∈µ≥
m since pm+1 does not violate µ=
m. (
) µm
y
Let pm violate µ=
m, but pm+1 does not violate µ=
m. By the if part of Property 9, we know
p∗∈µ≥
m since pm violates µ=
m. Also, by the contrapositive argument of the only if part of
Proposition 9 we know that p∗∈µ≥since pm+1 does not violate µ= pm violate µ=
m, but pm+1 does not violate µ=
m. By the if part of Property 9, we know
∈µ≥
m since pm violates µ=
m. Also, by the contrapositive argument of the only if part of Let p
violate µm, but p
does not violate µm. By the if part of Property 9, we know
p∗∈µ≥
m since pm violates µ=
m. SteeneckSarinEOLMapping_IJPR_appendix_v3.0 Appendix C. Additional tables and gures Table C1. Expressions required for the relationships in Table 2. Relationships
Expression
ρR = κ2
ρR = s1 + s2
ρR = κ1
ρR = s1 + cp
2
Π
′
0,1 = 0
ρRψ1 −cp
2(ψ1 −ψ2) = θ
Π
′
0,2 = 0
ρRψ2 + s1(ψ1 −ψ2) = θ
Π
′
1,2 = 0
s1ψ1 + cp
2ψ2 = θ
Π
′
2,3 = 0
s1ψ1 + s2ψ2 = θ Table C1. Expressions required for the relationships in Table 2. Relationships
Expression
ρR = κ2
ρR = s1 + s2
ρR = κ1
ρR = s1 + cp
2
Π
′
0,1 = 0
ρRψ1 −cp
2(ψ1 −ψ2) = θ
Π
′
0,2 = 0
ρRψ2 + s1(ψ1 −ψ2) = θ
Π
′
1,2 = 0
s1ψ1 + cp
2ψ2 = θ
Π
′
2,3 = 0
s1ψ1 + s2ψ2 = θ 15 15 International Journal of Production Research International Journal of Production Research SteeneckSarinEOLMapping_IJPR_appendix_v3.0 Table C2. Unconstrained optimal solutions for various special cases of EOL-CCPP-R
′
Problem
Unconstrained Optimal (pN ∗, pR∗)
EOL-CCPP-Rz
4
1
2
cN + 1 −(σz −θ)λ
, 1
2
cR + δ + ρR
z
EOL-CCPP-R5
1
2(cN + 1), 1
2
(cR+κl+δ)ψl−(σz−θ)
ψl
EOL-CCPP-R4-µ=
z , z = 1, . . . , K
1
2
(1+δλψz)((cR+ρR)λψz−(σz−θ)λ+cN)+(1+δλψz(2−(δ−2)λψz)
1+δλψz(2+λψz)
,
1
2
δ((1+λψz)(cN−(σz−θ)λ+(pR+cR)λψz)+(1+(δλψz−1+3δ)λψz)
1+δλψz(2+λψz)
EOL-CCPP-R4-µ=
K+1
1
2
cN + 1 −(σK −θ)λ
, 1
2δ(cN + 1 −(σK −θ)λ
EOL-CCPP-R5-µ=
K+1
1
2
cN + 1
, 1
2δ
cN + 1
EOL-CCPP-R4-h=
z , z = 1, . . . , K
1
2(cN + κz + cR −(σz −θ)λ + 1 −δ), cR + κz
EOL-CCPP-R5-h=
K
1
2
(cN+cR+κl+1−δ)ψl−(σK−θ)
ψl
, cR + κK
16 May 2, 2017 International Journal of Production Research List of Figures 1 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research SteeneckSarinEOLMapping_IJPR_appendix_v3.0 Figure C1. Example Πmax and ΠEOL. Figure C2. Πupdated
max
for example in Figure C1. Figure C1. Example Πmax and ΠEOL. Figure C2. Πupdated
max
for example in Figure C1. Figure C1. Example Πmax and ΠEOL. Figure C2. Πupdated
max
for example in Figure C1. Figure C2. Πupdated
max
for example in Figure C1. Figure C1. Example Πmax and ΠEOL. 2 2 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research SteeneckSarinEOLMapping_IJPR_appendix_v3.0 Figure C3. Appendix C. Additional tables and gures Case I of Property 2: Π for which Π
′
0,z ≤
Π
′
z−1,z. Figure C4. Case II of Property 2: Π for which Π
′
0,z ≥
Π
′
z,z+1. Figure C3. Case I of Property 2: Π for which Π
′
0,z ≤
Π
′
z−1,z. Figure C4. Case II of Property 2: Π for which Π
′
0,z ≥
Π
′
z,z+1. 3 3 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research Figure C5. Πmax
for which Π
′
m,m+1
≤
Π
′
0,m
≤
Π
′
m−1,m. Figure C6. Πmax for which Π
′
K−1,K ≥Π
′
0,K and thus
Π
′
S ≥Π
′
0,K. Figure C5. Πmax
for which Π
′
m,m+1
≤
Π
′
0,m
≤
Π
′
m−1,m. Figure C6. Πmax for which Π
′
K−1,K ≥Π
′
0,K and thus
Π
′
S ≥Π
′
0,K. Figure C6. Πmax for which Π
′
K−1,K ≥Π
′
0,K and thus
Π
′
S ≥Π
′
0,K. 4 4 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 May 2, 2017
International Journal of Production Research SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research Figure C7. Two-stage model. Figure C7. Two-stage model. Figure C7. Two-stage model. 5 5 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research Figure C8. System description for EOL options requiring disassembly (remanufacturing and part salvage/disposal). Figure C8. System description for EOL options requiring disassembly (remanufacturing and part salvage/disposal 6 6 May 2, 2017
International Journal of Production Research SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research Figure C9. Structures of the optimal EOL policies for disassembly and their respective shape of Πmax Figure C9. Structures of the optimal EOL policies for disassembly and their respective shape of Πmax 7 7 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research Figure C10. Strategies for No Collec-
tion and Salvage EOL policy types. Figure C11. Strategies for Remanufacturing EOL policy type with
various core availabilities, A. Figure C10. Strategies for No Collec-
tion and Salvage EOL policy types. gure C10. Strategies for No Collec-
n and Salvage EOL policy types. Figure C11. Strategies for Remanufacturing EOL policy type with
various core availabilities, A. Figure C11. Strategies for Remanufacturing EOL policy type with
various core availabilities, A. 8 8 International Journal of Production Research SteeneckSarinEOLMapping_IJPR_appendix_v3.0 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 Figure C12. Policies for Remanufacturing and Salvage EOL policy type with various core availabilities, A. Figure C12. Policies for Remanufacturing and Salvage EOL policy type with various core availabilities, A. Appendix C. Additional tables and gures 9 9 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research SteeneckSarinEOLMapping_IJPR_appendix_v3.0 Figure C13. EOL policy type with variation in ρR and θ for a 2-part product. Figure C13. EOL policy type with variation in ρR and θ for a 2-part product. 10 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 SteeneckSarinEOLMapping_IJPR_appendix_v3.0 International Journal of Production Research Figure C14. EOL policy type with variation in ψ1 and ψ2
for a 2-part product with Part 1 key. Figure C15. EOL policy type with variation in ψ1 and ψ2
for a 2-part product with Part 2 key. Figure C14. EOL policy type with variation in ψ1 and ψ2
for a 2-part product with Part 1 key. Figure C15. EOL policy type with variation in ψ1 and ψ2
for a 2-part product with Part 2 key. 11 11
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Long-term exposure to heavy physical work, disability pension due to musculoskeletal disorders and all-cause mortality: 20-year follow-up—introducing Helsinki Health Study job exposure matrix
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Abstract Purpose We developed a job exposure matrix (JEM) to study the association between long-term exposure to heavy physical
effort or heavy lifting and carrying at work with disability pension due to musculoskeletal disorders and premature all-cause
mortality.f Methods Exposure to heavy physical effort at work during 1996–2005 was estimated with JEM developed for this study
population, where the exposure was based on occupational titles of the participants. We included all employees of the City
of Helsinki, Finland, who had annual data of exposure for 8–10 years (1996–2005, n = 18387). The outcome variables were
register-based, and the follow-up was from 2006 until 2015. The risk estimates were evaluated using competing risk survival
analysis. Results There were 530 (3%) disability pension events due to musculoskeletal disorders during the 10-year follow-up. After
adjustment for sex, age, education and chronic diseases, employees in the second (SHR = 1.46, 95% CI 1.05–2.05), third
(SHR = 2.73, 95% CI 2.00–2.29), and the highest exposure quartile (SHR = 2.56, 95% CI 1.88–3.50) had a higher risk of
musculoskeletal disability pension than employees in the lowest quartile. A total of 110 (4%) men and 266 (2%) women died
during the follow-up. Men in the third quartile (SHR = 2.29, 95% CI 1.23–4.24), and women in the highest exposure quartile
(SHR = 1.54, 95% CI 0.99–2.41) had a higher risk of premature mortality than those in the lowest quartile. Conclusions Eight to ten years of exposure to heavy physical effort at work is strongly associated with disability pension Conclusions Eight to ten years of exposure to heavy physical effort at work is strongly associated with disability pension
due to musculoskeletal disorders. This exposure also increases the risk of premature mortality, particularly among men. Keywords Disability pension · Job exposure matrix · Mortality · Physical job demands Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00420-018-1393-5) contains
supplementary material, which is available to authorized users. International Archives of Occupational and Environmental Health (2019) 92:337–345
https://doi.org/10.1007/s00420-018-1393-5 International Archives of Occupational and Environmental Health (2019) 92:337–345
https://doi.org/10.1007/s00420-018-1393-5 International Archives of Occupational and Environmental Health (2019) 92:337–345
https://doi.org/10.1007/s00420-018-1393-5 International Archives of Occupational and Environmental Health (2019) 92:337–345
https://doi.org/10.1007/s00420-018-1393-5 ORIGINAL ARTICLE ORIGINAL ARTICLE Jenni Ervasti1,2 · Olli Pietiläinen1 · Ossi Rahkonen1 · Eero Lahelma1 · Anne Kouvonen3,4,5 · Tea Lallukka1,2 ·
Minna Mänty1,6 Received: 25 June 2018 / Accepted: 27 November 2018 / Published online: 3 December 2018
© The Author(s) 2018 * Jenni Ervasti
jenni.ervasti@ttl.fi Introduction A recent study with Danish reg-
ister cohort data found that lifting-years, but not kneeling- or
vibration-years, were associated with an increased risk of
all-cause disability pension (Sundstrup et al. 2017). How-
ever, the study did not separately examine musculoskeletal
disorder-related disability pensions. Exposure information In the HHS survey, the respond-
ents reported whether heavy physical effort or heavy lift-
ing and carrying were present in their work. The scale
was: 0 (does not occur); 1 (occurs, but does not bother);
2 (occurs, and bothers to a moderate degree); 3 (occurs
and bothers to a large degree). Value 0 was categorized as
“unexposed”, and all the other values as “exposed”. The
JEM estimate was calculated as the prevalence of exposure
(as percentage) in each occupational title.i Some of the studies on the association between physi-
cal workload and disability pension have used exposure
estimates derived from job exposure matrices (JEM) that
evaluate job exposures based on occupational titles. Each
occupation receives and exposure estimate based on survey
responses, face-to-face interviews, or on expert evaluations
(Kjellberg et al. 2016; Solovieva et al. 2012; Sundstrup
et al. 2017). Particularly for physical work exposures, these
matrices have shown rather high specificity and sensitiv-
ity, and validation studies support their use when individ-
ual exposures are unavailable (Dale et al. 2015; Rijs et al. 2014; Solovieva et al. 2012). However, the exposure values
(which originally are percentages, i.e., continuous variable)
are often dichotomized or otherwise grouped. This deci-
sion dilutes variation, and information is lost. Hence, in this
study, we kept the annual job exposure estimates as per-
centages, and calculated the average exposure during the
exposure follow-up. Occupational classification Occupational titles based
on employer’s register data were transformed to match
occupational titles based on coding of Statistics Finland
(Tilastokeskus 1999). The coding of occupations varies
from one to three or four digits. More digits indicate finer
classification: code ‘0’ groups all technical, scientific,
legal, humanistic, and artistic work; ‘00’ groups all tech-
nical work; ‘001’ groups all architects. We could match
an occupational title for 6789 baseline survey respond-
ents. A total of 132 different occupational titles were found
(3-digit classification). As there were 218 (3-digit) occupa-
tional titles in the codebook of Statistics Finland, our HHS
survey respondents represented 61% of all occupational
titles in municipal work. Introduction Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00420-018-1393-5) contains
supplementary material, which is available to authorized users. * Jenni Ervasti
jenni.ervasti@ttl.fi
1
Department of Public Health, University of Helsinki,
Helsinki, Finland
2
Finnish Institute of Occupational Health, PB 18,
00032 Helsinki, Finland
3
Faculty of Social Sciences, University of Helsinki, Helsinki,
Finland
4
SWPS University of Social Sciences and Humanities
in Wroclaw, Wroclaw, Poland
5
Administrative Data Research Centre‑Northern Ireland
(ADRC‑NI), Queen’s University Belfast, Belfast, UK
6
Laurea University of Applied Sciences, Vantaa, Finland Physically demanding work has been widely shown to be
a risk factor for disability pension (Karpansalo et al. 2002;
Krokstad et al. 2002; Labriola et al. 2009; Lahelma et al. 2012), and is linked specifically to a higher risk of disability
pension due to musculoskeletal disorders (Karkkainen et al. 2013; Lahelma et al. 2012). However, less is known about
how long-time continuous exposure to physically demanding
work affects the risk of disability pension. Two studies, one
among Finnish twins (Ropponen et al. 2014), and another
among two Swedish birth cohorts (Kjellberg et al. 2016),
showed that long-term exposure to physically demanding
work was associated with a higher risk of disability pension
due to musculoskeletal disorders. However, the measure of
long-term exposure was not based on annual follow-up of * Jenni Ervasti
jenni.ervasti@ttl.fi 1
Department of Public Health, University of Helsinki,
Helsinki, Finland 2
Finnish Institute of Occupational Health, PB 18,
00032 Helsinki, Finland 3
Faculty of Social Sciences, University of Helsinki, Helsinki,
Finland 4
SWPS University of Social Sciences and Humanities
in Wroclaw, Wroclaw, Poland 5
Administrative Data Research Centre‑Northern Ireland
(ADRC‑NI), Queen’s University Belfast, Belfast, UK 6
Laurea University of Applied Sciences, Vantaa, Finland (0121 International Archives of Occupational and Environmental Health (2019) 92:337–345 338 baseline data were derived from questionnaire surveys
conducted in 2000–2002 including employees reaching
the age of 40, 45, 50, 55 or 60 years each year. In total,
8960 employees (80% women) responded at baseline with
the response rate of 67%. The HHS protocol has been
approved by ethics committees of the health authorities of
the City of Helsinki and the Department of Public Health,
University of Helsinki, Finland (Lahelma et al. 2013). exposure, but on two separate measurement points 5–6 years
apart without information on potential changes in exposure
between the measurements. Introduction In addition to disability pension, heavy physical work
may increase the risk of premature death, i.e., death dur-
ing working age, particularly among men (Holtermann et al. 2009, 2010a, 2012). Thus, death should be viewed as com-
peting risk when evaluating the extent to which heavy physi-
cal work increases the risk of disability pension. The aim
of this study was to develop a new JEM based on the Hel-
sinki Health Study (HHS) survey data to assess long-term
exposures to physically demanding work. The HHS–JEM
was then used to build a prognostic model to estimate the
sub-distribution hazards of disability pension due to mus-
culoskeletal disorders and death among people with differ-
ent exposures to physically heavy work (Austin and Fine
2017; Putter et al. 2007). We used a competing risk model
to examine whether long-term exposure to heavy physical
effort estimated with JEM was associated with disability
pension due to musculoskeletal disorders and all-cause pre-
mature mortality. The HHS–JEM The JEM estimates were calculated sep-
arately for men (n = 1381) and women (n = 5378). Small
occupational groups were merged into larger (2 digit) clas-
sifications, if two experts (JE and MM) agreed in favor of
merging the occupations. Before occupational titles were
merged, we checked that the JEM estimates were similar
enough for the two groups. We omitted 66 (50%) occu-
pational titles with few respondents and which could not
be merged due to differential work tasks and occupational
exposure. In the final HHS–JEM, there were 40 occupational
titles (13 with 2-digit classification, and 27 with 3-digit clas-
sification), which covered 30% of all occupational titles in
municipal work. When considering only those occupations
that HHS survey respondent held, the HHS–JEM covered
50% of occupations. Men had JEM estimates for 21 occu-
pational titles, which were based on responses of 1168 men
(65% of all male respondents). Women had JEM values
for 36 occupational titles, which were based on responses
of 5189 women (72% of all female respondents). In total,
the HHS–JEM covered 71% of all survey respondents in
2000–2002. The minimum number of respondents for whom
the JEM estimates were calculated was 18 for men, and 24 1 3 Study population The study population were all employees of the City of Hel-
sinki between 1996 and 2005 (n = 118,122). For this cohort,
we had employer’s register data, including employees’ occu-
pational titles. We linked the HHS–JEM data for the City
of Helsinki employee cohort based on their occupational
titles. Using national personal identification numbers, we
were also able to link register data from the Finnish Centre
for Pensions covering all granted pensions based on disabil-
ity including ICD-10 coded diagnoses (International Statisti-
cal Classification of Diseases and Related Health Problems
(ICD-10) 1994), as well as old age pensions. In addition,
we had information on age, sex, education, and mortality
for all causes from the Statistics Finland. Information on
prescription medication purchases and special medication
reimbursements were from the Social Insurance Institution
of Finland. Information regarding notifications of diagnosed
malignant tumors were from the Institute of Statistical and
Epidemiological Cancer Research (the Finnish Cancer
Registry). The linkage of the JEM estimate (at least for one
out of 10 years) succeeded for a total of 98,834 employees. From this cohort, the eligible population were those who
were alive and not on disability or old age pension before
1st January 2006 (n = 87,130). We omitted employees with
less than 8 years of JEM estimates, and those without infor-
mation on covariates resulting in 18,387 employees for our
final analytical sample. Of the employees 84% were women
reflecting the sex distribution in the municipal sector. The
study design is further described in Fig. 1. Exposure We followed occupational exposure to heavy physical
effort for 10 years, from January 1, 1996 until December
31, 2005. Those with less than 8 observation-years during
the exposure follow-up were excluded. The annual exposure
was computed into the mean exposure during the exposure
follow-up time (percentage). For most participants (64%),
their occupational title did not change (i.e., the exposure was
on the same level throughout the exposure follow-up). For
36%, there were changes in exposure during the follow-up. The mean exposure variable was classified into quartiles as
follows: 0–25.9%, 26–64.9%, 65–82.6%, ≥ 82.7%. In sensi-
tivity analyses, we used continuous exposure variable. Compilation of the job exposure matrix (HHS–JEM) JEM population We used data from the Helsinki Health
Study (HHS), which focuses on health and working condi-
tions of employees of the City of Helsinki, Finland. The 1 3 1 3 339 International Archives of Occupational and Environmental Health (2019) 92:337–345 for women. The occupational titles and number of respond-
ents are detailed in Online Resource 1. Outcomes In Finland, a person who is unable to work is eligible to
receive a sickness absence benefit for a maximum of 1 year. After that, if the person’s work ability remains reduced at
least by 60% (remains unable to work), a full disability pen-
sion can be granted (either for a fixed period or as a perma-
nent disability pension). The decision on pension is based on
physician certificate and a medical diagnosis and is made by
an insurance institution. If part-time work is possible, part-
time disability pension can be granted when work ability is
reduced at least by 40%. We obtained register-based infor-
mation on the dates of granted disability pensions from the
Finnish Centre for Pensions (data on temporary, permanent,
full-time and part-time disability pensions) coded accord-
ing to the ICD-10 (International Statistical Classification
of Diseases and Related Health Problems (ICD-10) 1994). We examined disability pension due to musculoskeletal dis-
orders (M00-M99), which cover about 30% of all granted Fig. 1 Study design, variables and follow-up. HHS–JEM = job exposure matrix based on the Helsinki Health Study International Archives of Occupational and Environmental Health (2019) 92:337–345 340 95% confidence intervals (CI). The follow-up was until death
or the end of the follow-up (December 31, 2015), whichever
came first. pensions (Statistical yearbook of pensioners in Finland 2016
2017). Information on mortality (date of death) was retrieved
from Statistics Finland register of causes of death. The out-
come follow-up was 10 years, from January 1, 2006 until
December 31, 2015. We defined death and disability pension
due to other than musculoskeletal diagnosis as competing
event to disability pension due to musculoskeletal disorders. However, in the mortality analyses, disability pension was
not regarded as competing event to death. i
For disability pension due to musculoskeletal disorders,
we used Cox proportional hazards model, but with death and
disability pensions due to other than M00–M99 diagnoses
were treated as competing risks. Cases were censored in the
event of old-age pension, or when reaching the age of 63
after which disability pension can no longer be granted, or at
the end of the follow-up. Compared to the standard survival
analysis, where the follow-up of non-events terminates only
due to censoring, competing risk analysis considers compet-
ing events that prevent the event of interest from occurring. Outcomes Treating observations that experience competing events as
if they could later experience the event of interest overesti-
mates the probability of failure, and the bias is larger when
the competition due to frequent competing events is heavier
(Putter et al. 2007). Statistical analysis For general description of the data, we used frequency
tables, means and standard deviations. We tested effect mod-
ification by including interaction term ‘sex × exposure’ into
Cox proportional hazards models (time to disability pension
and time to death). As we observed statistically significant
(p < 0.001) interaction regarding mortality, those analyses
were stratified by sex. Results Of the participants (n = 18,387), 84%, were women. The
characteristics of the participants at the beginning of the
outcome follow-up period are described in Table 1 strati-
fied by sex. Men were slightly older and had higher level
of education than women. No difference was observed for
chronic disease prevalence between sexes. Women had more
exposure to heavy physical effort or lifting and carrying than
men. Covariates Age and educational level were obtained from Statistics Fin-
land. Educational level was classified into 0 (secondary edu-
cation or less); 1 (tertiary education, undergraduate); 2 (ter-
tiary education, graduate/doctoral degree). Chronic somatic
conditions were: cancer (diagnosed during 2003–2005 from
the Cancer Registry), and diabetes, cardiac failure, coronary
artery disease, stage 2 hypertension, rheumatoid arthritis,
asthma, Parkinson’s disease, epilepsy, uremia, bowel dis-
ease, multiple sclerosis, and diseases of pancreas as defined
through special medication reimbursement valid at the start
of the outcome follow-up on 1st January 2006. In addition,
we defined mental disorders from medication purchases with
the Anatomical Therapeutic Chemical Classification (ATC)
codes N05 (psycholeptics) and N06 (psychoanaleptics) dur-
ing 2003–2005. The presence of chronic disease was defined
as having at least one of these proxies for somatic or mental
condition. We tested whether the association was linear or non-linear
by including each exposure squared in addition to exposure
treated as a linear term. The results mainly supported non-
linear association. Thus, we show the results with classified
exposure variable (supporting non-linear association) as
main results, and as continuous variable (supporting linear
association) in sensitivity analysis (Online Resource 2). 3 Disability pension due to musculoskeletal disorders Figure 2 shows Kaplan–Meier curves for time to disabil-
ity pension due to musculoskeletal disorders stratified by
the average exposure to heavy physical effort. A total of
5–7% of employees with above median (3rd and 4th quartile)
exposure to heavy physical work ended up on musculoskel-
etal disorder-related disability pension during the 10-year
follow-up, whereas the corresponding percentage for those
with the lowest level of exposure was 2%. i
We used Kaplan–Meier estimator method to estimate
survival functions to compare time to disability pension
and death for the exposed and the unexposed, and to visu-
ally evaluate the assumption of proportional hazards. We
tested the proportional hazards assumption by including an
interaction of the exposure with the log of follow-up time. The interaction terms were non-significant (p values > 0.05)
justifying the proportional hazards assumption. The mean follow-up time for disability pension due to
musculoskeletal disorders was 8.5 years (SD 2.7). There
were 530 (3%) musculoskeletal disability pension events
during the follow-up. Compared to employees in the low-
est quartile of average level of exposure to heavy physical We used Cox proportional hazards models to examine the
association between exposure to heavy physical work and
incident mortality. The interaction test indicated effect modi-
fication by sex, and the analyses were stratified by sex. The
results were presented as subhazard ratios (SHR) and their 3 341 International Archives of Occupational and Environmental Health (2019) 92:337–345 effort, employees in all other exposure quartiles had a higher
risk of disability pension due to musculoskeletal disorders
(Table 2). The analysis with the linear exposure variable
showed a 1.15 (95% CI 1.10–1.20) times higher risk of mus-
culoskeletal disability pension for a 10%-unit increase in average exposure to heavy physical effort (Online Resource
2.)i 1 3f
risk of disability pension due to musculoskeletal disorders
(Table 2). The analysis with the linear exposure variable
showed a 1.15 (95% CI 1.10–1.20) times higher risk of mus-
culoskeletal disability pension for a 10%-unit increase in f
2.)
As a supplementary analysis, we further stratified the
outcome by sub-blocks of musculoskeletal diagnoses
(Online Resources 3 and 4). Fig. 2 Cumulative probabil-
ity of disability pension due
to musculoskeletal disorders
stratified by the average level
of 8–10 years of continuous
exposure to heavy physical
effort at work Disability pension due to musculoskeletal disorders Above median (3rd and 4th
Table 1 Characteristics of study
participants by sex (n = 18,387)
Men (n = 2870)
Women (n = 15,517)
%
Mean
SD
%
Mean
SD
Secondary education or lower
30
43
Tertiary education/undergraduate
35
37
Tertiary education/graduate
35
20
Chronic disease (at the beginning of the follow-up)
24
25
Age (at the beginning of the follow-up)
48.3
8.3
47.0
8.5
Exposure to heavy physical work/lifting and carrying
(during 1996–2005)
39.7
27.8
54.9
26.8
Fig. 2 Cumulative probabil-
ity of disability pension due
to musculoskeletal disorders
stratified by the average level
of 8–10 years of continuous
exposure to heavy physical
effort at work
Table 2 8–10 years of exposure
to heavy physical effort at
work and status at the end of
follow-up (n = 18,387)
a Adjusted for age, educational level, chronic disease and sex
b The interaction with sex was statistically significant (p < 0.001), and the analyses were stratified by sex
and adjusted for age, educational level, and chronic diseases
c 530 events of interest, 816 competing events, 17,034 censored
Disability pension due to
musculoskeletal disordera
Deatha,b
530 events (3%)
Men, 110 events (4%)
Women, 266 (2%)
SHRc
95% CI
SHR
95% CI
SHR
95% CI
Lowest quartile
1
1
1
2nd quartile
1.46
1.05–2.05
0.87
0.52–1.46
1.24
0.87–1.75
3rd quartile
2.73
2.00-3.72
2.29
1.23–4.24
1.16
0.79–1.71
Highest quartile
2.56
1.88–3.50
1.70
0.90–3.20
1.54
0.99–2.41 As a supplementary analysis, we further stratified the
outcome by sub-blocks of musculoskeletal diagnoses
(Online Resources 3 and 4). Above median (3rd and 4th Table 1 Characteristics of study
participants by sex (n = 18,387) mulative probabil-
bility pension due
oskeletal disorders i
by the average level
ears of continuous
o heavy physical
ork Fig. 2 Cumulative probabil-
ity of disability pension due
to musculoskeletal disorders
stratified by the average level
of 8–10 years of continuous
exposure to heavy physical
effort at work Table 2 8–10 years of exposure
to heavy physical effort at
work and status at the end of
follow-up (n = 18,387) Table 2 8–10 years of exposure
to heavy physical effort at
work and status at the end of
follow-up (n = 18,387) b The interaction with sex was statistically significant (p < 0.001), and the analyses were stratified by sex
and adjusted for age, educational level, and chronic diseases 1 3 3 International Archives of Occupational and Environmental Health (2019) 92:337–345 342 Risk of premature death quartile) exposure to heavy physical effort showed approx-
imately threefold risk for arthropathies, specifically for
arthrosis (four–fivefold risk). The risks for dorsopathies
and soft tissue disorders were also significantly elevated
for those with above median exposure (Online Resource
4). Figure 3 (Panel a for women and panel b for men) shows
Kaplan–Meier survival curves stratified by average expo-
sure to heavy physical effort. Among women, no difference
in premature mortality was observed by exposure to heavy
physical effort. Around 1.6–2.3% of women died during the 3 Cumulative probability of premature mortality stratified by the average level of 8–10 years of continuous exposure to heavy phyf
t at work among a women and b men Fig. 3 Cumulative probability of premature mortality stratified by the average level of 8–10 years of continuous exposure to heavy physical
effort at work among a women and b men 1 3 International Archives of Occupational and Environmental Health (2019) 92:337–345 343 10-year follow-up. Among men, a higher overall death rate
was observed. A total of 5–8% of men with above median
(3rd and 4th quartile) exposure to heavy physical work died
during the 10-year follow-up, whereas the corresponding
percentage with below median exposure was from 2 to 4%. studies. In this study, we could first follow the exposure for
10 years, and then follow the outcome for 10 more years. The risk for musculoskeletal disability pension was strongly
increased with 8–10 years of continuous exposure to physi-
cally demanding work. Moreover, we examined the relationship between
8–10 years of exposure to heavy physical effort and prema-
ture mortality in working age. Congruent to previous studies
(Holtermann et al. 2009, 2010a, b, 2012), men with heavy
physical work had an elevated risk of premature death. The
association between exposure to heavy physical effort and
mortality was significant among those with second highest
(third quartile) exposure, but not among those with the high-
est exposure. This may imply health selection, where only
very healthy men in physically demanding occupations con-
tinue to work for 8–10 years. We expanded earlier research
as we also studied this association among women, and found
a significant association in the highest exposure quartile. The mean follow-up time for premature death was
9.9 years (SD 0.7). There were 266 (2%) deaths among
women and 110 (4%) among men during the 10-year follow-
up. Discussion We compiled JEM estimates for various municipal sector
occupations regarding exposure to heavy physical effort
or heavy lifting and carrying. As a primary validation of
the JEM, the associations with disability pension due to
musculoskeletal disorders were examined. Confirming our
hypothesis, the study demonstrated an association between
8–10 years of continuous exposure to heavy physical effort
and disability pension due to musculoskeletal disorders. In
addition, we observed an increased risk of premature death
particularly among working age men who had 8–10 years
of exposure to heavy physical effort or lifting and carrying. Our results correspond to previous studies, which sup-
ports the validity of the municipal sector JEM (Karkkainen
et al. 2013; Karpansalo et al. 2002; Kjellberg et al. 2016;
Krokstad et al. 2002; Labriola et al. 2009; Lahelma et al. 2012; Ropponen et al. 2014; Sundstrup et al. 2017). How-
ever, we also add to the existing literature, because we meas-
ured the physical exposures continuously during 8–10 years
prior to the outcome follow-up period. The duration of the
exposure (i.e., how long participants have performed physi-
cally demanding tasks) has not been examined in previous We compiled JEM estimates for various municipal sector
occupations regarding exposure to heavy physical effort
or heavy lifting and carrying. As a primary validation of
the JEM, the associations with disability pension due to
musculoskeletal disorders were examined. Confirming our
hypothesis, the study demonstrated an association between
8–10 years of continuous exposure to heavy physical effort
and disability pension due to musculoskeletal disorders. In
addition, we observed an increased risk of premature death
particularly among working age men who had 8–10 years
of exposure to heavy physical effort or lifting and carrying. f
Our results correspond to previous studies, which sup-
ports the validity of the municipal sector JEM (Karkkainen
et al. 2013; Karpansalo et al. 2002; Kjellberg et al. 2016;
Krokstad et al. 2002; Labriola et al. 2009; Lahelma et al. 2012; Ropponen et al. 2014; Sundstrup et al. 2017). How-
ever, we also add to the existing literature, because we meas-
ured the physical exposures continuously during 8–10 years
prior to the outcome follow-up period. The duration of the
exposure (i.e., how long participants have performed physi-
cally demanding tasks) has not been examined in previous In most previous studies, the analysis of the risk of
disability pension has not considered competing events. Strengths and weaknesses A major strength of the current study was the 20-year time
frame, where we first followed the exposure for 10 years, and
then the outcome for another 10 years. JEM was based on
physical work exposure during 2000–2002. We generalized
the occupational exposures of 2000–2002 to 4 years before
and 3 years after (i.e., to 1996–2005) assuming that no major
changes in work and working conditions affecting physical
exposures in these occupations occurred during that time. Moreover, we kept the JEM estimates as continuous percent-
age variables (0–100%), and calculated the mean level of
exposure during 8–10 years for each participant. With this
approach, we could ascertain the duration of exposure, in
addition to include information about the variation in inten-
sity of exposure, should there have been a change in occupa-
tion and subsequent level of physical exposure. However, to
obtain a measure of long-term continuous exposure, we had
to omit all participants with less than 8 years of information
on occupation and subsequent occupational exposures. This
may have resulted in selection of fitter and healthier work-
ers who remained employed with the City of Helsinki (i.e.,
did not resign, die, or end up in disability pension until 31
December 2005), which may have led to underestimation
of the effect of exposure to physically demanding work on
disability pension and mortality. As our exposure estimate
was based on occupational title, we were unable to account
for work modification to less physically demanding within
the same occupational title. Nevertheless, a change into less
strenuous occupational title during the 10-year exposure
follow-up was accounted for. f
Among women, the highest exposure (> 82%) to heavy
physical effort was marginally associated with higher risk
of premature mortality (SHR = 1.54, 95% CI 0.99–2.41)
(Table 2). The SHR was 1.03 (0.98–1.09) for 10%-unit
increase in average exposure in the analysis with the linear
exposure variable in women (Online Resource 2). Risk of premature death Among men, the second highest exposure (65–82%) to
heavy physical effort was associated with a higher risk of
premature mortality (SHR = 2.29, 95% CI 1.24–4.26) com-
pared to employees in the lowest quartile of average level of
exposure (Table 2). The higher risk of premature mortality
among men was also observed when using dichotomized
exposure variable (median split): the hazard ratio was 2.11
(95% CI 1.27–3.49) among men who had above median
exposure to heavy physical effort compared to those with
below median level of exposure (data not shown in Tables). An analysis with linear exposure variable showed a 1.13
(95% CI 1.04–1.22) times higher risk of premature mortality
for a 10%-unit increase in average exposure to heavy physi-
cal effort (Online Resource 2). Conclusion Eight to ten years of continuous exposure to heavy physical
effort or heavy lifting and carrying at work was associated
with an increased risk of disability pension due to muscu-
loskeletal disorders. Furthermore, continuous exposure to
heavy physical effort or lifting and carrying was associated
with premature mortality, particularly among men. In addi-
tion to physically demanding work causing musculoskel-
etal disorders and injuries, these jobs may also have lower
possibilities to adjust work to accommodate reduced health
and functioning. Effective ways to accommodate physically
demanding work environment and work tasks to meet the
reduced physical functioning are therefore needed. yl
pp
p
We considered confounding through sex, age, education,
and chronic diseases, which were available from registers. We did not have information on health behaviors, including
physical fitness, body mass index, alcohol use, and smok-
ing. In an earlier study, the effect of physical risk factors
on disability pension was robust to adjustment for health
behaviors (Karpansalo et al. 2002). In some previous studies,
high levels of physical fitness have been observed to protect
from the adverse effects high physical work demands on pre-
mature mortality (Holtermann et al. 2010a, 2012). However,
it is very difficult to disentangle the effects of heavy physi-
cal effort at work from other risk factors that also pertain
among men doing heavy manual work, for example, high
blood pressure and smoking. Exposure to heavy physical
work may include other hazardous exposures as well (for
example, exposure to asbestos) or built-in socially struc-
tured inequality in health. This kind of mixed exposure to
physical, ergonomic, or psychosocial factors can produce
health consequences that are additive or synergistic (Mixed
Exposures Research Agenda. A Report by the NORA Mixed
Exposures Team 2004), and may explain the observed
increased risk of mortality. Acknowledgements Open access funding provided by University of
Helsinki including Helsinki University Central Hospital. Funding JE and MM are supported by the Finnish Work Environment
Fund (#115182), MM additionally by Juho Vainio foundation. AK is
supported by the Economic and Social Research Council (ESRC, #ES/
L007509/1). OR is supported by the Academy of Finland (#1294514)
and the Juho Vainio Foundation. TL is supported by the Academy of
Finland (#287488, #294096) and the Finnish Work Environment Fund
(#117308). Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of
interest. Open Access This article is distributed under the terms of the Crea-
tive Commons Attribution 4.0 International License (http://creativeco
mmons.org/licenses/by/4.0/), which permits unrestricted use, distribu-
tion, 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. Discussion We treated death and disability pension with other than 1 3 344 International Archives of Occupational and Environmental Health (2019) 92:337–345 musculoskeletal diagnosis as competing events to disability
pension due to musculoskeletal disorders.f adjustment for psychosocial factors (Friis et al. 2008; Kjell-
berg et al. 2016; Labriola et al. 2009; Lahelma et al. 2012). Measuring heavy physical effort at work with JEM can be
considered as a strength and as a weakness of the study. On
one hand, aggregate measures are not able to tap within-job
and individual variance in physical demands. On the other
hand, previous JEM validation studies have found that these
matrices have rather good validity, sensitivity and specificity,
particularly regarding physical exposures (Rijs et al. 2014;
Solovieva et al. 2012). Due to small number of respondents
in some occupational titles, we had to omit about 50% of
occupations from further analysis. This may have implica-
tion to generalizability. However, selecting only occupations
with enough respondents improves the validity of our meas-
ures, as they are not based on evaluations of few respond-
ents. Moreover, aggregation of self-reported exposure data
decreases the effects of recall bias and individual character-
istics including mood, personality, and health status, which
may influence individual appraisals of the exposure. References We did not adjust for psychosocial work environment fac-
tors. Occupation-specific aggregate measures of psychoso-
cial exposures, including psychological demands or social
support (i.e., psychosocial JEM), have shown poorer valid-
ity than physical exposures (Niedhammer et al. 2008; Rijs
et al. 2014; Solovieva et al. 2014). This is plausible, since
the level of, for example, coworker support may not be as
dependent from one’s occupation as it is from one’s work
unit, and even from one’s individual resources and the fit
between person and the work environment. Hence, we did
not compile a psychosocial JEM. Moreover, earlier studies
have shown that the effect of physical exposure is robust to Austin PC, Fine JP (2017) Practical recommendations for reporting
Fine-Gray model analyses for competing risk data. Stat Med
36:4391–4400. https://doi.org/10.1002/sim.7501 Dale AM et al (2015) General population job exposure matrix applied
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Environ Health 35:466–474 Lahelma E et al (2013) Cohort profile: the Helsinki health study. Int J
Epidemiol 42:722–730. https://doi.org/10.1093/ije/dys039 Mixed Exposures Research Agenda (2004) A report by the NORA
mixed exposures team Holtermann A, Mortensen OS, Burr H, Sogaard K, Gyntelberg F,
Suadicani P (2010a) Physical demands at work, physical fit-
ness, and 30-year ischaemic heart disease and all-cause mortality
in the Copenhagen Male Study Scand. J Work Environ Health
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psychosocial work factors: results from the national French. SUMER Surv Int Arch Occup Environ Health 82:87–97. https://
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Suadicani P (2010b) Physical work demands, hypertension sta-
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peting risks and multi-state models. Stat Med 26:2389–2430. https
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annhyg/met052 Holtermann A, Mortensen OS, Sogaard K, Gyntelberg F, Suadicani P
(2012) Risk factors for ischaemic heart disease mortality among
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prospective cohort study. BMJ Open 2:e000279. https://doi. org/10.1136/bmjopen-2011-000279i Ropponen A, Svedberg P, Koskenvuo M, Silventoinen K, Kaprio J
(2014) Physical work load and psychological stress of daily
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etal disorders. Scand J Public Health 42:370–376. https://doi. org/10.1177/1403494814525005 g
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matrix for psychosocial factors at work. PLoS One 9:e108987. https://doi.org/10.1371/journal.pone.0108987 Karpansalo M, Manninen P, Lakka TA, Kauhanen J, Rauramaa R,
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(2016) Long-term physical workload in middle age and disabil-
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3067771 Publisher’s Note Springer Nature remains neutral with regard to
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ity retirement: a longitudinal register linkage study. BMC Public
Health 12:309. https://doi.org/10.1186/1471-2458-12-309 1 3 1 3
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Design and realization of light absorbers using plasmonic nanoparticles
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To cite this version: Ludovic Escoubas, Miriam Carlberg, Judikaël Le Rouzo, Florent Pourcin, Jörg Ackermann, et al.. Design and realization of light absorbers using plasmonic nanoparticles. Progress in Quantum Elec-
tronics, 2019, 10.1016/j.pquantelec.2018.12.001. hal-01986953 Design and realization of light absorbers using
plasmonic nanoparticles Ludovic Escoubas, Miriam Carlberg, Judikaël Le Rouzo, Florent Pourcin,
Jörg Ackermann, Olivier Margeat, Clement Reynaud, David Duche,
Jean-Jacques Simon, Rose-Marie Sauvage, et al. Distributed under a Creative Commons Attribution 4.0 International License Design and realization of light absorbers using
plasmonic nanoparticles HAL Id: hal-01986953
https://amu.hal.science/hal-01986953v1
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The applications of light absorbers concern photodetectors, optical filters, solar applications or
flexible electronics. In this review, we will detail the application of such light absorbers and we
will develop the main demonstrations of the use of metallic nanoparticles embedded within a
host matrix to fabricate coatings aiming at harvesting light. We will explain how chemically syn-
thetized silver nanoparticles of various shapes (spheres, cubes, …) and sizes allow controlling
the optical properties of heterogeneous thin film layers. By coupling the optical characterizations
with computer modeling, we will describe how the nanoparticles behave both individually and
collectively. To control reflected and absorbed light by thin film layers containing nanoparticles
several points have to be addressed: the relation between the shape of the nanoparticle and the
absorptance of the layer, the interaction of light between nanoparticles and the collective behav-
ior of aggregates. U
∗Corresponding author.
Email address: ludovic.escoubas@im2np.fr (L. Escoubas) https://doi.org/10.1016/j.pquantelec.2018.12.001
Available online xxx
0079-6727/ © 2019. RO
Design and realization of light absorbers using plasmonic nanoparticles PRO
Miriam Carlberga, Judikael Le Rouzoa, Florent Pourcinb, Jorg Ackermannb,
ment Reynauda, David Duchea, Jean-Jacques Simona, Rose-Marie Sauvagec, PRO
Ludovic Escoubasa, ∗, Miriam Carlberga, Judikael Le Rouzoa, Florent Pourcinb, Jorg Ackermannb,
Olivier Margeatb, Clement Reynauda, David Duchea, Jean-Jacques Simona, Rose-Marie Sauvagec,
Gérard Bergincd a Aix Marseille Univ, Université de Toulon, CNRS, IM2NP, Marseille, France
b Aix Marseille Univ, CNRS, CINAM, Marseille, France
c DGA/DS/MRIS, 75015, Paris, France
d Thales Optronics, 78990, Elancourt, France a Aix Marseille Univ, Université de Toulon, CNRS, IM2NP, Marseille, France a Aix Marseille Univ, Université de Toulon, CNRS, IM2NP, Marseille, France
b Aix Marseille Univ, CNRS, CINAM, Marseille, France
c DGA/DS/MRIS, 75015, Paris, France
d Thales Optronics, 78990, Elancourt, France a Aix Marseille Univ, Université de Toulon, CNRS, IM2NP, Marseille, France
b Aix Marseille Univ, CNRS, CINAM, Marseille, France
c DGA/DS/MRIS, 75015, Paris, France
d Thales Optronics, 78990, Elancourt, France d Thales Optronics, 78990, Elancourt, France 1. Introduction UNCOR
Many applications require having at disposal or designing materials, which are able to absorb the light, either specifically
for some light incidences and spectral ranges chosen by users, or in broad-ranges of incidences and wavelengths. In most appli-
cations the efficiency of absorption must be maximal and must be obtained with the thinnest materials, very often in thin lay-
ers, and with the wish that these thin layers perfectly fit with the forms of the surfaces they cover. When one wishes to go be-
yond the absorption ranges usually found in the materials in their natural state, one possibility is to design new synthetic mate-
rials made of alloys of constituents, which are often of complex structures. The structuring of these materials, when the charac-
teristic dimensions are close to the wavelengths to be absorbed, greatly influences the properties of absorption in terms of spec-
tral range, of angular fields and of efficiency. To build such thin materials, which are ultra-absorbing and not available in nat-
ural state, resonance phenomena of light found in Fabry-Perot cavities or plasmonic effects are very useful. These plasmonic ef-
fects may appear in the gap between two thin metallic layers, and are called gap plasmons, or on the surface of structured materi-
als (called surface plasmons), or even directly at the level of nanoparticles of metal or degenerate semiconductor (called localized
plasmons). Thus, it is possible to obtain very strong light absorption with very small amount of material. Furthermore, associat https://doi.org/10.1016/j.pquantelec.2018.12.001
Available online xxx
0079-6727/ © 2019. L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx TED PROOF
Fig. 1. a) Sketch of a switchable perfect absorber device. Arrays of square aluminum (Al) nanoantennas are stacked above a spacer layer of the phase change material
GST-326 and an Al mirror. b) Amorphous to crystalline phase transition in GST. c) SEM images of two representative perfect absorber devices fabricated with antenna
side lengths of d=400nm and d=450nm @Wiley (extracted with permission from ref 7 - https://doi.org/10.1002/adma.201502023). TED
Fig. 1. a) Sketch of a switchable perfect absorber device. Arrays of square aluminum (Al) nanoantennas are stacked above a spacer layer of the phase change material
GST-326 and an Al mirror. b) Amorphous to crystalline phase transition in GST. 1. Introduction c) SEM images of two representative perfect absorber devices fabricated with antenna
side lengths of d=400nm and d=450nm @Wiley (extracted with permission from ref 7 - https://doi.org/10.1002/adma.201502023). RRECTE
Fig. 2. (a) Schematic of the dual-band perfect absorber structure and the incident polarization configuration. (b) A SEM image of the designed structure. @OSA (ex-
tracted with permission from ref 8 - https://doi.org/10.1364/OE.19.015221). RECT T ECTE RR
Fig. 2. (a) Schematic of the dual-band perfect absorber structure and the incident polarization configuration. (b) A SEM image of the designed structure. @OSA (ex-
tracted with permission from ref 8 - https://doi.org/10.1364/OE.19.015221). UNCORR
Fig. 3. Photograph of resonant color filters. @ACS Publications (extracted with permission from ref 10 - https://doi.org/10.1021/ph500410u). UN
Fig. 3. Photograph of resonant color filters. @ACS Publications (extracted with permission from ref 10 - https://doi.org/10.10 UN
Fig. 3. Photograph of resonant color filters. @ACS Publications (extracted with permission from ref 10 - https://doi.org/10.1021/ph500410u). UN
of resonant color filters. @ACS Publications (extracted with permission from ref 10 - https://doi.org/10.1021/ph500410u). U
ing optical resonators, whose dimensions are close to the wavelength, allows electromagnetic interactions to be obtained, which opens
new possibilities of controlling the light propagation in the materials. The aim of this article is firstly to describe the general working principles of light absorbers based on plasmonic effect,
mainly obtained from nanoparticles. In a second step, we discuss the synthesis routes of plasmonic nanoparticles and we show U
ing optical resonators, whose dimensions are close to the wavelength, allows electromagnetic interactions to be obtained, which opens
new possibilities of controlling the light propagation in the materials.i U
ing optical resonators, whose dimensions are close to the wavelength, allows electromagnetic interactions to be obtained, which opens
new possibilities of controlling the light propagation in the materials.i The aim of this article is firstly to describe the general working principles of light absorbers based on plasmonic effect,
mainly obtained from nanoparticles. In a second step, we discuss the synthesis routes of plasmonic nanoparticles and we show 2 2 L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx ROOF
Fig. 4. (a) Schematic of a MIM plasmonic rectenna - (b) SEM image of nanocubes self-assembled into a periodic holed polymer matrix using dithiol molecule linkers
localized between the base of the silver nanocubes and the gold surface. OO RO
Fig. 4. 1. Introduction (a) Schematic of a MIM plasmonic rectenna - (b) SEM image of nanocubes self-assembled into a periodic holed polymer matrix using dithiol molecule linkers
localized between the base of the silver nanocubes and the gold surface. ED TED PRO
Fig. 5. Schematic of a double-stacked MIM (i.e. MIMIM) photodetectors operating in the near-infrared (NIR) spectrum up to 1200nm wavelength and presented in
ref 17. The MIMIM comprised of metal nanoparticles (MNP), spacer insulator (Ispa), absorbing metal (Mabs), tunneling insulator (Itunnel) and bottom contact metal
(Mcont), (b) SEM image of the nanoparticles after annealing 10nm Silver film for 20minat 500°C and (c) the size distribution of particles from SEM image of part (b)
@Nature (extracted with permission from Ref. [17] - https://doi.org/10.1038/srep42349). ED PRO ED PRO TED
Fig. 5. Schematic of a double-stacked MIM (i.e. MIMIM) photodetectors operating in the near-infrared (NIR) spectrum up to 1200nm wavelength and presented in
ref 17. The MIMIM comprised of metal nanoparticles (MNP), spacer insulator (Ispa), absorbing metal (Mabs), tunneling insulator (Itunnel) and bottom contact metal
(Mcont), (b) SEM image of the nanoparticles after annealing 10nm Silver film for 20minat 500°C and (c) the size distribution of particles from SEM image of part (b)
@Nature (extracted with permission from Ref. [17] - https://doi.org/10.1038/srep42349). NCORRECT
Fig. 6. a) Specular measured and Transfer Matrix Method (TMM) computed reflectances of Ag nanocubes:PVP composite layer deposited on Si* (b) TEM picture of
silver nanocubes embedded in PVP c) Optical photograph of the Ag nanocubes:PVP composite on glass substrates. CORRECT CORRECT NC
Fig. 6. a) Specular measured and Transfer Matrix Method (TMM) computed reflectances of Ag nanocubes:PVP composite layer deposited on Si* (b) TEM picture of
silver nanocubes embedded in PVP c) Optical photograph of the Ag nanocubes:PVP composite on glass substrates. UN
how it is possible to control their shapes, which can be spherical, prismatic or cubic for example, and thus affecting the inter-
actions with the light. Then, we present analytical and numerical methods to model the interactions between these nanoparticles
and the light waves: Mie theory, Discrete Dipole Approximation (DDA), Finite Difference Time Domain (FDTD). The modeling of
the light propagation in these complex environments including nanoparticles of various forms requires the measurement of the
optical complex indices in order to implement them in the computation codes. O
2. Design of plasmonic light absorbers NCO
By definition, a light absorber is a device, whose optical properties are controlled. The specular and the diffuse reflectance, and
also the amount of light transmitted through, are minimized. The device absorbs the incident light at a particular wavelength or a
broad wavelength domain, ranging from the optical to the longest wavelength domain, and within the broadest solid angle for both
linear s and p polarization states. Such absorbers are of interest for many applications, depending on their optical properties, as will
be discussed in the following. Because of the large number of applications, extensive research has been done in order to define the
optimal design of a light absorber. 1. Introduction ORR
of two nanoparticles interacting in the Kerker's conditions or aggregates of particles (dimers, trimers, multimeres), whose 3D geome-
try is perfectly controlled to create spectral absorption bands on the demand. ORR
of two nanoparticles interacting in the Kerker's conditions or aggregates of particles (dimers, trimers, multimeres), whose 3D geome-
try is perfectly controlled to create spectral absorption bands on the demand. ORR
of two nanoparticles interacting in the Kerker's conditions or aggregates of particles (dimers, trimers, multimeres), whose 3D geome-
try is perfectly controlled to create spectral absorption bands on the demand. 1. Introduction So, we will explain how spectroscopic ellipsom-
etry measurement associated with Cauchy, Gaussian, or Lorentz models and the theory of effective medium allow extracting the
values of the optical complex indices of these composite materials. Finally, we will devote the last part of the article to present
achievements in which the light interactions between nanoparticles is exploited in very specific configurations: dimers consisting UN
how it is possible to control their shapes, which can be spherical, prismatic or cubic for example, and thus affecting the inter-
actions with the light. Then, we present analytical and numerical methods to model the interactions between these nanoparticles
and the light waves: Mie theory, Discrete Dipole Approximation (DDA), Finite Difference Time Domain (FDTD). The modeling of
the light propagation in these complex environments including nanoparticles of various forms requires the measurement of the
optical complex indices in order to implement them in the computation codes. So, we will explain how spectroscopic ellipsom-
etry measurement associated with Cauchy, Gaussian, or Lorentz models and the theory of effective medium allow extracting the
values of the optical complex indices of these composite materials. Finally, we will devote the last part of the article to present
achievements in which the light interactions between nanoparticles is exploited in very specific configurations: dimers consisting 3 ORRECTED PROOF
L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx
Fig. 7. Localized surface plasmon resonance (LSPR) frequency dependence on free carrier density and doping constraints @Nature (extracted with permission from ref
30 - https://doi.org/10.1038/nmat3004). Fig. 8. Seed and nanoparticle growth process. of two nanoparticles interacting in the Kerker's conditions or aggregates of particles (dimers, trimers, multimeres), whose 3D geome-
try is perfectly controlled to create spectral absorption bands on the demand. 2. Design of plasmonic light absorbersl L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx PROOF D P
Fig. 7. Localized surface plasmon resonance (LSPR) frequency dependence on free carrier density and doping constraints @Nature (extracted with permission from ref
30 - https://doi.org/10.1038/nmat3004). RRECTED
30 - https://doi.org/10.1038/nmat3004). Fig. 8. Seed and nanoparticle growth process. RRECTED
Fig. 8. Seed and nanoparticle growth process. f t
ti l
i t
ti
i
th K
k
'
diti
t
f
ti l
(di
t i
lti
)
h
3D RR
Fig. 8. Seed and nanoparticle growth process. RR
Fig. 8. Seed and nanoparticle growth process. UN
2.1. Metal insulator metal (MIM) structure U
A first highly-efficient absorber design concerns the metal-insulator-metal (MIM) structures [1–3]. The simplest MIM structure
is composed of two metallic layers separated by a dielectric layer. The bottom layer is thick to act as a reflector and avoid opti-
cal transmission. A simple MIM structure presents a single absorption band because there is a single resonant mode within the di-
electric cavity. The incident light is trapped inside the cavity. The position of the absorption band depends on the chosen metal
and dielectric materials. A typical choice for the metallic layer, in order for the device to absorb the light in the visible wave-
length band, is silver or gold. Typical dielectric spacers are Si, SiO2, AlO3 or TiO2. Depending on the aimed absorption band ED PROOF
L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx
Fig. 9. Schematic illustration of therapeutic nanoparticle platforms in preclinical development: (a) liposome, (b) polymer–drug conjugate, (c) polymeric nanoparticle,
(d) dendrimer, and (e) iron oxide nanoparticle. The red dots represent hydrophilic drugs and the blue dots represent hydrophobic drugs @Nature Publishing Group
(extracted with permission from ref 62 - https://doi.org/10.1038/sj.clpt.6100400). L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx ED
Fig. 9. Schematic illustration of therapeutic nanoparticle platforms in preclinical development: (a) liposome, (b) polymer–drug conjugate, (c) polymeric nanoparticle,
(d) dendrimer, and (e) iron oxide nanoparticle. The red dots represent hydrophilic drugs and the blue dots represent hydrophobic drugs @Nature Publishing Group
(extracted with permission from ref 62 - https://doi.org/10.1038/sj.clpt.6100400). RRECTE
Fig. 10. (a) Non uniform mesh and (b) TFSF disposition in the simulation window. RR
Fig. 10. (a) Non uniform mesh and (b) TFSF disposition in the simulation window. ORR
and applications, the suited materials and their thicknesses can be determined by computer simulations, such as finite difference time
domain (FDTD) method, and numerical calculations, such as transfer matrix method (TMM) [1,4]. COR
In order to absorb the light on a broader wavelength range, the number of layers can be increased to create multilayered MIM. Each dielectric cavity will be excited by a different wavelength of the incident light and therefore increase the absorption band of
the device. Recent reports show up to 90% absorption from 400 to 1640nm for multilayered thin film stacks [4]. The advantages of
multilayered MIM structures are undeniable, but the fabrication methods are an important factor in their industrial utilizations. UN
2.1. Metal insulator metal (MIM) structure NCO
y
p
Deposition techniques such as sputtering [3] or thermal evaporation, sol-gel/spin coating [1] or Langmuir-Blodgett [5] methods,
are of particular interest because of their simplicity and potential for low cost upscaling. Recent works have reported their ability to
achieve efficient absorbers for large angles of incidence with a low polarization sensitivity.i NC
g
g
p
y
Broadband multilayered MIM structure absorbers find applications in thermo and photovoltaic solar cells [6], radar technologies
and thermal imaging [7] (see Fig. 1).l UN
Experimental measurements show that the design of Fig. 1a exhibits low reflectance and hence very high resonant absorption
(A>90%) in the MWIR spectral range. Geometrical tunability of the resonance wavelength is achieved by varying the side length d of
the resonant square Al nanoantennas, with larger antennas corresponding to higher resonance wavelengths. When heating the sample
above the germanium antimony telluride (GST) crystallization temperature, the resonance undergoes a pronounced phase-change-in-
duced spectral redshift of up to 0.7μm while maintaining high absorbance. U
Another absorption mechanism used in MIM structures is based on plasmon resonances [8,9]. The incident electromagnetic wave
is coupled into the resonant dielectric gap in the form of a gap-plasmon and at the metal interface in the form of a surface-plasmon. Either the upper or the bottom metallic layer is structured to take advantage of the surface plasmon resonances. 5 L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx CTED PROOF
Fig. 11. Example of the FDTD computed amplitude of the electric field enhancement on a 35nm edge size silver cube in a PVP polymer matrix at λ=350nm. TED PROOF
Fig 11
Example of the FDTD computed amplitude of the electric field enhancement on a 35nm edge size silver cube in a PVP polymer matrix at λ=350nm TE
Fig. 11. Example of the FDTD computed amplitude of the electric field enhancement on a 35nm edge size silver cube in a PV CTE
Fig. 11. Example of the FDTD computed amplitude of the electric field enhancement on a 35nm edge size silver cube in a PVP polymer matrix at λ=350nm. NCORRECT
Fig. 12. Example of coupled resonance calculated by FDTD: electric field enhancement at the dipolar resonance wavelength of silver nanoprisms displayed in a periodic
squared pattern. The electromagnetic wave is travelling along x. NCORRECT NC
Fig. 12. Table 1 T
Table 1
Listing of the different optical models used to fit spectroscopic ellipsometry data adapted from Ref. [113]. UNCORRECT
Listing of the different optical models used to fit spectroscopic ellipsometry data adapted from Ref. [113]. Sample
Laws
Meaning
Ref
Ag in PVA
Lorentz
Lorentz oscillator for main LSPR
[101]
Ag in PVP
Lorentz + Cauchy
Lorentz oscillator for main LSPR and Cauchy for the polymer host
[112]
Au nanospheres on gold
substrate
Lorentz
Lorentz oscillator for main LSPR and Lorentz oscillator for background absorption
[103]
Ag nanospheres in Al2O3
Lorentz + Drude
[109]
Ag nanospheres and nanorods
on Si substrates
Lorentz + Tauc
Lorentz
Lorentz oscillator for main LSPR and Tauc-Lorentz for bulk silver
[110]
Au islands on glass substrate
Gauss
Gauss oscillator for main LSPR, Gauss oscillators for interband transitions of gold, Gauss
oscillator for inhomogeneous broadening of LSPR
[102]
Ag islands on glass substrate
Gauss + Tanguy
Gauss oscillators for main LSPR, Gauss oscillator for bulk plasmon resonance and Tanguy
oscillator for interband transitions
[111]
Fig. 15. (a) Complex refractive indices n and k of nanospheres and nanocubes blend in PVP and (b) the normalized extinction coefficient k compared to the one of
nanospheres in PVP and nanocubes in PVP. @OSA (extracted with permission from Ref. [116]). RRECT
Listing of the different optical models used to fit spectroscopic ellipsometry data adapted from Ref. [113]. Sample
Laws
Meaning
Ref
Ag in PVA
Lorentz
Lorentz oscillator for main LSPR
[101]
Ag in PVP
Lorentz + Cauchy
Lorentz oscillator for main LSPR and Cauchy for the polymer host
[112]
Au nanospheres on gold
substrate
Lorentz
Lorentz oscillator for main LSPR and Lorentz oscillator for background absorption
[103]
Ag nanospheres in Al2O3
Lorentz + Drude
[109]
Ag nanospheres and nanorods
on Si substrates
Lorentz + Tauc
Lorentz
Lorentz oscillator for main LSPR and Tauc-Lorentz for bulk silver
[110]
Au islands on glass substrate
Gauss
Gauss oscillator for main LSPR, Gauss oscillators for interband transitions of gold, Gauss
oscillator for inhomogeneous broadening of LSPR
[102]
Ag islands on glass substrate
Gauss + Tanguy
Gauss oscillators for main LSPR, Gauss oscillator for bulk plasmon resonance and Tanguy
oscillator for interband transitions
[111] UNCOR U
Fig. 15. (a) Complex refractive indices n and k of nanospheres and nanocubes blend in PVP and (b) the normalized extinction coefficient k compared to the one of
nanospheres in PVP and nanocubes in PVP. UN
2.1. Metal insulator metal (MIM) structure Example of coupled resonance calculated by FDTD: electric field enhancement at the dipolar resonance wavelength of silver nanoprisms displayed in a periodic
squared pattern. The electromagnetic wave is travelling along x. UN
As shown in Fig. 2, the structure is made from a film stack comprising: UN
As shown in Fig. 2, the structure is made from a film stack comprising: U
- a top metal layer composed of an elliptical gold nanodisk array, which geometrical parameters such as a, b and d can be tuned
individually to control the interaction with light. U
- a top metal layer composed of an elliptical gold nanodisk array, which geometrical parameters such as a, b and d can be tuned
individually to control the interaction with light. U
- a spacing dielectric SiO2, MgF2, or polymer layer with low permittivity to reach high absorption. The thickness of the dielectric layer
is of high importance as it influences the dipole resonance. By increasing the thickness, the resonance effect diminishes and the overall
absorption efficiency is reduced. 6 UNCORRECTED PROOF
L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx
Fig. 13. Simplified spectroscopic ellipsometry measurement setup. Fig. 14. Effective medium approach. Table 1
Listing of the different optical models used to fit spectroscopic ellipsometry data adapted from Ref. [113]. Sample
Laws
Meaning
Ref
Ag in PVA
Lorentz
Lorentz oscillator for main LSPR
[101]
Ag in PVP
Lorentz + Cauchy
Lorentz oscillator for main LSPR and Cauchy for the polymer host
[112]
Au nanospheres on gold
substrate
Lorentz
Lorentz oscillator for main LSPR and Lorentz oscillator for background absorption
[103]
Ag nanospheres in Al2O3
Lorentz + Drude
[109]
Ag nanospheres and nanorods
on Si substrates
Lorentz + Tauc
Lorentz
Lorentz oscillator for main LSPR and Tauc-Lorentz for bulk silver
[110]
Au islands on glass substrate
Gauss
Gauss oscillator for main LSPR, Gauss oscillators for interband transitions of gold, Gauss
oscillator for inhomogeneous broadening of LSPR
[102]
Ag islands on glass substrate
Gauss + Tanguy
Gauss oscillators for main LSPR, Gauss oscillator for bulk plasmon resonance and Tanguy
oscillator for interband transitions
[111] TED PROOF
oubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx
Fig. 13. Simplified spectroscopic ellipsometry measurement setup. Fig. 14. Effective medium approach. L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx R
Fig 13
Simplified spectroscopic ellipsometry measurement setup TED PROO
Fig. 13. UN
2.1. Metal insulator metal (MIM) structure Simplified spectroscopic ellipsometry measurement setup. Fig. 14. Effective medium approach. UNCORRECTED PRO
Fig. 13. Simplified spectroscopic ellipsometry measurement setup. Fig. 14. Effective medium approach. Table 1
Listing of the different optical models used to fit spectroscopic ellipsometry data adapted from Ref. [113]. Sample
Laws
Meaning
Ref
Ag in PVA
Lorentz
Lorentz oscillator for main LSPR
[101]
Ag in PVP
Lorentz + Cauchy
Lorentz oscillator for main LSPR and Cauchy for the polymer host
[112]
Au nanospheres on gold
substrate
Lorentz
Lorentz oscillator for main LSPR and Lorentz oscillator for background absorption
[103]
Ag nanospheres in Al2O3
Lorentz + Drude
[109]
Ag nanospheres and nanorods
on Si substrates
Lorentz + Tauc
Lorentz
Lorentz oscillator for main LSPR and Tauc-Lorentz for bulk silver
[110]
Au islands on glass substrate
Gauss
Gauss oscillator for main LSPR, Gauss oscillators for interband transitions of gold, Gauss
oscillator for inhomogeneous broadening of LSPR
[102]
Ag islands on glass substrate
Gauss + Tanguy
Gauss oscillators for main LSPR, Gauss oscillator for bulk plasmon resonance and Tanguy
oscillator for interband transitions
[111]
Fig. 15. (a) Complex refractive indices n and k of nanospheres and nanocubes blend in PVP and (b) the normalized extinction coefficient k compared to the one of
nanospheres in PVP and nanocubes in PVP. @OSA (extracted with permission from Ref. [116]). Fig. 13. Simplified spectroscopic ellipsometry measurement setup. TED PRO
Fig. 13. Simplified spectroscopic ellipsometry measurement setup. Fig. 14. Effective medium approach. TE
Fig. 14. Effective medium approach. TE
Table 1
Listing of the different optical models used to fit spectroscopic ellipsometry data adapted from Ref. [113]. TE
Table 1
Listing of the different optical models used to fit spectroscopic ellipsometry data adapted from Ref. [113]. Table 1 @OSA (extracted with permission from Ref. [116]). 7 7 ORRECTED PROOF
L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx
Fig. 16. Scheme of the considered geometry. The system is composed of two silicon nanoparticles located at a distance d between them. The particle sizes are such
that, while one nanoparticle has zero backscattering, the other one satisfied the MF scattering at the same incident wavelength (λ=700nm). The bottom figures show
the 3-D spatial distribution of the light scattering of each isolated nanoparticle, with blue being the lowest intensity and red being the highest intensity. Depending
on the incident direction, from right to the left (a) or vice versa (b), the light concentration on the gap drastically changes. @IEEE Photonics Society (extracted with
permission from Ref. [123] - https://doi.org/10.1109/JPHOT.2016.2577714). L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx PRO TED P RECTE ORR
Fig. 16. Scheme of the considered geometry. The system is composed of two silicon nanoparticles located at a distance d between them. The particle sizes are such
that, while one nanoparticle has zero backscattering, the other one satisfied the MF scattering at the same incident wavelength (λ=700nm). The bottom figures show
the 3-D spatial distribution of the light scattering of each isolated nanoparticle, with blue being the lowest intensity and red being the highest intensity. Depending
on the incident direction, from right to the left (a) or vice versa (b), the light concentration on the gap drastically changes. @IEEE Photonics Society (extracted with
permission from Ref. [123] - https://doi.org/10.1109/JPHOT.2016.2577714). OR
- a bottom gold layer deposited on top of the substrate CO
Fig. 2 (b) shows a SEM top-view image of the structure. Contrary to the component shown in Fig. 2, devices are generally highly
polarization dependent and the absorption band is narrow, which makes them promising candidates for sensing applications, col-
ored-optical filters [10] (see Fig. 3) and polarization detectors [11].ii CO
Fig. 2 (b) shows a SEM top-view image of the structure. Contrary to the component shown in Fig. 2, devices are generally highly
polarization dependent and the absorption band is narrow, which makes them promising candidates for sensing applications, col-
ored-optical filters [10] (see Fig. 3) and polarization detectors [11].ii NCi
As shown in Fig. 2.2. Plasmonic nanoparticles light absorbers ORRE
Plasmonic nanoparticles display localized surface plasmon resonances [14,15] (LSPRs), which induce a selective light absorption
and scattering depending on the material, size, shape and environment of the nanoparticle. Indeed, LSPRs excited in metallic nanopar-
ticles are non-propagating plasmon excitations. Since the size of a metallic nanoparticle is on the same scale of the penetration depth
of electromagnetic waves in metals (e.g., 20∼30nm for Ag and Au), the external field can penetrate the whole particle and shift
the conduction electrons with respect to the rigid ion lattice. Thus, the charges are separated and this charge separation results in
a restoring force and then an oscillation. The oscillation frequency is mainly related to effective electron mass, charge density, and
geometry of the particle, as well as the properties of the surrounding medium. The amplitude of the induced electromagnetic field is
much stronger than exciting fields (over 10 times). A comprehensive review on the applications of plasmonic effects to solar cells has
been published by Atwater and Polman [16]. The nanoparticles can be: NCO
- either deposited directly onto a thin dielectric spacer on a metallic substrate to fabricate a resonant absorbing MIM structure and the
light is then absorbed through surface plasmon resonances [17] (see Fig. 5) in the cavity between the metal layer and the nanopar-
ticles. The optical response of the deposited metasurfaces relies on the spacing and the nanocrystal size as well as nanoparticle
density. This allows a large parameter space to fine tune the optical response [18,19]. It is essential here to carefully choose the
underlying metal substrate as well as the thickness of the dielectric spacer to enhance optical couplings [20–22].i UN
- or embedded within a host matrix. In this second configuration, the light is absorbed by the localized surface plasmon reso-
nances of the nanoparticles inside the composite layer and the absorbed light can be locally converted in to heat [23]. The op-
tical properties of the light absorber are mainly controlled by the size, density and shape of the nanoparticles and are indepen-
dent of the choice of the substrate [24]. The role of the host materials, usually a transparent polymer, is to facilitate processing
of homogeneous films over large areas and to control spacing and organization of the plasmonic absorber inside the nanocom-
posite with dedicated optical properties. For example, Fig. Table 1 3, resonant optical filters can be fabricated from modified, asymmetric metal–insulator–metal (MIM) based
Fabry–Perot cavities including plasmonic, lossy ultrathin (∼30nm) metallic films used as the top metallic layer. Different colors can
be obtained by controlling the dielectric spacer thickness.i UN
Because they can be confined over nanometric areas at dielectric-metal interfaces or into nanocavities, surface plasmons can
create very high electric fields. This feature makes them particularly interesting for optical rectification purposes. One of the
promises of optical rectification is to enable the fabrication of devices that convert light into electricity without relying on the
photovoltaic effect. As a consequence, such a device – referred as optical rectennas (rectifying antenna) - would not be subject to
the so-called Schockley Queisser limit that bounds the efficiencies of PV solar cells to 33% for single junctions. The concept of
optical rectennas goes back to the 70's when Bailey [12] proposed that a nanoscale antenna coupled with a rectifier could har-
vest electromagnetic waves in the visible and infrared region. Recent work have already demonstrated power production origi-
nating from optical rectennas [13], but research in this field remains at stage of proof of concept. We have assembled very re-
cently rectennas solar cells composed of plasmonic nanocubes (see Fig. 4a) associated with rectifying self-assembled molecular
diodes that allow a plasmon cavity mode coupling between silver nanocubes and a gold plane. Thus, an electric field enhance 8 8 Progress in Quantum Electronics xxx (2019) xxx-xxx L. Escoubas et al. TED PROOF
L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx
Fig. 17. Distribution of the electric field in the incident and orthogonal planes (including the middle point of the gap) in the region between the nanoparticles consid-
ering an incident beam of (λ=700nm). The geometry and view are shown in the center. (a) and (b) corresponding to the configuration of Fig. 16a with gap distances
of d=375nm and d=200nm, respectively. (c) and (d) corresponding to the configuration of Fig. 16b with gap distances of d=120nm and d=445nm, respectively. @IEEE Photonics Society (extracted with permission from Ref. [123]- https://doi.org/10.1109/JPHOT.2016.2577714). TED
Fig. 17. Distribution of the electric field in the incident and orthogonal planes (including the middle point of the gap) in the region between the nanoparticles consid-
ering an incident beam of (λ=700nm). The geometry and view are shown in the center. (a) and (b) corresponding to the configuration of Fig. Table 1 16a with gap distances
of d=375nm and d=200nm, respectively. (c) and (d) corresponding to the configuration of Fig. 16b with gap distances of d=120nm and d=445nm, respectively. @IEEE Photonics Society (extracted with permission from Ref. [123]- https://doi.org/10.1109/JPHOT.2016.2577714). ECT
ment up to two orders of magnitude (intensity enhancement up to four orders of magnitude) is obtained that could enable the rectifi-
cation process without any applied bias. The nanocubes shown in Fig. 4 are self-assembled thanks to dithiol molecules. By choosing a
given molecule length, the gap thickness below the cube is controlled within a nanometer accuracy. A holed polymer matrix controls
the periodicity of the nanocubes array (see Fig. 4b), which has a crucial role in the optical absorption by the device. 2.2. Plasmonic nanoparticles light absorbers 6a shows the specular reflectance spectra of a PVP layer with em-
bedded Ag nanocubes. Deposited on Si, those nanocomposite layers produce a strong reflectance dip over the range of absorp 9 Progress in Quantum Electronics xxx (2019) xxx-xxx L. Escoubas et al. RRECTED PROOF
18. (A–C) Schematic and SEM images of nanocube assembly observed at embedding depths of 15nm, 42nm and 61nm of NCo. Scale bar=500nm. (D–F) Corre
ding statistical analysis showing the population distributions of monomers, dimers, trimers, and multimers in each assembly. (G–I) Corresponding optical extinctio
ra taken an incident illumination with a broadband white light source. The extinction spectrum of as-deposited NCo nanocubes prior to modular assembly is show
ference (black lines). @Royal Society of Chemistry (extracted with permission from Ref. [124] - 10.1039/C5FD00134J). PROOF RECTED PR
Fig. 18. (A–C) Schematic and SEM images of nanocube assembly observed at embedding depths of 15nm, 42nm and 61nm of NCo. Scale bar=500nm. (D–F) Corr
sponding statistical analysis showing the population distributions of monomers, dimers, trimers, and multimers in each assembly. (G–I) Corresponding optical extinctio RECTE RRE
Fig. 18. (A–C) Schematic and SEM images of nanocube assembly observed at embedding depths of 15nm, 42nm and 61nm of NCo. Scale bar=500nm. (D–F) Corre-
sponding statistical analysis showing the population distributions of monomers, dimers, trimers, and multimers in each assembly. (G–I) Corresponding optical extinction
spectra taken an incident illumination with a broadband white light source. The extinction spectrum of as-deposited NCo nanocubes prior to modular assembly is shown
for reference (black lines). @Royal Society of Chemistry (extracted with permission from Ref. [124] - 10.1039/C5FD00134J). UNCOR
Fig. 19. Schematic of a microbolometer membrane covered by a metamaterial consisting of a metal layer/dielectric (Si3Nx)/periodic metal patch structure. tion where the plasmonic absorption of the Ag nanocubes occurs, while spin coating on glass leads to composite layers in
which absorption is controlled by the amount of embedded cubes. The optical properties of such films depend not only on the
natural, size and shape of the metal nanocrystals, but are also function of the spacing and arrangement of the nanocrystals U
Fig. 19. Schematic of a microbolometer membrane covered by a metamaterial consisting of a metal layer/dielectric (Si3Nx)/periodic metal patch structure. tion where the plasmonic absorption of the Ag nanocubes occurs, while spin coating on glass leads to composite layers in
which absorption is controlled by the amount of embedded cubes. 2.2. Plasmonic nanoparticles light absorbers Thus degenerately do
me nanostructure, opening up the p
ing, nonlinear optics, and quantu
c MIM structures or by solely takin
ent optical phenomenon ROO
Fig. 20. Schematic of a metamaterial perfect absorber achieving near-unity optical absorption using ultrathin plasmonic nanostructures with thicknesses smaller than
the hot electron diffusion length. By integrating the metamaterial with a silicon substrate, a broadband and omnidirectional hot electron photodetector is obtained,
showing a very high photoresponsivity. Dimensions of the metamaterial perfect absorber L=185nm–195nm; P=340nm–360nm, respectively and H=135nm. within the embedding film [25]. Near close-packed configuration of nanocrystals and their relative orientation in this packing cre-
ate collective behaviors of unique optical signature [26,27]. Controlling aggregation and packing density of nanocrystals inside a
hostmatrix can be expected to generate a new class of high performance plasmonic light absorber. ROO
Fig. 20. Schematic of a metamaterial perfect absorber achieving near-unity optical absorption using ultrathin plasmonic nanostructures with thicknesses smaller than
the hot electron diffusion length. By integrating the metamaterial with a silicon substrate, a broadband and omnidirectional hot electron photodetector is obtained,
showing a very high photoresponsivity. Dimensions of the metamaterial perfect absorber L=185nm–195nm; P=340nm–360nm, respectively and H=135nm. within the embedding film [25]. Near close-packed configuration of nanocrystals and their relative orientation in this packing cre-
ate collective behaviors of unique optical signature [26,27]. Controlling aggregation and packing density of nanocrystals inside a
hostmatrix can be expected to generate a new class of high performance plasmonic light absorber. ROO
Fig. 20. Schematic of a metamaterial perfect absorber achieving near-unity optical absorption using ultrathin plasmonic nanostructures with thicknesses smaller than
the hot electron diffusion length. By integrating the metamaterial with a silicon substrate, a broadband and omnidirectional hot electron photodetector is obtained,
showing a very high photoresponsivity. Dimensions of the metamaterial perfect absorber L=185nm–195nm; P=340nm–360nm, respectively and H=135nm. within the embedding film [25]. Near close-packed configuration of nanocrystals and their relative orientation in this packing cre-
ate collective behaviors of unique optical signature [26,27]. Controlling aggregation and packing density of nanocrystals inside a
hostmatrix can be expected to generate a new class of high performance plasmonic light absorber. ED PR
The introduction of nanoparticles in the fluid of a thermal solar module increases the solar radiation absorption when compared
to the fluid only. 2.2. Plasmonic nanoparticles light absorbers The material and size of the nanoparticles are carefully chosen in order to maximize the absorption and minimize
the scattering of the nanoparticles in the spectral domain of interest. Indeed, the maximum solar irradiation intensity is located at
475nm. It has been reported, that the localized surface plasmon resonance absorption peak of core-shell Ag
TiO2 nanoparticles is
centered around 474nm, making them an ideal candidate to increase the light absorption at the maximum solar irradiation intensity
[28]. To increase the absorption on a larger wavelength band, from 250 to 1000nm, nanoparticles made of other materials can be
used. It has been shown, that homogeneous Ti and core-shell Ti
TiO2, Ni
NiO nanoparticles introduced in water at concentrations
of 109 - 1010cm−3 are suitable to increase the absorption [29]. CTED
LSPRs, can also be achieved in semiconductor quantum dots (QDs) with appreciable free carrier concentrations (see Fig. 7) allow-
ing active on-chip control of LSPR responses. As shown in Fig. 7, the LSPR frequency can be tuned from near infrared (NIR) to far
infrared (FIR) and even THz according the free carrier density and the nanosphere diameter. Thus degenerately doped semiconductor
QDs allow realization of LSPRs and quantum-confined excitons within the same nanostructure, opening up the possibility of strong
coupling of photonic and electronic modes, with implications for light harvesting, nonlinear optics, and quantum information pro-
cessing [30]. CT
Light absorbers are achieved by different means: MIM structures, plasmonic MIM structures or by solely taking advantage of the
optical properties of nanoparticles. Each described absorber is based on a different optical phenomenon. EC
The usage of plasmonic nanoparticles is of particular interest because of the large panel of optical properties they produce. In the
recent years, the research on nanoparticle production made huge advances and nowadays a large variety of nanoparticle are produced
chemically. This will be described in the following part. 2.2. Plasmonic nanoparticles light absorbers The optical properties of such films depend not only on the
natural, size and shape of the metal nanocrystals, but are also function of the spacing and arrangement of the nanocrystals U
Fig. 19. Schematic of a microbolometer membrane covered by a metamaterial consisting of a metal layer/dielectric (Si3Nx)/periodic metal patch structure. tion where the plasmonic absorption of the Ag nanocubes occurs, while spin coating on glass leads to composite layers in
which absorption is controlled by the amount of embedded cubes. The optical properties of such films depend not only on the
natural, size and shape of the metal nanocrystals, but are also function of the spacing and arrangement of the nanocrystals U
Fig. 19. Schematic of a microbolometer membrane covered by a metamaterial consisting of a metal layer/dielectric (Si3Nx)/periodic metal patch structure. tion where the plasmonic absorption of the Ag nanocubes occurs, while spin coating on glass leads to composite layers in
which absorption is controlled by the amount of embedded cubes. The optical properties of such films depend not only on the
natural, size and shape of the metal nanocrystals, but are also function of the spacing and arrangement of the nanocrystals 10 L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx ROOF
Fig. 20. Schematic of a metamaterial perfect absorber achieving near-unity optical absorption using ultrathin plasmonic nanostructures with thicknesses smaller than
the hot electron diffusion length. By integrating the metamaterial with a silicon substrate, a broadband and omnidirectional hot electron photodetector is obtained,
showing a very high photoresponsivity. Dimensions of the metamaterial perfect absorber L=185nm–195nm; P=340nm–360nm, respectively and H=135nm. within the embedding film [25]. Near close-packed configuration of nanocrystals and their relative orientation in this packing cre-
ate collective behaviors of unique optical signature [26,27]. Controlling aggregation and packing density of nanocrystals inside a
hostmatrix can be expected to generate a new class of high performance plasmonic light absorber. TED PROO
g ultrathin plasmonic nanostructures with
dband and omnidirectional hot electron ph
m–195nm; P=340nm–360nm, respectivel
tals and their relative orientation
regation and packing density of na
monic light absorber. reases the solar radiation absorpti
en in order to maximize the absorp
the maximum solar irradiation int
orption peak of core-shell Ag
Ti
absorption at the maximum solar i
00nm, nanoparticles made of oth
O nanoparticles introduced in wate
reciable free carrier concentration
quency can be tuned from near in
ere diameter. RE
3. Chemical synthesis of nanoparticles of various shapes and sizes Organic and inorganic reducing agents, such as sodium citrate, ascorbate
or sodium borohydride (NaBH4), are used to reduce silver and gold [41–43]. Changing the reaction temperature, stirring speed and
speed at which the solution is added induce a size and size dispersion change [59].i CTED
Nanocubes and nanoprisms can be achieved by a two-step seed based synthesis [42,43,46]. The first step produces spherical seeds
growing into the desired nanoparticle shape in the second step of the synthesis. Facet-specific capping agents or the solvent itself
induce crystallographic defects on the seeds. These defects then induce a face selective growth in the second step of the nanoparticle
growth. Indeed, the metal salt added in the second step of the synthesis will preferably be deposited on the other surfaces because
the defects are the sites of highest energy [43,46]. Nanocubes are achieved by adding PVP while growing the seeds. In this case, the
PVP protects the seeds from aggregating and, as described above, it preferentially binds to the {100} facets leading to a passivation
of these facets [42]. RECT
After synthesis, the nanoparticles are usually coated with additional surfactant to insure stability of size and shape and prevent
aggregation of particles in solution. Common surfactants are sodium citrates [46], sodium borohydride [46] or PVP [42,60,61]. They
are added in excess at the end of the synthesis to form a protective shell of a few nanometers around the nanoparticle. Aside the
protecting properties, interesting optical properties arise when the polymer shell around the metallic core is modified. Indeed, as plas-
monic responses of nanoparticles are environment sensitive, a 5nm thin layer of PVP around synthesized nanocubes can, for instance,
shift the absorption peaks wavelength of several tens of nanometers. Other examples occur using inorganic core/shell nanoparticles
and were applied for several applications in the biomedical domain [62] (see Fig. 9) such as imaging or sensing [54]. ORRE
It must be noted that, besides the most studied gold and silver nanoparticles, other plasmonic metallic materials have been devel-
oped. For instance, copper nanoparticles can be produced in large quantities by chemical synthesis for applications such as conductive
inks [60]. Aluminum also can be synthesized as nanospheres by hydrolysis [64] or as nanowires and nanocubes by plasma arc dis-
charge [65]. The major drawback of these materials, however, is their fast oxidation in presence of oxygen, even though the oxidation
rate can be reduced [66]. RE
3. Chemical synthesis of nanoparticles of various shapes and sizes ORR
The chemical and physical properties of nanoparticles depend on their material, size, shape and environment. Nanoparticles made
from noble metals such as silver and gold are studied for a long time for their extraordinary optical responses attributed to localized
plasmon resonances in the visible. As a classical example, the origin of the intriguing colors of the Lycurgus cup, made by ancient
Romans, are embedded silver and gold nanoparticles, but the synthesis process was not reported, or got lost over the centuries, mak-
ing us believe that it was rather an accidental than wanted process. Faraday conducted the first scientific study of synthesized noble
metal nanoparticles in 1857. He reduced gold chloride by phosphorus and analyzed their optical properties. Following this, the ability
to tune the optical properties of plasmonic nanoparticles leads the search on syntheses of nanoparticles of different sizes and shapes.i UNCO
The synthesis methods can be separated into “bottom up” and “top down” approaches. The first approach starts with precursor
material and through various reactions individual nanoparticles are produced. The produced nanoparticles present size dispersion, but
the quantity is rather high and it is easy to implement. The second approach, “top down”, requires a large amount of energy, which
will release nanoparticles from a target, e.g. laser ablation [31], arc discharge [32,33], chemical vapor condensation [33], ball milling
[33], hydrogen plasma [33]. The main advantage of the “top down” methods is the high quality and size homogeneity of produced
nanoparticles. However, the need for high energy makes the production expensive and mainly limited to applications where only a
small quantity of nanoparticles is needed. As a large quantity of nanoparticles is required to efficiently modify the optical properties
of thin film layers, this approach is not suitable in this case. On the contrary, chemical wet syntheses allow us to meet this criteria as
the wet synthesis process is scalable and economically attractive. This process is therefore described more in details in the following. UN
From the diversity of existing “bottom up” approaches, e.g. microemulsion [34], thermal decomposition [35], hydrothermal
synthesis [36,37], sol-gel [38], sonochemical [39], radiolysis [40], the most commonly used method is chemical reduction of
metallic ions by a chemical agent [41–43]. Since roman times, where church glasses were colored intentionally or not by metal 11 L. Escoubas et al. RE
3. Chemical synthesis of nanoparticles of various shapes and sizes Progress in Quantum Electronics xxx (2019) xxx-xxx OOF
lic dust, tremendous progress has been made in the understanding and developing of wet-chemical syntheses of various materials,
shapes and sizes [43]. The nanoparticle growth in chemical syntheses follows a model described in 1950 by LaMer [44] and schema-
tized on Fig. 8. Metal salts are dissolved in a solvent and slowly added to the reducing agent solution. On the nanometer scale, ho-
mogeneous nucleation occurs in a first step of the synthesis when the minimal concentration for nucleation is reached (Cmin). The
nucleus can then grow or vanish at any time depending on the critical radius in the system. Beyond this critical threshold, all formed
nuclei are stable and will grow but below this critical threshold, nuclei are instable and vanish. In the nanoparticle growth step, the
stable nuclei grow as long as further metal salts are added and as long as the concentration is above the minimal concentration for
heterogeneous growth on seeds (Cmin, seeds). The resulting solution changes then its color, as the nanoparticles grow. If the size dis-
persion is high, a small growth or size reduction might take place over time to make the nanoparticle more uniform. The produced
nanoparticles are said to be monodisperse in size when the standard deviation is equal or less than 5%. PROO
p
p
q
The most energetically favorable nanoparticle shape is a sphere. Other shapes are achieved by dividing the nanoparticle growth
into two or more synthesis steps. In the specific case of silver nanoparticles, facet-specific capping agent can be used to control the
shape evolution of silver nanoparticles in a seed-mediated synthesis. For instance, citrates and PVP were proven to selectively bind
to the (111) and (100) facets of silver seeds, respectively, stabilizing these facets more than others and thus favoring the formation
of silver nanoparticles with (111) or (100) facets exposed on the surface [45]. Following such strategies, silver nanoparticles could
be obtained with various shapes. A non-exhaustive list of produced shapes are: spheres [46], disks [47], plates [42], prisms [48],
dumbells [49,50], pyramids [51], cubes [42,43], cages [42], nanowires [42], nanoflowers [52–56], nanostars [57], multipods [58]. It seems that the only limit to nanoparticle synthesis is our imagination. Each of these nanoparticles has then different physical and
chemical properties. D P
For nanospheres, a single reduction step is required. RE
3. Chemical synthesis of nanoparticles of various shapes and sizes These materials also exhibit high losses at optical frequencies due to electron interband and intraband
transitions and their electron densities are not easily tuned. This motivates the search for other plasmonic materials such as doped
semiconductors with metal like behavior i.e. oxides, nitrides and chalcogenides [67–70]. COR
The diversity of produced nanoparticles leads to a diversity of chemical and especially optical properties. The optical properties of
the described nanoparticles can then either be experimentally characterized once produced or studied by computer modeling. In the
following chapter, we firstly describe computer modeling as a convenient tool to foresee the optical properties of the nanoparticles
and to validate the measured optical properties, obtained by spectroscopic ellipsometry for instance. In a second part, we describe the
spectroscopic ellipsometry measurement technique. 4.2. Mie theory CTE
The optical properties of plasmonic nanoparticles are numerically calculated using the Mie Theory [76]. Mie identified the need
for a theory linking the particle size and shape to the optical properties of a colloidal metal solution for particles much smaller than
the wavelength. He solved the Maxwell equations for spherical particles of sizes smaller than the wavelength by switching to spherical
coordinates, giving an analytical solution for the scattering and absorption of metal nanospheres in any media. The Mie theory can
then be applied to calculate the absorption and scattering cross sections. The dipolar absorption σabs and scattering σscat cross section
of a spherical particle depends on the polarizability α of the particle: RR
where the polarizability
depends on the volume V of the particle and the dielectric constants ε of the sphere and εm of
the medium. The derivation of the above equations is described in detail elsewhere [77].i RR
where the polarizability
depends on the volume V of the particle and the dielectric constants ε of the sphere and εm of
th
di
Th d
i
ti
f th
b
ti
i d
ib d i
d t il l
h
[77] RR
where the polarizability
depends on the volume V of the particle and the dielectric RR
the medium. The derivation of the above equations is described in detail elsewhere [7 COR
The calculation of the cross sections gives us a first insight on the resonance wavelength of the nanoparticle and whether the
nanoparticle will predominately absorb or scatter the incoming light at this wavelength. At the resonance wavelength, plasmonic
nanoparticles strongly interact with the incoming light, i.e. the cross section exceeds the geometrical size of the nanoparticle. This
simple calculation guides us in the choice of the right nanoparticle material and size with our application in mind. The knowledge of
the size of the nanoparticle and the dielectric constant of the nanoparticle and its medium are necessary for the calculation. The cross
sections of silver nanospheres are calculated by using the optical indices of bulk silver. NCO
It is worth noting that the Mie theory describes the optical behavior of a single nanosphere in a homogeneous surrounding
medium. The interaction and coupling between particles are not taken into account. The Mie theory is therefore useful for colloidal
solutions, in which the nanoparticles typically do not interact, and for thin film layers with low nanoparticle density. NC
4. Computer modeling and optical characterization of embedded nanoparticles UN
The optical properties of nanoparticles can be studied by different means. One common and convenient tool is computer modeling. Different simulation and numerical calculation solutions exist for this purpose and will be briefly described in the following: the Mie
theory is limited to spherical particles, while the discrete dipole approximation and the finite difference time domain methods can be
used for any shape.i U
In the following, finite difference time domain (FDTD) simulations are described in detail as it is a convenient software to an-
alyze the electric field enhancement on the nanoparticles and their optical properties. The geometry of the nanoparticle is freely
chosen, as is the environment medium. In particular, the distance between two, or more, neighboring particles can be studied. 12 L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx F
This feature is relevant to study aggregates of nanoparticles. Indeed, from a interparticle distance of a few nanometers, the enhanced
electric fields around the nanoparticles couple and consequently alter the optical properties.i F
This feature is relevant to study aggregates of nanoparticles. Indeed, from a interparticle distance of a few nanometers, the enhanced
electric fields around the nanoparticles couple and consequently alter the optical properties.i OFi
From an experimental point of view, spectroscopic ellipsometry of thin film layers is a powerful tool to obtain the complex optical
indices of the nanoparticles in their medium. The measurement principle is described in the second part of this chapter. The measure-
ment itself is straight forward, but an adequate diffusion model is required to derive the optical indices. 4.1. Computer modeling 4.1. Computer modeling ROO
Computer simulations and numerical calculations are powerful tools to design optical absorbers. They can be used to study differ-
ent design in order to optimize it and to confirm the measured optical properties. Calculations are cost effective and convenient tools,
especially when the complexity of the design increases. Furthermore, they give us insight on the physical phenomena happening at
the nanoscale. One example is the visualization of the electric field enhancement on a nanoparticle. RO
Different calculation methods, having their advantages and drawbacks, co-exist and must be chosen with care depending on the
application. An important feature in calculations and simulations is the material properties, which are either chosen from textbooks,
such as from Johnson and Christy [71] or Palik [72], or experimentally measured by spectroscopic ellipsometry.i PR
In simulation using finite element analysis, an important aspect for accurate results is the size and shape of the used mesh. De-
creasing the mesh size improves the results, but considerably increases the calculation time. Therefore, the mesh has to be carefully
chosen. D P
The calculation method is chosen depending on the application and the structure of the device. The optical properties of MIM struc-
tures can be obtained numerically by transfer matrix method (TMM) [73] and by electromagnetic computer simulations by full-wave
solver based on the finite element method. The optical properties of plasmonic nanoparticles are either numerically calculated by Mie
Theory, for spherical and ellipsoidal shapes, or simulated by time domain methods, such as FDTD [74], or frequency domain methods,
such as FEM [75]. U
where ξ0 and Χ are size dependent variables. 4.4. Finite difference time domain (FDTD) simulations 4.4. Finite difference time domain (FDTD) simulations CORRECT
Finite difference time domain (FDTD) simulations [86] allows to simulate the light interaction of particles with any shapes. This is
particularly interesting for non-spherical nanoparticles, such as nanocubes, nanoprisms or nanostars produced by facile chemical syn-
theses. The optical properties of single nanoparticles, i.e. the absorption, scattering and extinction cross sections, are easily computed
and the electric field enhancement on the nanoparticles are visualized. In order to compare the measured optical properties with the
simulation, the optical properties of nanoparticles distributed in thin film layer are calculated. In this configuration, the electromag-
netic coupling between the nanoparticles in the layer can be studied. FDTD simulations are a convenient tool to verify the optical
properties of complex samples, such as multilayers or structured surfaces. The main drawback of FDTD simulations is calculation
time. In order to increase the accuracy, especially for curved surfaces, very small mesh cells are chosen, leading to time consuming
computation. To counter this, a solution is to work with non-uniform meshed regions with a fine meshing around the nanoparticle,
as shown on Fig. 10a. A systematic two step analysis of nanoparticles can be efficiently used: first the nanoparticle alone is studied in
different media, such as water and polymer, then the nanoparticles are arranged in a periodic pattern. In the case of non-interacting
nanoparticles, in other words electromagnetically isolated particles from each other, the periodic pattern is sufficient. If the distance
between the nanoparticles is a few nanometers, the enhanced electric fields will couple and the localized plasmon resonance is af-
fected [87]. The study of the nanoparticle alone is mainly performed to obtain the absorption, scattering and extinction cross sections. For this, a total field scattered field source (TFSF) is used, as schematized on Fig. 10b. The electromagnetic radiation is along the blue
arrow and its polarization along the purple arrows. The absorption monitor is placed around the particle inside the source region,
region 1, and the scattering monitor is placed outside the source region, region 2. Outside the TFSF region, the incident fields are
subtracted from the total fields, i.e. only the field scattered by the particle remains.i Cii
The cross section is defined by the relation: UN
where P is the scattered (respectively absorbed) power and I is the source intensity. 4.3. Discrete dipole approximation (DDA) 4.3. Discrete dipole approximation (DDA) TED PRO
The absorption and scattering cross sections of arbitrary shaped nanoparticles can be numerically studied by using the discrete
dipole approximation (DDA) method. This method was first described in 1964 by DeVoe [81] to calculate the optical properties of
molecular aggregates and improved by Purcell and Pennymaker [82] to calculate the optical properties of interstellar dust. The DDA
method approximates the studied object with a cubic array of dipoles, each dipole having a defined polarizability. In other words, the
object is decomposed into a finite number of points for which the Maxwell equations are solved. The different dipole points interact,
i.e. they are electromagnetically coupled. Therefore, the DDA method is sometimes referred to as the coupled dipole approximation
(CDA). The accuracy of the calculation strongly depends on the number N of dipoles point chosen, especially for curved surfaces. Increasing N leads to accurate results and increases the computation time consequently. The DDA method is an accurate method to
calculate the absorption and scattering cross sections [83]. Dunklin et al. [84] recently showed, that the optical properties of different
densities of gold nanoparticles in polymer layers are successfully calculated by DDA. The method also allows a differentiation between
dipolar and quadrupolar contribution, which is not straightforward with FDTD simulation. Zhou et al. [85] used DDA calculations
to analyze the optical properties of silver nanocubes of edge sizes from 15 to 200nm. The physical origin of each resonance peak is
determined, i.e. dipolar and/or quadrupolar resonances, and their size dependence is analyzed. DDA calculations are a convenient
tool when used together with experimental measurements to identify the measured absorption and scattering peaks. 4.2. Mie theory An analytical
formula has been derived for ellipsoidal particles and is typically called Modified Long Wavelength Approximation (MLWA) [78–80]. The formula takes into account the two axes of the particle, which lead to two distinct resonance wavelengths. N
The polarizability for an ellipsoid of minor axis a and major axis b is: UN U
where ξ0 and Χ are size dependent variables. 13 L. Escoubas et al. L. Escoubas et al. L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx Progress in Quantum Electronics xxx (2019) xxx-xxx OO
The Mie theory offers simple equations to calculate the optical properties of nanospheres, but with the emergence of chemical and
lithography methods to produce differently shaped nanoparticles such as nanocubes and nanoprisms, the use of the Mie theory is not
sufficient anymore. For non-spherical nanoparticles, different methods are used. 4.4. Finite difference time domain (FDTD) simulations In the TFSF configuration, the σabs is simply the
complementary to the total cross section measured in the region 1. As only the scattered light reaches region 2, σscat is measured in
region 2. The extinction cross section is then the addition of the absorption and scattering cross sections: 14 14 L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx L. Escoubas et al. F
Generally, the normalized scattered (respectively absorbed) efficiency is used to remove the size dependence. The efficiency sim-
ply corresponds to the cross section normalized to the geometrical area A of the particle: OFi OF
Once the absorption and scattering peaks for every shape are computed, the electric field enhancement, around and in the particle,
are visualized to gain understanding on the origin and the nature of the peaks. ROO
Perfect silver 35nm edge size nanocubes in PVP matrix exhibit an absorption peak at the wavelength 350nm. Several hypotheses
are found in literature on the origin of this peak as shape dependent [88], quadrupole resonance [89], due to aggregates or to silver
interband transition [90]. Fig. 11 below is an example of the FDTD computed amplitude of the electric field enhancement on a 35nm
edge size silver cube in a PVP polymer matrix at λ=350nm. The enhancement is maximal at the corners of the cube. Therefore, we
can conclude that the peak is related to the shape of the particle.i PRO
Long et al. used the electric field enhancement visualization of silver nanospheres to investigate the lasing emission enhancement
at the exciting wavelengths. The simulations confirmed the coupling between the plasmonic resonance of the silver nanosphere and
the laser emission [91]. Sun et al. [92] analyzed the electric field enhancement of gold-silver core-shell nanorods deposited on dif-
ferent thicknesses of a PMMA layer. It was showed that increasing the thickness of the polymer layer has an influence on the electric
field enhancement and on the electric field distribution on the nanoparticle. The simulations allowed the authors to determine the
optimal PMMA thickness, 56nm, to maximize the electric field enhancement, 27-fold enhancement [92]. ED P
For systems where the nanoparticles do not interact, i.e. there is no electromagnetic coupling between the nanoparticles, the study
of the nanoparticle alone is sufficient. 4.4. Finite difference time domain (FDTD) simulations For systems where the nanoparticles interact with each other, such as in thin film layers of
randomly distributed nanoparticles, a second simulation step is necessary to study the effect of the coupling on the optical properties. Different simulation configurations are possible, as the nanoparticles can be distributed in a periodic lattice or pseudo-randomly. The
squared lattice distribution is the simplest to implement, as it can be automatically generated, and the fastest in calculation time, as
the calculation can be done on a single unit cell. Fig. 12 displays an example of a silver nanoprisms in a squared periodic pattern. The
distance between the particles is small enough for the enhanced electric fields to interact.i CTEi
For pseudo random distributions, different configurations have to be taken into account. Based on the periodic pattern of
nanoprisms displayed on Fig. 12, a pseudo random configuration could be obtained by either modifying the orientation of one or
more prisms. By turning a prism by 45° or 90°, the incident electromagnetic wave will not exalt the plasmonic resonance in the same
manner. An alternative consists on modifying the distance between the nanoparticles to have coupled interactions and uncoupled
interactions, i.e. nanoparticles behaving like single nanoparticles.i RECT
As an example of specific optical properties obtained from a random distribution of interacting nanoparticles, in a recent theo-
retical paper of B. X. Wang [93], a strong-backscattering phase function is studied. It is demonstrated that in particular conditions a
disordered medium composed of randomly distributed silicon nanoparticles exhibits a strongly negative scattering asymmetry in the
near infrared due to multiple light scattering. As the concentration of scattering particles rises, the backscattering is also enhanced. Predicting and controlling wave propagation in random particulate materials allows people to manipulate the scattering and absorp-
tion of radiation. It paves the way to applications such as imaging through turbid media or radiative cooling of coatings by efficient
reflection of incident solar power. RRl
To conclude, numerical studies are a convenient way to obtain fast results on the optical properties of nanoparticles and to study
their behavior either alone or when electromagnetic coupling occurs. 4.5. Characterization 4.5. Characterization UNCOR
Following the chosen approach, computations either precede experimental characterization or confirm them. Synthesized nanopar-
ticles can be optically characterized as prepared in solution and embedded in a polymer thin film layer. The optical measurements
of solutions and of thin films are similar. In the following, only thin films will be considered. In a first step the optical properties,
such as transmittance, total reflectance and diffuse reflectance, of the nanoparticles in different media are determined through spec-
trophotometric measurements. These quantities are only intensities and strongly dependent on the characteristics of the samples: the
thickness of the thin film layers, which changes with the deposition speed, the viscosity of the solution, the ambient temperature or
the substrate. Spectroscopic ellipsometry measurements are used to obtain the complex optical indices of the thin film layers, which
are independent on the thickness of the layer or the substrate. Spectroscopic ellipsometry is a powerful tool, which requires a general
understanding, i.e. the wavelength and broadening of the different absorption or reflection peaks of the samples, as the measured
data needs to be fitted with a dispersion model to obtain the optical indices. The general understanding is then either derived from
computation or spectrophotometric measurements. UN
4.6. Spectroscopic ellipsometry 4.7. The data fit CORREC
The optical model consists of mathematical laws, also called dispersion laws, describing the material of each layer. Once the right
optical model is found, iterations verifying the Kramers-Krönig relations are necessary to validate the consistency of the determined
indices [98]. Numerous dispersion laws, verifying the Kramers-Krönig relations, exist to account for multiple optical properties of
probed materials. Ground knowledge of the optical properties, as the absorbance, is therefore required to choose the right laws and
reduce the number of variables of the laws: the spectroscopic ellipsometry measurements are performed combined with spectropho-
tometer measurements or numerical calculations. The free variables are then used as fitting parameters to increase the agreement
of the calculated data with the experimental ones using the Levenberg-Marquardt method [99] as regression analysis. In the case
of visible light absorbing silver nanoparticles dispersed in a non-absorbing polymer layer, the dispersion model is composed of a
Cauchy law, accounting for the transparent non-absorbing polymer, and Gauss laws or Lorentz laws, accounting for the different ab-
sorption peaks of the plasmonic nanoparticles [100–103]. The suited optical model is chosen by analyzing the model that minimizes
the root-mean square error (RMSE) and maximize the coefficient of determination. Furthermore, the obtained indices will be used to
compute the reflectance R through a transfer matrix method. The computed R is then compared to the measured R to validate the
optical model. The difficulty in the data fit lies in the right choice of dispersion laws and in the number of parameters to vary for each
law: a model with one Cauchy law and one Gauss law has six variables. The Cauchy, Gauss and Lorentz laws are described in detail
in the following. UN
4.6. Spectroscopic ellipsometry an effective medium is sensed as sc PR
The embedded nanoparticles in the thin film layers are small compared to the wavelength of the light therefore the measurement
does not distinguish between the polymer and the nanoparticles, i.e. an effective medium is sensed as schematized in Fig. 14. PR
een the polymer and the nanoparticles, i.e. an effective medium is sensed as schematized in Fig. 14. TED P
The measured properties of the effective medium are an average of the properties of each material, i.e. the inclusions and the
polymer matrix. It is supposed to recreate the experimental values and simplify the calculations [95]. Si substrates are typically used
to maximize the optical index difference between the two layers. The substrate is assumed to be semi-infinite, i.e. there is no reflected
light at the backside of the substrate, and its optical indices are known. Layer 2 on Fig. 14 represents a homogeneous layer of effective
indices neff and keff, whose thickness is measured, for example, by a stylus profilometer. The described system has only two interfaces,
but this can also be generalized to a multilayered stack. In the case of a multilayered stack, the thickness of each deposited layer
has to be known. Furthermore, it is recommended, that the difference in optical indices of two neighboring layers should be large. If
the indices are similar, the phase difference between two layers will not be significant enough to be sensed. The angle at which the
measurement is suitably performed is chosen to be near the Brewster angle of the substrate to maximize the intensity of the reflected
light [96]. Typically, variable angle spectroscopic ellipsometry (VASE) increases the precision [97].ii UN
4.6. Spectroscopic ellipsometry U
Spectroscopic ellipsometry measurement is an indirect technique to determine either the complex optical indices or the thickness
of transparent or semi-transparent thin film layers. These two variables are linked: the knowledge of one is necessary to determine
the other. 15 L. Escoubas et al. L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx F
The knowledge of the complex refractive index allows computing the reflectance and transmittance of any layer thickness on any
substrate.i F
The complex refractive index
is the complex addition of the refractive index n and the extinction coefficient k:
Fur-
thermore, the complex refractive index is closely linked to the complex dielectric function:
[94]. complex refractive index
is the complex addition of the refractive index n and the extinction coefficien
e, the complex refractive index is closely linked to the complex dielectric function:
[94]. i
e, the complex refractive index is closely linked to the complex dielectric function:
[94]. OF
A spectroscopic ellipsometry setup is schematized on Fig. 13. The light emitted by the light source is linearly polarized by the
polarizer P. Upon reflecting onto the sample, the light becomes elliptically polarized.l OOl
To compare the reflected light with the incident light, the light is once again linearly polarized by passing through the analyzer
A before reaching the detector. In other words, the measurement consists of the analysis of the polarization change of a reflected
light beam by a thin film layer. This is translated into a change of Fresnel reflection coefficients rs and rp, which is measured by the
quantity ρ: PRO
where ψ symbolizes the amplitude change and Δ the phase difference of the reflected light compared to the incident light. Each inter-
face yields a different result.i PRO
where ψ symbolizes the amplitude change and Δ the phase difference of the reflected light compared to the incident light. Each inter-
face yields a different result.i PR
rticles in the thin film layers are small compared to the wavelength of the light therefore the measuremen PR
ded nanoparticles in the thin film layers are small compared to the wavelength of the light therefore the m
guish between the polymer and the nanoparticles, i.e. an effective medium is sensed as schematized in Fig. PR
The embedded nanoparticles in the thin film layers are small compared to the wavelength of the ligh
does not distinguish between the polymer and the nanoparticles, i.e. 4.10. Lorentz law ECT
Based on the simple model of a mass and spring system, the Lorentz model describes the classical theory of light-matter interaction
and the frequency dependent polarization due to bound charges. The incident electromagnetic field induces vibrations of the electrons
behaving as harmonic oscillators. EC
g
The Lorentz oscillator (LO) is characterized by an energy E0, an oscillator strength f and a broadening Γ RREC OR
The use of the Lorentz law therefore adds two variables to the analysis. OR
A constant ε∞common to all the complex dielectric functions, e.g. LO and GO, is added to describe the oscillators outside of the
measured spectral domain. This constant is another variable added to the optical model. 4.9. Gauss law 4.9. Gauss law RO
The Gauss law is defined as an oscillator centered at an energy E0, of amplitude Amp and broadening Br. The dielectric function is
then defined as: RO
ss law is defined as an oscillator centered at an energy E0, of amplitude Amp and broadening Br. The dielei
d as: RO
as an oscillator centered at an energy E0, of amplitude Amp and broadening Br. The dielectric function is D PR ED
where
is the full width at half maximum of the oscillator and D is the Dawson's integral [104] TED
When describing a localized surface plasmon resonance absorption peak with a Gauss oscillator (GO), the energy EO is related to
the measured plasmon resonance energy of the nanoparticles. Amp is related to the intensity and Br to the width of the absorption
peak. The use of the Gauss law therefore adds two variables to the analysis. 4.8. Cauchy law NC
The Cauchy law typically describes transparent materials: U UNC UNC U
where the parameter n∞is dimensionless and n(λ) tends to n∞at high energy, A and B characteriz U
where the parameter n∞is dimensionless and n(λ) tends to n∞at high energy, A and B characterize the curvature and the 16 16 Progress in Quantum Electronics xxx (2019) xxx-xxx L. Escoubas et al. F
amplitude in the visible and the UV respectively. The parameters C, D and E are similar to n∞, A and B for the extinction coefficient
k.i F
For non-absorbing materials, the extinction coefficient is simply set to zero over the whole spectral range. The parameters of a
non-absorbing Cauchy law, as used in the following, are: OOF O
The use of the Cauchy law therefore adds three variables to the analysis. OO
The use of the Cauchy law therefore adds three variables to the analysis. 4 9 G
l The use of the Cauchy law therefore adds three variables to the analysis. 4.11. Effective medium theory 4.11. Effective medium theory UNCO
When using an effective medium approach, the samples composed of nanoparticles embedded in a host material are considered
as a homogeneous material characterized by an effective medium εeff The Maxwell Garnett formula shown below, allows linking the
effective medium dielectric function to the dielectric functions of each material constituting the effective medium [105,106]. This
approach takes into account the first order approximation of the Rayleigh formulae. This simple theory, compared to other such as
described in Ref. [107], does not take into account the multiple scattering of the particles in the layer and the polarization of the
light. The multiple scattering induces macroscopic optical behavior and is therefore linked to a property of the effective medium. The
random orientation of the nanoparticles in the layers should make the optical properties polarization independent. For the results
presented in the fourth part of this article, the polymer host matrix is seen as a medium and the silver nanoparticles as inclusions
within the medium, therefore: U 17 17 L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx F
where δi is the volume fraction of the inclusions and εi the dielectric function of the inclusion, εm is the dielectric function of the
medium and εeff the dielectric function of the effective medium. For the equation to be valid, the volume fraction should not exceed
one third [81]. F
where δi is the volume fraction of the inclusions and εi the dielectric function of the inclusion, εm is the dielectric function of the
medium and εeff the dielectric function of the effective medium. For the equation to be valid, the volume fraction should not exceed
one third [81]. OF
Furthermore, the Maxwell-Garnett formula is only valid for spherical inclusions. For spheroids, a factor of depolarization has to be
taken into account [99,105,108]. Analytical expressions for other shapes, such as nanoprisms and nanocubes, have not been described
yet in the literature. ROO
To conclude, there are a variety of modeling tools to study the optical properties of nanoparticles. FDTD calculations are used for
nanoparticles of any shape in any medium, but it must be noted, that the calculation times are long. Then, the experimental character-
ization method of spectroscopic ellipsometry is described. The measurement is an indirect technique to determine the effective optical
indices of a layer. 5.1. Optical indices n and k D P
The knowledge of the optical indices of a thin film layer, determined through spectroscopic ellipsometry, allows a complete un-
derstanding of the interaction of light with the layer. In the scope of a highly efficient light absorber, the knowledge of the optical
indices of a single layer allows the calculation of multilayers to optimize the absorption. As described above, the determination of
the optical indices depends on the right choice of the dispersion model. Different samples and their adapted dispersion models are
discussed below. 4.11. Effective medium theory In order to obtain the optical indices, it is necessary to derive an optical model composed on several dispersion
laws. In the following chapter, silver nanospheres and nanocubes randomly embedded in a PVP layer are studied with a suited optical
model. 5. Optical properties of plasmonic nanoparticles absorbers 5.1. Optical indices n and k EC
5.3. Non-electromagnetically coupled nanospheres and nanocubes in PVP EC
5.3. Non-electromagnetically coupled nanospheres and nanocubes in PVP NCORRE
One interesting example concerns the spectroscopic ellipsometry characterization of a blend of silver nanospheres and silver
nanocubes embedded in a PVP thin film. The silver nanospheres in PVP and nanocubes in PVP are firstly characterized independently. Then both shapes, nanospheres and nanocubes, are randomly distributed within the same thin film. In the scope of spectroscopic ellip-
sometry measurements, the knowledge of the optical properties of each shape is necessary in order to choose an appropriated optical
model. Indeed, the model is composed of a Cauchy law, accounting for the optical properties of the non-absorbing host matrix (PVP
in this example), and several Lorentz laws centered at the localized plasmon resonance peaks of the nanospheres and nanocubes. Fig. 15a describes the optical indices of such a layer of nanospheres and nanocubes. The peak centered at 420nm is associated with the
dipolar resonance of the nanospheres, while dipolar resonance of the nanocubes is situated at 450nm. The peak centered at 350nm is
linked to the cubic shape of the nanocubes. The intensity of each peak is related to the concentration of each type of nanoparticles. For
example, by adding more nanocubes to the layer, the intensity of the peaks at 350nm and 450nm is increased. As the nanoparticles
in the layer are not electromagnetically interacting, the optical model used to fit the spectroscopic ellipsometry data is a simple ad-
dition of the model used for each geometry, nanosphere or nanocube. This is visualized on Fig. 15b, where the normalized extinction
coefficient of the layer containing nanospheres and nanocubes is compared to the individual extinction coefficients of nanospheres
and nanocubes in separated layers. Electromagnetic coupling between particles in a layer would occur if the distance between the
particles is in the order of a few nanometers. This can be achieved by increasing the concentration of nanoparticles in the layer or by
forming aggregates [114,115]. The electromagnetic coupling then induces a shifting of the plasmon resonances. UN
The values of the optical indices n and k (Fig. 15a) are directly linked to the concentration of nanoparticles within the PVP thin
film. By increasing the concentration, the absorption at the plasmonic resonance wavelength is increased. In this example, as shown in
Fig. 15b, using a blend with two differently shaped nanoparticles, nanospheres and nanocubes, allows a broadening of the absorption
from 400 to 600nm. 5.2. The dispersion models CTE
The spectroscopic ellipsometry measurements of noble metal nanoparticles on substrates and embedded within various dielectric
matrices are studied by several authors [100–103,109–111]. Concerning the non-absorbing host matrix, the use of a Cauchy law is
well established. This is not the case for the optical properties of noble metal nanoparticles as shown on Table 1, which lists the dif-
ferent laws used and their physical meaning. CT
overview of the different laws used and a guideline for determining the optical model. Depending on th
mple, the right model has to be chosen. CT
Table 1 gives a broad overview of the different laws used and a guideline for determining the opti
structure of the probed sample, the right model has to be chosen. EC
In the following, the model composed of a Cauchy law and several Lorentz laws is described in more EC
In the following, the model composed of a Cauchy law and several Lorentz laws is described in more detail. 5.4. Interaction of light between nanoparticles D PROOF
Controlling the scattered field of nanoparticles in interaction is of high interest for new applications needing dynamic devices such
as in optical communications and in the foreseen optical computing. Components such as the all-optical nano-switch based on the
accurate control of the interaction between neighboring scatterers, semiconductor or dielectric nanoparticles, have been predicted
and experimentally demonstrated [117–119]. The spatial distribution of the scattered fields of the nanoparticle and the distance be-
tween them allows controlling the electromagnetic interaction of the nanoparticles. Indeed, at the beginning of the 1980s, Kerker et
al. published the basis of the modeling of interacting scatterers in specific conditions [120]. Considering spherical particles exhibiting
both electric and magnetic responses, in the Rayleigh limit, they studied the relation between the scattering coefficients of the Mie
theory, which is a multipolar decomposition to calculate the scattering and absorption cross sections. This decomposition involves
coefficient associated to the electric behavior and other coefficients corresponding to the magnetic one. For dipole-like particles, only
the two first Mie coefficients, one electric and one magnetic, are not negligible. Kerker's conditions correspond to interferences of
the dipole scattered fields producing a zero scattering in either the forward or the backward direction. A directional control over the
global scattered field can be achieved using the coherent interaction between electric and magnetic resonances. The shape [121] and
the size [122] of the nanoparticles and their distance are the main parameters governing Kerker's conditions. In a recent paper by R. Vergaz [123], two nanoparticles satisfying Kerker's conditions in an optimal configuration are presented (see Figs. 16 and 17). Indeed
with this dimer of nanoparticles, two interferential effects are possible, one between the scattered field of each nanoparticle and the
background-incoming field and one between the scattered fields of each component of the dimer. Thus constructive and destructive
interferences appear depending on the distance between the nanoparticles. Then one can control the spatial distribution of light, and
more precisely obtain a maximum variation of light intensity in the gap region, by manipulating these interferences. This dimer con-
figuration can be used as the base for the design of the all-optical nano-switch. CTED
g
g
p
Fig. 16 describes the system composed of two spherical silicon nanoparticles separated by a distance d, one particle having a radius
R1 of 82nm and a second one with a radius R2 of 97nm. 5.5. Collective behavior of aggregates NCOR
One example of creating collective response of silver nanoparticle in a controlled way was presented by G. K. Laxminarayana et
al. [124]. A novel, modular approach to Ag nanoparticle self-assembly utilizes polymer templating to control meta-atom size and
geometry. Colloidal nano-crystals (NCs) are deposited onto the polymer support. They serve as the initial binding platform and are
called NCo. Using solvent or thermal annealing of the polymer allows then immobilizing and embedding the NCo into the polymer. Their exposed surfaces are chemically modified with a covalent molecular linker such as a dithiol. Finally, by introducing a second
particle (NC1), which reacts with the molecular linker, NC meta-atoms are formed. Repeating this protocol allows producing hierar-
chical or dendritic NC motifs (see Fig. 18A–C). Horizontal and vertical nanocube dimers were successfully fabricated with remarkably
high yield. As it can be seen in Fig. 18 (G-I), the assembly of the Ag nanocubes into aggregates of controlled number of cubes and
organization generates specific optical responses related to their collective properties. EC
5.3. Non-electromagnetically coupled nanospheres and nanocubes in PVP U
The knowledge of such optical indices allows one to design the structure depending on the applications. 18 Progress in Quantum Electronics xxx (2019) xxx-xxx L. Escoubas et al. L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx 5.4. Interaction of light between nanoparticles The radius has been chosen such that the first particle (R1=82nm) satisfies
the zero-backward (ZB) scattering condition (see Fig. 156 on the left), while the second one (R2=97nm) fulfills the minimum for-
ward scattering condition (MF) at the incident wavelength 700nm (see Fig. 16a on the right), but there is still an appreciable electric
field (red color of the sphere on the illuminated side). Thus the scattered radiation by the nanoparticles can be directed very selec-
tively, which allows controlling the overlapping of the fields in a dimer: as a function of the direction of illumination of the dimer, the
scattered light can be directed outwards or towards the gap between the nanoparticles. Thus, depending on the illumination side of
the dimer, a maximum or a minimum of the scattered field could be observed in gap. For high values of the gap “d”, nanoparticles are
considered as isolated and there is no interaction between them but smaller distances allow interferential phenomena to be obtained.i REC
In the configuration of Fig. 16a, with light impinging the particle with the largest radius (97nm) from the right, the electromag-
netic fields scattered by the particles in the gap are low and as seen in Fig. 17a and b, there is now interference phenomena in the
gap. On the other hand, Fig. 17c, which is corresponding to Fig. 16b with light impinging the particle with the smallest radius (82nm)
from the left, we observe strong electromagnetic fields scattered by the particles in the gap, but there is a destructive interference
phenomena in the gap. By changing the “d” value from 120nm to 445nm, constructive interference can be obtained in the gap as
shown in Fig. 17d. Progress in Quantum Electronics xxx (2019) xxx-xxx 6.2. Photodetection D PR
T. Maier and H. Brueckl [127] associate a microbolometer, made from a Si3Nx membrane, with a metamaterial absorbing incident
wavelengths in a resonance domain. The metamaterial is directly built above the microbolometer membrane (Fig. 19). It consists of
a metal layer/dielectric (Si3Nx)/periodic metal patch structure. The resonance peaks of this structure allow the microbolometer to
become wavelength selective by only optimizing the geometry of the metamaterial elements. This selectivity can be tuned between
2.9μm and 7.7μm by adjusting the dimensions of the metal patchs and their periodicity. The absorption peak can reach up to 88%. The structure is very weakly sensitive to the angle of incidence of radiation because the resonance mode used is of the gap-plasmon
type [128] (see part 1). In addition, the use of a metallic layer covering the entire surface changes the conduction heat in the structure
by increasing its heat capacity, which leads to an improvement of the response time of the microbolometer. ECTED
After excitation, surface plasmons can lose their energy in the form of photon re-emission or non-radiatively in the form of en-
ergetic electrons or 'hot electrons’. Recently, hot carriers (hot electrons) have sparked a strong interest because they can be useful
for many applications such as photodetection, photovoltaic devices, photocatalysis or surface imaging. Hot carriers are used typically
to the photodetection with a structure of Schottky type barrier consisting of a thin metallic layer in contact with a semiconductor
material. In the article by W. Li and J. Valentine [129], the authors study the addition of a perfectly absorbing metamaterial at the
top of the Schottky barrier (Fig. 20). Thus, they are able to strongly increase the photoresponsivity of the device and obtain a sili-
con photodetector sensitive in the infrared domain well below the silicon bandgap energy. In addition, the photodetector response is
broadband with a photoresponsivity larger than 1.8mA/W for wavelengths ranging from 1200nm to 1500nm and insensitive to the
polarization state through the use of square resonators. 6.1. Spectral filtering 6.1. Spectral filtering ROOF
It is possible to create artificial materials able to transmit the visible light and totally absorb the infrared (IR) one. Applications
concerns IR blocking plasmonic glass windows. In the articles presented by Y. Qin et al. [125] and L.V. Besteiro et al. [126], the
authors use a hexagonal matrix of polystyrene latex spherical nanoparticles (PSL) assembled on a flat silicon substrate. They first
perform a reactive ion etching (RIE) process to tune the PSL shape, followed by a magnetron sputtering process of a thin copper
or aluminum layer, covering the PSL. By then dissolving the PSL in toluene, they obtain a solution containing copper or aluminum
‘nanocups’. These nanocups, which metal thickness can be controlled by the sputtering time and curvature by the dimensions of the
polystyrene nanospheres, exhibit localized plasmons. By adjusting plasmonic absorption peaks, through the nanocup geometric para-
meters, authors can thus control the spectral transmission of the windows on which the nanocups are deposited and thus maintain
transparency properties in the visible domain while blocking IR. NC
6. Few applications of plasmonic light absorbers UN
Among the very many articles dealing with localized plasmons and surface plasmons, we highlight here a few applications of par-
ticular interest and dealing with spectral filtering and photodetection. 19 Progress in Quantum Electronics xxx (2019) xxx-xxx L. Escoubas et al. 7. Conclusion NCORRE
Beyond simply absorbing light and dissipating this energy into thin composite layers, new applications, such as the all-optical
nano-switch previously described, are emerging and are using the coupling of light with nanoparticles. These nanoparticles may ex-
hibit various shapes, be alone or apart from each other and therefore without electromagnetic interaction, or even very close in inter-
action at a controlled distance and so in resonant electromagnetic regime. They can be aggregated and their morphology controlled
by chemical routes, in dimer and multimer forms. They can be in interaction with the substrates and thus constituting Fabry-Perot
resonators. These nanoparticles can also be metallic and thus allowing localized plasmons to be excited or be used to generate gap
plasmons by the electromagnetic interaction with planar substrates. We also find dielectric nanoparticles presenting electric or mag-
netic responses under light excitation and leading to remarkable effects under certain conditions, for example in the Kerker's condi-
tions. Thus, by combining the expertise of chemists, able to find original synthesis routes to create, to shape, and to connect these
nanoparticles together or with substrates of different natures, and the analysis and models of physicists to understand the interaction
of light with these nano-objects, it is already possible to create original components, for example the rectenna or ‘hot electrons’ pho-
todetector which directly transforms the light energy into an electrical current. But in the future, it will be possible to create much
more complex objects, that we probably can not imagine yet, and whose operating principles will certainly rely on controlled assem-
blies of nanoparticles with not only specific optical and magnetic properties but also with quantum properties. Uncited references [63]. 20 L. Escoubas et al. Progress in Quantum Electronics xxx (2019) xxx-xxx Acknowledgments OF
This work was carried out thanks to the support of the A*MIDEX project (No. ANR-11-IDEX-0001-02) funded by the “Investisse-
ments d’Avenir” French Government program and managed by the French National Research Agency (ANR). We also thank the Di-
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https://openalex.org/W2547996674
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https://orca.cardiff.ac.uk/id/eprint/95063/3/SPILIOTI_95063_post-print.pdf
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English
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Media convergence and publicness: Towards a modular and iterative approach to online research ethics
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Applied linguistics review
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public-domain
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ORCA – Online Research @ Cardiff This is an Open Access document downloaded from ORCA, Cardiff University's
institutional repository:https://orca.cardiff.ac.uk/id/eprint/95063/ This is an Open Access document downloaded from ORCA, Cardiff University's
institutional repository:https://orca.cardiff.ac.uk/id/eprint/95063/ This is the author’s version of a work that was submitted to / accepted for
publication. This is the author’s version of a work that was submitted to / accepted for
publication. Citation for final published version: Publishers page: http://dx.doi.org/10.1515/applirev-2016-1035 Publishers page: http://dx.doi.org/10.1515/applirev-2016-1035 Please note:
Changes m ade as a result of publishing processes such as copy-editing, formatting
and page num bers m ay not be reflected in this version. For the definitive version of
this publication, please refer to the published source. You a re advised to consult the
publisher’s version if you wish to cite this paper. Citation for final published version: Spilioti, Tereza 2017. Media convergence and publicness: towards a modular and
iterative approach to online research ethics. Applied Linguistics Review 8 (2-3) , pp. 191-212. 10.1515/applirev-2016-1035 Spilioti, Tereza 2017. Media convergence and publicness: towards a modular and
iterative approach to online research ethics. Applied Linguistics Review 8 (2-3) , pp. 191-212. 10.1515/applirev-2016-1035 Publishers page: http://dx.doi.org/10.1515/applirev-2016-1035 Tereza Spilioti (Cardiff University) FINAL MANUSCRIPT, accepted September 2016 FINAL MANUSCRIPT, accepted September 2016 Abstract: The aim of the article is to build a bridge between assumptions about publicness and ethics in
traditional (mass) media research and similar issues pertaining to research ethics in so-called new media
environments. The article starts off with unpacking ‘publicness’ as defined in authoritative ethical guidelines that
regulate research on (and through) media. It points to the challenges media convergence - and, particularly, the
increasingly multimodal, multiauthored and multimedial content of websites - have brought to perceptions of
publicness, as previously understood in mass media research. With reference to language-focused research on
multilingual digital writing in such contexts, I critically engage with ethical tensions related to collecting and
analysing internet data, on the one hand, and presenting and publishing data extracts from new media contexts,
on the other. Drawing on modularity as a key organising principle of web design and discourse (Androutsopoulos
2010: 208; Pauwels 2012: 251), the article proposes a modular and iterative approach to research ethics that takes
into account the complex and fluid configuration of web environments and attends to the conditions of multiple
authorship and multiple publics that are increasingly typical of such contexts. Keywords: Media convergence, research ethics, publicness, privacy, consent, copyright, modularity, websites Please note: p
y
p p
This version is being m ade available in accordance with publisher policies. See
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publications m ade available in ORCA are retained by the copyright holders. Media convergence and publicness: Towards a modular and iterative approach
to online research ethics Tereza Spilioti (Cardiff University) 2. Mass media and the ‘public domain’: Research ethics Any empirical research endeavour involves information management, typically referred to as
data collection, analysis and presentation in academic discourse. In the discipline of applied
linguistics, in particular, such information can be intimately linked with the persons who
provide, produce and consume texts the researcher is interested in. It is this relationship
between the researcher and the persons researched that is primarily facilitated and regulated by
the key principles of research ethics, such as respect to human dignity, autonomy, and safety,
as well as protection from harm (BAAL 2016; AoIR 2012; ESRC 2015). Acknowledging all
stages of the research process, from initial design to dissemination (AoIR 2012: 3), we realise
that ethical considerations do not only target the process of data collection and analysis (i.e. informed consent to access, record, store and analyse language/texts) but they also concern the
process of data presentation and dissemination (i.e. consent or permission to quote and present
language/texts). With respect to the above ethical considerations, media discourse, available in various forms
of published press, radio, television and film, has attracted the attention of language and
communication scholars, as mass media are deemed to offer ample and convenient access to
language data (see Yates 1996: 30, for a similar argument about new media). With media
discourse being produced for purposes other than language research and arguably readily
accessible, research using such sources seems to kill two birds with one stone: eliminating any
influence on the data caused by the presence of the researcher - what Labov (1972) called the
‘observer’s paradox’ - and quickly passing through ethical scrutiny from institutional review
panels. Understanding mass media research as low - or no - risk of harming the persons researched
draws on a key premise that distinguishes personal data from material already available in the
public domain. To quote the UK’s Economic and Social Research Council framework for
research ethics, ‘While data collected and stored as a record at an individual level are considered personal data, material already
in the public domain are not. 1. Introduction The aim of this article is to build a bridge between assumptions about publicness and research
ethics in traditional (mass) media research (e.g. newspapers, TV and radio) and similar issues
pertaining to research in so-called ‘new media’ environments. The reason for doing so is two-
fold: first, discussions about online ethics are inevitably informed by perceptions of publicness
previously articulated in relation to research on mass media; and, second, convergence of
multiple modes and media in everyday communication makes ‘old-new’ dichotomies
particularly difficult to keep separate. By moving beyond the ‘old-new’ dichotomy in relation
to research ethics in media investigation, the article complements previous work that offers
historicised understandings of textual and mediated discourse practices (e.g. Herring 2004: 3,
Jones 2011: 336), genres (e.g. Heyd 2016: 89-90), and media ideologies (e.g. Thurlow 2013:
243; Spilioti 2016: 134). More specifically, understanding how publicness has been defined
and applied to the process of researching mass media discourse paves the way for
contextualising and critically revisiting ethical debates about collecting and analysing internet
data, on the one hand, and presenting and publishing data extracts from new media contexts,
on the other. Media convergence, as a key property of new media environments (cf. ‘convergent media
computer-mediated communication’ Herring 2013: 4), challenges assumptions about
publicness. In particular, the study of websites, which increasingly include multimodal,
multiauthored and multimedial content, questions any assumed clear-cut boundaries between
public and personal data. With reference to language-focused research on multilingual digital
writing in such contexts, I critically engage with the challenges to the process of ethical
decision-making and the potential risks for internet users of a priori assumed public-private
dichotomies. Drawing on modularity as a key organising principle of web design and discourse
(Androutsopoulos 2010: 208; Pauwels 2012: 251), the article proposes a modular and iterative
approach to research ethics that takes into account the complex and fluid configuration of web 1 environments and attends to the conditions of multiple authorship and multiple publics that are
increasingly typical of such contexts. 2. Mass media and the ‘public domain’: Research ethics For example, published biographies, newspaper accounts of an individual’s activities
and published minutes of a meeting would not be considered “personal data” requiring ethics review, nor would
interviews broadcast on radio or television or online, nor diaries or letters in the public domain.’ (ESRC 2015: 12) Unpacking the notion of ‘already available in the public domain’ as used for press and
broadcasting material, it appears that publicness, here, is understood in terms of three key
assumptions. First, publicness is associated with a domain, i.e. a space that makes such material
available and, thus, accessible to the wider public, including researchers (see also Giaxoglou
this issue). Second, publicness is conceived in terms of the material’s purpose and targeted
audience: material is produced to be consumed by the public, understood in the context of
broadcasting as a mass and unidentified audience (cf. the ‘broadcast audience’ Marwick and
boyd 2011: 128-129). Third, publicness is associated with the roles and relevant identities of
the authors of such media texts: for example, media professionals, politicians, and sports
figures are usually invoked in their professional or public roles in mass media contexts. 2 2 However, there are cases where the three aforementioned assumptions may not justify media
material as public. One is programmes that are referred to as ‘public or audience participation
programmes’ (Livingstone & Lunt 1994; Thornborrow 2015). Radio phone-ins and reality TV
shows (like Big Brother or the Jeremy Kyle Show) involve ordinary people (i.e. non-media or
non-expert professionals) who often disclose personal and sensitive information about
themselves. In such cases, researchers arguably deal with what is, essentially, mediated
personal data, if the latter is defined as ‘information relating to an identifiable living individual’
(ESRC 2015: 23). Thus far, though, such material is also considered low risk, given that ‘the
information contained in the personal data has been made public as a result of steps deliberately
taken by the data subject’ (UK Data Protection Act 1998). It can be argued that people who
willingly come forward, agree to be auditioned, enter into contractual agreements with media
organisations and put themselves in front of open microphones and TV cameras have made
decisions that involve assessing, as well as confronting, the publicness of such media
interactions. 2. Mass media and the ‘public domain’: Research ethics Although one can never be sure about the intentions of such individuals or their
understanding of the potential (harmful) impact of public sharing of their personal life on air,
researchers into media discourse have long considered such interactions as public. Here,
publicness also invokes assumptions about the speakers’ or authors’ agency in making such
material accessible in the public space and to be consumed by the public. Against this backdrop of current authoritative ethical guidelines, the media discourse
researcher does not need to seek informed consent for collecting and analysing such material. Her ethical stance involves primarily issues related to the process of data presentation and
dissemination, i.e. copyright and respect to intellectual property. In the case of press, TV and
radio, ownership of published material lies primarily with the media organisation. As a result,
appropriate attribution of material quoted or analysed, as well as respect for restrictions
regarding the legal reproduction of media content, constitute the main ethical concerns of the
media discourse analyst. To what extent, though, does increasing media convergence challenge
current perceptions of publicness and, thus, call for revisiting ethical decision-making in
language-focused media research? After defining media convergence, the following section
questions assumptions of publicness as material that are (i) accessible in a domain, (ii)
produced for a mass public, (iii) authored by public or professional figures and/or (iv) the result
of deliberate agentive decisions of individuals. 3. Media convergence: Questioning ‘old-new’ dichotomies and ‘publicness’ Media convergence is a key phenomenon in the contemporary mediascape. From a
technological perspective, convergence refers to the merging of previously distinct
technologies within a single device or media platform. As a sociocultural phenomenon, Jenkins
(2006: 2) defines convergence as ‘the flow of content across multiple media platforms, the
cooperation between multiple media industries, and the migratory behaviour of media
audiences who would go almost everywhere in search of the kinds of entertainment experiences
they wanted’. In other words, convergence resists dichotomies between ‘old’ (e.g. press and
broadcasting) and ‘new’ (e.g. email, online forums, Twitter, Facebook, etc) media, by
foregrounding the increasing integration of multiple media platforms and the mobility of
content across media environments. In the context of broadcasting media, for example, there
are three key ways in which intersections between ‘old’ and ‘new’ media manifest. Firstly,
public participation from TV and radio audiences is no longer limited to telephone calls or to
the odd appearances of lay people in talk shows; instead, broadcast programmes increasingly
rely on digital technologies for interaction with their audience and often embed emails, tweets, 3 and other digital texts in the flow of the TV or radio programme (Thornborrow 2015). Secondly, the public engages with ‘old’ media snippets across digital platforms through
commenting on, copying-and-pasting, liking, sharing, editing and remixing TV or radio
material (e.g. Page 2012, Georgakopoulou 2014). Thirdly, and primarily discussed in this
article, media industries work together to augment the inter-medial presence of news
corporations, TV and radio programmes through dedicated websites, Twitter and Facebook
profiles, used both for (re)broadcasting content and interacting with different audiences. The challenges media convergence raises for researchers studying language use online have
been repeatedly pointed out in the literature (e.g. Androutsopoulos 2011: 281; Herring 2013:
5; Bolander & Locher 2014: 16; Georgakopoulou & Spilioti 2016: 4). Among the key
methodological and theoretical issues noted are: (i) the need for a methodological framework
appropriate for analysis of ‘media coactivity’ (Herring 2013) or multitasking across and within
media; (ii) attending to multimodality, as the range of available semiotic resources and their
potential combinations have dramatically increased; and (iii) orienting to multiauthorship
processes, as multiple participants are involved in the production of media content and any
boundaries between media producers and consumers become increasingly blurred (cf. ‘prosumer’ Ritzer & Jurgenson 2010). 3. Media convergence: Questioning ‘old-new’ dichotomies and ‘publicness’ From a research ethics perspective, media convergence – together with the increasingly
multimedial, multimodal, and multiauthored environments it affords – resists assumptions
about media content as something available in a domain, i.e. given, static, located in a particular
space, and, thus, accessible or amenable for collection and recording. Instead, digital content
is mobile and moves – or, ‘flows’ in Jenkins’s (2006) words – across multiple sites for media
engagement and activity. How can one discern whether this circulating and mobile content has
been produced for a mass and unidentified public – if such a public exists in internet contexts? In a similar vein, how can one identify certain individuals as authors or discern the relevance
of their professional or public roles to the communicative context, especially in circumstances
of multiauthorship production where boundaries between content producers and consumers are
no longer fixed or static? Last, but not least, how easy or clear is it for the ethical researcher to
assess individual or deliberate agency in making personal content public, especially in
environments where, as we will see, social media plug-ins may create conditions of automatic
disclosure? These questions unsettle the key assumptions that arguably underpin established
distinctions between personal data and material available in the public domain, complicating
ethical decision-making, on the one hand, and questioning the scope of applying this distinction
to digital contexts, on the other. With reference to research on multilingual writing in digital environments, this article revisits
the above questions in the context of websites that bring together content produced by multiple
authors, for multiple publics and through combination of multiple modes and media. As will
be shown, assumptions about the publicness of perceived public facing websites can be
nuanced through attention to the ways in which individual areas, or modules, of a website are
organised, combined, and discursively constructed by – and for – different social actors. 4. Websites as sources for research on multilingual writing Unlike social network sites or personal websites as sources for language-focused research,
official websites run by institutions and professional organizations arguably represent a low
risk digital environment from the point of view of research ethics. With respect to criteria for 4 publicness, material embedded in such websites can be considered to be ‘already in the public
domain’ (ESRC 2015: 12), as the web domain is created and regulated by relevant professionals
(i.e. web designers and organization represented), with content produced for public or mass
consumption. Nevertheless, demonstrating shortcomings of the public-personal data distinction
even in these less controversial areas can be revealing of the need to radically rethink and move
beyond received dichotomies. As mentioned in the previous section, a key factor in challenging current ideas about publicness
and ethics is media convergence. In the context of broadcast organizations, convergence
between the so-called ‘old and new’ media is manifest, among others, in websites dedicated to
popular TV and radio programmes. Such websites typically have the following purposes: to
provide the programme’s audience with further opportunities to (re)consume broadcast
material, to enable them to engage with a wealth of other material relevant to the show’s topic
(e.g. alternative news sources) and to interact with media professionals (e.g. show presenters)
and other audience members. These purposes usually shape - and are fulfilled in - two distinct
communicative spaces: (i) an edited web space that features content designed and/or selected
for (re)broadcasting to TV/radio and web audiences (cf. section 5.1) and (ii) an interactive
space that includes messages posted by visitors to the website who often identify themselves
as audience members of the particular programme (cf. section 5.2). Content in both areas is
highly dynamic and volatile, with material and messages updated (i.e. added or deleted) and
reconfigured on a regular basis. From the perspective of language-focused research, the conditions of mobility,
multiauthorship, multimodality, and multimediality afforded in such environments often result
in the mobilization and, at times, strategic and reflexive configuration of multiple linguistic
resources, such as styles, registers, codes, etc (cf. Deumert 2014; Tagg 2015). For that reason,
the developing field of the sociolinguistics of writing acknowledges the central role of research
on digitally-mediated communication in reconceptualising writing (Lillis & McKinney 2013:
421-424) and pushing written multilingual discourse from the periphery to the core of
sociolinguistic research on multilingualism (Sebba 2013: 98-99). 4.1. ‘Greekophrenia’ and multilingual writing online interactive games,
links to external websites about recommended books) was not included; visitor comments were
nevertheless retained as a key feature in the sub-pages archiving the radio and TV episodes. Since December 2015, the comments section is no longer available and the main navigation
hyperlinks have changed, though with content from different media sources still featuring
across the website. Similar to other websites dedicated to TV and radio programmes, the
Greekophrenia webpages combine spaces for content (re)broadcast and interaction with/among
audience members, include material authored by (and for) different people, invite visitors to
engage with multiple activities (reading, listening, watching, gaming, commenting), and embed
volatile content that is added, and deleted, on a regular basis. In other words, content is far from
static resulting in ethical tensions for the researcher who deals with sources whose status of
public accessibility shifts through the different stages of the research process. For the language-focused researcher, the Greekophrenia website displays language hybridity
in the form of mixing Greek and English elements. Unlike the pattern of English/national
language bilingualism, identified by Androutsopoulos (2012) as ‘English-on-top’, in German
and Greek websites, English is represented through marked visual and graphic (e.g. typographic) resources, such as archaic typefaces and Greek alphabet letters. Parallel to the
radio stylizations of (Greek-accented) English, the website documents stylistic variation in
written representations of English and Greek evident not only in the edited web content but
also in comments by fans. In order to address such representations of written variation and
multilingual writing, my research focuses on how these resources are strategically deployed in
the different areas of the website and how they are used (reacted to, and interpreted) by the
range of content contributors to the website. In terms of research ethics, when one makes the transition from researching a broadcast radio
programme to examining webpages, particularly those containing comments from fans, is one
still dealing with material already available in the public domain? Considering that there are
usually two distinct communicative spaces in such websites, we might want to address this
question separately in each area. The edited web space includes content that is accessible to all
internet users and intended for public distribution and mass consumption. It also undergoes
processes of heavy editing, selection and artful presentation in line with the professional
practices and standards of relevant industries. 1 Since comments are no longer accessible or available, as they have disappeared from the site, I am offering,
here, constructed examples that echo the content and tone of messages appearing in that section: 4.1. ‘Greekophrenia’ and multilingual writing online To illustrate the challenges to ethical decision-making and the fuzzy boundaries of publicness
when studying multilingual writing in digital environments and, particularly, in websites
associated with ‘old’ media programmes, I draw on ongoing research on media representations
of language variation and hybridity. Previous research has shown that social representations of
stylistic variation which people find funny in media performances can be revealing of the socio-
cultural values associated with certain language and cultural practices at a given moment (e.g. Coupland 2001). In recent work, I focused on a satirical radio show (Ελληνοφρένεια, lit. translated as ‘Greekophrenia’) that is broadcast live on one of the Greek national radio stations
(Real FM). The show has a strong political edge, using banter and pranks to play with, ridicule
and criticize prominent political figures, institutions, widely held beliefs and stereotypes. In
my study of the radio show’s pranks targeting members of the public, I discussed how
perceived competence in English is increasingly becoming an index of social identity in the
contemporary Greek context, giving rise to stylizations of (Greek-accented) English as a
powerful device for subversion and humour (Spilioti forthcoming). Since 2007 when the show first aired on national radio, ‘Greekophrenia’ have expanded their
inter-medial presence: there is now a TV programme (with the same title) using satirical 5 sketches to comment on current political affairs. The show also has a Facebook profile page, a
Twitter account followed by 395,000 users (at the time of writing), and an official website
(www.ellinofreneianet.gr). The website is updated daily, as each TV and radio show is
uploaded after its live broadcast, and provides listeners (and viewers) with an online archive of
all aired shows. Typical of the volatile nature of digital environments, the web domain of
‘Greekophrenia’ has changed its overall design and user interface three times since 2012 when
I first turned my attention to the site. Initially, it hosted its archive, together with a wealth of
material from other websites, such as video documentaries, news articles, blogs, comments
from fans and visitors of the site, links to other web environments and simple interactive games,
where the goal was, for instance, to throw tomatoes at prominent political figures. In 2013, the
homepage of the website was revised and some of the initial content (e.g. ‘Congratulations on your show – the best satire on the Greek radio!’; ‘When will you upload the prank with the
old lady asking for Oprah Winfrey’s show?’; ‘Well done for taking the mick of this government – corruption!’. 4.1. ‘Greekophrenia’ and multilingual writing online The interactive space with visitors’ comments
can be understood as an alternative format enabling audience participation and interaction with
the programme’s producers/presenters. For example, comments that clearly address the radio
producers, in the case of the Greekophrenia website, are reminiscent of telephone calls by radio
listeners who often congratulate presenters on their programme.1 6 6 Following current guidelines regarding research on media discourse, it appears that both spaces
can be considered as belonging to the public domain. Both edited web content and comments
are embedded within a wider domain that is created and regulated by professionals, i.e. the
show’s producers/presenters and web designers, for mass consumption. In addition to the fact
that visitors deliberately post a comment, the topic of such comments also orients to the public
broadcast of the show, presenting striking similarities with spoken material one could research
without consent from the lay people participating in such programmes. So far, it appears that
questions about ethics in relation to web content on sites dedicated to TV or radio programmes
can be answered in terms of well established procedures for researching media (e.g. broadcast)
material. Tracing trajectories of practices such as audience participation and interaction with media
professionals across media is important because it offers a more contextualised understanding
of communicative topics, purposes and roles. This is particularly useful at the start of a project,
because it helps the researcher to avoid being trapped within the confines of the argument of
equating access with publicness and to resist being caught in the popular and a-historicised
hype of ‘newness’, whereby ‘new’ media involves ‘new’ procedures. But, as will be shown in
the following section, there is value in revising such initial claims by paying attention to the
individual building blocks of websites and developing gradually a modular and iterative
approach to ethics. 5.1. Ethics and edited web content: towards a modular approach Edited content on official websites of media institutions and professional organisations is
generally thought to belong to the producers and owners of the web domain. This approach to
web domain as an undifferentiated whole is challenged by research on web discourse that
foregrounds ‘modularity’ as a key organisational concept (e.g. Pauwels 2012: 251): content of
individual webpages is organised into building blocks or modules that have been selected and
combined, either advertently or inadvertently, into a coherent whole. As a result, in addition to
the aforementioned methodological issues pertaining to the study of writing, the identification
of modules that make up a website is useful for processes of ethical decision-making, as each
building block may have different conditions of authorship and, thus, perceived ownership of
creative content. In order to identify the relevant modules on the multimodal space of a web page, visual
elements can be combined with functional criteria. Visual elements (as suggested by Kress &
van Leeuwen 2006: 214-218), particularly lines that frame and separate spatially contiguous
texts, as well as colour, font and shape discontinuities that distinguish visually different areas
of the web domain, have been particularly useful for identifying areas likely to operate as
separate modules on edited web content on the Greekophrenia website. Drawing on visual
resources, the identification of relevant building blocks also takes into account the extent to
which the visually and spatially distinct modules fulfil various functions. With respect to
websites dedicated to TV / radio programmes, such functions orient to (i) information structure
or organization (menus, internal links, navigational tools), (ii) (re)broadcasting of media
material (external links and other embedded textual, audio and visual material), and (iii)
interactivity with site visitors (internal links and prompts to post comments). The application of these criteria to websites reveals that different modular parts entail varying
attention to copyright issues. For example, elements designed by the site developers, such as
the ‘Greekophrenia on the net’ logo, site menus and internal links, represent creative content
belonging to the site. Proper attribution and permission by the creators and owners of the
website to reproduce such content in published material is necessary, similar to practices used
for reproducing mass media material. 5. Opening Pandora’s box of ‘convergence’: Revisiting publicness in the research process What counts as data in a multi/bilingual website, together with the ethical issues raised by such
methodological decisions, is a reflexive process shaped and driven by one’s research question
and wider epistemology. Recent research on multilingualism and writing (Sebba 2013; Lee
2016: 121; Tagg & Seargeant 2016: 341) has stressed the multimodal aspect of such
phenomena, driven by a shift in sociolinguistic research towards writing as an (ideological)
semiotic process (Lillis & McKinney 2013; Maybin 2013: 554). This multimodal approach
involves taking into account the ‘visual contexts’ of multilingual discourse, ‘as a text
surrounded by other texts, potentially with differing font sizes, colours and styles all potentially
providing context for interpreting the content of the [multilingual] text’ (Sebba 2013: 102). This theoretical shift in the approach of the written word as a (multi)semiotic object has
methodological consequences: from the decontextualized written word as unit for analysis, the
focus shifts to the study of writing in multimodal ensembles, i.e. multimodal representations
or interactions that are ‘seen as a material outcome or trace of the social context, available
modes and modal affordances, the technology available and the agency of an individual’ (Jewitt
2013: 254-255). Following this approach, attention to users’ – and, at the same time, web
designers’ – strategic use of visual means to organise content on a webpage helps the researcher
to delineate multimodal analytical units within which multilingual writing can be explored in
relation to the surrounding linguistic, graphemic, and visual resources. Revisiting ethics
iteratively within and across such multimodal ensembles is paramount as the varying social
and material constraints at play in each unit impact upon perceived boundaries between public
and personal data. ‘Congratulations on your show – the best satire on the Greek radio!’; ‘When will you upload the prank with the
old lady asking for Oprah Winfrey’s show?’; ‘Well done for taking the mick of this government – corruption!’. 7 7 5.1. Ethics and edited web content: towards a modular approach In contrast, decisions about copyright permission become
less straightforward when considering other areas that embed and (re)broadcast, for example,
articles or news bites from journalists or bloggers who are not directly involved in the site
design and who probably produced such texts for different audiences. The multiauthored
composition of edited web content, together with the multimedial make up of the web area
embedding the show’s radio or TV episodes, present cases where web discourse involves texts
produced for different publics and exhibiting different conditions of media production (e.g. broadcast vs. internet). The challenges arising from convergence, here, concern the need to
clarify copyright issues related to the reproduction of creative content for presentation and
publication purposes. Contacting the site developers and owners and seeking permission for
reproduction in academic research is the starting point. Although it is advisable to contact web developers and owners of individual sites, digital
affordances for copying, pasting, embedding and circulating content often obscure ownership
and complicate copyright permission. In such cases, researchers can think of alternative ways
for (re)presentation of web material. In fact, what seems like an obstacle at first sight can be a
useful research tool. Publishing research on any type of discourse data involves a process of
selective presentation of data in order to illustrate and support the arguments made. Rather than
approaching potential issues with reproduction of multimodal and multiauthored discourse as 8 mere challenges, the time is ripe for developing appropriate methods for presenting such
sources where copyright clearance may not be adequate or logistically possible. Similar to
transcribing and presenting (multimodal) spoken discourse, arbitrariness of symbols and
selectivity in discourse (re)presentation are inherent properties of this part of the research
process. Research on multimodal and multilingual writing can draw on publishing practices of
discourse analytic research on artwork, where strict copyright restrictions do not allow the
reproduction of such material for publication purposes. Nevertheless, this does not hinder
researchers from examining and analysing such material as evident in Jaworski’s (2014) study
of multilingualism and heteroglossia in artwork by multimedia and performance artists. In such
cases, visual material can be (and are) represented through the use of diagrams (Jaworski 2014:
154). The following section makes use of a diagrammatic representation of web discourse in
order to discuss ethical and privacy issues raised by research on the interactive areas of websites
dedicated to TV and radio shows. 5.2. Multimedia convergence and interactivity: from copyright to privacy issues One of the most distinctive features of language-focused research on multimedia and
multilingual texts, such as those found on websites, concerns the meaning-making dynamics
associated with hybrid, heteroglossic and multivoiced environments (Deumert 2014: 120). Such environments are often afforded through social media plug-ins activated in traditional
web (1.0) environments to enhance participation and interactivity. On the Greekophrenia site,
Facebook plug-ins enable visitors and fans to post comments under embedded broadcast
material, such as past radio and TV episodes. Typical features of multimedia web environments can include (i) verbal and visual elements
(i.e. texts and pictures) designed by the site’s developers and owners; (ii) verbal and visual
elements designed by the social media platform developers and automatically embedded in the
website through plug-in services; and (iii) verbal and visual elements designed by members of
the public and posted on the host website. To illustrate how such elements are laid out on the
Greekophrenia website (from 2012 to 2015), the following diagram annotates material
originating in different domains, produced by different actors (site’s producers in bold; social
media web developers in italics; and members of the public in normal font) and, as will be
argued below, for different audiences. At the same time, information about the varying
linguistic and typographic resources mobilised in each area is provided. [insert diagram 1 here] [insert diagram 1 here] Similar to edited web content, the issue of how to deal with creative content designed by
different actors but integrated within the same digital space is also relevant here. The comments
section, though, foregrounds an additional complication: unlike the articles, extracts from the
broadcast radio/TV shows, and other creative content chosen for inclusion by the site’s
designers/owners, the comments section includes personal information that is automatically
generated from the integration of social media plug-ins. Whose permission does one need in
order to use such material for analysis, presentation or publication? In other words, to what
extent can we safely assume that automatically disclosed metadata, as well as comments
generated by users, in such interactive web areas are produced for a mass public, authored by
public/professional figures and/or the result of users’ deliberative acts (cf. section 2)? 9 9 Attention to the modular organisation of web spaces and the multiple semiotic resources
activated in such digital environments provides a more nuanced picture of interactive comment
areas on such sites. The diagram reveals the individual comments authored and posted by
multiple users, as well as the range of accompanying metadata that are automatically embedded
in that area of the website (see elements in italics, on the diagram). For example, the Facebook
plug-in automatically displays the profile picture of the commenter, their Facebook user name
and selected information from their personal profile (i.e. where they work, where they live,
school/university they went to). Ethical decision-making regarding analysis and dissemination
of such information raises key issues regarding the publicness of such material. With respect
to who such material is produced for, metadata information (profile picture, username, etc) is
arguably produced, first and foremost, for the Facebook audiences, rather than visitors to the
radio show’s website. Whether such information is made available to the site’s visitors as a
result of the commenter’s deliberate act is debatable as this type of personal metadata is
automatically transferred through the technological specificities of such plug-ins. More
specifically, based on my own experience with using the plug-in on the particular site, users do
not have control over the inclusion or exclusion of information, and they cannot post comments
as anonymous users. In addition, there is no alert informing commenters about the type of
metadata/information that will eventually appear in the comments section. [insert diagram 1 here] Although one could
argue that names and tone of voice also give and give off (in Goffman’s 1959 terms) personal
identification information about listeners who call radio stations to speak to their favourite
presenters, the lack of control by social media users over such transferable personal information
is a factor researchers need to take into account when making ethical decisions. According to current ethical and legal frameworks, the use of such personal identifiable
information without consent can be problematic as there is limited (or no) evidence that the
metadata became available as a result of steps deliberately taken by commenters to disclose
their profile picture or place of residence. In fact, the discussion of metadata in the context of
online ethics also demonstrates the limits of current ethical guidelines. The internet researcher
is often placed in the unenviable position of second-guessing participants’ intentions or
reconstructing the steps participants might have deliberately taken in order to post a comment. Participant observation (as I did in my study) and interviews with users can be useful tools for
gaining further insights about the process of participating in an online environment. Nevertheless, such tools are still limited as users can over/underreport their intentions and
platform interfaces are frequently updated, altering the steps users take to participate and
upload comments. One wonders whether ethical recommendations should shift their focus from
participants’ intentions to researchers’ responsibility over information inadvertently embedded
in the building blocks of a website. While the process of determining whether data is public
becomes increasingly elusive, we (as researchers) can be more sensitive to information we
make available to a range of publics through our research. Alertness and sensitivity to the
handling of metadata information is important, especially since, unlike tone of voice or accent
in recorded media talk, online information can be searched and linked to a person’s online (and
offline) profile (cf. Zimmer 2010: 319). In addition to metadata, the interactivity patterns observed in the comments exchanged between
visitors of institutional websites call for revisiting claims about publicness that draw on
assumptions about discourse produced for public consumption. The interactive affordances of
social media plug-ins (e.g. ‘reply’ or ‘like’ buttons) often result in exchanges of comments
between individual users. In the case of the Greekophrenia site, although the majority of
comments are directed towards the show and the site producers, there are cases where users
interact between themselves. [insert diagram 1 here] Such one-to-one publicly accessible exchanges often take the 10 form of overt disagreements as to what counts as good satire or appropriate humour. Approaching such material as targeting a mass and unidentified public is challenged by users’
strategic use of addressivity (referring to the other person’s username) as a means for
identifying intended addressee(s). These means for users’ negotiation of multiple audiences in
digital communication indicate varying orientation to different participation frameworks and
active engagement in discursively constructing spaces as more or less public (Tagg &
Seargeant 2016: 349; Marwick and boyd 2011). In such cases, public-private can be understood
as a continuum that is discursively constructed and negotiated by users, rather than a checklist,
as put forward current regulatory approaches to ethics (cf. Giaxoglou; and Mackenzie this
issue). Previous research on internet data (e.g. Bolander & Locher 2014) has also pointed out the
sensitivity of topics as an important factor in assessing the publicness of a communicative
space. Nevertheless, even topics that appear non-sensitive could still raise ethical concerns if
they are embedded in discourse activities that are face threatening. On the Greekophrenia
website, public debates about what counts as good satire tend to devolve into personal attacks
at another user’s political beliefs or ethnicity, through comments on another user’s choice of
profile picture or writing style. In other words, what starts as a public (or safe/ non-sensitive)
topic can often take the form of personal disagreement, foregrounding sensitive aspects related
to a person’s identity and resulting in highly-charged interactions which neither parties might
want exposed to external scrutiny. Given the fuzziness of ‘topic’, alternatives might include
the notion of discourse activity that focuses on what is done and achieved through discourse,
rather than what is said. Furthermore, participant observation in such sites provides researchers with valuable
information about control and regulation of information, informing claims about the extent to
which site owners may intervene in their professional or public role. In the case of
Greekophrenia, the scarce evidence of moderation from the show or site producers resists an
interpretation of the comments area as a space owned and regulated by the site developers and
owners. [insert diagram 1 here] Although the comments section falls under the spatial boundaries of the website as a
whole, close observation of the aforementioned disagreements and personal attacks reveals no
apparent intervention or moderation from the site developers, in the form of either deleting or
responding to posts. Assumptions about how content flows in highly convergent media
environments (afforded by social media plug-ins) should pay attention to each separate
building block, as control over content by website owners varies across the different modules
that make up a website. The modular approach undertaken in this section reveals that assumptions about publicness are
challenged by the different elements that are combined on a website. The use of social media
plug-ins to afford interactivity and participation blurs perceived boundaries between public and
private. More importantly, content travels across platforms, with personal and private
information, such as the user’s picture or location on their Facebook profile, becoming publicly
available as metadata that frame individual comments. Similarly, what starts as a public debate
on non-sensitive topics can soon take the form of personal attacks to specific individuals,
unsettling any clear-cut public-private distinctions and inviting the researcher to revisit such
decisions by focusing, for example, on unfolding discourse activities, rather than topics. 6. Towards a modular and iterative approach to ethics in applied linguistics 11 Challenges related to media convergence arise from the interplay of multiple modes, authors
and media material. Approaching a website as a single domain or space runs counter to the
modular make up of such communicative environments which, in turn, resists any blanket
ethics statement about the publicness of institutional websites. Instead, unpacking media
convergences and any associated ethical challenges foregrounds the need for a more
contextualised and process-based approach that addresses ethical issues at different junctures
of a research project and opens a dialogue between regulatory-driven ethical guidelines and
context-sensitive understandings of ethical dilemmas (AoIR 2012: 4-5). In the case of dealing with intersections of ‘old’ and ‘new’ media (as in the example of
radio/TV programmes’ websites), tracing trajectories of media practices enables the researcher
to enrich and contextualise understandings of publicness. It moves beyond equating publicness
with accessibility and adds further dimensions to understandings of the public domain, by
taking into account discussion topics (cf. Bolander & Locher 2014: 17; Buchanan 2011: 94;
Sveningsson 2004) and professional roles of media producers (cf. similar distinction between
amateur and commercial web environments in Androutsopoulos 2008: 9). At the same time, a
process-based approach involves revising ethical decisions during different research stages. The research questions and methodological premises shape the process of ethical decision-
making. For instance, methodological decisions about the study of multilingual writing online,
ranging from the written word as individual, decontextualized units to writing as ideological
(multi)semiotic process, have an impact on how the ethical researcher deals with informed
consent and copyright issues (cf. Page et al 2014: 60). In terms of consent for using web material for research purposes, a modular perspective to
ethical decision-making contributes to the development of a process-based approach to
research ethics (AoIR 2012). As shown in the article, websites consist of several, functionally
and spatially distinct, areas where conditions of multiauthorship and multimediality are
(re)configured in various ways. Different configurations involve varying copyright restrictions
and understandings of publicness across and within the pages of a website. Attention to the
design, multiple audiences, discourse activities, and interactivity patterns in each individual
area is important both for methodological (i.e. digital writing as hybrid, multisemiotic and
multivocal) and ethical reasons, as digital content is generated through various means. The
article also foregrounds the need for careful ethical consideration of metadata that might get
inadvertently collected by the internet researcher. 6. Towards a modular and iterative approach to ethics in applied linguistics Metadata, such as users’ profile picture and
other personal information, are often automatically embedded in websites that use social media
plugins. In this case, human agency and publicness as framed in ethical guidelines (i.e. as steps
deliberately taken by human subjects) need to be revisited in order to account for
technologically mediated agency that involves instances of automatic disclosure of personal
information. Together with a modular perspective, the process of ethical decision-making online is iterative. Understandings of publicness have been revisited across the different stages of the research
and across the different modules that make up the main web pages of the site. Concerns about
publicness extend to the current moment of writing the article and, thus, the stage of publishing
and disseminating research, particularly because visitors’ comments are no longer available on
the site. What sort of ethical decisions should one make when certain material no longer exists
in the public domain but only in the private record of the researcher? It seems that the process
of academic research is also caught up in the ethical conundrums of online scalability and
spreadability (boyd 2011: 48). It is a truism that the speed and scale at which online content is
circulated are unprecedented but academic research and publishing have always played their 12 own role in spreading and scaling up (certain) types of discourse. My response to this ethical
conundrum was to represent the different areas and scripts in a diagram, rather than including
screenshots or verbatim messages from the comments section. Although this would be a
questionable practice for publishing results and findings from a language-focused study, such
an instrumental approach to research presentation (i.e. moving beyond the publishing of
verbatim quotes if justified by the aims of the study) can be useful in digital contexts where
public-private discussions are muddying the waters of research ethics. The process of transforming recorded material in line with ethical commitments is well rooted
in research practices of transcribing and analysing spoken discourse (Ochs 1979). Compared
to collecting and transcribing speech, written discourse (e.g. written documents, newspaper
articles) lends itself conveniently to academic research. In other words, it deceives researchers
into thinking that writing is already available for scrutiny, i.e. given material or, to use the Latin
term, data (‘given’) ready to be studied and analysed (Markham 2013). 6. Towards a modular and iterative approach to ethics in applied linguistics The element of deceit
lies in the fact that this reasoning collapses together understandings about the materiality of the
sources (persistence of writing on a page vs. volatile speech) and epistemological assumptions
about texts and people as objects and ‘data subjects’, respectively (UK Data Protection Act
1998). Perceiving writing as a static object produced by data subjects shapes our stance towards
research on texts and people (cf. Cameron et al 1993: 87). Research on digital discourse carries
over these assumptions about written discourse data. Perhaps we need to stop thinking about
discourse data in terms of materiality and move beyond objectifying texts and people; then we
can embrace richness and complexity as inherent properties of doing research (in any setting,
face-to-face or mediated). This can take us a step closer to solutions that orient to research as
process and shed light on, rather than obscure, the nuances of communicative encounters,
online and offline. Acknowledgement: I would like to thank the anonymous reviewers for their insightful
comments and feedback, as well as Alison Wray and Caroline Tagg for their continuous
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Perspectives. Amsterdam/Philadelphia: John Benjamins. 29-46. Zimmer, M (2010) “But the data is already public”: On the ethics of research in Facebook. Ethics, Information and Technology 12: 313-325. 16 iagram 1. Modular representation of comments section (www.ellinofreneianet.gr 2012-2015) agram 1. Modular representation of comments section (www.ellinofreneianet.gr 2012-2015) 17 17
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AC Electrothermal Effect in Microfluidics: A Review
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Micromachines
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Received: 11 October 2019; Accepted: 28 October 2019; Published: 11 November 2019 Abstract: The electrothermal effect has been investigated extensively in microfluidics since the 1990s
and has been suggested as a promising technique for fluid manipulations in lab-on-a-chip devices. The purpose of this article is to provide a timely overview of the previous works conducted in
the AC electrothermal field to provide a comprehensive reference for researchers new to this field. First, electrokinetic phenomena are briefly introduced to show where the electrothermal effect stands,
comparatively, versus other mechanisms. Then, recent advances in the electrothermal field are
reviewed from different aspects and categorized to provide a better insight into the current state of
the literature. Results and achievements of different studies are compared, and recommendations are
made to help researchers weigh their options and decide on proper configuration and parameters. Keywords: electrothermal; microelectrode; microfluidics; micromixing; micropump Review
AC Electrothermal Effect in Microfluidics: A Review 1
Biomedical Engineering Graduate Program, Ryerson University, Toronto, ON M5B 2K3, Canada;
maryam.navi@ryerson.ca 2
Institute for Biomedical Engineering, Science and Technology (iBEST), St. Michael’s Hospital,
Toronto, ON M5B 1T8, Canada 3
Keenan Research Centre, St. Michael’s Hospital, Toronto, ON M5B 1T8, Canada
4
Biomedical Engineering Graduate Program, University of Calgary, Calgary, AB T2N 1N4, Canada;
thomas.lijnse@ucalgary.ca 5
Electrical and Computer Engineering Department, University of Calgary, Calgary, AB T2N 1N4, Canada
*
Correspondence: a1salari@ryerson.ca (A.S.); cdalton@ucalgary.ca (C.D.);
Tel.: +1-647-889-1276 (A.S.); +1-403-210-8464 (C.D.) †
These authors contributed equally to this work. Micromachines 2019, 10, 762; doi:10.3390/mi10110762 www.mdpi.com/journal/micromachines micromachines micromachines micromachines micromachines 1. Introduction Microfluidics is the precise control, and manipulation of fluids that are geometrically constrained
to small, typically sub-millimeter, manufactured systems. Over recent decades, microfluidics has
gained a great deal of attention in multiple fields, including medicine, chemistry, and biomedical
engineering, due to its ability to perform multiplexing, automation, and high-throughput screening
tasks. [1,2]. Due to the high surface-to-volume ratio of the fluid, and thus, the dominance of
surface forces over inertial forces (i.e., low Reynolds number), fluid flow generation is a major
challenge in microfluidic devices, as conventional pressure driven methods have poor efficiency
in such devices [3,4]. The mechanisms of micro scale manipulation of fluids and particles can be
categorized into two groups: mechanical, such as diaphragm-based devices, and non-mechanical, such
as electrokinetic-based techniques. The presence of moving parts increases the risk of mechanical
failure and can be incompatible with particulate flows, and thus, can limit the application of mechanical
pumps for lab-on-a-chip devices [1,4,5]. Non-mechanical strategies, however, do not have these
limitations. They can be integrated with microfluidic devices and also be used with particulate
fluids [4,6,7]. Examples of non-mechanical methods include ultrasonic, direct current (DC) charge
injection, and travelling wave driven electrohydrodynamic (EHD) micropumps [7–10]. Electrokinetics is a popular non-mechanical technique used for microfluidic fluid manipulation
applications owing to its simple design and electronic automation [3]. Electrokinetic phenomena result Micromachines 2019, 10, 762; doi:10.3390/mi10110762 2 of 27 Micromachines 2019, 10, 762 from the interaction of an external electric field and induced electric charges. DC electrokinetics (DCEK),
which has been studied over decades, requires relatively high voltages (i.e., on the order of several
kilovolts) to operate which can limit its application in lab-on-a-chip devices [4,11,12]. alternating
current (AC) electrokinetics (ACEK), however, which operates in low voltages (i.e., 1–20 Vrms), has led
to the development of devices being portable and capable of handling biofluids without engaging in
unwanted chemical reactions [13,14]. Furthermore, with non-uniform fluid flow streamlines generated
by ACEK, this mechanism can be used to mix fluids [6]. AC electrokinetics mainly includes the dielectrophoresis (DEP), AC electroosmosis (ACEO),
and AC electrothermal effects (ACET) [15], each of which is explained briefly in the following sections. There have been many substantive review articles on micropumps [16,17], electrohydrodynamics [18],
electrokinetics [3,19,20], and their subcategories [2,21], but a comprehensive review focused on the
electrothermal effect in microfluidics is still missing in the literature. 2.1. Dielectrophoresis Dielectrophoresis arises from the interaction between a dipole moment on a particle and a
non-uniform electric field [23]. If the particle has a polarizability higher than the surrounding medium,
the DEP force exerting on the particle will be towards regions with a high electric field (positive
DEP). For particles with lower polarizability, however, this force will be towards regions with a low
electric field (negative DEP). This is demonstrated by the Clausius–Mossotti factor, which specifies the
direction of the DEP force with respect to the electric field [24]. In addition to the permittivities of the
particle and the medium, the magnitude of DEP force is also a function of the particle size. DEP force
is directly related to the third power of the particle radius, and thus, is an ideal tool for separating,
concentrating, and sorting particles, cells, and viruses [25–31]. Moreover, since DEP force scales with
the gradient of the electric field, it decreases with the distance from electrodes, and the generated
velocity is inversely proportional to the third power of distance [13]. Therefore, DEP is not an effective
technique for handling particles of relatively small size (e.g., ≤1 µm) far from a strong electric field
(e.g., a few micrometers away) [32]. Two excellent reviews on dielectrophoresis are [33,34]. 1. Introduction This review intends to study
the advances in the utilization of the electrothermal effect in microfluidics from different aspects,
namely: the electric field, temperature field, and velocity field. In addition, channel properties,
conditions of numerical simulations, experimental setup, and applications of ACET effect are presented. Finally, we will mention the ongoing research directions and future potential opportunities for the
electrothermal effect. The majority of the publications cited throughout the manuscript that have
made major contributions are also summarized in Table A1. It should be noted that the study of the
electrothermal effect in other mechanisms such as ACEO is not in the scope of this paper and can
be found elsewhere [5,22]. Furthermore, strategies which are based on DC electric fields (e.g., DC
electrophoresis) or non-electrical (e.g., magnetic) forces are not discussed here. 2.3. AC Electrothermal Unlike ACEO and DEP, ACET has been shown to be very effective in biomedical applications
which involve high conductivity biofluids, such as blood, urine, and saliva [60]. This is due to the
fact that the ACET effect originates from a temperature gradient in the bulk of the fluid and not
the fluid-electrode interface (i.e., the EDL). Fluids with higher conductivities can generate stronger
microflows, and therefore, it is the most efficient electrokinetic mechanism for manipulating biological
fluids with conductivities above 0.7 S·m−1 [1,4,13,14,39,60–63]. Emerging in 1960s [64,65], ACET has been widely used for fluid manipulations over the years [7,66],
and is also referred to as induction EHD [67,68]. Despite similar flow patterns, the physics behind
ACET and ACEO are different. ACEO is the result of the interaction of an electric double layer at
the interface of the fluid-electrode and a non-uniform AC electric field, while ACET arises from the
interaction of a temperature gradient in the bulk of the fluid and a non-uniform AC electric field. The source of the temperature gradient may be internal (i.e., Joule heating) or external (e.g., strong
illumination, microheaters, etc.). Temperature gradients in the fluid lead to gradients in the electrical
properties of the fluid, i.e., conductivity and permittivity, which induce free charge density. An electric
force arising from the non-uniform electric field causes the free charges to move. As a result of shear
stress, the surrounding fluid is also dragged into motion which produces microflows. Unlike ACEO,
the ACET effect shows plateaus in force in a wide frequency range (10–1011 Hz) [39,69]. With ACET,
the fluid velocity is steadier and more predictable at different frequencies compared to ACEO and
DEP. In general, ACET flow can be generated in frequencies above 100 kHz and salt concentrations of
above 10−2 mol·dm−3, whereas ACEO is more common at low frequencies and salt concentrations
of 10−2 mol·dm−3 and below [62]. Despite the fact that fluid heating is crucial for the ACET effect,
ambient heat conduction helps dissipating energy so that the temperature rise in the bulk of the fluid
is typically low (∆T < 5 K), which is safe for biofluids [11,13,14,41]. The ACET force is proportional to
the temperature gradient |∇T| and not the temperature rise [1]. In order to generate AC electric fields required for inducing the electrothermal effect,
microfabricated electrode arrays are commonly used. 2.2. AC Electroosmosis AC electroosmosis is dependent on the formation of an electric double layer (EDL) at the interface
of a liquid and solid substrate [35–37]. At an interface of a solid object and electrolyte fluid, due to the
adsorption of ions onto the object surface it acquires charges, and as a result, an EDL forms inside the
fluid near the surface [3,38]. When an electric field is applied to this system, the charges in this layer
experience an electrostatic force, which can cause fluid motion. The rest of the fluid is then dragged
into motion due to viscous forces. Since the EDL thickness is inversely related to the fluid electrical conductivity, at relatively high
conductivities (e.g., 84 mS·m−1), the EDL thickness becomes very small (e.g., <1 nm), which makes
ACEO ineffective for the manipulations of biological fluids (1–2 S·m−1) [1,13,15,39–41]. In addition,
ACEO is frequency-dependent, and increasing the actuation frequency beyond 100 kHz causes the 3 of 27 Micromachines 2019, 10, 762 ACEO effect to become invisible, since at high frequencies, the electric double layer is unable to form,
and no fluid flow is generated [35]. Similarly, at very low frequencies, the double layer can completely
screen the electric field, and thus, no net flow can be generated. ACEO has been developed and used
in many forms to pump fluids or manipulate particles, namely biased ACEO for particle assembly and
micropumping [32,42], micropumping of fluids [43,44], Travelling wave ACEO [45] and asymmetric
ACEO micropumping [40], and DEP electrohydrodynamic particle trapping (i.e., ACEO in conjunction
with DEP) [46,47]. As ACEO is only effective in relatively low frequencies, it is more prone to bubble
generation and electrode deterioration resulting from electrochemical reactions, which can affect the
electric field distribution and eventually damage the device [39]. Despite these limitations, there
are many application-driven papers in the literature using ACEO [48–51], where modifications have
been suggested to enhance ACEO applicability in fluids with electric conductivities up to 0.1 S·m−1
under the actuation of high frequencies and voltages. These modifications include the utilization of
polarizable walls in induced-charge electroosmosis [52], AC faradic polarization [50], and nonlinear
electroosmosis on curved surfaces [53], to name a few. An advantage of ACET is that it can be used
for higher conductivities, i.e., over 1 S·m−1. More details of different strategies for implementation of
ACEO in microfluidics can be found in [54,55]. 2.2. AC Electroosmosis DEP and ACET effects can be combined [13,56–59] to
improve particle manipulations, as DEP has difficulty in manipulating submicron particles, and also is
weak in areas far from electrodes where ACET is strong [13,56]. 2.3. AC Electrothermal Employing a symmetric pair of electrodes at the
bottom of a microfluidic channel can induce two symmetric sets of microvortices above the electrodes,
and thus, no net flow can be generated [70]. For pumping applications, however, the electrode symmetry
needs to be broken. Since the electrothermal force is a function of the electric field and temperature
gradient, asymmetry may be achieved by manipulating either or both of these factors. This will 4 of 27 Micromachines 2019, 10, 762 be discussed in more details in the following sections. Typically, due to its simple implementation,
imposing geometry asymmetry to microelectrodes is the most common approach for breaking the
symmetry of microvortices. In addition, manipulating the temperature field with the help of external
heat sources, such as strong illumination [69,71–73], embedded microheaters [74,75], and heating
electrodes [1], can also be used for creating a net flow. Although a common ACET microdevice
implements an array of electrode pairs placed at the bottom of a microchannel with a rectangular
cross section, more complicated configurations with electrode arrays placed on the top, bottom,
and sidewalls of channels with different cross sections have also been studied [69,76–78]. Studies with
the use of grooves on the channel surface to induce further asymmetry and increase flow have also
been addressed, but fabrication of these designs suffers from serious challenges. Similar to other electrokinetic mechanisms, ACET suffers from some drawbacks, most of which
have been addressed to some extent in the literature, as will be shown in this paper. In microfluidic
devices, miniaturization can be hindered as the ACET effect originates from the bulk of the fluid
and decreasing the channel dimensions can decrease the volume of the fluid flowing inside the
channels [3,35]. In addition, ACET depends on the formation of temperature gradients, and therefore,
cannot be used with low conductivity fluids. As such, its application in conjunction with DEP, which
requires low conductivity fluids for efficient particle sorting, is limited [1,4,5]. Importantly, an excessive
temperature rise in fluids with high conductivities can cause the buoyancy force to dominate over the
ACET force [4]. The reason is that the ratio of electrothermal force to buoyancy force is proportional to
|∇T|/∆T. Thus, when ∆T > |∇T|, the buoyancy force becomes the dominant force. Finally, increasing
the actuation voltage to achieve high fluid velocity can lead to electrochemical reactions which can
limit the application of ACET effect on biofluids [1]. 2.3. AC Electrothermal There have been some reports on how to mitigate
this issue [79]. 3. Theory As stated in the previous section, the ACET effect results from the interaction of a non-uniform
electric field and a temperature gradient in the bulk of the fluid. The energy balance equation governs
the amount of Joule heating as follows [15]: k∇2T + 1
2 < σ|E|2 >= 0
(1) (1) where, k and σ are the thermal and electrical conductivities of the fluid, respectively, and E is the
electric field, which can be obtained from the Laplace equation in a homogeneous medium as below: where, k and σ are the thermal and electrical conductivities of the fluid, respectively, and E is the
electric field, which can be obtained from the Laplace equation in a homogeneous medium as below: ∇2V = 0
(2) (2) ∆T ≈σV2
k
(3) (3) Based on Equation (3), the temperature rise is directly proportional to the fluid electrical
conductivity and actuation voltage squared, which, in most applications, is the control parameter. Based on Equation (3), the temperature rise is directly proportional to the fluid electrical
conductivity and actuation voltage squared, which, in most applications, is the control parameter. The ratio of heat convection to heat conduction in a microchannel is very low (i.e., Peclet
≤0.07) [15,67,80]. Furthermore, it has been numerically shown that, compared to electrical forces,
natural convection in micro-channels can be neglected [15,80]. However, for cases with high thermal
Peclet numbers, heat convection cannot be neglected [81]. The temperature gradient in the fluid can Micromachines 2019, 10, 762 5 of 27 change the fluid properties, including permittivity ε and conductivity σ, and can be calculated as
follows [15]:
∂
! change the fluid properties, including permittivity ε and conductivity σ, and can be calculated as
follows [15]:
! ∇ε =
∂ε
∂T
! ∇T
(4)
∇σ =
∂σ
∂T
! ∇T
(5) (4) ∇σ =
∂σ
∂T
! ∇T
(5) (5) In most ACET applications, it is assumed that the rate of change of permittivity and conductivity
with the change in temperature is very small [15]. Otherwise, a temperature coefficient needs to
be defined to account for the changes in fluid properties [81,82]. As a result of this assumption,
the perturbed electric field can be neglected, and the charge convection can be assumed to be much
smaller than the charge conduction [15]. 3. Theory Micromachines 2019, 10, 762 6 of 27 With the above approximations, the electrothermal force can be simplified as below [15]: < FE >= 1
2
ε(α −β)
1 + (ωτ)2 (∇T·E)E −1
4εα|E|2∇T
(14) (14) where, τ = ε
σ is the charge relaxation time of the liquid and is in the range of 0.7–35 ns for conductivities
in the range of 0.02–1 S·m−1 [41,85]. As stated above, the first term represents the Coulomb force, and the
second term is the dielectric force. These forces act in different frequency ranges (i.e., the Coulomb force
dominates at low frequencies and dielectric force dominates at high frequencies) and are in different
directions [83]. Near a certain frequency, known as the cross-over frequency fc, the two forces compete,
and flow reversal can occur as a result of switching from a Coulomb force dominant to a dielectric
force dominant regime or vice versa [15]. The cross-over frequency can be calculated as below [15]: fc ≈
1
2πτ
2
1
σ
∂σ
∂T
1
ε
∂ε
∂T
1
2
(15) (15) For example, the cross over frequency for a biofluid with a conductivity of 1 S·m−1 is roughly
200 MHz [61]. ACET force, and thus ACET flow velocity, is higher (up to 11 times) at low frequencies and
has no dependence on frequency except for the transition region (i.e., near crossover frequency) [13,41,69]. p
q
y
p
g
(
q
y) [
]
By taking a closer look at Equation (11), as α, β, and ω are constants, we can conclude that
ρE ∝∇T·E [1]. Commonly, in electrokinetics, frequencies much lower than 10 MHz (usually around
200 kHz) are used, where 1 + (ωτ)2 ≈1 and dielectric force is negligible (i.e., the Coulomb force
is ~11 times larger than the dielectric force) [41]. At these frequencies, there is not enough time
for the double layer to form, and thus, the dielectric force is neglected [13]. As a result, the flow
direction is determined by the Coulomb force and Equation (14) is reduced to the first term on
the right hand side. With a similar argument, as α, β, and ωτ are constants, we can conclude that
|< FE >| ∝|E|2|∇T|, and according to Equation (1), when Joule heating is implemented, ∇T ∝E2,
and therefore, |< FE >| ∝|E|4 [69]. 3. Theory The change in electrical properties of the fluid leads to the generation of electrical charge density
as follows [15]: g
as follows [15]: ρE = ∇·(εE)
(6)
∂ρE
∂t + ∇·(σE) = 0
(7) (6) (7) where, ρE is the charge density. where, ρE is the charge density. where, ρE is the charge density. Under the effect of the electric field, there is a force applied to the charge density which is [15 fE = ρEE −1
2E2∇ε
(8) (8) The first term in Equation (8) is the Coulomb force and the second term is the dielectric force As charges move in the electric field, they drag the surrounding medium into motion. Therefore, microflows are generated in the fluid and are governed by: ∇p + η|∇|2u + fE = 0
(9) (9) where, η, p, and u are the dynamic viscosity, pressure, and velocity field, respectively. Furthermore, from
the conservation of mass for an incompressible fluid, we have: where, η, p, and u are the dynamic viscosity, pressure, and velocity field, respectively. Furthermore, from
the conservation of mass for an incompressible fluid, we have: ∇·u = 0
(10) (10) With an order of magnitude estimation from Equation (9), the flow velocity can be written
as |u| ≈< fE > · l2
η , where l is the characteristic length of device, which is usually the electrode
spacing [4,15,60]. g
Charge density can be calculated by combining Equations (6) and (7) as follows [83]: ρE =
σε
σ + iωε(α −β)(∇T·E)
(11) (11) where, ω is the angular frequency of the AC electric field, and: where, ω is the angular frequency of the AC electric field, and: where, ω is the angular frequency of the AC electric field, and: α = 1
ε
∂ε
∂T
! (12)
β = 1
σ
∂σ
∂T
! (13) (12) β = 1
σ
∂σ
∂T
! (13) (13) For aqueous solutions and temperatures around 293 K, α and β can be estimated as −0.4% K−1
and 2% K−1, respectively [84]. 3. Theory This means that electrothermal force is proportional to the fourth
power of the electric field. According to the order of magnitude estimation of velocity obtained
from Equation (9), ACET fluid velocity also has a quartic relationship with the actuation voltage. This means that a small increase in the electric field can cause a large increase in electrothermal force,
and thus, a significant increase in the resultant flow velocity. As stated above, since electrothermal and
electroosmotic flows have similar patterns, one way to distinguish them is to use this proportionality. Since electroosmotic velocity is proportional to the square of voltage, by plotting velocity against
applied voltage, the source of microflows can be revealed [14,15]. The theory discussed here is based on an uncoupled model developed by Ramos et al. in
1999 [15]. In such a model, an assumption of a small temperature rise ∆T < 5 K renders the change of
fluid properties and electric field with temperature insignificant. However, if the temperature rise is
considerably higher, then the fluid properties will change by temperature variations, and therefore,
a fully coupled model needs to be used [74,86–88]. Hong et al. developed a coupled model, compared it
with the classical model, and found that only at small temperature rises do the results of the two models
match [87]. More recently, pumping and mixing of non-Newtonian fluids have been studied [89–91]. 4.1. Introducing Asymmetry to Geometry To produce a strong electric field, microelectrodes are typically patterned on the inner surfaces
of microchannels [11,66]. Due to changes to the electric field and temperature gradient strengths,
by manipulating the electrode configuration, a wide variety of flow patterns and velocities can be
achieved [41]. Microelectrode arrays with different shapes have been reported, which can be categorized
into two-dimensional (2D) and three-dimensional (3D) geometries [41,85]. Micromachines 2019, 10, x
7 of 26 Due to the simplicity and ease of fabrication, 2D electrode geometries are the most common and
can be further categorized into asymmetric rectangular [4,41,69,92], orthogonal [4], meandering [62],
concentric [60], and triangular [56], with asymmetric rectangular being investigated the most in
the literature (Figure 1a–e). Imposing asymmetry on the electrode pairs induces asymmetry in the
resultant microflows, and therefore renders pumping action. Due to this feature of microelectrode
configuration, more heat is hypothesized to be generated near the narrow electrode [1]. In general,
in such a configuration, the width of the narrow electrode and the gap between electrodes in a pair are
the two major design parameters which govern the magnitude of the fluid velocity [41]. Due to the simplicity and ease of fabrication, 2D electrode geometries are the most common and
can be further categorized into asymmetric rectangular [4,41,69,92], orthogonal [4], meandering [62],
concentric [60], and triangular [56], with asymmetric rectangular being investigated the most in the
literature (Figure 1a–e). Imposing asymmetry on the electrode pairs induces asymmetry in the
resultant microflows, and therefore renders pumping action. Due to this feature of microelectrode
configuration, more heat is hypothesized to be generated near the narrow electrode [1]. In general, in
such a configuration, the width of the narrow electrode and the gap between electrodes in a pair are
the two major design parameters which govern the magnitude of the fluid velocity [41]. Figure 1. Schematic of different electrode geometries. (a–e) 2D Electrodes: (a) asymmetric rectangular,
(b) orthogonal, (c) meandering, (d) concentric, and (e) triangular. (f–i) 3D Electrodes: (f) 3D electrode
array, (g) microgrooved configuration, (h) electrodes facing each other, and (i) spiral design. Reproduced with permission from [56,60,62,69,77,85,93–95]. Yuan et al conducted a thorough study on the optimization of 2D rectangular electrode arra
Figure 1. Schematic of different electrode geometries. (a–e) 2D Electrodes: (a) asymmetric rectangular,
(b) orthogonal, (c) meandering, (d) concentric, and (e) triangular. 4. Electric Field In lab-on-a-chip applications, the maximum limit for actuation voltage is typically within the range
of 5–7 Vrms, beyond which electrochemical reactions can harm the nature of biofluids. Since, as stated
in the previous section, electrothermal force, and thus, fluid velocity is highly dependent upon the
applied voltage (i.e., quartic dependence), the increase in velocity of electrothermal based biomedical
microdevices is hindered accordingly. To overcome this issue, many studies have proposed and 7 of 27 Micromachines 2019, 10, 762 developed new ACET designs with modifications to the electric field, which include either changes to
the geometry of the electrode array or introducing asymmetry to the electric potential, using techniques
such as travelling wave, multiphase, DC biased, etc. In this section, different strategies proposed for
modifying the electric field are reviewed. 4.1. Introducing Asymmetry to Geometry (f–i) 3D Electrodes: (f) 3D
electrode array, (g) microgrooved configuration, (h) electrodes facing each other, and (i) spiral design. Reproduced with permission from [56,60,62,69,77,85,93–95]. Yuan et al conducted a thorough study on the optimization of 2D rectangular electrode array Figure 1. Schematic of different electrode geometries. (a–e) 2D Electrodes: (a) asymmetric rectangular,
(b) orthogonal, (c) meandering, (d) concentric, and (e) triangular. (f–i) 3D Electrodes: (f) 3D electrode
array, (g) microgrooved configuration, (h) electrodes facing each other, and (i) spiral design. Reproduced with permission from [56,60,62,69,77,85,93–95]. Figure 1. Schematic of different electrode geometries. (a–e) 2D Electrodes: (a) asymmetric rectangular,
(b) orthogonal, (c) meandering, (d) concentric, and (e) triangular. (f–i) 3D Electrodes: (f) 3D
electrode array, (g) microgrooved configuration, (h) electrodes facing each other, and (i) spiral design. Reproduced with permission from [56,60,62,69,77,85,93–95]. Yuan et al. conducted a thorough study on the optimization of 2D rectangular electrode arrays
and reported a set of ratios for the corresponding parameters [41]. 2D rectangular asymmetric arrays
Yuan et al. conducted a thorough study on the optimization of 2D rectangular electrode arrays
and reported a set of ratios for the corresponding parameters [41]. 2D rectangular asymmetric arrays Yuan et al. conducted a thorough study on the optimization of 2D rectangular electrode arrays
and reported a set of ratios for the corresponding parameters [41]. 2D rectangular asymmetric arrays
Yuan et al. conducted a thorough study on the optimization of 2D rectangular electrode arrays
and reported a set of ratios for the corresponding parameters [41]. 2D rectangular asymmetric arrays 8 of 27 Micromachines 2019, 10, 762 are used mostly for pumping applications, either alone or with other changes to electric or thermal
fields [1,74]. In pumping applications, one or multiple arrays of asymmetric microelectrode pairs
are placed perpendicular to the channel length in order to generate a net flow along the channel. In applications, where a lateral fluid mixing is also desirable, however, the electrode array needs to be
placed at an angle <90◦to the channel length [77,95]. In 2D electrode configurations, by decreasing the
gap between electrodes up to a certain point, the resultant fluid velocity can be increased as the strength
of the electric field increases. 4.1. Introducing Asymmetry to Geometry However, if the strength of the electric field is maintained at a constant,
increasing the gap between electrodes can enhance the resultant fluid flow, as increasing the gap allows
a larger volume of fluid bulk above the gap to experience the strong electric field [41]. One way to
enhance the ACET effect is to decrease the width of the narrow electrode, increasing the non-uniformity
of the electric field. However, if the narrow electrode is made too small, the strength of the electric field
can decrease accordingly, and, as electrothermal force is proportional to the fourth power of the electric
field, electrothermal force and, hence, velocity can decrease drastically [41]. As shown, the dimensions
and electrode geometry are of great importance to the electrothermal effect. In addition, the channel
size and cross section can also significantly affect the resultant flow rate [96]. More on this topic can be
found in Section 9. Orthogonal electrode arrays were proposed by Wu et al. for pumping applications
with velocities exceeding 1 mm·s−1 [4,93]. A meandering electrode array, with a sinusoidal electrode
gap, was proposed for rapid mixing of two biofluids [62]. Rapid mixing of high-conductivity (up to
22 S·m−1) fluids has been shown using concentric electrode designs [60]. A triangular electrode pattern
has also been suggested for micromixing [56]. The backward flow generated above the narrow electrode can negatively impact the use of
planar configurations of electrodes for pumping applications, as they cause the average flow in
the microchannel to slow down [85]. In 2000, Ajdari, suggested a 3D electrode configuration to
circumvent this problem [94] (Figure 1f). Expanding on the same idea, Du and Manoochehri suggested
a microgrooved channel (instead of 3D electrodes) to impose spatial asymmetry to the electrode
pairs [97] (Figure 1g). More recently, this effect has been numerically simulated with a number of
substrate configurations [98]. Manufacturing a channel with modified roughness of substrate surface
is easier than manufacturing 3D electrodes. In their work, different shapes of grooves are proposed
and experimentally studied. This study showed that by using the microgrooved structure, based
on the shape of the grooves, the pumping capacity increases by up to six-fold compared to a planar
configuration. In another work, they reported an optimized configuration of such a design [85]. They managed to increase the net flow rate by further suppressing backflows and shortening the
streamlines [85]. 4.1. Introducing Asymmetry to Geometry However, fabrication of such microgrooved structures is more complicated than
conventional microchannels with planar electrode design [69]. A variation of such configuration in
conjunction with opposing electrodes on the top surface is suggested for mixing applications [89,99]. A similar configuration is also reported where opposing castellated electrodes are utilized to eliminate
the ACET vortex and thereby enhance pumping [100]. A two-layer microelectrode configuration, where microelectrodes are facing each other, has been
proposed for particle trapping in order to investigate the DEP effect in conjunction with ACET [13]. In this electrode configuration, one electrode is placed on top and the other at the bottom of the
channel. A similar configuration is also used for studying Joule heating effects [80], as well as
for patterning of colloids when equipped with underlying microheaters [75]. Such an opposing
microelectrode configuration with different patterns is also reported for mixing fluids [77,89,101]
(Figure 1h). This configuration is also used in rapid electrokinetic patterning, where electrothermal
effects play a significant role in particle manipulation [102,103] More recently, studies have been carried out on utilizing electrodes on all walls of the microchannel
in different patterns [104,105]. Using a particular configuration of multiple electrode arrays on side
walls, simultaneous mixing and pumping of biofluids in one microchannel during a short time and
over a short distance is feasible [104]. In a very recent publication, a spiral electrode pattern has been 9 of 27 Micromachines 2019, 10, 762 proposed for simultaneous mixing and pumping which is capable of achieving a velocity of 400 µm·s−1
for a biofluid with a conductivity of 0.224 S·m−1 (Figure 1i) [95]. proposed for simultaneous mixing and pumping which is capable of achieving a velocity of 400 µm·s−1
for a biofluid with a conductivity of 0.224 S·m−1 (Figure 1i) [95]. 4.2. Introducing Asymmetry in Electric Potential In addition to an asymmetric electrode geometry, the electric field can also be modified to enhance
the electrothermal effect. Applying a DC potential to the electrode pairs in addition to the existing AC
potential, using a multiphase AC signal in the form of a travelling wave, and utilizing a two-phase
actuation system are examples of such modifications, each of which is briefly described here. 4.2.1. DC Biased The narrow electrodes in two adjacent pairs have
different polarities. The superposition of the two configurations enhances the electric field in region B. Reproduced with permission from [6,68,69]. 4.2.1. DC Biased Note that the direction of propagation of travelling wave is
opposite to the direction of fluid flow. (c) Two-phase asymmetric configuration. The narrow
electrodes in two adjacent pairs have different polarities. The superposition of the two configurations
enhances the electric field in region B. Reproduced with permission from [6,68,69]. Figure 2. Schematic of different mechanisms for imposing asymmetry to the electric field in alternating
current electrothermal effects (ACET) devices. (a) Direct current (DC) biased configuration. The left
electrode is always at a positive potential and subject to faradaic charging, while the right electrode is
always at a negative potential and subject to capacitive charging. (b) Travelling wave configuration. The induced temperature gradient generates a charge density which is moved with the travelling
electric field. Note that the direction of propagation of travelling wave is opposite to the direction of
fluid flow. (c) Two-phase asymmetric configuration. The narrow electrodes in two adjacent pairs have
different polarities. The superposition of the two configurations enhances the electric field in region B. Reproduced with permission from [6,68,69]. Figure 2. Schematic of different mechanisms for imposing asymmetry to the electric field in
alternating current electrothermal effects (ACET) devices. (a) Direct current (DC) biased
configuration. The left electrode is always at a positive potential and subject to faradaic charging,
while the right electrode is always at a negative potential and subject to capacitive charging. (b)
Travelling wave configuration. The induced temperature gradient generates a charge density which
is moved with the travelling electric field. Note that the direction of propagation of travelling wave is
opposite to the direction of fluid flow. (c) Two-phase asymmetric configuration. The narrow
electrodes in two adjacent pairs have different polarities. The superposition of the two configurations
enhances the electric field in region B. Reproduced with permission from [6,68,69]. Figure 2. Schematic of different mechanisms for imposing asymmetry to the electric field in alternating
current electrothermal effects (ACET) devices. (a) Direct current (DC) biased configuration. The left
electrode is always at a positive potential and subject to faradaic charging, while the right electrode is
always at a negative potential and subject to capacitive charging. (b) Travelling wave configuration. The induced temperature gradient generates a charge density which is moved with the travelling
electric field. Note that the direction of propagation of travelling wave is opposite to the direction of
fluid flow. (c) Two-phase asymmetric configuration. 4.2.1. DC Biased In this method, symmetric electrodes are used but one electrode is always at a positive potential
and the other is always at a negative potential subject to faradaic charging and capacitive charging,
respectively (Figure 2a) [6]. Hence, positive charges on both electrodes lead to unidirectional flow. The advantage of this configuration is that pumping action can be achieved by a symmetric pair
of electrodes. Micromachines 2019, 10, x
9 of 26 Figure 2. Schematic of different mechanisms for imposing asymmetry to the electric field in
alternating current electrothermal effects (ACET) devices. (a) Direct current (DC) biased
configuration. The left electrode is always at a positive potential and subject to faradaic charging,
while the right electrode is always at a negative potential and subject to capacitive charging. (b)
Travelling wave configuration. The induced temperature gradient generates a charge density which
is moved with the travelling electric field. Note that the direction of propagation of travelling wave is
opposite to the direction of fluid flow. (c) Two-phase asymmetric configuration. The narrow
electrodes in two adjacent pairs have different polarities. The superposition of the two configurations
enhances the electric field in region B. Reproduced with permission from [6,68,69]. Figure 2. Schematic of different mechanisms for imposing asymmetry to the electric field in alternating
current electrothermal effects (ACET) devices. (a) Direct current (DC) biased configuration. The left
electrode is always at a positive potential and subject to faradaic charging, while the right electrode is
always at a negative potential and subject to capacitive charging. (b) Travelling wave configuration. The induced temperature gradient generates a charge density which is moved with the travelling
electric field. Note that the direction of propagation of travelling wave is opposite to the direction of
fluid flow. (c) Two-phase asymmetric configuration. The narrow electrodes in two adjacent pairs have
different polarities. The superposition of the two configurations enhances the electric field in region B. Reproduced with permission from [6,68,69]. Figure 2. Schematic of different mechanisms for imposing asymmetry to the electric field in
alternating current electrothermal effects (ACET) devices. (a) Direct current (DC) biased
configuration. The left electrode is always at a positive potential and subject to faradaic charging,
while the right electrode is always at a negative potential and subject to capacitive charging. (b)
Travelling wave configuration. The induced temperature gradient generates a charge density which
is moved with the travelling electric field. 4.2.3. Two-Phase Actuation In 2011, Zhang et al. proposed a two-phase planar asymmetric electrode design which yielded an
increase of 25–50% in flow rate compared to conventional single-phase asymmetric configurations [69]. In this design, an AC signal of 0◦/180◦was applied to the narrow electrode in an asymmetric electrode
pair (Figure 2c). This led to an enhanced electric field. As the temperature gradient has a quadratic
relationship with the electric field, temperature, and therefore, flow rate increased accordingly. 4 2 2 Travelling Wave (TW)
4.2.2. Travelling Wave (TW) g
(
)
For the first time in 1966, Melcher observed pumping of fluids with very small conductivities,
by imposing a temperature gradient along the depth of the channel in conjunction with travelling
wave induction [64]. In 1967, Melcher and Firebaugh further investigated the phenomenon and
t d th
i
ti
[65]
For the first time in 1966, Melcher observed pumping of fluids with very small conductivities,
by imposing a temperature gradient along the depth of the channel in conjunction with travelling wave
induction [64]. In 1967, Melcher and Firebaugh further investigated the phenomenon and presented
the governing equations [65]. presented the governing equations [65]. Fuhr et al. studied a travelling wave induced micropump and modified the principles presented
by Melcher and Firebaugh [7,65]. Later, in 1994, the same group explained the mechanisms in which
temperature gradients (both externally applied and internally generated) are used in conjunction
with a travelling wave [66]. With an induced temperature gradient in the fluid bulk, free charges can
Fuhr et al. studied a travelling wave induced micropump and modified the principles presented
by Melcher and Firebaugh [7,65]. Later, in 1994, the same group explained the mechanisms in which
temperature gradients (both externally applied and internally generated) are used in conjunction with a
travelling wave [66]. With an induced temperature gradient in the fluid bulk, free charges can be generated, Micromachines 2019, 10, 762 10 of 27 and the travelling wave can move them along the channel above the microelectrodes causing fluid flow. This principle is further illustrated in Figure 2b. The mechanism of applying a temperature gradient in this
design can be external (i.e., bed heating) or self-generated (i.e., Joule heating). In 1992, Fuhr et al. experimentally investigated a high frequency travelling wave induced
micropump with self-generated temperature gradient, i.e., higher temperatures at the bottom of the
channel in the space between electrodes and lower temperatures at the top of the channel [7]. In this
case, the resultant fluid flow is in the opposite direction of the travelling wave, as also verified by Liu
et al., [68]. Their measurements of flow velocity show a quadratic relationship with voltage caused
by applying an external temperature gradient [13,106]. Following the study of Fuhr et al., several
numerical investigations of travelling wave induced electrothermal flow were also carried out [68,107]. Internal and External Heating In an ACET device, heating of the fluid is the source of thermal gradients which can eventually
lead to electrothermal microflows. Heating can be either internal (i.e., Joule heating) or external. Internal heating refers to Joule heating of the fluid upon activation of the electric field. In a
rectangular asymmetric electrode configuration, the temperature rise above the narrow electrode is
higher than that above the wide electrode. Joule heating scales with conductivity of the fluid and
applied voltage (Equation (1)). As a result, fluids with low conductivity need a higher electric field to
produce sufficient thermal gradients. However, at high applied voltages, electrochemical reactions can
occur, which can damage the fluid and electrodes. This issue can be resolved by applying an external
heat source and keeping the applied voltage low. A few methods have been proposed for external heating, including strong illumination [69,71,73],
integrated heating elements (ohmic heating element) [39], heating of the electrodes [1], and thin film
resistive heaters [74], the latter of which is shown to be portable and more efficient [74,75]. Incorporation of external heating allows the electrothermal flow to be controlled independently
of the electric field strength [39,74]. Therefore, the effect of fluid properties on the resultant flow is
minimized [39], and manipulation of low conductivity fluids with the electrothermal effect becomes
feasible [74]. In addition, by means of external heating, the electrothermal flow can be implemented in
conjunction with other electrokinetic methodologies, which work under a uniform electric field, such
as electrophoresis and electroosmosis [74,75]. External heating strategies also enable control of the
direction of fluid flow [1,71,106]. Green et al. performed a thorough study on strong illumination as an external heat source [71,106]. The resultant velocity field in the case of external heating can be obtained from the equation below [13,106]: |u| ≈3 × 10−3
εV2
η
! ∂T
∂y
1
σ
(16) (16) where, ∂T
∂y is the external thermal gradient along axis y. Unlike Joule heating, here the velocity has a
quadratic relationship with voltage, which was verified experimentally by Stubbe et al., where ohmic
heating elements were used [39]. Depending on the amount of heat flux introduced into the fluid bulk by the external heating,
the effect of voltage on the flow velocity changes. In 2013, Yuan et al. Internal and External Heating found that if the heat flux is 11 of 27 Micromachines 2019, 10, 762 small (around 104 W·m−2), it can be counteracted by the Joule heating of the fluid, and the relationship
between fluid velocity and voltage can be between quartic and quadratic [1]. However, if the heat flux
is relatively large and the fluid is mostly under the influence of external heating, the fluid velocity will
have a quadratic dependence on the voltage. Furthermore, thin film microheaters can be embedded in the substrate below the electrodes,
separated by an insulating layer [75]. Such a heating mechanism can yield a thermal efficiency of
close to 100% [74]. Velasco and Williams showed the application of thin film heaters for assembly
of colloids [75]. The geometry of the assembly is governed by the geometry of the array of
microheaters. Therefore, particles can be trapped in a larger area when compared to conventional
particle trapping techniques [13]. The assembly of 1 µm and 2 µm particles was achieved in the
frequency range of 1–200 kHz, while no particle aggregation was observed at other frequencies. Since the temperature gradient is the underlying mechanism, microheaters cannot be patterned close
to each other. Furthermore, with thin film microheaters, sharper temperature gradients exist near
the electrodes which result in a larger value of ∇T·E2 [74]. Compared to Joule heating, thin film
microheaters alone require only 40% of the power to produce the same flow rate. Without a change in
power, the flow rate can be increased to 250% of that of solely Joule heating [74]. Both the proximity of
thin film resistive heaters to each other and their distance to the regions of maximum electric field
strength determine the resultant flow regime [74,75]. The maxima of temperature gradient and electric
field need to occur in the same region in the bulk of the fluid in order to maximize the product of
∇T·E2 [74]. More recently, Williams and Green applied the same idea to a symmetric pair of electrodes,
which is more desirable for DEP applications, and carried out numerical simulations towards finding
an optimum location for the heater with respect to the electrodes [108]. In spite of a fundamental factor for generation of electrothermal flow, Joule heating can be an
unwanted effect in other electrokinetic devices [80]. Internal and External Heating For example, in a DEP based particle manipulation
device, Joule heating can harm the biological fluid and form electrothermal microflows taking particles
away from their intended spot of sorting or trapping [80,81]. For this reason, studies have been
conducted to gain a better insight into Joule heating [80] and its effects on insulator-based DEP
devices [81,109]. Almost all electrothermal designs studied in the literature are checked for excess temperature
rise due to Joule heating, as this can negatively impact their potential application with biofluids. For example, Du and Manoochehri compared the Joule heating in their proposed microgrooved and
planar configurations at a wide range of conductivities [85] and found no major difference between the
two structures. In devices with thin film microheaters, temperature rise is shown to be half of that of
Joule heating, which further corroborates the high efficiency of these devices [74]. Similar to Joule heating, external heating may not always lead to an enhanced electrothermal effect. Zhang et al. studied the effect of strong illumination coupled with their two-phase planar electrode
configuration [69]. Interestingly, they observed a decrease in velocity when a strong illumination
was applied. This observation was justified by assuming that the flow direction generated by the
illumination is opposite to that generated by Joule heating [83]. As mentioned earlier, for external
heating to be effective, heat flux introduced into the system must be significantly higher than the Joule
heating generated by the system. Not clearly stated by Williams [74], however, is that their reported
results show that by increasing the conductivity, the ratio of electrothermal force generated by thin
film heaters to that generated by Joule heating gradually decreases. This indicates that the coupled
electric and temperature fields need to be solved for higher temperature rises (>5 K) [82,86]. ACET flow velocity has a strong dependence on the applied voltage (u ∝V4, when no external
heating is applied). However, voltage can be increased up to a certain point, usually below 7 Vrms,
to avoid thermal damage to the biofluid [1,11,14,41,60,71,75]. To solve this problem, Yuan et al. introduced a thermally biased configuration, where one electrode in a pair is at a higher temperature
than the other one [1]. In this way, the temperature gradient in the fluid can be controlled independently
from the voltage. 6.1. Flow Velocity Almost all the work that has been performed dealing with the electrothermal effect is an attempt to
increase the ACET velocity in high conductivity fluids while avoiding significant increase in temperature
and voltage. Many studies have attempted to increase the electrothermal flow velocity by focusing on the
governing parameters mentioned in Equation (14) and have proposed new designs to increase the velocity
based on their interpretations of the formula. The maximum of the flow velocity reported in these studies
differs both in its magnitude and location (i.e., height/distance from channel bottom/electrodes). Since electrothermal velocity has a quadratic relationship with the temperature gradient, many
studies have focused on increasing this parameter of electrothermal force to increase the flow velocity. External heating has been proposed and thoroughly investigated for this purpose, which is discussed
in Section 5. By simplifying the equation of electrothermal force to |< FE >| = ξ(ω)|E|2|∇T|, Zhang et al. concluded that by increasing both the strength of electric field and the temperature gradient, a significant
increase in electrothermal force and thus fluid velocity could be obtained [69]. Taking advantage of
this combination, by developing a two-phase asymmetric planar electrode design in which a stronger
electric field is obtained, they demonstrated flow velocities reaching 25–50% higher than those of
conventional single-phase configurations. In this simplified equation, ξ(ω) is a function of frequency
and the angle between the vectors of applied field and temperature gradient. They also found that
fluid velocity is much higher at low frequencies, which was also verified by Williams who determined
that pumping rates at high frequencies (>fc) drop to 10% of that at low frequencies [74]. To accurately measure the electrothermal velocity field, usually micro particle image velocimetry
(micro-PIV) is used. The assumption of this method is that particles follow the fluid flow and are not
under the influence of any other forces [110–112]. Therefore, ACET velocity needs to be measured at a
height with a negligible DEP effect on particles [1]. At a distance close to electrode surface, the DEP
effect on particles is significantly high, which can cause them to be trapped at the electrode edges [1]. As a result, depending on the channel height and size of tracer particles, ACET velocity is typically
measured at a height of 10–50 µm above the electrode surface. Internal and External Heating 12 of 27 Micromachines 2019, 10, 762 At frequencies below 10 MHz, where the Coulomb force is the dominant force, the electrothermal
force can be simplified to FE = ρEE. Since, due to the limitations discussed above, there is an upper limit
for the applied electric field, the electrothermal force can be increased further by increasing the charge
density, ρE. As ρE ∝∇T·E, Yuan et al. attempted to increase the electrothermal force by increasing
the temperature gradient by means other than Joule heating [1]. In their design, unidirectional
flow was obtained with symmetric electrodes, making it possible for their configuration to be used
either as a micromixer or a micropump depending on the applied voltage, frequency, and heat flux. When imposing external heat flux to the narrow electrode in an asymmetric pair, they obtained a
velocity 5.7 times higher than that of a regular asymmetric ACET micropump. Their simulation results
show that by applying the heat flux to the wide electrode, the direction of the flow above the electrodes
can be reversed. This finding corroborates their hypothesis that the generated heat on the narrow
electrode is the primary cause of pumping in a conventional ACET device, and that external heating
has a strong influence on the pumping performance. 6.1. Flow Velocity A good control for this effect is carried
out by Wu et al., where, they found, by using particles with a size of ~500 nm, an order of magnitude
estimation yields that at ~5 Vrms particles move with a DEP-induced flow velocity of 0.11 µm·s−1 at the
height of 10 µm above the electrodes [4]. Since ACET velocity is often higher than 100 µm·s-1, DEP
velocity is negligible at this height. The highest electrothermal velocities reported are taken at a height
of 20–50 µm above the electrodes [1,13,92]. The lowest electrothermal velocity occurs very close to the
electrodes (~5 µm above electrodes in a microchannel with a height of 200 µm) [92]. These observations
are in agreement with the physics of the electrothermal effect as it is created in the bulk of the fluid. 13 of 27 Micromachines 2019, 10, 762 Increasing the number of microelectrode pairs has shown to result in increasing ACET fluid
flow of high conductivity biofluids at voltages above 4 Vrms [92]. In addition, flow velocities
measured experimentally are typically smaller than those predicted numerically, sometimes by orders
of magnitude, which can be justified to be related to the experimental conditions. Here, we discuss
some of the experimental issues reported in the literature. In their experimental study, Sigurdson et al. showed an electrothermal flow velocity of 100 µm·s−1,
which was reported at a lower voltage in a numerical study [14]. Since their simulations are performed
for a 2D geometry, they attributed this discrepancy to neglecting the out of plane heat transfer, which
is significant in devices with a small channel height (i.e., ~200 µm). In their later work, since they
encountered the same issue (i.e., observing an experimental velocity of 1.5 orders of magnitude lower
than the numerical result), they defined an effective voltage [61]. For this matter, a reduction coefficient
of 0.38 was introduced to correct the actuation voltage in the numerical simulations. With the modified
voltage, the velocity–voltage curve of numerical study closely matches the corresponding curve of the
experimental study. Studying the Joule heating effect, Williams et al. [80] defined a coefficient, Erel, for medium
conductivity, to account for the loss in applied electric field, which they attribute to the electrical
resistance of their electrode material (i.e., indium tin oxide (ITO)). Therefore, by using a more conductive
material for electrodes (e.g., gold), there will be less potential loss (i.e., larger value of Erel). 6.1. Flow Velocity Sin et
al. showed a significant deviation of experimentally measured fluid velocity from the theoretically
predicted one for fluids with conductivities on the order of 22 S·m−1 [60]. The reason for this discrepancy
was assumed to be the deterioration of the electrode surface due to electrochemical reactions at high
conductivities, which is not accounted for in the numerical model. The same argument is also reported
as the reason for deviation of experimental data from simulation in other studies [76,105]. With the
surface of the electrode being altered, the electric field, flow patterns, and velocities are no longer
predictable as the actual electric potential can be significantly decreased [76]. In addition to high
conductivity, increasing voltage above 5.5 Vrms is also reported to cause electrode deterioration [76]. Evaporation, and thus a change of the medium’s properties, is also mentioned as another possible
factor for this discrepancy. In addition, the buoyancy effect can play a role in causing this discrepancy,
since a high conductivity medium can experience high temperatures. The ratio of the electrothermal
force FE to buoyancy force FB can be approximated as the following [4]: FE
FB
= 7.95 × 10−12
∇T
∆T
·E2
rms
(17) (17) As suggested by this approximation, for buoyancy to be negligible, temperature gradient ∇T needs
to be much higher than temperature rise ∆T. As a result, for enhancing the ACET performance, device
elements (e.g., electrodes) with relatively high thermal conductivities need to be implemented [4]. 6.2. Direction of AC Electrothermal (ACET) Flow In micropumps with planar electrode configurations, two microvortices on both electrodes in
every pair are in competition to determine the direction of net flow [85]. In conventional rectangular
asymmetric configurations, since the fluid flow spends more time on the wider electrode, the direction
of ACET net flow is determined by the microvortices on the wider electrode [4]. It has been experimentally shown that the direction of flow can be controlled by external heating,
i.e., switching the heat flux between wide and narrow electrodes [1,71,106]. This is true for both
rectangular symmetric and asymmetric electrode configurations. Additionally, some studies have been
performed on the use of unique device configurations to easily toggle the direction of net flow and
introduce mixing [113,114], however, these models have not yet been rigorously validated. Micromachines 2019, 10, 762 14 of 27 6.3. Flow Reversal One major advantage of ACET is that its velocity is generally independent from frequency,
however, at around the crossover frequency, flow reversal occurs, and thus, lower velocities (~10–20%
of velocity of low frequencies) are generated [39,69,74,115]. One major advantage of ACET is that its velocity is generally independent from frequency,
however, at around the crossover frequency, flow reversal occurs, and thus, lower velocities (~10–20%
of velocity of low frequencies) are generated [39,69,74,115]. Flow reversal has also been observed in devices with external heating. In 2014, Liu et al. numerically investigated applying a temperature gradient along the channel length on rectangular
symmetric electrode arrays and compared it to the conventional asymmetric array [68]. They observed
unidirectional pumping in the direction from higher to lower temperatures at 100 kHz (i.e., from the
narrow electrode to wide electrode). They also observed that by increasing the frequency to 500 kHz,
the direction of flow in a symmetric array reverses but still maintains a unidirectional flow, whereas,
in the asymmetric array, the unidirectional flow turns to vortices with no pumping action. In general, factors other than frequency can also lead to flow reversal. For example, ACEO systems
can face flow reversal at higher voltages due to faradaic charging [116]. Transition between ACEO
and ACET mechanisms [93] and steric effect can also be important [117,118]. However, none of these
reasons can justify the flow reversal in the DC biased AC electrothermal device of Lian et al. [6], which
is still open for further investigation. Flow reversal is considered by most studies as a disadvantage in ACEK due to the uncontrollable
and unpredictable nature of this phenomenon. However, some studies have investigated controllable
flow reversal by tuning the actuation frequency, switching electric field, and/or applying external heat
sources [1,39,68,113,114]. 7. Application Most common applications of the electrothermal effect in micro systems include mixing and
pumping of fluids and particle manipulations [1,13,60,62]. A good example of using the electrothermal
effect for mixing is immunoassays [11,14,20,61,119–121]. Immunoassays are biochemical tests in which
the concentration of macromolecules (e.g., ligands or proteins) in a biofluid is measured by the use of
an antibody. The antibody is immobilized on a surface and then the biofluid of interest is introduced
above the surface. The concentration of macromolecules is measured by the number of macromolecules
attached to the antibodies. The key factor in this process is for macromolecules to contact the antibodies
repeatedly so that all possible bindings take place and lead to a precise sensing result. Since this process
is diffusion limited, incubation time can take hours [61]. By using the electrothermal effect, the chance
of macromolecules reaching the sensing area can be increased, thereby significantly decreasing the
response time, enhancing the bindings by seven to nine times within minutes [14,61]. Sigurdson et al. investigated this idea numerically and found that by applying a voltage of 6 Vrms, an increase of seven
times in the amount of bound antigen can be reached. It should be noted that electrothermal stirring
is effective only when Damkohler ≥100, i.e., when the process is diffusion limited and not reaction
limited. Therefore, electrothermal stirring is not significantly effective for reaction limited systems such
as DNA systems. Sigurdson et al. also found that the electrothermal stirring is especially effective in
the space above the electrode gap, where the velocity and the concentration gradient are high, making
it the optimum place for antibodies to be immobilized [11,14,61]. This was proven experimentally
in their later work [61]. Huang et al. found the optimum reaction site to be closer to the negative
electrode in a symmetric rectangular pair [122]. In this case, both Damkohler and Peclet numbers
need to be considered. Electrothermal stirring is best for mass transport limited regimes, where the
Peclet number is relatively low, and convection contributes more to the overall mass transport than
diffusion. Therefore, by utilizing the electrothermal effect, the required sample volume can be reduced,
leading to higher efficiency devices [14]. It has also been shown that in immunoassay applications,
electrothermal force can be a better choice compared to electroosmotic force, as electroosmotic force
may cause the antigen–antibody bounds to fall apart [14]. In 2012, Sasaki et al. 8. Substrate Material As electrodes are patterned on a substrate, and the substrate is one of the channel walls through
which the heat is dissipated, the choice of substrate material is of great importance [135]. The materials
typically used for the fabrication of microfluidic devices include silicon, glass, polydimethylsiloxane
(PDMS), and polymethylmethacrylate (PMMA). As a commonly used substrate material, glass has a
lower thermal conductivity compared to silicon and can yield higher temperature gradients, and thus,
higher rates of electrothermal flow [105]. Heat transfer through the substrate can be controlled by other
means too. For example, a thermoelectric cooler under the silicon substrate has been suggested for
maintaining the temperature gradient of interest in an ACET device [61]. As opposed to glass, silicon
has a higher heat transfer coefficient, and thus, is a good candidate for applications where excessive
temperature rise is undesirable [61,85,87,92,136]. Silicon is also admired for its precise geometrical
features and low surface roughness [85]. Compared to silicon, the thermal conductivity of PDMS is
much lower, causing the temperature rise in PDMS microchannels to be significantly high [92,136]. 7. Application used a meandering electrode configuration in a Y-shaped channel to mix
two high salt content fluids [62]. They reported a fivefold reduction in mixing time compared to 15 of 27 Micromachines 2019, 10, 762 diffusional mixing. In their study, the dependence of mixing index on salt concentration, frequency,
and mixing time was investigated. It was concluded that the meandering structure was suitable for salt
concentrations of 10−3 to 10−1 mol·dm−3, provided that the frequency lied in the range of 100–200 kHz,
which is typical for electrothermal devices. A long range ACET effect, where centimeter scale vortices
are generated, can also be used for mixing purposes [70]. Further development on electrothermal based immunoassays was carried out by Liu et al. in
2011 [11]. They decreased the incubation time from 30 min to 3 min by implementing ACET in their
immunoassay with the conventional asymmetric electrode array. A study on electrode geometry
on capacitive immunoassays showed that electrode geometry is an important component in high
electric field configurations with asymmetric geometries rendering higher detection efficiency [123]. Another study suggested that the placement of electrodes on the same surface as the reaction site
renders the most efficient configuration for immunoassays [121]. Selmi et al. studied the effect of
temperature on immunoassays with asymmetrical electrodes [124]. The use of a pulsed ACET flow
and amplitude modulated (AM) sine waves has also been reported for enhancing mixing efficiency in
immunoassays [125,126]. Electrothermal effect in conjunction with dielectrophoresis can be used for the trapping and
assembly of particles, patterning of colloids, and preconcentration of biological samples for detection
and characterization purposes [13,56,59,75,127–131]. Additional studies have shown the enhancement
of DNA hybridization with ACET configurations [132]. Recently, the concept of an AC electrothermal
micropump was applied to a cell-culture system (with culture media of conductivities up to 2 S·m−1)
towards the development of organ-on-a-chip and human-on-a-chip systems [63,133]. Recent studies have also shown that, although other ACEK effects such as DEP can enhance
trapping of biological samples, they render nonspecific responses [134]. 9. Channel Height Unlike ACEO, the origin of ACET is the charge density generated in the bulk of the fluid. Therefore, the height of the microfluidic channel plays an important role in the formation of microflows. The microflows will be suppressed when the channel height is too small (<200 µm in a typical
microfluidic ACET device) [13,76]. In contrast, ACEO devices utilize a thin layer of electric double
layer responsible for dragging the fluid bulk through the microchannel, meaning that reducing the
channel height causes the ACEO effect to be more effective. It has been shown that in two-phase actuation systems, the optimal channel heights are in the
range of 500–1000 µm [69]. In a planar configuration, the maximum velocity is reached at the height of
~500 µm, above which no significant difference in the flow rate can be observed [76,85]. In microgrooved
structures, however, it is shown that a velocity of five times higher than planar electrode configurations
can be reached at a significantly smaller channel height, i.e., ~50 µm, which is 10% of the optimal 16 of 27 Micromachines 2019, 10, 762 height in conventional and two-phase systems. This feature of the microgrooved configuration helps
further miniaturizing the ACET based devices. As mentioned above, miniaturization is hindered in
ACET devices due to dependence of the microflows on the channel height. With increasing the channel
height above 50 µm in a microgrooved configuration, the velocity drops but still stays at higher values
compared to a planar configuration. In the case of patterning electrodes both on the top and bottom of the microchannel in an ACET
device, increasing the channel height above 200 µm causes the velocity profile to become more similar
to that of an ACEO device [104]. A thorough study on the effect of channel height in micropumps can
be found in reference [76]. 10.1. Numerical Simulation 10.1. Numerical Simulation 10.1. Numerical Simulation •
Since the wavelength of the electric field is typically larger than the dimensions of the microchannel,
electrostatic assumptions can be made [41]. •
If electrodes are thin, they do not affect heat transfer [61]. Sufficiently thin electrodes (e.g., ~1200 Å
thick) can be assumed isothermal [13,106]. •
In typical ACET devices, the ratio of buoyancy force to electrothermal force, i.e.,
FB
FE
, is estimated to
be in the range of 7 × 10−4 −27 × 10−4 [41,85]. When buoyancy is included in the simulations, only a
0.1–0.8% decrease in flow velocity is obtained [41]. Therefore, the buoyancy effect can be neglected in
simulations. However, at large length scales and low voltages, it becomes important [41]. •
ACEO effect can be neglected at high frequencies and high conductivities [41]. •
2D and 3D simulations usually render the same results. For example, Yuan et al. [41] reported a
1.48% difference in velocities obtained from their 2D and 3D simulations. Therefore, 2D simulations
can help saving computational time [41]. •
2D and 3D simulations usually render the same results. For example, Yuan et al. [41] reported a
1.48% difference in velocities obtained from their 2D and 3D simulations. Therefore, 2D simulations
can help saving computational time [41]. •
While in most studies electric and thermal fields are considered independent, using the results
of Loire et al. [86], Williams [74] conducted numerical simulations with coupled electrical and
thermal fields as ∇2V = γ·∇V, where γ = −β∇T, instead of the conventional sequential method
i.e., ∇2V = 0. It was shown that when the temperature rise in a system is >5 K, the two fields can
no longer be considered independent [86]. •
New methods based on Lattice Boltzman were reported for studying ACET flows [137,13 10. Numerical and Experimental Settings In this section, some key points and influential factors in numerical simulation and experimental
setup of ACET devices are briefly reviewed. To assure that the same conditions are valid to apply to
other devices of interest, the readers are advised to refer to the articles associated with each statement. 10.2. Experimental Setup •
The lighting on the microscope, on which the ACET device is mounted, can play as an external heat
source and interfere with the experiments, and thus cause unreliable results. In order to reduce
the effects of microscope light, either it needs to be set at its lowest power [13] or a heat absorbing
filter between the device and the objective lens needs to be used [62]. Otherwise, illumination
effects must be taken into account as an external heat source. •
To reduce the effect of Brownian motion, the average of at least four velocity readings at each
voltage setting is recommended to be taken [13]. •
If the work involves study of temperature on DEP effect, a non-invasive method (i.e., with no particles
involved) must be used to measure temperature in the device. Laser-induced fluorescence (LIF)
thermometry, in which a dye is used to measure temperature, is recommended for this purpose [80]. F
i
l
it
t
t bilit
ti l
t b t
k d
t l
t th
i •
If the work involves study of temperature on DEP effect, a non-invasive method (i.e., with no particles
involved) must be used to measure temperature in the device. Laser-induced fluorescence (LIF)
thermometry, in which a dye is used to measure temperature, is recommended for this purpose [80]. •
For measuring velocity, to ensure repeatability, particles must be tracked over at least three pairs
of electrodes along the microchannel [41,76]. 17 of 27 Micromachines 2019, 10, 762 •
For generating effective electric field at the electrode surface, electrodes should be fabricated
relatively thin, e.g., 50–100 nm [32]. •
For generating effective electric field at the electrode surface, electrodes should be fabricated
relatively thin, e.g., 50–100 nm [32]. 11. Future Work Although utilizing the ACET effect for various biomedical applications has been investigated
extensively over the past two decades, more investigative work is yet to be carried out to further our
knowledge of the phenomenon. Existing drawbacks must be better addressed in order to facilitate
utilization of this effect in biomedical laboratory settings. For example, temperature rise, and its
detrimental effects on biological samples, is of great concern while using the ACET effect in microfluidic
devices. Substrate materials with high heat conductivities, which enable a low temperature rise while
keeping high voltages, must be investigated to achieve strong electrothermal flow. While plenty of novel and efficient electrode designs have been proposed for pumping and
mixing applications, the majority of such reports are numerical studies. A lack of experimental studies
that would reveal the hidden challenges in applying these novel strategies to real life applications
exists in the literature. Such lack of experimental works is mainly due to the limitations in microscale
fabrication and electrode degradation occurred at high voltages. To address such fabrication challenges,
techniques for low cost fabrication of prototypes of such complicated designs and investigation of
different electrode materials and coatings to withstand high voltages [79] are in great demand. Author Contributions: Writing—original draft preparation, M.N. and A.S.; writing—review and editing, M.N.,
A.S., T.L., and C.D.; supervision, A.S., C.D.; funding acquisition, C.D. Funding: This research was funded by a Canadian Natural Sciences and Engineering Research Council (NSERC)
Discovery Grant. Funding: This research was funded by a Canadian Natural Sciences and Engineering Research Council (NSERC)
Discovery Grant. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Appendix A The majority of the publications cited throughout the manuscript which have made major
contributions are summarized in Table A1. The majority of the publications cited throughout the manuscript which have made major
contributions are summarized in Table A1. Table A1. A short summary of relevant articles studying the electrothermal effect, useful for new
researchers in this field as a guide to what has been done and those who are working in the field. Article
Application
Achievement
Specific Observations
[71]
Mixing
Experimental study of
illumination-induced
electrothermal
The direction of force at high
frequencies is form hot regions
to cold regions while at low
frequencies the opposite is true. [14,61]
Mixing
Increasing the binding rate
and significantly decreasing
the incubation time to minutes
Binding rate increased by a
factor of nine compared to
diffusion-limited reaction
[4]
Pumping
Study of pumping for two
electrode configurations of
planar asymmetric and
orthogonal
Orthogonal configuration yields
higher velocities
[13]
Particle manipulation
and pumping
Manipulation of particles and
fluids of high conductivity at
low voltages using a parallel
plate and a planar asymmetric
electrode configuration
Velocity of 162 µm·s−1 18 of 27 Micromachines 2019, 10, 762 Table A1. Cont. Article
Application
Achievement
Specific Observations
[93]
Pumping
Numerical and experimental
investigation of flow reversal
in orthogonal electrodes
Change of flow patterns is a result
of change from alternating current
electrothermal (ACET) effects to
alternating current electroosmosis
(ACEO) phenomenon
[6]
Pumping
Applying asymmetry in
electric potentials in
conjunction with
spatial asymmetry
Velocity of 2500 µm·s−1
[62]
Mixing
Introducing meandering
electrode configuration with
electrothermal effect in a
Y-shaped channel
Fivefold reduction of the mixing
time of high salt content fluids
compared to
diffusion-limited methods
[85,97]
Pumping
Introducing microgrooved
electrode configuration
Five times increase in pumping
rate compared to conventional
planar configurations
[69]
Pumping
Introducing two-phase AC
signal configuration
25–50% faster flow rates in
two-phase configuration
compared to the conventional
single-phase configuration
[60]
Mixing
Introducing concentric
electrode design
Velocity of 70 µm·s−1
[11]
Mixing
Using asymmetric electrodes
for immunoassay
Ten times acceleration in
binding rate compared to
diffusion-limited method
(30 min vs. Appendix A 3 min)
[1]
Pumping
Thermally biased ACET
pumping using symmetric and
asymmetric electrodes
Velocity of 750 µm·s−1
[75]
Particle manipulation
Using parallel plate (opposing)
electrodes in conjunction with
thin film resistive heaters
Sorting between 1 µm and 2 µm
particles
[92]
Pumping
Study on the effect of the
number of electrode pairs over
channel length; asymmetric
planar electrodes
Increasing the number of
electrode pairs helps increase
the pumping efficiency
[105]
Pumping
Introducing electrodes both on
top and bottom of the
microchannel; asymmetric
planar electrodes
Opposing electrodes increase
the flow rate by 105%
[76]
Pumping
Multiple Array Electrothermal
Micropump (MAET) with
different actuation patterns
and cross sections
Flow rate of 16 × 106 µm3·s−1
[96]
Pumping
3D circular electrodes
Flow rate of 15 × 106 µm3·s−1
[104]
Mixing and pumping
Numerical investigation of
simultaneous pumping and
mixing by introducing
microelectrodes on side walls
of the microchannel
Mixing efficiency of 80% in
<3 min and over a length of
<600 µm Table A1. Cont. Table A1. Cont. 19 of 27 Micromachines 2019, 10, 762 Table A1. Cont. Table A1. Cont. Table A1. Cont. Article
Application
Achievement
Specific Observations
[135]
Pumping
Numerical study of multiple
array ACET channel
Flow rate of 16 × 106 µm3·s−1
[74,108]
Pumping
Study of using thin film
heaters for pumping
2.5 times faster flow rate with
thin film heaters compared to
Joule heating alone
[63]
Pumping
Application of ACET pumping
to cell culture on chip
Flow rate of 44.82 µL·h−1
[139]
Particle manipulation
Combining ACET and
dielectrophoresis (DEP) for
detection of circulating
cell-free DNA (cfDNA)
Detection of cfDNA in 10 min in
concentrations as low as
43 ng·mL−1
[140]
Pumping
Numerical and experimental
study of the effects of
conductivity and channel
height on ACET flow
A critical conductivity exists
below which there is no net flow
and there exists only
microvortices
[119]
Mixing
Quantum dot-linked
immunodiagnostic assay
coupled with ACET mixing
Reduction of detection time
from 3.5 h to 30 min using a
volume of 2 µL
[59]
Particle manipulation
Development of a
mathematical model for rapid
electrokinetic patterning (REP)
REP based on ACET and DEP
Increasing particle size results in
an increase in ratio of ACET to
DEP velocity and therefore
results in a lower
focusing performance
[73]
Mixing
Experimental study of light
actuated ACET flow
When AC frequency is above
liquid charge relaxation
frequency, natural convection is
above 35% of the ET flow. Appendix A [123]
Mixing
Numerical and experimental
comparison of immunoassay
performance when using
symmetric or
asymmetric electrodes
Symmetric and asymmetric
geometries render different
performance efficiencies only at
high electric fields
[102]
Particle manipulation
Numerical and experimental
study of electrode material
in REP
Titanium electrodes are more
efficient than conventionally
used indium tin oxide
(ITO) electrodes
[141]
Mixing
Numerical and experimental
study of AC biased concentric
electrodes in biosensors
Faster sensing speed compared
to diffusion-limited conditions
[142]
Mixing
Numerical and experimental
study of rotating asymmetric
electrode pair; Supplying
controlled drug concentration
to tumor cells
Mixing efficiency 89.12%
[70]
Mixing
Numerical and experimental
study of long-range fluid
motion induced by
ACET microvortices
Centimeter scale ACET vortices
are observed
[124]
Mixing
Numerical study of the effect
of temperature on binding
efficiency in immunoassays
Keeping external surfaces of the
microchannel at a constant
temperature improves the
binding efficiency 20 of 27 20 of 27 Micromachines 2019, 10, 762 Table A1. Cont. Table A1. Cont. Article
Application
Achievement
Specific Observations
[143]
Mixing
Numerical and
experimental-3D electrodes
embedded inside walls of
the channel
Mixing efficiency of 90%
[113]
Pumping
Numerical and experimental
study of bi-directional
micropump using asymmetric
planar electrodes
1500 µm·s−1 fluid velocity
[121]
Mixing
Numerical study of
electrothermal effect
in immunoassays
Placement of electrodes on the
same wall as the reaction surface
renders the best performance of
the biosensor
[126]
Mixing
Study of pulsed ACET flow for
detection of dilute samples of
small molecules
83% mixing efficiency over a
length of 400 µm
[125]
Mixing
Numerical investigation of
amplitude modulated
(AM) sinewave
100% mixing efficiency with
maximum 5.5 K temperature rise
[144]
Mixing
Numerical investigation of the
effect of ionic strength
on mixing
Mixing efficiency 90%
[134]
Pumping
Experimental study of an
immunoassay chip featuring
an ACET micropump
Reducing incubation time to
1 min vs. Appendix A hours in
conventional methods
[99]
Simultaneous pumping
and mixing
Numerical study of high
throughput mixing using
opposing asymmetric
microgrooved electrodes and
symmetric electrode pair
Mixing efficiency of 97.25%
[114]
Simultaneous pumping
and mixing
Numerical study of
bi-directional pumping and
mixing by switching electric
potential on planar electrodes
Mixing efficiency of 90%
Pumping velocity 90 µm·s−1
[90]
pumping
Numerical investigation of
pumping non-Newtonian
blood flow
Velocity of 0.02 m·s−1
[89]
Mixing
Numerical investigation of the
effect of shear dependent
viscosity on mixing efficiency
and flow rate using opposing
asymmetric microgrooved
electrodes and symmetric
electrode pair
In similar configurations,
dilatant fluids show better
mixing efficiency compared to
pseudoplastic fluids
[101]
Mixing
Study of arc electrodes in
ring-shaped microchamber
100% mixing efficiency at 8 V
[127]
Trapping
Using ACET and DEP to
preconcentrate and detect
E. Coli
Method can detect
concentrations two orders of
magnitude smaller than what is
possible with diffusion
limited methods Method can detect
concentrations two orders of
magnitude smaller than what is
possible with diffusion
limited methods 21 of 27 Micromachines 2019, 10, 762 Table A1. Cont. Table A1. Cont. Table A1. Cont. Appendix A Article
Application
Achievement
Specific Observations
[133]
Pumping
Using laser etching on ITO
glass to pattern electrodes for
pumping cell culture medium
in a 3D biomimetic liver
lobule model
2 µm·s−1 at 5.5 V
[100]
Pumping
Using castellated electrodes;
combined DEP and ACET
EHD for bioparticle delivery
Negative DEP prevents particles
from colliding with channel
surfaces; castellated electrodes
eliminate ACET vortices
[138]
Pumping
Combining ACET and
negative DEP for long range
cell transport and suspension
in high conductivity medium
DEP is essential for cell
suspension under ACET effect
[95]
Simultaneous pumping
and mixing
Numerical investigation of 3D
asymmetric spiral
microelectrode pair
Flow rate 440 µm·s−1
[91]
Pumping
Numerical investigation of the
effect of electrode
configuration on pumping
mechanism of non-Newtonian
blood flow
Ring shaped electrodes are the
optimal configuration for blood
flow pumping
[88]
Pumping, mixing,
and trapping
Study of 3D particle-fluid flow
under simultaneous effects of
ACET, thermal buoyancy (TB),
and DEP using multi-layered
electrodes
Long range vortices induced by
ACET and short-range
circulations induced by TB
[77]
Simultaneous pumping
and mixing
Introducing two opposing
microelectrode arrays placed
at an angle relative to
channel length
Mixing time reduced by 95%
compared to diffusion-limited
methods
[72]
Mixing
Study of light induced ACET
flow over electrodes of
different materials using
opposing electrodes
Electrodes with high optical
absorption rate and low thermal
conductivity are best for
effective light-induced heating
[58]
Comprehensive particle
and droplet
manipulation
Combining ACET and DEP
Particle transit time between
multiple branches 0.008 s;
droplet sorting purity 90%;
particle sorting purity 93% References References References
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144. Vafaie, R.H.; Madanpasandi, A. In-situ AC electroosmotic and thermal perturbation effects for wide range of
ionic strength. Aims Biophys. 2017, 4, 451–464. [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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Supplementary Figure from Proteolytic pan-RAS Cleavage Leads to Tumor Regression in Patient-derived Pancreatic Cancer Xenografts
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Proteolytic pan-RAS cleavage leads to tumor regression in patient-derived pancreatic cancer
xenografts Proteolytic pan-RAS cleavage leads to tumor regression in patient-derived pancreatic cancer
xenografts Vania Vidimar, Minyoung Park, Caleb K Stubbs, Nana K Ingram, Megan M Packer, Wenan
Qiang, Shanshan Zhang, Demirkan Gursel, Roman A Melnyk, and Karla JF Satchell Supplemental Figure S1. Assessment of HB-EGF levels in PDAC cell lines and crystal violet Supplemental Figure S1. Assessment of HB-EGF levels in PDAC cell lines and crystal violet Supplemental Figure S1. Assessment of HB-EGF levels in PDAC cell lines and crystal violet Supplemental Figure S1. Assessment of HB-EGF levels in PDAC cell lines and crystal violet cytotoxicity assay quantification. (A) Representative western blot image of HB-EGF expression
in six different KRAS-mutant PDAC cell lines. (B) PDAC cells treated with increasing doses of
RRSP-DTB and 10 nM of RRSP*-DTB for 72 hours were fixed and stained with crystal violet,
which was subsequently dissolved in 10% acetic acid and measured by absorbance at 590 nm. Bar
plots represent mean ± SD of three independent experiments (*p < 0.05, **p < 0.01, ****p <
0.0001 versus control 0 nM; one-way ANOVA followed by Dunnett’s multiple comparison test;
n = 3). Supplemental Figure S2. Monitoring of mouse weight during PDX studies. (A) and (B) Mouse
weight was recorded during PDX1 (A) and PDX2 (B) studies and plotted against time (days). A
B
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RRSP-DTB A A B Supplemental Figure S2. Monitoring of mouse weight during PDX studies. (A) and (B) Mouse Supplemental Figure S2. Monitoring of mouse weight during PDX studies Supplemental Figure S2. Monitoring of mouse weight during PDX studies. (A) and (B) Mouse
weight was recorded during PDX1 (A) and PDX2 (B) studies and plotted against time (days). weight was recorded during PDX1 (A) and PDX2 (B) studies and plotted against time (days). Supplemental Figure S3. Proteolytic pan-RAS cleavage leads to tumor regression in patient-derived pancreatic cancer
xenografts Whole tumor sections from PDX1 and PDX2 studies. (A) and (B)
t1
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day 81 Supplemental Figure S3. Whole tumor sections from PDX1 and PDX2 studies. (A) and (B)
Whole tumor sections from PDX1 (A) and PDX2 (B) studies stained with hematoxylin and eosin,
Masson’s trichrome staining, CK-19, Ki-67 and pERK antibodies from three mice per group
(saline and RRSP*-DTB) and all six mice in the RRSP*-DTB group. Scale bar = 10 mm. t1
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day 81 A B Supplemental Figure S3. Whole tumor sections from PDX1 and PDX2 studies. (A) and (B) Supplemental Figure S3. Whole tumor sections from PDX1 and PDX2 studies. (A) and (B) Whole tumor sections from PDX1 (A) and PDX2 (B) studies stained with hematoxylin and eosin,
Masson’s trichrome staining, CK-19, Ki-67 and pERK antibodies from three mice per group
(saline and RRSP*-DTB) and all six mice in the RRSP*-DTB group. Scale bar = 10 mm. Supplemental Figure S4. Immunohistochemical analysis of total RAS levels in PDAC PDX
tumors following treatment with RRSP-DTB. (A) Representative images of saline, RRSP*-DTB
and RRSP-DTB-treated PDAC PDX1 and PDX2 tumors stained with an anti-RAS antibody (scale
bar = 250 µm) and corresponding jitter plots representing the mean DAB intensity of RAS
antibody in the entire tumor sections. Data are means ± SEM (*p < 0.05, **p < 0.01; one-way
ANOVA, n = 3). Supplemental Figure S4. Immunohistochemical analysis of total RAS levels in Supplemental Figure S4. Immunohistochemical analysis of total RAS levels in PDAC PDX
tumors following treatment with RRSP-DTB. (A) Representative images of saline, RRSP*-DTB
and RRSP-DTB-treated PDAC PDX1 and PDX2 tumors stained with an anti-RAS antibody (scale
bar = 250 µm) and corresponding jitter plots representing the mean DAB intensity of RAS
antibody in the entire tumor sections. Data are means ± SEM (*p < 0.05, **p < 0.01; one-way
ANOVA, n = 3). Proteolytic pan-RAS cleavage leads to tumor regression in patient-derived pancreatic cancer
xenografts ANOVA, n = 3). Supplemental Figure 5. Immunohistochemical analysis of cleaved caspase 3 in PDAC PDX
tumors and caspase 3/7 activity in PDAC cell lines following treatment with RRSP-DTB (A) Supplemental Figure 5. Immunohistochemical analysis of cleaved caspase 3 in PDAC PDX
tumors and caspase 3/7 activity in PDAC cell lines following treatment with RRSP-DTB. (A)
Representative images of saline, RRSP*-DTB and RRSP-DTB-treated PDAC PDX1 and PDX2
tumors stained with an anti-cleaved caspase 3 (CC3) antibody (scale bar = 250 µm). A section
from human tonsils was used as positive control for CC3. (B) Activity of caspase 3/7 in PDAC
cell lines after 24- and 48-hour treatment with RRSP*-DTB and RRSP-DTB at the indicated
concentrations. Data are means ± SEM (*p < 0.05, **p < 0.01, ***p<0.001 vs corresponding
vehicle control; one-way ANOVA, n = 3). Supplemental Figure 5. Immunohistochemical analysis of cleaved caspase 3 in PDAC PDX
tumors and caspase 3/7 activity in PDAC cell lines following treatment with RRSP-DTB. (A)
Representative images of saline, RRSP*-DTB and RRSP-DTB-treated PDAC PDX1 and PDX2
tumors stained with an anti-cleaved caspase 3 (CC3) antibody (scale bar = 250 µm). A section
from human tonsils was used as positive control for CC3. (B) Activity of caspase 3/7 in PDAC
cell lines after 24- and 48-hour treatment with RRSP*-DTB and RRSP-DTB at the indicated
concentrations. Data are means ± SEM (*p < 0.05, **p < 0.01, ***p<0.001 vs corresponding
vehicle control; one-way ANOVA, n = 3). Supplemental Figure 5. Immunohistochemical analysis of cleaved caspase 3 in PDAC PDX tumors and caspase 3/7 activity in PDAC cell lines following treatment with RRSP-DTB. (A)
Representative images of saline, RRSP*-DTB and RRSP-DTB-treated PDAC PDX1 and PDX2
tumors stained with an anti-cleaved caspase 3 (CC3) antibody (scale bar = 250 µm). A section
from human tonsils was used as positive control for CC3. (B) Activity of caspase 3/7 in PDAC
cell lines after 24- and 48-hour treatment with RRSP*-DTB and RRSP-DTB at the indicated
concentrations. Data are means ± SEM (*p < 0.05, **p < 0.01, ***p<0.001 vs corresponding
vehicle control; one-way ANOVA, n = 3). Supplemental Figure 6. Western blot images of RAS levels in YAPC and KP-4 cell lines
treated with sera from mice previously exposed to RRSP-DTB. Proteolytic pan-RAS cleavage leads to tumor regression in patient-derived pancreatic cancer
xenografts (A) and (B) Western blots
showing total RAS levels in YAPC (A) and KP-4 (B) cells treated for 24 hours with serum from
immunocompetent mice treated with RRSP-DTB at 0.1 mg/kg or 0.5 mg/kg for 1 hour and 16
hours. Five mice for each treatment group were employed. Vinculin was used as loading control. m#1
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treated with sera from mice previously exposed to RRSP-DTB. (A) and (B) Western blots
showing total RAS levels in YAPC (A) and KP-4 (B) cells treated for 24 hours with serum from
immunocompetent mice treated with RRSP-DTB at 0.1 mg/kg or 0.5 mg/kg for 1 hour and 16
hours. Five mice for each treatment group were employed. Vinculin was used as loading control.
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FOGYASZTÓI MOTIVÁCIÓS VIZSGÁLATOK A DVSC LABDARÚGÓ CSAPATÁNAK BÉRLETESEI KÖRÉBEN
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Vol 3, No 1 (2020): Stadium -Hungarian Journal of Sport Sciences
https://doi.org/10.36439/SHJS/2020/1/5457
FOGYASZTÓI MOTIVÁCIÓS VIZSGÁLATOK A DVSC LABDARÚGÓ
CSAPATÁNAK BÉRLETESEI KÖRÉBEN
CONSUMER MOTIVATIONAL RESEARCH AMONG DVSC SOCCER CLUB’S SEASON TICKET
OWNERS
Komáromi Dominika Anna1, Bácsné Bába Éva1
1Debreceni
Egyetem, Gazdaságtudományi Kar, Sportgazdasági és –Menedzsment Intézet
Összefoglaló
Jelen tanulmányban a DVSC labdarúgó mérkőzéseit bérlettel látogató szurkolók szocio-demográfiai
jellemzőit és összetételét vizsgáltuk, illetve a debreceni közönség sportfogyasztását meghatározó
motivációs tényezőket mértük fel. A szakirodalmi források feldolgozása után elkészítettük a
mérőeszközt, mely szocio-demográfiai, mérkőzéslátogatási szokásokkal, létesítménnyel és
szolgáltatásokkal, illetve belépőkkel, jegyárakkal kapcsolatos kérdéseket tartalmazott. Az adatokat
személyes és online formában is gyűjtöttük, így összesen 49 fős mintát vizsgáltunk. Az eredmények
kiértékelése során kiderült, hogy a DVSC bérlettel rendelkező szurkolói úgy a csapat sikerét, mint
kudarcát sajátjukként élik meg. Összességében megállapítható, hogy az állandó belépővel
rendelkezők biztos résztvevőket jelentenek, számuk növelése kulcsfontosságú a klub számára, mely
a csapat és a szurkoló közötti kötelék létrehozásával és erősítésével valósítható meg.
Kulcsszavak: labdarúgó klub, bérletesek, sport fogyasztás, fogyasztói motiváció, mérkőzés
látogatási szokások
Abstract
In this research, we investigated socio-demographic attributes and structure of DVSC Soccer Club’s
season ticket owners and measured the motivational factors determining the sports consumption
of Debrecen’s audience. After the literature review, we developed a tool for the measurement of the
following factors: socio-demographic and match visiting habits, facilities and service, tickets and
prices. We collected the data of the 49 samples online and through personal meetings. By
evaluating the results, it became clear that fans of DVSC with season tickets experience the team’s
success and failure as their own. Overall, it can be stated that those who have permanent entrants
are surely visiting the matches. The growth of the season ticket owners is a key question for the
club, which is determined by the creation and improvement of the club-fan bond.
Keywords: soccer club, season ticket owners, sports consumption, consumer motivation, match
visiting habits
Vol 3, No 1 (2020): Stadium -Hungarian Journal of Sport Sciences
https://doi.org/10.36439/SHJS/2020/1/5457
BEVEZETÉS
Napjainkban a labdarúgás jelentős tömegeket mozgat meg, hazánkban még ma is az egyik
legnézettebb sportágak közé tartozik. A magyar labdarúgó bajnokság mérkőzésein
ugyanakkor az alacsony nézőszámok problémájával találkozunk, mely a debreceni
futballnak otthont adó Nagyerdei Stadion lelátóit sem kerülte el.
A DVSC NB1-es férfi labdarúgó csapatának mérkőzéseit gyerekkorom óta bérletesként
látogatom, egyetemi tanulmányaim alatt pedig betekintést nyerhettem a fogyasztói
magatartás vizsgálatok publikált eredményeibe. Ezek adtak indíttatást arra, hogy egy
speciális fogyasztói csoport, a DVSC labdarúgó csapat állandó belépővel rendelkező nézőit
tanulmányozzam.
A bérletes szurkolók általában jelentős hányadát képezik a közönségnek, az
összecsapásokat rendszeresen látogatók közé sorolhatóak, így felmérésük, jellemzőik és
motivációik megismerése mérvadó a nézőszámok emelésének érdekében.
A megfelelő szakirodalmi források összegyűjtése és feldolgozása után a DVSC magyar első
osztályban szereplő labdarúgó klub bérleteseinek összetétel és motiváció vizsgálatára
alkalmas mérőeszközömet kívánom elkészíteni. Célom, hogy feltárjam a különböző korú,
szociális helyzetű, képzettségű szurkolók mérkőzéslátogatási szokásait, ami alapján
javaslatok fogalmazhatóak meg az állandó belépővel rendelkezők számának bővítésére.
SZAKIRODALMI ÁTTEKINTÉS
A SPORTFOGYASZTÁS
A sporttermékek fogyasztása
Napjainkban a sport egyre fontosabb részét képezi mindennapi életünknek, a különböző
infrastrukturális fejlesztéseknek köszönhetően több lehetőségünk van sportolni, hazánk
pedig jelentős nemzetközi sporteseményeknek adhat otthont.
A sport, mint termék rendelkezik kereslettel és kínálattal, és hasonlóan bármilyen másik
jószághoz, hasznot és megoldást jelent a fogyasztók részére (NEULINGER, 2007).
A sporttermékek közé a szakirodalom a következő elemeket sorolja (MULLIN ET AL.
1993; HOFFMANN, 2000; SHANK, 2002; NEULINGER, 2007) : tárgyiasult elemek;
szolgáltatás; személy; szervezet; hely, helyszín; eszme, imázs.
Így elmondhatjuk a fentiek alapján, hogy egy sporttermék állhat egy elemből is, de lehet
akár több elem komplex szerveződése. Továbbá ezeknek a termékeknek a jellemzője,
hogy megfogható és megfoghatatlan összetevőkkel is rendelkeznek, megfogható termék
Vol 3, No 1 (2020): Stadium -Hungarian Journal of Sport Sciences
https://doi.org/10.36439/SHJS/2020/1/5457
például a belépőjegy, vagy valamilyen sporteszköz, megfoghatatlan pedig a klub, az imázs,
a sportesemény és az általa nyújtott élmény lehet (NEULINGER, 2007; SZABÓ, 2009).
Tehát a sporttermékek iránti kereslet, azaz a sportfogyasztók igényei, szükségletei ilyen
tárgyiasult és nem tárgyiasult elemekben határozhatóak meg.
A sportfogyasztás megvalósulhat aktív és passzív formában is, aktív fogyasztást jelent
valamilyen sporttevékenység végzése, passzív formája pedig sportesemény, mérkőzésen
nézőként való részvétel, vagy ezen események egyéb módon történő követése
(NEULINGER, 2007; SZABÓ, 2009). Esetünkben a sportfogyasztás passzív formájára
koncentrálunk, mely tehát különböző sporttal kapcsolatos rendezvények megtekintését
jelenti. Neulinger (2007) alapján a sportesemény: „Magában foglalja a sportjátszmákat,
mérkőzéseket, a sportcsarnokot (helyszínt), a sportolókkal, sportsztárokkal együtt”.
Nézői sportfogyasztás tekintetében elmondhatjuk, hogy ebben az esetben a megtekintett
vagy megtekinteni kívánt esemény által nyújtott élmény az, ami miatt bekövetkezik a
fogyasztás.
Természetesen egy sportesemény nem csak az általa nyújtott élményből és izgalomból áll,
mindez kiegészül többek között az esemény szereplőivel, a szervezőkkel, a helyszínül
szolgáló létesítménnyel, és ezek együttesen képezik azt a szolgáltatást,
szolgáltatáscsomagot, amelyet a sportfogyasztók megvásárolnak (NEULINGER, 2007). A
szervezők legfontosabb feladata tehát, hogy minden szinten kielégítsék és akár
felülmúlják a fogyasztók igényeit és elvárásait, és összességében olyan élményt
nyújtsanak – minőségben és színvonalban egyaránt –, amely a későbbiekben újabb
fogyasztást idézhet elő a nézők körében. A sportszervezetek számára egy olyan érték
megalkotása a cél, mely ösztönzi a vásárlást, jelentse ez akár a játékosok
sportteljesítményét, a létesítmény állapotát, a szolgáltatások minőségét is.
A sportfogyasztók köre
Neulinger (2007) szerint a sporttermékek iránti keresletet a piacon az alábbi szereplők
alkotják:
az egyén, aki sporttevékenységet végez vagy nézőként vesz részt,
a szponzorok és támogatók, akik gazdaságilag vagy egyéb hozzájárulással
finanszírozzák a működést,
és a sporteseményeket közvetíteni kívánó média.
A keresletnek jelen helyzetben azt a részét vizsgáljuk, melynek igénye élőben, helyszíni
nézőként megtekinteni az adott sporteseményt, esetünkben labdarúgó mérkőzést.
A sportfogyasztók tipizálása, szegmentálása kiemelten fontos annak érdekében, hogy a
szervezetek meg tudják határozni marketing tevékenységüket, segít az árképzésben, a
termékek piaci elhelyezésében és segít a különböző szegmensek eltérő igényeinek
Vol 3, No 1 (2020): Stadium -Hungarian Journal of Sport Sciences
https://doi.org/10.36439/SHJS/2020/1/5457
feltérképezésében, ezen szükségletek minél hatékonyabb kielégítésének céljából (Stewart
et al. 2003).
A legelső duális tipológiák egyikét Clarke (1978) hozta létre, aki megkülönböztette a
nézők két csoportját: a genuine szurkolók azok, akik a futballmérkőzéseket közösségi
élményként élik meg, ezzel szemben a corporate az a csoport, akik a mérkőzéseket hétvégi
kikapcsolódásnak, szórakozásnak tekintik. Lewis (2009) szintén duális megközelítése
szerint, amely az identifikáción alapult, a civic csoportba tartozók azok, akik a várossal, a
symbolic pedig azokat jelenti, akik magával a csapattal azonosulnak (Stewart et al. 2003).
Smith és Stewart (1999) a mérkőzéseket rendszeresen látogató, a csapathoz hűséges,
annak játékosaival és hagyományaival azonosuló szurkolókat passionate partisans -nek
nevezte. A champ followers azokat jelenti, akik kevésbé fanatikusak, és csak csapatuk jó
szereplésekor jelennek meg. A harmadik csoport a reclusive partisans, akik erősen
elköteleződtek a csapathoz, de ritkán látogatják a mérkőzéseket (STEWART et al. 2003).
A sportszervezetek számára elengedhetetlen célközönségük megismerése és megfelelő
szegmentálása (BÁCSNÉ et al., 2019; BALOGH et al., 2019c) annak érdekében, hogy
gazdaságilag megfelelően tudjanak működni, termékeiket a megfelelő helyen és időben,
egy adott szegmens számára értékesíteni tudják. A szurkolók rendkívül széles köre miatt
csoportosításuk nehéz feladat, a legtöbb tipizálás a hűségen, az azonosuláson és ezek
különböző erősségein alapszik.
A magyar labdarúgás
Hazánkban a labdarúgásnak jelentős hagyománya és történelme van. Bár nem ez a
legtöbb sikert hozó sportág, kijelenthető, hogy napjainkban is talán a legnagyobb
népszerűségnek örvend országunkban és a világban is.
Az elmúlt években több, NB1-es labdarúgó csapat számára is új stadion épült, ugyanakkor
általánosan a nézőszámok csökkenéséről lehet hallani. A magyarfutball.hu adatai alapján
ezt az állítást meg lehet cáfolni, hiszen az elmúlt évek statisztikái alapján az élvonalbeli
labdarúgó mérkőzések átlagos nézőszámai szezonról szezonra növekedtek, viszont az
kijelenthető, hogy a stadionok befogadóképességéhez és más első osztályú
bajnokságokhoz képest ezek a számok valóban alacsonyak (BALOGH et al., 2019b).
Az előző, azaz a 2019/2020-as idényben az NB1-ben 3300, a DVSC hazai mérkőzésein
3547 volt az átlag, a DVSC adatai az elmúlt 20 évben szinte mindig a bajnokság
nézőszámainak átlagai felett voltak (MAGYARFUTBALL, 2020).
A magyarfutball.hu statisztikái azt mutatják, hogy a DVSC hazai környezetben
megrendezett mérkőzései közül a 2019/2020-as szezonban az FTC elleni eredményezte
a legnagyobb, számszerit 7002 fős nézősereget. A legmagasabb nézőszámokat továbbá a
Paksi FC, az MTK és a DVTK elleni összecsapások hozták, ezzel is mutatva, hogy a hasonló
Vol 3, No 1 (2020): Stadium -Hungarian Journal of Sport Sciences
https://doi.org/10.36439/SHJS/2020/1/5457
szintű, illetve a nagy múltú fővárosi csapatok keltik a legnagyobb érdeklődést a szurkolók
körében.
A közönség jelentős részét alkotják és többnyire állandó fogyasztást biztosítanak azok,
akik bérlettel, azaz egész szezonra szóló érvényes belépőjeggyel rendelkeznek. A DVSC
futballcsapatának körülbelül 3000 bérletes szurkolója van, ennek a számadatnak a
bővítése fontos célja a klubnak, hiszen ők biztos bevételi forrást és folyamatos részvételt
garantálhatnak hazai környezetben.
A bérletesek fontosságát a DVSC is felismerte, hiszen az ilyen belépővel rendelkező
nézőinek különböző kedvezményeket ad, ilyen például a mérkőzések napján ingyenes
utazás a DKV járataival, engedmények az Állatkertbe és az Aquaticumba szóló jegyek
árából, illetve kedvezően kialakított családi, diák és nyugdíjas bérletárak (DVSC, 2020).
A DVSC a LokomotívBlog (2019) összefoglalója alapján a magyar NB1-ben szereplő
klubok közül az első három, legkedvezőbb bérletárakat alkalmazók körébe tartozik. A
legjobban megfizethető szezonális belépők tekintetében csak az Újpest FC és a
Mezőkövesd Zsóry FC előzi meg, ezzel is alátámasztva azt, hogy a klub erőteljes hangsúlyt
fektet a bérletesek számának növelésére, mind az árak kialakítását figyelembe véve, mind
pedig a bérletekkel járó kedvezmények vonatkozásában.
Motivációs skálák
Annak érdekében, hogy azon sportfogyasztók számát növelni tudjuk, akik a helyszínen, a
nézőtéren tekintik meg az adott sporteseményt, azaz a DVSC labdarúgó csapatának hazai
mérkőzéseit, meg kell vizsgálnunk, hogy melyek azok a motivációs tényezők, amelyek
kiváltják a fogyasztásnak ezen típusát, a különböző motivációs skálák pedig ehhez
nyújtanak nekünk segítséget.
A skálák közül a 2017-ben validált magyar nyelvű SPEEDE-H skálát, a nézőtéri
sportfogyasztás motivációit mérő eszközt (BALOGH et al., 2019a) választottuk, mely az
eredeti SPEED nyomán készült el, de olyan változtatásokkal egészült ki, amelyek lehetővé
teszik magyarországi alkalmazását. A SPEEDE-H skála hat faktort tartalmaz, minden
faktorhoz két item tartozik, így összesen tizenkét kérdés mentén méri a nézők motivációit.
A készítők a skála hat faktorát a következőképpen határozták meg: Társas kapcsolatok,
Teljesítmény,
Önbecsülés,
Izgalomkeresés,
Kikapcsolódás/Elszabadulás,
Esztétikum/Szépség (KAJOS et al. 2017).
Így tehát ezt a skálát alkalmasnak találtam a DVSC labdarúgó szakosztályának helyszíni
szurkolóinak, azon belül is a bérlettel rendelkezők fogyasztási motivációinak mérésére.
Vol 3, No 1 (2020): Stadium -Hungarian Journal of Sport Sciences
https://doi.org/10.36439/SHJS/2020/1/5457
ANYAG ÉS MÓDSZER
A bevezetésben felsorolt célok eléréséhez a kérdőíves felmérés módszerét választottuk,
mivel ezt találtuk a legalkalmasabbnak a DVSC labdarúgó csapat bérlettel rendelkező
szurkolóinak összetételének és motivációinak vizsgálatára.
A nézőtéri sportfogyasztók jellemzőinek és motivációinak tanulmányozása érdekében, és
a megfelelő szakirodalmi források feldolgozása alapján elkészítettünk egy kérdőívet, mely
a szükséges szociodemográfiai kérdéseken túl mérkőzéslátogatási szokásokkal,
létesítménnyel és szolgáltatásokkal, illetve belépőkkel, jegyárakkal kapcsolatos
kérdéseket tartalmaz. Ezek mellett rákérdeztünk a mérkőzéslátogatások, és azok
csökkenésének lehetséges okaira. Továbbá beépítettük a kérdőívbe a Kajos Attila,
Prisztóka Gyöngyvér és Paic Róbert által, 20l7-ben validált SPEEDE-H skálát.
Fontosnak tapasztalat lehet, a személyes megkérdezés, ezért 2019 novemberében és
decemberében összesen három alkalommal, a DVSC labdarúgó csapatának hazai
mérkőzései előtt összesen 13 emberrel sikerült személyesen kitöltetni a kérdőívet. A
megkérdezések során a szurkolók hozzáállása rendkívül pozitív és segítőkész volt,
szívesen osztották meg véleményüket a klubbal és a hazai alacsony nézőszámok
problémájával kapcsolatban.
A kérdőívet a továbbiakban interneten megosztottuk, így összesen 49 fős lett a minta. A
válaszadók között 46 férfi és 3 nő volt, tehát a kitöltők 94%-a férfi, 6%-a nő.
60 év felett 14 év alatt
14-20 év
4%
2%
10%
51-60 év
21-30 év
16%
12%
41-50 év
33%
31-40 év
23%
1.ábra: A minta életkor szerinti megoszlása
Forrás: Saját szerkesztés, 2020
Az életkor alapján, ahogyan az 1. ábráról leolvasható, legnagyobb arányban a 41-50 év
közöttiek (33%) szerepelnek. Őket követi sorrendben a 31-40 éves korosztály (23%),
majd az 51-60 évesek (16%), utánuk közel azonos százalékban a 21-30 (12%) és 14-20
Vol 3, No 1 (2020): Stadium -Hungarian Journal of Sport Sciences
https://doi.org/10.36439/SHJS/2020/1/5457
év köztiek (10%) következnek, a 14 év alattiak (4%) és a 60 évnél idősebbek (2%) az
utolsók a sorban..
Családi állapot tekintetében egyedül 24,5%-uk él, 1 fővel 20,4%, 2-4 fővel 49%, illetve 5
vagy annál több fővel 6,1% osztozik közös háztartáson. A minta legmagasabb iskolai
végzettség szerinti megoszlása a következőképpen alakul: kevesebb mint nyolc általánosa
10,2%-nak van, nyolc általánossal 6,1%, szakiskola/szakmunkás képesítéssel 4,1%,
érettségivel 44,9%, főiskolai végzettséggel 12,2% rendelkezik, míg az egyetemet
végzettek aránya 22,4%.
Továbbá havi jövedelmük nagyságára is rákérdeztünk, az erre érkezett válaszok alapján
a következő a sorrend az egyes kategóriák között: 1. 200 001 - 300 000 Ft (24,4%), 2.
300 001 - 400 000 Ft (19,5%), 3. 100 001 - 200 000 Ft (17,1%), 4. 100 000 Ft alatt (9,8%),
5. 400 001-500 000 Ft (9,8%), 6. 500 001 - 1 000 000 Ft (9,8%), 7. 1 000 000 Ft felett (2,4%).
EREDMÉNYEK
Először arra kerestük a választ, hogy a válaszolók mióta látogatják a DVSC
labdarúgócsapatának hazai mérkőzéseit. Az eredmények azt mutatják, hogy az előző
szezon, azaz a 2018/19-es idény óta csupán 2%-uk jár, 2-4 éve 8%-uk, 5-10 éve 12%-uk,
10-20 éve 27%-uk, 51%-uk több mint 20 éve tekinti meg a csapat hazai összecsapásait.
Ezekből az adatokból levonható az a következtetés, hogy minél régebb óta jár valaki
mérkőzésekre, annál nagyobb a valószínűsége annak, hogy bérlettel rendelkezik.
Egy szezonon belül a megkérdezettek 67%-a minden mérkőzésre (néhány kivételével),
20%-a 10-nél többre, 6-6 %-uk 5 – 10 közötti, illetve 5-nél kevesebb mérkőzésre látogat
ki.
A válaszadók 12,2%-a úgy nyilatkozott, hogy rendszeresen, 63%-a néhány alkalommal,
25%-a pedig nem jelenik meg a vendég lelátókon, az idegenbeli meccseken.
A kitöltők életüknek átlagosan 13. évében látták először helyszínen a DVSC mérkőzését.
Az első mérkőzésére a minta 55%-át szülei vitték el, 18%-át barátaik, 23% a
sport/labdarúgás szeretete miatt, a maradék 4% pedig egyéb okból látogatott ki. A média
motiváló erejét viszont senki sem jelölte meg.
Vol 3, No 1 (2020): Stadium -Hungarian Journal of Sport Sciences
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20-50 km-re
50-100 km-re
8%
2%
Több mint 100
km-re
6%
Kevessebb mint
20 km-re
8%
Debrecenben
lakom
76%
2. ábra: A válaszok megoszlása a lakóhely Debrecentől való távolsága alapján
Forrás: saját szerkesztés, 2020
A megkérdezettek nagy többsége a 2. ábra alapján Debrecenben és vonzáskörzetében él,
de előfordulnak olyanok, akik nagyobb távokat is megtesznek azért, hogy a helyszínen
lehessenek (a minta 6%-a 100 km-től messzebbről érkezik a mérkőzésekre).
A kitöltők legnagyobb gyakorisággal barátaikkal látogatják a DVSC mérkőzéseit, valamivel
kevesebben rokonaikkal, míg mindössze páran munkatársaikkal.
A Loki összecsapásaira a minta közel 60%-a saját gépjárműjével; gyalog 16%; barátok,
ismerősök, családtagok gépjárműjével 10%; a DKV járataival 8%; kerékpárral 6% érkezik
a színtérre. Két válaszlehetőség, a motorkerékpár és a vonat, távolsági busz (és DKV
járatai) nem kapott egyetlen jelölést sem. Ebből fakadóan arra a következtetésre lehet
jutni, hogy a klub intézkedésének - a mérkőzések napján a DKV járatain való ingyenes
utazási lehetőség - ellenére a legtöbben mégis gépjárművel közelítik meg a Nagyerdei
Stadiont.
Ehhez kapcsolódóan arra is fény derült, hogy a parkolási opciókat tekintve 63%-os az
elégedettség, míg 37% kifogásolja a stadion körüli megállási lehetőségeket.
A kitöltők 43%-a saját bevallása szerint 15 percnél kevesebb idő alatt ér ki a stadionba a
mérkőzések napján, 39%-nak 16-30 perc, 14%-nak 31-60 perc a kijutási ideje, míg 2-2%
a 61-90 percet és a 90 percnél többet utazók aránya, ebből is látszik, hogy vannak olyan
bérletesek, akik a nagyobb távolságok ellenére is megjelennek a nézőtéren.
Vol 3, No 1 (2020): Stadium -Hungarian Journal of Sport Sciences
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1. táblázat: A válaszadók aránya a Nagyerdei Stadion szektorai alapján
Szektor megnevezése
A szektorban helyet foglalók Százalékos
száma
megoszlás
7
(B-közép
14
A szektor
B
szektor
kivételével)
B- közép (B5, B6 szektorok)
C szektor
D szektor
VIP
Forrás: saját szerkesztés, 2020
12
4
1
11
14%
29%
24%
8%
2%
22%
A felvett adatok alapján az 1. táblázat azt mutatja, hogy a megkérdezettek stadionon belül
hol szoktak helyet foglalni.
A jegyek és bérletek árával kapcsolatban 96% elégedett volt, és csupán két válaszadó
alany, azaz 4% jelezte elégedetlenségét. Ez az adat azt is jól tükrözi, hogy a magyar
bajnokságon belül a DVSC kimondottan kedvezően határozza meg belépőinek árait. A
legnagyobb összeg, amit a válaszadók átlagosan kifizetnének egy jegyért, ami arra a helyre
szól, ahova általában szoktak ülni a beérkezett adatokat átlagolása alapján, a maximum
5000 Ft lett.
A mérkőzések helyszíni megtekintésének elképzelhető okaként a kitöltők több
válaszlehetőséget is megjelölhettek. A felvett adatokból kiderül, hogy az első helyen a
labdarúgás szeretete áll, második helyen holtversenyben a jó társaság és a jó hangulat
szerepel, míg a harmadik legtöbb voksot az azonosulás a csapattal kapta. Ennélfogva a
DVSC megkérdezett bérleteseinek nem csupán maga a labdarúgás jelent indítékot, hanem
a mérkőzéslátogatásaikat övező körülmények, azaz a társaság és a hangulat is befolyásolja
a fogyasztást, ezek mellett pedig erőteljes hatása van a csapattal való azonosulásnak, azaz
az ahhoz való kötődésnek, így kiemelten fontos lehet ezt a fajta szurkoló-játékos,
szurkoló-csapat kapcsolatot fenntartani, a két fél közötti kommunikációt erősíteni és
minél szorosabb köteléket kialakítani.
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2. táblázat: A SPEEDE-H skálával történő mérés eredményei
A SPEEDE-H skála faktorai és a hozzájuk tartozó
állítások
Társas kapcsolatok
Szeretem, hogy a mérkőzésen való részvétel lehetőséget
teremt az ismerkedésre.
Szeretem, hogy a mérkőzésen való részvétel lehetőséget
teremt rá, hogy beszélgessek másokkal.
Teljesítmény
A mérkőzést a játékosok által nyújtott teljesítmény miatt
szeretem nézni.
A mérkőzést a játékosok képességei/készségei miatt
nézem.
Önbecsülés
Ha a csapat nyer, én is nyerek.
A csapat győzelme sikerélményt nyújt számomra.
Izgalomkeresés
Szeretem a mérkőzések izgalmát.
Nagyon izgalmasnak találok egy mérkőzést.
Kikapcsolódás/Elszabadulás
A mérkőzésen való részvétel segít elmenekülni az
életemmel kapcsolatos feszültségek elől.
A mérkőzésen való részvétel segít elszakadni a
mindennapi élet rutinjától.
Esztétikum/Szépség
Szeretem a játék/sportág eleganciáját.
Szeretem a sportág szépségét.
Forrás: saját szerkesztés, 2020
A válaszok A válaszok
átlagai
átlagai
állításonk faktoronké
ént
nt
3,29
3,61
3,94
3
2,78
2,89
4,45
4,49
4,47
4,55
3,71
4,13
2,79
3,54
3,84
4,23
3,17
4,03
A nézőtéri sportfogyasztás motivációinak mérésére, a SPEEDE-H skálát alkalmaztuk, ahol
a válaszadóknak el kellett dönteniük, hogy a 6 faktorból álló SPEEDE-H skála állításai
mennyire igazak rájuk egy öt fokozatú Likert-skálán, ahol az „1” az egyáltalán nem, míg az
„5” a teljes mértékben igazat jelentette, a mérés eredményeit a 2. táblázat tartalmazza.
Az Önbecsülés 4,47-es értékével tekinthető az elsőszámú motivációs faktornak, tehát a
megkérdezettek nézőtéri sportfogyasztását a csapat győzelmének és sikerének saját
sikerként történő megélése ösztönzi leginkább. Az itemeket tekintve a legerősebb
motivációs tényező az Izgalomkeresés faktoron belül található, mégpedig a „Szeretem a
Vol 3, No 1 (2020): Stadium -Hungarian Journal of Sport Sciences
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mérkőzések izgalmát” állítás volt, 4,55-ös értékkel. Ennek értelmében a mérkőzések
(várható) izgalma az, amely elsődlegesen motiválja őket, viszont a faktor másik iteme már
jóval alacsonyabb, 3,71-es átlagot produkált, szóval voltaképpen az, hogy nagyon
izgalmasnak találnak egy mérkőzést, már kevésbé igaz rájuk. Eképpen annak ellenére,
miszerint nem különösebben találják a DVSC mérkőzéseit izgalmasnak, mégis a
bizonytalan végkimenetelből, a meccsekből adódó stressz és izgalom az, ami a legnagyobb
motivációja a bérletesek körében a helyszíni nézőtéri sportfogyasztásnak Debrecenben.
A legkisebb motivációt a skála Teljesítmény faktora adja, ami mentén arra a
következtetésre lehet jutni, hogy a DVSC bérleteseit a játékosok képességei és készségei,
valamint az általuk nyújtott teljesítmény befolyásolja legkevésbé fogyasztási döntésük
meghozatalában.
Bár legtöbben úgy nyilatkoztak, hogy nem változtak a mérkőzéslátogatási szokásaik, azok,
akik bevallásuk szerint a korábbiakhoz képest kevesebb alkalommal tekintenek meg
helyszínen mérkőzéseket, az alacsony játékszínvonalat jelölték meg elsődleges okként.
Második meghatározó ok a hangulat hiánya, harmadik helyen holtversenyben az MLSZ
korábbi, nem szurkolóbarát intézkedései (BALOGH-BÁCSNÉ, 2019) és a gyengébb szereplés
az elmúlt években elnevezésű okok állnak. Következésképp azok a bérletesek, akik
kevesebbszer tekintenek meg helyszínen hazai mérkőzéseket, ennek okát elsősorban a
rosszabb játékszínvonal és a jó teljesítmény hiányaként határozták meg.
A mintának közel 100%-a volt elégedett a helyszínül szolgáló sportlétesítménnyel, azaz a
Nagyerdei Stadionnal. Ugyanakkor a mérkőzések ideje alatt nyújtott szolgáltatások
vonatkozásában már más az arány, bár 59%-uk az elégedett volt ezzel, 41%-uk nem
találta kifogástalannak a szolgáltatásokat, ezen véleményüket pedig a lassú kiszolgálással
indokolták.
A kitöltők legnagyobb többsége a DVSC közönségének növelését a játékosok és a
szurkolók közötti kapcsolat erősítésével oldaná meg. Többször említették, hogy szívesen
találkoznának a pályán kívül is a csapat tagjaival, ami erősíthetné a kötődést a klub és a
szurkolók között, a bérlettel rendelkezők száma pedig emelkedhetne. Így kialakulhatna
egyfajta tisztelet és érdeklődés a játékosok és teljesítményük iránt, a szurkolók pedig
fontosnak éreznék magukat, ezért egy esetlegesen előforduló rosszabb játékminőség és
széria esetén is biztosítható lenne, hogy a lelátón helyet foglalók száma ne csökkenjen.
KÖVETKEZTETÉSEK ÉS JAVASLATOK
Az eredményekből arra lehet következtethetni, hogy a DVSC hazai labdarúgó
mérkőzéseire állandó belépővel rendelkezők legnagyobb többségében férfiak, több mint
felük 31 és 50 év közötti és közös háztartásban él 2-4 fővel, tehát az átlagbérletest
középkorú, családos férfiként lehetne meghatározni.
Vol 3, No 1 (2020): Stadium -Hungarian Journal of Sport Sciences
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Átlagosan 13 éves korukban látták életükben először a DVSC-t élőben, ahová
túlnyomórészt szüleikkel mentek el, így megállapítható, hogy a szülők döntően
meghatározhatják gyerekeik későbbi mérkőzéslátogatási szokásait, főleg ha ez fiatal
korukban történik meg.
Mivel a minta majdnem háromnegyede szinte az összes hazai mérkőzésen részt vesz
nézőként, ezért a bérletesek gyarapításának megcélzása fontos lépés a rendszeres
nézőtéri sportfogyasztás és a stabil nézőszámok eléréséhez. Ennek megvalósítására a klub
az egy szezonra szóló belépők magyar viszonylatban is kedvező áraival és a hozzájuk
tartozó kedvezményekkel már megtette első lépéseit.
Bár a klub a DKV-val való együttműködéssel támogatja a belépővel rendelkezők
helyszínre való kijutását, illetve tehermentesíteni próbálja a Nagyerdei Stadion
környékét, az eredmények azt mutatják, hogy a nézők nagy többségben ennek ellenére
továbbra is gépjárművel közlekednek, a válaszok alapján pedig szükség lehet a parkolási
lehetőségek bővítésére.
Az elsőszámú motivációs faktor a mérkőzések helyszíni megtekintéséhez az Önbecsülés
faktor volt, illetőleg a meccstől való távolmaradás legfőbb indoka az alacsony
játékszínvonal volt. Ebből kiderül, hogy a rosszabb szereplést és gyengébb teljesítményt
saját kudarcukként élhetik meg a szurkolók, mely érzés újbóli előfordulásának elkerülése
miatt csökkenhet a mérkőzéslátogatásaik száma, így ezzel is magyarázhatóak a nézőtéri
üres helyek.
A bérlettel rendelkezők között a fiatal korosztály, azaz a 20 év alattiak kis arányban
jelentek meg, így ezen fogyasztók megnyerése kifejezetten ajánlott, olyan okból is, hogy
ők a következő évtizedekben is biztos közönséget jelenthetnek a DVSC-nek.
Természetesen a bérletszámok növelésének egyik feltétele a klub iránti hűség és kötődés
kialakítása, mely a publikum és a csapat közötti szoros kapcsolat kiépítésével
kivitelezhetnek.
A publikáció elkészítését az EFOP-3.6.1-16-2016-00022 „Debrecen Venture Catapult
Program” projekt támogatta. A projekt az Európai Unió támogatásával, az Európai Szociális
Alap társfinanszírozásával valósult meg.
Vol 3, No 1 (2020): Stadium -Hungarian Journal of Sport Sciences
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IRODALOMJEGYZÉK
Balogh, R., Bácsné, Bába É. (2019). Sportszövetségi intézkedések attitűdvizsgálata. Acta
Carolus Robertus, 9, (1), 23-33.
Balogh, R., Bácsné, Bába É. (2019a). Az MSSC szurkolói motivációs skála adaptálása
egyetemi hallgatók körében. Studia Mundi – Economica, 6, (1), 3-10.
Balogh, R., Dajnoki, K., & Bácsné, Bába É. (2019b). Miért beteg a magyar futball még
mindig? – a magyar labdarúgás játékos piacának jellemzése. Jelenkori Társadalmi És
Gazdasági Folyamatok, 13, (3-4), 105-117.
Balogh, R., Molnár, A., Müller, A., Szabados, Gy. N., & Bácsné, Bába É. (2019c). A passzív
sportfogyasztás vizsgálatának tapasztalataiból. Acta Carolus Robertus, 9, (1), 35-48.
Bácsné, Bába É., Balogh, R., Bács, B. A., Molnár, A., Fenyves, V., & Müller, A. (2019). A
passzív sportfogyasztás motivációinak vizsgálata nemek tükrében. Economica (Szolnok,)
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https://openalex.org/W4322494779
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https://figshare.com/articles/journal_contribution/Island_Tourism_Vulnerable_or_Resistant_to_Overtourism_/22183729/1/files/39418852.pdf
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English
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Island Tourism: Vulnerable or Resistant to Overtourism?
| null | 2,023
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cc-by
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Copyright: © 2022 Butler and
Dodds. This article is distributed
under the terms of the Creative
Commons Attribution Li-
cense (CC BY 4.0), which per-
mits unrestricted use and distribu-
tion provided that the original
work is properly cited. Received: 24 February 2022
Accepted: 27 April 2022
Published: 29 April 2022 Academic Editor
Deborah Edwards, University of
Technology Sydney, Australia Academic Editor
Deborah Edwards, University of
Technology Sydney, Australia Richard W. Butler 1,* and Rachel Dodds 2 Richard W. Butler 1,* and Rachel Dodds 2 1 School of Business, University of Strathclyde, Glasgow, G4 0LG, UK; E-Mail: richard.butler@strath.ac.uk
2 School of Hospitality and Tourism Management, Toronto Metropolitan University, Toronto, ON M5B 2K3,
Canada; E-Mail: r2dodds@ryerson.ca 1 School of Business, University of Strathclyde, Glasgow, G4 0LG, UK; E-Mail: richard.butler@strath.ac.uk
2 School of Hospitality and Tourism Management, Toronto Metropolitan University, Toronto, ON M5B 2K3,
Canada; E-Mail: r2dodds@ryerson.ca * For correspondence. * For correspondence. Abstract Islands have long attracted tourists and some islands rank amongst the most visited
places in the world. Such popularity has created problems of overdevelopment and tourism at
unsustainable levels, leading to the phenomenon of overtourism. Traditionally islands could rely
on natural features to limit tourist numbers but this is increasingly not the case today, therefore,
this paper reviews how changes in attitude, access and media coverage have led to problems of
excessive visitation. The paper discusses the failure to create and implement appropriate policies
which might mitigate against such developments and notes the inherent long-term problems
many island authorities have traditionally faced when trying to improve economic conditions for
their residents. The paper concludes that more specific action in terms of policy goals and imple-
mentation are needed if islands are to avoid the issues of unsustainable development and over-
tourism currently being experienced in many mainland tourist destinations. Keywords islands; sustainable tourism; overtourism; vulnerability; pressures; development;
controls Highlights Sustain. 2022, 1, 54–64. https://doi.org/10.54175/hsustain1020005 Received: 24 February 2022
Accepted: 27 April 2022
Published: 29 April 2022 1. Introduction Islands have long been considered attractive tourism destinations, yet their size and geograph-
ical isolation have also often contributed to a lack of control over many issues that afflict them. Such issues have sometimes left islands vulnerable to external forces, and this paper explores the
specific issue of overtourism to islands in the light of characteristics of islands in general. It begins
with a discussion of both endogenous and exogenous forces that drive the development of islands,
focusing on the difficulties many island destinations face in controlling the level and type of tour-
ism development. The paper addresses the ways in which islands might control tourism develop-
ment, and in particular, the problem of overdevelopment. Contributory factors in the context of
islands include a small land area and limited opportunity to engage in alternative economic ac-
tivities, which can leave them often heavily dependent on tourism [1]. Islands are often also
heavily subject to external controls, both economic and political, thus suggesting that islands
could be considered more vulnerable than most tourist destinations to experience excessive de-
velopment and tourist numbers far beyond their capacity to control and manage. Overtourism
is one form of unsustainable tourism that has been recorded on islands in a variety of forms for
many years including excessive numbers of tourists and resident unrest over recent years [2–4],
suggesting that islands are particularly vulnerable to problems relating to unsustainable levels
and forms of tourism. Academic Editor
Deborah Edwards, University of
Technology Sydney, Australia 2. Island Vulnerability There are five key factors which place many islands as highly vulnerable locations, namely:
limited population, limited area, limited natural resources, lack of control and geographic loca-
tion. First, many islands are small, both in area and in population, which makes them potentially
vulnerable to a number of pressures, not least of which is often a significant number of visitors
[5]. A limited local population on a small land area means that what might be considered mod-
erate numbers of tourists elsewhere may be highly noticeable and therefore represent visibly in-
creased pressure on facilities and resources on a small island. Often these facilities and associated
infrastructure on islands have been developed and designed for a small number of local residents Highlights Sustain. 2022, 1, 54–64. https://doi.org/10.54175/hsustain1020005 https://www.hos.pub Highlights Sustain. 55 and thus any increased pressure, for example by visitors during the tourist season, can be partic-
ularly disturbing and apparent. This may result in undesired congestion as well as competition
[6]. A small resident population may also mean a limited range of goods and supplies, with re-
plenishment occurring at intervals rather than constantly as in more central large population
areas. This can potentially result in shortages if visitors, for example those in self-catering accom-
modation such as Airbnb, are competing with residents for such resources. In the case of small islands or SIDS (Small Island Development States), a limited or confined
area means the likelihood of contact between residents and visitors is high, and while this may
be welcomed in some island situations [7], especially when visitor numbers are small, this attitude
may change as visitor numbers increase [5,8]. It is not inevitable that residents will become less
positive towards tourism and tourists as visitor numbers increase [9], but such a situation cer-
tainly creates the possibility of a level of contact greater than that desired by residents and which
then is viewed as disturbance [10,11]. In large islands, where tourists may be spatially dispersed,
such pressure is less likely, but on small islands there is often simply nowhere else for tourists to
go, other than permanent residential settlements. 2. Island Vulnerability This can result in specific locations e.g., beaches
(some of the finest in the world are found on islands), cultural and heritage sites, scenic spots,
retail areas, parking sites, experiencing high tourist pressure as such locations are both centres of
attraction and sources of necessary purchases and use by the two populations [6,12]. Due to a
limited land area, tourist penetration is likely to occur in many parts of an island and thus, contact
between residents and visitors becomes unavoidable. Limited areas and limited populations also make small islands particularly vulnerable to tour-
ism development because of the likelihood of limited resources and thus few alternative forms of
economic development [13]. Island destinations, according to the UNWTO [14], are more de-
pendent on tourism than other destinations. For example, the Caribbean islands comprise one
of the most tourist intense regions in the world, with tourism contributing 15% of GDP on aver-
age [12] while in the Balearic Islands and Canary Islands tourism provides almost 35% of GDP
[15,16]. Historically, such problems as lack of employment and income sources were normally
dealt with by emigration and a subsistence level of existence, but in modern times island residents
are less willing to accept little or no economic development, and tourism is often seized upon as
a potential source of livelihood for jobs and income, if not a panacea for economic development
on a larger scale [17]. Thus, in many cases tourism has not only been welcomed but actively
sought and encouraged, sometimes with economic incentives. Islands with limited natural re-
sources are inevitably vulnerable to almost any form of development [18] and this situation tends
to encourage island decision-makers to allow development. This is often accompanied by a re-
luctance to pose any challenge to agents of development because of a fear that such opposition
may result in the loss of potential further growth, leading to economic recession [19]. The full
long-term implications of such development, including the loss of traditional sites or resources
used by residents and the prevention of alternative forms of development being initiated, may
not be appreciated for several or even many years, by which time adjustments and limitations
may be difficult to impose [20]. Another key issue is that many islands have little or no control over tourism to them and are
dependent on, and in reality, under the control of, off-island forces and agencies. 2. Island Vulnerability These include
international airlines, externally owned ferry companies, international hotel chains, and higher
levels of government [21]. Islands which are particularly vulnerable to the pressures of tourism
include those that are easily accessible from major close markets, i.e., 1–3 hours flying time, have
good reliable air service, are on major cruise ship routes, and have attractions that are unique,
or iconic. The Caribbean islands, the Balearics, Canaries, Malta, Cyprus, and Capri are exam-
ples of such islands and groups. Some South Pacific Islands that have a specific appeal because
of unique features (e.g., Easter Island or Pitcairn Island), are perhaps not as vulnerable to tourism
numbers as they are too far from markets and thus expensive and relatively difficult to reach,
although they are receiving visitors in increasing numbers in recent years [22]. However, those
with a very small resident population, such as Atutaki (Cook Islands) may still be overwhelmed
by the daily tourist flight of some 180 passengers. From these examples, we may conclude that
island vulnerability to the negative impacts of excessive numbers of tourism, such as overtourism,
reflects their geographical location, their accessibility and links to tourist markets, their local pop-
ulation (numbers and cohesion), their size, and their relative power or control over their own
development. https://www.hos.pub Highlights Sustain. 56 Another issue of control facing many small islands is that they may face loss of land and
facilities after natural disasters, not only from the actual disaster but from capitalistic opportunists
that present themselves once attractive sites become vulnerable [23]. When tourism is potentially
economically attractive, rebuilding for residents is not necessarily a goal of all those who control
an island after a hurricane or tsunami has happened [24]. Islands reliant on tourism may see the
local or national government evict residents from their land either by decree or under the guise
of rehabilitation with the intent of tourism development. There have been a number of such
examples in Sri Lanka [25], Barbuda [24]; and more generally in the Caribbean [26]. This last point of control is one which is a problem for many islands, often irrespective of
their size. By reason of being peripheral and apart from a mainland, many islands, individually
and in groups, are part of a nation state rather than being independent, and thus lack complete
political control over their own destiny [13,17,27]. 3. The Threat of Unwanted Change on Islands Although there has been much focus on the need to make tourism more sustainable, many
of the vulnerabilities of islands have led to issues of overdevelopment, unwanted change and even
overtourism. While overtourism, or at least the term, is a relatively new phenomenon, the exist-
ence of over development and excessive numbers of visitors is certainly not new [30]. There is
no question that stakeholder perceptions of more tourists in already busy locations have funda-
mentally shifted in recent years and although the scale of the problem in earlier times may have
been very different to what is experienced now, the nature of the problem has remained very
similar, namely, discontent from residents of destinations at the rate and scale of development
and visitation and also at the behaviour of at least some of the visitors. There has been a plethora of articles and books in the past few years on overtourism, discuss-
ing related issues globally [2–4,31,32]. The term overtourism can be defined as “the acceleration
and growth of tourism supply and demand, the use of tourism destinations’ natural ecological
goods, the destruction of their cultural attractions, and negative impacts on their social and eco-
nomic environments” [33]. While this is not the only definition, it is clear that the term includes:
large, perhaps excessive, numbers of tourists, inappropriate behaviour by tourists, inconven-
ience/disturbance for residents, and unwanted change in the physical and social environments
of destinations. Slogans, protests, acts of damage and threats to visitors have all been recorded
[34]. Despite the considerable attention that has been given to overtourism by many forms of
media because of the very visible expression of discontent being expressed by residents of desti-
nations on a scale not seen previously (e.g., [35,36]), it remains controversial. It is not clear if
overtourism is really anything more than over-development under a new name [37], whether it
is a media-generated term experiencing a rush of media popularity [38,39], whether it is simply
a management problem [40], or whether it is a common phenomenon that has been around for
a long time but has only just received widespread attention [41]. In the context of islands, it is important to resolve whether overtourism should be considered
in absolute terms, or whether it should be considered as a relative situation. If the latter is true,
it may be much more of a destination or site-specific issue. 2. Island Vulnerability Even being fully independent does not nec-
essarily mean an island has complete control over tourism development, because as mentioned
above, the means of access and many sources of investment capital lie in agencies that are exter-
nal to the island. However, islands and their archipelagos that are suitable for tourism develop-
ment are often subject to policies dictating the rate, type and scale of development that have been
established on the mainland or other parts of the specific state [27]. Such policies may continue
in force even when island populations would prefer less or no further growth in tourism or related
development, and steps to control and mitigate against undesirable development may not be
supported by higher levels of government [28,29]. 3. The Threat of Unwanted Change on Islands If so, then reflecting on local conditions
and characteristics should be considered. Small, thinly populated but easily accessible islands
would seem to be prime candidates to experience tourism at excessive levels beyond their carry-
ing capacity [42]. When discussing overtourism in islands, one key issue is the often-heavy dependence on off-
shore operators to bring tourism to an island and to promote that island, as local agencies may
not have adequate funding to do so. Such a dependence can create many problems for a number
of enterprises on an island, particularly small-scale accommodation operators [43], and place
island tourism operators in a position of not being able or willing to change their image without
a risk of losing the powerful suppliers of customers. As most agents of development are generally https://www.hos.pub Highlights Sustain. 57 in favour of continued expansion as long as there is a market, regardless of whether it suits a
destination or not, [44] this can bring about changes in the characteristics of visitors and ulti-
mately in the locations themselves [45]. The pressure on destinations to continue to develop and
grow, results in changes in places that have little opportunity to slow or halt expansion and almost
no chance of returning to a quiet existence, their original culture, and an unspoiled environment. This situation can be seen in a loss of cultural strength, and heritage, at least the living human
heritage, as shown in adulterated handicrafts and other goods produced by indigenous peoples
[46], and witnessed in island communities from the Arctic to the South Pacific [47]. One argument is that the appropriate application of sustainable development principles in
the form of sustainable tourism could alleviate or prevent overtourism occurring in islands in
particular [14], but the failure to implement sustainability has proven too widespread and polit-
ically unpalatable for this to be achieved [48]. The development of platforms such as Airbnb has
allowed private development on a small individual scale to become massive in an overall context
and result in overdevelopment in destinations, with such properties sometimes being more heav-
ily used than conventional hotels [49]. 3. The Threat of Unwanted Change on Islands These types of problems have been experienced in Mal-
lorca as well as in Greek islands [50] where problems associated with second homes, Airbnb,
time shares and condominium developments have suggested that plans claiming sustainable tour-
ism principles are mostly simply only claims and have not been successful in avoiding overtourism
[51]. It is important to observe, however, that overtourism is not necessarily the same as mass
tourism, therefore, it cannot be assumed that all busy destinations are automatically experiencing
overtourism or unsustainable tourism. Busy destinations, even on islands, are often successful
tourist destinations and large numbers of people do not always mean too many people. The term
overtourism should not be used automatically as a criticism of mass tourism, as mass tourism,
including having large scale resorts on an island, does not necessarily mean that overtourism
exists. Instead, it should be recognized that there may be some stakeholders who oppose tourism
in general at any level, so opposition may be relative in extent and pronouncement, thus there
needs to be care when using the term, particularly in relating it to the concept of sustainability. 4.2. Culture, Heritage Factors, and Population Factors Island destinations, in many respects, are the same as mainland destinations with regard to
the appeal of cultural and heritage attributes. Tourism has long been drawn to unique or different
cultures, to different culinary traditions, ways of dress, languages, architecture, historical remains,
sites of major events, both real and imaginary, even non-existent attractions [53]. Thus, the more
different, and in some cases, the more remote or less visited the island, the greater the attraction
to some tourists and to some tour companies, also raising the risk of ever-increasing development
and overtourism as knowledge about a place spreads. One example is the Faroe Islands which
was once relatively unknown, became popular very quickly. Where local populations are small
and often without significant powers of control overdevelopment, heritage in its many forms may
become subject to the impacts of overtourism, with subsequent loss of authenticity and ultimately
of the islands’ key attractions. Where the population is concentrated into one or two urban cen-
tres, development is more likely to be attracted to these specific locations which are often cultural
and historic centres, and therefore more attractive to tourists as attractions are likely to be in
close proximity to each other than when population is dispersed. Venice is perhaps the most
visible example of an island experiencing overtourism, with tourists attracted by its cultural- her-
itage attributes, and is often cited as losing or having already lost, not only much of its appeal,
but also a large proportion of its traditional population [54]. Here the key issue is how to limit,
mitigate or prevent overtourism and its effects while maintaining the quality and nature of life
for the residents of such islands Venice has faced these problems over the years with little success
[55] or indeed, with little being done successfully to prevent or mitigate the problems. Many
solutions have been discussed but few if any steps actually taken, and development outside but
adjoining Venice has accentuated the problem by increasing the number of day visitors without
the benefits that accrue from their staying overnight in the city. Venice suffers (or benefits de-
pending on one’s viewpoint) from proximity to major markets, an unequalled iconic image, a
high level of visibility in markets, while having none of the island characteristics which might
deter visitation, particularly as it is connected to its mainland by a causeway, allowing unlimited
constant access. 4. Discussion Unsustainable tourism in islands is therefore, often a result of a combination of political, ge-
ographical, cultural and physical characteristics that can make islands, particularly small islands,
vulnerable to the pressures from excessive tourism development when they are relatively poorly
equipped to prevent or limit such pressures (see Figure 1). Figure 1. Factors increasing or reducing island vulnerability to overtourism. Location
•Remoteness (over 8 hours)
•Irregular/infrequent access
Population
•Concentrated in few
locations
•Coastal, urban
•Dispersed, rural
Size
•Large size
Resources
•Abundant, mineral
Control
•Independent
•Control over ports/airports
Decreasing
vulnerability
Location
Population
Size
Resources
Control
•Proximity to markets (1-3
hours)
•Frequent air/sea access
•On Cruise ship itineraries
•Concentrated in few locations
•Coastal, urban
•Dispersed, rural
•Small (under 1000 sq.kms)
•Few/limited/agricultural
•Local or regional power only
•No control of means of access
Increasing
vulnerability
Bold text indicated increased importance •Proximity to markets (1-3
hours)
•Frequent air/sea access
•On Cruise ship itineraries •Concentrated in few locations
•Coastal, urban
•Dispersed, rural Decreasing
vulnerability Increasing
vulnerability •Small (under 1000 sq.kms) •Abundant, mineral •Independent
•Control over ports/airports •Local or regional power only
•No control of means of access Bold text indicated increased importance Figure 1. Factors increasing or reducing island vulnerability to overtourism. Figure 1. Factors increasing or reducing island vulnerability to overtourism. https://www.hos.pub Highlights Sustain. 58 4.1. Locational Characteristics Re markets 4.1. Locational Characteristics Re markets While location is a key importance for an island to attract tourism development in the first
place, it is also important with respect to the likelihood of such development becoming unsus-
tainable. Islands which are in close proximity to major existing or potential markets are most
vulnerable as a short relatively cheap journey is likely to attract large numbers of visitors, e.g.,
Jeju Island, only 60 miles from mainland Korea [5] or Cozumel, only 10 miles from mainland
Mexico. Being near to major markets often usually means lower costs in terms of importing ma-
terials and supplies and therefore less costly development than in isolated and remote islands. Remote locations, however, as illustrated by great distance from potential markets, and subject
to harsh physical conditions, can be seen as shielding some islands from excessive tourist devel-
opment and visitation. Some islands in the South Pacific, like Easter Island, while having unique
and iconic attractions, receive relatively few visitors because of the cost and difficulty of reaching
them, alhtoughalthough increasingly frequent air services are capable of changing this situation. St Helena, in the South Atlantic is one island that will face such a situation as a newly extended
airport comes into full operation, allowing relatively easy and quick access compared to the pre-
vious limited and lengthy sea ferry access [52]. Other islands, like Pitcairn, or Tokelua with no
cruise boat harbour and no commercial air service, benefit from their natural characteristics and
limited access maintaining the numbers of visitors at relatively sustainable levels. 4.2. Culture, Heritage Factors, and Population Factors 4.4. Physical and Environmental Attributes In recent years, growing interest in the physical world, driven greatly by organisations such
as National Geographic, and television programmes such Planet Earth and Blue Planet hosted
by celebrities such as Sir David Attenborough, has seen many islands increase in visitation. The
Galapagos Islands are a case in point. Relatively inaccessible for many years, on the “wrong”
side of South America to the major markets of Europe and much of North America, accessed
only via a country (Ecuador) that was not itself a major tourist destination, the islands are an
unlikely tourist destination. Despite the disadvantages of a required transhipment from Ecuador,
the absence of any cultural features of note, the high cost of access, limitations on group size and
behaviour, the requirements for the vast majority of tourists to go on a package tour, and the
minimum time involved for a visit, visitor numbers have increased rapidly over the past five
decades (from 11,000 visitors in 1979 to 271,000 in 2019) and exceeded the declared tourist
number limits (originally set at 25,000 in 1982) many times [56]. Growth of tourism has been
matched by resident population growth as the islands offer the potential of above average income
for Ecuadorian citizens living there compared to those on the mainland, in turn generating the
need for further development to support such residential growth. Thus, if the attractions of an
island are great enough, rare enough, or of sufficient interest to the increasingly social-media
conscious public, then tourist numbers will continue to grow and be driven in part by continued
mass exposure. Social media is undoubtedly a factor in stimulating increased visitation and po-
tentially being able to reduce visitation [3], but is something which is not unique to islands and
is far beyond their ability to control, leaving them as vulnerable to its affects as non-insular des-
tinations. Such trends are visible even in islands more remote than the Galapagos, with islands in the
far north of Canada and Norway now being visited by cruise ships and aircraft, as are islands in
Antarctica, with tourists attracted by sea and bird life, or in the case of Svalbard, polar bears. As
well as icebreakers, reinforced hulled cruise liners are making excursion to these islands, being
followed by luxury “yachts” carrying a few hundred passengers. 4.3. Political Power Structures and Control The political structure in islands may greatly influence the potential for overtourism. Islands
which are fully independent may have the best chance of being able to control the rate and nature
of tourism development, but even those face major problems of finding alternative sources of
employment and income if they reject or strictly limit tourism development. Those islands which
have extensive regional controls, Shetlands, Orkneys, Greenland, Canaries, and Balearics, for
example, may also have sufficient controls and powers, should they choose to use them, to man-
age and control tourism. The most vulnerable islands are those which are small, with limited
resources, populations and powers, such as Gozo (Malta), Koh Phi Phi (Thailand) or some of the https://www.hos.pub Highlights Sustain. 59 outer Canary Islands, the Azores and some Caribbean territories, which are subject to control
by authorities at several different higher levels of power and thus have little power over develop-
ment of any kind on their own small island. Pressure to allow airlines to operate services, for cruise ships to land passengers for short pe-
riods, developers to construct resorts and other facilities is always high and often next to impos-
sible to resist. All politicians, particularly local representatives, are subject to local pressures for
jobs and for income to residents and from taxes [19], and once tourism development begins, it
tends to take on a life of its own and becomes increasingly difficult to manage and control. For
that reason, the earlier the imposition of appropriate controls can be made, the greater the
chances of success in avoiding overtourism. Equally important, is the need to make well-thought
out and long-term decisions at the beginning of tourism development to ensure development
follows a desired path with respect to rate of development and type of development, as well as
any possible curbs on non-local ownership and also designation of areas to be protected from
development. Limits on level of air services and numbers of cruise ships allowed at any one time
are also better and more easily imposed at an early stage of development rather than later. 5. Controls to Mitigate and Prevent Overtourism If destinations are to avoid the problems of overtourism, or indeed unsustainable develop-
ment of any kind, the ways in which control over tourism may be exerted in the insular context
must be examined and strengthened. Considerable attention has been paid to overtourism which
raises the question of what actions could be taken to prevent or mitigate the effects of too many
tourists in a destination or whether concerns are really related to the easing of congestion and
not to excess numbers of tourists [4,31,57]. Many general mitigation and control issues become
very specific in the case of islands. A key issue for islands, as noted earlier, is that of who has power and control over key issues
and where that power is located, i.e., whether it is local or external to the island(s) affected. If
control is vested in a local or regional island authority, the introduction of mitigation measures
has much greater potential to be implemented than when such power belongs with other, gener-
ally external, agencies. Given that the problems of overtourism are primarily and usually experi-
enced at the local level, which may be a whole island in some cases, then it is logical to argue that
solutions are most likely to be found at the local level and it is at that level that actions should be
taken [58]. For example, the Gili islands in Indonesia face significant problems managing over-
tourism which is compounded by the larger controlling government body of Lombok having a
growth-oriented stance [59]. One approach that is being tried is to gain local ownership of key sites in order to make them
more resilient to tourism pressures, and thus be able to shield them from impacts and reduce
more general problems in the wider area. Such steps have been taken on the Isle of Skye, (Scot-
land) where residents have become concerned over inappropriate behaviour of tourists [60] and
excessive levels of use of locations without adequate facilities such as parking and toilets [61]. In
this case residents have used existing powers to buy out specific sites with a view to providing
toilets and car parking spaces to alleviate problems in these areas. 5. Controls to Mitigate and Prevent Overtourism Skye is an interesting case, as
until 1995 it could only be reached by ferries from the Scottish mainland, which had provided
some limits on tourist numbers, including no service on Sundays, when many establishments on
Skye were closed in observance of the Sabbath. When a bridge was opened in 1995 allowing
unlimited access (in terms of numbers and time), considerable opposition was expressed, both at
the availability of access on Sundays and the likely implications for changes in the nature of life
on the island. The issue of the loss of “islandness” [62] was also raised although this has not been
reflected in any apparent loss of appeal to tourists and users (locals and visitors) have risen in
numbers from 612,500 in 1995 to around 1,825,000 in 2019 [63]. The same issue of the loss of
the image and feeling of islandness has been raised elsewhere, for example, at a far larger scale,
when Prince Edward Island (Canada) was joined to the mainland by a bridge in 1997. In a more general sense, proactive management such as the provision of facilities and services
in some locations and not in others, provision of information and directions, and controlling of
behaviour in specific locations can all reduce the effects, if not the numbers of tourists. However,
all stakeholders must have the same goals in mind otherwise growth will trump demand as in the
case of the Caribbean [44]. The increasing use of social media to identify areas to visit [64] has
led to crowding at specific sites such as Maya Bay in Thailand [65] and the Mermaid pools in
New Zealand [66] and also to the development and maintenance of inappropriate behaviour. When residents have to resort to direct action to counter the effects of overtourism, it is an indi-
cation that all levels of government and the private sector have failed to develop or control tour-
ism appropriately and that alternative actions are required. Islands can often exert control over development, even when possessing only small popula-
tions, if they have the appropriate powers. The Shetland Isles, to the north of Great Britain,
gained such powers over development and the use of the shoreline when they argued for the need
for local control over oil and gas development in the 1970s [67]. 4.4. Physical and Environmental Attributes In such thinly populated or even
deserted environments, unsustainable tourism takes on a new face and operates at much lower
absolute levels of visitation than in locations like Venice or Mallorca, supporting the argument
that overtourism is essentially a relative concept. As with cultural heritage, the pressures of tour-
ism on rare and endangered species and environments are often not acknowledged or even no-
ticed until sometimes impacts have exceeded the level of self-correction and irreparable change
has occurred. Many such areas are relatively unprotected because of the absence of a permanent
human presence to enforce any restrictions on behaviour and operation of tourists and tourism. Self-regulation on cruise ship operations overall in particular are unlikely to be very effective,
despite the praiseworthy efforts of some companies. While in previous times the long distance
from markets and inhospitable climate and marine conditions deterred tourists, technological
improvements in vessels have been matched with changing logistical arrangements, such that
visitors now overcome the distance by flying to the southern tip of South America and then
boarding ships to go further south, thus saving many weeks sailing time. https://www.hos.pub Highlights Sustain. 60 5. Controls to Mitigate and Prevent Overtourism The acquisition of such powers
has enabled the island authority to limit and control the scale, nature and rate of all kinds of
development, including tourism, on the islands since, with considerable success. If such a small
island group (18,000 residents) could control and prevent development pursued by multi-national
companies supported by the national government, then controlling and directing tourism is also
clearly possible. As well as gaining such powers, however, it is necessary to present a united front
in terms of local opinions and preferences and to have strong and consistent leadership to present
and push such opinions in order to withstand pressures from external forces. In the case of tour-
ism, where many proponents may be small in scale and local in origin, gaining unanimity against https://www.hos.pub Highlights Sustain. 61 excessive development or visitor numbers is extremely difficult and rarely achieved. Even when
it does occur, it can be counter-acted by higher levels of government [29]. excessive development or visitor numbers is extremely difficult and rarely achieved. Even when
it does occur, it can be counter-acted by higher levels of government [29]. Control over means of access can be critical and this is one area in which islands may have
an advantage over mainland destinations as means of access may be limited and clearly identi-
fied. Simply stopping visitors coming to islands is not an effective or desirable way of dealing with
tourism which has become unsustainable. It would send a very strong message that tourism of
any kind is not welcome, and most islands do not wish to do this even though some islands in
Thailand, such as Maya Bay, Koh Khai Nok, Koh Khai Nui, Koh Tachai, and Koh Khai Nai
and in Iceland (Fjaðrárgljúfur Canyon) have done so. Limiting, or even reducing the frequency
and capacity of access, such as to the Seychelles [59] can be highly effective and does not neces-
sarily send a negative message to tourists. In the case of Pitcairn Island, the continued absence of
a landing site for cruise ships means passengers have to use the island’s open boats for transfer
from ship to shore and return, reducing the numbers making such excursions and limiting their
time ashore [22]. 5. Controls to Mitigate and Prevent Overtourism Keeping access by tourists limited and or expensive may be appropriate and is
often accepted by tourists if they appreciate the importance of giving priority to maintaining
access and egress for local residents. For example, the Guna communities in Panama have used
their ability to control resources to enable them to refuse to accept non-desirable forms of devel-
opment and to strengthen the resilience of their culture [68]. Another alternative action is to reduce or refocus promotional messages in order to change
or reduce demand. When appropriately done, such messages may make a destination more at-
tractive to desired markets and less attractive to inappropriate ones, for example, Benner [69]
discussed redirecting or “nudging” tourists to certain areas, and Araya Lopez recorded the ex-
pressions of opposition to overtourism in Barcelona as an indication of concern over such issues
[70]. National and regional level messages and images may be difficult to change as they are
decided at non-local levels and may be intended to produce different results. Locally focused
messages and images, especially those using social media, if correctly designed and packaged, can
be highly effective at not only passing on useful information but also redirecting visitation and
changing visitor behaviour [71]. Clearly such messages are not going to be successful in all cases
and may be beyond the capability of local destinations such as small islands, to create and send,
but it is becoming increasingly possible to reach large numbers of potential visitors at relatively
low cost through the many forms of social media. A number of other approaches to mitigating the growth of excessive tourism have been sug-
gested [40], although unfortunately many are relatively ineffective in the long term, focused spe-
cifically on urban destinations, and often only succeed in creating a new problem in another area
or at another time. Other suggestions such as adding or increasing fees or taxes (e.g., Galapogas,
Faroe Islands, Venice) are becoming increasingly common. Another suggestion is to educate
tourists (e.g., pamphlets in Aruba, in-flight videos in Kauai, the Palau pledge), however the suc-
cess of such initiatives over the long term has not been proven. Alternatively, suggesting tourists
visit alternative quiet areas, while possibly reducing pressure in high use locations, may serve only
to make previously little-visited areas also become subject to excessive use. 5. Controls to Mitigate and Prevent Overtourism By virtue of being
previously unused, such areas are very likely to not have adequate, if any, facilities to handle a
sudden increase in visitation. Such a “solution” also ignores the fact that highly popular spots are
highly popular for a reason, such as unusual attributes, and these will not be found in other
locations, resulting in disappointed visitors. This is particularly true for islands with specific
unique features, such as Galapagos. Attempting to shift demand from peak to off-season is ex-
tremely difficult to achieve because of seasonal climatic and also institutional limits on when
people can take vacations and when it is suitable to visit specific places. Encouraging tourists to
visit outside the main season may create other problems as facilities and services may not be open
then [3]. It has been found [72] that attempts to attract visitors out of season may in fact simply
create additional demand then and not diminish peak season visitation, which may mean locals
experience a longer period of disturbance as the peak season continues into what were shoulder
and rest seasons. Such extensions of the tourist season can have negative impacts on environ-
mental recovery from visitor use also. Other suggested solutions such as building replicas of lo-
cations experiencing overtourism [73] is clearly impractical in the case of small islands. 6. Conclusion For many years a considerable number of islands have been struggling to overcome natural
problems in order to allow greater numbers of tourists to reach them. Improving access by https://www.hos.pub Highlights Sustain. 62 increasing and improving transportation links to market have been the most common solution,
with gaining air transport service being a key factor in opening many islands to mass tourism. increasing and improving transportation links to market have been the most common solution,
with gaining air transport service being a key factor in opening many islands to mass tourism. In recent years, however, at least some islands have been seeking ways to limit tourist numbers
where they have reached unsustainable levels. Relying on inaccessibility has proven ineffective
in many cases as the tourism industry, often supported by national (and off-island) levels of gov-
ernment which favour increased levels of tourism and development overall. It is clear that islands
that have control over their own destiny in terms of means of access and level of development
are in a much stronger position to prevent overdevelopment. The political and power aspects of
tourism and general growth has often been ignored, leaving many small communities with little
or no power or even influence over developments of means of access and levels and types of
development. Good intentions count for little when the power to implement such intentions is
not available. Even where islands may have sustainable tourism policies and goals it has become
clear that inaction and failure to implement such policies [3,48], can lead fairly directly to over-
development and overtourism in many situations. While in past years islands’ natural attributes,
such as remote locations, limited attractions, absence from social media and difficulty of access
may have mitigated against overtourism occurring, now many islands are major tourist destina-
tions and clearly susceptible to excessive levels of visitation. Some islands and their residents are
content with, visitation by large numbers of tourists and the income gained from them, but in
other cases concerns are being expressed over excessive numbers and inappropriate behaviour
of some of these visitors. In some, perhaps many cases the problem of overtourism has been
exacerbated, or actually caused, by media discourse [74]. Inauthentic messages can create tourist
perceptions endorsing inappropriate behaviour which then becomes the norm, and also draws
attention to specific locations [75]. 6. Conclusion Being too successful in attracting tourists is not something
many destinations anticipated and few have made preparations for such an eventuality. There is
no immediate nor any single solution to this problem, and it clearly is not confined to islands,
although they may be the most vulnerable of destinations, and are often the least able to control
their own destinies. Author Contributions Conceptualization: R.W.B. and R.D.; Writing—original first draft: R.W.B.; Writing—review
and editing: R.W.B. and R.D.; Visualization: R.W.B. and R.D. Conceptualization: R.W.B. and R.D.; Writing—original first draft: R.W.B.; Writing—review
and editing: R.W.B. and R.D.; Visualization: R.W.B. and R.D. Conflicts of Interest The authors declare no conflict of interest. References 1. Alberts, A.; Baldachinno, G. Resilience and Tourism in Islands: Insights from the Caribbean. In Tourism and
Resilience, Butler, R.W., Ed.; CABI: Wallingford, UK, 2017; pp. 150–162. 2. Butler, R.W.; Dodds, R. Overcoming Overtourism: A Review of Failure Tourism Review. Tour. Rev. 2022, 77, 35–
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The Joint Effect of Maternal Marital Status and Type of Household Cooking Fuel on Child Nutritional Status in Sub-Saharan Africa: Analysis of Cross-Sectional Surveys on Children from 31 Countries
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Nutrients
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Citation: Amadu, I.; Seidu, A.-A.;
Duku, E.; Okyere, J.; Hagan, J.E., Jr.;
Hormenu, T.; Ahinkorah, B.O. The
Joint Effect of Maternal Marital Status
and Type of Household Cooking Fuel
on Child Nutritional Status in
Sub-Saharan Africa: Analysis of
Cross-Sectional Surveys on Children
from 31 Countries. Nutrients 2021, 13,
1541. https://doi.org/10.3390/
nu13051541
Academic Editor: Pietro Vajro
Received: 12 March 2021
Accepted: 28 April 2021
Published: 3 May 2021 p
6
Neurocognition and Action-Biomechanics-Research Group, Faculty of Psychology and Sport Sc
Bielefeld University Postfach 10 01 31 33501 Bielefeld Germany 6
Neurocognition and Action-Biomechanics-Research Group, Faculty of Psychology and Sport Sciences, 6
Neurocognition and Action-Biomechanics-Research Group, Faculty of Psychology and Sport Sciences
Bielefeld University, Postfach 10 01 31, 33501 Bielefeld, Germany g
p,
y
y
gy
p
,
Bielefeld University, Postfach 10 01 31, 33501 Bielefeld, Germany efeld University, Postfach 10 01 31, 33501 Bielefeld, Germany Citation: Amadu, I.; Seidu, A.-A.;
Duku, E.; Okyere, J.; Hagan, J.E., Jr.;
Hormenu, T.; Ahinkorah, B.O. The
Joint Effect of Maternal Marital Status
and Type of Household Cooking Fuel
on Child Nutritional Status in
Sub-Saharan Africa: Analysis of
Cross-Sectional Surveys on Children
from 31 Countries. Nutrients 2021, 13,
1541. https://doi.org/10.3390/
nu13051541 y
y
7
School of Public Health, Faculty of Health, University of Technology Sydney, Sydney, NSW 2007, Australia;
brightahinkorah@gmail.com *
Correspondence: elvis.hagan@ucc.edu.gh Abstract: The current study sought to investigate the joint effect of maternal marital status and type
of household cooking fuel on child nutritional status in sub-Saharan Africa. Data in the children’s
files of 31 sub-Saharan African countries were pooled from the Demographic and Health Surveys
collected between 2010 and 2019. The outcome variables were three child anthropometrics: stunting
(height-for-age z-scores); wasting (weight-for-height z-scores); and underweight (weight-for-age
z-scores). The joint effect of maternal marital status and type of household cooking fuel on child
nutritional status was examined using multilevel regression models. The results were presented
as adjusted odds ratios (aORs) at p < 0.05. The percentages of children who were stunted, wasted
and underweight in the 31 countries in sub-Saharan Africa were 31%, 8% and 17%, respectively. nutrients nutrients nutrients nutrients nutrients The Joint Effect of Maternal Marital Status and Type of
Household Cooking Fuel on Child Nutritional Status in
Sub-Saharan Africa: Analysis of Cross-Sectional Surveys on
Children from 31 Countries Iddrisu Amadu 1,2
, Abdul-Aziz Seidu 3,4
, Eric Duku 1,2
, Joshua Okyere 3
, John Elvis Hagan, Jr. 5,6,*
,
Thomas Hormenu 4
and Bright Opoku Ahinkorah 7 1
Africa Centre of Excellence in Coastal Resilience, University of Cape Coast, Cape Coast PMB TF0494, Ghana;
iddrisu.amadu@stu.ucc.edu.gh (I.A.); eric.duku@stu.ucc.edu.gh (E.D.) g
g
2
Department of Fisheries and Aquatic Sciences, College of Agriculture and Natural Sciences, School of
Biological Sciences, University of Cape Coast, Cape Coast PMB TF0494, Ghana 3
Department of Population and Health, College of Humanities and Legal Studies, University of Cape Coast,
Cape Coast PMB TF0494, Ghana; abdul-aziz.seidu@stu.ucc.edu.gh (A.-A.S.);
joshuaokyere54@gmail.com (J.O.) j
y
g
4
College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville, QLD 4811, Australia; thormenu@ucc.edu.gh 5
Department of Health, Physical Education, and Recreation, University of Cape Coast,
Cape Coast PMB TF0494, Ghana 1. Introduction Children are expected to receive the required nutritional needs to promote their
physical and cognitive development [1]. Hence, child nutrition can be thought of as a fun-
damental right of children, with several countries across the globe contributing significantly
towards the improvement of children’s nutritional status (CNS) [1]. Notwithstanding these
efforts to reduce child morbidity and mortality, CNS (manifesting as stunting, wasting, or
underweight) remains an obdurate public health concern, especially in sub-Saharan Africa
(SSA) [2]. Reports from UNICEF [3] indicate that SSA recorded the highest prevalence of
stunting (40%) and the second-highest prevalence of wasting (9%) worldwide in 2018. This
is a worrying development for SSA, and therefore calls for urgent research to explore the
underlying factors that result in the nutritional status of children within the region. Prioritizing CNS is integral to the health, and wellbeing of the child sincepoor CNS
may have deleterious repercussions (short and long term) on the individual. For in-
stance, some studies have revealed that stunted children are at higher risk of experienc-
ing poorer health, lower economic status, poor cognition, as well as lower educational
performance [4,5]. Existing evidence on CNS has largely focused on how characteristics such as the age
of the mother, poverty, and feeding practices [6–8] relate to CNS, with little attention given
to the potential relationship between maternal marital status and CNS. However, available
evidence suggests that there have been significant changes in the family structure, with
an increasing incidence of out-of-wedlock motherhood, divorce, and widowhood being
reported in SSA [9]. The effects of globalization, urbanization, and the HIV pandemic
have been cited as potential reasons, hence resulting in an increased proportion of single
mothers within the sub-region [9,10]. This proliferation of single mothers also arouses
concerns that a substantial proportion of children born are being raised in a single-mother
household [2]. Beyond the postulation that there may be an association between maternal marital
status and CNS, there is a growing interest within the public health discipline concerning
the role or association between household cooking fuel type and CNS [11–14]. Previous
studies have demonstrated the association between cooking fuel and children’s health. For instance, in a study conducted by Owili et al. [11], it was found that the odds of an
under-five child dying was higher for those whose households used charcoal and biomass
cooking fuel compared to those who used clean fuel.
On the joint effect of maternal marital status and type of household cooking fuel on stunting, we
found that compared to children born to married mothers who used clean household cooking fuel,
children born to single mothers who use unclean household cooking fuel, children born to single
women who use clean household cooking fuel, and children born to married women who used
unclean household cooking were more likely to be stunted. With wasting, children born to single
mothers who used unclean household cooking fuel and children born to married women who used
unclean household cooking fuel were more likely to be wasted compared to children born to married
mothers who used clean household cooking fuel. With underweight, we found that compared to
children born to married mothers who used clean household cooking fuel, children born to single
mothers who used unclean household cooking fuel, children born to single women who used clean
household cooking fuel and children born to married women who used unclean household cooking
were more likely to be underweight. It is imperative for the governments of the 31 sub-Saharan
African countries to double their efforts to end the use of unclean household cooking fuel. This goal
could be achieved by promoting clean household cooking fuel (e.g., electricity, gas, ethanol, solar,
etc.) through effective health education, and promotion programmes. The attention of policymakers
is drawn to the urgent need for children’s nutritional status policies and programmes (e.g., dietary
supplementation, increasing dietary diversity, improving agriculture and food security) to be targeted
towards at-risk sub-populations (i.e., single mothered households). Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. 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/nutrients Nutrients 2021, 13, 1541. https://doi.org/10.3390/nu13051541 2 of 16 Nutrients 2021, 13, 1541 Keywords: biomass fuel; child nutrition; single-motherhood; solar; sub-Saharan Africa 1. Introduction Thus, a lack of access to and non-use
of clean cooking fuel exacerbates under-five mortality. Other studies have also shown that
under-fives may die when exposed to solid fuel usually used in the household within the
sub-Saharan African context [15]. After an extensive literature search, we found no study in SSA that has explicitly ex-
amined the joint effect of maternal marital status and household cooking fuel type on CNS. Therefore, in the present study, we sought to contribute towards bridging the gap in the
literature by investigating the joint effect of maternal marital status and householdcooking
fuel type on CNSin SSA. Our study is timely and significant in facilitating SSA’s quest to achieve the Sustainable
Development Goals (SDG), particularly SDG 2.2, which envisions to end all forms of
malnutrition by 2030, and also ensure that by 2025, the internationally agreed targets on
stunting and wasting in children under five years of age are achieved [3]. 2.1. Data Source Data for this study were obtained from the Demographic and Health Surveys (DHS)
of 31 countries in SSA counducted from 2010 to 2019. The DHS Program has since 1984 Nutrients 2021, 13, 1541 3 of 16 3 of 16 assisted in the conduct of over 400 surveys in many low-and middle-income countries
around the world. These cross-sectional surveys provide nationally representative house-
hold data on various nutrition, population and health indicators in more than 90 countries. Standardized protocols and instruments are employed to gather data of children, women,
men and households. For this study, data in the children’s files were pooled from the DHS. The surveys employ a two-stage stratified sampling in selecting participants. The first
stage involves the selection of clusters, usually called enumeration areas (EAs), and the
second stage consists of the selection of households for the survey. To ensure consistency in
data collection across countries, the DHS use a standard questionnaire comparable across
countries for data collection, and the questionnaire is often translated into the major local
languages of the countries involved. To ensure validity of the translated questionnaires,
the DHS reports that the translated questionnaires, together with the version in English,
are pretested in English and the local dialect [16,17]. Figure 1 shows the countries included
in this study. We followed the Strengthening the Reporting of Observational Studies in Epi-
demiology’ (STROBE) statement in writing the manuscript. The dataset is freely accessible
for download at: https://dhsprogram.com/data/available-datasets.cfm (accessed on 3
February 2021). g
assisted in the conduct of over 400 surveys in many low-and middle-income countries
around the world. These cross-sectional surveys provide nationally representative house-
hold data on various nutrition, population and health indicators in more than 90 coun-
tries. Standardized protocols and instruments are employed to gather data of children,
women, men and households. For this study, data in the children’s files were pooled from
the DHS. The surveys employ a two-stage stratified sampling in selecting participants. The first stage involves the selection of clusters, usually called enumeration areas (EAs),
and the second stage consists of the selection of households for the survey. To ensure con-
sistency in data collection across countries, the DHS use a standard questionnaire compa-
rable across countries for data collection, and the questionnaire is often translated into the
major local languages of the countries involved. 2.1. Data Source To ensure validity of the translated ques-
tionnaires, the DHS reports that the translated questionnaires, together with the version
in English, are pretested in English and the local dialect [16,17]. Figure 1 shows the coun-
tries included in this study. We followed the Strengthening the Reporting of Observa-
tional Studies in Epidemiology’ (STROBE) statement in writing the manuscript. The da-
taset is freely accessible for download at: https://dhsprogram.com/data/available-da-
tasets.cfm. Figure 1. Map showing the 31 sub-Saharan African Countries
Figure 1. Map showing the 31 sub-Saharan African Countries. Figure 1. Map showing the 31 sub-Saharan African Countries
Figure 1. Map showing the 31 sub-Saharan African Countries. 2.2. Measures 2.2.1. Outcome Variables 2.2. Measures
2.2.1. Outcome Variables
The outcome variables are three child anthropometrics: stunting (height-for-age z-
scores); wasting (weight-for-height z-scores); and underweight (weight-for-age z-scores). These variables were defined and coded using the WHO child growth standard which is
followed by the DHS program [18]. The coding was done as follows: i. Stunting: children with height-for-age z-scores below minus 2 (−2.0) standard
deviations less than the mean on the WHO Child Growth Standards (moderately or i. Stunting: children with height-for-age z-scores below minus 2 (−2.0) standard
deviations less than the mean on the WHO Child Growth Standards (moderately or Nutrients 2021, 13, 1541 4 of 16 severely stunted) and children with height-for-age z-scores below minus 3 (−3.0)
standard deviations less than the mean on the WHO Child Growth Standards
(severely stunted) were combined to form the response group “Stunt” while those
height-for-age z-scores equal to or higher than minus 2 (−2.0) standard deviations
greater than the mean on the WHO Child Growth Standards were regarded as “not
a stunt”. ii. Wasting: children with weight-for-height z-scores below minus 2 (−2.0) standard
deviations less than the mean on the WHO Child Growth Standards (moderately
or severely wasting) and children with weight-for-height z-scores below minus 3
(−3.0) standard deviations less than the mean on the WHO Child Growth Standards
(severely wasting) were combined to form the response group “Wasting” while
those weight-for-height z-scores equal to or higher than minus 2 (−2.0) standard
deviations greater than the mean on the WHO Child Growth Standards were
regarded as “No wasting”. Wasting: children with weight-for-height z-scores below minus 2 (−2.0) standard
deviations less than the mean on the WHO Child Growth Standards (moderately
or severely wasting) and children with weight-for-height z-scores below minus 3
(−3.0) standard deviations less than the mean on the WHO Child Growth Standards
(severely wasting) were combined to form the response group “Wasting” while
those weight-for-height z-scores equal to or higher than minus 2 (−2.0) standard
deviations greater than the mean on the WHO Child Growth Standards were
regarded as “No wasting”. iii. 2.2.2. Key Predictor Variable The main predictor variables used were generated based on literature and poten-
tial contextual implications of findings. They were maternal marital status and type of
household cooking fuel. The variable “maternal marital status” was coded to produce
two responses as follows: never married, widowed and separated/divorced were coded
together as “Single” and married and living with a partner as “Married” [9]. For parsimony,
theoretical and contextual relevance, the variable “type of household cooking fuel” was
also coded into two response categories “Clean” and “Unclean” following previous stud-
ies [19,20]. Clean fuels included electricity, liquefied petroleum gas (LPG) and natural gas
while charcoal, firewood, grass/straw, dung, shrubs, agricultural crop waste represented
unclean cooking fuels [19,20]. The two variables “maternal marital status” and “type of
household cooking fuel” were then combined [19,20] to produce the variable “Maternal
marital status-Type of cooking fuel” with four (4) mutually exclusive categories: “Single
mother-clean” (single mothers living in a household that uses clean cooking fuel), “Single
mother-unclean” (single mothers living in households that uses unclean cooking fuel),
“Married -clean” (mothers who are married or living with a partner in a household that
uses clean cooking fuel” and “Married -unclean” (mothers who are married or living with
a partner in a household that uses unclean cooking fuel). To observe the effect of maternal
marital status and the type of household cooking fuel on the nutritional status of children
under the age of 5 years, married-clean is used as the reference group. 2.2.1. Outcome Variables g
g
Underweight: children with weight-for-age z-scores below minus 2 (−2.0) standard
deviations less than the mean on the WHO Child Growth Standards (moderately or
severely underweight) and children with weight-for-age z-scores below minus 3
(−3.0) standard deviations less than the mean on the WHO Child Growth Standards
(severely underweight) were combined to form the response group “Underweight”
while those weight-for-age z-scores equal to or higher than minus 2 (−2.0) standard
deviations greater than the mean on the WHO Child Growth Standards were
regarded as “Not underweight”. For each of these variables, “age out of plausible
limits”, “height out of plausible limits”, missing and “flagged” responses which
constituted, 13, 744, 1833 and 3285 respectively were deemed invalid and dropped. 2.2.3. Covariates In the analysis of the effect of maternal marital status and household cooking fuel
type on the nutritional status of children under age 5, three categories/clusters of variables
(individual level factors-child and mother’s characteristics, household characteristics, and
contextual factors) were considered ascovariates. The selection of these variables was
based on their significant associations with CNS in previous studies (6–8). Variables under
individual level factorsconsidered include the age of the child (0, 1, 2 and 4); sex of child
(female and male); birth order of child (1, 2 to 4, and 5 and above); and perceived size Nutrients 2021, 13, 1541 5 of 16 at birth (small, average and large) (see [21]). Other included maternal age (re-coded into
two categories “15–19” years and “20–49” years (see [22]); educational attainment (no
formal education, primary, secondary and higher); working status (yes and no); antenatal
visits during pregnancy (yes, no, and “Don’t know”); postnatal check within 2 months
(yes and no); and place of delivery (home, health facility, other). At the household level,
relevant variables included wealth status (recode as “poor”, “middle” and “rich”); the
age of household head (recoded as ages below 35 years “young adults”, between 35
and 55 years “middle-aged adults” and those above 55 years “old-aged adults”; sex
of household head (male and female); access to electricity (yes and no); type of toilet
facility (re-coded into “improved” and unimproved”; source of drinking water (re-coded as
“improved” and “unimproved” (see [23]); and access to media (yes, no) which was derived
from the three variables “access to television”, “radio” and “newspaper/magazine”. The
contextual factors considered are Urbanicity (rural and urban) and geographic region. The
variable “Country” was re-coded to generate “Geographic region” following the UN’s list
of countries and geographic regions in SSA. 2.3. Data Analyses Stata SE version 14.2 (StataCorp, College Station, TX, USA) was used for statistical
analyses of data. Descriptive statistics, including frequencies, percentages (weighted) and
95% confidence intervals (CIs) of percentages at p < 0.05 were used to summarize and
present the data in tables. To enhance visualization and appreciation of the distributions
of the outcome variables across the study countries, the data was integrated into a GIS
environment and presented in map images. This procedure was then followed with
a bivariate chi-square test of independence to determine the associations between the
outcome variables and each of the key predictor variables and covariates. Collinearity
diagnosis tests, including Variance Inflation Factors (VIF), Square VIF, Tolerance and R-
squared were conducted for the key predictor variables and covariates. The joint effect
of maternal marital status and type of household cooking fuel on CNS was examined
using six multilevel regression models for each of the outcome variables (stunting, wasting,
and underweight). The first model (Model 0) showed the variance in nutritional status
attributed to the clustering of the primary sampling units (PSUs), without the explanatory
variables. Model I contained only the key predictor variable (maternal marital status-type
of household cooking fuel). Model II and III controlled for the individual and household
level factors, respectively, while Model IV controlled for the contextual level factors. The
final model (Model V) controlled for all the the individual, household, and contextual level
factors. The Stata command “melogit” was used in fitting these models. We used Akaike’s
Information Criterion (AIC) tests for Model comparison. All the results were presented
using adjusted odds ratios (aOR) at 95% Confidence Interval (CI). To prevent potential
challenges of oversampling or under-sampling and clustering of samples emerging from
the multi-stage sampling technique used in the data collection, the weighting, cluster and
strata variables were used to adjust the effect sizes. 2.4. Ethical Approval For DHS reports, ethical clearance are sought from the Ethics Committee of ORC
Macro Inc. as well as Ethics Boards of partner institutions (e.g., Ministries of Health)
of the studied countries. The DHS protocols ensure that standards for the protection of
respondents’ privacy and confidentiality are adhered. Inner City Fund International also
make sure that the survey conforms with the United States Department of Health and
Human Services’ regulations for the respect of human subjects. This study used a secondary
data, hence, no further ethical approval was required. The datasets are freely available for
download in the public domain. Further information about the DHS data usage and ethical
standards is available at http://goo.gl/ny8T6X (accessed on 3 February 2021). Nutrients 2021, 13, 1541 6 of 16 3. Results
3.1. Descriptive Analysis on the Percentage of Children Who Were Stunted, Wasted and
Underweight in the 31 Countries in SSA
The study included 129,646 children under five from 31 sub-Saharan African countries. The percentage of children who were stunted, wasted, and underweight in the 31 countries
in SSA considered was 31%, 8% and 17%, respectively (see Table 1). 3. Results 3.1. Descriptive Analysis on the Percentage of Children Who Were Stunted, Wasted and
Underweight in the 31 Countries in SSA The study included 129,646 children under five from 31 sub-Saharan African countries. The percentage of children who were stunted, wasted, and underweight in the 31 countries
in SSA considered was 31%, 8% and 17%, respectively (see Table 1). The study included 129,646 children under five from 31 sub-Saharan African countries. The percentage of children who were stunted, wasted, and underweight in the 31 countries
in SSA considered was 31%, 8% and 17%, respectively (see Table 1). Table 1. Distribution of study variables from 2010 to 2019 DHS data of 31 sub-Saharan African countries. 2.4. Ethical Approval Variable
Weighted
N
Weighted %
Variable
Weighted
N
Weighted %
Key outcome variables
Household characteristics
Stunting
40,453
31
Wealth status
Wasting
10,770
8
Poor
54,734
42
Underweight
22,503
17
Middle
26,262
20
Rich
48,650
38
Key predictor variable
Age of household head
Maternal marital status
Young-adults
55,803
43
Single
14,071
11
Middle-aged adults
56,748
44
Married
2645
2
Old-aged adults
17,094
13
Type of cooking fuel
102,192
79
Sex of household head
Clean
Unclean
10,725
8
Male
103,254
80
Child characteristics
Female
26,392
20
Age
Access to electricity
0
39,906
31
No
89,364
69
1
36,828
28
Yes
40,272
31
2
26,214
20
Type of toilet facility
3
16,128
12
Improved
55,247
43
4
10,571
8
Unimproved
74,375
57
Sex
Source of drinking water
Male
65,442
50
Improved
85,103
66
Female
64,204
50
Unimproved
44,531
34
Birth order
Type of cooking fuel
1
25,421
20
Unclean
116,263
90
2 to 4
62,426
48
Clean
13,369
10
5 and above
41,799
32
Access to media (tv/radio/newspaper)
Perceived size at birth
No
45,684
35
Large
43,855
34
Yes
83,962
65
Average
64,360
50
Contextual factors
Small
21,422
17
Urbanicity
Weight at birth
Urban
43,412
33
Underweight
7180
6
Rural
86,234
67
Normal
72,089
56
Country
Not taken
50,377
39
Angola
7384
6
Stunting
Benin
15,857
12
Severely/moderately
stunting
40,454
31
Burkina Faso
8908
7
No stunting
89,192
69
Burundi
4174
3
Wasting
Cameroon
2936
2
Severely/moderately wasting
10,770
8
Chad
6114
5
No wasting
118,876
92
Comoros
1523
1
Underweight
Congo
2620
2
Not underweight
107,136.99
83
Cote d’Ivoire
2190
2
Underweight
22,503.33
17
DR Congo
4842
4
Mother’s characteristics
Ethiopia
6585
5
Maternal marital status
Gabon
1998
2
single
16,718
13
Gambia
746
1
Married
112,928
87
Ghana
1908
1
Maternal Age
Guinea
2291
2
15–19
9230
7
Kenya
6082
5
20–49
120,416
93
Lesotho
1018
1
Educational attainment
Liberia
2035
2
No education
54,150
42
Malawi
4035
3
Primary
40,339
31
Mali
5785
4
Secondary
31,071
24
Namibia
1155
1
Higher
4086
3
Nigeria
7502
6 Table 1. Distribution of study variables from 2010 to 2019 DHS data of 31 sub-Saharan African countries. Nutrients 2021, 13, 1541 7 of 16 Table 1. Cont. 2.4. Ethical Approval Variable
Weighted
N
Weighted %
Variable
Weighted
N
Weighted %
Working status
Rwanda
4424
3
No
46,034
36
Senegal
1989
2
Yes
83,505
64
Siera Leone
3052
2
Antenatal visits during
pregnancy
South Africa
877
1
No
13,711
11
Tanzania
6088
5
Yes
113,765
88
Togo
2227
2
Dont’ know
2153
2
Uganda
2900
2
Postnatal check within 2
months
Zambia
6402
5
No
75,893
59
Zimbabwe
4000
3
Yes
53,753
41
Geographic region
Place of delivery
Western Africa
54,489
42
Home
40,300
31
Eastern Africa
42,213
33
Health facility
87,846
68
Central Africa
25,894
20
Other
1494
1
South Africa
7051
5
Total
129,646 Table 1. Cont. The prevalence of stunting varied across countries and sub-regions, with the highest
prevalence of stunting found in Burundi (44.5%–51.7%), while the lowest prevalence
was found in Congo, Gabon, Namibia, and Ghana (15.4%–22.7%) (see Figure 2A). In
terms of sub-region, the highest prevalence of wasting was observed in Eastern Africa
(34.6%) (see Figure 3A). The prevalence of wasting also varied across countries and sub-
regions. The highest prevalence of wasting was found in Burundi, Burkina Faso and
Chad (15.7%–19.1%), whereas the lowest prevalence was seen in South Africa, Lesotho,
Zimbabwe, Zambia, Malawi, Kenya, Uganda, Rwanda, Gabon, Congo, and Cameroon
(1.6%–5.1%) (see Figure 2B). Across sub-regions, wasting was more predominant in Western
Africa (9.6%) (see Figure 2B). The highest prevalence of underweight was found in Burundi,
Burkina Faso and Chad (24.0%–28.8%) (see Figure 2C) while the lowest prevalence was
noted in South Africa, Lesotho, Zimbabwe, Kenya, and Gabon (4.8%–9.6%) (see Figure 2C). Central/Middle Africa had the highest prevalence of underweight children across
sub-regions (18.74%) (see Figure 3C). Central/Middle Africa had the highest prevalence of underweight children acro
sub-regions (18.74%) (see Figure 3C). In terms of the association between the key predictor variables, covariates and stunting,
wasting, and underweight, we found significant associations between all the key predictor
variables, covariates and stunting, except thematernal marital status and maternal age. Apart from age of the household head, all the independent variables had significant
associations with wasting. With underweight, all the independent variables had significant
associations, except the maternal age(see Table 2). 8 of 16
8 of 17 Nutrients 2021, 13, 1541
Nutrients 2021, 13, x FOR Figure 2 Maps showing the prevalence of stunting, wasting and underweight from 2010 to 2019 DHS across the 31 Sub-
Saharan African Countries. Figure 2. 2.4. Ethical Approval Maps showing the prevalence of stunting, wasting and underweight from 2010 to 2019 DHS across the 31 Sub-
Saharan African Countries. (A) Stunting (%) (B) Wasting (%) (C) Underweight (%). Figure 2 Maps showing the prevalence of stunting, wasting and underweight from 2010 to 2019 DHS across the 31 Sub-
Saharan African Countries. Figure 2 Maps showing the prevalence of stunting, wasting and underweight from 2010 to 2019 DHS across the 31 Sub-
Saharan African Countries. Figure 2. Maps showing the prevalence of stunting, wasting and underweight from 2010 to 2019 DHS across the 31 Sub-
Saharan African Countries. (A) Stunting (%) (B) Wasting (%) (C) Underweight (%). Figure 3. Maps showing the prevalence of stunting, wasting and undernutrition from 2010 to 2019 DHS across the 31
Sub-Saharan African Countries. (A) Stunting (%) (B) Wasting (%) (C) Underweight (%). Figure 3. Maps showing the prevalence of stunting, wasting and undernutrition from 2010 to 2019 DHS across the 31
Sub-Saharan African Countries. (A) Stunting (%) (B) Wasting (%) (C) Underweight (%). 9 of 16 Nutrients 2021, 13, 1541 Table 2. Association between stunting, wasting and underweight and characteristics of a child, mother and household, and
contextual factors from 2010 to 2019 DHS across the 31 Sub-Saharan African Countries. 2.4. Ethical Approval Independent Variables
Stunting
(Weighted %)
95% CI
p-Value
Wasting
(Weighted %)
95% CI
p-Value
Underweight
(Weighted %)
95% CI
p-Value
Key predictor
variables
Maternal marital
status
0.382
<0.001
<0.001
Single
31.3
30.5–32.0
6.4
6.0–6.7
15.5
15.0–16.1
Married
31.2
30.9–31.5
8.6
8.4–8.8
17.6
17.4–17.9
Type of cooking fuel
<0.001
<0.001
<0.001
Unclean
32.6
32.4–32.9
8.7
8.5–8.9
18.3
18.1–18.5
Clean
18.8
18.1–18.4
5.0
4.6–5.3
9
8.5–9.5
Child characteristics
Age of child
<0.001
<0.001
<0.001
0
16.8
16.4–17.2
11.1
10.8–11.4
13.56
13.2–13.0
1
36.3
35.8–36.8
9.7
9.4–10.0
19.97
19.6–20.4
2
43.5
42.9–44.1
6.2
5.9–6.5
20.59
20.1–21.1
3
37.2
36.438.0
4.3
4.0–4.6
16.84
16.3–17.4
4
28.2
27.3–29.0
4.4
4.0–4.8
15.35
14.7–16.1
Sex of child
<0.001
<0.001
<0.001
Male
34.0
33.6–34.4
9.2
8.9–9.4
18.84
18.5–19.1
Female
28.3
28.0–28.7
7.5
7.2–7.7
15.85
15.6–16.1
Birth order
<0.001
<0.001
<0.001
1
29.5
28.9–30.0
7.5
7.2–7.8
15.23
14.8–15.7
2 to 4
30.0
29.4–30.1
7.9
7.7–8.2
16.15
15.9–16.4
5 and above
34.4
33.9–34.8
9.3
9.1–9.6
20.45
20.0–20.8
Perceived size at birth
<0.001
<0.001
<0.001
Large
26.7
26.3–27.1
6.4
6.2–6.6
12.93
12.6–13.2
Average
31.4
31.0–31.8
8.3
8.1–8.5
17.02
16.7–17.3
Small
39.8
39.1–40.4
12.2
11.7–12.6
27.45
26.9–28.0
Mother’s
characteristics
Maternal age
0.604
<0.001
0.189
15–19
31.0
30.0–31.9
9.6
9.0–10.2
17.68
16.9–18.5
20–49
31.2
31.0–31.5
8.2
8.1–8.4
17.33
17.1–17.5
Educational
attainment
<0.001
<0.001
<0.001
No education
36.6
36.2–37.0
11.5
11.3–11.8
24.02
23.7–24.3
Primary
32.6
32.1–33.0
6.4
6.2–6.7
15.1
14.8–15.5
Secondary
22.6
22.2–23.1
5.6
5.3–5.9
10.19
9.9–10.5
Higher
10.9
10.0–11.9
4.6
4.0–5.3
5.81
5.1–6.6
Working status
0.017
<0.001
<0.001
No
30.8
30.3–31.2
9.6
9.4–9.9
18.16
17.8–18.5
Yes
31.4
31.1–31.8
7.6
7.4–7.7
16.91
16.7–17.3
Antenatal visits
during pregnancy
<0.001
<0.001
<0.001
No
41.7
41.0–42.6
12.5
12.0–13.1
28.42
27.7–29.2
Yes
30.0
29.8–30.3
7.8
7.7–8.0
16.1
15.9–16.3
Dont’know
26.3
24.4–28.2
7.6
6.5–8.8
13.07
11.7–14.6
Postnatal check within
2 months
<0.001
0.748
<0.001
No
33.0
32.6–33.3
8.3
8.1–8.5
18.54
18.3–18.8
Yes
28.7
28.3–29.1
8.4
8.1–8.6
15.69
15.4–16.0
Place of delivery
<0.001
<0.001
<0.001
Home
38.5
38.1–39.0
11.2
11.0–11.5
24.9
24.5–25.3
Health facility
27.8
27.5–28.1
7.0
6.8–7.1
13.87
13.6–14.1
Other
34.8
32.4–37.3
7.5
6.2–9.0
19.26
17.3–21.4
Household
characteristics
Wealth status
<0.001
<0.001
<0.001
Poor
37.2
36.8–37.6
9.4
9.1–9.6
21.28
20.9–21.6
Middle
32.6
32.0–33.2
8.2
7.9–8.6
17.8
17.3–18.3
Rich
23.7
23.4–24.1
7.2
6.9–7.4
12.71
12.4–13.0
Age of household
head
0.680
0.791
<0.001
Young-adults
31.5
31.1–32.0
8.2
7.9–8.4
16.8
16.5–17.1
Middle-aged adults
30.9
30.5–31.3
8.5
8.2–8.7
17.83
17.5–18.1
Old-aged adults
31.1
30.4–31.8
8.3
7.9–8.7
17.61
17.0–18.2
Sex of household head
<0.05
<0.001
<0.001
Male
31.4
31.1–31.7
8.7
8.5–8.9
17.78
17.5–18.0
Female
30.5
30.0–31.0
6.9
6.6–7.3
15.72
15.3–16.2
Access to electricity
<0.001
<0.001
<0.001
No
35.3
35.0–35.6
9.2
9.0–9.4
20.01
19.7–20.3
Yes
22.1
21.7–22.5
6.3
6.0–6.5
11.47
11.1–11.8
Type of toilet facility
<0.001
<0.001
<0.001
Improved
26.8
26.4–27.2
6.4
6.2–6.6
13.07
12.8–13.3
Unimproved
34.5
34.1–34.8
9.7
9.5–9.9
20.54
20.3–20.8
Source of drinking
water
<0.001
<0.05
<0.001
Improved
30.1
29.8–30.4
8.2
8.0–8.4
16.64
16.4–16.9
Unimproved
33.4
32.9–33.8
8.5
8.3–8.8
18.72
18.4–19.1 Table 2. 2.4. Ethical Approval With underweight, we found that compared to children born to married mothers
who use clean household cooking fuel, children born to single mothers who use unclean
household cooking fuel (aOR = 1.41; 95% CI = 1.28–1.55), children born to single women
who use clean household cooking fuel (aOR = 1.33; 95% CI = 1.14–1.55) and children born
to married women who use unclean household cooking (aOR = 1.33; 95% CI = 1.22–1.45)
were more likely to be underweight (Table 3, Model V). Table 3. Multilevel logistic regression results on joint effect of maternal marital status and type of household cooking fuel
on childhood stunting from 2010 to 2019 DHS across the 31 Sub-Saharan African Countries. Key Predictor Variable
Model 0
Model I
Model II
Model III
Model IV
Model V
OR (95% CI)
aOR (95% CI)
aOR (95% CI)
aOR (95% CI)
aOR (95% CI)
Fixed effects
Maternal marital
status-Cooking fuel
Married-clean
1
1
1
1
1
Single unclean
2.22 ***
(2.08–2.36)
1.65 ***
(1.54–1.77)
1.49 ***
(1.39–1.60)
1.83 ***
(1.71–1.95)
1.27 ***
(1.17–1.47)
1 25 ***
1 21 ***
1 22 ***
1 23 *** Table 2. Cont. Independent Variables
Stunting
(Weighted %)
95% CI
p-Value
Wasting
(Weighted %)
95% CI
p-Value
Underweight
(Weighted %)
95% CI
p-Value
Access to media
(tv/radio/newspaper)
<0.001
<0.001
<0.001
No
37.9
37.4–38.3
10.0
9.7–10.2
22.43
22.0–22.8
Yes
27.6
27.3–27.9
7.4
7.2–7.6
14.6
14.4–14.8
Contextual factors
Urbanicity
<0.001
<0.001
<0.001
Urban
23.6
23.2–24.0
6.6
6.3–6.8
12.12
11.8–12.4
Rural
35.0
34.7–35.4
9.2
9.0–9.4
20
19.7–20.3
Geographic region
<0.001
<0.001
<0.001
Western Africa
29.0
28.6–29.4
9.6
9.4–9.8
18.55
18.2–18.9
Eastern Africa
34.6
34.1–35.0
7.1
6.9–7.4
16.44
16.1–16.8
Central Africa
31.8
31.3–32.4
8.6
8.3–8.9
18.74
18.3–19.2
South Africa
25.8
24.7–26.8
4.4
3.9–4.9
8.59
8.0–9.3
3.2. Multivariate Analysis on the Joint Effect of Single-Motherhood and Unclean Household Table 2. Cont. 3.2. Multivariate Analysis on the Joint Effect of Single-Motherhood and Unclean Household
Cooking Fuel Use on Child Nutritional Status Tables 3–5 show the results from the multilevel logistic regression analysis on the
joint effect of maternal marital status and household cooking fuel type on CNS. The last
models (Model V) of each table indicate the joint effect of maternal marital status and
household cooking fuel type on CNS, while controlling for individual level factors, house-
hold characteristics, and contextual factors. 2.4. Ethical Approval Association between stunting, wasting and underweight and characteristics of a child, mother and household, and
contextual factors from 2010 to 2019 DHS across the 31 Sub-Saharan African Countries. Nutrients 2021, 13, 1541 10 of 16 Table 2. Cont. Independent Variables
Stunting
(Weighted %)
95% CI
p-Value
Wasting
(Weighted %)
95% CI
p-Value
Underweight
(Weighted %)
95% CI
p-Value
Access to media
(tv/radio/newspaper)
<0.001
<0.001
<0.001
No
37.9
37.4–38.3
10.0
9.7–10.2
22.43
22.0–22.8
Yes
27.6
27.3–27.9
7.4
7.2–7.6
14.6
14.4–14.8
Contextual factors
Urbanicity
<0.001
<0.001
<0.001
Urban
23.6
23.2–24.0
6.6
6.3–6.8
12.12
11.8–12.4
Rural
35.0
34.7–35.4
9.2
9.0–9.4
20
19.7–20.3
Geographic region
<0.001
<0.001
<0.001
Western Africa
29.0
28.6–29.4
9.6
9.4–9.8
18.55
18.2–18.9
Eastern Africa
34.6
34.1–35.0
7.1
6.9–7.4
16.44
16.1–16.8
Central Africa
31.8
31.3–32.4
8.6
8.3–8.9
18.74
18.3–19.2
South Africa
25.8
24.7–26.8
4.4
3.9–4.9
8.59
8.0–9.3
3.2. Multivariate Analysis on the Joint Effect of Single-Motherhood and Unclean Household
Cooking Fuel Use on Child Nutritional Status
Tables 3–5 show the results from the multilevel logistic regression analysis on the
joint effect of maternal marital status and household cooking fuel type on CNS. The last
models (Model V) of each table indicate the joint effect of maternal marital status and
household cooking fuel type on CNS, while controlling for individual level factors, house-
hold characteristics, and contextual factors. On the joint effect of maternal marital status
and type of household cooking fuel on stunting, we found that compared to children born
to married mothers who use clean household cooking fuel, children born to single mothers
who use unclean household cooking fuel (aOR = 1.27; 95% CI = 1.17–1.47), children born
to single women who use clean household cooking fuel (aOR = 1.18; 95% CI = 1.05–1.32)
and children born to married women who use unclean household cooking (aOR = 1.25;
95% CI = 1.17–1.33) were more likely to be stunted (Table 3, Model V). With wasting,
children born to single mothers who used unclean household cooking fuel (aOR = 1.17;
95% CI = 1.03–1.33) and children born to married women who use unclean household
cooking fuel (aOR = 1.24; 95% CI = 1.11–1.39) were more likely to be wasted compared
to children born to married mothers who used clean household cooking fuel (Table 4,
Model V). 2.4. Ethical Approval On the joint effect of maternal marital status
and type of household cooking fuel on stunting, we found that compared to children born
to married mothers who use clean household cooking fuel, children born to single mothers
who use unclean household cooking fuel (aOR = 1.27; 95% CI = 1.17–1.47), children born
to single women who use clean household cooking fuel (aOR = 1.18; 95% CI = 1.05–1.32)
and children born to married women who use unclean household cooking (aOR = 1.25;
95% CI = 1.17–1.33) were more likely to be stunted (Table 3, Model V). With wasting,
children born to single mothers who used unclean household cooking fuel (aOR = 1.17;
95% CI = 1.03–1.33) and children born to married women who use unclean household
cooking fuel (aOR = 1.24; 95% CI = 1.11–1.39) were more likely to be wasted compared
to children born to married mothers who used clean household cooking fuel (Table 4,
Model V). With underweight, we found that compared to children born to married mothers
who use clean household cooking fuel, children born to single mothers who use unclean
household cooking fuel (aOR = 1.41; 95% CI = 1.28–1.55), children born to single women
who use clean household cooking fuel (aOR = 1.33; 95% CI = 1.14–1.55) and children born
to married women who use unclean household cooking (aOR = 1.33; 95% CI = 1.22–1.45)
were more likely to be underweight (Table 3, Model V). Table 3. Multilevel logistic regression results on joint effect of maternal marital status and type of household cooking fuel
on childhood stunting from 2010 to 2019 DHS across the 31 Sub-Saharan African Countries. Key Predictor Variable
Model 0
Model I
Model II
Model III
Model IV
Model V
OR (95% CI)
aOR (95% CI)
aOR (95% CI)
aOR (95% CI)
aOR (95% CI)
Fixed effects
Maternal marital
status-Cooking fuel
Married-clean
1
1
1
1
1
Single unclean
2.22 ***
(2.08–2.36)
1.65 ***
(1.54–1.77)
1.49 ***
(1.39–1.60)
1.83 ***
(1.71–1.95)
1.27 ***
(1.17–1.47)
Single clean
1.25 ***
(1.12–1.39)
1.21 ***
(1.08–1.36)
1.22 ***
(1.09–1.36)
1.23 ***
(1.10–1.37)
1.18 * (1.05–1.32)
Married-unclean
2.25 ***
(2.03–2.26)
1.49 ***
(1.41–1.59)
0.04 ***
(1.32–1.48)
1.76 ***
(1.66–1.87)
1.25 ***
(1.17–1.33) Table 3. Multilevel logistic regression results on joint effect of maternal marital status and type of househ
on childhood stunting from 2010 to 2019 DHS across the 31 Sub-Saharan African Countries. 2.4. Ethical Approval * p < 0.05; *** p < 0.001; Model 0: Empty model with no independent variables; Model I: Joint effect of maternal marital status and household
cooking fuel type on stunting; Model II: Included individual level characteristics (age of child in years, sex of child, birth order and
perceived size of child at birth, maternal age, educational attainment, working status, antenatal visits during pregnancy, postnatal check
within 2 months and place of delivery) as covariates; Model III: Included household characteristics (wealth status, age of household head,
sex of household head, access to electricity, type of toilet facility, source of drinking water and access to media) as covariates; Model IV:
Included contextual factors (urbanicity and geographic region) as covariates; Model V: Included individual, household and contextual level
characteristics as covariates Exponentiated coefficients; 95% confidence intervals in brackets; cOR: crude odds ratios; aOR adjusted odds
ratios; CI Confidence Interval; 1 = Reference category; PSU = Primary Sampling Unit; ICC = Intra-Class Correlation; LR Test = Likelihood
ratio Test; AIC = Akaike’s Information Criterion. ratios; CI Confidence Interval; 1 = Reference category; PSU = Primary Sampling Unit; ICC = Intra-Class Correlation; LR Test = Likelihood
ratio Test; AIC = Akaike’s Information Criterion. Table 4. Multilevel logistic regression results on joint effect of maternal marital status and type of household cooking fuel
on childhood wasting from 2010 to 2019 DHS across the 31 Sub-Saharan African Countries. Key Predictor Variable
Model 0
Model I
Model II
Model III
Model IV
Model V
OR (95% CI)
aOR (95% CI)
aOR (95% CI)
aOR (95% CI)
aOR (95% CI)
Fixed effects ratio Test; AIC = Akaike’s Information Criterion. Table 4. Multilevel logistic regression results on joint effect of maternal marital status and type of household cooking fuel
on childhood wasting from 2010 to 2019 DHS across the 31 Sub-Saharan African Countries. Table 4. Multilevel logistic regression results on joint effect of maternal marital status and type of household cooking fuel
on childhood wasting from 2010 to 2019 DHS across the 31 Sub-Saharan African Countries. Table 4. Multilevel logistic regression results on joint effect of maternal marital status and type of household cooking fuel
on childhood wasting from 2010 to 2019 DHS across the 31 Sub-Saharan African Countries. Table 4. 2.4. Ethical Approval Key Predictor Variable
Model 0
Model I
Model II
Model III
Model IV
Model V
OR (95% CI)
aOR (95% CI)
aOR (95% CI)
aOR (95% CI)
aOR (95% CI)
Fixed effects
Maternal marital
status-Cooking fuel
Married-clean
1
1
1
1
1
Single unclean
1.46 ***
(1.30–1.63)
1.23 **
(1.09–1.39)
1.12 (0.99–1.27)
1.32 ***
(1.17–1.49)
1.17 * (1.03–1.33)
Single clean
0.92 (0.74–1.14)
0.91 (0.73–1.13)
0.94 (0.75–1.17)
0.97 (0.78–1.20)
0.97 (0.78–1.21)
Married-unclean
1.98 ***
(1.80–2.19)
1.35 ***
(1.22–1.50)
1.45 ***
(1.30–1.61)
1.69 ***
(1.54–1.88)
1.24 ***
(1.11–1.39)
Random effects
PSU Variance (95% CI)
0.04 (0.03–0.06)
0.4 (0.03–0.06)
0.04 (0.03–0.05)
0.04 (0.03–0.05)
0.04 (0.03–0.5)
0.04 (0.03–0.5)
ICC
0.012
0.013
0.012
0.012
0.013
0.012
LR Test
84.12 ***
86.79 ***
78.58 ***
82.1 ***
86.54 ***
77.54 ***
Wild χ2
Reference
302.86 ***
3430.13 ***
813.45 ***
633.06 ***
3623.65 ***
Model fitness
Log-likelihood
−37,598.7
−37,423.4
−35,695.6
−37,145.5
−37,243.6
−35,562.1
AIC
75,201.42
74,856.82
71,439.27
74,319.03
74,505.22
71,198.25
Number of clusters
1608
1608
1608
1608
1608
1608
* p < 0.05; ** p < 0.01; *** p < 0.001; Model 0: Empty model with no independent variables; Model I: Joint effect of maternal marital status and
household cooking fuel type on wasting; Model II: Included individual level characteristics (age of child in years, sex of child, birth order
and perceived size of child at birth, maternal age, educational attainment, working status, antenatal visits during pregnancy, postnatal
check within 2 months and place of delivery) as covariates; Model III: Included household characteristics (wealth status, age of household
head, sex of household head, access to electricity, type of toilet facility, source of drinking water and access to media) as covariates; Model IV:
Included contextual factors (urbanicity and geographic region) as covariates; Model V: Included individual, household and contextual level
h
t
i ti
i t
E
ti t d
ffii
t
95%
fid
i t
l i
b
k t
OR
d
dd
ti
OR dj
t d dd Table 3. Cont. 2.4. Ethical Approval vel logistic regression results on joint effect of maternal marital status and type of household cooking fuel
unting from 2010 to 2019 DHS across the 31 Sub-Saharan African Countries. Nutrients 2021, 13, 1541 11 of 16 Table 3. Cont. Key Predictor Variable
Model 0
Model I
Model II
Model III
Model IV
Model V
OR (95% CI)
aOR (95% CI)
aOR (95% CI)
aOR (95% CI)
aOR (95% CI)
Random effects
PSU Variance (95% CI)
0.02 (0.18–0.03)
0.02 (0.02–0.3)
0.02 (0.02–0.03)
0.02 (0.01–0.03)
0.02 (0.01–0.03)
0.02 (0.01–0.03)
ICC
0.007
0.007
0.006
0.006
0.006
0.006
LR Test
161.74 ***
141.62 ***
111.82 ***
118.11 ***
121.89 ***
105.02 ***
Wild χ2
Reference
864.24 ***
10,324.15 ***
2958.64 ***
2061.47 ***
11,264.12 ***
Model fitness
Log-likelihood
−81,022.8
−80,532.1
−74,861.3
−79,409.4
−79,911.5
−74,213.7
AIC
162,049.6
161,074.3
149,770.6
158,846.9
159,841
148,501.4
Number of clusters
1608
1608
1608
1608
1608
1608
* p < 0.05; *** p < 0.001; Model 0: Empty model with no independent variables; Model I: Joint effect of maternal marital status and household
cooking fuel type on stunting; Model II: Included individual level characteristics (age of child in years, sex of child, birth order and
perceived size of child at birth, maternal age, educational attainment, working status, antenatal visits during pregnancy, postnatal check
within 2 months and place of delivery) as covariates; Model III: Included household characteristics (wealth status, age of household head,
sex of household head, access to electricity, type of toilet facility, source of drinking water and access to media) as covariates; Model IV:
Included contextual factors (urbanicity and geographic region) as covariates; Model V: Included individual, household and contextual level
characteristics as covariates Exponentiated coefficients; 95% confidence intervals in brackets; cOR: crude odds ratios; aOR adjusted odds
ratios; CI Confidence Interval; 1 = Reference category; PSU = Primary Sampling Unit; ICC = Intra-Class Correlation; LR Test = Likelihood
ratio Test; AIC = Akaike’s Information Criterion. Table 3. Cont. 2.4. Ethical Approval Key Predictor Variable
Model 0
Model I
Model II
Model III
Model IV
Model V
OR (95% CI)
aOR (95% CI)
aOR (95% CI)
aOR (95% CI)
aOR (95% CI)
Random effects
PSU Variance (95% CI)
0.02 (0.18–0.03)
0.02 (0.02–0.3)
0.02 (0.02–0.03)
0.02 (0.01–0.03)
0.02 (0.01–0.03)
0.02 (0.01–0.03)
ICC
0.007
0.007
0.006
0.006
0.006
0.006
LR Test
161.74 ***
141.62 ***
111.82 ***
118.11 ***
121.89 ***
105.02 ***
Wild χ2
Reference
864.24 ***
10,324.15 ***
2958.64 ***
2061.47 ***
11,264.12 ***
Model fitness
Log-likelihood
−81,022.8
−80,532.1
−74,861.3
−79,409.4
−79,911.5
−74,213.7
AIC
162,049.6
161,074.3
149,770.6
158,846.9
159,841
148,501.4
Number of clusters
1608
1608
1608
1608
1608
1608
* p < 0.05; *** p < 0.001; Model 0: Empty model with no independent variables; Model I: Joint effect of maternal marital status and household
cooking fuel type on stunting; Model II: Included individual level characteristics (age of child in years, sex of child, birth order and
perceived size of child at birth, maternal age, educational attainment, working status, antenatal visits during pregnancy, postnatal check
within 2 months and place of delivery) as covariates; Model III: Included household characteristics (wealth status, age of household head,
sex of household head, access to electricity, type of toilet facility, source of drinking water and access to media) as covariates; Model IV:
Included contextual factors (urbanicity and geographic region) as covariates; Model V: Included individual, household and contextual level
characteristics as covariates Exponentiated coefficients; 95% confidence intervals in brackets; cOR: crude odds ratios; aOR adjusted odds
ratios; CI Confidence Interval; 1 = Reference category; PSU = Primary Sampling Unit; ICC = Intra-Class Correlation; LR Test = Likelihood
ratio Test; AIC = Akaike’s Information Criterion. Table 4. Multilevel logistic regression results on joint effect of maternal marital status and type of household cooking fuel
on childhood wasting from 2010 to 2019 DHS across the 31 Sub-Saharan African Countries. 2.4. Ethical Approval Multilevel logistic regression results on joint effect of maternal marital status and type of household cooking fuel
on childhood wasting from 2010 to 2019 DHS across the 31 Sub-Saharan African Countries. Key Predictor Variable
Model 0
Model I
Model II
Model III
Model IV
Model V
OR (95% CI)
aOR (95% CI)
aOR (95% CI)
aOR (95% CI)
aOR (95% CI)
Fixed effects
Maternal marital
status-Cooking fuel
Married-clean
1
1
1
1
1
Single unclean
1.46 ***
(1.30–1.63)
1.23 **
(1.09–1.39)
1.12 (0.99–1.27)
1.32 ***
(1.17–1.49)
1.17 * (1.03–1.33)
Single clean
0.92 (0.74–1.14)
0.91 (0.73–1.13)
0.94 (0.75–1.17)
0.97 (0.78–1.20)
0.97 (0.78–1.21)
Married-unclean
1.98 ***
(1.80–2.19)
1.35 ***
(1.22–1.50)
1.45 ***
(1.30–1.61)
1.69 ***
(1.54–1.88)
1.24 ***
(1.11–1.39)
Random effects
PSU Variance (95% CI)
0.04 (0.03–0.06)
0.4 (0.03–0.06)
0.04 (0.03–0.05)
0.04 (0.03–0.05)
0.04 (0.03–0.5)
0.04 (0.03–0.5)
ICC
0.012
0.013
0.012
0.012
0.013
0.012
LR Test
84.12 ***
86.79 ***
78.58 ***
82.1 ***
86.54 ***
77.54 ***
Wild χ2
Reference
302.86 ***
3430.13 ***
813.45 ***
633.06 ***
3623.65 ***
Model fitness
Log-likelihood
−37,598.7
−37,423.4
−35,695.6
−37,145.5
−37,243.6
−35,562.1
AIC
75,201.42
74,856.82
71,439.27
74,319.03
74,505.22
71,198.25
Number of clusters
1608
1608
1608
1608
1608
1608
* p < 0.05; ** p < 0.01; *** p < 0.001; Model 0: Empty model with no independent variables; Model I: Joint effect of maternal marital status and
household cooking fuel type on wasting; Model II: Included individual level characteristics (age of child in years, sex of child, birth order
and perceived size of child at birth, maternal age, educational attainment, working status, antenatal visits during pregnancy, postnatal
check within 2 months and place of delivery) as covariates; Model III: Included household characteristics (wealth status, age of household
head, sex of household head, access to electricity, type of toilet facility, source of drinking water and access to media) as covariates; Model IV:
Included contextual factors (urbanicity and geographic region) as covariates; Model V: Included individual, household and contextual level
characteristics as covariates; Exponentiated coefficients; 95% confidence intervals in brackets; cOR: crude odds ratios; aOR adjusted odds
ratios; CI Confidence Interval; 1 = Reference category; PSU = Primary Sampling Unit; ICC = Intra-Class Correlation; LR Test = Likelihood
ratio Test; AIC = Akaike’s Information Criterion. 2.4. Ethical Approval * p < 0.05; ** p < 0.01; *** p < 0.001; Model 0: Empty model with no independent variables; Model I: Joint effect of maternal marital status and
household cooking fuel type on wasting; Model II: Included individual level characteristics (age of child in years, sex of child, birth order
and perceived size of child at birth, maternal age, educational attainment, working status, antenatal visits during pregnancy, postnatal
check within 2 months and place of delivery) as covariates; Model III: Included household characteristics (wealth status, age of household
head, sex of household head, access to electricity, type of toilet facility, source of drinking water and access to media) as covariates; Model IV:
Included contextual factors (urbanicity and geographic region) as covariates; Model V: Included individual, household and contextual level
characteristics as covariates; Exponentiated coefficients; 95% confidence intervals in brackets; cOR: crude odds ratios; aOR adjusted odds
ratios; CI Confidence Interval; 1 = Reference category; PSU = Primary Sampling Unit; ICC = Intra-Class Correlation; LR Test = Likelihood
ratio Test; AIC = Akaike’s Information Criterion. 12 of 16 12 of 16 Nutrients 2021, 13, 1541 Table 5. Multilevel logistic regression results on joint effect of maternal marital status and type of household cooking fuel
on childhood underweight from 2010 to 2019 DHS across the 31 Sub-Saharan African Countries. 2.4. Ethical Approval Key Predictor Variable
Model 0
Model I
Model II
Model III
Model IV
Model V
OR (95% CI)
aOR (95% CI)
aOR (95% CI)
aOR (95% CI)
aOR (95% CI)
Fixed effects
Marital
status-Cooking fuel
Married-clean
1
1
1
1
1
Single unclean
2.26 ***
(2.08–2.47)
1.62 ***
(1.48–1.77)
1.46 ***
(1.32–1.60)
1.87 ***
(1.71–2.04)
1.41 ***
(1.28–1.55)
Single clean
1.21 ***
(1.13–1.52)
1.28 * (1.10–1.50)
1.29 * (1.11–1.50)
1.38 ***
(1.19–1.61)
1.33 ***
(1.14–1.55)
Married-unclean
2.60 ***
(2.41–2.80)
1.56 ***
(1.44–1.69)
1.61 ***
(1.48–1.74)
2.01 ***
(1.86–2.18)
1.33 ***
(1.22–1.45)
Random effects
PSU Variance (95% CI)
0.03 (0.02–0.04)
0.03 (0.02–0.04)
0.02 (0.02–0.03)
0.03 (0.02–0.03)
0.03 (0.02–0.04)
0.02 (0.02–0.03)
ICC
0.009
0.009
0.007
0.008
0.008
0.007
LR Test
124.1 ***
118.46 ***
76.82 ***
101.51 ***
115.42 ***
80.61 ***
Wild χ2
Reference
707.76 ***
6725.19 ***
2423.9 ***
1821.05 ***
7154.32 ***
Model fitness
Log-likelihood
−60,841.6
−60,404.4
−57,078.2
−59,474.5
−59,780.2
−56,749.8
AIC
121,687.2
120,818.7
114,204.4
118,977
119,578.4
113,573.6
Number of clusters
1608
1608
1608
1608
1608
1608
* p < 0.05; *** p < 0.001; Model 0: Empty model with no independent variables; Model I: Joint effect of maternal marital status and household
cooking fuel type on underweight; Model II: Included individual level characteristics (age of child in years, sex of child, birth order and
perceived size of child at birth, maternal age, educational attainment, working status, antenatal visits during pregnancy, postnatal check
within 2 months and place of delivery) as covariates; Model III: Included household characteristics (wealth status, age of household head,
sex of household head, access to electricity, type of toilet facility, source of drinking water and access to media) as covariates; Model IV:
Included contextual factors (urbanicity and geographic region) as covariates; Model V: Included individual, household and contextual level
characteristics as covariates; Exponentiated coefficients; 95% confidence intervals in brackets; cOR: crude odds ratios; aOR adjusted odds
ratios; CI Confidence Interval; 1 = Reference category; PSU = Primary Sampling Unit; ICC = Intra-Class Correlation; LR Test = Likelihood
ratio Test; AIC = Akaike’s Information Criterion. Table 5. Multilevel logistic regression results on joint effect of maternal marital status and type of household cooking fuel
on childhood underweight from 2010 to 2019 DHS across the 31 Sub-Saharan African Countries. 4. Discussion Improving the health status of children has become an important global health is-
sue. International organisations and individual countries have concentrated efforts and
strategies to improve child nutritional status [1]. Yet, there are still a substantial proportion
of children who are stunted, underweight or wasted [3]. Therefore, this study aimed to
examine the joint effect of maternal marital status and household cooking fuel type on CNS
in SSA. The results indicated that there were some significant inter-country variations con-
cerning the nutritional status of children. Consistently, our findings showed that Burundi
had the worst children’s nutritional status (stunting, wasting, and underweight). This
indicates that the indicators of child nutritional status in Burundi were poorer than those of
other countries in SSA. Previous studies have found a high prevalence of stunting, wasting,
and underweight among children in Burundi [24–26]. Besides Burundi, the prevalence of
wasting and underweight was highest in Burkina Faso and Chad. This finding is in line
with previous evidence that shows that wasting and underweight are most prevalent in
Burkina Faso [27] and Chad [28]. We found that the prevalence of stunting is lowest in Ghana. This finding is consistent
with previous outcomes in the country [6]. The identified pattern could be explained by
the various strategies adopted by the government of Ghana to alleviate poverty and ensure
food security for households to meet the children’s nutritional requirements. Notable Nutrients 2021, 13, 1541 13 of 16 13 of 16 among these interventions are the capitation grant and the school feeding programme, as
well as the Livelihoods Empowerment Against Poverty Programme (LEAP) [29]. Concerning the joint effect of maternal marital status and type of household cooking
fuel use on stunting, we found that compared to children born to married mothers who use
clean household cooking fuel, children born to single mothers who use unclean household
cooking fuel, single mothers who use clean household cooking fuel, and married mothers
who use unclean household cooking fuel were more likely to be stunted. This finding
corroborates recent studies (e.g., [30]) that the likelihood of childhood stunting among
children living in households using unclean cooking fuel was significantly higher than
those living in households using clean cooking fuel. For example, Balietti and [31] found
increased odds of stunting among children whose household used unclean cooking fuel. 4. Discussion Unclean cooking fuel coupled with poor ventilation increases household air pollution
(HAP), which exacerbates the likelihood of stunting among children [32]. These result in
respiratory infections that lead to the activation of the immune system to fight off disease-
causing agents and consume metabolic energy, which will no longer be available for other
functions of the metabolism. Child growth can be impaired, leading to stunting [33]. The
association between use of unclean cooking fuel and stunting could also be explained from
the perspective that, during pregnancy, mothers who used biofuel in the form of wood and
dung have a higher risk of delivering small-for-gestational-age infants, with this outcome
persisting during the growth of the child, and later causing stunting [34]. As found in this
study, the use of unclean household cooking fuel influences stunting, together with single
motherhood. Hence, children borrn to single mothers are also more likely to be stunted,
irrespective of the type of household cooking fuel used by their mothers. The findings
on the association between single motherhood and stunting is supported by previous
studies [35–37]. Other findings also showed that children born to single mothers who used unclean
household cooking fuel, single mothers who use clean household cooking fuel and married
mothers who use unclean household cooking fuel were more likely to be underweight
compared to children born to married mothers who used clean household cooking fuel. Thus, being a child born to a single mother who uses unclean household cooking fuel, a
married mother who use unclean household cooking fuel and single mother who uses
a clean household cooking fuel increases your likelihood of being underweight [12–14]. Generally, single mothers face substantial socio-economic stress and hardship in many
parts of SSA [38]. These challenges limit the capacity of single mothers to provide quality
child care for their children compared to married women [39]. As such, single mothers
usually find it difficult to juggle between work and providing the nutritional needs of
their children, hence, the higher likelihood of underweight among children born to single
mothered households. The result is further supported by [40] which shows that the use of
clean cooking fuel significantly reduces childhood underweight and mortality by 14–31%. 4. Discussion g
g
y
g
y
y
We also found that, compared to children born to married mothers who use clean
household cooking fuel, those born to single and married mothers who use unclean
household cooking fuel are more likely to be wasted. Hence, the use of unclean household
fuel has significant effect on childhood stunting, irrespective of marital status. Similar
finfings are evidenced in prior studies [12,41]. The effect of unclean cooking fuel and
wasting could be attributed to the higher likelihood of delivering small-for-gestational-age
infants among mothers who use unclean household cooking fuel, as this later can cause
wasting [34]. 4.1. Strength and Limitations The study has several strengths. First, the use of a valid survey and rigorous statistical
methods emphasise the trustworthiness and robustness of our findings. Additonally,
the use of nationally representative data ensures that our findings are generalizable and
replicable in the studied 31 sub-Saharan African countries. Moreover, current findings
contribute to bridging the gaps in child nutrition literature which previous studies did Nutrients 2021, 13, 1541 14 of 16 14 of 16 not consider. Notwithstanding these strengths, the study has some limitations. Therefore,
interpretations and inferences made from our findings must be considered in light of
these limitations. First, childhood nutritional status, maternal marital status and use of unclean cooking
fuel interact in a complex system in SSA influenced by cultural beliefs. However, because
the study relied on secondary data, the analysis was limited to only variables that were in
the datasets. Further, the DHS employs cross-sectional designs, hence causal effects cannot
be ascribed to the noted outcomes. However, only associative effects can be ascribed to
the noted outcomes. The key predictor variables were self-reported by the mothers, and
therefore, responses given are subject to recall biases and other social desirability concerns. Relatedly, self-reported cooking fuel type is only a crude proxy for exposure to household
air pollution and, therefore, may not completely capture the relationship between exposure
to particulate matter or carbon monoxide and health outcomes. Finally, the pooling of the
data may be affected by heterogeneity across countries or regions. 4.2. Practical Implications Practically, these findings demonstrate the need to invest in clean household cooking
fuel. It is imperative for the governments of the 31 sub-Saharan African countries to double
their efforts to end the use of unclean household cooking fuel. This could be achieved by
going beyond just increasing access and affordability of clean cooking fuels (e.g., electricity,
gas, ethanol, solar) by promoting their use through effective health education and health
promotion programmes. Findings also draw the attention of policymakers to the urgent
need for CNS policies and programmes (e.g., dietary supplementation, increasing dietary
diversity, improving agriculture and food security) to be targeted towards at-risk sub-
populations (i.e., single mothered households). Given the sub-regional variations noted
in the current study, appropriate context-specific interventions are required to address
household cooking fuel challenges and issues related to stunting, wasting and underweight
among children in the studied 31 sub-Saharan African countries. References 1. Remans, R.; Flynn, D.F.B.; Declerck, F.; Diru, W.; Fanzo, J.; Gaynor, K.; Lambrecht, I.; Mudiope, J.; Mutuo, P.K.; Nkhoma, P.; et al. Assessing nutritional diversity of cropping systems in African villages. PLoS ONE 2011, 6, e21235. [CrossRef] g
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2. Ntoimo, L.F.C.; Odimegwu, C.O. Health effects of single motherhood on children in sub-Saharan Afric
BMC Public Health 2014, 14, 1145. [CrossRef] 3. UNICEF. Briefing Note Series on SDG Global Indicators Related to Children. 2018. Available online: File:///C:/Users/iddri/
AppData/Local/Temp/SDG-briefing-note-1_introduction.pdf (accessed on 6 March 2021). 4. De Onis, M.; Blössner, M.; Borghi, E. Prevalence and trends of stunting among pre-school children, 1990–2020. Public Health Nutr. 2012, 15, 142–148. [CrossRef] 5. Devlin, K. Stunting limits learning and future earnings of children. World J. 2012, 40, 26. 5. Devlin, K. Stunting limits learning and future earnings of children. World J. 2012, 40, 26. 6. Darteh, E.K.M.; Acquah, E.; Kumi-Kyereme, A. Correlates of stunting among children in Ghana. BMC Public Health 2014, 14, 504. [CrossRef] [PubMed] 6. Darteh, E.K.M.; Acquah, E.; Kumi-Kyereme, A. Correlates of stunting among children in Ghana. BM
[CrossRef] [PubMed] 7. Goudet, S.M.; Kimani-Murage, E.W.; Wekesah, F.; Wanjohi, M.; Griffiths, P.L.; Bogin, B.; Madise, N.J. How does poverty affect
children’s nutritional status in Nairobi slums? A qualitative study of the root causes of undernutrition. Public Health Nutr. 2016,
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status of children aged 6–23 months in rural Northern Ghana? BMC Public Health 2015, 15, 1–12. [Cros 9. Clark, S.; Hamplová, D. Single motherhood and child mortality in sub-Saharan Africa: A life course perspective. Demography
2013, 50, 1521–1549. [CrossRef] [PubMed] 10. Odimegwu, C.; Somefun, O.D.; De Wet, N. Contextual determinants of family dissolution in sub-Saharan Africa. Dev. S. Afr. 2017, 34, 721–737. [CrossRef] 11. Owili, P.O.; Muga, M.A.; Pan, W.-C.; Kuo, H.-W. Cooking fuel and risk of under-five mortality in 23 Sub-Saharan African
countries: A population-based study. Int. J. Environ. Health Res. 2017, 27, 191–204. [CrossRef] [PubMed] y
12. Obasohan, P.E.; Walters, S.J.; Jacques, R.; Khatab, K. Risk factors associated with malnutrition among children under-five years in
sub-Saharan African countries: A scoping review. Int. J. Environ. Res. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References Public Health 2020, 17, 8782. [CrossRef] 13. Wali, N.; Agho, K.E.; Renzaho, A.M. factors associated with stunting among children under 5 years in fi
(2014–2018): Analysis of demographic health surveys. Nutrients 2020, 12, 3875. [CrossRef] y
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14. Tanwi, T.S.; Chakrabarty, S.; Hasanuzzaman, S. Double burden of malnutrition among ever-married women in Bangladesh: A
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15. Ezeh, O.K.; Agho, K.E.; Dibley, M.J.; Hall, J.J.; Page, A.N. The effect of solid fuel use on childhood mortality in Nigeria: Evidence
from the 2013 cross-sectional household survey. Environ. Health 2014, 13, 1–10. [CrossRef] 16. Corsi, D.J.; Neuman, M.; E Finlay, J.; Subramanian, S.V. Demographic and health surveys: A profile. Int. J. Epidemiol. 2012, 41,
1602–1613. [CrossRef] 17. Rutstein, S.O.; Rojas, G. Guide to DHS Statistics; ORC Macro: Calverton, MD, USA, 2006; Volume 38. 18. DHS. Guide to DHS Statistics DHS-7: Nutritional Status. 2021. Available online: https://dhsprogram.com/data/Guide-to-DHS-
Statistics/Nutritional_Status.htm (accessed on 11 March 2021). 19. Armah, F.A.; Ekumah, B.; Yawson, D.O.; Odoi, J.O.; Afitiri, A.-R.; Nyieku, F.E. Predictive probabilities of access to clean cooking:
Evidence from the demographic and health surveys in 31 countries in sub-Saharan Africa. Environ. Justice 2019, 12, 118–131. [CrossRef] [
]
20. Amegah, A.K.; Boachie, J.; Näyhä, S.; Jaakkola, J.J.K. Association of biomass fuel use with reduced body weight of adult Ghanaian
women. J. Expo. Sci. Environ. Epidemiol. 2020, 30, 670–679. [CrossRef] [PubMed] 21. Yaya, S.; Uthman, O.A.; Ekholuenetale, M.; Bishwajit, G.; Adjiwanou, V. Effects of birth spacing on adverse childhood health
t
E id
f
34
t i
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Af i
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t l M d 2019 33 1 8 [C
R f] [P bM d] 21. Yaya, S.; Uthman, O.A.; Ekholuenetale, M.; Bishwajit, G.; Adjiwanou, V. Effects of birth spacing on adverse childhood health
outcomes: Evidence from 34 countries in sub-Saharan Africa. J. Matern. Neonatal Med. 2019, 33, 1–8. [CrossRef] [PubMed] 22. Amare, Z.Y.; Ahmed, M.E.; Mehari, A.B. Determinants of nutritional status among children under age 5 in Ethiopia: Further
analysis of the 2016 Ethiopia demographic and health survey. Glob. Health 2019, 15, 1–11. [CrossRef] [PubMed] 22. Amare, Z.Y.; Ahmed, M.E.; Mehari, A.B. Determinants of nutritional status among children under age 5 in Ethiopia: Further
analysis of the 2016 Ethiopia demographic and health survey. Glob. Health 2019, 15, 1–11. [CrossRef] [PubMed] analysis of the 2016 Ethiopia demographic and health survey. Glob. 5. Conclusions The current study sought to examine the joint effect of maternal marital status and
household cooking fuel type on child nutritional status in SSA. We found that maternal
marital status and household cooking fuel type have a joint effect on childhood stunting,
wasting and underweight. Future studies could explore the cultural dimension to this
association between being a single mother, the use of unclean cooking fuel, and child
nutritional status in SSA. Undertaking such studies would be instrumental in informing
appropriate culturally sensitive strategies that can effectively improve CNS in SSA. Author Contributions: I.A. and B.O.A. developed the study concept. I.A. and E.D. performed
the data analysis. A.-A.S., E.D., J.O., J.E.H.J., T.H. and B.O.A. drafted and revised the manuscript
critically for its intellectual content. All authors have read and agreed to the published version of
the manuscript. Funding: The authors sincerely thank Bielefeld University, Germany for providing financial support
through the Institutional Open Access Publication Fund for the article processing charge. Institutional Review Board Statement: The study was conducted using data provided by the
Demographic and Health Surveys (DHS) Program. The DHS Program uses protocols reviewed
and approved by the ICF Institutional Review Board (IRB). Also, IRB’s in host countries review
contry-specific DHS survey protocols to ensure they comply with local laws on inclusion of minors
and as well as adults in its surveys. Informed Consent Statement: The DHS Program seeks written consent from all individuals aged
18 years and above. For those aged below 18 years, written consent is obtained from parent(s) or
guardian(s) before inclusion in the study. Data Availability Statement: The dataset is available on the following website: http://goo.gl/ny8
T6X (accessed on 3 February 2021). 15 of 16 15 of 16 Nutrients 2021, 13, 1541 Acknowledgments: We acknowledge the DHS Program for making the data sets freely accessible
upon request. Acknowledgments: We acknowledge the DHS Program for making the data sets freely accessible
upon request. References Health 2019, 15, 1–11. [CrossRef] [PubMed]
23
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Burundian children aged 6-23 months: Evidence from a national cross-sectional household survey, 2014. BMC Pediatr. 2017, 17,
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28. Ekholuenetale, M.; Tudeme, G.; Onikan, A.; Ekholuenetale, C.E. Socioeconomic inequalities in hidden hunger, undernutrition,
and overweight among under-five children in 35 sub-Saharan Africa countries. J. Egypt. Public Health Assoc. 2020, 95, 1–15. [CrossRef] 29. United Nations. The Millennium Development Goals Report; United Nations: New York, NY, USA, 2015. 30. Islam, S.; Rana, J.; Mohanty, S.K. Cooking, smoking, and stunting: Effects of household air pollution sources on childhood growth
in India. Indoor Air 2021, 31, 229–249. [CrossRef] 31. Balietti, A.; Datta, S. The Impact of Indoor Solid Fuel Use on the Stunting of Indian Children. 2017. Available online: http:
//www.ancabalietti.net/wp-content/uploads/2017/04/Datta_Balietti_March2017.pdf (accessed on 3 March 2021). 32. Upadhyay, A.K.; Singh, A.; Kumar, K.; Singh, A. Impact of indoor air pollution from the use of solid fue
threatening respiratory illnesses in children in India. BMC Public Health 2015, 15, 1–9. [CrossRef] [PubM 33. Schlaudecker, E.P.; Steinhoff, M.C.; Moore, S.R. Interactions of diarrhea, pneumonia, and malnutrition in childhood: Recent
evidence from developing countries. Curr. Opin. Infect. Dis. 2011, 24, 496. [CrossRef] 34. Tielsch, J.M.; Katz, J.; Thulasiraj, R.D.; Coles, C.L.; Sheeladevi, S.; Yanik, E.L.; Rahmathullah, L. References Exposure to indoor biomass fuel
and tobacco smoke and risk of adverse reproductive outcomes, mortality, respiratory morbidity and growth among newborn
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The immatures of three Neotropical species of Palpomyia Meigen (Diptera, Culicomorpha, Ceratopogonidae)
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INTRODUCTION in the tibialis group, and the remaining six in the
distincta group: P. mapuche Spinelli, Grogan &
Ronderos, P. marinoi Spinelli, Grogan & Ronderos,
P. patagonica Ingram & Macfi e, P. septentrionalis
Spinelli, Grogan & Ronderos, P. subfuscula
Ingram & Macfi e and P. yamana Spinelli, Grogan
& Ronderos. Of these species only the pupa of
P. subaspera is known, described by Grogan &
Wirth (1979) from Nearctic specimens. Palpomyia Meigen, a worldwide genus of
predaceous midges of the tribe Palpomyiini,
includes 283 species. Of these, 46 are recorded
for the Neotropical region by Borkent &
Spinelli (2007), and three more were recently
described for this region: P. ryszardi by Spinelli
& Ronderos (2013) and P. amazonensis and P. lanceolata by Feijó (Almeida et al. 2017). They are
relatively common inhabitants of streams, lakes
and ponds, swamps, marshes and sphagnum
bogs (Grogan & Wirth 1975, 1979), and their
preimaginales stages are poorly known. So
far, immatures are known only for fi ve species
that inhabit the Neotropics: P. guarani Lane, P. lacustris Lane, Forattini & Rabello, P. ryszardi,
P. subaspera (Coquillett) and P. wirthi Lane,
Forattini & Rabello. During a recent survey carried out in
northwestern Argentinean Patagonia, larvae and
pupae of P. mapuche and pupae of P. subaspera
and P. subfuscula were collected. The purpose
of this paper is to describe the immatures of P. mapuche and P. subfuscula and to redescribe
the pupa of P. subaspera with modern standards. DANIELLE ANJOS-SANTOS, FLORENTINA DÍAZ, GUSTAVO R. SPINELLI &
MARÍA M. RONDEROS Abstract: The fi rst description of the fourth instar larva and pupa of Palpomyia mapuche
Spinelli, Grogan & Ronderos and the pupa of P. subfuscula Ingram & Macfi e are provided,
as well as the redescription of the pupa of P. subaspera (Coquillett). Studied specimens
were collected in lotic environments of Argentinian Patagonia, in Neuquén and Chubut
Provinces. The described stages were examined and illustrated with a phase-contrast
microscope. The larva was examined using a scanning electron microscope. Data on the
bionomics for P. mapuche and new records for the three species are provided. Key words: Biting midges, immature stages, larva, Palpomyiini, pupa. BIOLOGICAL SCIENCES The immatures of three Neotropical
species of Palpomyia Meigen (Diptera,
Culicomorpha, Ceratopogonidae) An Acad Bras Cienc (2020) 92(Suppl. 2): e20190718 DOI 10.1590/0001-3765202020190718
Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences
Printed ISSN 0001-3765 I Online ISSN 1678-2690
www.scielo.br/aabc | www.fb.com/aabcjournal An Acad Bras Cienc (2020) 92(Suppl. 2): e20190718 DOI 10.1590/0001-3765202020190718
Anais da Academia Brasileira de Ciências | Annals of the Brazilian Academy of Sciences
Printed ISSN 0001-3765 I Online ISSN 1678-2690
www.scielo.br/aabc | www.fb.com/aabcjournal An Acad Bras Cienc (2020) 92(Suppl. 2) MATERIALS AND METHODS 2, 3) with medium-sized, stout seta; maxillary
palpus (Figs. 2, 3) elongated, slightly flattened,
with 3 subapical papillae. Hypostoma (Figs. 1–3)
smooth on medial margin, flanked by fine tooth. Epipharynx (Figs. 5, 8, 11) gently massive, with 2
combs: ventral comb with 9–10 stout, pointed
teeth, dorsal comb with 6 long, pointed teeth
on posterior edge; lateral arms elongate; small,
curved auxiliary sclerites near lateral arms; LAW
0.048–0.050 (0.049, n = 2) mm, DCW 0.023–0.025
(0.024, n = 2) mm. Hypopharynx (Figs. 5, 7–8, 10)
elongate, thin, gently sclerotized, arms slender. Thoracic pigmentation uniformly pale yellowish. Caudal segment (Fig. 4) about 2.3 times as long
as wide, with two pairs of long, stout setae “o”,
two pairs of long, thin setae “i”; two pairs of long,
thin setae “l1”, one pair of long, stout setae “l2”,
one pair of medium-sized, thin setae “v”. CSL
0.65–0.70 (0.68, n = 2) mm, CSW 0.27–0.32 (0.30, n
= 2) mm, CSR 2.03–2.60 (2.32, n = 2), OL 0.26–0.30 MATERIALS AND METHODS 1–3, 7, 9) hooked,
strongly sclerotized, apical tooth long, sensory
pit and one medium-sized, thin seta present
on the aboral surface; with prominent point of
articulation, deep fossa mandibularis on ectal
surface (Fig. 9); MDL 0.06–0.08 (0.07, n = 2) mm,
MDW 0.040–0.044 (0.041, n = 2) mm. Maxilla (Fig. 3) galeolacinia with lacinial sclerite 1 (Figs. 2, 3)
with one long, thin seta, lacinial sclerite 2 (Figs. 2, 3) with medium-sized, stout seta; maxillary
palpus (Figs. 2, 3) elongated, slightly flattened,
with 3 subapical papillae. Hypostoma (Figs. 1–3)
smooth on medial margin, flanked by fine tooth. Epipharynx (Figs. 5, 8, 11) gently massive, with 2
combs: ventral comb with 9–10 stout, pointed
teeth, dorsal comb with 6 long, pointed teeth
on posterior edge; lateral arms elongate; small,
curved auxiliary sclerites near lateral arms; LAW
0.048–0.050 (0.049, n = 2) mm, DCW 0.023–0.025
(0.024, n = 2) mm. Hypopharynx (Figs. 5, 7–8, 10)
elongate, thin, gently sclerotized, arms slender. Thoracic pigmentation uniformly pale yellowish. Caudal segment (Fig. 4) about 2.3 times as long
as wide, with two pairs of long, stout setae “o”,
two pairs of long, thin setae “i”; two pairs of long,
thin setae “l1”, one pair of long, stout setae “l2”,
one pair of medium-sized, thin setae “v”. CSL
0.65–0.70 (0.68, n = 2) mm, CSW 0.27–0.32 (0.30, n
= 2) mm, CSR 2.03–2.60 (2.32, n = 2), OL 0.26–0.30 0.104 (n = 2) mm; SGR 1.35–1.83 (1.59, n = 2). Setae
simple, medium-sized to long, setae “x”, “s”, “u”
and “o” branched, chaetotaxy as in Figs. 1–2, 5–6. Antenna cylindrical, medium-sized, length 0.02
mm. Labrum (Figs. 1, 2, 5) longer than broad,
not extending beyond hypostoma, palatum with
three pairs of anterolateral sensilla styloconica
(Figs. 1–3), two pairs of sensilla trichoidea (Figs. 2–3) and one pair of campaniform sensillum (Fig. 2); messors (Figs. 2–3) stout, curved, sclerotized,
situated away from mandibles; palatal bar (Fig. 2) triangular, situated immediately posterior
to messors. Mandible (Figs. 1–3, 7, 9) hooked,
strongly sclerotized, apical tooth long, sensory
pit and one medium-sized, thin seta present
on the aboral surface; with prominent point of
articulation, deep fossa mandibularis on ectal
surface (Fig. 9); MDL 0.06–0.08 (0.07, n = 2) mm,
MDW 0.040–0.044 (0.041, n = 2) mm. Maxilla (Fig. 3) galeolacinia with lacinial sclerite 1 (Figs. 2, 3)
with one long, thin seta, lacinial sclerite 2 (Figs. MATERIALS AND METHODS Spinelli et al. (2009) recognized eight
species from Patagonia. Two of them, P. aculeata
Ingram & Macfi e and P. subaspera, are included Larvae and pupae were collected from the
bordering vegetation of two streams in Chubut
Province, one located in the Subantartic forest An Acad Bras Cienc (2020) 92(Suppl. 2) DANIELLE ANJOS-SANTOS et al. IMMATURES OF Palpomyia and the other in the Patagonian steppe, and from
the bordering mud of a river in the steppe of the
Neuquén Province. In all cases, the substrate was
removed with the aid of a strainer and transferred
to a white tray where immatures were collected
with a pipette. Larvae were placed in individual
containers with water and substrate from their
natural environment. Pupae were isolated in a
vial with a drop of water, and observed daily
until adult emergence. Adults were allowed
to harden for 24 h before being preserved in
ethanol to ensure their complete pigmentation. For detailed examination with phase-contrast
microscope, larval and pupal exuviae and adults
were mounted in Canada balsam following
the technique described by Borkent & Spinelli
(2007). Photomicrographs were taken with a
Leica EC3 digital camera, through a Leica DM
500 microscope. Illustrations were drawn with
Adobe illustrator CC®. Larvae were also examined
using scanning electron microscopy (SEM) (JOEL
2000) following the technique of Ronderos et al. (2000, 2008). Measurements were taken with a
(BCM) Leitz Wetzlar binocular microscope. The
temperature of the water and air were measured
with an alcohol thermometer in degrees Celsius. For larval terms see Anjos-Santos et al. (2017);
for pupal terms, see Borkent (2014). Studied
specimens are deposited in the collection of the
Museo de La Plata, La Plata, Argentina (MLPA). 0.104 (n = 2) mm; SGR 1.35–1.83 (1.59, n = 2). Setae
simple, medium-sized to long, setae “x”, “s”, “u”
and “o” branched, chaetotaxy as in Figs. 1–2, 5–6. Antenna cylindrical, medium-sized, length 0.02
mm. Labrum (Figs. 1, 2, 5) longer than broad,
not extending beyond hypostoma, palatum with
three pairs of anterolateral sensilla styloconica
(Figs. 1–3), two pairs of sensilla trichoidea (Figs. 2–3) and one pair of campaniform sensillum (Fig. 2); messors (Figs. 2–3) stout, curved, sclerotized,
situated away from mandibles; palatal bar (Fig. 2) triangular, situated immediately posterior
to messors. Mandible (Figs. distincta group Palpomyia mapuche Spinelli, Grogan & Ronderos
Spinelli et al. 2009: 50 (female, male);
Borkent 2016: 170 (in online World catalog). Description of fourth instar larva (Figs. 1–11). Head capsule (Figs. 1–2, 5–6) pale brown, about
2.8 times longer than wide, apex slightly bent
ventrally, HL 0.42 (n = 2) mm; HW 0.14–0.19 (0.17,
n = 2) mm, HR 2.21–3.00 (2.61, n = 2) mm; SGW An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 2 | 13 DANIELLE ANJOS-SANTOS et al. IMMATURES OF Palpomyia Figures 1-4. Palpomyia m
Spinelli, Grogan & Rond
instar larva (SEM). 1. hea
chaetotaxy, frontoventr
head capsule detail, ant
view; 3. mouthparts, ant
view; 4. caudal segment
view. Scale 0.05 mm. Antennae (AN); collar (C
galeolacinea (GL); hypos
labrum (LB); lacinial scl
lacinial sclerite 2 (LC2);
(MD); messors (MS); max
maxillary palpus (MP); p
bar (PB); palatum (PL); s
campaniformia (SCa); se
styloconica (ss); sensilla
(st). Head capsule chaet
o, parahypostomal seta
mesolateral setae; v, po
setae; w, anterolateral s
parantennal setae; y, ve
Caudal segment chaeto
inner seta; “l1”, first late
second lateral seta; “o”,
“v”, ventral setae. Figures 1-4. Palpomyia mapuche
Spinelli, Grogan & Ronderos, fourth
instar larva (SEM). 1. head capsule,
chaetotaxy, frontoventral view; 2. head capsule detail, anterofrontal
view; 3. mouthparts, anterofrontal
view; 4. caudal segment, lateral
view. Scale 0.05 mm. Antennae (AN); collar (CO);
galeolacinea (GL); hypostoma (HY);
labrum (LB); lacinial sclerite 1 (LC1);
lacinial sclerite 2 (LC2); mandible
(MD); messors (MS); maxilla (MX);
maxillary palpus (MP); palatal
bar (PB); palatum (PL); sensilla
campaniformia (SCa); sensilla
styloconica (ss); sensilla trichoidea
(st). Head capsule chaetotaxy:
o, parahypostomal setae; u,
mesolateral setae; v, posterolateral
setae; w, anterolateral setae; x,
parantennal setae; y, ventral setae. Caudal segment chaetotaxy: “i”,
inner seta; “l1”, first lateral seta; “l2”,
second lateral seta; “o”, outer seta;
“v”, ventral setae. Figures 1-4. Palpomyia mapuche
Spinelli, Grogan & Ronderos, fourth
instar larva (SEM). 1. head capsule,
chaetotaxy, frontoventral view; 2. head capsule detail, anterofrontal
view; 3. mouthparts, anterofrontal
view; 4. caudal segment, lateral
view. Scale 0.05 mm. Antennae (AN); collar (CO);
galeolacinea (GL); hypostoma (HY);
labrum (LB); lacinial sclerite 1 (LC1);
lacinial sclerite 2 (LC2); mandible
(MD); messors (MS); maxilla (MX);
maxillary palpus (MP); palatal
bar (PB); palatum (PL); sensilla
campaniformia (SCa); sensilla
styloconica (ss); sensilla trichoidea
(st). Head capsule chaetotaxy:
o, parahypostomal setae; u,
mesolateral setae; v, posterolateral
setae; w, anterolateral setae; x,
parantennal setae; y, ventral setae. An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 3 | 13 distincta group Caudal segment chaetotaxy: “i”,
inner seta; “l1”, first lateral seta; “l2”,
second lateral seta; “o”, outer seta;
“v”, ventral setae. thin seta, located on rounded small tubercle,
DA-2-H campaniform sensillum; DAL 0.24–0.26
(0.25, n = 5) mm; DAW 0.25–0.27 (0.26, n = 5) mm;
DAW/DAL 0.97–1.04 (1.01, n = 5); two dorsolateral
cephalic sclerites (Fig. 26): DL-1-H short, stout
seta, DL-2-H campaniform sensillum; clypeal/
labrals (Fig. 25): CL-1-H medium-sized thin
seta, CL-2- H long, thin seta; oculars (Fig. 25):
O-1-H short, stout seta, O-2-H campaniform
sensillum, O-3-H long, thin seta. Cephalothorax
rectangular, surface predominantly smooth with
small spinules on mesonotum, between bases
of respiratory organs. Length of cephalothorax
1.55–1.67 (1.60, n = 4) mm, width 1.05–1.19 (1.10, n
= 3) mm. Thorax: Respiratory organ (Figs. 12, 14, Description of female pupa (Figs. 12–15,
25–28, 38–39). Habitus as in Fig. 14. Exuviae
pale brown. Total length 4.12–4.65 (4.34, n = 4)
mm. Head: Dorsal apotome (Fig. 13) with disc 2
times broader than long, bearing rounded small
tubercles, anterior margin covered with stout
rounded spinules; posterior margin slightly
concave; posterolateral margin with broad raised
areas, bearing two dorsal apotomal sensilla;
antenna extending posteriorly to midleg (Fig. 14); mouthparts (Fig. 25) with mandible well
developed; palpus extending to posterolateral
margin of labium; labium separated medially
by labrum; apex of labrum slightly rounded;
sensilla: dorsal apotomals (Fig. 13): DA-1-H long, An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 3 | 13 DANIELLE ANJOS-SANTOS et al. IMMATURES OF Palpomyia 27) smooth, brown, about 3.80–4.04 (3.96, n = 5)
times longer than broad, apex rounded, with row
of 12–15 pores closely abutting at apex; pedicel
(Figs. 12, 27) slender, P 0.020–0.024 (0.021, n = 5)
mm; RO length 0.208–0.228 (0.215, n = 5) mm, RO
width 0.052–0.060 (0.054, n = 5) mm; P/RO 0.095–
0.109 (0.097, n = 5); sensilla: three anteromedials
(Fig. 27): AM-1-T, AM-2-T long, stout setae; AM-
3-T campaniform sensillum; one anterolateral
(Fig. 27): AL-1-T short, stout seta; dorsals (Fig. 28): D-1-T, D-2-T, D-4-T, long, thin setae, D-3-T
campaniform sensillum, D-5-T minute seta,
all on small rounded tubercle, except D-3-T;
supraalar (SA-2-T) campaniform sensillum;
metathoracic (Fig. 38): M-3-T campaniform sensillum, near anterior margin of metathorax. Abdomen: segments covered with small
spicules, segments with simple setae; segment
9 (Figs. distincta group 14, 39): D-2-IV, D-3-IV medium-sized, thin
seta, D-4-IV, D-7-IV campaniform sensilla, D-5-
IV short, stout seta, D-8-IV medium-sized, stout
seta, D-9-IV long, thin seta; L-1-IV, L-2-IV, L-4-IV
medium-sized, stout setae, L-3-IV long, thin seta,
all on small or medium-sized tubercles; V-5-IV,
V-7-IV medium-sized, stout setae, V-6-IV long, posterior sensilla: D-4-I, D-7-I campaniform
sensilla, D-5-I short, stout seta, D-8-I medium-
sized, stout seta, D-9-I long, thin seta; 3 lateral
sensilla: L-1-I long, thin seta, L-2-I medium-
sized, stout setae, L-3-I minute seta; segment 4
(Figs. 14, 39): D-2-IV, D-3-IV medium-sized, thin
seta, D-4-IV, D-7-IV campaniform sensilla, D-5-
IV short, stout seta, D-8-IV medium-sized, stout
seta, D-9-IV long, thin seta; L-1-IV, L-2-IV, L-4-IV
medium-sized, stout setae, L-3-IV long, thin seta,
all on small or medium-sized tubercles; V-5-IV,
V-7-IV medium-sized, stout setae, V-6-IV long, thin seta, all on small tubercles; segment 9 (Figs. 14–15) with D-5-IX, D-6-IX campaniform sensilla. 14 15) with D 5 IX, D 6 IX campaniform sensilla. Description of male pupa (Figs. 24, 40). Similar to female with usual sexual differences:
Total length 3.74–3.96 (3.83, n = 4) mm. Dorsal
apotome (Fig. 24) with distal margin slightly
rounded, DAL 0.24–0.27 (0.26, n = 3) mm; DAW
0.22–0.27 (0.24, n = 3) mm, DAW/DAL 0.85–1.00
(0.93, n = 3). Cephalothorax: length 1.22–1.33
(1.29, n = 4) mm, width 0.92–1.04 (0.99, n = 3) mm. Respiratory organ 3.57–4.08 (3.78, n = 4) times
longer than broad, P 0.016–0.020 (0.018, n = 4)
mm; RO length 0.18–0.20 (0.19, n = 4) mm, RO An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 5 | 13
Figures 12-23. Female pupa. 12-15. Palpomyia mapuche Spinelli, Grogan
& Ronderos. 16–19. Palpomyia
subfuscula Ingram & Macfie. 20-23. Palpomyia subaspera (Coquillett). 12, 16, 20. respiratory organ. 13,
17, 21. dorsal apotome. 14, 18, 22. habitus. 15, 19, 23. Segment 9. Scale
0.05 mm. Antenna (AN); dorsal apotome
sensilla (DA-1-H, DA-2-H); dorsal
sensilla of segment 9 (D-5-IX);
pedicel (P); pore (p); respiratory
organ (RO); segment 1 (1st seg.);
segment 4 (4th seg.); segment 9 (Seg. 9); terminal process (TP). Figures 12-23. Female pupa. 12-15. Palpomyia mapuche Spinelli, Grogan
& Ronderos. 16–19. Palpomyia
subfuscula Ingram & Macfie. 20-23. Palpomyia subaspera (Coquillett). 12, 16, 20. respiratory organ. 13,
17, 21. dorsal apotome. 14, 18, 22. habitus. 15, 19, 23. Segment 9. Scale
0.05 mm. distincta group 14–15) approximately 1.5 longer than
broad, length 0.28–0.33 (0.30, n = 5) mm, width
0.18–0.22 (0.20, n = 5) mm; dorsal and ventral
surface covered with pointed spicules; ventral
surface with one small, circular mark located
on a wide bare, mesal area; terminal process
(Figs. 14–15) moderately short, nearly straight,
base wide, with few pointed spicules, extreme
tip somewhat dark, length 0.09–0.12 (0.10, n = 5)
mm, width 0.03–0.05 (0.04, n = 5) mm; sensilla:
tergite 1 (Figs. 14, 38) with two anteromesals:
D-2-I short, stout seta, D-3-I long, thin seta; 5 Figures 5-11. Palpomyia mapuche
Spinelli, Grogan & Ronderos,
fourth instar larva. 5, 7-8. photomicrographs. 6, 9-11. draw
illustrations. 5. head capsule, ventral
view; 6. head capsule, dorsal view;
7. detail of anterior portion of head
capsule and hypopharynx, ventral
view; 8, 11. epipharynx, ventral view;
9. left mandible, ventral view; 10. hypopharynx, ventral view. Scale
0.05 mm. An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 4 | 13
Figures 5-11. Palpo
Spinelli, Grogan &
fourth instar larva
photomicrographs
illustrations. 5. hea
view; 6. head caps
7. detail of anterio
capsule and hypop
view; 8, 11. epiphar
9. left mandible, ve
hypopharynx, vent
0.05 mm. Collar (CO); dorsal
mandible (MD); fos
(MF); labrum (LB);
ventral comb (VC). chaetotaxy: j, colar
pits; o, parahypost
p, posterior perifro
postfrontal setae;
pits; s, anteroperif
prefrontal setae; x
setae; z, frontal pit An Acad Bras Cienc (2020) 92(Suppl. 2 Collar (CO); dorsal comb (DC);
mandible (MD); fossa mandibularis
(MF); labrum (LB); palatum (PL);
ventral comb (VC). Head capsule
chaetotaxy: j, colar pits; k, pronotal
pits; o, parahypostomal setae;
p, posterior perifrontal setae; q,
postfrontal setae; r, postnotal
pits; s, anteroperifrontal setae; t,
prefrontal setae; x, parantennal
setae; z, frontal pits. An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 4 | 13 DANIELLE ANJOS-SANTOS et al. IMMATURES OF Palpomyia thin seta, all on small tubercles; segment 9 (Figs. 14–15) with D-5-IX, D-6-IX campaniform sensilla. posterior sensilla: D-4-I, D-7-I campaniform
sensilla, D-5-I short, stout seta, D-8-I medium-
sized, stout seta, D-9-I long, thin seta; 3 lateral
sensilla: L-1-I long, thin seta, L-2-I medium-
sized, stout setae, L-3-I minute seta; segment 4
(Figs. distincta group Antenna (AN); dorsal apotome
sensilla (DA-1-H, DA-2-H); dorsal
sensilla of segment 9 (D-5-IX);
pedicel (P); pore (p); respiratory
organ (RO); segment 1 (1st seg.);
segment 4 (4th seg.); segment 9 (Seg. 9); terminal process (TP). An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 5 | 13 DANIELLE ANJOS-SANTOS et al. IMMATURES OF Palpomyia width 0.05–0.06 (0.05, n = 4); P/RO 0.08–0.11 (0.10,
n = 4). Segment 9 (Fig. 40) dorsal surface covered
anteriorly with pointed spicules, length 0.26–
0.29 (0.27, n = 4) mm, width 0.15–0.21 (0.18, n = 4)
mm; terminal process (Fig. 40) length 0.09–0.11
(0.10, n = 4) mm, width 0.03–0.06 (0.04, n = 4) mm;
genital lobe (Fig. 40) globose, short, not reaching
posterior margin of segment, surface smooth. Material examined. Argentina, Chubut,
Parque Nacional Los Alerces, Pucon Pai, unnamed
stream, 42°49ʹ33.5ʹʹS; 71°36ʹ’44.1ʹʹW, 547 m, 20-I-
2016, adults emerged in laboratory 23-I-2016, D. Anjos-Santos and P. Pessacq, 2 females, 1 male
(with pupal exuviae); same data except 29-XII-
2016, adult emerged 01-I-2017, D. Anjos-Santos
and P. Pessacq, 1 female (with pupal exuvium);
same data except adult emerged 02-I-2017, D. Anjos-Santos and P. Pessacq, 1 male (with pupal
exuvium); same data except adult emerged Distribution. Argentina (western Neuquén,
Río Negro and Chubut Provinces); Chile (Valdivia
Province). An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 6 | 13
Figures 24-37. Palpomyia mapuche
Spinelli, Grogan & Ronderos;
Palpomyia subfuscula Ingram
& Macfie; Palpomyia subaspera
(Coquillett). 24. Palpomyia mapuche
Spinelli, Grogan & Ronderos, male
pupa. 25-28. Palpomyia mapuche
Spinelli, Grogan & Ronderos, female
pupa. 29-32. Palpomyia subfuscula
Ingram & Macfie, female pupa. 33. Palpomyia subaspera (Coquillett),
male pupa. 34-37. Palpomyia
subaspera (Coquillett), female pupa. 24, 33. dorsal apotome, dorsal view;
25, 29, 34. mouthparts, ventral view;
26, 30, 35. dorsolateral cephalic
sclerites, dorsolateral view; 27, 31,
36. anterolateral sensillum and
anteromedial sensilla, dorsolateral
view; 28, 32, 37. dorsal sensilla and
supraalar sensillum, dorsal view. Scale 0.05 mm. Anterolateral sensillum (AL-1-T);
anteromedial sensilla (AM-1-T,
AM-2-T, AM-3-T); clypeal/labral
sensilla (CL-1-H, CL-2-H); dorsal
apotome sensilla (DA-1-H, DA-2-H);
dorsal sensilla (D-1-T, D-2-T, D-3-T,
D-4-T, D-5-T); dorsolateral cephalic
sclerite sensilla (DL-1-H, DL-2-H);
respiratory organ (RO); pedicel (P);
ocular sensilla (O-1-H, O-2-H, O-3-H);
supraalar sensillum (SA-2-T). Figures 24-37. Palpomyia mapuche
Spinelli, Grogan & Ronderos;
Palpomyia subfuscula Ingram
& Macfie; Palpomyia subaspera
(Coquillett). 24. Palpomyia mapuche
Spinelli, Grogan & Ronderos, male
pupa. 25-28. Palpomyia mapuche
Spinelli, Grogan & Ronderos, female
pupa. 29-32. distincta group Palpomyia subfuscula
Ingram & Macfie, female pupa. 33. Palpomyia subaspera (Coquillett),
male pupa. 34-37. Palpomyia
subaspera (Coquillett), female pupa. 24, 33. dorsal apotome, dorsal view;
25, 29, 34. mouthparts, ventral view;
26, 30, 35. dorsolateral cephalic
sclerites, dorsolateral view; 27, 31,
36. anterolateral sensillum and
anteromedial sensilla, dorsolateral
view; 28, 32, 37. dorsal sensilla and
supraalar sensillum, dorsal view. Scale 0.05 mm. Anterolateral sensillum (AL-1-T);
anteromedial sensilla (AM-1-T,
AM-2-T, AM-3-T); clypeal/labral
sensilla (CL-1-H, CL-2-H); dorsal
apotome sensilla (DA-1-H, DA-2-H);
dorsal sensilla (D-1-T, D-2-T, D-3-T,
D-4-T, D-5-T); dorsolateral cephalic
sclerite sensilla (DL-1-H, DL-2-H);
respiratory organ (RO); pedicel (P);
ocular sensilla (O-1-H, O-2-H, O-3-H);
supraalar sensillum (SA-2-T). An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 6 | 13 DANIELLE ANJOS-SANTOS et al. IMMATURES OF Palpomyia Description of female pupa. Habitus as
in Fig. 18. Exuviae brownish. Total length 3.74
mm. Head: Dorsal apotome (Fig. 17) with disc
1.9 times broader than long, bearing rounded
small tubercle, anterior margin covered with
stout rounded spinules; posterior margin
slightly concave; posterolateral margin with
broad raised areas, bearing two dorsal apotomal
sensilla; antenna extending posteriorly to
midleg; mouthparts (Fig. 29) with mandible well
developed; palpus extending to posterolateral
margin of labium; labium separated medially by
labrum; apex of labrum nearly straight; sensilla:
dorsal apotomals (Fig. 17): DA-1-H long, stout
seta, located on rounded small tubercle, DA-2-H
campaniform sensillum; DAL 0. 22 mm; DAW 0.21
mm; DAW/DAL 0.95; two dorsolateral cephalic
sclerites (Fig. 30): DL-1-H short, stout seta, DL-
2-H campaniform sensillum; clypeal/labrals (Fig. 29): CL-1-H medium-sized, thin seta CL-2- H long,
thin seta; oculars (Fig. 29): O-1-H medium-sized,
thin seta, O-2-H campaniform sensillum, O-3-H
short, thin seta. Cephalothorax rectangular,
surface of mesonotum with small spinules
between bases of respiratory organs. Length of
cephalothorax 1.56 mm, width 1.11 mm. Thorax:
Respiratory organ (Figs. 16, 18, 31) smooth, pale
brown except distal 1/4 darker, about 4.42 times
longer than broad, rounded apex, with simple
row of 18–20 pores closely abutting at apex;
pedicel (Figs. 16, 31) slender, P 0.02 mm; RO
length 0.23 mm, RO width 0.05 mm; P/RO 0.10;
sensilla: three anteromedials (Fig. 31): AM-1-T
long, stout seta, AM-2-T long, thin seta; AM-3-T
campaniform sensillum; anterolateral (Fig. 31):
AL-1-T short, stout seta; dorsals (Fig. 32): D-1-T,
D-2-T, D-4-T, long, thin setae, D-3-T campaniform
sensillum, D-5-T short, thin seta, all on small
rounded tubercle except D-3-T; supraalar (SA-
2-T) campaniform sensillum; metathoracic
(Fig. distincta group 41): M-3-T campaniform sensillum, near
anterior margin of metathorax. Abdomen: 04-I-2017, D. Anjos-Santos and P. Pessacq, 1
male (with pupal exuvium); same data except
adult emerged 06-I-2017, D. Anjos-Santos and
P. Pessacq, 1 male (with pupal exuvium); same
data except pupa emerged in laboratory 01-I-
2017, adult emerged 09-I-2017, D. Anjos-Santos
and P. Pessacq, 1 female (with larval and pupal
exuviae); same data except pupa emerged in
laboratory 02-I-2017, adult emerged 10-I-2017,
D. Anjos-Santos and P. Pessacq, 1 female (with
larval and pupal exuviae). Bionomics. Larvae and pupae were collected
in an unnamed stream located in the Parque
Nacional Los Alerces in western Chubut Province. The stream flows into the Futalaufquen lake
and is surrounded by Subantarctic Nothofagus
forest. Immatures were collected in a puddle of
water with muddy bottom, decomposing organic
matter and filamentous algae formed among
the roots of a tree, on the bank of the stream. The water temperature ranged between 13–14
°C, and the air temperature between 28–32 °C. Under laboratory conditions, the larvae took
around 5 days to reach the pupal stage, and 9
days to complete its development to the adult
stage. Specimens collected as pupae completed
their development in 4–9 days in the laboratory. Palpomyia subfuscula Ingram & Macfie
(Figs.16–
19, 29–32, 41–42) Palpomyia subfuscula Ingram & Macfie
(Figs.16–
19, 29–32, 41–42) 19, 29–32, 41–42) Palpomyia subfuscula Ingram & Macfie 1931: 216
(female; Argentina); Wirth 1974: 55 (in catalog of
species south of USA); Borkent & Wirth 1997: 134
(in World catalog); Spinelli 1998: 326 (in list of
Argentinean species); Borkent & Spinelli 2000:
64 (in catalog of species south of USA); Borkent
& Spinelli 2007: 97 (in Neotropical synopsis);
Spinelli & Marino 2009: 205 (in list of Patagonian
species); Spinelli et al. 2009: 60 (in revision of
Patagonian species, diagnosis, description, key,
distribution); Borkent 2016: 172 (in online World
catalog). An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 7 | 13 DANIELLE ANJOS-SANTOS et al. IMMATURES OF Palpomyia segments covered with small spicules, segments
with simple setae; segment 9 (Figs. 18–19)
approximately 1.3 longer than width, length 0.31
mm, width 0.24 mm; dorsal and ventral surface
covered with pointed spicules; ventral surface
with one small, circular mark located on a wide
bare, mesal area; terminal process (Figs. 18–19)
moderately short, nearly straight, base wide,
smooth, extreme tips somewhat dark, length
0.11 mm, width 0.05 mm; sensilla: tergite 1 (Figs. 18, 41) with two anteromesals: D-2-I short, stout
seta, D-3-I long, thin seta; 5 posterior sensilla:
D-4-I, D-7-I campaniform sensilla, D-5-I short,
stout seta, D-8-I short, thin seta, D-9-I long,
thin seta; 3 lateral sensilla: L-1-I long, thin seta,
L-2-I, L-3-I short, stout setae; segment 4 (Figs. 18, 42): D-2-IV short, stout seta, D-3-IV long, thin
seta, D-4-IV, D-7-IV campaniform sensilla, D-5-
IV short, stout seta, D-8-IV medium-sized, stout
seta, D-9-IV long, thin seta; L-1-IV, L-2-IV, L-4-IV
medium-sized, stout setae, L-3-IV long, thin seta,
all on small rectangular tubercles; V-5-IV, V-7-
IV short, stout setae, V-6-IV long, thin seta, all
on small subquadrangular tubercles; segment
9 (Figs. 18–19) with D-5-IX, D-6-IX campaniform
sensilla. USA species); Johannsen 1952: 166 (in key); Snow
et al. 1957: 34 (habitat notes); Wirth 1965: 140
(distribution). Palpomyia subaspera: Grogan & Wirth
1975: 10 (lectotype designation; redescription);
Grogan & Wirth 1979: 23 (description, adults,
pupa; distribution; P. essigi as synonym); Spinelli
& Wirth 1993: 68 (P. maculicrus as synonym);
Borkent & Wirth 1997: 134 (in World catalog);
Spinelli 1998: 326 (in list of Argentinean species);
Borkent & Spinelli 2000: 64 (in catalog of species
south of USA); Borkent & Spinelli 2007: 96 (in
Neotropical synopsis); Spinelli et al. Palpomyia subfuscula Ingram & Macfie
(Figs.16–
19, 29–32, 41–42) 2009: 46
(revision of patagonian species, diagnosis,
description, key, distribution); Borkent 2016: 172
(in online World catalog); Cazorla et al. 2018: 8
(in list of Punta Lara Reserve). Palpomyia essigi Wirth 1952: 225 (female,
male; California); Wirth 1965: 140 (distribution).i Palpomyia maculicrus Ingram & Macfie 1931:
230 (female; Argentina); Wirth 1974: 55 (in catalog
of species south of USA). Redescription of female pupa (Figs. 20–
23, 33–37, 43–44). Habitus as in Fig. 22. Exuviae
brownish. Total length 4.02 mm. Head: Dorsal
apotome (Fig. 21) with disc 2 times broader
than long, covered with stout spines, anterior
margin triangular, posterior margin concave,
posterolateral margin with broad raised areas,
bearing two dorsal apotomal sensilla; antenna
extending posteriorly to midleg; mouthparts
(Fig. 34) with mandible well developed; palpus
extending to posterolateral margin of labium;
labium separated medially by labrum; apex of
labrum truncated; sensilla: dorsal apotomals
(Fig. 21): DA-1-H elongate, thin seta, located on
rounded small tubercle, DA-2-H campaniform
sensillum; DAL 0.23 mm; DAW 0.24 mm; DAW/DAL
1.04; two dorsolateral cephalic sclerites (Fig. 35):
DL-1-H short, stout seta, DL-2-H campaniform
sensillum; clypeal/labrals (Fig. 34): CL-1-H short,
thin seta, CL-2-H medium-sized, thin seta; Distribution. Argentina (Neuquén, Río
Negro and Chubut Provinces); Chile (Ñuble,
Concepción, Valdivia, Casa Pangue, Chiloe and
Mechuque Islands). Material examined. Argentina, Neuquén,
Parque Nacional Nahuel Huapi, Río Cuyín
Manzano, 40°44ʹ13ʹʹS; 71°09ʹ17ʹʹW, 760 m, 06-II-
2009, A. Siri, 1 female (with pupal exuvium). An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 9 | 13 tibialis group Palpomyia subaspera (Coquillett)
(Figs. 20–23, 33–37, 43–45)
Ceratopogon subasper Coquillett 1901: 606
(female; USA). Palpomyia subaspera (Coquillett)
(Figs. 20–23, 33–37, 43–45) ( g
,
,
)
Ceratopogon subasper Coquillett 1901: 606
(female; USA). Palpomyia subasper: Malloch 1914: 22
(combination); Johannsen 1943: 784 (in list of An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 8 | 13 DANIELLE ANJOS-SANTOS et al. IMMATURES OF Palpomyia Figures 38-45. 38-39. Palpomyia
mapuche Spinelli, Grogan &
Ronderos, female pupa. 40. Palpomyia mapuche Spinelli,
Grogan & Ronderos, male pupa. 41-42. Palpomyia subfuscula Ingram
& Macfie, female pupa. 43-44. Palpomyia subaspera (Coquillett),
female pupa. 45. Palpomyia
subaspera (Coquillett), male pupa. 38, 41, 43. metathoracics and tergite
1 chaetotaxy, dorsal view; 39, 42,
44. segment 4 chaetotaxy, dorsal
and ventral view. 40, 45. segment 9,
ventral view. Scale 0.05 mm. Dorsal sensilla of segment 1 (D-2-I,
D-3-I, D-4-I, D-5-I, D-7-I, D-8-I, D-9-I);
dorsal sensilla of segment 4 (D-2-IV,
D-3-IV, D-4-IV, D-5-IV, D-7-IV, D-8-IV,
D-9-IV); dorsal sensilla of segment
9 (D-5-IX, D-6-IX); genital lobe
(GL); lateral sensilla of segment 1
(L-1-I, L-2-I, L-3-I); lateral sensilla of
segment 4 (L-1-IV, L-2-IV, L-3-IV, L-4-
IV); metathoracic sensillum (M-3-T);
terminal process (TP); ventral
sensilla of segment 4 (V-5-IV, V-6-IV,
V-7-IV). Figures 38-45. 38-39. Palpomyia
mapuche Spinelli, Grogan &
Ronderos, female pupa. 40. Palpomyia mapuche Spinelli,
Grogan & Ronderos, male pupa. 41-42. Palpomyia subfuscula Ingram
& Macfie, female pupa. 43-44. Palpomyia subaspera (Coquillett),
female pupa. 45. Palpomyia
subaspera (Coquillett), male pupa. 38, 41, 43. metathoracics and tergite
1 chaetotaxy, dorsal view; 39, 42,
44. segment 4 chaetotaxy, dorsal
and ventral view. 40, 45. segment 9,
ventral view. Scale 0.05 mm. 1
4
a
v
D
D
d
D
D
9 Dorsal sensilla of segment 1 (D-2-I,
D-3-I, D-4-I, D-5-I, D-7-I, D-8-I, D-9-I);
dorsal sensilla of segment 4 (D-2-IV,
D-3-IV, D-4-IV, D-5-IV, D-7-IV, D-8-IV,
D-9-IV); dorsal sensilla of segment
9 (D-5-IX, D-6-IX); genital lobe
(GL); lateral sensilla of segment 1
(L-1-I, L-2-I, L-3-I); lateral sensilla of
segment 4 (L-1-IV, L-2-IV, L-3-IV, L-4-
IV); metathoracic sensillum (M-3-T);
terminal process (TP); ventral
sensilla of segment 4 (V-5-IV, V-6-IV,
V-7-IV). oculars (Fig. 34): O-1-H, O-3-H short, stout setae,
O-2-H campaniform sensillum. Cephalothorax
rectangular, surface predominantly smooth with
small spinules on mesonotum, between bases
of respiratory organs. Length of cephalothorax
1.50 mm, width 1.01 mm. Thorax: Respiratory
organ (Figs. TAXONOMIC DISCUSSION Palpomyia mapuche and P. subfuscula belong
to the distincta group and P. subaspera to the
tibialis group, as they were defined by Grogan
& Wirth (1975). The first two species are herein
compared to each other and to their congeners
P. guarani, and P. subaspera with P. ryszardi. The larva of P. mapuche is very similar to
P. guarani by virtue of the labrum longer than
broad, the maxillary palpus long and cylindrical
with three apical papillae, the hypostoma finely
toothed, the epipharynx with curved auxiliary
sclerites, and the elongate and thin hypopharynx
lacking fringe. However, P. guarani differs by
the following combination of characters: the
head capsule is smaller (HL 0.30–0.32 mm) and
its setae are simple, the mandible has one
short and strong tooth and bears stout seta
on the aboral surface, the dorsal comb of the
epipharynx is armed with 2 lanceolate and stout
teeth interrupted by 1–2 shorter ones while
the ventral comb is unarmed, and the caudal
segment bears 6 pairs of setae (4 long, 2 thinner
ones). Ronderos et al. (2004) in the description
of the larva of P. guarani, incorrectly mentioned
that the messors are thin and omitted to
describe the palatar bar. A detailed revision of
the larva and pupa of P. guarani revealed that
the messors are stout and the palatal bar is
triangular, as they are herein described for P. mapuche. Redescription of male pupa (Figs. 33, 45). Similar to female with usual sexual differences:
Total length 3.11–3.95 (3.57, n = 4) mm. Dorsal
apotome (Fig. 33) pointed, ventral line of
weakness; DAL 0.21–0.24 (0.23, n = 2) mm; DAW
0.24–0.25 (0.25, n = 2) mm, DAW/DAL 1.11–1.62
(1.37, n = 2). Cephalothorax: length 1.26–1.44 (1.35,
n = 4) mm, width 0.86–0.95 (0.91, n = 2) mm. Respiratory organ 4.5–5.0 (4.8, n = 4) times longer
than broad, P 0.020–0.024 (0.021, n = 4) mm; RO
length 0.18–0.20 (0.19, n = 4) mm, RO width 0.04
(n = 3); P/RO 0.10–0.12 (0.11, n = 4). Segment 9 (Fig. 45) ventral surface with anterior band bearing
few pointed spicules, length 0.30–0.33 (0.32, n =
4) mm, width 0.17–0.24 (0.21, n = 3) mm; terminal
process length 0.10–0.14 (0.13, n = 4) mm, width
0.06–0.07 (0.06, n = 3) mm; genital lobe (Fig. 45),
longer than broad, surpassing base of terminal
process. Distribution. tibialis group 20, 22, 36) smooth, pale brown, about
3.77 times longer than broad, with rounded
apex, with simple row of 14 pores closely
abutting at apex; pedicel slender (Figs. 20, 36),
P 0.02 mm; RO length 0.20 mm, RO width 0.05
mm; P/RO 0.10; sensilla: three anteromedials
(Fig. 36): AM-1-T, AM-2-T long, stout setae, AM-
3-T campaniform sensillum; one anterolateral (Fig. 36): AL-1-T short, curved, stout seta; dorsals
(Fig. 37): D-1-T, D-2-T, D-4-T, D-5-T long, thin setae,
D-3-T campaniform sensillum, all on small
rounded tubercle; metathoracic (Fig. 43): M-3-T
campaniform sensillum, near anterior margin of
metathorax. Abdomen: segments with anterior
and posterior bands bearing small spicules;
with simple setae; segment 9 (Figs. 22–23)
approximately 1.6 longer than broad, length 0.31
mm, width 0.20 mm; dorsal and ventral surface
bearing band of pointed spicules on anterior
margin; dorsal and ventral surface smooth;
ventral surface with one small, ventral surface
with one small, star-shaped mark located on a (Fig. 36): AL-1-T short, curved, stout seta; dorsals
(Fig. 37): D-1-T, D-2-T, D-4-T, D-5-T long, thin setae,
D-3-T campaniform sensillum, all on small
rounded tubercle; metathoracic (Fig. 43): M-3-T
campaniform sensillum, near anterior margin of
metathorax. Abdomen: segments with anterior
and posterior bands bearing small spicules;
with simple setae; segment 9 (Figs. 22–23)
approximately 1.6 longer than broad, length 0.31
mm, width 0.20 mm; dorsal and ventral surface
bearing band of pointed spicules on anterior
margin; dorsal and ventral surface smooth;
ventral surface with one small, ventral surface
with one small, star-shaped mark located on a oculars (Fig. 34): O-1-H, O-3-H short, stout setae,
O-2-H campaniform sensillum. Cephalothorax
rectangular, surface predominantly smooth with
small spinules on mesonotum, between bases
of respiratory organs. Length of cephalothorax
1.50 mm, width 1.01 mm. Thorax: Respiratory
organ (Figs. 20, 22, 36) smooth, pale brown, about
3.77 times longer than broad, with rounded
apex, with simple row of 14 pores closely
abutting at apex; pedicel slender (Figs. 20, 36),
P 0.02 mm; RO length 0.20 mm, RO width 0.05
mm; P/RO 0.10; sensilla: three anteromedials
(Fig. 36): AM-1-T, AM-2-T long, stout setae, AM-
3-T campaniform sensillum; one anterolateral An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 9 | 13 DANIELLE ANJOS-SANTOS et al. IMMATURES OF Palpomyia wide bare, mesal area; terminal process (Figs. 22–
23) moderately short, nearly straight, base wide,
smooth, extreme tips somewhat dark, slightly
curved; length 0.13 mm, width 0.06 mm; sensilla:
tergite 1 (Figs. tibialis group 22, 43) with two anteromesals:
D-2-I short, stout seta, D-3-I long, thin seta; 5
posterior sensilla: D-5-I short, stout seta, D-4-I,
D-7-I campaniform sensilla, D-8-I short, stout
seta, D-9-I long, thin seta; 3 lateral sensilla: L-1-I
long, thin seta, on rounded small tubercle, L-2-I
minute seta, L-3-I short, stout seta on cylindrical
tubercle; segment 4 (Figs. 22, 44): D-2-IV short,
stout seta, D-3-IV short, thin seta, D-4-IV, D-7-IV
campaniform sensilla, D-5-IV short, stout seta,
D-8-IV minute seta, D-9-IV short, thin seta, all
on small rounded tubercles; L-1-IV, L-2-IV, L-4-
IV short, stout setae, L-3-IV short, thin seta, all
on small triangular tubercles; V-5-IV minute
seta, V-6-IV, V-7-IV short, thin setae, all on small
tubercles; segment 9 (Figs. 22–23) with D-5-IX,
D-6-IX campaniform sensilla. Florida), Mexico, Haiti, Cuba, Paraguay, Chile and
Argentina (Misiones, Buenos Aires, Río Negro
and Chubut Provinces). Florida), Mexico, Haiti, Cuba, Paraguay, Chile and
Argentina (Misiones, Buenos Aires, Río Negro
and Chubut Provinces). Material examined. Argentina, Chubut, Ruta
Provincial 35, 33 km SW Cushamen, unnamed
stream, 42°26’10.5”S; 70°30’31”W, 520 m, 10-XII-
2006, G. Rossi, 1 female, 4 males (with pupal
exuviae). An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 10 | 13 REFERENCES ALMEIDA JF, FARIAS ES, ALENCAR RB & PESSOA FAC. 2017. Description of two new species of Palpomyia Meigen
(Diptera: Ceratopogonidae) from the Brazilian Amazon. EntomoBrasilis 10: 118-112. In the redescription of the pupa of P. guarani
by Ronderos et al. (2004), the authors used the
terms proposed by Huerta et al. (2001). In this
study we homologate this terminology with the
one proposed by Borkent (2014). The pupa of
P. guarani is distinguished from P. mapuche by
the smaller dorsal apotome (DAL 0.08–0.11 mm),
the longer and stouter pedicel of the respiratory
organ which bears 10-12 pores, the presence
of two long and subequal dorsolateral setae,
one clypeal labral sensilla and two oculars,
the D-5-IV of the fourth segment represented
by a minute seta, and the straight and parallel
terminal processes of the caudal segment. ANJOS-SANTOS D, CAZORLA CG & RONDEROS MM. 2017. First
description of immature stages and adult females
of Stilobezzia (Acanthohelea) megatheca (Diptera:
Ceratopogonidae) from Patagonia. Ann Limnol - Int J Lim
53: 175-188. BORKENT A. 2014. The pupae of the biting midges of the
world (Diptera: Ceratopogonidae), with a generic key
and analysis of the phylogenetic relationships between
genera. Zootaxa 3879: 1-327. BORKENT A. 2014. The pupae of the biting midges of the
world (Diptera: Ceratopogonidae), with a generic key
and analysis of the phylogenetic relationships between
genera. Zootaxa 3879: 1-327. BORKENT A. 2016. World species of biting midges
(Diptera: Ceratopogonidae). http://wwx.inhs.illinois.edu/
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p. Last update May 16, 2016. Finally, the pupa of P. ryszardi differs from
P. subaspera by the shorter dorsal apotomal
sensilla D-1-H, the presence of one anteromedial
sensillum as pore, the medium-sized clypeal/
labrals, the presence of two oculars, the
medium-sized D-2-I and D-3-I on the first
abdominal tergite, the fourth segment with
D-5-IV represented by a minute seta, and the
genital lobe not reaching the posterior margin
of segment. We would like to remark that in the
original description, Spinelli & Ronderos (2013),
incorrectly indicate the anterolateral sensilla
(DL-1-H, DL-2-H) as dorsolateral sensilla (AL-1-T,
AL-2-T) and the sensilla DL-1-H, DL-2-H mentioned
are, in fact, the antennal sensilla. Besides, the
D-7-IV described as a seta was omitted in Fig. 23
of the mentioned publication. BORKENT A & SPINELLI GR. 2000. Catalogue of the New
World biting midges south of the United States of
America (Diptera: Ceratopogonidae). Contrib Entomol Int
4: 1-107. BORKENT A & SPINELLI GR. 2007. Neotropical
Ceratopogonidae (Diptera: Insecta). Acknowledgments We are grateful to Drs Pablo Pessacq, Augusto Siri and
Mariano Donato for their help in the field work, and
to PhD Miguel Archangelsky for the critical reading of
the manuscript. This work was funded by the Consejo
Nacional de Investigaciones Científicas y Técnicas
(CONICET, Argentina), PIP114 201101 00405 and PIP 0305. This is the contribution number 148 of LIESA (CIEMEP). TAXONOMIC DISCUSSION Canada (Alberta to Ontario),
United States of America (south to California, An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 10 | 13 DANIELLE ANJOS-SANTOS et al. IMMATURES OF Palpomyia The pupa of P. subfuscula differs from that
of P. mapuche by the longer and stouter DA-1-H,
the medium-sized and thinner O-1-H and the
shorter and thinner O-3-H; the respiratory organ
is pale brown except its distal 1/4 darker, with 18
pores. Besides, in the thorax the AM-2-T is longer
and thinner, in the first segment the D-5-I and
D-8-I are shorter and stouter, and in the fourth
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E-mail: danianjos_santos@comahue-conicet.gob.ar Correspondence to: Danielle Anjos-Santos
E-mail: danianjos_santos@comahue-conicet.gob.ar E-mail: danianjos_santos@comahue-conicet.gob.ar An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 13 | 13 SNOW WE, PICKARD E & MOORE JB. 1957 The Heleidae of the
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of the Patagonian predaceous midges of the genus An Acad Bras Cienc (2020) 92(Suppl. 2) e20190718 12 | 13 IMMATURES OF Palpomyia DANIELLE ANJOS-SANTOS et al. 1Laboratorio de Investigaciones en Ecología y Sistemática
Animal, (CIEMEP, UNPSJB), CONICET-CCT-PATAGONIA NORTE,
Gral. Roca 780, Esquel (9200), Chubut, Argentina
2Centro de Estudios Parasitológicos y de Vectores/
CEPAVE, CONICET-CCT-LA PLATA, Boulevard 120 s/n
e/61 y 62, La Plata (1900), Buenos Aires, Argentina
3Instituto de Limnología “Dr. Raúl Ringuelet” (ILPLA),
CONICET-CCT-LA PLATA, Boulevard 120 s/n e/Avda. 60
y calle 64, La Plata (1900), Buenos Aires, Argentina
4División Entomología, Museo de La Plata, UNLP-FCNYM, Paseo
del Bosque s/n, La Plata (1900), Buenos Aires, Argentina Author contributions DAS performed the fieldwork, mounted part of the material,
made all the drawings, worked the digital images, made
the MEB and drawing plates; DAS and FD performed most
of the morphological studies, wrote the manuscript and
manuscript revision; GRS identified the species; MMR made
photomicrographs and plates; MMR and GRS review the
manuscript.
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An automatically contamination-avoiding technique for intracorporeal esophagojejunostomy using a transorally inserted anvil during laparoscopic total gastrectomy for gastric cancer
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World journal of surgical oncology
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Hu et al. World Journal of Surgical Oncology (2015) 13:154
DOI 10.1186/s12957-015-0563-0 Hu et al. World Journal of Surgical Oncology (2015) 13:154
DOI 10.1186/s12957-015-0563-0 WORLD JOURNAL OF
SURGICAL ONCOLOGY Abstract Background: Intracorporeal Roux-en-Y esophagojejunostomy during laparoscopic total gastrectomy for gastric cancer
remains a challenging manipulation due to the uncontrolled direction of the jejunal side or unintended embedded
tissues, although several methods have been introduced. In this study, we simplified the procedure based on a surgical
string fixing technique using a transorally inserted anvil (OrVil™; Covidien Ltd., Mansfield, MA, USA). Methods: From March 2012 to September 2013, 14 consecutive patients underwent simplified intracorporeal
Roux-en-Y esophagojejunostomy using OrVil™during laparoscopic total gastrectomy for gastric cancer at our
hospital. Clinicopathologic characteristics and surgical outcomes of these patients were retrospectively analyzed. Results: All of the procedures were successful completed with no complication or conversion to open surgery. The mean
overall operative time was 193.8 ± 41.8 min, whereas the mean reconstruction time was 32.6 ± 4.6 min. The mean
estimated blood loss was 105.7 ± 65.4 ml. The mean diameter of anastomosis measured by upper gastrointestinal
contrast X-ray test at 1 month after operation was 2.3 cm. During a median follow-up period of 12 months, neither local
recurrence nor anastomosis-related morbidity was observed. Conclusions: Our preliminary results suggested that this automatically contamination-avoiding technique based on a
surgical-string-fixing strategy using OrVil™during laparoscopic total gastrectomy for gastric cancer might be feasible and
safe and provide a simple solution for intracorporeal Roux-en-Y esophagojejunostomy. Keywords: Gastric cancer, Laparoscopy, Esophagojejunostomy, Gastrectomy © 2015 Hu et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated. Open Access An automatically contamination-avoiding
technique for intracorporeal esophagojejunostomy
using a transorally inserted anvil during
laparoscopic total gastrectomy for gastric cancer Yan-Feng Hu1†, Da Wang1,2†, Tian Lin1, Ting-Yu Mou1, Hao Liu1, Tao Chen1, Zhen-Wei Deng1, X
Jiang Yu1* and Guo-Xin Li1* Yan-Feng Hu1†, Da Wang1,2†, Tian Lin1, Ting-Yu Mou1, Hao Liu1, Tao Chen1, Zhen-Wei Deng1, Xin Lu1,
Jiang Yu1* and Guo-Xin Li1* * Correspondence: balbc@163.com; gzliguoxin@163.com
†Equal contributors
1Department of General Surgery, Nanfang Hospital, Southern Medical
University, No. 1838 The North Guangzhou Avenue, Guangzhou 510515,
Guangdong, China
Full list of author information is available at the end of the article Background recently [1-4]. Among those methods, circular anasto-
mosis following transabdominal insertion of an anvil with
purse-string suture is the most frequently used pattern
[1,4]. However, the difficulties in terms of laparoscopically
creating a purse-string suture and fixing the anvil at the
esophageal stump always exist, increasing the potential
risk of complications and prolonging the operation time. Extracorporeal Roux-en-Y esophagojejunostomy during
laparoscopy-assisted total gastrectomy for gastric cancer is
one of the most challenging manipulations even in experi-
enced hands. To overcome the surgical difficulty and
maximize the clinical benefits from minimally invasive
surgery in the management of patients with proximal gas-
tric cancer, intracorporeal esophagojejunostomy, including
either circular or linear anastomosis, has been introduced To avoid these difficulties mentioned above, a transo-
rally inserted anvil (OrVil™; Covidien Ltd., Mansfield, MA,
USA) device has been developed in recent years [5-7]. Pre-
vious reports [6,8] have shown its technical feasibility and
potential advantages; however, the course of approxima-
tion between the anvil and center rod does not always
proceed safely by intracorporeal laparoscopic view due to * Correspondence: balbc@163.com; gzliguoxin@163.com
†Equal contributors
1Department of General Surgery, Nanfang Hospital, Southern Medical
University, No. 1838 The North Guangzhou Avenue, Guangzhou 510515,
Guangdong, China
Full list of author information is available at the end of the article Page 2 of 7 Page 2 of 7 Hu et al. World Journal of Surgical Oncology (2015) 13:154 the uncontrolled direction of the jejunal side or unintended
embedded tissues [7,8]. Thus, we attempted to simplify this
technique by adopting several tips with inexpensive and
available devices and evaluated its surgical safety in the
present study. Figure 1 Placement of the trocars. The left upper port site will be extended
transversely to an incision length of 3 to 4 cm for extraction of the specimen
and insertion of the circular stapler before anastomosis. RUP, right upper
port; LUP, left upper port; RLP, right lower port; LLP, left lower port. Methods
Patients Between March 2012 and September 2013, 14 consecu-
tive patients with proximal gastric adenocarcinoma
underwent simplified intracorporeal Roux-en-Y esopha-
gojejunostomy using a transorally inserted anvil system
(OrVil™; Covidien, Mansfield, MA, USA) during laparo-
scopic total gastrectomy at our hospital. These patients
followed a stepwise postoperative management proto-
col for diet resume from water to other liquids to semi-
fluids to normal food when the patient can tolerate the
diet satisfactorily and is free from anastomotic compli-
cation as early as possible after surgery and received
upper gastrointestinal contrast X-ray check of esopha-
gojejunostomy at postoperative 1 month. Clinicopatho-
logic characteristics and surgical outcomes of these
patients based on a prospectively maintained database
[9] were then retrospectively analyzed. The present
study was approved by the Ethics Committee of Nan-
fang Hospital (No. 2013087A). Figure 1 Placement of the trocars. The left upper port site will be extended
transversely to an incision length of 3 to 4 cm for extraction of the specimen
and insertion of the circular stapler before anastomosis. RUP, right upper
port; LUP, left upper port; RLP, right lower port; LLP, left lower port. Figure 2 Minilaparotomy at the left upper quadrant for specimen retrieval and stapler insertion. Figure 2 Minilaparotomy at the left upper quadrant for specimen retrieval and stapler insertion. Hu et al. World Journal of Surgical Oncology (2015) 13:154 Page 3 of 7 a
b
c
Figure 3 Placement of the anvil head. (a) The tube of the anvil head was inserted transorally (picture downloaded from the website of Covidien). (b) A small hole was made at the esophageal stump. (c) The thread was cut after fixation of the anvil head. c a b b c a Figure 3 Placement of the anvil head. (a) The tube of the anvil head was inserted transorally (picture downloaded from the website of Covidien). (b) A small hole was made at the esophageal stump. (c) The thread was cut after fixation of the anvil head. camera holder stood between the legs of the patient. Trocar placement is shown in Figure 1. Procedure before anastomosis Under general anesthesia, the patient was placed in the
supine position with legs slightly apart (relaxed dorsal
lithotomy position). The operator and assistant stood on
the patient’s left and right side, respectively, and the To obtain better exposure of the operative field, the falci-
form ligament and liver were suspended to the abdominal
wall using Hyung’s strategy [4]. After dissection of the a
b
d
c
e
f
Figure 4 A self-made single-site access system. (a) The circular stapler passed through the glove. (b) The stapler, jejunal stump, and loop were
fixed by the silk string. (c) A slipknot was made to fix the shaft. (d) Establishment of the pneumoperitoneum by the self-made single-site access
system. (e) Schematic of making a slipknot using a silk string. (f) Schematic of making a surgical knot to the center rod. Red arrow points to the
silk suture. The knot was released automatically during firing of the stapler without additional cutting. b a b a c c d f e Figure 4 A self-made single-site access system. (a) The circular stapler passed through the glove. (b) The stapler, jejunal stump, and loop were
fixed by the silk string. (c) A slipknot was made to fix the shaft. (d) Establishment of the pneumoperitoneum by the self-made single-site access
system. (e) Schematic of making a slipknot using a silk string. (f) Schematic of making a surgical knot to the center rod. Red arrow points to the
silk suture. The knot was released automatically during firing of the stapler without additional cutting. Page 4 of 7 Hu et al. World Journal of Surgical Oncology (2015) 13:154 anvil was then released (Figure 3c). The tube was re-
moved to expose the rod of the anvil sufficiently. At that
time, the insertion of the anvil was completed. regional lymph nodes, the duodenal bulb was transected
using a linear stapler laparoscopically, followed by transec-
tion of the distal esophagus with a proper distance from the
lesion using a linear stapler with flexible articulation. Next,
the entire specimen was removed through a 3- to 4-cm
minilaparotomy incision extending to the left upper port
site, where a wound protector (Beijing HangTian KaDi
Technology R & D Institute, Beijing, China) was used
(Figure 2). Insertion of the anvil transorally A small hole was created at one angle of the esophageal
stump laparoscopically. Concurrently, an anesthetist inserted
the tube of the anvil (OrVil™) transorally until the tip of the
tube passed through the small hole at the esophageal stump
(Figure 3a, b). A laparoscopic grasper was then used to
slowly drag the tube until the anvil rod came into view. Approximation between the anvil and center rod
The anvil and circular stapler were connected, and anasto-
mosis was performed under laparoscopic view directly Modification and tips of the intracorporeal
esophagojejunostomy The jejunum was transected 15 cm away from the
Treitz’s ligament using a linear stapler laparoscopically. A surgical glove, a minilaparotomy wound protector,
and the shaft of the stapler were integrated as a self-
made single-site access system (Figure 4). The stapler
was then positioned within the jejunal loop from the
distal jejunal stump. Both the jejunal end and loop were
anchored on the main unit with a silk string to create a
slipknot to fix the center rod to prevent separation and
avulsion (Figure 4). Discussion Laparoscopic total gastrectomy becomes significantly
challenging when the reconstruction procedure involves
intracorporeal esophagojejunostomy because it is diffi-
cult to perform a proper purse-string suture at the
esophageal stump and insert an anvil into the esophageal
lumen [4]. Even more troublesome is applying the stapl-
ing device appropriately under limited laparoscopic
views, particularly in obese patients [7]. Some reports
have introduced solutions to improve the procedure, but Approximation between the anvil and center rod The tube was lightly secured to fix the anvil at the
proper position. The thread connecting the tube and The anvil and circular stapler were connected, and anasto-
mosis was performed under laparoscopic view directly a
b
d
c
e
f
Figure 5 Intracorporeal anastomotic technique using a circular stapler. (a) Connection of the shaft and anvil. (b) Approximation of the shaft and
anvil. The knot was released automatically during firing of the stapler. (c) The stapler was carefully removed. (d) The jejunal stump was closed
using a linear stapler. (e) Schematic of approximation between the anvil and center rod. (f) Schematic of automatically removing an anchoring
string during firing. a a c e Figure 5 Intracorporeal anastomotic technique using a circular stapler. (a) Connection of the shaft and anvil. (b) Approximation of the shaft and
anvil. The knot was released automatically during firing of the stapler. (c) The stapler was carefully removed. (d) The jejunal stump was closed
using a linear stapler. (e) Schematic of approximation between the anvil and center rod. (f) Schematic of automatically removing an anchoring
string during firing. Figure 5 Intracorporeal anastomotic technique using a circular stapler. (a) Connection of the shaft and anvil. (b) Approximation of the shaft and
anvil. The knot was released automatically during firing of the stapler. (c) The stapler was carefully removed. (d) The jejunal stump was closed
using a linear stapler. (e) Schematic of approximation between the anvil and center rod. (f) Schematic of automatically removing an anchoring
string during firing. Hu et al. World Journal of Surgical Oncology (2015) 13:154 Hu et al. World Journal of Surgical Oncology (2015) 13:154 Page 5 of 7 Hu et al. World Journal of Surgical Oncology (2015) 13:154 Results (Figure 5). Moderation by rotating the stapler was easily
performed to make a fine alignment, precisely controlling
the direction of the jejunal side with little concern about
tearing embedded tissues. After confirming that the bilat-
eral crura of the diaphragm were not embedded in the
stapler (the anastomotic stoma was satisfactory), the stap-
ler was fired and loosened. Simultaneously, an anchoring
string on the small bowel and the circular stapler were re-
moved automatically without additional cutting. The cir-
cular stapler was removed gently from the anastomotic
bowel, and the quality of the anastomosis was checked by
identifying the donuts on the stapler. The jejunal stump
was closed 2 to 3 cm away from the esophagojejunal site
using a linear stapler. After side-to-side jejunojejunostomy
was performed by linear stapler laparoscopically, the con-
tinuity of the digestive tract was completed. The clinicopathologic features and operative results of the
patients are shown in Table 1. The mean overall operation
time was 193.8 ± 41.8 min. The mean reconstruction time
was 32.6 ± 4.6 min. No intraoperative complication was ob-
served. The mean first flatus time and hospital duration
were 3.29 ± 0.73 and 8.7 ± 3.2 days, respectively. The mean
diameter of anastomosis measured by upper gastrointes-
tinal contrast X-ray test at 1 month after operation was
2.3 cm (Figure 6). During a median follow-up period of
12 months, neither local recurrence nor anastomosis-
related morbidity was observed. Statistical analysis Demographic, operative, and clinicopathologic data of
the patients are expressed as mean ± standard deviation
or median (range) if they are continuous variables. Table 1 Clinicopathologic characteristics and operative
results of the patients
Variable
Value
Demography
Male/female, n
10/4
Age, years
59.0 ± 12.1
Body mass index, kg/m2
23.4 ± 2.2
Operation
Overall operation time, min
193.8 ± 41.8
Overall reconstruction time, min
32.6 ± 4.6
Time for anvil placement, min
8.4 ± 4.0
Time for esophagojejunostomy, min
23.8 ± 5.2
Estimated blood loss, ml
105.7 ± 65.4
Pathology
Stagea I/II/III, n
1/3/10
Proximal margin, cm
3.6 ± 1.7
No. of retrieved lymph nodes
33.9 ± 18.1
Immediate postoperative course
First flatus, mean, days
3.3 ± 0.7
Hospital duration, mean, days
8.7 ± 3.2
Mortality, n
0
Short-term anastomosis-related complications, n
0
Esophagojejunostomy diameter, cm
2.3 ± 0.3
Follow-up
Follow-up period (median, range), months
12 (6 to 24)
Long-term complications, n
0
Values are expressed as mean ± standard deviation unless otherwise indicated. aAccording to the American Joint Committee on Cancer seventh edition. Table 1 Clinicopathologic characteristics and operative
results of the patients Table 1 Clinicopathologic characteristics and operative
res lts of the patients Figure 6 Upper gastrointestinal contrast X-ray check of
esophagojejunostomy at postoperative 1 month. Long-term complications, n Figure 6 Upper gastrointestinal contrast X-ray check of
esophagojejunostomy at postoperative 1 month. Hu et al. World Journal of Surgical Oncology (2015) 13:154 Page 6 of 7 jejunal stump. A diameter of 25-mm anvil is the most
common choice at our institution for Chinese patients,
while a relatively larger size would be applied casually. Meanwhile, intraoperative examination and postopera-
tive early oral intake was also recommended [12]. In our
daily clinical practice, generally, a stepwise management
protocol for diet resume from water to other liquids to
semi-fluids to normal food should be carried out when
the patient can tolerate the diet satisfactorily and is free
from anastomotic complication as early as possible after
surgery. Theoretically, adding the solid food appropriately
in terms of volume and frequency might be helpful to dilate
the anastomotic site. Besides, routine upper gastrointestinal
contrast X-ray test at postoperative 1 month is recom-
mended for early detection of stricture. problems remain such as the requirement for special ex-
pensive instruments that are not always available or add-
itional skills [4,10,11]. Authors’ contributions
d
b
d YFH and DW contributed equally to this work and should be considered
co-first authors. DW and YFH wrote the paper. YFH, DW, TL, TYM, HL, CT,
ZWD, and XL collected and analyzed the data. JY and GXL designed the
study and reviewed the article. All authors read and approved the final
manuscript. Acknowledgements This work was supported by the Research Fund of Public welfare in Health
Industry (No. 201402015), the Major Program of Science and Technology
Program of Guangzhou (No. 201300000087), the National Key Technology R
& D Program (no. 2013BAI05B00), and the Development Center for Medical
Science and Technology, Ministry of Health of China (no. W2011WAI44), and
the Key Clinical Specialty Discipline Construction Program. Statistical analysis A linear stapler has been chosen
by some surgeons instead of a circular one to perform
esophagojejunostomy laparoscopically that unsatisfactor-
ily overcomes the limitation of requiring a certain
esophageal length and an intracorporeal hand-suture [3]. To solve these problems and overcome the limitations,
we describe herein a simplified technique for intracorporeal
esophagojejunostomy using a circular stapler. No special
technique is required, and the simplified procedure can be
easily performed. Its safety and feasibility has been sug-
gested in a series of 14 patients, of whom none suffered
postoperative anastomosis-related complications. Transoral insertion of an anvil (OrVil™) provides a so-
lution for secure anvil placement in the esophagus. With
an anesthesiologist’s help, pharyngeal or esophageal in-
jury can be effectively avoided. Our method to manipu-
late the process is to open a small incision at the
alternative edge of the esophageal stump after dividing
the esophagus horizontally using a linear stapler and se-
curing the anvil with a pair of grasping forceps after
introducing the anvil tube into view. Thus, the anvil is
guaranteed to be placed at an appropriate position with-
out tearing the esophageal stump facilitating subsequent
operations. Conclusions Our
initial
findings
show
that
this
automatically
contamination-avoiding technique based on a surgical-
string-fixing strategy is technically feasible and could
provide a simple solution for intracorporeal Roux-en-Y eso-
phagojejunostomy. However, a perspective study is needed
to evaluate the surgical safety of this technique. Competing interests
Th
h
d
l
h Competing interests
The authors declare that they have no competing interests. p
With similar methods in the recently published litera-
ture [7], problems such as distortion of the Roux limb or
mesenterium and slipping of the esophagojejunal anasto-
motic site into the low mediastinum have not yet met
ideal solutions. As presented above, fixation of the circu-
lar stapler and glove was followed by fixation of the je-
junal loop and glove, causing the stapler-glove-jejunal
loop to integrate and form a self-made single-site access
system that prevents slippage of the jejunal loop out of
the circular stapler during the intracorporeal procedure. Additionally, flexible mobilization is enabled to make a
fine alignment, ensuring optimal functioning of the re-
constructive digestive tract. The small bowel was more
easily held backward to prevent it from becoming
trapped between the anvil and circular stapler. The
sealed space, established by turning over the glove edge
to seal off the wound protector, allowed for a clearer vis-
ual field for the laparoscopic operation. Thus, tissue in-
jury caused by excessive traction or slight errors can be
minimized. Moreover, the anchoring suture could be cut
along with the donut during the scalper firing and sepa-
rated automatically without additional cutting when the
main unit was pulled back. Thus, the simplified tech-
nique does not significantly create excessive steps and
prolong the overall operative time. To prevent possible
anastomotic stenosis, selection of the proper anvil size
of circular stapler should be considered carefully, which
usually depends on both the diameter of esophageal and Received: 5 November 2014 Accepted: 26 March 2015 Received: 5 November 2014 Accepted: 26 March 2015 Author details
1 1Department of General Surgery, Nanfang Hospital, Southern Medical
University, No. 1838 The North Guangzhou Avenue, Guangzhou 510515,
Guangdong, China. 2The Key Laboratory of Cancer Prevention and
Intervention, China National Ministry of Education, Department of Surgical
Oncology, The Second Affiliated Hospital of Zhejiang University School of
Medicine, 88 Jie-Fang Road, Hangzhou 310009, Zhejiang Province, China. Hu et al. World Journal of Surgical Oncology (2015) 13:154 Hu et al. World Journal of Surgical Oncology (2015) 13:154 4. Kim HI, Cho I, Jang DS, Hyung WJ. Intracorporeal esophagojejunostomy
using a circular stapler with a new purse-string suture technique during
laparoscopic total gastrectomy. J Am Coll Surg. 2013;216:e11–6. 4. Kim HI, Cho I, Jang DS, Hyung WJ. Intracorporeal esophagojejunostomy
using a circular stapler with a new purse-string suture technique during
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the transorally inserted anvil (OrVil™) after laparoscopy-assisted total gastrectomy. Surg Endosc. 2011;25:1300–5. 5. Kunisaki C, Makino H, Oshima T, Fujii S, Kimura J, Takagawa R, et al. Application of
the transorally inserted anvil (OrVil™) after laparoscopy-assisted total gastrectomy. Surg Endosc. 2011;25:1300–5. g
6. Mou T, Hu Y, Yu J, Wang Y, Zhao L, Li G. Clinical value of transorally inserted
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surgery. World J Surg. 1992;16:541–4. Submit your next manuscript to BioMed Central
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and take full advantage of:
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• Research which is freely available for redistribution
Submit your manuscript at
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https://openalex.org/W2605895611
|
https://www.ann-geophys.net/35/683/2017/angeo-35-683-2017.pdf
|
English
| null |
Causal kinetic equation of non-equilibrium plasmas
|
Annales geophysicae
| 2,017
|
cc-by
| 5,920
|
Causal kinetic equation of non-equilibrium plasmas Correspondence to: Rudolf A. Treumann (art@geophysik.uni-muenchen.de) Abstract. Statistical plasma theory far from thermal equilib-
rium is subject to Liouville’s equation, which is at the base
of the BBGKY hierarchical approach to plasma kinetic the-
ory, from which, in the absence of collisions, Vlasov’s equa-
tion follows. It is also at the base of Klimontovich’s approach
which includes single-particle effects like spontaneous emis-
sion. All these theories have been applied to plasmas with
admirable success even though they suffer from a funda-
mental omission in their use of the electrodynamic equa-
tions in the description of the highly dynamic interactions
in many-particle conglomerations. In the following we ex-
tend this theory to taking into account that the interaction
between particles separated from each other at a distance re-
quires the transport of information. Action needs to be trans-
ported and thus, in the spirit of the direct-interaction theory
as developed by Wheeler and Feynman (1945), requires time. This is done by reference to the retarded potentials. We de-
rive the fundamental causal Liouville equation for the phase
space density of a system composed of a very large num-
ber of charged particles. Applying the approach of Klimon-
tovich (1967), we obtain the retarded time evolution equation
of the one-particle distribution function in plasmas, which
replaces Klimontovich’s equation in cases when the direct-
interaction effects have to be taken into account. This be-
comes important in all systems where the distance between
two points |1q| ∼ct is comparable to the product of obser-
vation time and light velocity, a situation which is typical in
cosmic physics and astrophysics. Ann. Geophys., 35, 683–690, 2017
www.ann-geophys.net/35/683/2017/
doi:10.5194/angeo-35-683-2017
© Author(s) 2017. CC Attribution 3.0 License. Ann. Geophys., 35, 683–690, 2017
www.ann-geophys.net/35/683/2017/
doi:10.5194/angeo-35-683-2017
© Author(s) 2017. CC Attribution 3.0 License. Causal kinetic equation of non-equilibrium plasmas
Rudolf A. Treumann1,2 and Wolfgang Baumjohann3
1Department of Geophysics and Environmental Sciences, Ludwig-Maximilians-Universität München, Munich, Germany
2International Space Science Institute Bern, Bern, Switzerland
3Space Research Institute, Austrian Academy of Sciences, Graz, Austria
Correspondence to: Rudolf A. Treumann (art@geophysik.uni-muenchen.de)
Received: 20 April 2017 – Accepted: 2 May 2017 – Published: 24 May 2017 Published by Copernicus Publications on behalf of the European Geosciences Union. R. A. Treumann and W. Baumjohann: Causal Kinetic Theory the signal-emitting particles to the signal-receiving parti-
cles under consideration, i.e. the absorbers and reactors. Their sources are the charge and current densities ρm
a (q,t),
jm(q,t), which are assumed to be known at any instant t
in all space points q. Obtaining this knowledge is impossi-
ble as it requires instantaneous measurements at time t of all
positions q and momenta p of the particles present in real
space. Instead, the information must be synchronized among
all locations. This is taken care of in the Liénard–Wiechert
potentials, which explicitly account for the transport of infor-
mation from point q′ to point q. In this case in the Lorentz
gauge ρm
a (q,t) = ea
Na
X
i=1
δ
q −qai(t)
= ea
Z
d3p Na(p,q,t),
jm
a (q,t) = ea
ma
Na
X
i=1
pai(t) δ
q −qai(t)
= ea
ma
Z
d3p p Na(p,q,t),
(2 = ea
ma
Z
d3p p Na(p,q,t),
(2) (2) where the exact 6-D phase space density is defined through Na(p,q,t) =
Na
X
i=1
δ
p −pai(t)
δ
q −qai(t)
(3) Em = −∇qφm(q,t) −∂tAm(q,t),
Bm = ∇q ∧Am(q,t),
(6) (3) (6) and qai(t),pai(t) are the spatial and momentum phase space
trajectories which the particle ai performs in the phase space
under the action of the complete microscopic electromag-
netic field (E,B)m, which it feels at its location q = qai(t) at
time t. Liouville’s equation for the exact phase space density
can then be written in the form the correct scalar and vector potentials are to be expressed by
the retarded charge and current densities φm(q,t) =
1
4πϵ0
X
a
Z
d3q′ ρm
a (q′,t′)
|q −q′| ,
Am(q,t) = µ0
4π
X
a
Z
d3q′ jm
a (q′,t′)
|q −q′|
(7) (7) ∂Na
∂t
+ p
ma
· ∇q Na + ea
h
Em(q,t)
+ p
ma
∧Bm(q,t)
i
· ∂Na
∂p
= 0. (4) taken at the retarded time t′ = t −|q −q′|/c,
q ̸= q′
(8) (4) (8) of arrival of all the signals emitted at t′ from all the particles
at spatial distance |q−q′| from the location of particle ai at q
and at time t. This also implies that in the expressions for the
charge and current densities Na →Na(p,q′,t′) is a function
of the retarded time t′. R. A. Treumann and W. Baumjohann: Causal Kinetic Theory This is Klimontovich’s equation for the exact microscopic
phase space density Na(p,q,t) in 6-D phase space (Klimon-
tovich, 1967). It is a tautology because it does not say any-
thing other than that particle number is conserved along all
the dynamical orbits of the particles in phase space under
the action of their mutual electromagnetic fields. The micro-
scopic fields it contains are given by Maxwell’s equations in
differential form: Since all particles serve both as field sources and actors,
excluding their self-interaction, the use of the instantaneous
fields ignores the time-consuming signal transport and thus
cannot be correct. It is an approximation only that holds for
comparably small volumes such that, in the expression for
the retarded time, the spatial difference can be neglected. Thus, the restriction on the distance between particles is that
|1q| ≪c1t. (9) ∇q ∧Bm = µ0jm + µ0ϵ0∂tEm,
∇q · Bm = 0,
∇q ∧Em = −∂tBm,
∇q · Em = 1
ϵ0
X
a
ρm
a . (5) (5) (9) |1q| ≪c1t. The solution of this set of equations is not possible as it re-
quires knowledge of all microscopic particle orbits. One can,
however introduce some coarse graining procedure and de-
fine integrated distribution functions which ultimately reduce
the system to a set of equations known as Klimontovich–
Vlasov equations for a one-particle phase space distribution
in the presence of the average electromagnetic fields. This
procedure is very efficient, and we will follow it below in a
modified version. Clearly, this condition will readily be violated in large vol-
umes of cosmic and astrophysical size, where one must refer
to the above precise potentials and the fields resulting from
them in reference to the Lorentz gauge. For single-particle–particle interactions, this problem has
been discussed in depth in seminal papers by Wheeler and
Feynman (1945, 1949). They showed that in a closed sys-
tem where no information is lost to the outside eliminating
any self-interaction of a particle with its proper electromag-
netic field implies that the fields are properly described via
retarded potentials only as done above. These potentials ac-
count for the emission of a signal by one particle and the ab-
sorption of the signal after some travel time by the target par-
ticle, causing this particle to interact. The emitted signals be-
long to advanced potentials which, when correctly included, 1
Introduction The starting point of (classical) kinetic theory is Liou-
ville’s equation. Written in terms of the Na-particle Hamilto-
nian HNa(q,p,t) and defining the 6-D phase space density
Na(q,p,t) of species a, both functions of space q and mo-
mentum p, it becomes ˙Na ≡∂tNa +
HNa,Na
= 0,
(1) (1) where it is assumed that the particle number Na of species a
is conserved (along all dynamical phase space orbits). Oth-
erwise the right-hand side would contain the difference of
number sources and losses Sa −La. This equation, under the
assumptions made, is completely general, applying to any
system consisting of Na ≫1 particles in interaction with an
external as well as with their mutual fields, of which they
function as sources. These fields are contained in the Hamil-
tonian and act via the Poisson bracket [...]. In view of an application to plasmas, the relevant field
is the electromagnetic field E,B, with the particles carry-
ing electric charges ea = ∓e ≡ae (with a = −, +) being the
sources of the field. For simplicity, in the following, we re-
strict ourselves to electrons and ions (protons) of mass ma,
and gravity can be neglected on all scales small enough
for the electromagnetic fields to dominate. We also assume
global quasi-neutrality and the absence of any external fields. Then E,B = (E,B)m is the set of microscopic electromag-
netic fields produced solely by the microscopic charge and
current densities of the interacting particle components a,
which serve as their sources: AnGeo Communicates Keywords. Space plasma physics (General or miscella-
neous) Published by Copernicus Publications on behalf of the European Geosciences Union. R. A. Treumann and W. Baumjohann: Causal Kinetic Theory 684 www.ann-geophys.net/35/683/2017/ R. A. Treumann and W. Baumjohann: Causal Kinetic Theory subtract out, thereby restoring the required real-world causal-
ity. It is incorrect to assume that the information arrives mi-
croscopically instantaneously at the target, causing this to
act. The electromagnetic fields following from the Lorentz
gauge in the microscopic domain are These expressions are implicit for both the charge and cur-
rent densities. In order to relate them to the exact microscopic
phase space distribution Na as defined in Eq. (3), one refers
to the representations (Eq. 2) of the charge and current densi-
ties. This shows that the functional dependence of the phase
space density is itself implicit. It depends on itself, taken at
all the positions q′ and retarded times t′. Em(q,t)=
1
4πϵ0
Z
d3q′
ρm(q′,t′)
|q −q′|3
+∂t ρm(q′,t′)
c|q −q′|2
(q −q′) −∂t jm(q′,t′)
c2|q −q′|
,
Bm(q,t)= µ0
4π
Z
d3q′
jm(q′,t′)
|q −q′|3 + ∂t jm(q′,t′)
c|q −q′|2
∧
q −q′,
(10) The proper way of dealing with this problem is to focus on
the microscopic picture for as long as possible. There, all the
charged particles can be imagined as moving in a vacuum as
long as the medium is sufficiently dilute. By progressing to
a coarse-grained picture one may afterwards advance to con-
sidering a more continuous medium in which ultimately the
propagation properties of the signals will become modified
by the collective properties of the matter. (10) which were first given, independently, by Panofsky and
Phillips (1962) and Jefimenko (1966). One should note that
in these expressions the charge ρm and current densities jm
are summed over all particle species a. With these results it is convenient to express the micro-
scopic electromagnetic fields through the microscopic phase
space densities of the particle species: This explicit representation of the microscopic fields ac-
counts properly for the time delay between the signal emit-
ted from the total compound of primed particles to arrive at
the location q of the particle under consideration. Since the
microscopic charge and current densities are functionals of
the phase space density, these expressions contain the latter,
though in a more involved manner than when using the dif-
ferential forms of the electrodynamic equations, which do
not show where and whether the retardation of the signal is
taken into account. It is clear from these expressions that par-
ticles which are far away from the target do not affect it. 2
Effect of retardation The problem of the above equations is that they do not
account for the fact that the electromagnetic signal of the
presence and motion of the particles is transferred from www.ann-geophys.net/35/683/2017/ Ann. Geophys., 35, 683–690, 2017 685 R. A. Treumann and W. Baumjohann: Causal Kinetic Theory The
main effect will always come from close neighbours. Em(q,t) =
X
a
ea
4πϵ0
Z
d3p d3q′
Na(p,q′,t′)
|q −q′|3
+ ∂t Na(p,q′,t′)
c|q −q′|2
(q −q′) −p ∂t Na(p,q′,t′)
c2|q −q′|
,
(13)
Bm(q,t) =
X
a
eaµ0
4π
Z
d3p d3q′
pNa(p,q′,t′)
|q −q′|3
+
+ p ∂t Na(p,q′,t′)
∧
q −q′
(14) (14) These are the expressions of the electromagnetic field which
have to be used in the microscopic Liouville Eq. (4) for the
microscopic N-particle phase space density. Not only do they
couple the different particle species, thus leading to a cou-
pling between their phase space distributions, they also make
each microscopic distribution Na a functional of the distri-
butions taken at all different phase space locations which are
causally accessible via their retarded times of signal propa-
gation t′. Clearly, this is a substantial complication, which is
introduced into kinetic theory by the requirement of causal-
ity. www.ann-geophys.net/35/683/2017/ 3
Retarded charge and current densities Taking the divergence of the microscopic electric field and
the curl of the microscopic magnetic field, one readily reads
the correct microscopic charge and current densities when
comparing the expressions with the microscopic Maxwell
equations: ρm(q,t) = 1
4π
X
a
∇q ·
Z
d3q′
ρm
a (q′,t′)
|q −q′|3
+ ∂t ρm
a (q′,t′)
c|q −q′|2
q −q′
−∂t jm
a (q′,t′)
c2|q −q′|
,
(11)
jm(q,t)= 1
4π
X
a
∇q ∧
Z
d3q′
jm
a (q′,t′)
|q −q′|3 + ∂t jm
a (q′,t′)
c|q −q′|2
∧
q −q′
−∂t
Z
d3q′
ρm
a (q′,t′)
|q −q′|3
+ ∂t ρm
a (q′,t′)
c|q −q′|2
q −q′
−∂t jm
a (q′,t′)
c2|q −q′|
. (12) It is quite inconvenient to deal with all microscopic phase
space densities. We would rather have separate equations for
them. This can be achieved when observing that Eq. (4) is an
equation for Na. Thus, putting a →b in the last expressions,
which means that we sum over all particle species b including
also b = a (with self-interaction excluded by the definition of
the retarded time), we have (11) (12) These are the correct forms of the charge and current
densities summed over species a which have to be used in
Maxwell’s equations in order to account for the retarded
transfer of information between the particles in the plasma. 4
Average distribution functions 1
ϵ0
Nb(p′,q′,t′)
|q −q′|3
+ ∂t Nb(p′,q′,t′)
c|q −q′|2
q −q′
−p′ ∂t Nb(p′,q′,t′)
c2|q −q′|
−µ0
ma
p ∧
q −q′
∧
p′Nb(p′,q′,t′)
|q −q′|3
+ p′ ∂t Nb(p′,q′,t′)
c|q −q′|2
Dealing with the causal N-particle kinetic Eq. (15) is im-
practical. One wants to reduce it to an equation for a one-
particle distribution function in 6-D phase space for indis-
tinguishable particles of sort a. This is done by integrating
out in Eq. (3) all particle coordinates i > 1. Defining phase
space coordinates x = (p,q), xai(t) =
pai(t),qai(t)
, the
N-particle density becomes
· ∂
∂p Na
p,q,t
= 0. (15) (15) Na(x,t) =
Na
X
i=1
δ
x −xai(t)
. (16) (16) Here Na(p,q,t), while Nb(p′,q′,t′) depends on the dummy
coordinates of all particles b of integration and on the re-
tarded time t′ = t −|q −q′|/c, q ̸= q′. Thus, in the q′ in-
tegration all particles of sort a are also included, with the
exception of the particle located at q = q′ at time t. Following Klimontovich (1967), let us define the one-
particle distribution of sort a of indistinguishable particles
by The above Eq. (15) is the causal Liouville equation act-
ing on the microscopic Na-particle phase space density
Na(p,q,t) in the presence of a large number of charged par-
ticles interacting via their self-consistently generated elec-
tromagnetic fields. It extends Klimontovich’s equation to the
correct inclusion of the retardation effect of transmission of
information between the particles via electromagnetic fields. fa(xa1,t) = Va
Z
fNd6xa2...d6x6
aNa×
×
Y
b̸=a
d6xb1...d6x6
bNb. (17) (17) The N-particle probability distribution fN depends on all
the particle coordinates in phase space which have been in-
tegrated out in the last expression, including xa1, and V is
the spatial volume of particle a1, i.e. the volume all indistin-
guishable particles occupy. With its help the averaged phase
space density yields directly The N-particle probability distribution fN depends on all
the particle coordinates in phase space which have been in-
tegrated out in the last expression, including xa1, and V is
the spatial volume of particle a1, i.e. the volume all indistin-
guishable particles occupy. With its help the averaged phase
space density yields directly The inclusion of information transport between the inter-
acting particles substantially complicates the basic kinetic
equation. R. A. Treumann and W. Baumjohann: Causal Kinetic Theory tributions, we will consider Liouville’s equation without ex-
plicit reference to the fields. ∂Na
∂t
+ p
ma
· ∇qNa +
X
b
eaeb
4π
Z
d3p′ d3q′
1
ϵ0
Nb(p′,q′,t′)
|q −q′|3
+ ∂t Nb(p′,q′,t′)
c|q −q′|2
q −q′
−p′ ∂t Nb(p′,q′,t′)
c2|q −q′|
−µ0
ma
p ∧
q −q′
∧
p′Nb(p′,q′,t′)
|q −q′|3
+ p′ ∂t Nb(p′,q′,t′)
c|q −q′|2
· ∂
∂p Na
p,q,t
= 0. ( 4
Average distribution functions It causes a delay in response, and thus refers to a
natural measuring process in which the particles are not only
generators of the electromagnetic field but also measure its
effect over a causal distances accessible to them. The delay
must thus necessarily cause decorrelation of the response. Na
Va
fa(p,q,t) =
Na(p,q,t)
. (18) (18) There is another complication with this picture which
comes into play when considering large compounds of par-
ticles rather than single particles. Single charged particles
are assumed to move in the vacuum; the signal propaga-
tion between them takes place at light speed c. Immersed in
the comparably dense environment of all the other charged
particles, any light or radiation experiences radiation trans-
port, which is dominated by scattering, reflection, transmis-
sion and absorption, processes that occur due to the active
response of the environment to the presence of radiation and
depend on the capabilities of the medium to let electromag-
netic signals pass. In these processes various proper elec-
tromagnetic modes excited in the medium become involved. These are solutions of the dispersion properties of the matter. Hence, correctly accounting for the signal transport becomes
rather involved. For this reason the theory even in this com-
plex version applies to sufficiently dilute media to allow the
assumption of signal propagation in a vacuum. Here, the right-hand side is the ensemble-averaged one-
particle phase space density ⟨Na(p,q,t)⟩, which is a func-
tional only of the indistinguishable dynamics of the particles
indexed by i = 1. Accordingly, averaging the product of two
phase space densities Na(x,t) and Nb(x′,t) yields Na(x,t)Nb(x′,t)
= naδabδ(x −x′)fa(x,t)
+ nanbfab(x,x′,t),
(19) (19) where the partial densities are defined as na = Na/Va, nb =
Nb/Vb and fab(x,x′,t) is the two-particle distribution func-
tion (Klimontovich, 1967). In the same way, higher-order av-
erage products of phase space densities can be reduced to
sums of distribution functions. This procedure must be applied to the causal N-particle
kinetic Eq. (15). This is a formidable task if using the N-
particle kinetic equation in its explicit form. As stated ear-
lier, it is more convenient to remain with the implicit ver-
sions of the Lorentz gauge (Eq. 6) and the retarded potentials In the following we will proceed along the same lines as
Klimontovich (1967) but will in the end refer to the above
field equations. www.ann-geophys.net/35/683/2017/ Ann. Geophys., 35, 683–690, 2017 686 5
Causal one-particle kinetic equation (24) These expressions are to be used in the Lorentz gauge (Eq. 6)
when expressing the electromagnetic fields in the N-particle
kinetic Eq. (4). Formally, this is the same as if we used
Eq. (15) directly in deriving the corresponding causal equa-
tion for the one-particle distribution function of indistin-
guishable particles of sort a. It is only the electromagnetic
fields in Eq. (4) which depend on the retarded time. There-
fore, one can formally calculate the average to obtain The interaction term on the right-hand side arises from the
various interparticle collisions which are mediated by the
electromagnetic field. From the above definition of the fluc-
tuations and correlations, it is given by Ca(p,q,t) ≡1
na
X
b
nbeaeb
4πϵ0
Z
d3p′d3q′
∇q +
p′
mbc2
∂
∂t
−
1
mbc2 ∇q ∧p′
· ∂
∂p
δNa(p,q,t) δNb(p′,q′,tR)
|q −q′|
. (25) ∂
Na
∂t
+ p
ma
· ∇q
Na
+ ea
h
Em(q,t) + p
ma
∧Bm(q,t)
i
· ∂Na
∂p
= 0. (21) (25) The last term in this equation contains particles of kind a and
b as well as the retarded time coordinate. Nevertheless, by
carefully ordering the different contributions and variables
of integration one can bring it into a more convenient form. For this we indicate all integration variables by primes ′ and
rename the retarded time variable by a superscript R. Then
t′ →tR = t −|q−q′|/c. After expressing the last term in an-
gular brackets for the average phase space density, this yields
: Formally, these expressions, as claimed in the previous
sections, are rather similar to those which, for the non-
retarded interactions, had already been obtained by Klimon-
tovich (1967), with the main difference being that here they
are written in terms of the full electromagnetic field and con-
tain the spatial integration over all the remote particle space. Referring to the full electromagnetic fields is necessary be-
cause of their role in the information transport and mainte-
nance of causality in absorber theory. The expressions above
are, however, very different from Klimontovich’s because
they account for the necessary causal relation between the
interacting particles, which is contained in their dependence
on the retarded time tR, by which the particles respond to the
transport of information. 4
Average distribution functions This means that in defining the average dis- Ann. Geophys., 35, 683–690, 2017 www.ann-geophys.net/35/683/2017/ 687 R. A. Treumann and W. Baumjohann: Causal Kinetic Theory the product of the fluctuations (Klimontovich, 1967) the product of the fluctuations (Klimontovich, 1967) in which we replace the charge end current densities by the
general expressions given in Eq. (2). From Eq. (7), this yields δNa(x,t)δNb(x′,t)
= nanbgab(x,x′,t)
+ naδabδ(x −x′)fa,
(23) φm(q,t) =
X
a
ea
4πϵ0
Z
d3p d3q′ Na(p,q′,t′)
|q −q′|
,
Am(q,t) =
X
a
eaµ0
4π
Z
d3p d3q′ p Na(p,q′,t′)
|q −q′|
, (23) (20) we finally arrive at the desired causal kinetic equation: ∂fa
∂t + p
ma
· ∇qfa −
X
b
nbeaeb
4πϵ0
Z
d3p′d3q′
∇q +
p′
mbc2
∂
∂t
−
1
mbc2 ∇q ∧p′
· ∂
∂p
fa(p,q,t)fb(p′,q′,tR)
|q −q′|
= Ca(p,q,t). (24) with the time taken as the retarded time t′ thus depending on
the spatial coordinate q′, which is to be integrated out. 6.1
Remarks The one-particle kinetic equation (Eq. 24) obtained here
is fundamental to all electromagnetic plasma interactions. Since these are electromagnetic, the purely electrostatic ap-
proximation when applied must be justified separately. This
is not easy because in a strictly electrostatic approach the
field response is instantaneous, which contradicts electrody-
namics and relativity, on which it is based. It can be upheld if
the information transport occurs by electrostatic waves only
but still requires some assumption about the brevity of time
delay. This assumption is that the electrostatic fluctuations
occur on a vastly longer timescale than the travel time of
light from the remotest position of particles. Thus, one re-
stricts oneself to sufficiently small plasma volumes in which
the information transport may occur without some remark-
able delay. Ca(x,t) ≡
X
b
nbeaeb
4πϵ0
Z
d3p′d3q′
∇q +
p′
mbc2
∂
∂t
−
1
mbc2 ∇q ∧p′
·
∂
∂p
gab(x,x′,tR)
|q −q′|
. (26) (26) This is the general causal collision integral term including the
interactions between particles indexed by i = 1 and i = 2. From all these expressions one can again obtain an equa-
tion for the fluctuation of phase space density δNa as well as
for the fluctuating fields expressed through the space charge
density and current fluctuations. Under such conditions Klimontovich–Vlasov theory ap-
plies, and the complications introduced by reference to the
retarded time can be neglected. On the other hand, in very
large volumes like in cosmical and astrophysical applications
transport of information is provided by radiation transport
and becomes rather slow. Hence, remote volumes will not
respond immediately and not even within light-propagation
time, which can then be treated again in the simplified the-
ory. With knowledge of the collision term on the right or some
of its approximations, Eq. (24) provides the basis for a lin-
earized kinetic theory to any order, including particle and
time-retarded interaction effects. For this, one defines the
fluctuations of the one-particle distribution function in the
usual way as δfa = fa −¯fa,
(27) (27) δfa = fa −¯fa, where
¯fa is the one-particle “equilibrium” distribution
around which the fluctuations occur. The distribution is ei-
ther an equilibrium solution of the stationary kinetic Eq. 6
Discussion where the average refers to the integration over all particle
space i > 1, and all quantities still depend on the retarded
time tR which requires integration with respect to q′. It is,
however, more convenient to make use of the representation
via the correlation function, in which case, from Eqs. (23)
and (25) we have 6.1
Remarks (24)
or some of its large-scale solutions with scales exceeding
those of the fluctuations such that the average of the fluc-
tuation taken over these scales 1f a = 0 vanishes. Neglect-
ing the collision term by putting Ca = 0 and subtracting the
fluctuation averaged kinetic equation, the causal collisionless
kinetic equation for the fluctuations then becomes However, the current investigation is necessary as a clar-
ification of two points: Firstly, that the interaction among
different volumes in plasma in principle cannot be consid-
ered to occur instantaneously. Secondly, the inclusion of re-
tarded times gives a clue to the direction of time – as briefly
discussed below – which in many-particle systems has only
one direction, forward. Events are delayed by information
transport and thus decorrelate even though they become rel-
ativistically synchronized by accounting for the information
transport. This should necessarily contribute to dissipation
because information becomes diffused by passing across the
plasma from one particle to another. ∂δfa
∂t
+ p
ma
· ∇qδfa −
X
b
nbeaeb
4πϵ0
Z
d3p′d3q′
×
∇q +
p′
mbc2
∂
∂t
−
1
mbc2 ∇q ∧p′
· ∂
∂p
×δfa(x,t) fb(x′,tR) + fa(x,t) δfb(x′,tR) −δfaδfb
|q −q′|
= 0. (28) R. A. Treumann and W. Baumjohann: Causal Kinetic Theory (δE,δB) = (E,B)m −⟨(E,B)m⟩. This yields
Ca(x,t) = −ea
na
Z
d3q′d3p′
∂
∂p
δEδNa
−p
ma
·
∂
∂p ∧
δBδNa
, pointed out that even when dropping the collision term on
the right in a Klimontovich–Vlasov approach in linear the-
ory, the retardation effect remains in the third term on the
left-hand side in Eq. (24), which is the lowest-order electro-
magnetic field-charged particle interaction term. 5
Causal one-particle kinetic equation As a result of this response, the spa-
tial integral appearing in these expressions contains an inte-
gration over tR and thus also the primed space coordinate q′. This complicates the calculation substantially and in an ana-
lytical treatment possibly requires the introduction of further
approximations. Nevertheless, the above final equation with
the implicitly given collision term extends Klimontovich’s
theory to the explicit reference to causality. ∂
Na
∂t
+ p
ma
· ∇q
Na
−
X
b
eaeb
4πϵ0
Z
d3p′d3q′
∇q +
p′
mbc2
∂
∂t
−
1
mbc2 ∇q ∧p′
· ∂
∂p
Na(p,q,t)Nb(p′,q′,tR)
|q −q′|
= 0. (22) (22) In this version of the phase space (ensemble) averaged equa-
tion for the time and one-particle phase space evolution of
the (ensemble) averaged one-particle phase space density
⟨Na(p,q,t)⟩, the retarded time appears only in the averaged
product. This equation is the master equation for construct-
ing the kinetic equation for the particle distribution func-
tion. Defining the fluctuation of particle number density as
δNa(x,t) = Na(x,t) −
Na(x,t)
and referring to the corre-
lation function gab(x,x′,t) defined through the average of Referring to Eq. (22) the collision term can also be ex-
pressed via the fluctuations of the phase space density δNa =
Na −⟨Na⟩and the fluctuations of the electromagnetic fields www.ann-geophys.net/35/683/2017/ Ann. Geophys., 35, 683–690, 2017 Ann. Geophys., 35, 683–690, 2017 688 R. A. Treumann and W. Baumjohann: Causal Kinetic Theory R. A. Treumann and W. Baumjohann: Causal Kinetic Theory (1967) in his fundamental approach; it also required refer-
ence to the famous absorber theory (Wheeler and Feynman,
1945, 1949) and the Liénard–Wiechert potentials on which it
was based. The result is, however, substantially more compli-
cated than Klimontovich’s by the fact that the retarded time
itself depends on the primed space coordinate q′, which is
an integration variable, and on space itself. This complicates
any calculation. The main physical consequence is, however,
that the retardation effect restricts applications to the domain
which in observation time can be accessed by the propaga-
tion of light. For example, the observation of plasma waves
in the Earth’s foreshock at a frequency of ω/2π ∼30 kHz
implies that the observed source of emission must have been
located in a region of distance 1x < 10 km. Though this is
not a severe restriction for Langmuir waves, which are lo-
cally excited, application to astrophysical conditions is more
interesting. The mechanism of the modulation of solar ra-
dio emissions at, say, 300 MHz with a frequency of 10 Hz is
restricted to a region substantially smaller than 30 000 km,
which in any theory of such a mechanism must be taken
into account in the calculation. In galactic astrophysics, typ-
ical scales are several parsecs, referring to times of a few
lightyears, which sets bounds on plasma mechanisms which
could be evoked to participate in a causal relation. integrated response of the charge and current densities at lo-
cation q and time t to the variation in the corresponding den-
sities at all locations q′ and t′ takes account of causality and
thus of the direction of time. Ignoring the effect of time retar-
dation, the original Liouville equation is clearly symmetric in
time. It does not distinguish between processes proceeding
forward and backward in time. This is one of the big prob-
lems in physics, which possibly only resolves on a macro-
scopic level. When making reference to signal retardation in
absorber theory, this symmetry might be broken from the re-
tarded time Eq. (8) as suggested. By replacing t →−t, one
has (29) t′ = −
t + |q −q′|/c
,
(29) and thus, with constant velocity of light c, the negative re-
tarded time t′ →−t′ becomes advanced. Competing interests. The authors declare that they have no conflict
of interest. Competing interests. The authors declare that they have no conflict
of interest. 6.2
Direction of time Reference to the retarded potentials and the effect of emis-
sion and absorption implies a distinction between advanced
and retarded effects. This in itself unexpectedly brings up the
problem of direction of time, this time not in electrodynam-
ics like in absorber theory, but also and directly in the mi-
croscopic theory of phase space evolution. The delayed and This equation contains the correlations of the fluctuations
δfaδfb, which are neglected in a linearized theory. Clearly, the above equations resemble the well-known
approach to plasma kinetic theory. It should, however, be www.ann-geophys.net/35/683/2017/ Ann. Geophys., 35, 683–690, 2017 Ann. Geophys., 35, 683–690, 2017 689 www.ann-geophys.net/35/683/2017/ Data availability. No data sets were used in this article. Data availability. No data sets were used in this article. 6.3
Conclusions The present investigation extends Klimontovich’s approach
to kinetic plasma theory to the inclusion of signal retarda-
tion effects. It applies to systems of indistinguishable charged
particles interacting via their self-consistent electromagnetic
fields. One can trivially extend it to the presence of external
fields like stationary or variable magnetic fields caused by
external sources. Acknowledgement. This work was part of a Visiting Scientist Pro-
gramme at the International Space Science Institute Bern in 2007. The interest of the ISSI Directorate is acknowledged, as, and in par-
ticular, is the friendly hospitality of the ISSI staff. Thanks are di-
rected to the ISSI system administrator S. Saliba for technical sup-
port and to the librarians Andrea Fischer and Irmela Schweizer for
access to the library and literature. A number of points may be worth mentioning. First, the re-
sult looks simple as it seems that simple replacement of time
with retarded time would have been sufficient to obtain it. This is true, but it is not proof for the result’s correctness. For
this reason we have chosen to follow the derivation step by
step, which is the usual way of confirming a hypothesis. This
required using the basic equations derived by Klimontovich The topical editor, E. Roussos, thanks P. Yoon for help in eval-
uating this paper. Ann. Geophys., 35, 683–690, 2017 Ann. Geophys., 35, 683–690, 2017 R. A. Treumann and W. Baumjohann: Causal Kinetic Theory In order to restore
retardation as required by the Wheeler–Feynman absorber
theory, one needs to redefine the velocity of light as c →−c. In a time-symmetric many-particle theory, the negative time
direction would come into accord with absorber theory only
under the requirement that time velocity c is negative there,
i.e. one has to take the negative root c = −1/√ϵ0µ0. There
is no obvious reason why this should be imposed, and it thus
becomes a philosophical question. Should c be considered
the inverse positive or negative root of the product of suscep-
tibilities of the vacuum, or should c be interpreted as a pos-
itive speed, the speed of light, with reference to a distance
travelled by time in either positive or negative time? The same procedure may also be applied to other classi-
cal fields since in all interactions the transport of information
from the agent to the absorber takes time. This is the case in
gases where sound waves or gravity waves can be excited and
these transport the information from one fluid element to an-
other place to affect the dynamics of other elements. In these
cases it is not the photons but phonons that transport energy
and information. Application to these systems lies outside the
intention of the present work. This question cannot be answered a priori. Absorber the-
ory is restored in the second case in the causal many-particle
theory. When considering the vacuum as a medium in which
the dispersion of electromagnetic waves is described by a
dispersion relation ω2 = k2/ϵ0µ0, interpreting this as the
relation between photon energy and momentum, one has
ℏω = ±ℏk/√µ0ϵ0. Since photon energies should be real and
positive, a negative sign of the root implies negative wave
numbers or negative photon momenta and thus also spatial
inversion. R. A. Treumann and W. Baumjohann: Causal Kinetic Theory R. A. Treumann and W. Baumjohann: Causal Kinetic Theory www.ann-geophys.net/35/683/2017/ Ann. Geophys., 35, 683–690, 2017 www.ann-geophys.net/35/683/2017/ www.ann-geophys.net/35/683/2017/ 690 References Wheeler, J. A. and Feynman, R. P.: Interaction with the absorber
as the mechanism of radiation, Rev. Mod. Phys., 17, 157–161,
doi:10.1103/RevModPhys.17.157, 1945. Jefimenko, O. D.: Electricity and Magnetism: An Introduction to
the Theory of Electric and Magnetic Fields, Appleton-Century-
Crofts, New York, 1966. Wheeler, J. A. and Feynman, R. P.: Classical electrodynamics in
terms of direct interparticle action, Rev. Mod. Phys., 21, 425–
433, doi:10.1103/RevModPhys.21.425, 1949. Klimontovich, Y. L.: The Statistical Theory of Non-equilibrium
Processes in a Plasma, The MIT Press, Cambridge, MA, 1967. Panofsky, W. K. H. and Phillips, M.: Classical Electricity and Mag-
netism, Addison-Wesley, New York, 1962. Ann. Geophys., 35, 683–690, 2017 www.ann-geophys.net/35/683/2017/
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The Input Allocation and Potato Production on Small-Scale Farming in Pegunungan Arfak Regency
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Jurnal Manajemen & Agribisnis, Vol. 20 No. 1, March 2023
Permalink/DOI: http://dx.doi.org/10.17358/jma.20.1.79 Jurnal Manajemen & Agribisnis, Vol. 20 No. 1, March 2023
Permalink/DOI: http://dx.doi.org/10.17358/jma.20.1.79 Available online at
http://journal.ipb.ac.id/index.php/jmagr http://journal.ipb.ac.id/index.php/jmagr 1 Corresponding author:
Email: ta.pattiasina@unipa.ac.id INPUT ALLOCATION AND POTATO PRODUCTION ON SMALL-SCALE FARMING IN
PEGUNUNGAN ARFAK REGENCY Trees A. Pattiasina*)1, Rita Nurmalina**), Harianto**), Anna Fariyanti**) Trees A. Pattiasina*)1, Rita Nurmalina**), Harianto**), Anna Fariyanti**)
*)Study Program of Agribusiness, Faculty of Agriculture, Universitas Papua
Jl. Gunung Salju Amban, Manokwari,Papua Barat 98314, Indonesia
**)Department of Agribusiness, Faculty of Economics and Management, IPB University
Jl. Agatis, Campus of IPB Darmaga Bogor 16680, Indonesia *)Study Program of Agribusiness, Faculty of Agriculture, Universitas Papua
Jl. Gunung Salju Amban, Manokwari,Papua Barat 98314, Indonesia
**)Department of Agribusiness, Faculty of Economics and Management, IPB University
Jl. Agatis, Campus of IPB Darmaga Bogor 16680, Indonesia Abstract: Small-scale potato farming in the Arfak Mountains needs attention in allocating
inputs for efficient farming. This study aims to describe the allocation of the use of potato
farming inputs, analyze the factors that affect potato production, and analyze the level of
technical efficiency of potato farming in the Arfak Mountains. Arfak Mountains Regency was
chosen purposively as the research location. Data collection starts from December 2021 to
February 2022. Sampling was done by snowball sampling with a sample of 140 respondents. The data used in this study is cross-section data which was analyzed using the Stochastic
Frontier Cobb Douglas production function approach. The results showed that the average
farmer in the Arfak Mountains controlled 0.07 hectares of land for potato farming, with a seed
requirement of 22 kg per hectare. The factors that affect potato production are land, seeds, and
female labor. The level of technical efficiency of potato farming is 73 percent. Improvements
in farming management and extension assistance for good agricultural practices and the use
of technology according to farmers' local wisdom need to be carried out to increase potato
production and productivity. Copyright © 2023, ISSN: 1693-5853/E-ISSN: 2407-2524 Copyright © 2023, ISSN: 1693-5853/E-ISSN: 2407-2
1 Corresponding author:
Email: ta.pattiasina@unipa.ac.id INTRODUCTION Arfak Mountains and the Department of Food Crops
and Horticulture of West Papua Province show that
potato production in the Arfak Mountains during the
period 2018-2020 continued to decline with an average
of 47.49 tons. The decline in production indicates the
low productivity of potatoes. Potato productivity in the
Arfak Mountains for the last three years (2018-2020)
tends to fluctuate with an average of 1.48 tons/hectare. This productivity is very low compared to potato
productivity in Java, Sumatera, and North Sulawesi,
where the average productivity ranges from 10-22 tons
per/hectare (Central Bureau of Statistics, 2016-2020). Potato is one of the horticultural commodities that have
great potential to be developed in Indonesia because
it can be a source of income for the community and
farmers, both small, medium, and large (Ali et al. 2020). Further, (Mulyono et al. 2018) said that the
potential of potatoes as a source of carbohydrates and
the provision of sustainable food to achieve resilience
had made potatoes included in the superior national
commodity and received attention in their development
and cultivation. Potato production from 2015 to 2019 tends to increase,
but the rate of increase in production is relatively low,
at 2.03 percent. Meanwhile, the average productivity
of potatoes is 17.97 tons/ha. Although productivity
tends to be high, the rate of increase in productivity
is low compared to the production level. Therefore, it
is necessary to increase productivity. This is because
the productivity standard has not been achieved, which
is 20 tons/ha (BPS, 2017-2020). Based on the results
of the National Socio-Economic Survey in Quarter 1
of 2013-2018, potato consumption increased in 2013-
2016 and decreased in 2017 and 2018 with the average
level of potato consumption in Indonesia being 2.23
kg/capita/year or equivalent to 0.04 gram/capita/week
and the average growth throughout 2014-2018 was
13.95 percent. The increase in consumption should
also be accompanied by the fulfillment of consumption
needs from domestic production so that imports can be
reduced and the community’s needs can be met (Arifin
et al. 2021). The potato farming system in the Arfak Mountains is
still traditional on a small scale. The reason is that the
average land use is less than 0.1 hectares, so farmers
plant less. Second, the technology used is simple and
has not been managed intensively by applying good
cultivation techniques. Farmers are still practicing
shifting agriculture using a slash-and-burn system. Article history: Article history:
Received
8 October 2022
Revised
31 October 2022
Accepted
14 November 2022
Available online
31 March 2023
This is an open access
article under the CC BY
license Article history:
Received
8 October 2022
Revised
31 October 2022
Accepted
14 November 2022
Available online
31 March 2023
This is an open access
article under the CC BY
license Received
8 October 2022
Revised
31 October 2022
Accepted
14 November 2022 Keywords: local knowledge, low input, potato, small-scale farming, technical efficiency Abstrak: Usahatani kentang skala kecil di Pegunungan Arfak perlu mendapat perhatian
dalam pengalokasian input agar diperoleh usahatani yang efisien. Penelitian ini bertujuan
mendeskripsikan alokasi penggunaan input usahatani kentang, menganalisis faktor-faktor
yang memengaruhi produksi kentang, dan menganalisis tingkat efisiensi teknis usahatani
kentang di Pegunungan Arfak. Kabupaten Pegunungan Arfak dipilih secara purposive
sebagai lokasi penelitian. Pengambilan data dimulai Desember 2021 hingga Februari
2022. Pengambilan sampel dilakukan secara snowball sampling dengan jumlah sampel
140 responden. Data yang digunakan dalam penelitian ini adalah data cross section yang
dianalisis menggunakan pendekatan fungsi produksi Stochastic Frontier Cobb-Douglas. Hasil penelitian menunjukkan rata-rata petani di Pegunungan Arfak menguasai lahan untuk
usahatani kentang sebesar 0.07 hektar, kebutuhan bibit 22 kg per hektar. Faktor-faktor
yang memengaruhi produksi kentang adalah lahan, bibit, dan tenaga kerja wanita. Tingkat
efisiensi teknis usahatani kentang adalah 73 persen. Perbaikan manajemen usahatani dan
pendampingan penyuluh untuk praktek pertanian yang baik serta penggunaan teknologi
sesuai kearifan lokal petani perlu dilakukan untuk meningkatkan produksi dan produktivitas
kentang. Kata kunci: pengetahuan lokal, input rendah, kentang, usahatani skala kecil, efisiensi teknis 79 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 INTRODUCTION 20 No.1, March 2023 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 were conducted to obtain information about the socio-
economic characteristics of farmers (age, gender,
education level, and farming experience) and the
characteristics of farmers’ farming activities (land area,
land status, use of seeds, use of labor, and production). The data obtained will then be tabulated and analyzed
quantitatively and qualitatively. Based on the
objectives, it will be explained descriptively related to
the use of inputs according to the existing conditions
of potato farming. Second, as was done by (Battese
and Coelli, 1995), (Najjuma et al. 2016), (Dube et al. 2018), (Andaregie & Astatkie, 2020) who uses the
Cobb-Douglas stochastic frontier production function
to determine technical efficiency. This study uses the
stochastic frontier Cobb-Douglas production function
approach to analyze the factors that affect potato
farming production and technical efficiency. Stochastic
frontier analysis has implications for the choice of
functional form. The stochastic frontier production
function analysis can be used to measure and estimate
the technical efficiency of potato farming from the
input side and the factors that influence it. The Cobb-
Douglas production function model is empirically
proven to be relevant for research in the agricultural
sector. The Cobb-Douglas production function model
is as follows: the application of cultivation techniques and good
technology in areas with good access to markets to
provide inputs and outputs. This distinguishes it from
potato farming activities in the Arfak Mountains,
where farmers with local knowledge grow potatoes
in small quantities but still sell them to earn income
even though market access is far away. Therefore,
this study aims to describe the allocation of the use of
potato farming inputs, analyze the factors that affect
potato production, and analyze the level of technical
efficiency of potato farming in the Arfak Mountains. The research results are expected to be the material for
formulating agricultural policies, especially improving
the performance of potato farming in areas with
extensive but commercial farming characteristics. METHODS Arfak Mountains Regency was chosen as the research
location because it is the center of potato farming in
West Papua Province. Furthermore, three districts
were selected, namely Hingk District, Anggi District,
and Sururey District purposively with consideration of
high production and potential for potato development
with LQ > 1 (Sagrim et al. 2017). Data collection is
carried out in December 2021-February 2022. The
next stage is to determine the sample farmers using the
snowball sampling method which is a non-probability
sampling method when the total population is unknown
(Johnson, 2014). This is true in the Arfak Mountains
District, where obtaining a permanent list of farmers
is difficult because the list of farmers owned by the
Regional Agriculture Service is incomplete and not
up-to-date, so sample selection from the list may result
in bias (DiGaetano, 2013). Based on this technique,
the researcher’s initial process first identifies several
members of the population (initial sample) based on
criteria, namely farmers who are still farming potatoes
from each district selected through the Village Head,
Tribal Head, and the Department of Agriculture. Furthermore, the selected farmers will appoint or
invite other friends to be used as samples, increasing
the number of samples. The number of samples taken
from the three districts is 140 respondents. The data
used in this study were cross-sectional data at the
household level who engaged in potato farming which
was obtained through observation and open interviews
using a questionnaire. Observations and interviews Yi LnYi=β0+β1lnX1+β2lnX2+β3lnX3+β4lnX4+ei where: Yi is potato production, measured in units (kg),
X1 is area of potato farming (ha), X2 is number of seeds
used (kg), X3 is number of male workers (HOK), X4 is
number of female workers (HOK), β0 is intercept, βi is
vector of parameter to be observed, and ei is residual. Potato productivity in Arfak Mountains Regency
tends to be low and fluctuates. This can be caused
by internal factors such as the use of inputs (seeds,
without fertilization, use of labor) production that is not
appropriate so that efficiency is not achieved. External
factors consist of social and ecological factors. The
Arfak Mountains Regency has a topography of >
80 percent steep. The local wisdom adopted by the
community in carrying out their farming can also affect
the efficiency and productivity of potato farming. Thus,
the sign of the expected parameter is β1, β2, β3, β4 >
0. INTRODUCTION ,
simple equipment, and less responsive to innovation. However, farmers continue to pursue farming with
traditional patterns because they are supported by socio-
cultural values and the local knowledge of farmers
(Mulyadi dan Iyai, 2016). This is an obstacle in farming
activities because farmers prefer to implement farming
activities based on hereditary experiences, such as not
using fertilizers and drugs. The indication is farmers'
ability in cultivation techniques, and the allocation of
input use results in low production and productivity. Whereas the use of inputs plays an important role in
producing production. (Esmael, 2017) wrote that low
productivity was mainly due to the lack of superior
potato varieties, the lack of certified potato seeds, and
the managerial ability of farmers to manage potato
farming. The low productivity of potatoes in the Arfak
Mountains indicates the need to increase production
efficiency to reach its potential. Widanage et al. 2022
stated that technical efficiency describes the maximum
output level through the optimal allocation of resources. However, potatoes in the Arfak Mountains became a
commercial product as a source of farmers' income
(Yuminarti et al. 2018) even though farmers had to
travel > 100 km in extreme geographical conditions to
access the market. The potato-producing center in West Papua is the Arfak
Mountains Regency. Based on the results of interviews,
in the era of 2000s, in the Arfak Mountains, potatoes
became one of the crops that could increase farmers'
livelihoods and incomes because they gave high yields
at harvest. Currently, potatoes are still the main food crop
along with other sweet potato commodities, although
the introduction of rice has begun to shift the role of
tubers as a food source (Mulyadi, 2012), (Yuminarti et
al. 2018). This is done because potatoes and other tuber
crops can produce yields without intensive cultivation
and only use low-production inputs (Yaku et al. 2019). Thus, potatoes need attention in their development to
meet local and inter-regional food needs so that they
are no longer dependent on inter-island products due
to geographical conditions and relatively high transport
costs. Data from the Central Bureau of Statistics of the Several studies related to small-scale potato production
and technical efficiency have been carried out, such
as by Nyagaka et al. 2010, Wassihun et al. 2019,
Gulak and Obi-Egbedi, 2021, and Tolno et al. 2016. These analyses are applied to potato farming with 80 Jurnal Manajemen & Agribisnis,
Vol. Use of Inputs and Production of Potato Farming Farmers in the Arfak Mountains Regency generally
apply traditional agriculture so that production inputs
are limited and do not even use production inputs to
preserve the environment for generations. For potato
commodities, farmers do not use fertilizers and drugs
in the production process because farmers carry out
farming activities based on the experience of their
parents and do not want to accept innovations on the
grounds of crop failure. In this study, the production
inputs used are land, seeds, and labor. METHODS The estimation of the production function uses the
Frontier 4.1 program using the Maximum Likelihood
Estimator (MLE) method. where: Yi is potato production, measured in units (kg),
X1 is area of potato farming (ha), X2 is number of seeds
used (kg), X3 is number of male workers (HOK), X4 is
number of female workers (HOK), β0 is intercept, βi is
vector of parameter to be observed, and ei is residual. i
Potato productivity in Arfak Mountains Regency
tends to be low and fluctuates. This can be caused
by internal factors such as the use of inputs (seeds,
without fertilization, use of labor) production that is not
appropriate so that efficiency is not achieved. External
factors consist of social and ecological factors. The
Arfak Mountains Regency has a topography of >
80 percent steep. The local wisdom adopted by the
community in carrying out their farming can also affect
the efficiency and productivity of potato farming. Thus,
the sign of the expected parameter is β1, β2, β3, β4 >
0. The estimation of the production function uses the
Frontier 4.1 program using the Maximum Likelihood
Estimator (MLE) method. 81 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 RESULT RESULT owned by a farmer but also belongs to the farmer and
his relatives. The average garden area cleared is > 1
hectare. When the land is opened for cultivation, each
farmer will cultivate according to the area to be planted
so that the status of the previously communal land
(farmers and relatives) belongs to the farmer. The type
of land ownership determines the type of innovation
and development that farmers will carry out on their
land (Taiy et al. 2017). So, farmers will carry out
permanent long-term farming activities on their land. Sururey District farmers only grow potatoes in their
gardens because the land is used specifically for other
horticultural crops such as carrots, leeks, and shallots. In contrast to the gardens in the Hingk and Anggi
districts, which are located at the foot of the mountain
with an average slope of > 30 percent, the gardens in
the Sururey District are located on flat slopes, not far
from the house. Land tenure One of the production inputs that are the basis for
farming is the area of land cultivated by farmers, which
is the most important natural resource in farming
activities. Based on the results of surveys and interviews
with farmers, the average area of land cultivated by
potato farmers in the Arfak Mountains Regency is
0.07 hectares. Land tenure by farmers is very narrow
(between 0.05 - 0.1 hectares), with distribution in each
sample district. Hingk District farmers control 0.09
hectares of land, Anggi District farmers control 0.056
hectares of land, and Sururey District farmers' average
land tenure is 0.052 hectares. This can be one of the
obstacles in increasing production because the area or
narrowness of the business area has implications for
the high and low agricultural production produced. (Taiy et al. 2017) write that land size is positively
correlated with productivity. Farmers with relatively
large holdings are expected to benefit from economies
of scale. The same study by (Tolno et al. 2016) found
that most farmers in Guinea cultivate potatoes for
less than 1 hectare with an average of 0.89 hectares. Increasing farm size can increase production if the land
is used effectively, requiring appropriate agricultural
practices and inputs. In line with this, (Nyagaka et al. 2010) also wrote that Kenyan farmers allocated 0.34
hectares of land for potato farming on average. Seeds Male workers are more involved when land clearing
activities include tree felling, clearing fallen trees,
making fences, and loosening the soil. Women workers
play a role from land clearing to marketing activities. The workload of women is greater than that of men. The
average workload is calculated based on the number of
working days (HOK) equivalent to 8 hours of work in
one day. The amount of men’s work per planting season
for potato farming is 36.74 HOK, while the women’s
work is 48.53 HOK. This result is quite different from
the research conducted by (Nyagaka et al. 2010) and
(Tiruneh et al. 2017), that the average workload for
potato farming is between 150 - 300 HOK. farmer groups sometimes receive seed assistance from
the Department of Agriculture every year, they are not
used by farmers because there is no assistance from
extension workers. Farmers will apply technology
or innovation if there is continuous assistance from
extension workers or related agencies. The same thing
was written by (Tolno et al. 2016) that access to superior
seeds is an obstacle to production for potato farmers
in Guinea, so farmers also use seeds from previous
harvests and from informal systems or organizations. Based on the survey results for the last planting season,
the need for potato seeds for the next planting season is
not too much because it is in accordance with the size
of the land used. The preparation of seeds carried out
by farmers is approximately 2-5 nokens with varying
weights ranging from 5 kg - 20 kg per noken. The
average need for potato seeds per growing season is
22 kg per hectare . When compared between districts,
the need for seeds in Hingk District is higher for each
planting season, which is 27 kg per hectare. This is
because the land used to grow potatoes is larger than
in Anggi District and Sururey District, where the seed
requirements are only 12 kg per hectare and 17 kg per
hectare, respectively. The need for seeds in the Arfak
Mountains is very low when compared to the small-
scale potato farming conducted by (Nyagaka et al. 2010), (Tiruneh et al. 2017), (Tolno et al. 2016) and
(Maryanto et al. 2018) which need for seeds is between
500 - 2200 kg per hectare. Seeds The results of farmer interviews provide information
that potato farmers in the Arfak Mountains use seeds
obtained from previous harvests. Many of the seeds
planted by farmers are granola varieties, but the
farmers themselves do not know the varieties of seeds
used because they have been used for more than four
planting periods. Seedlings obtained from potato
cultivation for generations and with unclear varieties
and their derivatives will provide more tubers but are
economically unprofitable because the tubers are small,
causing a decrease in tuber quality and sensitivity to
pests and plant growth diseases (Syafri et al. 2005). Potato seeds are traditionally stored in “noken” bags
and hung on the outside wall of the house. According
to farmers, this method of storage is relatively easy,
but the quality of the seeds is low because stored seeds
can be contaminated with various diseases that are
transmitted through tubers for generations. This can
result in decreased productivity. Although potato is a
perishable commodity, they can be a climate change
adaptation strategy with good storage because it ensures
a sustainable supply of potatoes and seeds (Taiy et al. 2017). Furthermore (Sayaka dan Hestina, 2011) stated
that the use of certified seeds could increase farmers’
yields and profits compared to using uncertified seeds. Farmers in Hingk District and Sururey District have
never obtained potato seeds with quality standards
because obtaining these seeds requires a large amount
of capital because they are relatively expensive. While
some farmers in Anggi District who are members of The agricultural land cultivated by farmers for potatoes
in Hingk and Anggi Districts is divided into yard and
garden land. This is done because the existing yards
are relatively limited, so farmers open other business
areas which are located quite far from their homes with
1-2 hours on foot. Farmland owned by farmers is land
with communal ownership status. So, it is not only 82 Jurnal Manajemen & Agribisnis,
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Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 clear land (Tim Unipa, 2015). In further analysis, the
workforce is divided into male workers (TKP) and
female workers (TKW). This division is based on the
consideration that there are different roles between TKP
and TKW in farming activities in the Arfak Mountains. Seeds The need for seeds is low
because the area of land used to grow potatoes has also
been getting smaller in the last year. Farmers are tired
of planting potatoes because they are always attacked
by diseases when the plants are one month old and it
happens again and again. There has been no action
from the relevant agencies and the farmers themselves
are reluctant to report this incident. The recommended
potato seed requirement issued by the Research
Institute for Vegetable Crops (Balitsa) for an area of
one hectare is around 1,200 kg (with tuber size of 30 g/
knol) (Setiawati et al. 2007). Overall, women’s total workload is higher than men’s
in each study area. The largest employment in Hingk
District was 37.89 percent for land clearing activities. This is because the land-clearing process carried out
using the slash-and-burn system requires more labor
and takes longer. This was followed by marketing
activities of 25.02 percent. (Dube et al. 2018) wrote
that the use of labor for potato farming in Ethiopia is
for land clearing, weeding, and harvesting activities. Unlike in Anggi and Sururey Districts, marketing
activities require more work than other activities. The
time required for marketing activities is approximately
one week, depending on the products produced. The
least amount of work is for planting and post-harvest
activities. Planting usually takes 1-2 working days and
can be done by the farmer’s own family. Likewise,
the time for post-harvest activities is carried out 1-2
days. For maintenance, farmers do not do it every
day, especially for gardens located at the foot of the
mountain; except for those in their yards, they usually
only control whether there are pests and diseases. Labor Based on the survey results obtained data the average
potato production in Arfak Mountains Regency based
on research data is 150 kg or the equivalent of 0.15
tons. The low production is due to the reduced area of
business land and many potato plants that fail to grow
due to late blight. Sururey District has the lowest potato
production at only 106 kg, followed by Anggi District
at 116 kg. Failure to grow potato plants has occurred The labor used in potato farming comes from workers
in the family. Labor outside the family is only used
when clearing land, planting, and harvesting when
labor is unavailable. The number of outside workers
used depends on the area of land being managed. The
external workers used are those who still have kinship/
family relations or siblings with farmers who want to 83 Jurnal Manajemen & Agribisnis,
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Vol. 20 No.1, March 2023 for about one year. This reduces the motivation of
farmers to plant potatoes in large land areas due to
trauma to plants that rot when they are one month
old. Meanwhile, the average potato production in
Hingk District reaches 180 kg. There are still many
potato farmers in Hingk District, and potatoes are
currently the main crop grown by farmers. However,
traditional cultivation techniques left to nature make
production low. The average production in the Arfak
Mountains is very low compared to potato production
in other developing countries, which is between 2-17
tons (Andaregie & Astatkie, 2020), (Wassihun et al. 2019), (Tiruneh et al. 2017). When viewed from the
productivity point of view, the average productivity of
potatoes in the Arfak Mountains is very low, only 0.24
kg/ha, and this result is lower than the average potato
productivity for the last three years (2018-2020), which
is 1.48 kg/ha. The estimation results in Table 1 show that the value
of the Generalized-Likelihood Ratio (LR) test (15.60)
is greater than the value of the palm table code at
the level of = 5 percent (2.706), meaning that it is
statistically significant. This indicates that the LR test
accepts H1 that in this model, there is an effect or case
of inefficiency. Labor This means that almost all variations in
the output of the frontier production function can be
considered as the achievement of technical efficiency
related to managerial issues in farm management. So,
the LR test explains the existence of farmers’ technical
efficiency and inefficiency in the potato production
process. In other words, potato farming activities are
influenced by technical efficiency. The sigma-squared parameter value (σ2) shows the
total variance of the two components, namely the
inefficiency effect (ui) and the noise effect (vi). The
calculation results obtained that the value of 2 is 0.229,
significantly different at the level of = 1 percent, so it
can be concluded that the model used is correct and
the errors ui and vi normally spread according to the
desired assumptions. The calculation results can
be seen that the estimated coefficient of gamma (γ)
of 0.884 is not significantly different from zero or
significantly affects the level of = 1 percent. This figure
shows that 88.4 percent of the variation in yield among
the sample farmers is due to differences in technical
efficiency, and the remaining 11.6 percent is due to
external influences such as climate, pest and disease
attacks, and modeling errors. This shows that the effect
of technical inefficiency is a significant factor in the
variability of output. Factors Affecting Potato Production 2018) Based on table 1, the three presumed parameters have
been signed in accordance with expectations, namely
land area, seeds, and female labor, where the parameter
(β) is positive. Variables that affect the level of = 1
percent are land area and seeds, while female workers
influence the level of = 5 percent. Meanwhile, male
workers have a negative sign and have no effect on
production. The value of the regression coefficient (β)
in the production function of Cobb-Douglas shows the
value of elasticity. If farmers aim to maximize profits,
then farmers must produce in a rational area, i.e., when
the elasticity is between zero and one (Debertin, 1986). The sum of the elasticity values of the production
factors results in the Return to Scale (RTS) value,
namely the business’s economic scale, which is the
production response to changes in the input used. The calculation results show that the RTS value in the
production function is 1.009, which is greater than 1
(RTS>1). This means that farming is in the Increasing
Return to Scale (IRTS) condition that increasing the use
of input combinations will increase production yields
by a percentage greater than the percentage increase
in inputs. When viewed between districts, Hingk and
Anggi also have RTS values > 1, which are 1,094 and
1,586, respectively. Meanwhile, in Sururey District,
the RTS value is < 1 (0.89). This means that the farm
is in a Decreasing Return to Scale (DRTS) condition
that increases the use of a combination of inputs will
increase production yields by a smaller percentage than
the percentage increase in inputs. The variable number of seeds is the most responsive
production factor, with the first elasticity (0.520)
followed by land and female labor. The seed variety
that farmers use is granola, even though it is obtained
from previous harvests and has been more than four
generations of planting period. In this case, the seed
becomes a limiting factor in potato production. Farmers
do not buy potato seeds because they are expensive,
especially certified ones. Apart from the price factor,
farmers are not brave enough to try new seeds for fear
of failure, or the plants will not grow. Farmers are
more confident in the seeds produced from generation
to generation. Factors Affecting Potato Production In accordance with what has been mentioned in
the research method, the Stochastic Frontier Cobb
Douglas production function model with Maximum
Likelihood Estimator (MLE) estimation is used in this
study. Referring to equation 1 to estimate the overall
parameters (βi) and intercept (β0) and the gamma value
to estimate how much influence the variances vi and ui
have in determining the production model. The results
of the estimation of the production function model
using the MLE method are presented in Table 1. Table 1. Estimating the production function of potato farming in Arfak Mountains Regency, 2022
Variable
Combined
Hingk
Anggi
Sururey
Estimated parameters
Estimated parameters
Estimated parameters
Estimated parameters
Constant
3.992
3.578
1.823
2.934
Land area (X1)
0.317**
0.316**
0.181
0.413**
Seed (X2)
0.520**
0.483**
0.910**
0.213**
Male workforce (X3)
-0.010
0.141*
0.148
-0.070
Female workforce (X4)
0.182*
0.157*
0.374*
0.967**
Sigma-squared (σ2)
0.229
0.079
0.621
0.187
Gamma (γ)
0.884
0.884
0.999
0.999
LR Test
15.61
3.58
10.81
16.78
Information: * significant at level = 5% (0.05); ** significant at level = 1% (0.01) 84 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis,
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Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 the Arfak Mountains has resulted in a lot of potato land
being converted to non-agricultural use. In contrast to
Hingk District and Sururey District, where land is very
influential in increasing production, in Anggi District,
although the sign is positive, land does not significantly
increase production. This means that land is no longer
the main input in production. Anggi District, as the
capital of the Arfak Mountains Regency, made many
changes due to development. Land conversion is
visible, where land that used to be planted with potatoes
has been turned into buildings or left unplanted. In
addition to these factors, the saturation of farmers
growing potatoes appears when the plant does not
provide optimal results. Then farmers will switch to
other crops on potato fields. In this district, there are
not many farmers growing potatoes. Therefore, efforts
to increase productivity need to be emphasized by
policymakers. The results of research that produce land
area significantly affect production as carried out by
(Widayati, 2017), (Maryanto et al. 2018), and (Tristya
et al. Factors Affecting Potato Production The same is true for Kenyan farmers in
potato production, where there is no access to quality
and certified potato seeds and a structured seed supply
and distribution system. High prices and not being
available on time are obstacles (Taiy et al. 2017). The
highest seed elasticity value is in Anggi District (0.91)
because farmers in Anggi often receive assistance from
the government, there are farmer groups, and assistance
from extension workers. This is what makes it different
from other districts. Farmers who are members of farmer
groups often receive seed assistance once or twice a
year so that potato plants become better, as indicated
by large production. Farmers who use superior seeds
will increase their output and market surplus (Tolno et
al. 2016). (Tristya et al. 2018) stated that seeds play an
important role in optimizing production. The elasticity of the frontier production function for
inputs that have a significant effect on potato production
is land area, number of seeds and female labor with
values of 0.317, 0.520, and 0.182, respectively. If each
of these inputs is added by 10 percent, ceteris paribus,
it will increase potato production by 3.17 percent,
5.20 percent, and 18.2 percent, respectively. This
figure shows that potato production is very responsive
to land area, seeds, and female labor. Especially for
land, it becomes important in increasing production
considering that the productivity level of potatoes
is still very low. Farmers who allocate more land for
potatoes will increase production if done with good
management to increase farmers’ income (Tolno et
al. 2016). (Bukul, 2018) wrote that access to fertile
land should encourage farmers to increase potato
production. The results showed that farmers’ land
tenure in the Arfak Mountains for potato farming was
relatively small (less than 0.5 hectares), while there was
sufficient land available. The ongoing development in 85 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 al. 2010), (Wassihun et al. 2019), and (Andaregie &
Astatkie, 2020), which found that inefficient potato
farmers had an estimated technical efficiency value of
between 20-30 percent. The average value of technical
efficiency in the Arfak Mountains is 73 percent. This means that farmers can obtain 73 percent of the
potential output at the given input level. On average,
farmers are relatively efficient, but about 27 percent of
the output obtained by farmers is lost due to inefficiency
in production. Factors Affecting Potato Production This means that farmers could achieve
frontier output of up to 27 percent with the adoption of
better technology and management techniques (Gulak
and Obi-Egbedi, 2021), (Nyagaka et al. 2010), (Dube
et al. 2018). Thus, farmers with a low level of technical
efficiency can have the opportunity to increase
production according to farmers who have a high
level of technical efficiency with current technology
(Asmara et al. 2016). The results of this study are in
line with the findings (Nyagaka et al. 2010), (Wassihun
et al. 2019), (Andaregie & Astatkie, 2020), (Rizkiyah et
al. 2014), and (Maryanto et al. 2018) which stated that
the average technical efficiency of potatoes is between
50-85 percent. In contrast to the male workforce, the opposite result
is shown in the female workforce variable, which
generally gives a significant effect on the level of
significance (α) = 5 percent. The sign of a positive
parameter with an elasticity value of 0.182. This value
means a 10 percent increase in labor will increase
potato production by 18.2 percent. The role of female
workers who have the most influence on production is
in the District of Sururey with an elasticity value of
0.967 and is significant at the level of = 1 percent. This
means that a 10 percent increase in labor will increase
potato production by 96.7 percent. This indicates that
women are most involved in farming activities. The
survey results show the role of women in farming is
greater (51.65 percent), starting from land processing
activities to marketing. The male profession with the
main job as a civil servant or other occupations causes
the role of men in farming to be small. Especially in
marketing activities for Sururey and Anggi Districts,
it is the women’s job. In contrast to Olagunju (2007),
the role of male workers dominates farming activities. Female workers are more involved in planting,
fertilizing, maintaining, and marketing activities. CONCLUSIONS AND RECOMMENDATIONS Teknis Usahatani Kentang di Kabupaten Gowa
Sulawesi Selatan. Forum Agribisnis 11(1): 65–
74. https://doi.org/10.29244/fagb.11.1.65-74. Conclusions Asmara A, Purnamadewi YL, Lubis D. 2016. Keragaan
Produksi Susu dan Efisiensi Usaha Peternakan
Sapi Perah Rakyat di Indonesia. Jurnal
Manajemen Dan Agribisnis 13(1): 14–25. https://doi.org/10.17358/jma.13.1.14. Farmers in the Arfak Mountains control 0.07 hectares
of land for potato farming, use of 22 kg of seeds per
hectare, and the workload of men per growing season
is 36.74 HOK and female workers 48.53 HOK. The
average potato production is 150 kg with a productivity
level of 0.24 kg/ha. Factors that affect production are
land, seeds, and female labor. The elasticity value
generated in the production function is 1.009, namely
in the Increasing Return to Scale condition. The results
also show variation in technical efficiency among
potato farmers, with an average technical efficiency
level of 73 percent, implying there is potential to
increase efficiency by 27 percent. [BPS] Badan Pusat Statistik 2017-2020. Statistik
Indonesia. Jakarta: BPS. [BPS] Badan Pusat Statistik Kabupaten Pegunungan
Arfak 2016-2018. Pegunungan Arfak Dalam
Angka. Sorong: Pegunungan Arfak Papua Barat. Battese GE, Coelli TJ. 1995. A model for technical
inefficiency effects in a stochastic frontier
production function for panel data. Empirical
Economics
20(2):
325–332. https://doi. org/10.1007/BF01205442. Technical Efficiency Small-scale potato farming implemented by local
farmers determines the level of production and
productivity. The local hereditary knowledge held
by farmers in cultivating potatoes causes farmers
to be unable to allocate the use of inputs to achieve
efficient farming. The existing situation of potato
farming in the Arfak Mountains illustrates the inability
of farmers to allocate inputs even though the analysis
results show efficient farming. The capacity of farmers
must be increased with assistance from related parties
(government and private sector) to increase farmers’
managerial capacity. Technical efficiency is analyzed by using the stochastic
frontier production function model. The distribution of
the technical efficiency of potato farming can be seen
in Table 2, obtained from the analysis of the stochastic
frontier production function. The level of technical efficiency shows that most potato
farmers produce at technical efficiency between 70 –
90 percent. The least efficient farmers only achieve a
technical efficiency level of 23 percent, and the most
efficient farmers produce at a technical efficiency level
of 95 percent. This distribution is not different when
compared to the results of research by (Nyagaka et Table 2. Distribution of respondent farmers based on the level of technical efficiency of potato farming in Arfak
Mountains Regency, 2022
Technical Efficiency Rate (%)
Number of Respondents (Persons)
Percentage (%)
< 0.70
49
35.00
0.70 - 0.90
82
58.57
> 0.90
9
6.43
Total
140
100.00
Maximum
0.95
Minimum
0.23
Avarage
0.73 spondent farmers based on the level of technical efficiency of potato farming in Arfak
2022 Table 2. Distribution of respondent farmers based on the level of technical efficiency of pota 86 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 Recommendations Bukul BB. 2018. Factors Affecting Smallholder
Farmers Potato Production in Shashemene
District, West Arsi Zone, Oromia National
Regional State, Ethiopia. Food Science and
Quality Management 76: 66–72. Intensive assistance from extension workers needs to be
done, especially for good agricultural practices, the use
of technology according to the local wisdom of local
farmers, and the formation of farmer groups. Further
research can consider aspects of commercialization
to increase productivity and efficiency as well as the
sustainability of small-scale potato farming in the
Arfak Mountains. Debertin DL. 1986. Agricultural production economics. Macmillan. DiGaetano R. 2013. Sample Frame and Related
Sample Design Issues for Surveys of Physicians
and Physician Practices. Evaluation and the
Health Professions 36(3): 296–329. https://doi. org/10.1177/0163278713496566 FUNDING STATEMENT: This research did not
receive any specific grant from funding agencies in the
public, commercial, or not - for - profit sectors. Dube AK, Burhan O, Amanuel A, Diriba I, Ahmed,A. 2018. Technical efficiency and profitability of
potato production by smallholder farmers: The
case of Dinsho District, Bale Zone of Ethiopia. Journal of Development and Agricultural
Economics
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225–235. https://doi. org/10.5897/jdae2017.0890 CONFLICTS OF INTEREST: The authors declare
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farming in the Western Highlands of Guatemala. Economia Agro-Alimentare, 24(1). https://doi. org/10.3280/ecag2022oa13227.i Sagrim M, Sumule AI, Iyai DA, Baransano M. 2017. Prime potency of agriculture commodities on
highland of Arfak Mountains Regency, Papua
Barat. Jurnal Ilmu Pertanian Indonesia 22(3): Widayati T. 2017. Analisis efisiensi penggunaan faktor
produksi pada usahatani kentang di kawasan 88 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 Jurnal Manajemen & Agribisnis,
Vol. 20 No.1, March 2023 Dieng Jawa Tengah. Didalam: Optimalisasi Tata
Kelola Organisasi Dalam Upaya Peningkatan
Daya Saing Dan Iklim Investasi. Semarang:
Fakultas Ekonomika dan Bisnis. hlm 173–193. Dieng Jawa Tengah. Didalam: Optimalisasi Tata
Kelola Organisasi Dalam Upaya Peningkatan
Daya Saing Dan Iklim Investasi. Semarang:
Fakultas Ekonomika dan Bisnis. hlm 173–193. Yuminarti U, Darwanto DH, Jamhari J, Subejo S. 2018. Studi komparasi praktik perladangan berpindah
dan pertanian menetap untuk mendukung
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usahatani kentang di Kabupaten Pegunungan
Arfak). Jurnal Ketahanan Nasional 24(2): 215-
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115. 89
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Description of Lagocheirus delestali n. sp. (Coleoptera: Cerambycidae) from the Reserva Biológica Alberto Manuel Brenes, Alajuela, Costa Rica
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* Corresponding author: esteban@inia.es
Received: 07-03-07; Accepted: 08-10-07. Introduction Lagocheirus Dejean belongs to the tribe Acanthocinini
which is one of the most diverse tribes in the family
Cerambycidae. The distribution of species of Lagocheirus
is American, although their greatest richness is princi-
pally in Mesoamerica. Descripción de Lagocheirus delestali n.sp. (Coleoptera: Cerambycidae), nueva especie de la Reserva Biológica
Alberto Manuel Brenes de Alajuela en Costa Rica Se describe una nueva especie del género Lagocheirus Dejean, Lagocheirus delestali Toledo & Esteban a partir de
especímenes colectados en la Reserva Biológica Alberto Manuel Brenes, Alajuela, Costa Rica. Se describe una nueva especie del género Lagocheirus Dejean, Lagocheirus delestali Toledo & Esteban a partir de
especímenes colectados en la Reserva Biológica Alberto Manuel Brenes, Alajuela, Costa Rica. Palabras clave adicionales: Cerambycidae, Costa Rica, Lagocheirus, Lamiinae, nueva especie. p
g
,
j
,
Palabras clave adicionales: Cerambycidae, Costa Rica, Lagocheirus, Lamiinae, nueva collection of material of an unknown species belonging
to the genus Lagocheirus. Description of Lagocheirus delestali n. sp.
(Coleoptera: Cerambycidae) from the Reserva Biológica
Alberto Manuel Brenes, Alajuela, Costa Rica V. H. Toledo1 and J. R. Esteban Durán2*
1 CEAMISH. Universidad Autónoma del Estado de Morelos. Mexico
2 Departamento de Protección Vegetal. INIA. Ctra. A Coruña, km 7,5. 28040 Madrid. Spain V. H. Toledo1 and J. R. Esteban Durán2*
1 CEAMISH. Universidad Autónoma del Estado de Morelos. Mexico
2 Departamento de Protección Vegetal. INIA. Ctra. A Coruña, km 7,5. 28040 Madrid. Spain Abstract A new species of the genus Lagocheirus Dejean, Lagocheirus delestali Toledo & Esteban is described from speci-
mens collected from the Reserva Biológica Alberto Manuel Brenes, Alajuela, Costa Rica during 2006 and 2007. Additional key words: Cerambycidae, Costa Rica, Lagocheirus, Lamiinae, new species. A new species of the genus Lagocheirus Dejean, Lagocheirus delestali Toledo & Esteban is described from speci-
mens collected from the Reserva Biológica Alberto Manuel Brenes, Alajuela, Costa Rica during 2006 and 2007. Additi
l k
d
C
b
id
C
t Ri
L
h i
L
ii
i Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)
Available online at www.inia.es/sjar Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)
Available online at www.inia.es/sjar Spanish Journal of Agricultural Research 2008, 6 (Special issue), 26-29
ISSN: 1695-971-X Systematics Lagocheirus delestaliToledo & Esteban, new species. Figure 2. Holotype male. Detail of pronotum and scutellum. Diagnosis collected was deposited at INIA and in the Museo de
Insectos (UCR). The following combination of morphological
characters can distinguish this species from others:
elytra with dark brown to black pubescence in the basal
half, antennal segment III and IV biannulate, red
brown. Material and Methods The species was obtained from the Reserva Biológica
Alberto Manuel Brenes, a wild protected area created
on 1 June 1975, as the San Ramón Forest Reserve. The
reserve is administrated by the UCR and MINAE (Mi-
nisterio de Ambiente y Energía) of Costa Rica. The
reserve covers 7,800 ha, and 90% of it corresponds to
the San Lorencito River Basin. The area is limited to
the North by Arenal-Monteverde and the Cloud Forest
Reserve; to the East and South with primary and secon-
dary forest, and to the West with the forest of Cidral
in the locality of Miramar. The genus has been revised twice, firstly by Dillon
in 1957, and more recently by Toledo (1998), 10 years
ago, and only included species from Mexico and
Central America. Lately new distributional records and
a new species from Jamaica were added to knowledge
of the genus Lagocheirus Dejean (Toledo and Hovore,
2005). An Entomological Biodiversity Project between
the Instituto Nacional de Investigación y Tecnología
Agraria y Alimentaria (INIA, Spain) and the University
of Costa Rica (UCR, San José, Costa Rica) has involved
entomological explorations, from 2003, at the Reserva
Biológica Alberto Manuel Brenes, Costa Rica, with The insect specimens were attracted to the station
lights, or to a light trap located about 100 m from the
Station. The light trap had two kinds of lamps, one
was mercury vapour (400 watts) at 4 m high and there
were two further mercury vapour (125 watts) lamps
placed against a vertical white sheet. The material Description of Lagocheirus delestali n. sp. (Coleoptera: Cerambycidae) 27 Figure 1. Lagocheirus delestali. A) Holotype male. B) Paratype 1 female. B A B Figure 1. Lagocheirus delestali. A) Holotype male. B) Paratype 1 female. Type material Holotype male labelled: Costa Rica, Alajuela, Esta-
ción de la Reserva Biológica Alberto Manuel Brenes,
Alt. 850 m, 21 de abril de 2006, José Rafael Esteban
Durán (José Rafael Esteban Durán collection, deposited
in INIA, Madrid, Spain). Paratypes: three females, same
data as holotype except: 26 de enero de 2006, Marco
Antonio Zumbado; Trampa de luz, 2 de mayo de 2006,
José Rafael Esteban Durán; 30 de abril de 2006, José
Rafael Esteban Durán (Museo de Insectos de la
Universidad de Costa Rica); cebo luminoso (400 + 2 ×
× 125 W. Hg.), and one male 22-III-2007, cebo luminoso
(400 + 2 × 125 W. Hg.) José Esteban, leg. (Museo de
Insectos de la UCR). Figure 2. Holotype male. Detail of pronotum and scutellum. Span J Agric Res (2008), 6 (Special issue J. M. Malpica), 26-29 28 Figure 3. Holotype male, ventral view. Figure 4. Holotype male. Detail of 6th and 7th antenal segments. Figure 4. Holotype male. Detail of 6th and 7th antenal segments. terior and posteriorly by a whitish-beige vitta that con-
tinues to the suture, and with a whitish-beige macula
of pubescence on the suture near the apex; pro- and
mesosternum (Fig. 3) with whitish brown sparse irre-
gularity, metasternum almost glabrous at the anterior
half and at middle and densely clothed with whitish
brown pubescence at the apical half. Abdomen irregu-
larly clothed with whitish brown pubescence at the
sides and the margin of the segments. Legs with short,
whitish-beige pubescence, tibia annulate basal and
medially with reddish brown pubescence; tarsus with
dense beige pubescence; claws black. Figure 3. Holotype male, ventral view. Head. Front slightly convex; longitudinal median
line extending from epistoma to occiput; antennal tu-
bercles slightly prominent and divergent, eyes with
lower lobe wider than long and longer than genae;
upper interocular space broader than upper eyes lobes;
antennae with segment VIII extending beyond elytral
apices, scape with dense, scattered punctures, scape
shorter than segment III and slightly longer than seg-
ment IV, segment VI with apical appendix, shorter than
the width of the segment, with an apical set of long
black setae (Fig. 4), segments V to XI gradually de-
creasing in length. Pronotum 1.2 times broader than
long, base broader than apex; disk with prominent
subconical tubercles, apical tubercles slightly prominent, Phenology basal tubercles prominent and rounded, median discal
tubercle small and elongated, lateral tubercles prominent,
subconical, unarmed at apex; disk with small punctures
around tubercles, basal and apical depressions with
coarse punctures. Scutellum subtriangular, apically
emarginate. Elytra 1.5 times longer than broad; basal
gibbosity scarcely evident, with a single prominent basal
tubercle; humeral angles coarsely granulate; disk den-
sely, coarsely granulate-punctate on basal one-third,
punctures sub-equal granules, punctures becoming
simple, finer, and less dense to the apex; each elytron
vaguely tricostate, base with three rows of elevated and
dense tufts of black setae; apices slightly rounded. Prosternal process 1.7 times the width of a coxal cavity;
mesosternal process almost as wide as coxal cavity,
coarsely punctate laterally (Fig. 5); metasternum sparse
and finely punctate. Abdomen sparsely, finely punctate;
first sternite slightly longer than two to four, which are
sub-equal in length, fifth sternite tapered, emarginate
at apex. Legs regularly, finely punctate. The flight period of the specimen is from January
to May. It basically coincides with the period of acti-
vity of adults of other species of this genus which have
been collected in this ecosystem from December to
June. Description Male (Fig. 1A). Length: 21 mm; humeral width: 9.1mm. Form robust, elongated, slightly sub-depressed; inte-
gument dark brown to piceous; pubescence dense, very
short, appressed, white-yellowish, beige, reddish-brown
and black. Head regularly with reddish-brown pubes-
cence; upper interocular space with sparse, irregular
pubescence and long black setae; antennae with sparse,
short, appressed reddish-brown pubescence, segments
III and IV whitish-brown biannulate, segments V to
VII with central whitish-beige annulate. Pronotum with
a black basal vitta, extending from basal margin to the
base of basal tubercles, median lateral black vitta ex-
tending from basal margin to the base of lateral tuber-
cles (Fig. 2). Scutellum clothed with blackish pubes-
cence, with lateral margins reddish-brown. Elytra with
dark-brown to black pubescence in basal half, and
whitish-beige, dark brown and black pubescence in the
apical half; basal half limited apically by a transverse
whitish-beige vitta extending in a zig-zag from lateral
side to the elytral suture and extending to the apex,
apical third with a transverse black vitta extending in
a zig-zag from lateral side to near suture, limited an- Figure 5. Holotype male. Detail of mesosternum. Figure 5. Holotype male. Detail of mesosternum. Description of Lagocheirus delestali n. sp. (Coleoptera: Cerambycidae) 29 This work was financed by Project INIA AT07-001. This work was financed by Project INIA AT07-001. This work was financed by Project INIA AT07-001. References DILLON L.S., 1957. Revision of the neotropical Acantho-
cinini (Coleoptera: Cerambycidae) II. The genus Lago-
cheirus. Bull Br Mus (Nat Hist) Entomol 6(6), 137-166. Etymology We dedicate this beautiful species to Dr. D. Pedro
Del Estal Padillo, Professor of Entomology, Escuela
Técnica Superior de Ingenieros Agrónomos, Universidad
Politécnica de Madrid (Spain), for his contribution to
knowledge of the entomological fauna of the Reserva
Biológica Alberto Manuel Brenes, Costa Rica. Acknowledgments Female. Similar to the male, except for the antennae
with the VI segment without an apical projection and
IX extending beyond elytral apices. Length: 15-20 mm
(Fig. 1B). Remarks This species closely resembles Lagocheirus plantaris
indisctinctus Dillon & Dillon, but can be distinguished
from it because L. delestali has lateral elytra margins
in parallel until 3/5 near the apex; antennae with seg-
ment II longer; pronotum with lateral tubercles sub-
conic and more acute at the apex and scutellum with a
thin line of brown pubescence at the lateral margin. TOLEDO V.H., 1998. Revisión taxonómica del género
Lagocheirus Dejean para México y Centroamérica (Co-
leoptera: Cerambycidae). Fol Entomol Mex 101, 1-58. [In
Spanish]. TOLEDO V.H., HOVORE F.T., 2005. Notes on the genus
Lagocheirus Dejean: records and descriptions (Coleoptera:
Cerambycidae: Lamiinae: Acanthocinini). Zootaxa 1021,
29-36.
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Supratentorial and Infratentorial Lesions in Spinocerebellar Ataxia Type 3
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Keywords: spinocerebellar ataxia type 3 (SCA3), supratentorial involvement, fractal dimension, morphological
changes, MRI Supratentorial and Infratentorial
Lesions in Spinocerebellar Ataxia
Type 3 Po-Shan Wang 1,2,3, Yu-Te Wu 1,2,4, Tzu-Yun Wang 4, Hsiu-Mei Wu 5, Bing-Wen Soong 1,6,7,8*
and Chi-Wen Jao 1,2,9* 1 Brain Research Center, National Yang-Ming University, Taipei, Taiwan, 2 Institute of Biophotonics, National Yang-Ming
University, Taipei, Taiwan, 3 Department of Neurology, Taipei Municipal Gan-Dau Hospital, Taipei, Taiwan, 4 Department of
Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan, 5 Department of Radiology,
Taipei Veterans General Hospital, Taipei, Taiwan, 6 Taipei Neuroscience Institute, Taipei Medical University, Taipei, Taiwan,
7 Department of Neurology, Taipei Medical University-Shuang Ho Hospital, New Taipei City, Taiwan, 8 Department of
Neurology, Taipei Veterans General Hospital, Taipei, Taiwan, 9 Department of Neurology, Shin-Kong Wu Ho Su Memorial
Hospital, Taipei, Taiwan Background:
Spinocerebellar
ataxia
type
3
(SCA)
is
a
cerebellum-dominant
degenerative disorder that is characterized primarily by infratentorial damage, although
less severe supratentorial involvement may contribute to the clinical manifestation. These
impairments may result from the efferent loss of the cerebellar cortex and degeneration
of the cerebral cortex. ORIGINAL RESEARCH
published: 03 March 2020
doi: 10.3389/fneur.2020.00124 Edited by:
Salem Hannoun,
American University of
Beirut, Lebanon Method: We used the three-dimensional fractal dimension (3D-FD) method to quantify
the morphological changes in the supratentorial regions and assessed atrophy in the
relatively focal regions in patients with SCA3. A total of 48 patients with SCA3 and 50
sex- and age-matched healthy individuals, as the control group, participated in this study. The 3D-FD method was proposed to distinguish 97 automatic anatomical label regions
of gray matter (left cerebrum: 45, right cerebrum: 45, cerebellum: 7) between healthy
individuals and patients with SCA3. Reviewed by:
Roberto Rodríguez-Labrada,
Cuban Public Health Ministry, Cuba
Hong Jiang,
Central South University, China
Beisha Tang,
Central South University, China Reviewed by:
Roberto Rodríguez-Labrada,
Cuban Public Health Ministry, Cuba
Hong Jiang,
Central South University, China
Beisha Tang,
Central South University, China *Correspondence:
Bing-Wen Soong
bwsoong@gmail.com
Chi-Wen Jao
c3665810@ms24.hinet.net Results:
Patients with SCA3 exhibited reduced brain complexity within both the
traditional olivopontocerebellar atrophy (OPCA) pattern and specific supratentorial
regions. The study results confirmed the extensive involvement of extracerebellar regions
in SCA3. The atrophied regions of SCA3 in infratentorial and supratentorial cortex
showed a wide range of overlapped areas as in two functional cortexes, namely
cerebellum-related cortex and basal ganglia-related cortex. Specialty section:
This article was submitted to
Applied Neuroimaging,
a section of the journal
Frontiers in Neurology Conclusions: Our results found that the atrophy of the SCA3 are not only limited in the
infratentorial regions. Both cerebellar related cortex and basal ganglia related cortex were
affected in the disease process of SCA3. Our findings might correlate to the common
symptoms of SCA3, such as ataxia, Parkinsonism, dysarthria, and dysmetria. SCA3
should no longer be considered a disease limited to the cerebellum and its connections;
rather, it should be considered a pathology affecting the whole brain. Received: 06 November 2019
Accepted: 04 February 2020
Published: 03 March 2020 Participants Diffusion tensor imaging (DTI) can facilitate the visualization
and characterization of white matter (WM), and DTI is also
an efficient method for study of SCA3 (15). SCA3 had been
verified as a WM dominant atrophy neurodegenerative disease
(16). Previous studies showed patients with SCA3 demonstrate
decreases in fractional anisotropy (FA) in the areas of cerebellum
and brainstem, but increases in radial diffusivity (RD) in the
cerebellum, brainstem, thalamus, and frontal and temporal lobes
(17). Another DTI study identified widespread FA reduction in
the bilateral cerebral-frontal, -parietal, -temporal, and -occipital
WM; cerebellar WM; the thalamus; and the brainstem in
patients with SCA3 (16). Moreover, mean diffusivity (MD)
increases were detected in a similar, widely overlapping
pattern
in
bilateral
cerebral-frontal,
-parietal,
-temporal,
and -occipital WM; cerebellar WM; the thalamus; and the
brainstem (16). p
A total of 48 patients with SCA3 and 50 sex- and age-
matched healthy individuals, as the control group, participated
in this study. This study was conducted in accordance
with the Declaration of Helsinki and was approved by the
Institutional Review Board of Taipei Veterans General Hospital. All participants provided written informed consent before
participating in this study. All participants were recruited from
the Department of Radiology, Taipei Veterans General Hospital. The SARA (scale for the assessment and rating of ataxia) was
applied for clinical assessment of ataxia of patients with SCA3
(19). The symptoms of Parkinson’s disease progress in SCA3
were measured by using Modified Hoehn and Yahr staging (20). The SARA score, which was a rating of the severity of ataxia
symptoms ranging from 0 to 40, was used as a reference to
indicate the progression of clinical severity in comparison with
the cerebellar degeneration. A self-reported age of onset, the age
at which the patients showed the first sign of any ataxic symptom,
was acquired from each patient (21). The CAG repeat length of
each SCA3 patient was determined by polymerase chain reaction,
as described previously (22). Table 1 presents the demographic,
clinical, and MR imaging data of both groups. Patients with SCA3
met the inclusion criteria if they had progressive and otherwise
unexplained ataxia and tested positive for the SCA3 genotype. The disease duration in patients with SCA3 was 8.89 ± 6.432
years. Those in the control group had no central nervous system
disease and did not exhibit any neurological abnormalities during
the study period. Citation: Wang P-S, Wu Y-T, Wang T-Y,
Wu H-M, Soong B-W and Jao C-W
(2020) Supratentorial and Infratentorial
Lesions in Spinocerebellar Ataxia Type
3. Front. Neurol. 11:124. doi: 10.3389/fneur.2020.00124 March 2020 | Volume 11 | Article 124 Frontiers in Neurology | www.frontiersin.org 1 Supratentorial and Infratentorial Lesions in SCA3 Wang et al. TABLE 1 | Demographic, clinical, and MR image data of the control group and the
patient group. Characteristic
Group
Controls
(N = 50)
SCA3 Patients
(N = 48)
p-value
Sex (F/M)
25/25
21/27
0.535a
Age (years)†
48.24 ± 13.956
48.13 ± 11.747
0.516b
Duration (years)†
–
8.89 ± 6.432
–
SARA†
–
14 ± 8.103
–
H & Y staging†
–
2.88 ± 1.19
–
Cerebral
atrophy/cerebellar atrophy
observed through visual
inspection
–
9/39
–
CAG repeat length
–
73.2 ± 4.2
CAG < 74/CAG
> 74 = 26/22
–
SARA, Scale for the Assessment and Rating of Ataxia; MMSE, Mini-Mental State
Examination; H & Y staging, Hoehn and Yahr Staging Scale. aPearson’s chi-square test
(χ2 = 0.384); b2-tailed 2-sample t-test. †Continuous variables are expressed as mean
± standard deviation. TABLE 1 | Demographic, clinical, and MR image data of the control group and the
patient group. INTRODUCTION Spinocerebellar
ataxia
type
3
(SCA3)
is
an
inherited
neurodegenerative disorder caused by CAG expansion in the
coding region of chromosome 14q32.1 (1, 2). Clinically, patients
with SCA3 exhibit cerebellar syndrome, Parkinsonism, ataxic
gait, dysarthria, dysmetria, nystagmus, peripheral neuropathy,
pyramidal, and extrapyramidal manifestations (3–5). Patients
with SCA3 also suffered from emotional impairments, such as
depression and anxiety (6, 7). These higher-order dysfunctions
of SCA3 suggest the further involvement of extracerebellar
structures rather than the conventional “olivopontocerebellar”
pattern of neurodegeneration (8–12). Additionally, the SCA3
disorder has been described as cerebellar cognitive affective
syndrome (CCAS) (6, 8). Studies have used positron emission tomography (PET)
and single-photon emission computed tomography to identify
subclinical abnormalities in the cerebral cortex (13, 14). However,
these studies reported the disruption of the cerebrocerebellar
pathway cannot completely explain the cognitive and affective
impairment in patients with SCA3. For example, visuospatial
deficits in patients with SCA3 are markedly associated with the
parietal lobe and are less connected to the cerebellum. Few studies
have focused on the role of supratentorial regions in SCA3. The
quantification of the degeneration in supratentorial regions may
be crucial for evaluating dysfunction in patients with SCA3. In neuroimaging studies, measuring regional cortical atrophy is
crucial for evaluating its association with cognitive impairment
and emotional dysfunction. SARA, Scale for the Assessment and Rating of Ataxia; MMSE, Mini-Mental State
Examination; H & Y staging, Hoehn and Yahr Staging Scale. aPearson’s chi-square test
(χ2 = 0.384); b2-tailed 2-sample t-test. †Continuous variables are expressed as mean
± standard deviation. SARA, Scale for the Assessment and Rating of Ataxia; MMSE, Mini-Mental State
Examination; H & Y staging, Hoehn and Yahr Staging Scale. aPearson’s chi-square test
(χ2 = 0.384); b2-tailed 2-sample t-test. †Continuous variables are expressed as mean
± standard deviation. between motor-related impairment and supratentorial regions
atrophy in patients with SCA3. Frontiers in Neurology | www.frontiersin.org Participants An experienced neuroradiologist examined the In this study, we assessed atrophy in relatively focal regions
and explained supratentorial involvement in SCA3. This study
used the fractal dimension (FD) method, which has the advantage
of producing results with minimal variation (18), to quantify
cortical morphological changes and measure the regional cortical
atrophy of supratentorial regions. In the present study, we
measured the 3D-FD values of the 97 segments of the gray matter
from the entire brain for each participant. We further compare
these gray matter lesions results with DTI analysis of white matter
lesions in SCA3 of our recent study. We anticipated the assessed
GM lesions regions in this study are related to WM lesions
regions from DTI study, and the result may verify the association March 2020 | Volume 11 | Article 124 2 Wang et al. Supratentorial and Infratentorial Lesions in SCA3 FIGURE 1 | (A–E) Procedure of the image process and statistical analysis. FIGURE 1 | (A–E) Procedure of the image process and statistical analysis. FIGURE 1 | (A–E) Procedure of the image process and statistical analysis. was used to normalize each T1-weighted image toward the
JHU_MNI_SS_T_ss T1 template. T1- and T2-weighted images of the control group to ensure the
absence of uncovered signs of another neurological disease or
unexpected abnormalities. Frontiers in Neurology | www.frontiersin.org Magnetic Resonance Imaging
Data Acquisition and Processing The image data processes were performed using the SPM8
toolbox
(Wellcome
Department
of
Cognitive
Neurology,
Institute of Neurology, University College London, London,
UK,
http://www.fil.ion.ucl.ac.uk/spm/)
and
the
IBASPM
toolbox (Individual Brain Atlases using Statistical Parametric
Mapping,
http://www.thomaskoenig.ch/Lester/ibaspm.htm),
both run using MATLAB 2010 software (Mathworks, Natick,
MA, USA). Axial MR human brain images covering the entire cerebrum
and cerebellum were acquired using a 1.5-T Vision Siemens
scanner (Erlangen, Germany). Participants were scanned with
a circularly polarized head coil to obtain T1-weighted images
(TR = 14.4 ms; TE = 5.5 ms; matrix size: 256 × 256; 1.5 mm
axial slices; FOV = 256 × 256 mm2; voxel size, 1.0 ×
1.0 × 1.5 mm3, number of slice = 128). The acquired T1-
weighted images of each participant were reformatted into an
axial image and converted to an analysis format by MRIcro
software (Chris Rorden, University of Nottingham, UK; www. sph.sc.edu/comd/rorden/mricro.html). Figure 1 illustrates the
data processing and statistical analysis flowchart. To improve
the accuracy of brain tissue extraction, an automated skull-
stripping function was applied to image volumes using the brain
extraction tool in MRIcro software (Figure 1B). The subsequent
processes were performed using DiffeoMap (Li, X.; Jiang, H.;
and Mori, S.; Johns Hopkins University, www.MriStudio.org). In this procedure, a 12-parameter affine transformation (23) The procedure involves three steps after normalization: (1)
segmentation of the normalized image into gray matter, white
matter, and cerebral spinal fluid in native space, (2) parcellation
of gray matter into 116 regions based on the anatomical labeling
of the Montreal Neurological Institute (MNI) anatomical atlas
(24), and (3) transformation of gray matter images into the
MNI space and the anatomical alignment of each voxel of gray
matter to the 116 automatic anatomical label structures using
IBASPM (24), left cerebrum: 45 regions, right cerebrum: 45
regions, cerebellum: 26 regions; (Figure 1C). The 26 regions of
the cerebellum were merged into seven regions according to
their anatomical structures, and the volumes of the 97 labeled March 2020 | Volume 11 | Article 124 Frontiers in Neurology | www.frontiersin.org 3 Supratentorial and Infratentorial Lesions in SCA3 Wang et al. FIGURE 2 | Comparison of 3D-FD values in each lobe between the healthy group and the SCA3 group. The 3D-FD value in each lobe of SCA3 showed significantly
lower the control group. RESULTS (45 for each cerebral hemisphere and seven for cerebellum,
Supplementary Table 1) brain structures were extracted for each
person (Figure 1C). Statistical Analysis y
The sex- and age-related differences between the groups were
measured using Pearson’s chi-square test (χ2 = 0.384, p =
0.535) and the 2-tailed 2-sample t-test (p = 0.516), separately. Linear regression was applied to remove effects of age and
gender. We adopted a 2-tailed t-test to determine whether a
significant difference existed between the control and patient
groups regarding the 3D-FD values of each cortical region. The significance of the results was based on the false discovery
rate (FDR-corrected p = 0.05) (28). The magnitude of the
association between the 3D-FD value of individual brain regions
and clinical features, such as disease duration and SARA,
was determined through Pearson’s r measurement (Figure 1E). These analyses were conducted using the Statistics Toolbox in
MATLAB 2010. Patients With SCA3 Revealed Significant
Lesions in Typical Infratentorial Lesions
Regions The FD method was originally proposed by Mandelbrot for
the quantification of the shape-related complexity of objects
into a single numerical value (25). The FD method is used for
the topological measurement of complexity; a higher FD value
represents greater topological complexity of the tissue under
study (18, 26). Many neurologists have demonstrated that the
FD value can serve as a quantitative measure for accurately
describing the morphological complexity of the cerebral folding
(18, 26, 27). Because FD analysis is based on a logarithmic scale,
even a small increase in the FD value may correspond to a
considerable increase in complexity (18). This study adopted
the 3D box-counting method to measure the 3D-FD values of
the 97 segments of gray matter from the entire brain for each
participant (Figure 1D). A related study details the algorithm for
the box-counting method (18). Experienced neuroradiologists reported the MRI findings for
patients with SCA3 (Table 1). Figure 2 illustrates the comparison
of 3D-FD values in each lobe between the healthy group and the
SCA3 group. Globally, patients with SCA3 exhibited significantly
decreased 3D-FD values in every lobe, and the cerebellum was
associated with the most substantial reduction in 3D-FD value
(Figure 2). The 3D-FD values of the cerebellar cortex of SCA3
showed significant correlation with their SARA scores (r =
−0.3346; p = 0.023). We further parcellated the cerebral and
cerebellar cortex into 97 regions and quantified atrophy in each
parcellated region. Patients with SCA3 had typical infratentorial
lesions regions, including pontine nuclei, cerebellar cortex, and
inferior olives. The details of the significantly decreased 3D-FD
values of atrophy regions between the healthy group and the
SCA3 group are summarized in Table 2. Magnetic Resonance Imaging
Data Acquisition and Processing The p-value of significant difference of cerebellum, frontal lobe, parietal lobe, and occipital lobe are smaller than 0.01, and for other lobes are
smaller than 0.05. FIGURE 2 | Comparison of 3D-FD values in each lobe between the healthy group and the SCA3 group. The 3D-FD value in each lobe of SCA3 showed significantly
lower the control group. The p-value of significant difference of cerebellum, frontal lobe, parietal lobe, and occipital lobe are smaller than 0.01, and for other lobes are
smaller than 0.05. Patients With SCA3 Revealed Cerebral
Lateralized Supratentorial Lesions Beside the infratentorial lesions, lesions in supratentorial regions
were also observed in all patients with SCA3. They showed
widespread lesions in 39 cerebral parcellated regions (p < 0.05),
including frontal lobe, parietal lobe, occipital lobe, and temporal
lobe. They revealed lateralized atrophy, and predominantly
in the left hemisphere (right/left: 12/27). Especially in the
occipital and temporal lobes, patients with SCA3 revealed
significantly decreased 3D-FD values only in the left hemisphere. The significant atrophied regions in frontal regions were the
premotor cortex (left precentral gyrus, bilateral superior frontal
gyrus, and left-middle frontal gyrus), supplementary motor March 2020 | Volume 11 | Article 124 Frontiers in Neurology | www.frontiersin.org 4 Supratentorial and Infratentorial Lesions in SCA3 Wang et al. TABLE 2 | Significant atrophied regions in patients with SCA3 (p < 0.05). Patients With SCA3 Revealed Cerebral
Lateralized Supratentorial Lesions Region (L/R)
Controls
SCA3
Cerebellar cortex
Entire
2.56 ± 0.02
2.53 ± 0.04
Anterior lobe (L)
2.17 ± 0.04
2.11 ± 0.07
Anterior lobe (R)
2.15 ± 0.04
2.03 ± 0.08
Posterior lobe (L)
2.47 ± 0.03
2.45 ± 0.04
Posterior lobe (R)
2.48 ± 0.03
2.44 ± 0.04
Vermis
2.15 ± 0.05
2.12 ± 0.04
Frontal lobe
Precentral gyrus (L)
2.15 ± 0.07
2.07 ± 0.07
Superior frontal gyrus (L)
2.08 ± 0.03
2.05 ± 0.05
Superior frontal gyrus (R)
2.13 ± 0.04
2.10 ± 0.06
Middle frontal gyrus (L)
2.28 ± 0.04
2.25 ± 0.04
Orbitofrontal cortex (superior-medial) (L)
2.11 ± 0.04
2.08 ± 0.05
Orbitofrontal cortex (superior-medial) (R)
2.14 ± 0.04
2.10 ± 0.05
Inferior frontal gyrus (opercular) (R)
2.10 ± 0.05
2.07 ± 0.05
Inferior frontal gyrus (triangular) (L)
2.27 ± 0.04
2.23 ± 0.05
Supplementary motor area (L)
2.19 ± 0.05
2.14 ± 0.05
Superior frontal gyrus (medial) (L)
2.17 ± 0.04
2.12 ± 0.06
Superior frontal gyrus (medial) (R)
2.09 ± 0.05
2.06 ± 0.07
Paracentral lobule (L)
2.04 ± 0.07
1.99 ± 0.08
Paracentral lobule (R)
1.98 ± 0.07
1.92 ± 0.08
Parietal lobe
Post-central gyrus (L)
2.17 ± 0.05
2.10 ± 0.06
Superior parietal gyrus (L)
2.10 ± 0.06
2.03 ± 0.06
Superior parietal gyrus (R)
2.08 ± 0.06
2.04 ± 0.07
Inferior parietal gyrus (L)
2.19 ± 0.07
2.09 ± 0.08
Supramarginal gyrus (L)
2.11 ± 0.05
2.04 ± 0.06
Angular gyrus (L)
2.12 ± 0.07
2.00 ± 0.09
Precuneus (L)
2.21 ± 0.03
2.17 ± 0.05
Precuneus (R)
2.14 ± 0.04
2.10 ± 0.04
Temporal lobe
Superior temporal gyrus (L)
2.181 ± 0.051
2.120 ± 0.053
Middle temporal gyrus (L)
2.336 ± 0.034
2.303 ± 0.048
Occipital lobe
Calcarine fissure and surrounding cortex (L)
2.25 ± 0.04
2.22 ± 0.04
Cuneus (L)
2.13 ± 0.04
2.11 ± 0.04
Lingual gyrus (L)
2.20 ± 0.04
2.17 ± 0.05
Superior occipital gyrus (L)
1.95 ± 0.06
1.89 ± 0.08
Middle occipital gyrus (L)
2.19 ± 0.05
2.12 ± 0.07
Temporal lobe
Superior temporal gyrus (L)
2.18 ± 0.05
2.12 ± 0.05
Middle temporal gyrus (L)
2.34 ± 0.03
2.30 ± 0.05
Limbic
Posterior cingulate gyrus (L)
1.99 ± 0.05
1.95 ± 0.05
Parahippocampal gyrus (R)
2.16 ± 0.03
2.14 ± 0.03
Subcortical regions
Caudate nucleus (R)
2.09 ± 0.04
2.04 ± 0.05
Lenticular nucleus, putamen (L)
2.11 ± 0.08
2.07 ± 0.05
Amygdala (R)
1.94 ± 0.04
1.97 ± 0.04
Caudate nucleus (L)
2.08 ± 0.05
2.05 ± 0.05
Brain regions with significant difference (P < 0.01) in 3D-FD values between control and
patients with SCA3. Frontal lobe In Figure 6, we found many cerebral atrophied regions
included
cerebellum,
cerebellar–thalamocortical,
and
basal
ganglia–thalamocortical link circuits that implied patients
with
SCA3
revealed
cerebellar–thalamocortical
and
basal
ganglia–thalamocortical damaged in these pathways. Patients With SCA3 Revealed Cerebral
Lateralized Supratentorial Lesions Significant difference under a corrected threshold of FDR = 0.05, The cortex (left supplementary motor area), and primary motor
cortex (left precentral gyrus) (Figure 3). Other atrophied regions were in the left inferior frontal
gyrus (opercular), left inferior frontal gyrus (triangular), bilateral
orbitofrontal cortex (superior-medial), bilateral superior frontal
gyrus (medial), and bilateral paracentral lobule. In parietal lobe,
decreased FD values were detected in the left postcentral gyrus,
left supramarginal gyrus, left angular gyrus, bilateral superior
parietal gyrus, and bilateral precuneus of patients with SCA3
(Figure 4). In the occipital lobe, SCA3 patients revealed significant lower
FD values in the calcarine fissure and the surrounding cortex,
cuneus, lingual gyrus, and the superior and middle occipital gyri
in the left hemisphere (Figure 5A, blue color). In the temporal
lobe, regions with decreased FD values were in the left-superior
temporal and left-middle temporal gyri of patients with SCA3
(Figure 5A, purple color). The 3D-FD values in the left-posterior
cingulate gyrus of the limbic region were also significantly
decreased in patients with SCA3 (Figures 5B,D, green color). Regions of significant atrophy were observed in the basal ganglia,
including the bilateral caudate nucleus and left putamen, in
patients with SCA3 (Figures 5B,D, aqua blue color). In the
right-outer occipital lobe, no significant atrophied regions were
observed in patients with SCA3 (Figure 5C). DISCUSSION Patients with SCA3had significantly decreased 3D-FD values in frontal regions, including the premotor cortex (left
precentral gyrus, bilateral superior frontal gyrus, and left-middle frontal gyrus), supplementary motor cortex (left supplementary motor area), and primary motor cortex
(left precentral gyrus). Other atrophied regions were observed in the left inferior frontal gyrus (opercular), left inferior frontal gyrus (triangular), bilateral orbitofrontal
cortex (superior-1 medial), bilateral superior frontal gyrus (medial), and bilateral paracentral lobule in patients with SCA3. All the significantly decreased 3D-FD values
regions are illustrated in red. FIGURE 4 | Regions within the parietal lobe indicating a decrease in 3D-FD value in patients with SCA3 compared with controls. (A) Left-outer view, (B) left-inner
view, (C) right-outer view, and (D) right-inner view. Compared with controls, patients with SCA3had significantly lower 3D-FD values in the left postcentral gyrus, left
supramarginal gyrus, left angular gyrus, bilateral superior parietal gyrus, and bilateral precuneus of the parietal lobe. All the significantly decreased 3D-FD values
regions are illustrated in yellow. FIGURE 4 | Regions within the parietal lobe indicating a decrease in 3D-FD value in patients with SCA3 compared with controls. (A) Left-outer view, (B) left-inner
view, (C) right-outer view, and (D) right-inner view. Compared with controls, patients with SCA3had significantly lower 3D-FD values in the left postcentral gyrus, left
supramarginal gyrus, left angular gyrus, bilateral superior parietal gyrus, and bilateral precuneus of the parietal lobe. All the significantly decreased 3D-FD values
regions are illustrated in yellow FIGURE 5 | Regions within the occipital lobe, temporal lobe, and subcortical lobe indicating significantly lower 3D-FD values in patients with SCA3 compared with
controls. (A) Left-outer view, (B) left-inner view, (C) right-outer view, and (D) right-inner view. The blue regions show significantly lower 3D-FD values in the occipital
lobe, including the calcarine fissure and the surrounding cortex, cuneus, and lingual gyrus and the superior and middle occipital gyrus in the left hemisphere. Purple
regions denote lower 3D-FD values in the left-superior temporal gyrus and left-middle temporal gyrus. Aqua blue regions represent lower 3D-FD values in subcortical
regions, including the bilateral caudate and left putamen. Green regions represent significantly lower 3D-FD values in the left-posterior cingulate gyrus and
parahippocampal gyrus of the limbic system. FIGURE 5 | Regions within the occipital lobe, temporal lobe, and subcortical lobe indicating significantly lower 3D-FD values in patients with SCA3 compared with
controls. DISCUSSION Clinically, patients with SCA3 exhibit cerebellar syndrome and
parkinsonism (3–5). The atrophied regions of infratentorial
and supratentorial cortex listed in Table 2, and showed a wide
range of overlapped areas as in two functional regions, namely
cerebellum- related (CB-related) cortex (29) and basal ganglia-
related (BG-related) cortex (30). The CB-related cortex include
the cerebellum, prefrontal, sensorimotor cortices, prefrontal
cortex, and temporal lobe, and BG-related cortex include primary
motor cortex, supplementary motor area, premotor cortex, and
basal ganglia. Our results revealed consistent with previous
conventional neuroimaging studies that the cerebellum is the
most affected and atrophied region in patients with SCA3 (4,
31, 32). Additionally, the 3D-FD value in the cerebellar cortex of
patients with SCA3 had a significantly negative correlation with
SARA scores (r = −0.3346; p = 0.023) as Rezende et al. had ever
reported (33). In the infratentorial regions, patients with SCA3 had a
significant decreased 3D-FD values in putamen and caudate. Neuropathological studies have shown neuronal loss in the
putamen and caudate, which may lead to basal ganglia atrophy
in association with Parkinsonism (34). Parkinsonian features
are the prevalent phenotype of the SCA3 mutation and are
usually accompanied by basal ganglia symptoms (35–37). In
necropsy studies, the involvement of the basal ganglia is common March 2020 | Volume 11 | Article 124 5 Frontiers in Neurology | www.frontiersin.org Supratentorial and Infratentorial Lesions in SCA3 Wang et al. FIGURE 3 | Regions within the frontal lobe indicating lower 3D-FD values in patients with SCA3 compared with controls. (A) Left-outer view, (B) left-inner view,
(C) right-outer view, and (D) right-inner view. Patients with SCA3had significantly decreased 3D-FD values in frontal regions, including the premotor cortex (left
precentral gyrus, bilateral superior frontal gyrus, and left-middle frontal gyrus), supplementary motor cortex (left supplementary motor area), and primary motor cortex
(left precentral gyrus). Other atrophied regions were observed in the left inferior frontal gyrus (opercular), left inferior frontal gyrus (triangular), bilateral orbitofrontal
cortex (superior-1 medial), bilateral superior frontal gyrus (medial), and bilateral paracentral lobule in patients with SCA3. All the significantly decreased 3D-FD values
regions are illustrated in red. FIGURE 3 | Regions within the frontal lobe indicating lower 3D-FD values in patients with SCA3 compared with controls. (A) Left-outer view, (B) left-inner view,
(C) right-outer view, and (D) right-inner view. Frontiers in Neurology | www.frontiersin.org DISCUSSION (A) Left-outer view, (B) left-inner view, (C) right-outer view, and (D) right-inner view. The blue regions show significantly lower 3D-FD values in the occipital
lobe, including the calcarine fissure and the surrounding cortex, cuneus, and lingual gyrus and the superior and middle occipital gyrus in the left hemisphere. Purple
regions denote lower 3D-FD values in the left-superior temporal gyrus and left-middle temporal gyrus. Aqua blue regions represent lower 3D-FD values in subcortical
regions, including the bilateral caudate and left putamen. Green regions represent significantly lower 3D-FD values in the left-posterior cingulate gyrus and
parahippocampal gyrus of the limbic system. in patients with SCA3. Functional imaging using PET studies
has also demonstrated early functional decline of the caudate
and putamen in patients with SCA3. Additionally, patients
with SCA3 had a significantly smaller basal ganglia volume
(35). However, related studies have focused only on the basal
ganglia and infratentorial regions. In Figure 6, we verified that
SCA3 may not only have the basal ganglia lesions but also
the cerebellar–thalamocortical and basal ganglia–thalamocortical
pathways damage. control, such as muscle force and direction of reach. Damage
in these regions may induce ataxia of gait, stance, and limb, in
addition to movement decomposition (38, 39). The reduced 3D-
FD values of cerebellar cortex and other motor-related cerebral
regions in patients with SCA3 may explain the presence of clinical
symptoms. Additionally, in the basal ganglia–thalamocortical
loop, patients with SCA3 exhibit significantly decreased 3D-
FD values in the frontal and primary motor cortexes. The
primary motor cortex is also a major destination for basal ganglia
output (40). Disturbances of the basal ganglia–thalamocortical
loop may also contribute to Parkinsonian motor dysfunction
(41). The results of this study emphasize the morphological
changes in the basal ganglia–thalamocortical loop in patients Patients with SCA3 exhibit significantly decreased 3D-FD
values of the premotor cortex and supplementary motor cortex
in their cerebellar–thalamocortical loop. The premotor cortex
and supplementary motor area are associated with movement March 2020 | Volume 11 | Article 124 Frontiers in Neurology | www.frontiersin.org 6 Supratentorial and Infratentorial Lesions in SCA3 Wang et al. FIGURE 6 | SCA3 is involved in both the cerebellar–thalamocortical and basal ganglia–thalamocortical pathways. (A) Left-outer view, (B) left-inner view, (C) right-outer
view, and (D) right-inner view. In the cerebellar–thalamocortical loop, patients with SCA3 exhibit significantly lower 3D-FD values in the cerebellar, premotor, and
supplementary motor cortexes. DISCUSSION The decreased 3D-FD values in the cerebellar cortex and other motor-related cerebral regions in patients with SCA3 provide an
explanation for the presence of clinical symptoms. In the basal ganglia–thalamocortical loop, patients with SCA3 exhibit significantly lower 3D-FD values in the frontal
cortex, primary motor cortex, putamen, and caudate. These results reveal morphological changes in the basal ganglia–thalamocortical loop in patients with SCA3. FIGURE 6 | SCA3 is involved in both the cerebellar–thalamocortical and basal ganglia–thalamocortical pathways. (A) Left-outer view, (B) left-inner view, (C) right-outer
view, and (D) right-inner view. In the cerebellar–thalamocortical loop, patients with SCA3 exhibit significantly lower 3D-FD values in the cerebellar, premotor, and
supplementary motor cortexes. The decreased 3D-FD values in the cerebellar cortex and other motor-related cerebral regions in patients with SCA3 provide an
explanation for the presence of clinical symptoms. In the basal ganglia–thalamocortical loop, patients with SCA3 exhibit significantly lower 3D-FD values in the frontal
cortex, primary motor cortex, putamen, and caudate. These results reveal morphological changes in the basal ganglia–thalamocortical loop in patients with SCA3. with SCA3, which previous neuroimaging studies have failed
to address. association between supratentorial atrophy and motor-related
involvement in patients with SCA3. Our results found significant
degeneration of the cerebral cortex in specific regions, which
maybe cerebellar related or basal ganglia related. Our findings
might correlate to the common symptoms of SCA3, such as
ataxia, Parkinsonism, dysarthria, and dysmetria. SCA3 should no
longer be considered a disease limited to the cerebellum and its
connections; rather, it should be considered a pathology affecting
the whole brain. Recently, we used diffusion tensor imaging (DTI) to measure
water molecular diffusion of white matter (WM) alteration in
SCA3 (15). We found SCA3 revealed widespread white matter
lesions in the bilateral cerebral-frontal, -parietal, -temporal, and
-occipital WM; cerebellar WM; the thalamus, and the brainstem. Identically, in this study we observed the SCA3 showed almost in
the same parcellated regions of these WM lesions that revealed
GM lesions. These current findings confirm the involvement of
supratentorial regions in patients with SCA3. Braga et al. reported
similar findings, demonstrating that SCA3 causes CCAS through
the impairment of executive functions, verbal fluency, abstract
reasoning, and working memory (8). In neuropathological
studies, it was revealed that CCAS results from the disruption
of the cerebellar circuit, which is associated with the prefrontal,
superior parietal, superior temporal, and limbic cortexes (42,
43). ETHICS STATEMENT The studies involving human participants were reviewed and
approved by Institutional Review Board of Taipei Veterans
General Hospital. The patients/participants provided their
written informed consent to participate in this study. FUNDING This study was funded by the Ministry of Science and
Technology,
Taiwan
(MOST108-2634-F-010-002);
National
Health Research Institutes, Taiwan (NHRI-EX106-10526EI);
National Yang-Ming University and Shin-Kong Wu-Ho Su
Memorial Hospital Research Program (107GB006). DISCUSSION Studies have confirmed the role of the cerebellum in
cognitive function; however, cognitive, affective, and visuospatial
functions are directly associated with cortical degeneration. The significantly atrophied areas of the supratentorial regions
in patients with SCA3 were more extensive than the CCAS-
associated regions. The atrophy of the supratentorial cortex may
be the primary factor associated with the dysfunction of the ataxia
and Parkinsonism-related regions. These findings suggest that
SCA3 should no longer be considered a disease limited to the
cerebellum and its connections; rather, it should be considered
a pathology affecting the whole brain. DATA AVAILABILITY STATEMENT All datasets generated for this study are included in the
article/Supplementary Material. AUTHOR CONTRIBUTIONS P-SW and Y-TW organized the research project, reviewed and
critiqued the manuscript. T-YW wrote manuscript. H-MW
accessed research data. B-WS organized the research and
performed data collection. C-WJ performed the statistical
analysis, wrote, and revised the manuscript. However, this study still had certain limitations. The main
limitations in this study are the lack of specific cognitive
evaluations and clinical parameters for assessing CCAS in SCA. Further investigations are warranted regarding the correlation
between the present results and clinical data, such as functional
MRI data. This may help provide a more precise method for
assessing the association between cognitive with motor-related
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article distributed under the terms of the Creative Commons Attribution License (CC
BY). The use, distribution or reproduction in other forums is permitted, provided
the original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these
terms. Copyright © 2020 Wang, Wu, Wang, Wu, Soong and Jao. 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
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Bladder Cancer Pathologic Primary Tumor TNM Finding v7
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Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Bladder Cancer Pathologic Primary Tumor
TNM Finding v7 National Cancer Institute National Cancer Institute Qeios ID: 1A4SD5 · https://doi.org/10.32388/1A4SD5 Source National Cancer Institute. Bladder Cancer Pathologic Primary Tumor TNM Finding v7. NCI Thesaurus. Code C89351. A pathologic finding about one or more characteristics of bladder cancer, following the
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Long-term cognitive outcomes among unselected ventilated and non-ventilated ICU patients
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© The Author(s). 2017 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: Cognitive dysfunction is an important long-term complication of critical illness associated with
reduced quality of life, increase in healthcare costs, and institutionalization. Delirium, an acute form of brain
dysfunction that is common during critical illness has been shown to be associated with long-term cognitive
dysfunction. The aim of this prospective cohort study was to estimate the prevalence and severity of cognitive
dysfunction in an unselected population of medical and surgical ICU patients. Methods: This prospective observational cohort study included all adult patients admitted to the surgical
(13 beds) and medical (32 beds) ICUs of a tertiary hospital over a 12-month period. Patients with impaired
cognition were excluded. At least 3 months after hospital discharge, patients were assessed for cognition using
a validated battery of tests and were classified as having no cognitive impairment, mild to moderate cognitive
impairment, or severe cognitive impairment. Results: Four hundred thirteen patients were tested an average of 11 (3–18) months after discharge. Fifty-five
(13.3%) patients included in the follow-up cohort had delirium. Cognitive impairment was identified in 206
(49.9%) patients, 120 (29.1%) patients had mild or moderate cognitive impairment, and 86 (20%) patients had
severe cognitive dysfunction. Conclusions: This investigation in an unselected and lower severity population of critically ill patients
demonstrates that cognitive dysfunction is a frequent and severe long-term complication. Keywords: Critical illness, Intensive care unit, Follow-up, Cognitive dysfunction, Delirium, Out Long-term cognitive outcomes among
unselected ventilated and non-ventilated
ICU patients José Raimundo A. de Azevedo*, Widlani Sousa Montenegro, Djane Pereira Rodrigues, Suellen C. de C. Souza,
Vanessa F. S. Araujo, Margareth Pereira de Paula, Patricia H. C. P. Prazeres, Adenilde da Luz Leitão
and Adriana V. N. Mendonça * Correspondence: jrazevedo47@gmail.com
Intensive Care Unit, Hospital São Domingos, Av. Jerônimo de Albuquerque,
540, Bequimao, São Luis, MA 65060-645, Brazil de Azevedo et al. Journal of Intensive Care (2017) 5:18
DOI 10.1186/s40560-017-0213-4 de Azevedo et al. Journal of Intensive Care (2017) 5:18
DOI 10.1186/s40560-017-0213-4 Open Access Results From March 2014 to February 2015, 724 patients were
enrolled in the clinical trial; 53 patients died during
hospitalization and 4 had other criteria for exclusion
(1 had a large stroke before discharge and 3 had car-
diac arrest with suspected anoxic brain injury during
their ICU stay). The remaining 667 patients were eli-
gible for the cohort. Fifty-four (8.0%) patients died be-
fore follow-up testing. One hundred forty-five (21.7%)
were lost for follow-up (55 of them lived outside the
São Luis Island) and 55 (8.1%) patients refused to
undergo the cognition assessment. The remaining 413
patients were tested an average of 11 (3–18) months
after discharge. Table 1 shows that demographic and
clinical data were comparable between the in-hospital
and the follow-up group. Written informed consent was obtained from the pa-
tient or next of kin. Patients underwent two daily evaluations for delirium
(morning and evening) with the use of the Confusion
Assessment Method for the ICU (CAM-ICU), a diag-
nostic tool for determining the presence of delirium
based on four features: acute changes or fluctuation in
mental status, inattention, disorganized thinking, and
altered level of consciousness [10]. Level of conscious-
ness was evaluated with the use of the Richmond
Agitation-Sedation Scale (RASS). Scores range from −5
(unarousable) to +4 (agitated) [11]. The duration of de-
lirium was defined as the number of days in which the
patient had at least one of the two daily evaluations of
CAM-ICU positive for delirium during the ICU length
of stay. p g
p
Ninety-two patients (12.3%) of the in-hospital cohort
were submitted to mechanical ventilation for a median
of 4 days (interquartile range, 2–10) compared to 51
(13.4%) with a median duration of 3 days (interquartile
range, 1.2–6.7) in the follow-up cohort. Sepsis and septic
shock were the admission diagnosis in 77 (10.6%) of the
patients of in-hospital cohort and 37 (9.0%) of the
follow-up
cohort
patients. Two
hundred
sixty-nine
(65.2%) of the evaluations were done in the patients’
homes. One hundred forty-four patients (34.8%) were
evaluated in the psychology follow-up clinic. Fifty-five
(13.3%) patients included in the follow-up cohort had
delirium. Methods This prospective observational cohort study was ap-
proved by the Research Ethics Committee of Federal
University of Maranhao-Brazil-under number 990.167. Included in the study were all adult patients admitted to
the surgical (13 beds) and medical (32 beds) ICUs of a
tertiary hospital over a 12-month period (from March
2014 to February 2015). We excluded patients with pre-
existing cognitive dysfunction due to neurodegenerative
disease or central nervous system traumatic or vascular
disease; patients admitted to the ICU after cardiac arrest
with suspected anoxic brain injury; patients for whom
follow-up would be difficult due to active substance
abuse, psychotic disorder, or residence outside Sao Luis
Island (827 km2, five counties); patients who could not
be reliably assessed for delirium owing to blindness,
deafness or language deficit, and patients for whom in-
formed consent could not be obtained. Patients deter-
mined by the psychologist on ICU admission to have
evidence of a preexisting cognitive dysfunction were also
excluded. Background
h
l shown to be associated with long-term cognitive dys-
function [4, 6]. The large number of patients being treated annually in
intensive care units (ICUs) and the improved care of
these patients has resulted in a growing number of
critical illness survivors, many of who are left with sig-
nificant morbidities [1–3]. An important long-term
complication of critical illness is cognitive dysfunction,
which is associated with a reduced quality of life and
an increase in healthcare costs and institutionalization
[4, 5]. Delirium, an acute form of brain dysfunction
that is common during critical illnesses, has been Currently, cognitive dysfunction associated with crit-
ical illness is inserted in a broader syndrome context of
post intensive care syndrome (PICS), which includes
psychiatric and physical dysfunction. Although exact
data is not available, it is estimated that at least half of
patients discharged from ICUs have at least one mani-
festation of PICS [7, 8]. In a study of 293 patients that
survived ICU admission, 44% required assistance by
their community nurse, and a negative impact on family
income was reported by one third to one half of patients
and families of survivors [9]. * Correspondence: jrazevedo47@gmail.com
Intensive Care Unit, Hospital São Domingos, Av. Jerônimo de Albuquerque,
540, Bequimao, São Luis, MA 65060-645, Brazil Page 2 of 5 Page 2 of 5 de Azevedo et al. Journal of Intensive Care (2017) 5:18 The aim of this prospective cohort study was to esti-
mate the prevalence and severity of cognitive dysfunc-
tion
in
an
unselected population
of
medical
and
surgical patients and to evaluate if delirium duration
was an independent determinant of the severity of cog-
nitive dysfunction. applied by trained nurses and psychologists and inter-
preted by a neuropsychologist (MPP). Each patient’s
cognitive test scores were converted to T scores using
age-specific and education-specific normative data. We
classified patients as having mild or moderate impair-
ment if they had either two cognitive test scores 1.5
standard deviation (SD) below the mean or one cogni-
tive test score 2 SD below the mean; we classified pa-
tients as having severe cognitive impairment if they had
three or more cognitive test scores 1.5 SD below the
mean or two or more cognitive test scores 2 SD below
the mean. Statistical analysis Demographic and clinical characteristics of the in-
hospital cohort and follow-up cohort were examined
using median and interquartile range and proportions
for categorical variables. To determine whether the dur-
ation of delirium was an independent predictor of long-
term cognitive impairment, we used multiple non-linear
regression analysis to analyze the association between
days of delirium and summary scores of cognitive per-
formance at follow-up, adjusting for the following covar-
iates: age, education, APACHE IV score, and sepsis. Results Cognitive impairment was identified in 206 At least 3 months after hospital discharge, patients
were assessed for cognition using a validated battery of
tests including: (1) the forward and backward digit span
to assess attention and memory [12]; (2) the Rey Audi-
tory Verbal Learning Test to assess verbal memory
[13]; (3) the clock-drawing test to evaluate executive
functions [14]; (4) the verbal fluency test to assess lan-
guage [15]; and the Mini-Mental State Examination to
assess global mental status [16]. All these tests have
been validated for the Brazilian population and were de Azevedo et al. Journal of Intensive Care (2017) 5:18 Page 3 of 5 Page 3 of 5 Page 3 of 5 Table 1 Demographic and clinical characteristics of the patients
In-hospital cohort
(n = 724)
Follow-up cohort
(n = 413)
Age, year
Median
59
57
IQR
47–73
46–72
Male sex
n (%)
374 (51.7)
206 (50.1)
Education, year
Median
11
11
IQR
11–14
11–14
APACHE IV score
Median
35
32
IQR
23–53
21–48
SOFA score at
Enrollment
Median
1
1
IQR
0–3
1–3
Mechanical ventilation
No. of patients (%)
92 (12.3%)
51 (13.4%)
No. of days
Median
4
3
IQR
2–10
1.2–6.7
Use of sedative agent
No. of patients (%)
88 (12.1%)
44 (10.7%)
Diagnosis at admission
n (%)
AMI, CHF, Arrhytmia
146 (20.2)
98 (23.7)
Acute respiratory failurea
109 (15.1)
58 (14.0)
Other surgical proceduresb
99 (13.7)
56 (13.6)
Neurologic disease
93 (12.8)
53 (12.8)
Sepsis, septic shock
77 (10.6)
37 (9.0)
Digestive surgery
69 (9.6)
37 (9.0)
Digestive disease
67 (9.3)
39 (9.4)
Other diagnosis
64 (8.7)
35 (8.5)
Delirium
No. of patients (%)
80 (11.6)
55 (13.3)
No. of days
Median
4
3
IQR
2–5
2–5
Duration at ICU stay
Median
11
10
IQR
6–23
5–19
aAcute respiratory failure included acute respiratory distress syndrome,
pneumonia, acute exacerbation of chronic pulmonary disease, asthma,
pulmonary edema, and embolism
bOther surgical procedures included orthopedic, vascular, and urologic surgery Table 1 Demographic and clinical characteristics of the patients
In-hospital cohort
(n = 724)
Follow-up cohort
(n = 413) (49.9%) patients; 120 (29.1%) had mild or moderate cog-
nitive impairment and 86 (20%) severe cognitive dys-
function (Table 2). (
)
Eleven (34.3%) patients who had delirium for 3 days
or more presented with severe cognitive dysfunction
(p = 0.17). Results In logistic regression analysis, a duration of
delirium for 3 days or more was not an independent
predictor
of
cognitive
dysfunction
(p = 0.76). In
addition to delirium, we evaluated other factors that
could affect post intensive care cognitive dysfunction:
gender, ICU LOS, hospital LOS, years of education,
APACHE IV score, SOFA score at enrollment, and
Charlson comorbidity index (CCI). In univariate ana-
lysis variables SOFA score, years of education, and CCI
were significant (Fig. 1). When evaluated in the multi-
variate logistic regression analysis only CCI had a bor-
derline significance (p = 0.06). Discussion For a long time, the success of therapeutic interventions
in critically ill patients was evaluated by the in-hospital
mortality rate. However, it is becoming increasingly evi-
dent that hospital discharge as an endpoint needs to be
reevaluated. Critically ill patients often develop a series
of long-term complications during and after their hos-
pital stay, including cognitive impairment, psychological
disorders, and muscle weakness. These changes can per-
sist for years and seriously affect the quality of life [5, 9–
19]. Prolonged cognitive dysfunction is one of the most
prominent manifestations of PICS [7, 8]. We studied a very different group of patients than
what has been investigated before [4, 6, 18, 19]. Here,
we sought not to study the sickest of the sick ICU pa-
tients, but on the contrary, we chose this unselected
and most lower severity of illness group to determine
in this group what the rates of long-term cognitive im-
pairment are. It is surprising and alarming that we have
found such high rates of a wide spectrum of cognitive
impairment in a group of patients whom most ICU cli-
nicians would never suspect this degree of long-term
cognitive impairment. Furthermore, since this group
was of such a lower severity of illness, it was not alarm-
ing that they experienced most less acute brain dys-
function. In fact, with this low prevalence of delirium
[only about 1 in 10 in comparison to upwards of 7 out Table 2 Cognitive outcomes during follow-up
Follow-up assessment
(n = 413)
No impairment
207 (50.1)
Mild/moderate
120 (29.1)
Impairment
Severe impairment
86 (20.8) Table 2 Cognitive outcomes during follow-up de Azevedo et al. Journal of Intensive Care (2017) 5:18 Page 4 of 5 Page 4 of 5 duration of delirium, reducing the possibility that pa-
tients who were sicker were underrepresented. This is in
contrast to a large, multicenter, prospective cohort study
that exhibited clear differences between the patients that
completed the neurocognitive testing and those that did
not [4]. Fig. 1 Enrollment and follow-up Our study has limitations. The single-center design
limits the generalization of results to other similar pop-
ulations. Admissions to the ICU are not elective in
most cases. Thus, evaluation of the presence of pre-
existing cognitive dysfunction has limitations. Discussion As nearly 1 in 3 of our patients
had either cardiac or neurological injury, both of which
can pose major immediate problems to the brain in
terms of perfusion, this is a very important consider-
ation especially since this is the major difference be-
tween who we enrolled and studied versus previous
cohorts looking at long-term cognitive impairment in
ICU survivors [6, 18, 19]. Availability of data and materials
h d
h
l y
The dataset supporting the conclusions of the article is included within the
article. Acknowledgements
Not applicable. Acknowledgements
Not applicable. This study reinforces the findings of previous studies
that showed a high prevalence of cognitive dysfunction
in critically ill patients [1, 3–6]. More
important,
however, was the finding of a 50% incidence of cognitive
impairment (20% severe), in an unselected low severity
of illness group of clinical and surgical patients. Most
other studies evaluated specific nosologies such as acute
respiratory distress syndrome, older adults, and patients
admitted to medical ICU [1, 17–19]. The studies of
Girard et al. [6] and Pandariphande et al. [4] which
demonstrated a close relationship between cognitive
dysfunction and delirium analyzed only patients under-
going mechanical ventilation [6] and patients with
respiratory failure and cardiogenic and septic shock [4]. Funding Not applicable. Discussion Although
we excluded patients determined by a psychologist on
ICU admission to have evidence of preexisting cogni-
tive dysfunction, it is possible, nevertheless, that pa-
tients with mild cognitive dysfunction were included in
the study. of 10 in sicker ICU cohorts [4]], it is not surprising that
our analyses did not see a relationship between delir-
ium and long-term cognitive impairment, though this
analysis is severely under-powered and thus may repre-
sent a type II error. This should not be taken by the
readership to be a statement that delirium (acute brain
dysfunction in the ICU) is not a risk factor for long-
term cognitive impairment but rather that our investi-
gation was simply unable to assess this risk factor. However, this makes it even more interesting that we
still found such a high amount of long-term cognitive
impairment, which raises the specter that other risk
factors besides delirium must also be considered and
should be investigated. As nearly 1 in 3 of our patients
had either cardiac or neurological injury, both of which
can pose major immediate problems to the brain in
terms of perfusion, this is a very important consider-
ation especially since this is the major difference be-
tween who we enrolled and studied versus previous
cohorts looking at long-term cognitive impairment in
ICU survivors [6, 18, 19]. of 10 in sicker ICU cohorts [4]], it is not surprising that
our analyses did not see a relationship between delir-
ium and long-term cognitive impairment, though this
analysis is severely under-powered and thus may repre-
sent a type II error. This should not be taken by the
readership to be a statement that delirium (acute brain
dysfunction in the ICU) is not a risk factor for long-
term cognitive impairment but rather that our investi-
gation was simply unable to assess this risk factor. However, this makes it even more interesting that we
still found such a high amount of long-term cognitive
impairment, which raises the specter that other risk
factors besides delirium must also be considered and
should be investigated. Conclusions To our knowledge, this prospective observational cohort
study is the first investigation in an unselected and lower
severity population of critically ill medical and surgical
patients to demonstrate that cognitive dysfunction is a
frequent and severe long-term complication in survivors
of critical illness. The results of this study reinforce the evidence that
many patients who survive critical illness evolve after
discharge with a chronic condition characterized by cog-
nitive dysfunction and also muscle dysfunction and psy-
chiatric disorders, the post intensive care syndrome
(PICS). Prevention of PICS begins during the patient’s
stay in the ICU and needs to continue after discharge
with a multidisciplinary approach involving the patient
and their family. References 1. Hopkins RO, Weaver LK, Pope D, Orme JF, Bigler ED, Larson-Lohr V. Neuropsycological sequelae and impaired health status in survivors of severe
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method for the intensive care unit (CAM-ICU). JAMA. 2001;286:2703–10. 11. Sessler CN, Gosnell MS, Grap MJ, et al. The Richmond Agitation-Sedation
Scales: validity and reliability in adult intensive care unit patients. Am J
Respir Crit Care Med. 2002;166:1338–44. 12. Powell DH, Hiatt MD. Auditory and visual recall of forward and backward
digit spans. Percept Mot Skills. 1996;82:1099–103. 12. Powell DH, Hiatt MD. Auditory and visual recall of forward and backward
digit spans. Percept Mot Skills. 1996;82:1099–103. 13. Malloy-Diniz LF, Lasmar VA, Gazinelli Lde SR, Fuentes D, Salgado JV. The Rey
auditory-verbal learning test: applicability for the Brazilian elderly
population. Consent for publication
Written informed consent was obtained from the patient or next of kin. Consent for publication
Written informed consent was obtained from the patient or next of kin. Consent for publication
Written informed consent was obtained from the patient or next of kin. Received: 17 November 2016 Accepted: 3 February 2017 Received: 17 November 2016 Accepted: 3 February 2017 Authors’ contributions
bl f JRAA was responsible for study design, data analysis, statistical analysis, and
critical revision of the manuscript for important intellectual content. WSM
was responsible for study design, data analysis, statistical analysis, and critical
revision of the manuscript for important intellectual content. DPR was
responsible for study design, acquisitions of the data, interpretation of the
results, and revision of the manuscript for important intellectual content. SCCS contributed to the study design, acquisitions of the data, and
interpretation of the results. VFSA contributed to acquisition of the data,
interpretation of the results, and drafting of the manuscript. MPP contributed
to the study design, coordination of data acquisition, interpretation of the
data, and critical revision of the manuscript for important intellectual
content. PHCPP contributed to acquisition of the data and interpretation of
the result. All contributed to acquisition of the data and interpretation of the
results. AVNM contributed to acquisition of the data and interpretation of
the results. All authors read and approved the final manuscript. One strength of this study is that in spite of being a
cohort the in-hospital population did not differ signifi-
cantly from the follow-up population in terms of age,
years of formal education, and severity, incidence, and Page 5 of 5 Page 5 of 5 de Azevedo et al. Journal of Intensive Care (2017) 5:18 Competing interests Competing interests Competing interests
The authors declare that they have no competing interests. Ethics approval and consent to participate
h
b
l
h
d This prospective observational cohort study was approved by the Research Ethics
Committee of Federal University of Maranhao-Brazil-under number 990.167. This prospective observational cohort study was approved by the Research Ethics
Committee of Federal University of Maranhao-Brazil-under number 990.167. Written informed consent was obtained from the patient or next of kin. Written informed consent was obtained from the patient or next of kin. References Rev Bras Psiquiatr. 2007;29:324–9. 14. Freedman MI, Leach I, Kaplan E, Winocur G, Shulman KJ, Delis DC, editors. Clock drawing. Oxford: Oxford Uviversity Press; 1994. Clock drawing. Oxford: Oxford Uviversity Press; 1994. 15. Lezak M, Houieson DB, Bigler E, Tranel D. Neuropsicological assessment. 5th
ed. New York: Oxford University Press; 2012. 16. Folstein MF, Folstein SE, McHugh PR. Mini-Mental State: a practical method
for grading the cognitive state of patients for clinician. J Psychiatr Res. 1975;
12:189–98. Submit your next manuscript to BioMed Central
and we will help you at every step: 17. Davidson TA, Caldwell ES, Curtis JR, Hudson LD, Steinberg KP. Reduced
quality of life in survivors of acute respiratory distress syndrome compared
with critically ill control patients. JAMA. 1999;281:354–60. Submit your next manuscript to BioMed Central
and we will help you at every step: Submit your next manuscript to BioMed Central
and we will help you at every step: • We accept pre-submission inquiries
• Our selector tool helps you to find the most relevant journal
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Submit your manuscript at
www.biomedcentral.com/submit
and we will help you at every step: 18. Iwashyna TJ, Ely EW, Smith DM, Langa KM. Long-term cognitive impairment and
functional disability among survivors of severe sepsis. JAMA. 2010;304:1787–94. 19. Herridge MS, Tansey CM, Matté A, Tomlinson G, Diaz-Granados N, Cooper A,
et al. Functional disability 5 years after acute respiratory distress syndrome. N Engl J Med. 2011;364:1293–304.
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The nitrobenzoxadiazole derivative MC3181 blocks melanoma invasion and metastasis
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Oncotarget
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cc-by
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Anastasia De Luca1,*, Debora Carpanese2,*, Maria Cristina Rapanotti3, Tara Mayte
Suarez Viguria3, Maria Antonietta Forgione4, Dante Rotili4, Chiara Fulci1, Egidio
Iorio5, Luigi Quintieri6, Sergio Chimenti7, Luca Bianchi7, Antonio Rosato2,8, Anna
Maria Caccuri1 Correspondence to: Anna Maria Caccuri, email: caccuri@uniroma2.it
Antonio Rosato, email: antonio.rosato@unipd.it Correspondence to: Anna Maria Caccuri, email: caccuri@uniroma2.it
Antonio Rosato, email: antonio.rosato@unipd.it Accepted: December 27, 2016 Published: January 17, 2017 Accepted: December 27, 2016 Published: January 17, 2017 Published: January 17, 2017 Received: July 25, 2016 ABSTRACT The novel nitrobenzoxadiazole (NBD) derivative MC3181 is endowed with
remarkable therapeutic activity in mice bearing both sensitive and vemurafenib-
resistant human melanoma xenografts. Here, we report that subtoxic concentrations
of this compound significantly reduced invasiveness of BRAF-V600D mutated WM115
and WM266.4 melanoma cell lines derived from the primary lesion and related skin
metastasis of the same patient, respectively. The strong antimetastatic activity of
MC3181 was observed in both 2D monolayer cultures and 3D multicellular tumor
spheroids, and confirmed in vivo by the significant decrease in the number of B16-F10
melanoma lung metastases in drug-treated mice. Our data also show that MC3181
affects the lactate production in the high glycolytic WM266.4 cell line. To unveil
the MC3181 mechanism of action, we analyzed the ability of MC3181 to affect the
degree of activation of different MAPK pathways, as well as the expression/activity
levels of several proteins involved in angiogenesis, invasion, and survival (i.e. AP2,
MCAM/MUC18, N-cadherin, VEGF and MMP-2). Our data disclosed both a decrease
of the phospho-active form of JNK and an increased expression of the transcription
factor AP2, events that occur in the very early phase of drug treatment and may be
responsible of the antimetastatic effects of MC3181. www.impactjournals.com/oncotarget/ www.impactjournals.com/oncotarget/ Research Paper The nitrobenzoxadiazole derivative MC3181 blocks melanoma
invasion and metastasis
p Anastasia De Luca1,*, Debora Carpanese2,*, Maria Cristina Rapanotti3, Tara Mayte
Suarez Viguria3, Maria Antonietta Forgione4, Dante Rotili4, Chiara Fulci1, Egidio
Iorio5, Luigi Quintieri6, Sergio Chimenti7, Luca Bianchi7, Antonio Rosato2,8, Anna
Maria Caccuri1 INTRODUCTION of the basement membrane by neoplastic proliferating
cells, their migration and stroma invasion to enter the
vasculature, and finally their extravasation and adhesion
in distant organs to form a secondary tumor [1–3]. Every
phase of this complex scenario can be rate-limiting and
offers potential target for therapy. Malignant melanoma progresses through a multi-
step process switching from dysplasia to radial growth
phase, to invasive vertical growth phase, and subsequently
to distant metastases. These switches implicate the breach www.impactjournals.com/oncotarget Antiproliferative activity of MC3181 and
NBDHEX on WM115 and WM266.4 2D
monolayer cultures and 3D multicellular tumor
spheroids The antitumor efficacy of MC3181 was tested in
vitro on 2D WM115 and WM266.4 human melanoma cell
cultures, and compared with NBDHEX, temozolomide
(TMZ) and vemurafenib (VMF). The concentration–
response profiles (Supplementary Figure 1) fulfill the
IC50 values reported in Table 1. Of note, the IC50 values
calculated for MC3181 are in the low micromolar range
(1.0–1.3 μM), and close to those obtained for both
NBDHEX and vemurafenib (VMF), whereas TMZ is at
least 600 times less effective. In contrast with current clinical trials targeting
multiple signaling pathways involved in cell proliferation,
we recently reported that the nitrobenzoxadiazole
derivatives (NBDs) NBDHEX [6-((7-nitrobenzo[c]
[1,2,5]oxadiazol-4-yl)thio)hexan-1-ol] and its more water-
soluble analogue, MC3181 [2-(2-(2-((7-nitrobenzo[c]
[1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol], exert
a potent antitumor activity through the activation of
different MAPK pathways. These compounds represent a
new class of antitumor agents exhibiting an outstanding
therapeutic activity together with an extremely non-toxic
profile in human cutaneous melanoma mouse xenografts. NBDHEX and MC3181 inhibit glutathione transferase
P1-1 (GSTP1-1), and disrupt the GSTP1-1/JNK1 and
GSTP1-1/TRAF2 complexes, thereby causing prolonged
tumor cell cycle arrest and apoptosis. Therefore, these
drugs may constitute a new effective strategy for the
treatment of BRAF-mutated human melanomas, capable
of overcoming the resistance to vemurafenib [5, 6]. Next, we decided to evaluate the effect of MC3181
and its parent drug, NBDHEX, on 3D multicellular
tumor spheroids, which are more precise in mimicking
the complex organization of tumor tissue in vivo [7]. Spheroids were treated with graded concentrations of
MC3181 (Figure 1b and 1d) or NBDHEX (Figure 1c and
1e), and IC50 values were obtained by analyzing both cell
viability (MTS) and growth rate. A schematic diagram
for treatment schedule and analysis (cell imaging and
viability assay) is shown in Figure 1a. We noticed that
WM266.4 spheroids grew faster increasing their volume
25 times at the end of the experiment (day 17, Figure 1d
and 1e), whilst the WM115 counterparts augmented only
8 times (Figure 1b and 1c). The IC50 values of MC3181
on WM266.4 spheroids were in the low micromolar range
(0.5–7.7 μM, Table 2), comparable at both 48 hours and
17 days, and similar to those obtained with NBDHEX. In contrast, 48 hours treatment with both MC3181 and
NBDHEX caused flaking of WM115 spheroids and
formation of poorly defined contours that did not allow
an accurate measurement of spheroids’ diameter (data
not shown). Antiproliferative activity of MC3181 and
NBDHEX on WM115 and WM266.4 2D
monolayer cultures and 3D multicellular tumor
spheroids Additionally, after 17 days of treatment, the
spheroids’ viability dropped more slowly compared to the
spheroids’ volume, resulting in loss of linear relationship
between viability and cell number (Table 2). A similar
event has been already reported and explained by the
occurrence of cell cycle arrest [8]. On this ground, we were prompted to test the
antimetastatic properties of the more soluble NBD
derivative, MC3181, against melanoma cells lines derived
from the primary lesion (WM115) and a skin metastasis
(WM266.4) of a single patient harboring a BRAF-V600D
mutation. Our data revealed that subcytotoxic doses of
MC3181 exert a strong antimetastatic activity in vitro. To understand the mechanism(s) underling this effect, we
measured the expression/activity levels of key proteins
involved in melanoma cells migration and invasion,
and focused on signaling pathways that modulate these
proteins. Moreover, using the B16-F10 melanoma mouse
model of metastasis, we demonstrated that the oral
administration of MC3181 exerts a potent antimetastatic
effect, significantly suppressing and/or delaying lung RESULTS Antiproliferative activity of MC3181 and
NBDHEX on WM115 and WM266.4 2D
monolayer cultures and 3D multicellular tumor
spheroids www.impactjournals.com/oncotarget Oncotarget 15520 Therapeutically, one of the most recent and
promising approaches is immunotherapy that involves
the use of immune checkpoint inhibitors enabling the
interruption of T-cell pathways responsible for immune
down-regulation or tolerance, such as the anti-CTLA-4
monoclonal antibody (mAb) ipilimumab, along with
the anti-PD-1 mAbs pembrolizumab and nivolumab. Another important class of drugs is represented by low
molecular weight compounds interfering with signaling
pathways involved in the dysregulation of cell growth
and proliferation; this is the case of vemurafenib and
dabrafenib, which target mutated v-raf murine sarcoma
viral oncogene homolog B1 (BRAF), the selective
inhibitors of extracellular signal-regulated kinase
(ERK), as well as trametinib and cobimetinib targeting
mitogen-activated protein kinase (MAPK)/ERK Kinase
(MEK) proteins [4]. While these strategies provide a
potential initial clinical benefit, they delay but not prevent
patient mortality due to the ability of tumor to rapidly
acquire resistance to drugs and/or to activate alternative
proliferation pathways. metastasis in the absence of detectable sign of toxicity. These data provide evidence to further support the
potential of MC3181 as a novel therapeutic to treat
metastatic melanoma. MC3181 inhibits invasion of human melanoma
cells in vitro 50
We analyzed cell proliferation and cell cycle at
different time points after the addition of MC3181. A
decrease in the WM115 growth rate was observed after
24 hours incubation with both 0.26 and 1.3 μM MC3181
(Supplementary Figure 2a), which corresponds to a
significant arrest in the G2/M phase of the cell cycle
(Supplementary Figure 2b). In contrast, WM266.4
showed only a slight decrease of cell proliferation after 48
hours treatment with the highest MC3181 dose (1.0 μM)
(Supplementary Figure 2c). In accordance, the WM266.4
cell cycle was only slightly affected by treatment with 1.0
μM MC3181 (Supplementary Figure 2d). Another aspect of tumor progression is the ability
of tumor cells to invade basement membranes and
connective tissue leading to the possible formation of
distant metastases. We investigated the migratory and
invasive potential of both primary tumor (WM115) and
metastasis-derived (WM266.4) melanoma cells after 48
hours treatment with MC3181. This compound efficiently
suppressed invasion in both cell lines, without any effect
on their migration (Figure 3a, 3b, 3f and 3g). Of note, a
concentration of MC3181 corresponding to 1/25 of its IC50
value (0.04 μM) was capable of inducing 75% reduction of
WM266.4 invasion index (Figure 3h), while an equiactive
concentration of MC3181 (0.05 μM) reduced the invasion
index of WM115 by approximately 30% (Figure 3c). However, 60% inhibition of WM115 invasion index was
obtained with 0.26 and 1.3 μM. Further, we evaluated
the antimetastatic properties of MC3181 in a third tumor
cell line, namely the BRAF-V600E-mutated SK-MEL-5
human melanoma cell line, which has been established
from an axillary lymph node metastasis. Based on the
finding that in the SRB assay MC3181 exhibited an IC50
value of 1.6 ± 0.1 μM towards SK-MEL-5 cells (data not
shown), invasion/migration assays were carried out using
the following drug concentrations: 1.60 μM (i.e., the IC50);
0.32 μM (i.e., 1/5 of IC50); and 0.06 μM (i.e., 1/25 of the
IC50) (Supplementary Figure 3). The invasive potential
of SK-MEL-5 cells was reduced by approximately 30%
by 0.32 μM MC3181 whereas, a 60% inhibition was
recorded at a drug concentration of 1.6 μM. Furthermore,
MC3181 significantly depressed migration of SK-MEL-5
cells (Supplementary Figure 3a and 3b). Of note, a 50%
reduction of cell migration was achieved with 0.32 and 1.6
μM MC3181. Since the drug inhibited both SK-MEL-5
cell invasion and migration, no change of the invasion
index occurred (Supplementary Figure 3c). Effect of low concentrations of MC3181 on cell
proliferation and cell cycle Since MC3181 and NBDHEX showed comparable
activity in both WM115 and WM266.4 cells, we focused
on the antimetastatic efficacy of the more water soluble www.impactjournals.com/oncotarget Oncotarget 15521 Table 1: Evaluation of the antiproliferative (SRB assay) effects of MC3181, NBDHEX, VMF and TMZ on WM115
and WM266.4 2D monolayer cultures
Cell line
IC50 ± SD (μM)
MC3181
NBDHEX
VMF
TMZ
WM115
1.28 ± 0.02
1.99 ± 0.01
2.47 ± 0.19
1196 ± 103
WM266.4
1.07 ± 0.04
0.94 ± 0.04
0.89 ± 0.01
800 ± 5 MC3181 was sufficient to induce 40% reduction of cell
adhesion to collagen (Figure 2d), but a significant effect
(80% inhibition) on gelatin adhesion was evident only
with 1.0 μM MC3181 (Figure 2e). Finally, MC3181 did
not show any significant inhibitory effect on tumor cell
adhesion to Matrigel (Figure 2f). NBD derivative, namely MC3181. We investigated
three different concentrations of MC3181 for each cell
line: WM115 and WM266.4 cells were treated with
MC3181 concentrations of 1.3 and 1.0 μM respectively
(corresponding to their IC50 values), 0.26 and 0.20 μM
respectively (corresponding to 1/5 of their IC50 values) and
0.05 and 0.04 μM respectively (corresponding to 1/25 of
their IC50 values). MC3181 treatment affects the adhesion
properties of both WM115 and WM266.4 cells The successful dissemination of tumor cells and the
formation of new tumor foci require cancer cells adhesion
and detachment from components of the extracellular
matrix (ECM) and basement membrane (BM). Therefore,
we analyzed the effect of MC3181 on the adhesion
properties of WM115 and WM266.4 cells to different
ECM components, i.e. type I collagen, Matrigel and
gelatin [9, 10]. The primary tumor-derived WM115 cells showed
poor basal adhesion on both gelatin and Matrigel
(Figure 2b and 2c). The treatment with 0.26-1.3 μM
MC3181 reduced to approximately 50% their adhesion
to collagen (Figure 2a), whilst a concentration-dependent
effect was observed on gelatin (Figure 2b), reaching an
adhesion inhibition of 84% at 1.3 μM MC3181. This drug
concentration was also able to abolish (96%) the adhesion
of WM115 on Matrigel (Figure 2c). The skin metastasis-derived WM266.4 cells showed
excellent adhesion properties on all the substrates tested
(Figure 2d-2f). However, the sensitivity to MC3181
was clearly lower than that of WM115. Indeed, 0.04 μM www.impactjournals.com/oncotarget Oncotarget 15522 gure 1: MC3181 and NBDHEX concentration-dependent inhibition of tumor spheroid growth. a. Schematic illust
mor spheroid growth kinetics and compound treatment procedures. Spheroids were treated with drug or drug vehicle 4 days after cel
y 0); 50% medium replenishment was performed on days 2, 4, 6, 10 and 14. b-c. WM115 and d-e. WM266.4 spheroids treated wit
ncentrations of MC3181 (b and d) or NBDHEX (c and e). Control spheroids were treated with vehicle. Spheroid growth kinetics (
aluated by phase contrast imaging at day 2, 6, 10, 14 and 17, whereas the concentration-response curves relative to the MTS as
heroid volume analysis were obtained after 48 hours (center) and 17 days (right) of drug treatment. Phase contrast images (10X magn
digital magnification) correspond to 17 days treated spheroids. Scale bar: 100 μm. Values are means ± SD (n = 12). Figure 1: MC3181 and NBDHEX concentration-dependent inhibition of tumor spheroid growth. a. Schematic illustration of
tumor spheroid growth kinetics and compound treatment procedures. Spheroids were treated with drug or drug vehicle 4 days after cell plating
(day 0); 50% medium replenishment was performed on days 2, 4, 6, 10 and 14. b-c. WM115 and d-e. WM266.4 spheroids treated with graded
concentrations of MC3181 (b and d) or NBDHEX (c and e). Control spheroids were treated with vehicle. MC3181 treatment affects the adhesion
properties of both WM115 and WM266.4 cells Spheroid growth kinetics (left) was
evaluated by phase contrast imaging at day 2, 6, 10, 14 and 17, whereas the concentration-response curves relative to the MTS assays and
spheroid volume analysis were obtained after 48 hours (center) and 17 days (right) of drug treatment. Phase contrast images (10X magnification,
3X digital magnification) correspond to 17 days treated spheroids. Scale bar: 100 μm. Values are means ± SD (n = 12). MC3181 affects invadopodia formation in
WM115 and WM266.4 melanoma cell lines The strong effect of MC3181 prompted us to
investigate the possible effect of the drug on the formation
of invadopodia, protrusions emanating from the surface
of cells and characterized by a high proteolitic activity
towards the ECM. We examined the ability of WM115
and WM266.4 cells to degrade fluorescein-gelatin by
observing the loss of fluorescence of the labeled gelatin
and its correspondence with phalloidin structure, which
identifies invadopodia puncta (Figure 4a and 4d). In
addition, the presence of invadopodia was confirmed
by measuring the fluorescence intensity of TRITC-
phalloidin (red line) and fluorescein-gelatin (green line)
along an arbitrary line that crossed through neighboring
invadopodia structures. Figures 4b for WM115 and
4e for WM266.4 show a drastic decrease of the green
fluorescence intensity in correspondence with an increase
in the red fluorescence intensity of TRITC-phalloidin. Confocal microscopy analysis revealed a significant As shown in Supplementary Figure 4, we found a
progressive decrease in the intracellular levels of lactate
in the high glycolytic WM266.4 cells, while we did not
found other metabolic changes in the same cells (see
Supplementary Tables 1A and 1B). No evident effects on
metabolome were found in WM115 cells treated at both
low and high concentrations of MC3181. MC3181 treatment reduces MMP-2 intracellular
activity decrease in the number of cells with active invadopodia
following treatment with concentrations of MC3181
corresponding to 1/5 and 1/25 of its IC50 value in these cell
lines (Figure 4c and 4f). Moreover, the highest MC3181
concentrations (i.e., 1.3 and 1.0 μM for WM115 and
WM266.4, respectively) completely inhibited adhesion
of the cells to the fluorescein-gelatin layer. Under these
conditions, it was not possible to estimate the percentage
of cells with active invadopodia. We next examined MMP-2 activity in WM115 and
WM266.4 melanoma cells by gelatin zymography (Figure
3d, 3e, 3i and 3l). Densitometric analysis showed that
treatment of both cell lines with equiactive concentrations
of MC3181 induced a reduction of the MMP-2 activity
in a concentration-dependent manner. In particular, a
significant inhibition of MMP-2 proteolytic activity was
observed in WM115 and WM266.4 cells treated with a
concentration of MC3181 corresponding to the IC50 value
in these cell lines (i.e., 1.3 and 1.0 μM, respectively)
(Figure 3e and 3l). Effects of MC3181 treatment on intracellular
metabolome of WM115 and WM266.4 melanoma
cells A comprehensive analysis of the effects of
intracellular metabolome was carried out by quantitative
1H-NMR spectroscopy on WM115 and WM266.4 cells
following MC3181 treatment for 48 hours. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 15523 Table 2: Evaluation of the cytotoxic (MTS assay) and antiproliferative (volume analysis) effects of MC3181 and
NBDHEX on WM115 and WM266.4 3D multicellular tumor spheroids Table 2: Evaluation of the cytotoxic (MTS assay) and antiproliferative (volume analysis) effects of MC3181 and
NBDHEX on WM115 and WM266.4 3D multicellular tumor spheroids
IC50 ± SD (μM)
WM115
48 hours
17 Days
Volume
MTS
Volume
MTS
MC3181
n.d. 3.22 ± 0.19
0.53 ± 0.04
2.19 ± 0.15
NBDHEX
n.d. 1.44 ± 0.08
0.37 ± 0.09
6.27 ± 0.94
WM266.4
48 hours
17 Days
Volume
MTS
Volume
MTS
MC3181
1.67 ± 0.26
7.67 ± 1.00
1.10 ± 0.23
2.41 ± 0.40
NBDHEX
2.84 ± 0.19
2.44 ± 0.43
1.22 ± 0.33
3.60 ± 0.51 Table 2: Evaluation of the cytotoxic (MTS assay) and antiproliferative (volume analysis) effects of MC3181 and
NBDHEX on WM115 and WM266.4 3D multicellular tumor spheroids Table 2: Evaluation of the cytotoxic (MTS assay) and antiproliferative (volume analysis) effects of MC3181 and
NBDHEX on WM115 and WM266.4 3D multicellular tumor spheroids Analysis of the effect of MC3181 treatment
on the mRNA expression of a panel of genes
involved in melanoma progression and
metastatization Qualitative RT-PCR was carried out to characterize
WM115 and WM266.4 cell lines for the expression of
a panel of genes, including the pro-angiogenic factors
vascular endothelial growth factor (VEGF) and the basic
fibroblast growth factor (FGF2); the matrix-metallo- www.impactjournals.com/oncotarget Oncotarget 15524 Figure 2: Effect of MC3181 on the adhesion of human melanoma cells to different ECM components. WM115 cells and
WM266.4 cells (3x105/ml) were applied to individual coated wells with 200 μl of a, d. collagen (7.5 μg/ml), b, e. gelatin (0.1%) or c, f. Matrigel (0.2 mg/ml), in the absence and in the presence of increasing MC3181 concentrations, and incubated for 30 minutes at 37°C in
5% CO2. Cells were then fixed and colored with crystal violet. Images (20X magnification, 3X digital magnification) were obtained by
phase contrast microscopy. The absorbance values of wells were measured at 580 nm after dissolving crystal violet with 100% methanol. The results were expressed as the mean percentage of cell adhesion ± SD versus control, repeated in triplicate; *P < 0.05, **P < 0.005 and
*** P < 0.0005 vs control. Figure 2: Effect of MC3181 on the adhesion of human melanoma cells to different ECM components. WM115 cells and
WM266.4 cells (3x105/ml) were applied to individual coated wells with 200 μl of a, d. collagen (7.5 μg/ml), b, e. gelatin (0.1%) or c, f. Matrigel (0.2 mg/ml), in the absence and in the presence of increasing MC3181 concentrations, and incubated for 30 minutes at 37°C in
5% CO2. Cells were then fixed and colored with crystal violet. Images (20X magnification, 3X digital magnification) were obtained by
phase contrast microscopy. The absorbance values of wells were measured at 580 nm after dissolving crystal violet with 100% methanol. The results were expressed as the mean percentage of cell adhesion ± SD versus control, repeated in triplicate; *P < 0.05, **P < 0.005 and
*** P < 0.0005 vs control. Figure 2: Effect of MC3181 on the adhesion of human melanoma cells to different ECM components. WM115 cells and
WM266.4 cells (3x105/ml) were applied to individual coated wells with 200 μl of a, d. collagen (7.5 μg/ml), b, e. gelatin (0.1%) or c, f. Matrigel (0.2 mg/ml), in the absence and in the presence of increasing MC3181 concentrations, and incubated for 30 minutes at 37°C in
5% CO2. Cells were then fixed and colored with crystal violet. Analysis of the effect of MC3181 treatment
on the mRNA expression of a panel of genes
involved in melanoma progression and
metastatization Images (20X magnification, 3X digital magnification) were obtained by
phase contrast microscopy. The absorbance values of wells were measured at 580 nm after dissolving crystal violet with 100% methanol. The results were expressed as the mean percentage of cell adhesion ± SD versus control, repeated in triplicate; *P < 0.05, **P < 0.005 and
*** P < 0.0005 vs control. www.impactjournals.com/oncotarget Oncotarget 15525 Figure 3: MC3181 blocks WM115 and WM266.4 melanoma cells invasion and inhibits MMP2 activity. Cell lines were
assayed for in vitro invasion and migration using Boyden chamber without coating (migration) or coated with 5 μg of Matrigel. After
48 hours of treatment with graded MC3181 concentrations, migrated and invaded cells per field were stained with crystal violet and
counted. Representative phase contrast images (10X magnification, 3X digital magnification) of a. WM115 and f. WM266.4 are shown. Migrated/Invaded b. WM115 and g. WM266.4 cells. The invasion index of c. WM115 and h. WM266.4 cells was calculated as the invasion
percentage of treated cells divided by the invasion percentage of non-treated cells (see equations 2 and 3 in “Materials and Methods”
section). Intracellular MMP-2 activity was measured by gelatin zymography assay on d. WM115 and i. WM266.4 cells treated with graded
MC3181 concentrations for up to 48 hours. ImageJ quantification of 3 independent experiments of gelatin zymography performed on e. WM115 and l. WM266.4 cells. The control has been settled as the 100%, and results were expressed as the mean percentage of MMP-2
activity ± SD vs control. **P < 0.005 and ***P < 0.0005 vs control. Figure 3: MC3181 blocks WM115 and WM266.4 melanoma cells invasion and inhibits MMP2 activity. Cell lines were
assayed for in vitro invasion and migration using Boyden chamber without coating (migration) or coated with 5 μg of Matrigel. After
48 hours of treatment with graded MC3181 concentrations, migrated and invaded cells per field were stained with crystal violet and
counted. Representative phase contrast images (10X magnification, 3X digital magnification) of a. WM115 and f. WM266.4 are shown. Migrated/Invaded b. WM115 and g. WM266.4 cells. The invasion index of c. WM115 and h. WM266.4 cells was calculated as the invasion
percentage of treated cells divided by the invasion percentage of non-treated cells (see equations 2 and 3 in “Materials and Methods”
section). Intracellular MMP-2 activity was measured by gelatin zymography assay on d. WM115 and i. WM266.4 cells treated with graded
MC3181 concentrations for up to 48 hours. ImageJ quantification of 3 independent experiments of gelatin zymography performed on e. WM115 and l. WM266.4 cells. The control has been settled as the 100%, and results were expressed as the mean percentage of MMP-2
activity ± SD vs control. **P < 0.005 and ***P < 0.0005 vs control. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 15526 in Supplementary Figure 5. Afterwards, we analyzed
the mRNA expression of these genes following
treatment with equiactive concentrations of MC3181
(corresponding to its IC50 value, 1/5 and 1/25 of its IC50
value in these cell lines). CDH5, VEGF, FGF2, MMP-
2, MMP-9 and both the MCAM/MUC 18 Long and
Short isoforms mRNA were still present at 24 hours in
WM115 cells, while they drastically decreased at 48
hours after treatment with all the MC3181 concentrations
tested (Figure 5a, left). Interestingly, 9 hours of drug
exposure were sufficient to induce a drastic decrease
of the CDH2 mRNA (Figure 5a, left). Conversely, a
strong reduction of mRNA expression of all genes
could be observed in WM266.4 cells after 48 hours of proteinases 2 and 9 (MMP-2 and MMP-9); the cell-cell
adhesion molecules E-cadherin (CDH1), N-cadherin
(CDH2), Ve-cadherin (CDH5), and both the long and short
isoforms of the endothelial antigen MCAM/MUC18 [11]. To validate our expression panel, we used two melanoma
cell lines as positive control (M10 and M14) and β2-
microglobulin as housekeeping. RT-PCR documented mRNA expression in all the
melanoma cell lines analyzed (WM115, WM266.4, and
the positive controls M10 and M14) for VEGF, FGF2,
CDH2, CDH5, MMP-2, MMP-9, and the two isoforms
of MCAM/MUC18. The mRNA expression levels of all
the genes tested in WM115 and WM266.4 cell lines are
shown in Figure 5a, and a representative gel is reported Figure 4: Invadopodia matrix degradation activity. WM115 and WM266.4 cells were seeded on Fluorescein-Gelatin coated
chamber slides for 5 hours, and then fixed and stained for TRITC-Phalloidin. a. WM115 and d. WM266.4 representative confocal images
showing invadopodia as focal cytoplasmic concentrations of Phalloidin that overlap with areas of gelatin clearing (dark holes in the matrix)
within the merged image, as indicated by arrows. Fluorescein-Gelatin images were pseudocolored in gray. Images were acquired with
a 40X magnification oil immersion objective and a digital magnification 3X is shown. Scale bar = 10 μm. b. WM115 and e. WM266.4
representative fluorescence intensity plot showing co-localization of TRITC-Phalloidin (red line) and gelatin degradation (green line). Fluorescence intensity was measured at each image pixel along an arbitrary line that crossed through an invadopodia structure. Percentage
of c. WM115 and f. WM266.4 cells with active invadopodia was obtained by counting approximately 100 cells in 3 fields. Data shown are
means ± SD of three independent experiments. www.impactjournals.com/oncotarget *P < 0.05 and **P < 0.005 vs control. Figure 4: Invadopodia matrix degradation activity. WM115 and WM266.4 cells were seeded on Fluorescein-Gelatin coated
chamber slides for 5 hours, and then fixed and stained for TRITC-Phalloidin. a. WM115 and d. WM266.4 representative confocal images
showing invadopodia as focal cytoplasmic concentrations of Phalloidin that overlap with areas of gelatin clearing (dark holes in the matrix)
within the merged image, as indicated by arrows. Fluorescein-Gelatin images were pseudocolored in gray. Images were acquired with
a 40X magnification oil immersion objective and a digital magnification 3X is shown. Scale bar = 10 μm. b. WM115 and e. WM266.4
representative fluorescence intensity plot showing co-localization of TRITC-Phalloidin (red line) and gelatin degradation (green line). Fluorescence intensity was measured at each image pixel along an arbitrary line that crossed through an invadopodia structure. Percentage
of c. WM115 and f. WM266.4 cells with active invadopodia was obtained by counting approximately 100 cells in 3 fields. Data shown are
means ± SD of three independent experiments. *P < 0.05 and **P < 0.005 vs control. www.impactjournals.com/oncotarget Oncotarget 15527 On
15528
tjournals.com/oncotarget
RT-PCR analysis of the mRNA expression of cancer progression related genes. a. Total RNA from un
reated cells was extracted, reverse-transcribed, amplified by PCR, and submitted to electrophoresis on a 1.8% agaro
and Methods” section). The graphs show the relative intensity of the PCR products vs β2-microglobulin, obtained by
ftware: -■-ctrl; -◊- 0.05 and 0.04 μM MC3181 (WM115 and WM266.4, respectively); -●- 0.26 and 0.20 μM MC318
66.4, respectively) and -∆- 1.3 and 1.0 μM MC3181 (WM115 and WM266.4, respectively). b. Relative intensity o
om WM266.4 cells treated for 48 hours with equiactive concentrations of MC3181 and VMF (1.0 μM vs 0.9 μM, res Figure 5: RT-PCR analysis of the mRNA expression of cancer progression related genes. a. Total RNA from untreated and
MC3181-treated cells was extracted, reverse-transcribed, amplified by PCR, and submitted to electrophoresis on a 1.8% agarose gel (see
“Materials and Methods” section). The graphs show the relative intensity of the PCR products vs β2-microglobulin, obtained by the ImageJ
analysis software: -■-ctrl; -◊- 0.05 and 0.04 μM MC3181 (WM115 and WM266.4, respectively); -●- 0.26 and 0.20 μM MC3181 (WM115
and WM266.4, respectively) and -∆- 1.3 and 1.0 μM MC3181 (WM115 and WM266.4, respectively). b. www.impactjournals.com/oncotarget Relative intensity of the PCR
products from WM266.4 cells treated for 48 hours with equiactive concentrations of MC3181 and VMF (1.0 μM vs 0.9 μM, respectively). www.impactjournals.com/oncotarget Oncotarget 15528 treatment only with the highest concentration (1.0 μM) of
MC3181 (Figure 5a, right). We did not detect the presence
of CDH1 mRNA in both WM115 and WM266.4 cell lines,
either before or after MC3181 treatment (data not shown). Finally, we decided to perform a preliminary
comparison of the efficacy of equiactive concentrations
of MC3181 and VMF, corresponding to their IC50
values (1.0 and 0.9 μM, respectively), on the mRNA
expression. Interestingly, we found that MC3181 treatment
outperformed VMF in the suppression of the genes
analyzed after 48 hours, with the only exception of CDH5
(Figure 5b). treatment only with the highest concentration (1.0 μM) of
MC3181 (Figure 5a, right). We did not detect the presence
of CDH1 mRNA in both WM115 and WM266.4 cell lines,
either before or after MC3181 treatment (data not shown). able to invade the collagen matrix, and therefore subsequent
experiments were performed only on WM266.4 cells. Also
in this case, the compound was able to significantly reduce
cells invasion into collagen, in a dose dependent manner
(Figure 6). Twenty-four hour treatment with 0.04, 0.20 and
1.0 μM MC3181 reduced the distance invaded by WM266.4
cells by about 40, 60 and 80%, respectively, compared
to the control. The effect obtained with 0.20 and 1.0 μM
MC3181 remained constant over the 48 hours incubation
period, while spheroids treated with 0.04 μM MC3181
showed a recovery of their invasive potential. Finally, we decided to perform a preliminary
comparison of the efficacy of equiactive concentrations
of MC3181 and VMF, corresponding to their IC50
values (1.0 and 0.9 μM, respectively), on the mRNA
expression. Interestingly, we found that MC3181 treatment
outperformed VMF in the suppression of the genes
analyzed after 48 hours, with the only exception of CDH5
(Figure 5b). MC3181 reduces the expression of proteins
involved in tumor invasion and angiogenesis
and increases the level of the AP2 transcription
factor Then, we checked the expression levels of
several proteins involved in the extravasation process. Treatment of WM266.4 cells with 1.0 μM MC3181
caused a sustained increase of the transcription factor
AP2 (activating enhancer binding protein 2), starting
within 30 minutes after addition of the drug (Figure
7d and 7e). Furthermore, 1.0 μM MC3181 caused a
significant and sustained decrease in the expression of the
adhesion molecules MCAM/MUC18 (CD146), VEGF
and N-cadherin (Figure 7d, 7f, 7g and 7h). Of note, the
expression of the latter protein was significantly affected
also by lower concentrations of MC3181. Autophagy is not involved in the anti-metastatic
effect of MC3181 We previously reported [6] the high antiproliferative
activity of MC3181 against a wide panel of melanoma
cells in vitro. Here, we confirmed this potent and
concentration-dependent ability in both 2D monolayer
and 3D multicellular spheroids cultures. Moreover,
since cell adhesion is a fundamental step involved in
the physiological processes of proliferation, invasion,
as well as the pathology of neoplastic transformation
and metastasis, we were prompted to investigate the
capacity of MC3181 to hinder this phenomenon in both
2D and 3D-cultures. The drug successfully affected
the adhesion properties of cell cultures, and interfered
also with invadopodia formation. Moreover, MC3181
caused a concentration-dependent decrease of lactate
levels in the high glycolytic WM266.4 cells. MC3181
acted selectively to inhibit lactate formation without
affecting intracellular metabolites involved in glucose,
phospholipid and energetic metabolism of this cell line. Indeed, an increase in intracellular lactate (Warburg
metabolic phenotype) causes a consistent acidification
of the tumor microenvironment triggering epithelial–
mesenchymal transition (EMT) in melanoma cells and
promoting invasion and metastasis through expression,
activation and secretion of proteolytic enzymes [13]. Consequently, the ability of MC3181 to counteract the
formation of an acidic microenvironment may represent a
crucial step in the suppression of metastatic phenotype of
WM266.4 cells. Overall, these data are paralleled by the
in vivo antimetastatic activity observed against the highly
metastatic B16-F10 murine melanoma cells. As autophagy seems to be involved in tumor cell
motility and invasion [12], we investigated the impact
of subtoxic concentrations of MC3181 on the basal
autophagy of WM266.4 cells. Western blot analysis
showed that a 48-hours treatment with low concentrations
of MC3181 did not significantly affect the levels of the
autophagosome-associated LC3-II protein (Supplementary
Figure 7). Thus, the anti-metastatic activity of MC3181 is
not attributable to autophagy modulation. DISCUSSION The ability of cancer cells to migrate, invade, and
metastasize to other organs represents the most lethal
aspect of melanoma, as well as the major hurdle to
successful therapeutic intervention. In the present study, we demonstrated that subtoxic
concentrations of MC3181 are effective in reducing
the invasiveness of both two BRAF-V600D-mutated
melanoma cell lines (namely, WM115 and WM266.4) as
well as of the BRAF-V600E-mutated human melanoma
cell line SK-MEL-5. MC3181 inhibits WM266.4 3D-multicellular
tumor spheroid invasion into collagen type I We found that MC3181 was able
to induce a significant reduction of P-JNK levels in the
metastasis-derived melanoma cell line, starting within
30 minutes after addition of the drug (Figure 7a). This
reduction persisted for 48 hours in the presence of 1 μM
MC3181. Of note, when WM266.4 cells were treated
with higher MC3181 concentrations (i.e. 2.0 and 4.0 μM)
we observed a persistent JNK activation that paralleled
an increase of apoptotic cells, as previously reported
(Supplementary Figure 6) [5, 6]. Phospho-activation of
p38 was also significantly affected by 1.0 μM MC3181
up to 48 hours (P-p38, Figure 7c). As regards ERK
activation, the levels of P-ERK in drug treated cells were
not significantly different from those of control cells, at all
of the investigated MC3181 concentrations (Figure 7b). MC3181 treatment reduced by more than 30% the number
of metastatic lung nodules, as compared to untreated mice
(Figure 8), thus supporting the concept that the drug may
be particularly active in preventing melanoma invasion
and metastasis. MC3181 inhibits WM266.4 3D-multicellular
tumor spheroid invasion into collagen type I To gain insight about the molecular mechanisms,
we analyzed the effect of MC3181 treatment on proteins
involved in WM266.4 cell invasion. Since activation
of the MAPK/ERK pathway is a frequent event in
tumorigenesis, we investigated the activation level of Tumor invasion was further analyzed in WM115 and
WM266.4 cells grown as 3D-multicellular tumor spheroids
and embedded in type I collagen. Under these experimental
conditions, spheroids formed by WM115 cells were not Figure 6: MC3181 inhibits WM266.4 spheroids invasion into type I collagen. 3D melanoma spheroids were embedded in a
collagen matrix and treated with 0.04, 0.20 and 1.0 μM MC3181. The distance invaded by WM266.4 cells was monitored by phase contrast
imaging (10X magnification, 3X digital magnification), over a 48 hours incubation period. Scale bar: 100 μm. The graph shows the mean
distance invaded by spheroids ± SD; sample number: n = 10 for every condition; *P < 0.05, **P < 0.005 and ***P < 0.0005 vs the respective
time-matched control. Figure 6: MC3181 inhibits WM266.4 spheroids invasion into type I collagen. 3D melanoma spheroids were embedded in a
collagen matrix and treated with 0.04, 0.20 and 1.0 μM MC3181. The distance invaded by WM266.4 cells was monitored by phase contrast
imaging (10X magnification, 3X digital magnification), over a 48 hours incubation period. Scale bar: 100 μm. The graph shows the mean
distance invaded by spheroids ± SD; sample number: n = 10 for every condition; *P < 0.05, **P < 0.005 and ***P < 0.0005 vs the respective
time-matched control. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 15529 JNK, ERK and p38. We found that MC3181 was able
to induce a significant reduction of P-JNK levels in the
metastasis-derived melanoma cell line, starting within
30 minutes after addition of the drug (Figure 7a). This
reduction persisted for 48 hours in the presence of 1 μM
MC3181. Of note, when WM266.4 cells were treated
with higher MC3181 concentrations (i.e. 2.0 and 4.0 μM)
we observed a persistent JNK activation that paralleled
an increase of apoptotic cells, as previously reported
(Supplementary Figure 6) [5, 6]. Phospho-activation of
p38 was also significantly affected by 1.0 μM MC3181
up to 48 hours (P-p38, Figure 7c). As regards ERK
activation, the levels of P-ERK in drug treated cells were
not significantly different from those of control cells, at all
of the investigated MC3181 concentrations (Figure 7b). JNK, ERK and p38. MC3181 oral administration reduces melanoma-
induced lung metastases To gain insight into the mechanisms of action
underlying these results, we investigated the effects of
MC3181 on the expression of genes involved in melanoma
progression and metastatization, at both mRNA and
protein levels. In particular, we focused our analysis
on the transcription factor AP2, known to regulate the
developmental choice between growth and differentiation
in several embryonic tissues, and whose expression To assess the potential antimetastatic effect of
MC3181 in vivo, B6 mice were injected i.v. with syngeneic
melanoma cells at day 0, and treated 8 hours later, and
subsequently on a daily base, with an oral administration
of the drug. Two weeks after tumor cell injection, animals
were sacrificed, lungs explanted and metastases counted. www.impactjournals.com/oncotarget Oncotarget 15530 Figure 7: MC3181 causes a persistent decrease of JNK/p38 phospho-activation and of the expression of proteins
involved in melanoma invasion. Immunoblot and densitometric analysis of a. P-JNK, b. P-ERK, c. P-p38 in absence (-□-) and in
presence of MC3181 0.04 μM (-◊-), 0.20 μM (-○-) and 1.0 μM (-●-). The data revealed a significant decrease of P-JNK after only 30
minutes treatment with 1.0 μM MC3181 (***P < 0.0005 vs control). A sustained decrease of the phospho-active form of p38 was also
observed at the highest MC3181 concentration (1.0 μM, *P < 0.05 vs control). Immunoblot of d. AP-2, MCAM/MUC18 (CD146) and
VEGF. Actin was used as loading control. Densitometric analysis revealed a prolonged (up to 24 hours) increase of e. AP2 expression after
treatment with 1.0 μM MC3181 (*P < 0.05 vs control) that paralleled the persistent decrease of the expression levels of f. MCAM/MUC18
and g. VEGF (***P < 0.0005 vs control) caused by 1.0 μM MC3181. All subtoxic concentrations of MC3181 (from 0.04 to 1.0 μM) were
also able to efficiently reduce the h. N-cadherin expression up to 48 hours in WM266.4 cells (**P < 0.005 and ***P < 0.0005 vs control). Phosphorylated and non-phosphorylated proteins levels were quantitated by densitometry and normalized to their respective β-actin bands; Figure 7: MC3181 causes a persistent decrease of JNK/p38 phospho activation and of the expression of proteins s a persistent decrease of JNK/p38 phospho-activation and of the expression of proteins
i Figure 7: MC3181 causes a persistent decrease of JNK/p38 phospho-activation and of the expression of proteins
involved in melanoma invasion. Immunoblot and densitometric analysis of a. P-JNK, b. P-ERK, c. MC3181 oral administration reduces melanoma-
induced lung metastases P-p38 in absence (-□-) and in
presence of MC3181 0.04 μM (-◊-), 0.20 μM (-○-) and 1.0 μM (-●-). The data revealed a significant decrease of P-JNK after only 30
minutes treatment with 1.0 μM MC3181 (***P < 0.0005 vs control). A sustained decrease of the phospho-active form of p38 was also
observed at the highest MC3181 concentration (1.0 μM, *P < 0.05 vs control). Immunoblot of d. AP-2, MCAM/MUC18 (CD146) and
VEGF. Actin was used as loading control. Densitometric analysis revealed a prolonged (up to 24 hours) increase of e. AP2 expression after
treatment with 1.0 μM MC3181 (*P < 0.05 vs control) that paralleled the persistent decrease of the expression levels of f. MCAM/MUC18
and g. VEGF (***P < 0.0005 vs control) caused by 1.0 μM MC3181. All subtoxic concentrations of MC3181 (from 0.04 to 1.0 μM) were
also able to efficiently reduce the h. N-cadherin expression up to 48 hours in WM266.4 cells (**P < 0.005 and ***P < 0.0005 vs control). Phosphorylated and non-phosphorylated proteins levels were quantitated by densitometry and normalized to their respective β-actin bands;
data, presented as arbitrary units, represent means ± SD of three independent experiments. The x-axis is in logarithmic scale. www.impactjournals.com/oncotarget Oncotarget 15531 We previously reported that cytotoxic amounts
of NBDHEX and MC3181 can disrupt the interaction
between GSTP1-1 and TRAF2, leading to a prolonged
activation of different MAPK pathways and eventually
cell death [6, 29]. This is in contrast with the fact that
activation of MAPKs is involved in the control of cell
migration and invasion by regulating the expression and
activation of MMPs [30]; in particular, the activating
mutations in the Ras/Raf/MEK/ERK proteins result in
constitutive signaling that promote the oncogenic behavior
of melanomas. Moreover, the MAP kinase JNK plays a
role in the acquisition of the metastatic phenotype, acting
at multiple levels. JNK interacts with and phosphorylates
paxillin leading to an increased formation of invadopodia
in gliomas [31], and JNK inhibition in melanoma by
α-solanine significantly reduces cell migration and
invasion [32]. Surprisingly, we found that subtoxic
amounts of MC3181 not only did not activate ERK
but also caused a significant and sustained decrease of
the phospho-active forms of JNK and p38. This event
occurred in the very early phase of drug treatment, and
paralleled the increase in the AP2 expression. Nonetheless,
the potential involvement of alternative MC3181 targets
cannot be ruled out. Cell adhesion assay Overall, the results of the present study are
particularly relevant considering the high unmet need for
more effective and safer systemic therapies for metastatic
melanoma. A 48-well plate was coated with 200 μL of collagen
(7.5 μg/ml), gelatin (0.1%), or Matrigel (0.2 mg/ml),
and incubated for 2 hours at room temperature (RT). After PBS washes, plates were blocked by incubation
for 30 minutes at 37°C in cell culture medium. Human
melanoma cells (5×104 cell/well) were suspended in
complete medium in the absence or presence of graded
concentrations of MC3181, then plated onto the pre-coated
wells, and allowed to adhere to the different substrates for
30 minutes at 37°C. Non-adhering cells were washed off
with PBS, and the adhering cells were fixed with cold
methanol (Sigma-Aldrich) and stained with 0.5% crystal
violet. Digital photos of 3 random fields were taken at
magnification 20X (3X digital camera) with an inverted
microscope (Nikon ECLIPSE TS100). After imaging, the
absorbance of the solubilized dye (in 100% methanol)
was measured at 580 nm. The data are expressed as the
mean percentages ± SD from at least three independent
experiments. Cell culture and treatment The human melanoma cell lines WM115, WM266.4,
SK-MEL-5, M10, and M14; the Ehlers-Danlos syndrome
(EDS) derived fibroblast cell line; and the mouse
melanoma cell line B16-F10 were obtained from the
American Type Culture Collection (ATCC, Manassas,
VA). WM115, WM266.4, M10, and M14 cell line were
cultured in RPMI-1640 (EuroClone, Milan, Italy) whilst
SK-MEL-5 were cultured in DMEM/F12 (Sigma-
Aldrich) supplemented with 10% fetal bovine serum
(FBS, v/v) (EuroClone), 2 mM L-glutamine, 100 U/ml
penicillin and 100 mg/ml streptomycin (Lonza, Basel,
Switzerland). B16-F10 cells were cultured in DMEM MC3181 oral administration reduces melanoma-
induced lung metastases All cell lines were maintained at
37 °C in a 5% CO2 humidified atmosphere. The molecular alterations reported above may also
explain the effect of MC3181 treatment on the amount of
lactate present in the metastatic WM266.4 cancer cells. Gao et al. [33] found higher nuclear levels of pyruvate
kinase isoform M2 (PKM2) in WM266.4 cells, as
compared to those recorded in the parent WM115 cells. Nuclear PKM2 could act as a coactivator of β-catenin to
induce the expression of c-Myc, which in turn promotes
the expression of glycolytic genes. In particular, c-Myc
transcriptionally induces the expression of both glucose
transporter 1 and lactate dehydrogenase A, which are
required for glucose uptake and conversion of pyruvate
to lactate, respectively [34]. c-Myc transcription is also
regulated by the JNK/AP1 pathway [35], and the inhibition
of this pathway causes a decrease in the lactate production
[36]. Furthermore, the transcription factor AP-2 directly
interacts with c-Myc and inhibits its binding to DNA [37]. Therefore, we may hypothesize that both inhibition of
JNK phospho-activation and increment of AP2 levels may
be responsible for the decrease of lactate production in
WM266.4 cells upon treatment with MC3181. 2i
For in vitro antiproliferative studies, WM115
(2.5×104 cells/cm2), WM266.4 cells (1.25×104 cells/cm2)
and SK-MEL-5 (2.5×104 cells/cm2) were seeded in 96-
well plates and the IC50 values for each compound was
then evaluated by the SRB assay as previously reported
[5, 6]. 2
For in vitro antiproliferative studies, WM115
(2.5×104 cells/cm2), WM266.4 cells (1.25×104 cells/cm2)
and SK-MEL-5 (2.5×104 cells/cm2) were seeded in 96-
well plates and the IC50 values for each compound was
then evaluated by the SRB assay as previously reported
[5, 6]. The effects of MC3181 on cell growth and cell
cycle were evaluated in WM115 and WM266.4 cell lines
(2.5×104 and 1.25×104 cells/cm2, respectively) seeded in
75 cm2 flask (Corning B.V. Life Sciences, Amsterdam,
Netherlands). Forty-eight hours after plating, cells were
exposed to equiactive concentrations of MC3181 (0.05,
0.26 and 1.3 μM for WM115, and 0.04, 0.20 and 1.0 μM
for WM266.4), harvested at different time points, counted
using a Neubauer Chamber (after 1:1 dilution in Trypan
Blue), and analyzed by a FACSCalibur instrument (BD
Bioscence, San Jose, CA, USA). Flow cytometric data
were analyzed by FlowJo 8.8.6 software (Tree Stare, Inc,
Ashland, OR, USA). Drugs NBDHEX and MC3181 were synthesized as
previously reported [6, 38]. For in vitro studies, NBDHEX,
MC3181, temozolomide (TMZ; Sigma-Aldrich, Milan,
Italy) and vemurafenib (VMF; Selleckem, Munich,
Germany) were dissolved in DMSO. Before use, each
compound was diluted to the appropriate concentration in
complete culture medium; the final DMSO concentration
never exceeded 0.05% (v/v). For in vivo studies, MC3181
was dissolved in phosphate-buffered saline (PBS). MC3181 oral administration reduces melanoma-
induced lung metastases However, when the amount of
MC3181 was increased to concentrations higher than its
IC50 value, we confirmed the previously reported evidence levels inversely correlate with melanoma progression in
human primary melanoma specimens [14–21]. Of note,
MC3181 caused an early increase in the expression levels
of AP2 in the metastatic cell line, together with a decrease
of N-cadherin, MCAM/MUC18 and VEGF. In fact,
this transcription factor acts as a tumor suppressor, and
its loss results in the up-regulation of the expression of
several genes involved in the acquisition of the metastatic
phenotype [16, 22–24]. In particular, N-cadherin favors the interaction
of melanoma cells with other cell types expressing
N-cadherin, such as fibroblasts or vascular endothelial
cells [25, 26], thus fostering the access of tumor cells
to the vasculature and the formation of metastases [27]. This process is also promoted by the proteolytic activity
of matrix metalloproteinase MMP-2, which degrades
collagen IV [28], a major constituent of ECM, and favors
melanoma cells to cross the basal lamina and migrate
to their secondary sites of growth. Indeed, we showed
here that MC3181 also induced a significant reduction
of MMP-2 activity. These data are also supported by the
evidence that MC3181 treatment decreases the number of
cells with active invadopodia. Thus, MC3181 treatment
appears to force melanoma towards a less aggressive
phenotype lowering the expression of several proteins
involved in invasion and angiogenesis. Figure 8: Antimetastatic effect of MC3181 in vivo. On day 0, C57BL/6 mice were injected i.v. with 1×105 Syngeneic B16-F10
melanoma cells. Eight hours later, they were randomly assigned to an experimental group (n = 6) and received orally MC3181 dissolved in
PBS. Control group received PBS only. Mice were sacrificed on day 14, and lung metastatic nodules counted with the aid of a dissecting
microscope. P = 0.001. Figure 8: Antimetastatic effect of MC3181 in vivo. On day 0, C57BL/6 mice were injected i.v. with 1×105 Syngeneic B16-F10
melanoma cells. Eight hours later, they were randomly assigned to an experimental group (n = 6) and received orally MC3181 dissolved in
PBS. Control group received PBS only. Mice were sacrificed on day 14, and lung metastatic nodules counted with the aid of a dissecting
microscope. P = 0.001. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 15532 of a persistent phospho-activation of JNK driving cell
death. supplemented with the above-mentioned reagents and 50
mM β-mercaptoethanol. RNA isolation and reverse transcriptase-
polymerase chain reaction (RT-PCR) WM115 and WM266.4 cells were plated in
75cm2 flask (2.5×104 cells/cm2 and 1.25×104 cells/cm2,
respectively). Forty-eight hours after seeding, cells were
incubated with increasing concentrations of MC3181
for 9, 24, and 48 hours. Two human melanoma cell lines
(M10 and M14) were used as positive control, while
the negative counterpart is represented by the Ehlers-
Danlos syndrome (EDS) derived fibroblast cell line. Both control and WM115 and WM266.4 samples were
detached by trypsinization, washed twice with PBS,
centrifuged for 15 minutes at 1200 x g (4°C), and then
frozen at -70°C in a Guanidine-Iso-Thiocyanate solution
(2x106 cells/sample). RNA extraction was performed as
described by Chomczyńsky and Sacchi [41], with slight
modifications, and resuspended in distilled sterile water. RNA purity and concentration were determined both
spectrophotometrically and electrophoretically. WM115 and WM266.4 cells (2.0×104/cm2) were
seeded on 8-well chamber slides previously coated with
Fluorescein-Gelatin following manufacturer’s protocol
(QCMTM Gelatin Invadopodia Assay Green, Millipore). After 5 hours incubation in absence and in presence of
graded MC3181 concentrations (0.05, 0.26 and 1.3 μM
for WM115, 0.04, 0.20 and 1.0 μM for WM266.4), cells
were fixed with 3.7% formaldehyde, washed in PBS,
permeabilized by fluorescent staining buffer (2% blocking
serum and 0.25% Triton X-100 in DPBS without Ca2+ and
Mg2+), and then stained for TRITC-Phalloidin and DAPI
following manufacturer’s instruction. Fluorescence was
detected using a Fluoview 1000 Olympus (Opera Zerbo,
Milan, Italy) system equipped with an Olympus IX-81
inverted microscope. Acquisitions were performed with a
40X magnification oil immersion objective (NA 1.42, WD
0.15 mm). Cells with active invadopodia were defined as
cells characterized by dot like structures. The Z-optical
section series, obtained beginning from the nuclear apex
and progressing down in 0.48 μM (at least 22 planes),
were converted to maximum projection images to avoid
subjectivity in the choice of the plane to be analysed. Gelatin degradation was visualized as darker areas on the
coverslip due to proteolytic removal of the Fluorescein-
Gelatin. Almost 100-150 cells/sample were analysed in
merged images by the ImageJ software. p
p
y
p
y
For qualitative RT-PCR analysis, we designed
an expression panel including different pro-angiogenic
factors, cell-cell adhesion molecules, and matrix-metallo
proteinases. Of note, we analyzed two out of the 6 possible
transcripts of the melanoma adhesion molecule MCAM/
MUC 18 gene: the short isoform, widely expressed by
endothelial cells, and the long isoform, more melanoma-
specific [11, 42–44]. Gelatin zymography WM115 and WM266.4 cells (2×106) were treated
with different amounts of MC3181 (0.05, 0.26 and
1.3 μM for WM115, and 0.04, 0.20 and 1.0 μM for
WM266.4) for 9, 24 or 48 hours, and then lysed in Lysis
Buffer [25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1%
Nonidet-P-40, 1 mM PMSF, pH 7.5] (Sigma-Aldrich,
Milan, Italy). After determination of protein concentration
by the Lowry colorimetric assay, 20 μg of proteins were
loaded, under non-reducing conditions, on 10% SDS- www.impactjournals.com/oncotarget Oncotarget 15533 ethanol 70% according to an established protocol [39]. Briefly, samples were ultra-sonicated at 20 kHz by a MSE
ultrasonic disintegrator Mk2 (Crawley) and centrifuged at
14000 x g for 30 min. Supernatants were lyophilized twice
in a RVT 4104 Savant lyophilizer (Mildford), and the
residue resuspended in 0.7 ml D2O (Sigma-Aldrich, St. Louis, MO, USA) containing 0.1 mM 3-(trimethylsilyl)-
propionic-2,2,3,3-d4 acid sodium salt (TSP) as internal
standard. High-resolution NMR experiments (25°C) were
performed at 9.4T (Bruker AVANCE). 1H-NMR spectra
of cell extracts were acquired using 90° flip angle, 30 s
repetition time, 32K time domain data points and 128
transients [39, 40]. polyacrylamide gels containing 0.1% gelatin (Serva
ELECTROPHORESIS, Heidelberg, Germany). After
electrophoresis, gels were incubated in the Renaturing
Solution [2.5% Triton X-100 (Sigma-Aldrich)] for 30
minutes at room temperature, and then incubated in the
developing buffer [50 mM Tris-HCl (pH 7.8), 200 mM
NaCl, 5 mM CaCl2, 0.02% Triton X-100] overnight, at
37°C. Gels were then stained with 0.5% Comassie blu
R250 (Sigma-Aldrich) for 1 hour, and destained with 10%
methanol and 5% acetic acid (Sigma-Aldrich). Activity
was obtained by analysing the clear areas in the gels with
the ImageJ software. The data are expressed as the mean
percentages ± SD from three independent experiments. RNA isolation and reverse transcriptase-
polymerase chain reaction (RT-PCR) Two micrograms of total RNA and
2.5 units of Moloney Murine Leukemia virus reverse
transcriptase [45] (Applied BioSystems, Roche Molecular
Systems, Inc., Branchburh, New Jersey, USA) were applied
in all RT-PCR experiment, according to the manufacturer’s
instructions. For the generation of the first strand cDNA,
the reaction mix contained 2.5 μM oligo d(T)16, 5 mM
MgCl2, 1 μM dNTPs, 1 unit of RNase Inhibitor (Applied
BioSystems) during 1 h incubation at 42°C. A 2μl aliquot
of cDNA was used for single step PCR for all genes, with
the exception of MCAM/MUC 18 that included subsequent
nested PCR. Primer sequences and PCR conditions
are reported in detail in Supplementary Information. The resulting nested products (25 μl) were analyzed
on a 1.8% agarose gel. RNA integrity was checked Western blot analysis Spheroid growth is expressed as percentage of the volume
measured in the control spheroid at the beginning of
the treatment (day 0). Values are reported as means ±
SD of three separate experiments, each performed in
quadruplicate. At different time points after treatment, WM115
and WM266.4 cells were harvested, washed in PBS and
suspended in lysis buffer containing 50 mM Tris–HCl
(pH 7.4), 1 mM EDTA, 1 mM EGTA, 1% Triton X-100,
10 mM NaF, 1 mM Na3VO4, and protease inhibitors
(Sigma–Aldrich). After 30 minutes incubation on ice,
the samples were centrifuged at 13.000×g for 20 min
(4°C), after which the protein concentration of the
supernatant was determined using the Lowry colorimetric
assay. Proteins (50 μg) were separated on 12% SDS-
polyacrylamide gel and transferred onto an Immobilon-
PVDF Transfer Membrane (Millipore, Billerica, MA). For
immunodetection, the following primary antibodies were
used: anti-phospho-JNK (Thr183/Tyr185) (Cell Signaling,
Beverly, MA, USA), anti-JNK (Cell Signaling), anti-
phospho-ERK1&2 (Tyr185/187; Invitrogen, Camarillo,
CA), anti-ERK1&2 (Santa Cruz Biotechnology, Santa
Cruz, CA), anti-phospho-p38 (Thr180/Thr182; Cell
Signaling), anti-p38 (Cell Signaling), anti-AP2 (OriGene,
Rockville, MD, USA), anti-CD146 (OriGene), anti- MTS assays were performed using the CellTiter
Aqueous OneSolution kit (Promega Milan, Italy)
according to the manufacturer's instructions. Spheroids
viability is expressed as percentage of the absorbance
measured in the control cells. Results are presented as
means ± SD of three separate experiments, each performed
in quadruplicate. High-resolution 1H-NMR analyses of cell extracts WM115 (2.5×104 cells/cm2) and WM266.4 cells
(1.25×104 cells/cm2) were plated in 150 cm2 flasks and, 48
hours after seeding, were treated with equiactive MC3181
concentrations (0.26 and 1.3 μM for WM115, 0.20 and
1.0 μM for WM266.4). Cells were trypsinized 48 hours
after treatment, counted, and assessed for viability and
membrane integrity by trypan blue staining. Following
washes with ice-cold PBS, cell pellets were resuspended
in 0.5 ml of ice-cold twice-distilled water. Aqueous
extracts (from 10 x 106 cells/sample) were prepared in www.impactjournals.com/oncotarget Oncotarget 15534 was filled with complete medium supplemented with 20%
FBS. For the invasion assay, the transwell membranes
were previously coated with 5 μg of Matrigel (BD
Biosciences, Milan, Italy). After 48-hour incubation
(37°C; 5% CO2), cells adherent to the underside of the
filters were fixed and permeabilized with 70% ethanol,
washed with PBS, stained with 0.25% crystal violet (Serva
ELECTROPHORESIS), and counted. Four random fields
at magnification 20X (3X digital camera) were counted. Percent invasion and invasion index were calculated using
the following equations: electrophoretically, and quality of cDNA was controlled
by amplification of housekeeping genes such as β2-
microglobulin. The level of gene expression was evaluated
by densitometric analysis through the ImageJ software, and
normalized with the PCR product of the housekeeping β2-
microglobulin gene, coamplified in the same experiment. All RT-PCR experiments were performed in triplicate. Under these conditions, gross quantitative estimations of
mRNA expression could be detected. Generation and analysis of 3D multicellular
tumor spheroids Eq. (2)
Invasion
Mean of cells invading through
Matrigel coated insert membrane
Mean of cells migrating through
control insert membrane
X
%
100
=
Eq. (3)
Invasion Index
% Invasion test cells
% Invasion control cells
=
Results represent the means ± SD of three independent Spheroids were generated through the liquid-
overlay technique. Briefly, 100 μl/well of cell suspension
at optimized densities (0.5×104 cells/ml for WM115 and
0.5×103 for WM266.4), were dispensed onto 96-well flat-
bottomed plates (Corning B.V. Life Sciences), pre-coated
with 1.5% agarose (wt/vol, Serva ELECTROPHORESIS)
and incubated 4 days at 37°C, in a 5% CO2 humidified
atmosphere. Spheroids treatment was performed by
adding MC3181 and NBDHEX at a final concentration
ranging from 0.005 to 50 μM (final incubation volume,
200 μl). Fifty percent of the medium in each well was
replenished 48 hours after treatment and at day, 4, 6, 10
and 14. Spheroid size was measured up to 17 days by
phase contrast imaging (10X magnification, 3X digital
magnification) using a Nikon ECLIPSE TS100 inverted
microscope (Nikon Instruments S.p.A, Florence, Italy),
equipped with a digital camera. Images were analyzed by
the ImageJ software. The radius of each tumor spheroid
was used to calculate the volume (μm3): Eq. (3)
Invasion Index
% Invasion test cells
% Invasion control cells
= Results represent the means ± SD of three independent
experiments. In the spheroid invasion assay, tumor spheroids
of about 330 μm diameter were transferred in a 96-well
U-bottomed plate and embedded in 0.5 mg/ml of type I
collagen. After collagen solidification, 100 μl of culture
medium was added to the top. When present, MC3181
(0.04-1 μM) was added to both the type I collagen and the
overlaid medium. Images were captured with the inverted
microscope Nikon ECLIPSE TS100 after 24 and 48 hours
MC3181 incubation and analyzed through the ImageJ
software. Values are expressed as means ± SD of two
independent experiments, each performed in quintuplicate. Eq. (1) V = 4/3 π r3 Migration and invasion assay of 2D monolayer
cultures and 3D multicellular tumor spheroids Cell migration was performed using Boyden
chambers with an 8.0 μm pore size (Corning). WM266.4,
WM115 (5×104 cell/well) and SK-MEL-5 cells (7.5×104
cell/well) were suspended in FBS-free media and loaded
into the upper chamber, in the absence or presence of
increasing MC3181 concentrations. The lower chamber www.impactjournals.com/oncotarget In vivo murine melanoma lung metastasis model
and treatment A.M.C. was supported by a Consolidate the
Foundations 2015 grant; A.D.L was supported by a
fellowship from Fondazione Umberto Veronesi; D.R. was
supported by AIRC-TRIDEO 2015 (Id.17515) and PRIN
2012 (prot. 2012CTAYSY) grants. Procedures involving animals and their care were
in conformity with Institutional Guidelines (D.L. 116/92
and subsequent implementing circulars), and experimental
protocols were approved by the local Ethical Committee of
Padova University (CEASA). During in vivo experiments,
animals in all experimental groups were examined
daily for a decrease in physical activity and other signs
of disease or drug toxicity; severely ill animals were
euthanized by carbon dioxide overdose. www.impactjournals.com/oncotarget Oncotarget 15535 Romano of University of Rome ‘Tor Vergata’ for imaging
acquisition. Romano of University of Rome ‘Tor Vergata’ for imaging
acquisition. VEGF (OriGene), anti-N-cadherin (Abcam, Cambridge,
UK), anti-LC3 (Novus Biologicals, Co, USA) and anti-β
actin (Sigma-Aldrich) as loading control. Anti-rabbit or
anti-mouse secondary antibodies (Cell Signaling) were
revealed with the ECL LiteAblot Extend (EuroClone). Band intensities were measured by the ImageJ software. Data are presented as means of arbitrary units ± SD
resulting from three independent experiments. VEGF (OriGene), anti-N-cadherin (Abcam, Cambridge,
UK), anti-LC3 (Novus Biologicals, Co, USA) and anti-β
actin (Sigma-Aldrich) as loading control. Anti-rabbit or
anti-mouse secondary antibodies (Cell Signaling) were
revealed with the ECL LiteAblot Extend (EuroClone). Band intensities were measured by the ImageJ software. Data are presented as means of arbitrary units ± SD
resulting from three independent experiments. Statistical analysis Statistical analyses have been performed using the
Statistical Package for the Social Sciences Windows,
version 15.0 (SPSS, Chicago, Illinois, USA). Descriptive
statistics consisted of the mean ± SD for parameters
with gaussian distributions (Kolgomorov-Smirnov test). The equality of the variances was confirmed by the
Levene's Test. ANOVA one-way followed by Dunnett's
test was used for multiple comparison among treatment
groups (>2) and control. A P value < 0.05 was considered
statistically significant. 6. De Luca A, Rotili D, Carpanese D, Lenoci A, Calderan
L, Scimeca M, Mai A, Bonanno E, Rosato A, Geroni C,
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Predicting preferred prey of Sumatran tigers <i>Panthera tigris sumatrae</i> via spatio-temporal overlap
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Introduction I
nterspecific interactions are important aspects of com-
munity ecology, affecting the functional ecology of eco-
systems and dictating the ecological niches inhabited by
species (Begon et al., ). Such interactions can be diffi-
cult to assess, however, particularly for cryptic species such
as wild carnivores (Allen et al., ; Saggiomo et al., ). Carnivore–prey encounter rates are dependent on spatial
and temporal overlap, and high encounter rates are often in-
dicative of prey preference (Holling, ; Fortin et al., ). Data on spatio-temporal overlap of carnivores with poten-
tial prey species may thus facilitate inference of prey prefer-
ences and patterns of interspecific interactions, providing
insights into ecosystem functions that can inform effective
conservation. Camera trapping is a non-invasive method
that is increasingly being used to monitor wildlife and
provides data on species richness, behaviour, and spatio-
temporal activity (Swanson et al., ; Rich et al., ;
Allen et al., ). I The tiger Panthera tigris is categorized as Endangered
throughout its range, with four subspecies probably extinct
in the wild (Seidensticker, ; Goodrich et al., ). The Sumatran tiger Panthera tigris sumatrae is Critically
Endangered (Linkie et al., ), as are many other species
on the Indonesian island of Sumatra (O’Brien & Kinnaird,
; Pusparini et al., ). Prey abundance can have strong
effects on the abundance and population density of tigers
(Karanth et al., ; Barber-Meyer et al., ), and tiger
declines have been linked to declines of prey in Russia and
India (Miquelle et al., ; Ramakrishnan et al., ). The
prey preferences of tigers are unknown in many areas, but
such knowledge is important to inform conservation planning
and ensure sufficient prey is available in areas critical to tiger
conservation. Information about the Sumatran tiger’s diet is
limited (e.g. O’Brien et al., ; Linkie & Ridout, ), and
data on spatial and temporal activity patterns and overlap
between tigers and potential prey could improve our under-
standing of the subspecies’ prey preferences. Predicting preferred prey of Sumatran tigers
Panthera tigris sumatrae via spatio-temporal overlap M A X I M I L I A N L . A L L E N , M A R S Y A C . S I B A R A N I and M I H A Abstract Encounter rates of carnivores with prey are
dependent on spatial and temporal overlap, and are often
highest with their preferred prey. The Critically Endangered
Sumatran tiger Panthera tigris sumatrae is dependent on
prey populations, but little is known about its prey prefer-
ences. We collected camera-trap data for years (–)
in Bukit Barisan Selatan National Park, Sumatra, to investi-
gate spatial and temporal overlap of tigers with potential
prey species. We also developed a novel method to predict
predator–prey encounter rates and potential prey preferences
from camera-trap data. We documented at least individual
tigers, with an overall detection rate of .detections/
trap nights. Tigers exhibited a diurnal activity pattern and
had highest temporal overlap with wild boar Sus scrofa and
pig-tailed macaques Macaca nemestrina, but highest spatial
overlap with wild boar and sambar deer Rusa unicolor. We
created a spatial and temporal composite score and three
additional composite scores with adjustments for the spatial
overlap and preferred prey mass. Wild boars ranked highest
for all composite scores, followed by sambar deer, and both
are known as preferred tiger prey in other areas. Spatial and
temporal overlaps are often considered as separate indices,
but a composite score may facilitate better predictions of en-
counter rates and potential prey preferences. Our findings sug-
gest that prey management efforts in this area should focus
on wild boar and sambar deer, to ensure a robust prey base for
this Critically Endangered tiger population. 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.
Oryx, 2021, 55(2), 197–203 © The Author(s), 2020. Published by Cambridge University Press on behalf of Fauna & Flora International
doi:10.1017/S0030605319000577
ttps://doi.org/10.1017/S0030605319000577 Published online by Cambridge University Press Introduction Keywords Activity patterns, composite score, Panthera
tigris, prey preference, spatial overlap, Sumatra, temporal
overlap, tiger Supplementary material for this article is available at
doi.org/./S Bukit Barisan Selatan National Park is one of the largest
protected areas on the island of Sumatra, and is critical
for the conservation of the Sumatran tiger and other
species of conservation concern, including the Critically
Endangered Sumatran rhinoceros Dicerorhinus sumatren-
sis and Sumatran elephant Elephas maximus sumatranus
(O’Brien & Kinnaird, ; Pusparini et al., ). The Park
provides relatively abundant tiger habitat, but threat levels
are moderate to high because of inadequate conservation MAXIMILIAN L. ALLEN (Corresponding author,
orcid.org/0000-0001-8976-
889X) Illinois Natural History Survey, University of Illinois, 1816S. Oak
Street, Champaign, Illinois 61820, USA. E-mail maxallen@illinois.edu MARSYA C. SIBARANI Wildlife Conservation Society—Indonesia Program, Bogor,
West Java, Indonesia MIHA KROFEL (
orcid.org/0000-0002-2010-5219) Department of Forestry,
Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia Received January . Revision requested April . Accepted May . First published online March . Received January . Revision requested April . A
t d
M
Fi t
bli h d
li
M
h Accepted May . First published online March . 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-u
distribution, and reproduction in any medium, provided the original work is properly cited. Oryx, 2021, 55(2), 197–203 © The Author(s), 2020. Published by Cambridge University Press on behalf of Fauna & Flora International
doi:10.1017/S0030605319000577
https://doi.org/10.1017/S0030605319000577 Published online by Cambridge University Press M. L. Allen et al. 198 FIG. 1 The study site with camera-trap locations of both arrays
within Bukit Barisan Selatan National Park on the island of
Sumatra, Indonesia. measures (Sanderson et al., ). Given the Park’s importance
for tiger conservation, it is important to understand the spe-
cies’ ecology in this area. Previous studies of activity patterns
of Sumatran tigers in the Park produced conflicting results;
O’Brien et al. () reported tigers had a diurnal activity
pattern, whereas Pusparini et al. () reported a crepuscular
activity pattern with highest activity levels near dawn. The
tigers’ prey preferences and prey abundance in the area are
also unknown. To inform conservation efforts, we investigated tiger
spatio-temporal overlap with potential prey species, using
years of camera-trap data from an area of the Park with little
human activity. Study area Our study site is in Bukit Barisan Selatan National Park, in
the South Barisan Range ecosystem on the Indonesian is-
land of Sumatra (Fig. ). The Park is the third largest pro-
tected area (,km) on Sumatra (O’Brien & Kinnaird,
), spanning two provinces: Lampung and Bengkulu. Topography ranges from coastal plains and lowland rain-
forest at sea level in the southern peninsula of the Park
to mountains up to ,m in the central and northern
parts (Pusparini et al., ). The Park contains montane,
lowland tropical, coastal and mangrove forests. Annual
rainfall is ,–,mm, most of which falls in the mon-
soon season (November–May), and annual temperatures
are –°C (O’Brien et al., ). The Park contains a high
diversity of wildlife, with tigers and other species listed
in the CITES Appendices and categorized as Endangered
or Critically Endangered on the IUCN Red List. designed our survey to monitor multiple species effectively
(Rich et al., ). We set two arrays of camera traps,
using the Network’s protocols (TEAM Network, ), in
sites chosen for accessibility for long-term repeated surveys,
all in lowland forests at –m altitude. We placed camera
traps in each array at a density of per km(Fig. ), and
the arrays covered a total of .km. We deployed the
camera-trap arrays in the dry season (April–July) of each
year during –, with array in operation during
April–May and array during June–July, with the aim to
complete at least sampling days for each camera trap. We positioned camera traps near animal trails and/or
places used regularly by wildlife, to maximize detections. We placed camera traps –cm off the ground, with no
refractory period between images. Introduction Our objectives were to () document the
minimum number of individual tigers in the area, () deter-
mine the temporal and spatial overlap of tigers with six po-
tential prey species, and () create a composite score from the
indices of temporal and spatial overlap as a novel method to
predict predator–prey encounter rates and determine poten-
tial prey preference. In our analyses we included all potential
tiger prey species present in the study area, based on a review
of tiger dietary studies (Hayward et al., ). In line with
known prey preferences of tigers across their range, we ex-
pected sambar deer Rusa unicolor and wild boar Sus scrofa
to have the highest composite spatio-temporal score. FIG. 1 The study site with camera-trap locations of both arrays
within Bukit Barisan Selatan National Park on the island of
Sumatra, Indonesia. Methods To avoid pseudo-replication, we considered consecutive
photo captures of the same species as independent events
only if they occurred after an interval of . min
(Rovero & Zimmermann, ). We calculated the number
of independent captures for each species, but combined
both mouse deer species (greater mouse deer Tragulus
napu and lesser mouse deer Tragulus kanchil) in one cat-
egory because they share similar characteristics as potential 2), 197–203 © The Author(s), 2020. Published by Cambridge University Press on behalf of Fauna & Flora International
doi:10.1017/S003060531900 Camera trapping We expected that this simple composite score could be
improved by giving additional weight to some variables or
including other variables in the score. We therefore calcu-
lated three additional composite scores to determine how
different weighting of overlap scores or the inclusion of ad-
ditional variables affect the preference rankings of potential
prey. p
g
p
g
We used kernel density estimation to determine activ-
ity patterns and quantify overlap among species (Ridout &
Linkie, ). We reviewed potential prey species for tigers
(Hayward et al., ) and analysed those in our study area
with . detection events, which included the greater and
lesser mouse deer (n = ), Malay tapir Tapirus indicus
(n = ), pig-tailed macaque Macaca nemestrina (n = ),
red muntjac Muntiacus muntjac (n = ), sambar deer
(n = ), and wild boar (n = ). We first converted the
time of each event into a radians measurement for analysis. We then used the overlap package (Meredith & Ridout, )
in R ..(R Core Team, ) to fit the data to a circular
kernel density, and estimated the activity level at each time
period from the distribution of the kernel density using a
Δoverlap value based on our sample sizes. We used the
overlapEst function to estimate the degree of overlap in activ-
ity patterns between tigers and the potential prey species. We
calculated confidence intervals (CI) by bootstrapping ,
estimates of activity for each species, and then using the
bootEst and bootCI functions to estimate the % CI for the
overlap between tigers and each potential prey species. Firstly, we assigned more weight to the spatial overlap
value, because spatial overlap with prey is an important as-
pect of niche selection and resource partitioning for carnivores
(Schoener, ; du Preez et al., ) and may better reflect
prey species being sought out, compared to temporal overlap. We calculated the spatial adjusted composite score as: spatial adjusted
composite score = (spatial overlap × 0.6) +
(temporal overlap × 0.4) Secondly, we considered a composite score that also in-
cluded prey mass, with a higher mass adjustment value (spa-
tial and temporal composite score × .) for prey within the
preferred size range of tigers (–kg; Hayward et al.,
) and a lower value (spatial and temporal composite
score × .) for potential prey outside this range. Camera trapping We set camera traps in the Park as part of the Tropical
Ecology and Assessment Monitoring Network, which col-
lects long-term biodiversity data in tropical environments
globally to guide conservation actions. Our goal was to
monitor the terrestrial vertebrate community, and we Oryx, 2021, 55(2), 197–203 © The Author(s), 2020. Published by Cambridge University Press on behalf of Fauna & Flora International
doi:10.1017/S0030605319000577 https://doi.org/10.1017/S0030605319000577 Published online by Cambridge University Press Preferred prey of Sumatran tigers 199 tiger prey and it was difficult to distinguish between them on
camera-trap images (O’Brien et al., ). potential prey species. The upper right quadrant of the plot
(high spatial and temporal overlap) indicates the most en-
countered and potentially most preferred prey species, where-
as the upper left (high temporal but low spatial overlap) and
lower right (low temporal but high spatial overlap) quadrants
would indicate potential alternative prey that were encoun-
tered opportunistically in space or time. The lower left quad-
rant (low spatial and temporal overlap) would indicate species
rarely encountered and probably not preferred. We used a relative abundance index as a proxy for tiger
abundance, because this is a more accurate proxy for
abundance than occupancy values (Parsons et al., ). We calculated the index as: relative
abundance index = (detection events/trap nights) × 100 relative
abundance index = (detection events/trap nights) × 100 Finally, we calculated the mean of the spatial and tem-
poral overlap values for each prey species to create a spa-
tial and temporal composite score (Song et al., ). This
allowed us to rank the potential prey species, with higher
scores indicating higher encounter rate and potentially high-
er preference. for each camera trap, to determine detection events per
trap nights (e.g. Allen et al., ), and averaged the values
from all camera traps to determine an overall mean for the
study area. We used the stripe patterns of individual tigers to
identify the minimum number of individuals, separately for
photographs of the right and left flanks. for each camera trap, to determine detection events per
trap nights (e.g. Allen et al., ), and averaged the values
from all camera traps to determine an overall mean for the
study area. We used the stripe patterns of individual tigers to
identify the minimum number of individuals, separately for
photographs of the right and left flanks. https://doi.org/10.1017/S0030605319000577 Published online by Cambridge University Press Oryx, 2021, 55(2), 197–203 © The Author(s), 2020. Published by Cambridge University Press on behalf of Fauna & Flora International
doi:10.1017/S0030605319000577 https://doi.org/10.1017/S0030605319000577 Published online by Cambridge University Press Camera trapping We ob-
tained prey mass values from Nowak () and used %
of the mean weight of adult females to account for young
animals being eaten (Hayward et al., ). We then calcu-
lated the mass adjusted composite score as: To determine spatial overlap with potential prey species
we used the methods of Ngoprasert et al. (). We first cal-
culated the relative abundance index for each prey species,
as for tigers, and then scaled the index for each prey spe-
cies at each camera-trap site to continuous probability values
of –(Ngoprasert et al., ). We then created a logistic
regression for each prey species using data from each
camera-trap location. In the logistic regression we used
tiger presence as the dependent variable and prey probabil-
ity values as the independent variable. We then compared
spatial overlap of prey species using the area under the
curve (AUC) of receiver operating characteristic plots
(Fielding & Bell, ), and quantified the spatial overlap
of tigers with individual prey species as the AUC values,
which range from .(random) to .(perfect fit). mass adjusted
composite score = (spatial overlap × temporal overlap) ×
mass adjustment Thirdly, we calculated a spatial and mass adjusted com-
posite score as: spatial and mass
adjusted composite score =
((spatial overlap × 0.6) +
(temporal overlap × 0.4)) ×
mass adjustment spatial and mass adjusted composite score We then ranked potential prey species based on each
composite score, with higher scores indicating higher en-
counter rate and potentially higher preference. To determine which prey species may be preferred we
plotted the spatial and temporal overlap of tigers with each https://doi.org/10.1017/S0030605319000577 Published online by Cambridge University Press 30605319000577 Published online by Cambridge University Press M. L. Allen et al. 200 TABLE 1 The indices of potential prey species of tigers Panthera tigris sumatrae in Bukit Barisan Selatan National Park, Sumatra, including
relative abundance (detection events/trap nights), temporal overlap, spatial overlap, and composite scores. Higher composite scores
indicate greater encounter rates and potential prey preference. Species are listed in order of their spatial and temporal composite score. Results We had camera traps operating during –for a
total of ,trap nights. We obtained ,photographs
of species, including mammals. We documented all six
potential tiger prey species in all years of the study. We
captured photographs of people times in study years
(= , = , = , = ), and one domestic
dog in . We recorded a total of tiger captures (= , = ,
= , = , = , = , = ), with an overall
relative abundance of .± SE .detections/trap nights
per camera trap. We identified at least individual tigers in
left flank photographs and eight individuals in right flank
photographs (Supplementary Material ). Camera trapping Composite scores
Species
Relative
abundance
Temporal
overlap
Spatial
overlap
Spatial &
temporal
Spatial
adjusted
Prey mass
adjusted
Spatial & prey
mass adjusted
Wild boar Sus scrofa
3.15
0.80
0.71
0.76
0.77
0.83
0.84
Sambar Rusa unicolor
1.02
0.70
0.66
0.68
0.67
0.75
0.74
Pig-tailed macaque
Macaca nemestrina
4.32
0.76
0.60
0.68
0.66
0.61
0.60
Red muntjac
Muntiacus muntjac
7.10
0.68
0.57
0.63
0.62
0.56
0.56
Mouse deer1
3.39
0.62
0.53
0.58
0.57
0.52
0.51
Tapir Tapirus indicus
0.85
0.43
0.52
0.48
0.48
0.52
0.53
Greater mouse deer Tragulus napu and lesser mouse deer Tragulus kanchil. Greater mouse deer Tragulus napu and lesser mouse deer Tragulus kanchil. score, the prey mass adjusted score and the spatial and
prey mass adjusted score, with scores –% higher than
the next species (Table ). As for the spatial and tempo-
ral composite score, sambar and pig-tailed macaques also
ranked second and third, respectively, for these additional
composite scores. The spatial adjusted composite scores
for sambar and pig-tailed macaque were similar (sambar
scored .% higher than pig-tailed macaque), but the differ-
ence was greater for the prey mass adjusted score and the
spatial and prey mass adjusted score (sambar scored %
higher for both of these scores). Discussion The tiger is an important flagship species for conservation,
but remains threatened throughout its range (Seidensticker,
; Walston et al., ; Sibarani et al., ). Sumatran
tigers are categorized as Critically Endangered (Linkie et al.,
) and tiger populations in Bukit Barisan Selatan Na-
tional Park and other areas are threatened by encroach-
ment and habitat destruction (O’Brien & Kinnaird, ;
Pusparini et al., ), and by poaching of tigers and/or
their prey (Linkie et al., , ). Effective conservation
is dependent on collaboration between countries, govern-
ment agencies, local communities, and scientific organiza-
tions. The Tropical Ecology and Assessment Monitoring
Network is focused on open sharing of scientific data and
can be used as a model for data sharing amongst scientists
and other stakeholders for conservation. We documented ,captures of potential prey species,
with red muntjacs being recorded most frequently, followed
by pig-tailed macaques and mouse deer (Table ). Tigers
exhibited a diurnal activity pattern (Fig. ) and had the high-
est temporal overlap with wild boar, followed by pig-tailed
macaques and sambar (Fig. , Table ). The highest spatial
overlap was also with wild boar, followed by sambar and
pig-tailed macaques (Table ). When plotting the values indicating spatial and temporal
overlap of tigers with potential prey species, wild boar and
sambar deer fell in the upper right quadrant, suggesting they
are potentially preferred prey. Tapirs were in the lower left
quadrant, indicating they were probably not preferred, and
the other prey species were in the upper left (high temporal
but low spatial overlap), indicating potential alternative prey
(Fig. ). We found tigers exhibited diurnal activity patterns, and
we created a composite score of spatial and temporal overlap
with potential prey species to provide insights into tiger prey
preferences, which can inform conservation (e.g. Karanth
et al., ; Barber-Meyer et al., ). Previous studies in
the study area suggested tigers have a diurnal activity pat-
tern (O’Brien et al., ), or a crepuscular pattern with
highest activity near dawn (Pusparini et al., ). The g
Tigers had the greatest spatio-temporal overlap with wild
boar, with a spatial and temporal composite score of .,
which is % higher than the species with the second highest
scores (sambar and pig-tailed macaques, both .; Table ). The additional composite scores produced a similar
ranking to the spatial and temporal composite score. Oryx, 2021, 55(2), 197–203 © The Author(s), 2020. Published by Cambridge University Press on behalf of Fauna & Flora International
doi:10.1017/S0030605319000577 https://doi.org/10.1017/S0030605319000577 Published online by Cambridge University Press Discussion FIG. 2 The temporal activity
(including % confidence
intervals) and overlap of the
kernel activity density of tigers
and potential prey species:
(a) mouse deer (including
greater mouse deer Tragulus
napu and lesser mouse deer
Tragulus kanchil), (b) pig-
tailed macaque Macaca
nemestrina, (c) red muntjac
Muntiacus muntjac, (d) sambar
deer Rusa unicolor, (e) tapir
Tapirus indicus, (f) wild boar
Sus scrofa. Tiger activity is
represented as solid lines and
prey activity as dotted lines,
with their temporal overlap
shown as the shaded area. FIG. 2 The temporal activity
(including % confidence
intervals) and overlap of the
kernel activity density of tigers
and potential prey species:
(a) mouse deer (including
greater mouse deer Tragulus
napu and lesser mouse deer
Tragulus kanchil), (b) pig-
tailed macaque Macaca
nemestrina, (c) red muntjac
Muntiacus muntjac, (d) sambar
deer Rusa unicolor, (e) tapir
Tapirus indicus, (f) wild boar
Sus scrofa. Tiger activity is
represented as solid lines and
prey activity as dotted lines,
with their temporal overlap
shown as the shaded area. behaviour, or varying degrees of interactions with humans
or other species in different parts of the Park. For example,
Pusparini et al. () reported high rates of illegal human
activity (photographic captures of humans with guns) and
relative tiger abundances (relative abundance index = .)
that were an order of magnitude higher than in our study
(relative abundance index = .). This may have caused ti-
gers to change their activity patterns to avoid threats posed
by humans (e.g. Clinchy et al., ). Further research is re-
quired to ascertain reasons for these conflicting results from
the same Park and subpopulation. A high degree of spatio-temporal overlap does not
necessarily indicate prey preference but it suggests po-
tential for high encounter rates between carnivores and
their prey, which is a key component of prey preference
(Holling, ; Fortin et al., ). Temporal overlap has
been posited as a way of determining prey preferences
(Linkie & Ridout, ), but probably provides an incom-
plete picture if spatial overlap is not included (e.g. O’Brien
et al., ). Other factors to be considered include prey
body size and potential avoidance strategies by prey species. We created four composite index scores that included both
temporal and spatial overlap. Oryx, 2021, 55(2), 197–203 © The Author(s), 2020. Published by Cambridge University Press on behalf of Fauna & Flora International
doi:10.1017/S0030605319000577 Discussion Wild
boar ranked highest for the spatial adjusted composite Tigers had the greatest spatio-temporal overlap with wild
boar, with a spatial and temporal composite score of .,
which is % higher than the species with the second highest
scores (sambar and pig-tailed macaques, both .; Table ). The additional composite scores produced a similar
ranking to the spatial and temporal composite score. Wild
boar ranked highest for the spatial adjusted composite https://doi.org/10.1017/S0030605319000577 Published online by Cambridge University Press Preferred prey of Sumatran tigers 201 behaviour, or varying degrees of interactions with human
or other species in different parts of the Park. For example
Pusparini et al. () reported high rates of illegal human
activity (photographic captures of humans with guns) and
relative tiger abundances (relative abundance index = .
that were an order of magnitude higher than in our study
(relative abundance index = .). This may have caused ti
gers to change their activity patterns to avoid threats posed
by humans (e.g. Clinchy et al., ). Further research is re
quired to ascertain reasons for these conflicting results from
the same Park and subpopulation. A high degree of spatio-temporal overlap does no
necessarily indicate prey preference but it suggests po
tential for high encounter rates between carnivores and
their prey, which is a key component of prey preference
(Holling, ; Fortin et al., ). Temporal overlap ha
been posited as a way of determining prey preference
(Linkie & Ridout, ), but probably provides an incom
plete picture if spatial overlap is not included (e.g. O’Brien
et al., ). Other factors to be considered include prey
body size and potential avoidance strategies by prey species
We created four composite index scores that included both
FIG. 2 The temporal activity
(including % confidence
intervals) and overlap of the
kernel activity density of tigers
and potential prey species:
(a) mouse deer (including
greater mouse deer Tragulus
napu and lesser mouse deer
Tragulus kanchil), (b) pig-
tailed macaque Macaca
nemestrina, (c) red muntjac
Muntiacus muntjac, (d) samba
deer Rusa unicolor, (e) tapir
Tapirus indicus, (f) wild boar
Sus scrofa. Tiger activity is
represented as solid lines and
prey activity as dotted lines,
with their temporal overlap
shown as the shaded area. FIG. 3 The spatial (area under curve; AUC) and temporal (kernel
density) overlap of tigers with potential prey species plotted
together (with axes scaled to the reported values for ease of
comparison). Discussion Each composite score appears
to accurately rank prey preference of tigers in Bukit Barisan
Selatan National Park, with wild boar and sambar deer
being the most preferred, as predicted based on the find-
ings of Hayward et al. (). The spatial and temporal FIG. 3 The spatial (area under curve; AUC) and temporal (kernel
density) overlap of tigers with potential prey species plotted
together (with axes scaled to the reported values for ease of
comparison). sample size of tiger captures in our study was lower than in
both previous studies, but our findings appear to confirm
the diurnal activity pattern observed by O’Brien et al. (). Reasons for the observed differences could be
different sampling techniques, variation in individual tiger https://doi.org/10.1017/S0030605319000577 Published online by Cambridge University Press M. L. Allen et al. 202 should be conducted in other systems with known prey pre-
ferences of carnivores (e.g. from dietary analyses) to further
evaluate the accuracy of this method, assess general applic-
ability of the method, and further interpret the observed
relationships. composite score was effective, but the scores including pre-
ferred prey mass better separated sambar and pig-tailed
macaques as potential prey species for tigers. We used small
(%) adjustments to create the adjusted composite scores,
and future studies in areas with known prey preferences
could conduct sensitivity analyses to determine ideal weight-
ing adjustments for spatial and temporal composite scores
to determine prey preference. Acknowledgements All data used in this study were collected by
the Tropical Ecology Assessment and Monitoring Network, a collab-
oration between Conservation International, the Missouri Botanical
Garden, the Smithsonian Institution and the Wildlife Conservation
Society. The work was partially funded by these institutions, the
Gordon and Betty Moore Foundation, the Illinois Natural History
Survey, the Slovenian Research Agency (P4-0059), and other donors. Monitoring activities were managed by the Wildlife Conservation
Society in collaboration with the Bukit Barisan Selatan National Park
and the Ministry of Environment and Forestry, Republic of Indonesia. We thank all field staff and forest rangers involved in camera-trap
deployment, and W. Marthy for help in the field and coordination. y
The different composite scores indicated mostly the same
ranking amongst potential prey species, with the only excep-
tion being tapirs ranking slightly higher than mouse deer in
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LOVERIDGE, A.J. Conflicts of interest None. Ethical standards This research abided by the Oryx guidelines on
ethical standards. The research was based on passive monitoring,
and did not involve human subjects, experimentation with animals
and/or collection of specimens. Discussion Wild boar
ranked highest in all cases, followed by sambar deer, and
these two species are frequently the preferred prey of tigers
throughout their range (Seidensticker & McDougal, ;
Hayward et al., ; Basak et al., ). The greater overlap
and composite scores for wild boar in this study site could
be a result of wild boar having a relative abundance
c. three times greater than sambar deer. Species with higher
abundance are probably more widely distributed across
the landscape, which could inflate their spatial overlap
with predators and potentially overestimate prey preference. Red muntjac, pig-tailed macaque and mouse deer were
indicated as potential alternative prey species with inter-
mediate composite index scores and a position in the
upper left quadrant on the spatial and temporal overlap
plot (Fig. ), which indicates high temporal but low spatial
overlap with tigers. Tapirs had low spatial and temporal
overlap with tigers, which corresponds to published data
and suggests they may be non-preferred prey of tigers
(Hayward et al., ). Based on these results we suggest
that conservation efforts in the area should be focused on
wild boar and sambar deer, to ensure a robust prey base
for this tiger population. We found evidence of illegal snares
set for sambar deer in the Park, suggesting conservation
actions may be necessary. Author contributions Study concept: all authors; data collection:
MCS; statistical analyses: MLA; writing: MLA; revisions: all authors. https://doi.org/10.1017/S0030605319000577 Published online by Cambridge University Press Oryx, 2021, 55(2), 197–203 © The Author(s), 2020. Published by Cambridge University Press on behalf of Fauna & Flora International
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https://openalex.org/W4291020122
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https://researchonline.lshtm.ac.uk/id/eprint/4669583/1/Hollingworth-etal-2022-Antihypertensive-medicine-use-differs-between-Ghana-and-Nigeria.pdf
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Antihypertensive medicine use differs between Ghana and Nigeria
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BMC cardiovascular disorders
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© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Non-communicable diseases are a growing burden in many African countries; cardiovascular disease is
the main disease. Antihypertensive medicines (AHM) are a common treatment option but we know little about com-
munity use in most low- and medium-income countries (LMIC). We aimed to describe the use of antihypertensive
medicines (AHM) in Ghana and Nigeria using a novel data source. Methods: We used data from mPharma—a health and pharmaceutical company which distributes pharmaceuticals
to hospital and retail pharmacies. We extracted data using the anatomical therapeutic chemical (ATC) classification
codes and calculated use in defined daily doses and explored patterns by class, medicines, dose, and originator or
generic product. Results: AHM use differed between Ghana and Nigeria. The most used classes in Ghana were angiotensin receptor
blockers (ARB) followed by calcium channel blockers (CCB) and angiotensin-converting-enzyme inhibitors (ACEi). The
five most used products were 16 mg candesartan, 30 mg nifedipine, 10 mg lisinopril, 5 mg amlodipine and 50 mg
losartan. In Nigeria ARB, CCB and diuretics were widely used; the top five products were 50 mg losartan, 10 mg lisino-
pril, 30 mg nifedipine, 40 mg furosemide, and 5 mg amlodipine. More originator products were used in Ghana than
Nigeria. Conclusion: The differences between Ghana and Nigeria may result from a combination of medical, contextual and
policy evidence and reflect factors related to clinical guidance (e.g. standard treatment guidelines), accessibility to
prescribers and the role of community pharmacies, and structure of the health system and universal health coverage
including funding for medicines. We show the feasibility of using novel data sources to gain insights on medicines use
in the community. Keywords: Antihypertensive medicines, Hypertension, Ghana, Nigeria, Pharmacoepidemiology, (PubMed MESH
terms) Keywords: Antihypertensive medicines, Hypertension, Ghana, Nigeria, Pharmacoepidemiology, (
terms) ntihypertensive medicines, Hypertension, Ghana, Nigeria, Pharmacoepidemiology, (PubMed MESH diminished quality of life, and poor social development
in both high- and low-resourced countries [1, 2]. In
the latter, NCDs are growing swiftly; 41 million people
die each year (i.e. 71% of all deaths globally). Each year,
more than 15 million people aged between 30 and 69 die
from a NCD where 85% of these so-called ‘premature’
deaths occur in low-resourced countries [3]. Four NCDs
account for most (> 80%) premature NCD deaths: cardio-
vascular diseases (CVD 17·9 million deaths annually) is Antihypertensive medicine use differs
between Ghana and Nigeria Samantha A. Hollingworth1,2* , Daniel Ankrah3 , Benjamin S. C. Uzochukwu4 , Chinyere C. Okeke4 ,
Francis Ruiz5 and Emily Thacher6 Background Non-communicable diseases (NCD) are the leading
public health challenges globally in the twenty-first cen-
tury resulting in ill health and death, economic loss, *Correspondence: s.hollingworth@uq.edu.au 1 School of Pharmacy, The University of Queensland, 20 Cornwall St,
Woolloongabba, QLD 4102, Australia
Full list of author information is available at the end of the article 1 School of Pharmacy, The University of Queensland, 20 Cornwall St,
Woolloongabba, QLD 4102, Australia
Full list of author information is available at the end of the article Hollingworth et al. BMC Cardiovascular Disorders (2022) 22:368
https://doi.org/10.1186/s12872-022-02799-z Data analysis
Th d The data were extracted based on the Anatomical Ther-
apeutic Chemical (ATC) classification codes [18] of
antihypertensive medicines. These included antihyper-
tensives (C02), diuretics (C03), peripheral vasodilators
(C04), vasoprotectives (C05) beta blocking agents (C07),
calcium channel blockers (C08) and agents acting on the
renin-angiotensin system (C09). Each product was noted
for its medicine class (e.g. a calcium channel blockers)
medicine (e.g. amlodipine) and dose formulation (e.g. 5 mg tablets). The medicine classes included: centrally-
acting adrenergic agent (AAC); peripherally-acting adr-
energic agent (AAP); alpha adrenergic blocker (AB);
angiotensin converting enzyme inhibitor (ACEi); angio-
tensin receptor blocker (ARB); beta blocker (BB); calcium
channel blocker (CCB); diuretic (DU); and statin (ST in
combination products). We excluded the use of hydrala-
zine because it is not used as a first line medicine and
only used for hypertensive emergencies where labetalol
is contraindicated. We extracted data on sales volume for
each month between 1 January 2016 to 31 October 2020. Africa represents almost half of all people with
high blood pressure in the world (46%) exceeding the
proportion of deaths in low-resourced countries (40%)
[7]. The overall prevalence of hypertension in Nige-
ria ranges from 8 to 46% depending on the study target
population, type of measurement, and threshold value for
defining hypertension [8, 9]. The prevalence is similar in
men and women (7.9–50.2% vs. 3.5–68.8%, respectively)
and in the urban (8.1–42.0%) and rural setting (13.5–
46.4%). The pooled prevalence increased from 8.6% in
the only study in the 1970s (1970–1979) to 22.5% (2000–
2011) [9]. It also varies across the geo-political zones [10]. In Ghana the pooled prevalence of hypertension from a
recent meta-analysis was 27.0% (95% CI 24.0–30.0%); it
was double in the southern coastal (28%, 95% CI 24.0–
31.0%) and middle geo-ecological areas (29%, 95% CI
25.0–33.0%) compared to the northern areas (13%, 95%
CI 7.0–21.0%) [11]. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hollingworth et al. BMC Cardiovascular Disorders (2022) 22:368 Hollingworth et al. BMC Cardiovascular Disorders Page 2 of 9 medicines. This study was granted exemption from ethics
review by the University of Queensland, Australia (Ref no
2020000453, 4 March 2020). The research uses only exist-
ing collections of data that contain only non-identifiable
data and is of negligible risk. The parties (SH, University
of Queensland) and mPharma (ET) signed a non-disclo-
sure agreement due to the commercial nature of the data. Only mPharma employees had access to the raw data,
while SH analysed the de-identifiable data extracted at
aggregated level. double that of the next group with most deaths i.e. can-
cers (9·0 million) with substantial burdens from respira-
tory diseases (3·9 million), and diabetes (1·6 million) [4]. Hypertension—untreated or uncontrolled—is the sin-
gle largest contributor to CVD causing stroke, heart
failure, and coronary artery disease; it is also a major con-
tributor to kidney disease [5]. Most guidelines recommend that hypertension is diag-
nosed when a person’s systolic blood pressure (SBP) in
the office or clinic is ≥ 140 mm Hg and or their diastolic
blood pressure (DBP) is ≥ 90 mm Hg following repeated
examination [6]. Results We used data from mPharma (https://mpharma.com/)—
a health and pharmaceutical company based in Accra,
Ghana. They distribute pharmaceuticals to hospitals and
retail pharmacies and have aggregated data on distributed Data analysis
Th d For countries with constrained health budgets, every
effort needs to be made to reduce patients’ reliance on
costly medical treatments, including for NCDs, for which
medicines are often the mainstay of treatment to reduce
associated morbidity and premature mortality [12–14]
The two main treatment approaches for hypertension are
lifestyle modifications (more fruits and vegetables, less
fatty food, less salt, more exercise exercise) and antihy-
pertensive medicines (AHM) comprising several thera-
peutic groups.h To standardise the amount of drug dispensed, we used
the defined daily dose (DDD) metric from the WHO Col-
laborating Centre for Drug Statistics Methodology [18]. This DDD is the average daily maintenance dose for a
typical 70 kg adult when used for its main indication. The DDD is useful for comparing medicines use across
patient populations and accounts for dose formulation
(e.g. tablets and capsules) and quantity within a regular
pack (e.g. 28 tablets). We calculated the DDD use using
a modified formula: [N (dispensed prescriptions) × M
(mass of dose) × Q (quantity of pack size)]/DDD. There are several studies of hypertension in Ghana [15]
and Nigeria but few on the use of AHM; most are con-
fined to hospital audits [16], with one considering com-
munity use [17]. We aimed to describe the community
use of antihypertensive medicines in Ghana and Nigeria
using a novel data source. We examined the data by country (Ghana and Nige-
ria), medicine class (e.g. calcium channel blockers, CCB),
medicine (e.g. nifedipine), dose (e.g. 5 mg), and product
type (originator brand or generic). We descriptively ana-
lysed the data using Microsoft Office Excel for Office 365. Use by medicine
Th
fi The five most used medicines in Ghana were cande-
sartan, nifedipine, lisinopril, amlodipine, and losartan
accounting for 79% of the top ten medicines (Table 2). The five most used medicines in Nigeria were losar-
tan, lisinopril, nifedipine, furosemide, and amlodipine
accounting for 74% of the top ten medicines (Table 2). The use of combination-class products was over
tenfold higher in Nigeria (7.7%) than Ghana (0.6%,
Table 1). There were only two combination classes
used in Ghana: a CCB plus a statin (94.4% of all com-
bination use) and BB plus diuretic (remaining 5.6% of
all combination use). In Nigeria, combination prod-
ucts accounted for 7.7% of all class products used; one
class group—a double diuretics combination i.e. ami-
loride plus hydrochlorothiazide—accounted for four
fifths (80.9%) of all combination use. There was some
use of a CCB + ARB + diuretic (10.2% of combination
use; amlodipine plus valsartan plus hydrochlorothi-
azide), CCB + ARB (4.6 of all combination use; amlodi-
pine plus telmisartan), and BB plus a diuretic (3.9%
of all combination use; atenolol plus chlorthalidone,
Table 1). The least used combination products included The most widely used ARB in Ghana was candesartan
(68.6 of all ARB) followed by losartan (24.3% of all ARB). Conversely, in Nigeria, losartan accounted for more
than half of use (55.1% of all ARB) followed by valsartan
(25.5% of all ARB) and telmisartan (15.7% of all ARB). Of
the CCB used in Ghana, nifedipine was the most widely
used (51.7% of all CCB) followed by amlodipine (32.8%)
and felodipine (15.6%, Table 2). In Nigeria two CCBs
were used almost exclusively: nifedipine (50.8% of all
CCB) and amlodipine (49.1%, Table 2). In both Ghana
and Nigeria the most commonly-used ACEi was lisino-
pril (99.4% of all ACEi in Ghana, 81.3% in Nigeria) and
ramipril (0.6% of all ACEi in Ghana, 17.3% in Nigeria,
Table 2).hf The use of diuretics was substantially different across
the two countries. The preferred medicines in Ghana
were indapamide (90.4% of all DU), bendroflumethiazide
(6.9% of all DU) and furosemide (2.7% of all DU, Table 2)
whereas in Nigeria, the preferred DUs were furosemide
(61.2% of all DU), hydrochlorothiazide (22.8% of all DU),
and indapamide (11.4% of all DU, Table 2). Use by class In Ghana, single-medicine products
were preferred (99.4% of total) than Nigeria (92.3% of
total). Three single-class groups accounted for 84.7% of
all use in Ghana (ARB, CCB and ACEi) and 68.0% of all
use in Nigeria (ARB, CCB, DU). (39.2%) followed by CCB (32.1%), and ACEi (13.4%,
Table 1). There was low use of diuretics (7.5%) and BB
(6.7%). In Nigeria, however, the most used classes were
ARB (26.7%), CCB (22.8%), diuretics (18.5%) and ACEi
(14.6%, Table 1). In Ghana, single-medicine products
were preferred (99.4% of total) than Nigeria (92.3% of
total). Three single-class groups accounted for 84.7% of
all use in Ghana (ARB, CCB and ACEi) and 68.0% of all
use in Nigeria (ARB, CCB, DU).h Use by class Three single-class groups accounted for 84.7% of
all use in Ghana (ARB, CCB and ACEi) and 68.0% of all
use in Nigeria (ARB, CCB, DU). The use of combination-class products was over
tenfold higher in Nigeria (7.7%) than Ghana (0.6%,
Table 1). There were only two combination classes
used in Ghana: a CCB plus a statin (94.4% of all com-
bination use) and BB plus diuretic (remaining 5.6% of
all combination use). In Nigeria, combination prod-
ucts accounted for 7.7% of all class products used; one
class group—a double diuretics combination i.e. ami-
loride plus hydrochlorothiazide—accounted for four
fifths (80.9%) of all combination use. There was some
use of a CCB + ARB + diuretic (10.2% of combination
use; amlodipine plus valsartan plus hydrochlorothi-
azide), CCB + ARB (4.6 of all combination use; amlodi-
pine plus telmisartan), and BB plus a diuretic (3.9%
of all combination use; atenolol plus chlorthalidone,
Table 1). The least used combination products included
Table 1 Use of antihypertensive medicines by class (defined
daily dose [DDD] and proportion [%]) for single and combination
products in Ghana and Nigeria
ARB Angiotensin receptor blockers, CCB calcium channel blocker, ACEI
angiotensin-converting enzyme inhibitor, BB beta-blocker, DU Diuretic, AAP,
AAC and ST are not conventional abbreviations. AAC will involve centrally acting
drugs like methyldopa
Ghana
Nigeria
Use (DDD)
%
Use (DDD)
%
Single
ARB
380,029
39.2
940,546
26.7
CCB
310,645
32.1
803,845
22.8
ACEi
129,783
13.4
514,422
14.6
DU
72,272
7.5
651,408
18.5
BB
65,393
6.7
211,322
6.0
AAC
4,528
0.5
107,321
3.0
AAP
–
0
23,466
0.7
Total
962,650
99.4
3,252,329
92.3
Combination
DU + DU
0
218,619
6.2
BB + DU
342
0.0
10,421
0.3
CCB + ARB
0
12,360
0.4
CCB + ARB + DU
0
27,665
1.3
CCB + ST
5813
0.6
1290
0.0
ACEi + ARB
2
0.0
Total
6155
0.6
270,357
7.7 Page 3 of 9 a CCB + statin (amlodipine plus atorvastatin), and
ACEi + ARB (ramipril + felodipine, Table 1). a CCB + statin (amlodipine plus atorvastatin), and
ACEi + ARB (ramipril + felodipine, Table 1). a CCB + statin (amlodipine plus atorvastatin), and
ACEi + ARB (ramipril + felodipine, Table 1). (39.2%) followed by CCB (32.1%), and ACEi (13.4%,
Table 1). There was low use of diuretics (7.5%) and BB
(6.7%). In Nigeria, however, the most used classes were
ARB (26.7%), CCB (22.8%), diuretics (18.5%) and ACEi
(14.6%, Table 1). Use by medicine
Th
fi In Ghana,
three medicines accounted for most BB use: carvedilol
(44.8% of all BB), atenolol (41.0% of all BB); and metopro-
lol (12.1% of all BB) but the pattern was different in Nige-
ria where the most widely used BBs were atenolol (57.5%
of all BB), metoprolol (19.3% of all BB), and carvedilol
(10.6% of all BB, Table 2). Table 1 Use of antihypertensive medicines by class (defined
daily dose [DDD] and proportion [%]) for single and combination
products in Ghana and Nigeria
ARB Angiotensin receptor blockers, CCB calcium channel blocker, ACEI
angiotensin-converting enzyme inhibitor, BB beta-blocker, DU Diuretic, AAP,
AAC and ST are not conventional abbreviations. AAC will involve centrally acting
drugs like methyldopa
Ghana
Nigeria
Use (DDD)
%
Use (DDD)
%
Single
ARB
380,029
39.2
940,546
26.7
CCB
310,645
32.1
803,845
22.8
ACEi
129,783
13.4
514,422
14.6
DU
72,272
7.5
651,408
18.5
BB
65,393
6.7
211,322
6.0
AAC
4,528
0.5
107,321
3.0
AAP
–
0
23,466
0.7
Total
962,650
99.4
3,252,329
92.3
Combination
DU + DU
0
218,619
6.2
BB + DU
342
0.0
10,421
0.3
CCB + ARB
0
12,360
0.4
CCB + ARB + DU
0
27,665
1.3
CCB + ST
5813
0.6
1290
0.0
ACEi + ARB
2
0.0
Total
6155
0.6
270,357
7.7 Table 1 Use of antihypertensive medicines by class (defined
daily dose [DDD] and proportion [%]) for single and combination
products in Ghana and Nigeria Use by class AHM use differed between Ghana and Nigeria. The
most used single-medicine classes in Ghana were ARB Hollingworth et al. BMC Cardiovascular Disorders (2
(39.2%) followed by CCB (32.1%), and A
Table 1). There was low use of diuretics (
(6.7%). In Nigeria, however, the most used
ARB (26.7%), CCB (22.8%), diuretics (18.5
(14.6%, Table 1). In Ghana, single-medic
were preferred (99.4% of total) than Nige
total). Three single-class groups accounted
all use in Ghana (ARB, CCB and ACEi) an
use in Nigeria (ARB, CCB, DU). The use of combination-class produc
tenfold higher in Nigeria (7.7%) than G
Table 1). There were only two combin
used in Ghana: a CCB plus a statin (94.4%
bination use) and BB plus diuretic (rema
all combination use). In Nigeria, combi
ucts accounted for 7.7% of all class produ
class group—a double diuretics combina
loride plus hydrochlorothiazide—accoun
fifths (80.9%) of all combination use. The
use of a CCB + ARB + diuretic (10.2% of
use; amlodipine plus valsartan plus hy
azide), CCB + ARB (4.6 of all combination
pine plus telmisartan), and BB plus a d
of all combination use; atenolol plus ch
Table 1). The least used combination prod
Table 1 Use of antihypertensive medicines by
daily dose [DDD] and proportion [%]) for single an
products in Ghana and Nigeria
ARB Angiotensin receptor blockers, CCB calcium channel bloc
angiotensin-converting enzyme inhibitor, BB beta-blocker, DU
AAC and ST are not conventional abbreviations. AAC will invo
drugs like methyldopa
Ghana
Nige
Use (DDD)
%
Use (
Single
ARB
380,029
39.2
940
CCB
310,645
32.1
803
ACEi
129,783
13.4
514
DU
72,272
7.5
651
BB
65,393
6.7
211
AAC
4,528
0.5
107
AAP
–
0
23
Total
962,650
99.4
3,252
Combination
DU + DU
0
218
BB + DU
342
0.0
10
CCB + ARB
0
12
CCB + ARB + DU
0
27
CCB + ST
5813
0.6
ACEi + ARB
Total
6155
0.6
270 Hollingworth et al. BMC Cardiovascular Disorders (2022) 22:368 Hollingworth et al. BMC Cardiovascular Disorders (2022) 22:368
(39.2%) followed by CCB (32.1%), and ACEi (13.4%,
Table 1). There was low use of diuretics (7.5%) and BB
(6.7%). In Nigeria, however, the most used classes were
ARB (26.7%), CCB (22.8%), diuretics (18.5%) and ACEi
(14.6%, Table 1). In Ghana, single-medicine products
were preferred (99.4% of total) than Nigeria (92.3% of
total). Use by dose formulationi In Ghana, the five most used single-medicine dose prod-
ucts were candesartan 16 mg (16.0% of all single product
use), nifedipine 30 mg (12.4%), amlodipine 5 mg (10.6%,
only dose used), candesartan 8 mg (8.7%), and losartan
50 mg (7.8%, Table 3). The top ten medicine dose prod-
ucts constituted 80.5% of all single medicine products
used. In Nigeria, the five most used single-medicine dose
products were furosemide 40 mg (12.2% of all single
product use), amlodipine 5 mg (12.1%, only dose used),
losartan 50 mg (8.3%), nifedipine 30 mg (8.2%), and lisin-
opril 10 mg (7.2%, Table 3). The top ten medicine dose
products constituted 71.6% of all single medicine prod-
ucts used. Hollingworth et al. BMC Cardiovascular Disorders (2022) 22:368 Page 4 of 9 Table 2 Use of antihypertensive medicines by medicines (defined daily dose [DDD] and proportion within class [%]) for single
products in Ghana and Nigeria plus top ten ranking and proportion Table 2 Use of antihypertensive medicines by medicines (defined daily dose [DDD] and proportion within class [%]) for single
products in Ghana and Nigeria plus top ten ranking and proportion yp
y
(i
y
[
]
p
p
[ ])
g
products in Ghana and Nigeria plus top ten ranking and proportion
ARB Angiotensin receptor blockers, CCB calcium channel blocker, ACEI angiotensin-converting enzyme inhibitor, BB beta-blocker, DU Diuretic, AAP, AAC and ST are no
conventional abbreviations. Use by dose formulationi AAC will involve centrally acting drugs like methyldopa
a Amiloride + HCT was ranked #7 but removed as it is a combination product
Class
Medicine
Ghana
Nigeria
Use (DDD)
% within class
Rank
% of top 10
Use (DDD)
% within class
Rank
% of top 10
ARB
Candesartan
260,561
68.6%
1
27.7
33,203
3.5%
Losartan
92,384
24.3%
5
9.8
517,910
55.1%
1
17.8
Valsartan
27,084
7.1%
9
2.9
239,830
25.5%
6
8.3
Telmisartan
–
0
147,807
15.7%
8
5.1
Irbesartan
–
0
1796
0.2%
Total
380,029
940,546
CCB
Nifedipine
160,490
51.7%
2
17.1
408,500
50.8%
3
14.1
Amlodipine
101,778
32.8%
4
10.8
394,731
49.1%
5
13.6
Felodipine
48,377
15.6%
7
5.1
600
0.1%
Nimodipine
–
0
15
0.0%
Total
310,645
803,845
ACEi
Lisinopril
129,004
99.4%
3
13.7
418,073
81.3%
2
14.4
Ramipril
779
0.6%
88,831
17.3%
Perindopril
–
0.0%
7170
1.4%
Captopril
–
0.0%
178
0.0%
Enalapril
–
0.0%
170
0.0%
Total
129,783
514,422
DU
Indapamide
65,332
90.4%
6
6.9
74,382
11.4%
Furosemide
1968
2.7%
398,932
61.2%
4
13.7
Hydrochlorothiazide
–
0.0%
148,244
22.8%
7a
5.1
Bendroflumethiazide
4972
6.9%
21,731
3.3%
Metolazone
–
0.0%
5202
0.8%
Torasemide
–
0.0%
2917
0.4%
Total
72,272
651,408
BB
Atenolol
26,810
41.0%
10
2.8
121,556
57.5%
9
4.2
Carvedilol
29,275
44.8%
8
3.0
22,484
10.6%
6
8.7
Metoprolol
7911
12.1%
40,816
19.3%
Propanolol
21
0.0%
11,274
5.3%
Bisoprolol
1377
2.1%
7016
3.3%
Labetalol
–
0.0%
8177
3.9%
Total
65,393
211,322
AAC
Methyldopa
4528
100
107,321
100
10
3.7 ARB Angiotensin receptor blockers, CCB calcium channel blocker, ACEI angiotensin-converting enzyme inhibitor, BB beta-blocker, DU Diuretic, AAP, AAC and ST are not
conventional abbreviations. AAC will involve centrally acting drugs like methyldopa
a Amiloride + HCT was ranked #7 but removed as it is a combination product was nine times that of Ghana (9.6%, Table 4). The use of
CCB in Nigeria was almost exclusively generic (97.9%)
but lower in Ghana (59.4%). In four classes—BB, ARB,
ACEI, and diuretics—the use of originator brands pre-
vailed in Ghana whereas the use of generic products
dominated in all five classes in Nigeria (Table 4). Use by product typeh The use of generic products (across all single and com-
bination class products) dominated in Nigeria (78.4%)
but they were less often used in Ghana (21.6%; data not
shown). The use of generic products for the five main classes of
single products dominated in Nigeria (average 94.3%) but
they were less often used in Ghana (average 30.8%). The
proportional use of generic ARBs was 90.3% in Nigeria
but only 24.5% in Ghana—a difference of almost four-
fold; the proportional use of diuretics in Nigeria (88.1%) Discussion Dispensed use of antihypertensives varied between
Ghana and Nigeria. The most used medicine class in
Ghana was ARB followed by CCB and ACEi. The five Hollingworth et al. Discussion BMC Cardiovascular Disorders (2022) 22:368 Page 5 of 9 Table 3 Use of antihypertensive medicines by dose product: use in defined daily dose [DDD] and proportion within all single-
medicine dose products [%] in Ghana and Nigeria Table 3 Use of antihypertensive medicines by dose product: use in defined daily dose [DDD] and proportion within all single-
medicine dose products [%] in Ghana and Nigeria Table 3 Use of antihypertensive medicines by dose product: use in defined daily dose [DDD] and proportion within all single-
medicine dose products [%] in Ghana and Nigeria
HCT hydrochlorothiazide
No
Ghana
Nigeria
Medicine
Dose (mg)
Use (DDD)
% all use
Medicine
Dose (mg)
Use (DDD)
% all use
1
Candesartan
16
153,812
16
Furosemide
40
397,284
12.2
2
Nifedipine
30
119,532
12.4
Amlodipine
5
394,230
12.1
3
Amlodipine
5
101,778
10.6
Losartan
50
271,533
8.3
4
Candesartan
8
83,653
8.7
Nifedipine
30
265,585
8.2
5
Losartan
50
75,340
7.8
Lisinopril
10
234,870
7.2
6
Lisinopril
10
72,950
7.6
Losartan
100
194,982
6
7
Indapamide
1.5
65,332
6.8
Lisinopril
20
149,234
4.6
8
Nifedipine
20
38,309
4
HCT
50
148,244
4.6
9
Lisinopril
20
37,944
3.9
Nifedipine
20
142,915
4.4
10
Atenolol
50
26,014
2.7
Valsartan
160
128,352
3.9
11
Candesartan
32
23,096
2.4
Valsartan
80
111,478
3.4
12
Lisinopril
5
18,110
1.9
Methyldopa
250
107,321
3.3
13
Valsartan
160
17,784
1.8
Telmisartan
80
92,180
2.8
14
Losartan
100
17,044
1.8
Indapamide
1.5
74,382
2.3
15
Valsartan
80
7720
0.8
Atenolol
50
73,747
2.3
16
Nifedipine
10
2648
0.3
Telmisartan
40
55,627
1.7
17
Methyldopa
500
2345
0.2
Losartan
25
51,395
1.6
18
Methyldopa
250
2184
0.2
Lisinopril
5
33,969
1
19
Furosemide
40
1951
0.2
Atenolol
100
31,380
1
20
Valsartan
320
1580
0.2
Candesartan
16
21,002
0.6
21
Atenolol
100
795
0.1
Atenolol
25
16,429
0.5
22
Furosemide
20
17
0
Candesartan
8
12,201
0.4
23
Atenolol
25
1
0
Furosemide
20
1648
0.1
24
Losartan
25
–
0
Amlodipine
2.5
501
0
25
Amlodipine
2.5
–
0
Valsartan
320
–
0
26
HCT
50
–
0
Nifedipine
10
–
0
27
Telmisartan
80
–
0
Methyldopa
500
–
0
28
Telmisartan
40
–
0
Candesartan
32
–
0
Total
962,650
3,252,329
Top 10 (% all use)
80.5
71.6 HCT hydrochlorothiazide second most used medicines, but the third for Ghana was
ACEi while for Nigeria it was diuretics. Discussion This difference
may be attributed to the high cost of ACEi in Nigeria
which makes it more difficult to afford, despite its proven
advantages in terms of lowering blood pressure over diu-
retics [19]. most used medicines in Ghana were candesartan, nifedi-
pine, lisinopril, amlodipine, and losartan accounting for
79% of the top ten medicines. The five most used prod-
ucts in Ghana were candesartan 16 mg, nifedipine 30 mg,
amlodipine 5 mg, candesartan 8 mg, and losartan 50 mg. The most used medicine class in Nigeria was ARB fol-
lowed by CCB, then diuretics. The five most used medi-
cines were losartan, lisinopril, nifedipine, furosemide,
and amlodipine accounting for 74% of the top ten medi-
cines. In Nigeria the top five products were furosem-
ide 40 mg, amlodipine 5 mg, losartan 50 mg, nifedipine
30 mg, and lisinopril 10 mg. More generic products were
used in Nigeria than Ghana. AHM use was broadly simi-
lar for both countries—ARB and CCB were the first and A recent economic evaluation and budget impact anal-
ysis on the main antihypertensive therapeutic groups
used for uncomplicated essential hypertension in a Gha-
naian population [20] found that diuretics were more
cost-effective than ACEi, ARB, or BB for first-line man-
agement of essential hypertension; this result was driven
by the greater reduction in stroke incidence with diu-
retics. CCB were more effective than diuretics but were Page 6 of 9
Hollingworth et al. BMC Cardiovascular Disorders (2022) 22:368 Hollingworth et al. Discussion BMC Cardiovascular Disorders (2022) 22:368 Page 6 of 9 Table 4 Use of antihypertensive medicines for the five main classes (defined daily dose [DDD]) and product type—generic
or originator (proportion within class that is generic [%])—for single products in Ghana and Nigeria and combined countries plus
difference between Nigeria and Ghana Table 4 Use of antihypertensive medicines for the five main classes (defined daily dose [DDD]) and product type—generic
or originator (proportion within class that is generic [%])—for single products in Ghana and Nigeria and combined countries plus
difference between Nigeria and Ghana ARB Angiotensin receptor blockers, CCB calcium channel blocker, ACEI angiotensin-converting enzyme inhibitor, BB beta-blocker, DU Diuretic
Class
Type
Ghana
Nigeria
Nigeria/Ghana
Use (DDD)
% generic
Use (DDD)
% generic
% generic
ARB
Generic
92,971
24.5
849,568
90.3
3.7
Originator
287,058
90,978
Total
380,029
940,546
CCB
Generic
184,622
59.4
787,031
97.9
1.6
Originator
126,023
16,814
Total
310,645
803,845
ACEi
Generic
24,534
18.9
501,055
97.4
5.2
Originator
105,249
13,367
Total
129,783
514,422
DU
Generic
6940
9.6
574,109
88.1
9.2
Originator
65,332
77,299
Total
72,272
651,408
BB
Generic
27,122
41.5
206,229
97.6
2.4
Originator
38,271
5093
Total
65,393
211,322 associated with higher costs. Nevertheless, both thiazide
diuretics and CCB were regarded as cost-effective treat-
ment options in Ghana, consistent with evidence also
available from Nigeria [21]. Notably the 2017 Ghanaian
Standard Treatment Guidelines stipulate a preference
for TZDs and CCBs, and recommends against the use of
ACEi as first line drugs for uncomplicated hypertension
in black patients [22]. Despite these recommendations,
we found that the most used medicine class in Ghana was
ARB followed by CCB and ACEi. Data from the NHIS
showed that CCB, followed by diuretics, are the most
predominantly used in public health facilities in Ghana
[23]. When choosing antihypertensive drug treatment for
adults of black African or African-Caribbean family ori-
gin, the National Institute of Health and Care Excellence
(NICE) in the UK says to consider an ARB, in preference
to an ACE inhibitor [24].h The higher level of use of originator products may reflect
prescriber and or patient preferences for these products
perhaps linked to delayed NHIS reimbursement to pro-
viders, thus encouraging out-of-pocket expenditure, or
related to concerns about quality [25]. In addition, it may
also highlight the need for strengthened reimbursement
and procurement policies—informed by health technol-
ogy assessment (HTA)—to help ensure medicine costs
better reflect their ‘value-based’ price. B Angiotensin receptor blockers, CCB calcium channel blocker, ACEI angiotensin-converting enzyme inhibitor, BB beta-blocker, DU Diuretic Discussion The use of generic products reduced expenditure and
out-of-pocket payments by hypertensive patients with-
out affecting medicine utilisation [50]. This might
also explain the dominance of generic use in Nige-
ria due to patient preference: the out-of-pocket pay-
ment for healthcare was 71.5% in Nigeria (based on
national health accounts [51]) but only 40% in Ghana
[52]. The AHM use in Ghana is broadly consistent
with two other recent studies from Ghana using claims
data from the NHIS (where CCB were the most widely
used, preferred additional treatment was a diuretic)
[23] and private health insurance companies [53]. designed for research purposes so there may be some
elements that are not available for analysis. Secondly,
we do not have an adequate population denominator
to ascertain absolute quantities of medicines use as per
the DDD metric [18]. Thirdly, these are aggregated data
and we cannot ascertain the indications for use as these
AHM can be used to treat several CVD conditions nor
the likely concomitant use of single AHM products at the
same time. Studies of AHM use in Nigeria have been inconsistent;
most studies showed that CCBs, diuretics and BB are the
most used AHM [28–31] but one study showed a pref-
erence for ACEIs, either as single or combination prod-
ucts [32]. The former three studies were based in single
hospitals with few participants, whereas the latter study
[32] was community-based with a large sample size that
likely captures the AHM use irrespective of where they
received their prescriptions (e.g. hospital, clinic). The use
of particular AHM classes may be attributed to cost, pre-
scriber’s preference, a reluctance to change medication,
and perceived advantages of each class.h p
g
The use of combination AHM products is more com-
mon in Nigeria than Ghana. This observation is sup-
ported by other studies that show Nigerians prefer
combination therapy; most study participants used com-
bination therapy but only less than 20% [28], 5% [29] and
13% [32] of the study participants used monotherapy. The concomitant use of single AHM products can reduce
the burden of adverse reactions of a single AHM used
in high doses, in reducing cases of drug resistance, and
in gaining better blood pressure control, although the
cost might be higher and adherence more difficult. It
also conforms to recommendations from the American
Heart Association23 and to the recently revised Nigerian
National Standard Treatment Guidelines [33]. Discussion In that regard the
recently launched Ghana Strategy for Health Technology
Assessment (2020) sees HTA as a strategic instrument to
inform the selection, pricing and procurement of phar-
maceuticals as well as other health technologies, at least
for the publicly -funded sector [26]. The use of combination products was tenfold lower
in Ghana than Nigeria. Clinically, combination treat-
ment may be preferred when existing monotherapy is
not achieving adequate control and there is a reluctance
to increase doses because of the risk of side effects [27]. It is not clear why the use of combination products were
higher in Nigeria but some factors would likely be the
availability of such products, out-of-pocket costs, patient
and prescriber preferences, and the local epidemiology of
hypertension. This warrants further investigation. The discordance between guidelines and practice may
reflect, in part, that guidelines can be slow to influence
practice if not supported by an active implementation
strategy. The previous Ghanaian guidelines (2010) did
not indicate any clinically-informed medicine prefer-
ences for managing uncomplicated essential hyperten-
sion, leaving the choice of first-line treatment open for
the five main classes of AHM. In addition, the Ghanaian
NHIS did not provide incentives to encourage the use of
lower-priced formulations or better-value AHM classes. This appears to be an ongoing issue, given the relatively
low levels of generic AHM use revealed in our study. h
This is one of the first studies that we know of to report
on the use of AHM at the community level in Ghana
and Nigeria using a novel data source. We acknowledge
three main limitations of our approach. Firstly, the data
are extracted from a commercial source, which was not Hollingworth et al. BMC Cardiovascular Disorders (2022) 22:368 Hollingworth et al. BMC Cardiovascular Disorders Page 7 of 9 originator products are more expensive than generic
products as they are imported [46]. Furthermore, there
may be a wide range of costs for generic products in
low and middle income countries (LMIC) which may
be even more expensive than in higher income coun-
tries. People in LMIC disproportionately buy expen-
sive branded generic medicines rather than cheaper
unbranded medicines [47]. Although there are good
reasons to support the local production of generic
(i.e. off-patent) medicines [48], some prescribers and
patients might trust originator products more [49]. Funding
h
d This study received no specific funding and was funded from existing sala-
ries. FR is supported by the International Decision Support Initiative, which is
funded by the Bill and Melinda Gates Foundation (OPP1202541). 10. Odili AN, Chori BS, Danladi B, Nwakile PC, Okoye IC, Abdullahi U, et al. Prevalence, awareness, treatment and control of hypertension in nigeria:
data from a nationwide survey 2017. Glob Heart. 2020;15(1):47. Discussion BMC Cardiovascular Disorders (2022) 22:368 Hollingworth et al. BMC Cardiovascular Disorders (2022) 22:368 Page 8 of 9 Declarations 13. Abubakar I, Obansa S. An estimate of average cost of hypertension and
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k
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Agyeman at mPharma. y
7. Nulu S, Aronow WS, Frishman WH. Hypertension in sub-Saharan Africa: a
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only existing collections of data that contain only non-identifiable data and
is of negligible risk. Administrative permissions were required from mPharma
to access the extracted aggregated data from mPharma. The parties (SH, Uni-
versity of Queensland) and mPharma (ET) signed a non-disclosure agreement
due to the commercial nature of the data. Only mPharma employees (ET) had
access to the raw data, while SH analysed the de-identifiable data provided by
mPharma. Research involving these human data were performed in accord-
ance with the Declaration of Helsinki. 14. Chalkidou K, Claxton K, Silverman R, Yadav P. Value-based tiered pricing
for universal health coverage: an idea worth revisiting. Gates Open Res. 2020;4:16. 15. Atinyi R, Takramah W, Axame WK, Owusu R, Parbey PA, Takase M, et al. Prevalence and awareness of hypertension among urban and rural adults
in the Keta municipality, Ghana. J Med Res. 2017;3(3):155–63. 16. Sarfo FS, Mobula LM, Burnham G, Ansong D, Plange-Rhule J, Sarfo-
Kantanka O, et al. Factors associated with uncontrolled blood pressure
among Ghanaians: evidence from a multicenter hospital-based study. PLoS ONE. 2018;13(3):e0193494. Author contributions SH and ET designed the study. ET obtained the data and SH analysed the data. SH and ET developed the manuscript. DA, BSCU, CO and FR interpreted the
data and provided expert advice on clinical aspects and health systems. All
authors read, revised, and approved the paper before its final submission. 8. Adeloye D, Basquill C, Aderemi AV, Thompson JY, Obi FA. An estimate of
the prevalence of hypertension in Nigeria: a systematic review and meta-
analysis. J Hypertens. 2015;33(2):230–42. 8. Adeloye D, Basquill C, Aderemi AV, Thompson JY, Obi FA. An estimate of
the prevalence of hypertension in Nigeria: a systematic review and meta-
analysis. J Hypertens. 2015;33(2):230–42. 9. Ogah OS, Okpechi I, Chukwuonye II, Akinyemi JO, Onwubere BJ, Falase
AO, et al. Blood pressure, prevalence of hypertension and hypertension
related complications in Nigerian Africans: a review. World J Cardiol. 2012;4(12):327–40. Discussion Recent
Nigerian studies have shown low adherence to concomi-
tant use of single AHM products (1% [34], 4.1% [35], 8.9%
[36], and 31.8% [37]) and this is consistent with a study
that compared AHM adherence in Nigeria and Ghana
[38]. On the contrary, adherence to AHM was found to
be high in a Ghanaian study (89.2%, but not stated if fixed
dose combination or concomitant single products) [39]
and as reported by stakeholders in a qualitative study
[40]. We emphasise the need for tailored adherence edu-
cation and counselling for patients using concomitant
AHM. p
p
Both countries adhered to the guidelines for the
treatment of hypertension; they are using CCB, diu-
retics, ACEI, ARB, and BB medicines. The Ghana STG
are not directive on class; any of the five classes of
major antihypertensive drugs can be used as first-line
treatment [22]. The Nigerian STG on the other hand
stipulates the class of the first line medicines and the
subsequent lines of medicines to be used [33]. The dif-
ferences in selecting a particular AHM from the vari-
ous classes may be largely due to prescriber preference
and availability. The patterns of AHM use in Ghana are
not consistent with the economic evaluation of AHM
showing that DU are more cost-effective than CCB
[20]. HTA can help to reduce costs within national or
jurisdiction-based health insurance systems within the
context of universal health coverage [54, 55].f We have revealed some interesting differences in
AHM use in Ghana and Nigeria over five years. In
future, it would be beneficial to examine AHM use over
time to better understand how guidelines, for exam-
ple, may influence prescriber preferences and medi-
cines within a class. The economic evaluation of AHM
in Ghana [20] could be replicated in Nigeria to better
inform medicine use. We could also use data on medi-
cines use to calculate potential savings (and overspend)
given the patterns of AHM and the cost-effectiveness
of particular AHM classes. Patient level data (e.g. from
mPharma and NHIS) can provide powerful insights
into the rational—or otherwise—use of medicines. Nigerians use more generic products in the com-
munity whereas Ghanaians prefer originator products;
consistent with other studies [41–43]. In Nigeria, the
guidelines [22] and the essential medicine lists [44]
contain only generic names, as does the Ghanaian
NHIS medicine list [45]. In both Ghana and Nigeria, Hollingworth et al. Received: 10 March 2022 Accepted: 19 May 2022 Author details 1 School of Pharmacy, The University of Queensland, 20 Cornwall St, Wool-
loongabba, QLD 4102, Australia. 2 Faculty of Pharmacy and Pharmaceutical
Sciences, Kwame Nkrumah University of Science and Technology, Kumasi,
Ghana. 3 Department of Pharmacy, Korle Bu Teaching Hospital, Accra, Ghana. 4 Department of Community Medicine, College of Medicine, University
of Nigeria, Enugu, Nigeria. 5 International Decision Support Initiative, London
School of Hygiene and Tropical Medicine, London, UK. 6 Seattle, USA. 19. Reisin E, Graves JW, Yamal JM, Barzilay JI, Pressel SL, Einhorn PT, et al. Blood pressure control and cardiovascular outcomes in normal-weight,
overweight, and obese hypertensive patients treated with three different
antihypertensives in ALLHAT. J Hypertens. 2014;32(7):1503–13 (discus-
sion 13). p
g
ET was an employee of mPharma between 2019 and 2021. The remaining
authors declared that they do not have any conflicts of interest. yp
sion 13). Bull World Health Organ. 2020;98(7):507–8. 49. Hamill H, Hampshire K, Mariwah S, Amoako-Sakyi D, Kyei A, Castelli M. Managing uncertainty in medicine quality in Ghana: the cognitive and
affective basis of trust in a high-risk, low-regulation context. Soc Sci Med. 2019;234:112369. 28. Adejumo O, Okaka E, Iyawe I. Prescription pattern of antihypertensive
medications and blood pressure control among hypertensive outpa-
tients at the University of Benin Teaching Hospital in Benin City, Nigeria. Malawi Med J J Med Assoc Malawi. 2017;29(2):113–7. 50. de Jager H, Suleman F. The impact of generics and generic reference pric-
ing on candesartan and rosuvastatin utilisation, price and expenditure in
South Africa. Int J Clin Pharm. 2019;41(1):81–7. 29. Bakare OQ, Akinyinka MR, Goodman O, Kuyinu YA, Wright OK, Adeniran
A, et al. Antihypertensive use, prescription patterns, and cost of
medications in a Teaching Hospital in Lagos, Nigeria. Niger J Clin Pract. 2016;19(5):668–72. 51. Federal Ministry of Health Nigeria. National health accounts. 2017. Avail-
able from: https://www.health.gov.ng/doc/FINAL-VERSION-NHA-2017.
pdf. 30. Ojji DB, Ajayi SO, Mamven MH, Alfa J, Albertino D. Pattern of prescription
of anti-hypertensive medications in a tertiary health care facility in Abuja,
Nigeria. Ethn Dis. 2013;23(4):480–3. 52. Akweongo P, Aikins M, Wyss K, Salari P, Tediosi F. Insured clients out-of-
pocket payments for health care under the national health insurance
scheme in Ghana. BMC Health Serv Res. 2021;21(1):440. 31. Olanrewaju TO, Aderibigbe A, Busari OA, Sanya EO. Antihypertensive
drug utilization and conformity to guidelines in a sub-Saharan African
hypertensive population. Int J Clin Pharmacol Ther. 2010;48(1):68–75. 53. Marfo AF, Marfo JS, Plange-Rhule J, Hollingworth S. Trends in antihyper-
tensive use in Ghana. J Hypertension. 2022. Submitted. 32. Mijinyawa Muhammad S, Yusuf MS, Mohammed H, Saidu H, Sulaiman BA,
Uloko AE. Choice of antihypertensive medications among physicians and
its impact on blood pressure control among Nigerians living with hyper-
tension. Niger J Med J Natl Assoc Resid Dr Nigeria. 2016;25(3):220–5. 54. Hollingworth S, Gyansa-Lutterodt M, Dsane-Selby L, Nonvignon J,
Lopert R, Gad M, et al. Implementing health technology assessment in
Ghana to support universal health coverage: building relationships that
focus on people, policy, and process. Int J Technol Assess Health Care. 2020;36(1):8–11. 33. Federal Ministry of Health Nigeria. Standard treatment guidelines Nigeria,
2nd edn. 2017, https://www.medbox.org/document/nigeria-standard-
treatment-guidelines#GO. Accessed 23 Sept 2020. 55. Uzochukwu BSC, Okeke C, O’Brien N, Ruiz F, Sombie I, Hollingworth S. yp
sion 13). 20. Gad M, Lord J, Chalkidou K, Asare B, Lutterodt MG, Ruiz F. Supporting
the development of evidence-informed policy options: an economic
evaluation of hypertension management in Ghana. Value Health J Int Soc
Pharmacoecon Outcomes Res. 2020;23(2):171–9. 21. Ekwunife OI, Okafor CE, Ezenduka CC, Udeogaranya PO. Cost-utility analy-
sis of antihypertensive medications in Nigeria: a decision analysis. Cost Eff
Resour Alloc C/E. 2013;11(1):2. Page 9 of 9 Hollingworth et al. BMC Cardiovascular Disorders (2022) 22:368 22. Ministry of Health Republic of Ghana. Standard treatment guidelines. 7th
ed. Accra: Ministry of Health Republic of Ghana; 2017. perceptions and attitudes of physicians. Expert Rev Pharmacoecon
Outcomes Res. 2016;16(5):639–50. 43. Sarfo F, Mobula L, Arthur L, Plange-Rhule J, Burnham G, Sablah J, et al. Differential pricing of medicines to improve access to medicines for
hypertension and diabetes control in Ghana: The Ghana Access and
Affordability Program, a multi-center prospective trial [version 1; peer
review: 2 not approved]. Gates Open Research. 2019;3:1515. 23. Donneyong MM, Chang T-J, Pottegård A, Ankrah D, Asenso-Boadi F,
Addo-Cobbiah V, et al. Prevalence and quality of antihypertensive therapy
among hypertension patients enrolled in the Ghana National Health
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diagnosis and management. NICE Guideline (NG136] London: NICE;
2019. Available from: https://www.nice.org.uk/guidance/ng136/chapter/
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from: http://www.nhis.gov.gh/medlist.aspx. 25. Koduah A, Nonvignon J, Colson A, Kurdi A, Morton A, van der Meer R,
et al. Health systems, population and patient challenges for achieving
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affordability of cardiovascular, diabetes, and global medicines in Abuja,
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tion in global health procurement. Linked Immunisation Action Network. 2019. 26. Ministry of Health Republic of Ghana. Ghana strategy for health technol-
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Secretariat, editor. Accra: MoH; 2020. 27. European Medicines Agency. Guideline on clinical investigation of
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ence and 24-h blood pressure in apparently uncontrolled hypertensive
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for asthma, diabetes and hypertension in eight counties of Kenya. Trop
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Odigwe CO. Adherence to antihypertensive medication and its correlates
among individuals with hypertension in a semi-urban community of
Southern Nigeria. J Med Biomed Res. 2015;14(1):5–17. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. 38. Boima V, Ademola AD, Odusola AO, Agyekum F, Nwafor CE, Cole H, et al. Factors associated with medication nonadherence among hypertensives
in Ghana and Nigeria. Int J Hypertens. 2015;2015:205716. 39. Sarkodie E, Afriyie DK, Hutton-Nyameaye A, Amponsah SK. Adherence
to drug therapy among hypertensive patients attending two district
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O, et al. The prescribing of generic medicines in Nigeria: knowledge,
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Using evaluated AquaCrop and Response Surface Method to determine optimum irrigation water and seeding density of wheat growing in a sprinkler irrigation system
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Research Article Page 1/32 Page 1/32 Abstract This study used AquaCrop to predict wheat grain yield under different irrigation and seeding densities. Experimental data from two successive growing seasons during 2004–2006 was used for model calibration
and validation. After calibration, the model was used to predict grain yield for 47 years (1975–2021) with five
seeding densities (120, 80, 160, 200, and 240 kg ha-1) and four irrigation schedules (7-, 10-, 13-, and 16-days
interval). Predicted data were used to identify the optimal seeding density and irrigation water level. AquaCrop's
simulations of grain yield, biomass, soil water content, evapotranspiration, and canopy cover were promising. Under extreme water stress, the model produced less reliable results. The RSM method determined the optimal
seeding density and irrigation schedule to maximize crop yield and income per hectare. Results showed that
747, 198, and 747 mm of irrigation water and 211, 188, and 208 kg ha-1 of seeding density maximized wheat
yield, water productivity, and profit per unit area, respectively. Additionally, 350 and 1230 mm of irrigation and
rainfall and 162 and 212 kg ha-1 of seeding density were found to maximize water productivity and profit per
unit area. Overall, this study demonstrates that the AquaCrop model can be used to accurately estimate wheat
grain yield under different irrigation intensities and seeding densities, which can inform decisions on optimal
irrigation and seeding practices for maximizing crop yield and profit. This study used AquaCrop to predict wheat grain yield under different irrigation and seeding densities. 1. Introduction For human survival, wheat is more vital than any other crop. Wheat is Iran's most profitable crop and a staple in
the country's diet (Ghahremaninejad et al. 2021). The rising human population has also increased wheat
demand (Igrejas and Branlard 2020). Water is the most crucial factor in producing agricultural goods and
services. High water stress and increased frequency and intensity of droughts (Yin and Xu 2020), primarily
driven by climate change dynamics, have reduced freshwater resources in arid and semi-arid regions like Iran
(Nazari et al. 2018; Vema et al. 2022). Plant evapotranspiration has a relatively direct relationship with the amount of crop production (Tamimi et al. 2022). For this reason, irrigation should be adjusted, so that crop yield does not decrease significantly besides
reducing the amount of irrigation water (Zarei et al. 2020; Pardo et al. 2022). This can be achieved by using a
deficit irrigation strategy (Allakonon et al. 2022; Li et al. 2022). For effective deficit irrigation implementation, a
comprehensive examination of the yield-to-applied-water relationship is required (Akhtar et al. 2013; Zhou et al. 2020). To maximize crop productivity under deficit irrigation, crop modeling is valuable for evaluating and
creating new irrigation scheduling strategies (Ding et al. 2021; Kheir et al. 2021). Different crop models such as CERES-wheat (Otter and Ritchie 1985), WOFOST (van Diepen et al. 1989), STICS
(Brisson et al. 2003), and CropSyst (Stöckle et al. 2003) are being used for simulation of crop growth and yield
(Bai et al. 2021; Si et al. 2021; Hafiza et al. 2022). These models require extensive data sets for simulations. They are sophisticated and require advanced modeling skills for their operation. Their input parameters are not
readily available variables and are much more familiar to scientists than to end users (Toumi et al. 2016). To
resolve these problems, the Food and Agriculture Organization (FAO) Water Unit has created a model called
AquaCrop (Raes et al. 2009; Steduto et al. 2009). This user-friendly and practitioner-oriented model, which
simulates the yield response to water of numerous herbaceous crops, aims to strike a compromise between
accuracy, simplicity, and robustness (Salman et al., 2021). AquaCrop has been calibrated and validated in Different crop models such as CERES-wheat (Otter and Ritchie 1985), WOFOST (van Diepen et al. 1989), STICS
(Brisson et al. 2003), and CropSyst (Stöckle et al. 2003) are being used for simulation of crop growth and yield
(Bai et al. 1. Introduction 2021; Si et al. 2021; Hafiza et al. 2022). These models require extensive data sets for simulations. Page 2/32 Page 2/32 many studies for several crops, such as wheat (Shirazi et al. 2021; Amiri et al. 2022; Dercas et al. 2022; Zhang
et al. 2022), sunflower (Khaleghi et al. 2022), maize (Feng et al. 2022), quinoa (Geerts et al. 2009), sugar beet
(Malik et al. 2017), cotton (Li et al. 2018, 2019; Masasi et al. 2020). Wheat growth and yield under varied
watering circumstances have been simulated in multiple experiments using the AquaCrop model. When tested
under varying conditions of irrigation intensity and salinity (Mohammadi et al. 2016; Zhai et al. 2022), the
model was found to reliably predict grain production, water usage efficiency, and other crop metrics. Maximum
crop yields and profits were achieved by using the model to fine-tune irrigation timing and establish ideal water
and sowing rates. In agriculture, crop density is a major influencer of both crop yield and water efficiency. Crop yields can be
increased by planting at the optimal density, but can be decreased by overplanting owing to competition for
nutrients and water. A higher LAI can be achieved by increasing plant crop density, which causes the crop's
canopy to become denser. Transpiration, or the evaporation of water from plant leaves, is crucial for plant
growth and, if controlled properly, can improve water use efficiency (Gao et al. 2021; Dai et al. 2022). Increasing
seeding density increases crop demand for water and nutrients because more leaves and stems are produced
per area of land (Zhang et al. 2023). This leads to higher crop water consumption and lower water productivity. Low light levels, decreased air movement, and increased disease pressure are all microclimatic challenges that
can be caused by overcrowding plants (Li et al. 2022). So, it's important to use well-established models like the
Aquacrop model to find optimal plant densities that promote effective use of available water while enhancing
crop yield and quality without compromising water productivity. Furthermore, the availability of water resources
is crucial to the impact of crop density on output. Maximizing productivity and water use efficiency in semi-arid
environments requires careful management of irrigation water and crop density. When it comes to modelling
crop development and water use in water-limited regions, the AquaCrop model is an invaluable tool. 1. Introduction With this
model, farmers may anticipate their harvest and make informed decisions about irrigation and crop density. The optimal irrigation and crop density levels that maximize production and water use efficiency can be
determined by combining AquaCrop with response surface methods. This paper describes a study that makes use of the AquaCrop model and response surface approach to
establish guidelines for irrigating and planting wheat in a semiarid environment. The purpose of this research
is to compare the effectiveness of various crop densities and irrigation regimes on wheat yield and water
efficiency in a sprinkler irrigation system. As a result, this study aimed to see how well the AquaCrop model
predicted various wheat crops parameters like evaporation-transpiration, leaf area index, soil moisture, grain
yield, and total dry matter in a sprinkler irrigation system at different irrigation water levels and seeding density. The amount of irrigation water and seeding density that results in the highest grain yield, profit per unit area,
and water productivity were then calculated. 2.1. Study area Located at 1810 meters above sea level, the study was carried out in the Badjgah region of Fars province, Iran
(29° 54′ 36″ N to 29° 57′ 42″ N latitude and 53° 27′ 36″ E to 53° 29′ 42″ E longitude). Badjgah map is shown in
Fig. 1. A semi-arid climate prevails there, with warm and dry summers and cold and wet winters. The area Page 3/32 receives an average of 300 mm of precipitation annually, most of which falls between October and May. Wheat, a major staple crop in the area, is grown extensively in the study area. Wheat is planted in late October
or early November, with harvest occurring in late May or early June. Most farmers in the study area use
conventional irrigation techniques like furrow irrigation, which rely on a combination of surface and
groundwater sources for watering crops. Due to the sandy loam soil's low water-holding capacity, proper
irrigation management is essential for maximizing regional wheat harvests. The semi-arid Badjgah region, with
its favorable irrigation and climate conditions, is an ideal testing ground for the AquaCrop wheat yield
prediction model. receives an average of 300 mm of precipitation annually, most of whi
Wheat, a major staple crop in the area, is grown extensively in the stud
or early November, with harvest occurring in late May or early June. M
conventional irrigation techniques like furrow irrigation, which rely on
groundwater sources for watering crops. Due to the sandy loam soil's
irrigation management is essential for maximizing regional wheat har
its favorable irrigation and climate conditions, is an ideal testing grou
prediction model. 2.2. Field experiment In a field experiment, the effect of irrigation level and seeding density on the yield of winter wheat (Triticum
aestivum L.) cv Shiraz was investigated. The soil's physical and chemical characteristics in the study plots are
displayed in Table 1. The experiment was conducted over two consecutive growing seasons in 2004–2005 and
2005–2006. The experiment was set up as a randomized complete block. The treatments involved three
distinct levels of irrigation (severe deficit irrigation (rain-fed), moderate deficit irrigation, and full irrigation) and
four distinct densities of planting seeds. Seed densities of 80, 120, 160, and 200 kg ha− 1 were used in the
initial year of the experiment (2004–2005). In the second year of the experiment (2005–2006) 120, 160, 200
and 240 kg of seed. Table 1
Physical and chemical soil characteristics of the study area
Soil
depth
(cm)
EC
(dS.m−
1)
pH
OM
(%)
Sand
(%)
Silt
%))
Clay
(%)
ρb
(g
cm− 3)
PWP
(cm3
cm− 3)
FC
(cm3
cm− 3)
SAT
(cm3
cm− 3)
0–30
0.59
8
2
35
35
30
1.58
16
31
49
30–54
0.4
8.2
-
23
38
39
1.83
19
33
46
54–112
0.52
8
1
21
39
40
-
19
33
46
112–
158
0.5
8.1
-
19
48
25
-
-
-
-
158–
180
0.56
8.4
1
33
51
16
-
-
-
-
EC: Electrical conductivity; OM: Organic matter; ρb: Soil bulk density; PWP: Permanent wilting point; FC:
Field capacity; SAT: Soil saturation water content Table 1 EC: Electrical conductivity; OM: Organic matter; ρb: Soil bulk density; PWP: Permanent wilting point; FC:
Field capacity; SAT: Soil saturation water content EC: Electrical conductivity; OM: Organic matter; ρb: Soil bulk density; PWP: Permanent wilting point; FC:
Field capacity; SAT: Soil saturation water content In this field experiment, a Line-source sprinkler system was used for irrigation. The system had a primary
pipeline diameter of 63 mm, 11 sprinklers per 6 m lateral, and was operated at 4.5–5 bar. Each sprinkler wetted
a 28-meter square with a flow rate of 0.18 liters per second. The system's design would apply water evenly
throughout the experimental field. Based on the sprinkler-spraying pattern, as the distance from the pipe
increases, the volume of water that infiltrates the soil also decreases, thereby enabling the desired application Page 4/32 Page 4/32 of water treatment. 2.2. Field experiment The winter wheat crop was irrigated at three different levels based on their distance from
the pipeline: severe deficit irrigation (rain-fed, 11 m), moderate deficit (6 m), and full irrigation (1 m). Apart from the initial water provided for uniform plant establishment and a small amount of incidental water
from droplets moved by wind during irrigation, the severe deficit irrigation treatments did not receive any
additional water except for rainfall. The irrigation application rate for every irrigation event was determined by
evaluating the soil water deficit at a distance of 1 meter from the primary pipeline. Rainwater harvesting dishes
were utilized to measure the amount of water applied during the mild deficit irrigation treatment. The sprinkler
system was activated early in the night to reduce water loss from evaporation and wind drift. Before the
experiment, the system was calibrated to ensure precise water application rates throughout the field. The soil
water content before each irrigation event was measured using the neutron scattering method. The irrigation
depth was chosen based on the lack of water in the soil relative to field capacity conditions near the main
pipeline. The depth of the roots was estimated using the Borg and Grims (1986) model and the corresponding
water depth was calculated. Table 2 shows the water applied in various irrigation treatments on various days
following planting and cumulative rain occurred between irrigation events for both crop years. The following equations were used to convert the leaf area index to canopy cover (CC) (Goudriaan and Van
Laar 2012): CC = 1 −exp(−K ∗LAI) CC = 1 −exp(−K ∗LAI) 1 K = min(1, 1.43 ∗LAI −0.5) K = min(1, 1.43 ∗LAI −0.5) 2 2 where K is the light extinction coefficient, and LAI is the leaf area index. where K is the light extinction coefficient, and LAI is the leaf area index. where K is the light extinction coefficient, and LAI is the leaf area index. Table 2. Applied irrigation water depth (mm) and rainfall (mm) for different water treatments in 2004-2005 and
2005-2006 growing seasons. 2.2. Field experiment Page 5/32 Page 5/32 2005-2006
2004-2005
Rainfall
severe
deficiency
Mild
deficiency
Wet
Rainfall
severe
deficiency
Mild
deficiency
Wet
Days
after
planting
-
43
43
43
-
43
43
43
0
-
-
-
356*
0
0
0
58
225
-
-
-
68
-
-
-
211
13
63
118
139
128
2
59
121
-
-
-
147
-
-
-
8
58
103
151
0
31
95
-
-
-
162
-
-
-
6
63
121
166
15
0
42
110
-
-
-
176
-
-
-
4.5
5
64
118
178
0
43
113
-
-
-
190
-
-
-
12
62
122
192
13
62
131
-
-
-
198
15
67
141
-
-
-
210
-
-
-
6
62
139
215
*Accumulated rainfall until 58 days after planting *Accumulated rainfall until 58 days after planting 2.3. Aquacrop model A detailed description of the AquaCrop model is given by Raes et al. (2009) and Steduto et al. (2009). The
AquaCrop model can assist in how environmental conditions affect crop growth and yield. The principal
outputs include biomass production, water productivity, yield, and soil water balance components, such as
evapotranspiration, runoff, deep percolation, and soil water content. Biomass production is essential since the
amount of dry matter accumulated over time measures the crop's growth and productivity (Van Gaelen et al. 2013). Another critical outcome is water productivity, which is the quantity of biomass generated per amount
of water needed to cultivate the crop. This conclusion is constructive for determining the most efficient water
management and irrigation techniques. Considering the dynamic interplay between plant physiology,
environmental conditions, and management techniques, AquaCrop simulates plant development and yield
(Raes et al. 2012). 2.4. Model calibration and evaluation The AquaCrop model was calibrated using wheat plant measurement data from the first year of the study
(2004–2005) and validated using data from the second year (2005–2006) to ensure reliable predictions. Page 6/32 Page 6/32 Adjustments were made to the model's settings based on various soil-plant variables, such as grain yield, dry
matter, evapotranspiration, leaf area index, and soil moisture. Similarly, these parameters were used in a model
validation to compare predicted values with actual measurements, providing a comprehensive evaluation of
the model's accuracy. In addition, the Penman-Montieth method (Allen et al. 1998) was used to estimate the
reference evapotranspiration. The collected information was then summarized and displayed graphically in
Fig. 2, giving readers a bird's-eye view of seasonal mean reference evapotranspiration rates. Table 3 displays the AquaCrop wheat parameters. According to the AquaCrop user manual appendix, a few are
conservative (Raes et al., 2009). This set of parameters was used in the validation to evaluate AquaCrop's
functionality and reliability. These parameters include canopy cover growth and decline coefficients, crop
coefficients for transpiration at the full canopy, normalized water productivity (WP*) for biomass, soil water
depletion thresholds for inhibition of leaf growth and stomatal conductance, and coefficients for adjusting the
HI for inhibition of leaf growth and stomatal conductance. Some crop parameters, such as yield, are not
generalizable because they are cultivar-specific or are affected by management or environmental factors. These parameters, typically specified by users and estimated using measured data from a cropping season
experiment in 2004-2005 (Table 3, Calibrated ones), aid in predicting system performance. Using crop
measurement data, it is possible to tailor the model's required crop characteristics to a specific cultivar. The
AquaCrop model requires regular canopy cover calibration. The canopy cover was monitored in the field
throughout the growing season to track its development. We used the model's features to estimate the initial
canopy cover based on the sowing rate, seed weight, seed number, and assumed germination rate (CCo). The
canopy expansion rates were automatically estimated after entering the emergence, maximum canopy cover,
senescence, and maturity dates. The flowering date, stage length, reference HI, and HI build-up period were used to calculate grain yield. Table 3
shows the FAO's suggested and calibrated values for crop parameters (Raes et al., 2009). Experiment data were
used to calculate the values of non-conservative parameters. 2.4. Model calibration and evaluation All these values are within the FAO's
recommended range except for the reference harvest index. The difference could be explained by the fact that
this cultivar is a short-season variety developed for use in warmer climates. After calibration, winter wheat's ideal irrigation water level and seeding density in the study area were
determined using the AquaCrop model. The model was used to simulate the results under various scenarios to
find the optimal seeding density and frequency of irrigation for maximum grain yield. Their effects were
evaluated for five different planting densities (80, 120, 160, 200, and 240 kg ha-1) and four different irrigation
intervals (7, 10, 13, and 16 days). Over 47 years, from 1975 to 2021, we ran these simulations. We found the
best seeding density and irrigation schedule to achieve maximum yield and water productivity by analyzing the
simulated grain yield under varying conditions. Page 7/32 Table 3 Table 3
Calibrated winter wheat parameters of AquaCrop used in this study
Parameter
Unit
Value
Base temperature
°C
0
Cut off temperature
°C
30
Crop coefficient when canopy is complete (KcTr,x)
-
1.0
Canopy decline coefficient (CDC)
%/day
3.1
Soil water depletion factor for canopy expansion (Pupper)
-
0.2
Soil water depletion factor for canopy expansion (Plower)
-
0.65
Shape factor for water stress coefficient for stomatal control
-
5
Soil water depletion factor for canopy senescence
-
0.7
Shape factor for water stress coefficient for canopy senescence
-
2.5
Maximum canopy cover (CCx)
-
100
Reference harvest index (HIo)
%
16
Minimum effective rooting depth
m
0.1
Maximum effective rooting depth
m
1.3
Canopy growth coefficient (CGC)
%/day
2.9
Time from sowing to emergence
Day
13
Time from sowing to maximum rooting depth
Day
192
Time from sowing to start senescence
Day
225
Time from sowing to maturity
Day
248
Time from sowing to flowering
Day
178
Length of the flowering stage
Day
28
Seeding rate (kg ha− 1)
Plants per hectare
Normalized water
productivity (WP*, g m− 2)
80
2026667
14
120
3040000
14
160
4053333
20
200
5066667
21
240
6080000
18 2.5. Statistical analyses To compare the simulated and observed values, the normalized root means square error (NRMSE), the
agreement index (d), and mean relative error (MRE) statistical indices were used. The formulas for these
measures are as follows: (3) MRE = × 100
∑
p
i=1( )
si−mi
mi
p (6) where RMSE is the root mean square error, M is the average of the measured values, p is the number of
observations, m is the measured value, and s is the value simulated by the model. The excellent value for
NRMSE is less than 10%; the range of 10–20% indicates a good prediction, and 20–30% suggests a fair
prediction. The model performance is poor if its value exceeds 30% (Jamieson et al. 1991; Mahbod et al. 2015). The closer the d value is to one, the better the model's prediction. where RMSE is the root mean square error, M is the average of the measured values, p is the number of
observations, m is the measured value, and s is the value simulated by the model. The excellent value for
NRMSE is less than 10%; the range of 10–20% indicates a good prediction, and 20–30% suggests a fair
prediction. The model performance is poor if its value exceeds 30% (Jamieson et al. 1991; Mahbod et al. 2015). The closer the d value is to one, the better the model's prediction. Various statistical techniques, both parametric and nonparametric, can be used to analyze trends within time
series data. In the current study, the normality of the predicted annual yield from 1975 to 2021, as estimated by
the AquaCrop model, was assessed. Based on the results of the normality test, either the Mann-Kendall or
Spearman's Rho correlation coefficient tests were employed to analyze the yield trends. A positive correlation
coefficient value indicates an increasing trend in yield over time, while a negative value indicates a decreasing
trend. When there are numerous independent variables, the Wilks lambda test is typically employed to examine if
there are statistically significant differences between groups. The goal is to use predetermined variables to
foretell which individuals will be assigned to which group (Rencher and Christensen, 2002). Through this
analysis, we can see if the categories' differences matter. This test is instrumental in the environmental
sciences, engineering, and agriculture, frequently using numerous variables (Rezaali et al. 2020). 2.5. Statistical analyses The Wilks
lambda test can help researchers zero in on the most crucial variables in explaining observed group
differences, leading to more robust predictive models. This study used the statistical software SPSS version 26
to perform the Wilks lambda test (SPSS 2019). Page 9/32
Multiple comparisons of means frequently make use of the Duncan's test, a post hoc statistical technique. After an analysis of variance, the Duncan's test calculates the difference between each set of means; if the Multiple comparisons of means frequently make use of the Duncan's test, a post hoc statistical technique. After an analysis of variance, the Duncan's test calculates the difference between each set of means; if the Page 9/32 difference is statistically significant, the means are said to be significantly different (Duncan 1955). The test
ranks the means and identifies the categories with substantially different means. This makes it possible to
determine whether set has the highest or lowest mean. The Duncan's test is widely applied in diverse fields of
agricultural and environmental sciences (e.g., Moradi et al., 2022). 2.7. Economy functions Production costs are classified as either fixed or variable. Fixed costs include land rent, planting operations,
equipment, and irrigation (rain) system design, whereas variable costs include seeds, applied water, fuel, labor,
and product transportation. Data from the Fars Province Regional Agriculture Service (http://shiraz-fajo.ir) were
used to calculate the cost function. Wheat has a fixed cost of 86647763 Rials (Rls) per hectare. Wheat seed
costs 42297 Rls kg− 1, irrigation water costs 8046.6 Rls m− 3, and pumping energy costs 990 Rls m− 3. Wheat
costs 80000000 Rials per tonne. (155000 Rls in the studied year = 1 United States Dollar) The net income per unit land is calculated as follows: (7)i (W , D) = PC×Y (W , D) - C (W, D) where
is the net income in Rials per hectare;
is the price of wheat in Rials per tonne; W is the
amount of irrigation water in m3 ha− 1; D is the seeding density in kg ha− 1;
is the grain yield in tonnes
per hectare; and
is the total cost in Rials per hectare. Both irrigation frequency and seeding density
affect Y and C. The cost function is the sum of variable costs and fixed costs, which is formulated as follows:
i(W, D)
PC
Y(W, D)
C(W, D) C (W, D) = 86647763 + 17946.6W + 42297D C (W, D) = 86647763 + 17946.6W + 42297D 8 Where 86647763 is the fixed cost (Rls ha− 1), 17946.6 is the water cost (Rls m− 3) and 42297 is the seed cost
(Rls kg− 1). 2 8 W
d
i i Where 86647763 is the fixed cost (Rls ha− 1), 17946.6 is the water cost (Rls m− 3) and 42297 is the seed cost
(Rls kg− 1). 2.6. Response surface methodology The response surface methodology (RSM) is a statistical tool used in experimental design and optimization
that can be used to study the relationship between an independent variable and an interesting response. To get
the intended result, it is usually used to locate the ideal balance between all the independent factors (Kleijnen
2014). To ascertain which values of one or more independent variables will result in the desired response is the
aim of the response surface methodology. With this method of optimization, a number of trials are scheduled
to collect data on the response variable as the independent variables are altered over a range of potential
values.. Response surface approach is an effective technique for streamlining processes and improving
product quality because it identifies what values of independent variables create the desired response (Khuri
2006). Response surface methodology was used to find the best combination of irrigation water and plant
seeding density by using the RSM package (Lenth 2010) in R software. The response surface methodology (RSM) is a statistical tool used in
that can be used to study the relationship between an independent va
the intended result, it is usually used to locate the ideal balance betwe
2014). To ascertain which values of one or more independent variable
aim of the response surface methodology. With this method of optim
to collect data on the response variable as the independent variables
values.. Response surface approach is an effective technique for stre
product quality because it identifies what values of independent varia
2006). Response surface methodology was used to find the best com
seeding density by using the RSM package (Lenth 2010) in R softwar 3.1. Model calibration After careful calibration and evaluation of the AquaCrop model, the results presented in Fig. 3 reveal promising
predictions for soil moisture content, evapotranspiration, and crop yield. Notably, the NRMSE of 28% for soil
moisture content suggests the model accurately predicted this parameter, despite moderate compatibility with
measured data (d = 0.69) and a slight overestimation (MRE = 0.11) overall. However, the model's accuracy in
predicting low soil moisture was poor (NRMSE > 30%, not shown). Previous studies have also reported
discrepancies between observed and simulated soil water content in AquaCrop, but no clear patterns of
overestimation or underestimation have emerged (Tsakmakis et al. 2019; Ahmadi et al. 2022). These
differences could be attributed to the inaccurate partitioning of soil water loss between soil evaporation and
crop transpiration in the model, leading to overestimation or underestimation of soil water content.(Sandhu
and Irmak 2019; Lu et al. 2021; Huang et al. 2022). AquaCrop simulations have been found to perform better in
fully irrigated environments than in soil water stress conditions, as the model tends to accurately model soil
water content in the absence of water stress but performs less well when water stress levels rise.(Mkhabela
and Bullock 2012; Ahmadi et al. 2015; Masasi et al. 2019, 2020). These discrepancies could also be due to
spatial variability in soil and the use of a hypothetical linear root water extraction pattern (Ahmadi et al. 2022). As such, additional root growth calibration parameters and a more realistic root water extraction pattern have
been proposed as ways to improve AquaCrop's simulations of soil water content (Ahmadi et al. 2015, 2022;
Tsakmakis et al. 2019). On the other hand, the AquaCrop model demonstrated good accuracy in predicting
evapotranspiration and plant cover percentage (NRMSE = 18% and 10%, respectively) with high compatibility
(d = 0.96 and 0.54, respectively). In the AquaCrop model, total dry matter is determined by the product of the cumulative transpiration during the
growing season and the normalized water productivity (WP*, Steduto et al., 2009). Using a single WP* value for
all planting densities resulted in discrepancies between the observed and simulated dry matter (data not
shown). On the other hand, several field experiments have provided evidence that the relationship between
seeding density and both dry matter and yield can be accurately described by a parabolic curve. 2.8. Water productivity Water productivity—crop yield per unit of water consumed—measures agricultural water efficiency. A crop's
water productivity is its ability to yield a certain amount of dry matter or grain yield from a given volume of Page 10/32 Page 10/32 water. Water productivity depends on seeding density and irrigation. Density affects plant water and nutrient
needs and may reduce water productivity due to resource competition. However, optimal irrigation can
maintain soil moisture at appropriate levels for plant growth, while excessive or insufficient water can reduce
water productivity. In this investigation, we used two methods to quantify water productivity. The ratio of yield
to applied water was determined in the first approach, while in the second approach, the ratio of yield to total
rainfall plus applied water was determined. 3.1. Model calibration The parabolic
curve includes three parts: relatively rapid growth (upward part of the curve), slow growth and zero growth
(around the maximum point of the curve), and declining growth (downward part of the curve) (Jin-hua et al.,
2020; Khan et al., 2017; Assefa et al. 2016; Turgut et al., 2005). Grain yield commonly reaches a maximum at
appropriate seeding density, after which it declines due to reduced growth of the lateral branch and pod
formation on lower branches (French 2016; Hu et al., 2017). Earlier studies by Ahmadi et al. (2022), Heng et al. (2009) and Sandhu and Irmak (2019) showed that harvest index, maximum canopy cover and WP* values can
vary depending on crop varieties, planting densities, weather conditions, geographical location, and other
factors. Ahmadi et al. (2022) used two different WP* values for the first half of the growing season until Page 11/32 Page 11/32 flowering and the latter half after flowering for potato. In this study, WP* was assumed to be a function of
seeding density. The relationship between WP* and seeding density was determined during the calibration
stage and is presented in Fig. 4. Results indicated that there was no significant change in WP* with an increase in seeding density from 80 kg
ha-1 to 120 kg ha-1 seeds. However, a significant increase in WP* is observed when the seeding density was
further increased to 160 kg ha-1. Interestingly, WP* exhibited a sinusoidal pattern with a decrease at a seeding
density of 250 kg ha-1. These findings provide valuable insights into the optimal seeding density for enhancing
WP*, which can have practical implications for improving water management strategies in agricultural
production. Based on the relationship between WP* and seeding density, the AquaCrop model performed well
in predicting dry matter for irrigated treatments (NRMSE = 10%, d = 0.84) and demonstrated good accuracy in
predicting grain yield (NRMSE = 18%, d = 0.82) with a slight overestimation (MRE = 0.03). However, the model
poorly estimated above-ground dry matter and grain yield in severe deficit irrigation (rain-fed) treatments, and
therefore, these parameters were not included in the model evaluation (data not shown). This finding is
consistent with prior literature, as reported by Ahmadi et al. (2022), Masasi et al. (2019), Ranjbar et al. (2019),
and Mousavizadeh et al. 3.1. Model calibration (2016), which have shown a higher discrepancy between the simulated parameters by
the AquaCrop and the observations under severe drought conditions. Major reasons recommended by
researchers to decrease discrepancy between simulated results by AquCrop and those observed value,
including using a single crop coefficient instead dual crop coefficient, not considering physiological responses
of crops to water stress, using a root water extraction pattern of 40%, 30%, 20%, 10%, which may not apply to
all plants and stress intensities, not considering effect of other stress such as heat and light intensity dominant
in hot and semi-arid or arid area on plant growth and yield (Ahmadi et al. 2022; Shahrokhnia and Wu, 2021;
Ahmadi et al., 2017). 3.2. Model validation After calibration of the AquaCrop model, as seen in Fig. 5, the model demonstrated acceptable to excellent
accuracy in predicting key parameters, such as soil moisture content, evapotranspiration, and crop yield. Despite moderate compatibility with measured data, the NRMSE of 28% for soil moisture content suggests that
the model predicted this parameter with acceptable accuracy. However, poor accuracy was observed in
predicting low soil moisture (NRMSE > 30%, not shown). Notably, the AquaCrop model exhibited excellent
accuracy in predicting evapotranspiration and canopy cover percentage (NRMSE = 8%), while good predicting
dry matter (NRMSE = 10%) and grain yield (NRMSE = 19%) for irrigated treatments with slight overestimation. These results confirm the calibration process's success and highlight the AquaCrop model's importance as a
valuable tool for predicting soil crop parameters with acceptable to excellent accuracy. Furthermore, the
validation results have further strengthened the model's reliability in predicting key parameters and provided
additional evidence of its effectiveness. 3.3. Winter wheat simulation results This study employed the AquaCrop model to predict dry matter and wheat grain yield for different crop
densities and irrigation intervals from 1975 to 2021, as presented in Fig. 6. The figure clearly depicts that the
predicted yield increased as the plant seeding density increased to 200 kg ha− 1, after which the yield decreased
as the seeding density increased to 250 kg ha− 1. Moreover, the figure also reveals that as the seeding density Page 12/32 Page 12/32 increased, the difference between grain yield at irrigation levels increased, indicating that irrigation significantly
impacted crop yield at higher densities. Additionally, with higher irrigation levels, the deviation of grain yield
across the studied years decreased, implying that a higher irrigation frequency reduced the impact of
environmental factors on plant production. These findings provide valuable insights into the optimal seeding
density and irrigation interval for maximizing wheat grain yield while minimizing water usage and the impact
of environmental factors. Trend of predicted yield data series from 1975 to 2021 was evaluated statistically at 5% significance level
using Spearman’s rho and Pearson and non-parametric and parametric tests, respectively (Fig. 7). Based on the
statistical test for the annually predicted yield, treatments with 80 kg ha− 1 seeding densities and 7- and 10-day
irrigation intervals and 160 kg ha− 1 seeding densities and 10-day irrigation interval had a significant increasing
trend, while the treatment with a seeding density of 240 kg ha− 1 and a 7-day irrigation interval had a significant
decreasing trend. Across all seeding densities, the 16-day irrigation interval had a negative value and a
decreasing trend. These results suggest that the choice of seeding density and irrigation interval can have a
significant impact on crop yield, and that careful management of these factors can lead to increased
productivity. The SPSS Wilks lambda test was used to analyze the interplay between crop density, irrigation frequency, and
total irrigation volume on yield and profit per hectare. The dependent variables were the yield and profit per
hectare, while the seeding density and the frequency with which they were watered were the independent
variables. Table 4 details the statistical characteristics of the Wilks lambda test. With a p-value less than 0.05
in the final column, we can infer that crop density, irrigation interval, and irrigation amount significantly affect
crop yield and profit per unit area. 3.3. Winter wheat simulation results Therefore, seeding density and irrigation interval significantly influence crop
yield and profit per unit area. Table 4
Results of Wilk's lambda test. Factor
Value
F
degree of freedom
p-value
Irrigation
0.997
20.869
1
0
Seeding density
0.279
580.011
4
0
irrigation interval
0.503
265.691
3
0
irrigation interval*seeding density
0.749
25.136
12
0 This study used the Duncan's test in SPSS 23 to examine how changing crop densities (from 80 to 240 kg ha−
1) affected crop yield and profit per unit area. The analysis (Table 5) showed that 200 kilograms of seeds per
hectare resulted in the highest crop yield and profit per unit area. The highest crop density (240 kg ha− 1) was Page 13/32 Page 13/32 discovered to not lead to the highest yield or profit per unit area. Thus, 200 kg ha− 1 was the optimum crop
density for optimizing yield and financial gain. Table 5
Results of Duncan's test to examine effect of changing crop densities on crop yield and profit per unit area. Seeding
density
(kg ha−
1)
(kg ha− 1)Grain yield
80
1.9439
120
2.6552
240
2.7217
160
3.0021
200
3.2410
sig
1.000
1.000
1.000
1.000
1.000
(Rls ha− 1) profit per unit area
80
25917685.1957
120
77339370.1087
240
83053497.7174
160
107770152.9457
200
125497324.3370
sig
1.000
1.000
1.000
1.000
1.000 In addition, the Duncan's test results for the optimal irrigation interval are presented in Table 6, which resulted
in the highest crop yield and profit per unit area. The outcomes of the table highlight the irrigation intervals of 7
to 16 days, respectively, as the most optimal. It was found that the irrigation interval of 7 days resulted in the
highest crop yield and profit per unit area. These results highlight the significance of meticulously considering
crop density and irrigation to maximize crop yield and financial gain. In addition, the Duncan's test results for the optimal irrigation interval are presented in Table 6, which resulted
in the highest crop yield and profit per unit area. The outcomes of the table highlight the irrigation intervals of 7
to 16 days, respectively, as the most optimal. It was found that the irrigation interval of 7 days resulted in the
highest crop yield and profit per unit area. These results highlight the significance of meticulously considering
crop density and irrigation to maximize crop yield and financial gain. 3.3. Winter wheat simulation results In addition, the Duncan's test results for the optimal irrigation interval are presented in Table 6, which resulted
in the highest crop yield and profit per unit area. The outcomes of the table highlight the irrigation intervals of 7
to 16 days, respectively, as the most optimal. It was found that the irrigation interval of 7 days resulted in the
highest crop yield and profit per unit area. These results highlight the significance of meticulously considering
crop density and irrigation to maximize crop yield and financial gain. Page 14/32 Table 6
Results of Duncan's test to examine effect of irrigation interval on crop yield and profit per unit area. Irrigation interval (day)
(kg ha− 1)Grain yield
16
2.719
13
2.5082
10
2.8784
7
3.3926
sig
1.000
1.000
1.000
1.000
(Rls ha− 1) profit per unit area
16
42550511.8174
13
70147786.3043
10
94197968.6957
7
128766157.4261
sig
1.000
1.000
1.000
1.000
3.4. Optimal depth of irrigation water and crop density
The Duncan's test is a useful tool for comparing different treatments and determining which one leads to the 3.4. Optimal depth of irrigation water and crop density The Duncan's test is a useful tool for comparing different treatments and determining which one leads to the
highest yield and profit per unit area. However, it cannot determine the optimal amount of irrigation water and
seeding density. To address this limitation, the RSM method used to determine the optimal seeding density and
applied water in this study. In the case of applied water, two scenarios were analyzed. The first scenario only
considered water from irrigation systems, while the second scenario considered the sum of irrigation water and
rainfall. The results of the RSM analyses are presented in Figs. 8, 9, and 10. Figure 8 depicts water productivity in terms of the quantity of water used and crop density. It shows that water
productivity has improved as crop densities have grown and water consumption has decreased. When only
irrigation water is considered, the optimal density is 188.3 kg ha− 1, and the optimal irrigation water depth is
198 mm. Figure 8 shows that at a density of 162.4 kg ha− 1 and a total of 350 mm of irrigation + rainfall, the
maximum value of water productivity was obtained at around 0.45 kg m− 3, resulting in the highest water
productivity. Similarly, calculations were made to determine the optimum crop density and irrigation water usage for the
maximum profit per hectare (Fig. 9). While the profit per unit area increases with increasing crop density up to
207.7 kg ha− 1 when irrigation water is considered, it decreases with increasing density beyond that point. In
addition, the profit per hectare declines as the amount of irrigation water used rises above 747 mm. Therefore,
a density of 207.7 kg ha− 1 with 747 mm of irrigation water yields the highest profit per hectare (Fig. 9). Using a
combination of precipitation and irrigation in RSM analysis lowered water productivity values to the 0.15–0.45 Page 15/32 Page 15/32 range. Additionally, the profit per area unit increased with increasing cultivation density up to 212.5 kg of seeds
per hectare but then decreased with further increases in density. Similarly, if the sum of irrigation water and
rainfall is greater than 1230 mm, the profit per hectare declines. Overall, the results showed that the highest
profit per hectare was achieved with a density of 212.5 kg and total irrigation water + rainfall of 1230 mm (Fig. 9). range. Conclusion In conclusion, this study demonstrates the promise of the AquaCrop model as a useful resource for forecasting
soil crop parameters with satisfactory to outstanding precision. Reasonable estimates for soil moisture,
evapotranspiration, and crop production have resulted from the model's careful calibration and thorough
review. The NRMSE results imply that the model predicted the parameters with reasonable accuracy, despite
the model's compatibility with measured data being modest in some circumstances. Also, the model's good
accuracy in predicting dry matter and grain yield for irrigated treatments with moderate overestimation and
remarkable accuracy in predicting evapotranspiration and canopy cover percentage demonstrate its efficacy. Also, the study elucidates the optimum seeding density and irrigation intervals for maximizing water
productivity and agricultural yield with minimal water use and negative environmental impact. Increasing
seeding density up to about 200 kg ha− 1 can increase yields while increasing it beyond that can reduce them. As population densities increase, so does the importance of regular irrigation in maximizing food productivity
and mitigating the effects of environmental conditions on plant growth. More statistical support for the importance of crop density and irrigation frequency on agricultural production
and profit per unit area is provided. The findings suggest that crop density, irrigation frequency, and irrigation
volume considerably influence agricultural production and profit per hectare. The research also revealed that
200 kg ha− 1 was the optimal crop density for optimizing output and financial advantage. This research shows that optimizing crop yield, water productivity, and profit per hectare requires thoughtful
planning of crop density and irrigation management systems. This study's findings shed light on the optimum
seeding density and irrigation intervals for maximizing water productivity and crop yield with minimal water
use and environmental effect. Other factors, such as soil type and climate, may also influence crop production
and water productivity, which could be the subject of future investigation. 3.4. Optimal depth of irrigation water and crop density Additionally, the profit per area unit increased with increasing cultivation density up to 212.5 kg of seeds
per hectare but then decreased with further increases in density. Similarly, if the sum of irrigation water and
rainfall is greater than 1230 mm, the profit per hectare declines. Overall, the results showed that the highest
profit per hectare was achieved with a density of 212.5 kg and total irrigation water + rainfall of 1230 mm (Fig. 9). Crop yield can be maximized using the density and optimal irrigation water shown in Fig. 10. According to the
findings, a total of 747 mm of irrigation resulted in the highest product yield. Full irrigation achieved the highest
yield at a seeding density of 210.9 kg ha− 1. Figure 10 reveals that increasing the density of cultivation up to
210.9 kg ha− 1increases the yield while increasing it beyond that causes a decrease in yield and that increasing
the amount of irrigation to more than 747 mm also causes a decrease in yield. C
l
i Competing interest The authors have no relevant financial or non-financial interests to disclose. Data Availability Available from the corresponding author upon request. The authors declare that no funds, grants, or other support were received during the preparation of this
manuscript. Author Contributions All authors contributed to the study conception and design. Field experiment was carried out by Farhad
Partojou. Crop modeling and statistical analysis were done by Majid Habibagahi and Mohammad Reza
Mahmoudi. The first draft was written by Mehdi Mahbod. Ali Shabani conceived the original idea, supervised
the work and prepared final edition of the manuscript. Consent to Publish All authors consented to publish this study Ethical Approval Ethical Approval The authors confirm that the submission is original work and not published elsewhere Page 16/32 Page 16/32 Page 16/32 Consent to Participate Consent to Participate All authors consented to participate in this study. References 1. Ahmadi SH, Ghorra MRR, Sepaskhah AR (2022) Parameterizing the AquaCrop model for potato growth
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improving grain yield and water productivity. Agric Water Manag 266:107580. https://doi.org/10.1016/J.AGWAT.2022.107580 75. Zhang C, Xie Z, Wang Q, et al (2022) AquaCrop modeling to explore optimal irrigation of winter wheat for
improving grain yield and water productivity. Agric Water Manag 266:107580. https://doi.org/10.1016/J.AGWAT.2022.107580 76. Zhang F, Zhang D, Li L, et al (2023) Effect of Planting Density on Canopy Structure, Microenvironment, and
Yields of Uniformly Sown Winter Wheat. Agronomy 13:870 76. Zhang F, Zhang D, Li L, et al (2023) Effect of Planting Density on Canopy Structure, Microenvironment, and
Yields of Uniformly Sown Winter Wheat. Agronomy 13:870 77. Zhou H, Chen J, Wang F, et al (2020) An integrated irrigation strategy for water-saving and quality-
improving of cash crops: Theory and practice in China. Agric Water Manag 241:106331. https://doi.org/10.1016/J.AGWAT.2020.106331 77. Zhou H, Chen J, Wang F, et al (2020) An integrated irrigation strategy for water-saving and quality-
improving of cash crops: Theory and practice in China. Agric Water Manag 241:106331. https://doi.org/10.1016/J.AGWAT.2020.106331 Figures Page 22/32 Page 22/32 Page 22/32 Figure 1 Figure 1 Location of the experimental field. Location of the experimental field. Page 23/32 Page 23/32 re 2
thly reference crop evapotranspiration for Badjgah in (a) 2004-2005 and (b) 200 Figure 2 Monthly reference crop evapotranspiration for Badjgah in (a) 2004-2005 and (b) 2005-2006 growing seasons. Monthly reference crop evapotranspiration for Badjgah in (a) 2004-2005 and (b) 2005-2006 growing seasons. Monthly reference crop evapotranspiration for Badjgah in (a) 2004-2005 and (b) 2005-2006 growing seasons. Page 24/32 Page 24/32 Figure 3
Scatter plot of measured vs predicted values of (a) soil water content, (b) evapotranspira
(d) above-ground biomass, and (e) grain yield by the AquaCrop model in the stage of cali Figure 3 Scatter plot of measured vs predicted values of (a) soil water content, (b) evapotranspiration, (c) canopy cover,
(d) above-ground biomass, and (e) grain yield by the AquaCrop model in the stage of calibration. Page 25/32 Figure 4
Normalized water productivity coefficient in different crop densities. Figure 4 Normalized water productivity coefficient in different crop densities. Normalized water productivity coefficient in different crop densities. Page 26/32 Figure 5
Scatter plot of measured vs predicted values of (a) soil water content, (b) evapotran
(d) above ground biomass, and (e) grain yield by the AquaCrop model in validation. Figure 5 Scatter plot of measured vs predicted values of (a) soil water content, (b) evapotranspiration, (c) canopy cover,
(d) above ground biomass, and (e) grain yield by the AquaCrop model in validation. Page 27/32 Figure 6 Figure 6 Figure 6 Predicted winter wheat grain yield for seeding density of (a) 80, (b) 120, (c) 160, (d) 200, and (e) 240 kg ha-1
and different irrigation intervals during 1975-2020. Predicted winter wheat grain yield for seeding density of (a) 80, (b) 120, (c) 160, (d) 200, and (e) 240 kg ha-1
and different irrigation intervals during 1975-2020. Predicted winter wheat grain yield for seeding density of (a) 80, (b) 120, (c) 160, (d) 200, and (e) 240 kg ha-1
and different irrigation intervals during 1975-2020. Page 28/32 Figure 7 Trend of predicted yield data series from 1975 to 2021 using Spearman’s rho and Pearson, non-parametric and
parametric tests (* statistically significant trends at significance level of 0.05). Trend of predicted yield data series from 1975 to 2021 using Spearman’s rho and Pearson, non-parametric and
parametric tests (* statistically significant trends at significance level of 0.05). Page 29/32 Page 29/32 gure 8
lationship between water productivity (kg m-3) and irrigation water, irrigation water + rainfall a
nsity Figure 8 Relationship between water productivity (kg m-3) and irrigation water, irrigation water + rainfall and seeding
density Relationship between water productivity (kg m-3) and irrigation water, irrigation water + rainfall and seeding
density Page 30/32 e 9
ionship between profit per hectare (Rls ha-1) and irrigation water, irrigation water +
ty Figure 9 Figure 9 Relationship between profit per hectare (Rls ha-1) and irrigation water, irrigation water + rainfall and seeding
density Relationship between profit per hectare (Rls ha-1) and irrigation water, irrigation water + rainfall and seeding
density Page 31/32 Page 31/32 Figure 10
Relationship between crop yield (t ha-1) and irrigation water, irrigation water + rainfall and seeding density Figure 10 Relationship between crop yield (t ha-1) and irrigation water, irrigation water + rainfall and seeding density Relationship between crop yield (t ha-1) and irrigation water, irrigation water + rainfall and seeding density Relationship between crop yield (t ha-1) and irrigation water, irrigation water + rainfall and seeding density Page 32/32 Page 32/32 Page 32/32
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An Upper-Limb Power-Assist Exoskeleton Using Proportional Myoelectric Control
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An Upper-Limb Power-Assist Exoskeleton Using Proportional
Myoelectric Control Zhichuan Tang 1, Kejun Zhang 1,*, Shouqian Sun 1, Zenggui Gao 1, Lekai Zhang 1 and
Zhongliang Yang 2 1 College of Computer Science and Technology, Zhejiang University, Hangzhou 310027, China;
E-Mails: ttzzcc@163.com (Z.T.); ssq@zju.edu.cn (S.S.); gzg@zju.edu.cn (Z.G.);
zlkzhang@gmail.com (L.Z.) 1 College of Computer Science and Technology, Zhejiang University, Hangzhou 310027, China;
E-Mails: ttzzcc@163.com (Z.T.); ssq@zju.edu.cn (S.S.); gzg@zju.edu.cn (Z.G.);
zlkzhang@gmail.com (L.Z.) 1 College of Computer Science and Technology, Zhejiang University, Hangzhou 310027, China;
E-Mails: ttzzcc@163.com (Z.T.); ssq@zju.edu.cn (S.S.); gzg@zju.edu.cn (Z.G.);
zlkzhang@gmail.com (L.Z.) 2 College of Mechanical Engineering, Donghua University, Shanghai 201620, China;
E-Mail: yzl@dhu.edu.cn * Author to whom correspondence should be addressed; E-Mail: channy@zju.edu.cn;
Tel.: +86-571-8795-3695. * Author to whom correspondence should be addressed; E-Mail: channy@zju.edu.cn;
Tel.: +86-571-8795-3695. * Author to whom correspondence should be addressed; E-Mail: channy@zju.edu.cn;
Tel.: +86-571-8795-3695. Received: 12 November 2013; in revised form: 17 February 2014 / Accepted: 20 March 2014 /
Published: 10 April 2014 Received: 12 November 2013; in revised form: 17 February 2014 / Accepted: 20 March 2014 /
Published: 10 April 2014 Received: 12 November 2013; in revised form: 17 February 2014 / Accepted: 20 March 2014 /
Published: 10 April 2014 Abstract: We developed an upper-limb power-assist exoskeleton actuated by pneumatic
muscles. The exoskeleton included two metal links: a nylon joint, four size-adjustable
carbon fiber bracers, a potentiometer and two pneumatic muscles. The proportional
myoelectric control method was proposed to control the exoskeleton according to the user’s
motion intention in real time. With the feature extraction procedure and the classification
(back-propagation neural network), an electromyogram (EMG)-angle model was constructed
to be used for pattern recognition. Six healthy subjects performed elbow flexion-extension
movements under four experimental conditions: (1) holding a 1-kg load, wearing the
exoskeleton, but with no actuation and for different periods (2-s, 4-s and 8-s periods);
(2) holding a 1-kg load, without wearing the exoskeleton, for a fixed period; (3) holding
a 1-kg load, wearing the exoskeleton, but with no actuation, for a fixed period; (4) holding
a 1-kg load, wearing the exoskeleton under proportional myoelectric control, for a fixed
period. The EMG signals of the biceps brachii, the brachioradialis, the triceps brachii and
the anconeus and the angle of the elbow were collected. The control scheme’s reliability
and power-assist effectiveness were evaluated in the experiments. Sensors 2014, 14, 6677-6694; doi:10.3390/s140406677 Sensors 2014, 14, 6677-6694; doi:10.3390/s140406677 Article
An Upper-Limb Power-Assist Exoskeleton Using Proportional
Myoelectric Control
Zhichuan Tang 1, Kejun Zhang 1,*, Shouqian Sun 1, Zenggui Gao 1, Lekai Zhang 1 and
Zhongliang Yang 2
1 College of Computer Science and Technology, Zhejiang University, Hangzhou 310027, China;
E-Mails: ttzzcc@163.com (Z.T.); ssq@zju.edu.cn (S.S.); gzg@zju.edu.cn (Z.G.);
zlkzhang@gmail.com (L.Z.)
2 College of Mechanical Engineering, Donghua University, Shanghai 201620, China;
E-Mail: yzl@dhu.edu.cn
* Author to whom correspondence should be addressed; E-Mail: channy@zju.edu.cn;
Tel.: +86-571-8795-3695. Received: 12 November 2013; in revised form: 17 February 2014 / Accepted: 20 March 2014 /
Published: 10 April 2014 1. Introduction In order to assist physically disabled or elderly people, to increase the strength of the upper limb
and for self-rehabilitation purposes, various upper-limb power-assist exoskeletons and robots have been
developed [1–9]. Kiguchi [1] proposed an electromyogram (EMG)-based impedance control method
to control an upper-limb power-assist robot, which was simple and adaptable to any user. The results
showed that the robot had an effective power-assist performance when users performed some aiming
motions. Yagi [2] discussed an upper-limb power-assist system to assist workers with lifting a 30-kg
rice bag without inducing lower back pain. The system used a pneumatic actuator to support shoulder
and elbow movement. Su [3] presented electromyogram (EMG)-based neural network control of an
upper-limb power-assist exoskeleton robot, which could predict the user’s motion intention precisely. A four degrees-of-freedom system actuated by pneumatic muscles on the shoulder, elbow and wrist was
built to assist the patients with achieving therapy at home or in the clinic [4], which was safe and easy to
use. Rosen [5] constructed an exoskeleton structure, including two links and two joints, to demonstrate
the feasibility of using an EMG-based control. In most previous studies, surface electromyogram (EMG), as one of the neurological signals, is
often used for the control signal of the power-assist exoskeletons, since it directly reflects the user’s
muscle activity level in real time [3,6–9]. However, most EMG control methods belong to on-off
control (constant speed in one direction or full stop) [10–13]. The control method could not understand
or estimate the user’s motion intention in real time. However, real-time control is important, which
could make the exoskeleton augment the power-assist performance effectively [14]. It is especially
important for controlling an upper-limb power-assist exoskeleton, since the motion of the upper limb
is complex in daily life. Proportional myoelectric control is different from on-off control, as it is a
continuous and real-time control method to be used to control prostheses, orthosis and power-assist
exoskeletons [15–18]. Ferris [19–21] built an ankle-foot orthosis actuated by two pneumatic muscles
for studying human walking and assisting gait rehabilitation using proportional myoelectric control. Additionally, there were two other improved versions that offer torques to the knee and hip joints. Fougner [22,23] developed an upper limb prosthesis to evaluate the effects of limb position on
pattern recognition by proportional myoelectric control. Some related tests of wrist rotation and hand
opening/closing were done in this study. Keywords:
upper limb; power-assist exoskeleton; proportional myoelectric control;
pneumatic muscles; motion intention Keywords:
upper limb; power-assist exoskeleton; proportional myoelectric control;
pneumatic muscles; motion intention An Upper-Limb Power-Assist Exoskeleton Using Proportional
Myoelectric Control The results indicated that
the exoskeleton could be controlled by the user’s motion intention in real time and that it
was useful for augmenting arm performance with neurological signal control, which could
be applied to assist in elbow rehabilitation after neurological injury. 6678 Sensors 2014, 14 Sensors 2014, 14 2.1. Subjects Six male subjects (age = 25 ± 3 years, height = 171.0 ± 5.4 cm, weight = 62.1 ± 6.0 kg) participated
in this study. All subjects had a medical examination to eliminate any musculoskeletal and nerve
diseases. Before the experiment, all subjects were requested not to participate in any upper-limb activities
that would lead to fatigue. Sensors 2014, 14 Sensors 2014, 14 6679 the on-off control; (2) it is difficult to control an upper-limb exoskeleton in real time; (3) traditional
control methods are not continuous, which makes user operation and adaption difficult; and (4) most of
the traditional control methods belong to passive control. In order to solve these problems effectively,
an upper-limb power-assist exoskeleton actuated by pneumatic muscles using proportional myoelectric
control was constructed. An experiment protocol was established, including collecting the EMG signals
and the elbow angle, which was used for recording data for the following experiments. With the feature
extraction procedure and the classification (back-propagation neural network), an EMG-angle model
was built to be used for pattern recognition. The angle that was predicted could be transferred to the
control signals of the actuators (pneumatic muscles) according to the corresponding equations. The
mapping from the input signal (EMG signal) to the output signal (voltage value) was set up. The
upper-limb power-assist exoskeleton’s control scheme and the power-assist effectiveness were evaluated
and compared in four experiments. This paper is organized as follows. In Section 2, we describe the hardware of the upper-limb
power-assist exoskeleton and the experimental methods. Section 3 presents the results of the experiments
and some data analyses. The reliability of the control scheme and the positive effectiveness of the
power-assist were found in experimental data. Section 4 explains and discusses the experimental results. Section 5 draws our conclusions. 1. Introduction Muceli [24] proposed a proportional control strategy that could
be practically applied in amputees for the real-time control of multiple degrees-of-freedom. Artificial
neural networks were used as the control strategy to estimate the position of the complex wrist and
hand movement. Pistohl [25] studied efficient ways of high-dimensional proportional myoelectric
control according to many degrees-of-freedom hand prostheses and found some control principles in
prosthetic applications. In general, some of difficulties in controlling upper-limb power-assist exoskeleton based on EMG
signals have been as follows: (1) traditional control methods do not predict users’ motion intention, like 2.2. Hardware We fabricated an upper-limb power-assist exoskeleton for the subject’s right arm, as shown in
Figure 1. It consisted of two metal links corresponding to the arm limbs (the upper arm and the
lower arm), a nylon joint corresponding to the elbow joint, four size-adjustable carbon fiber bracers, a
potentiometer and two pneumatic muscles. The two links could adjust in the length for different subjects. The length of the upper and lower arm links were from 25 to 30 cm and from 20 to 25 cm, respectively. The joint was a 6-cm diameter nylon axis. As a one degree-of-freedom mechanism, the shoulder had a
specific angle (θ1) from 0◦to 180◦, and the angle (θ2) range of the elbow joint could move from 0◦to
145◦(the average human anthropometric boundaries) [26]. The load of the exoskeleton was 2.1 kg. 6680 Sensors 2014, 14 e 1. The overview of the upper-limb power-assist exoskeleton. Figure 1. The overview of the upper-limb power-assist exoskeleton. Figure 1. The overview of the upper-limb power-assist exoskeleton. The exoskeleton was fixed to the arm using four size-adjustable carbon fiber bracers (two for the
upper arm and two for the lower arm). There were cotton linings clinging to the inside of the bracers,
which allowed subjects feel comfortable in their movement. There was a potentiometer that consisted of
a rotation axis, and a fixed end positioned the center of the nylon joint. The rotation axis connected to
the nylon joint, and the fixed end connected to the upper arm link. The exoskeleton was fixed to the arm using four size-adjustable carbon fiber bracers (two for the
upper arm and two for the lower arm). There were cotton linings clinging to the inside of the bracers,
which allowed subjects feel comfortable in their movement. There was a potentiometer that consisted of
a rotation axis, and a fixed end positioned the center of the nylon joint. The rotation axis connected to
the nylon joint, and the fixed end connected to the upper arm link. The exoskeleton was fixed to the arm using four size-adjustable carbon fiber bracers (two for the
upper arm and two for the lower arm). There were cotton linings clinging to the inside of the bracers,
which allowed subjects feel comfortable in their movement. There was a potentiometer that consisted of
a rotation axis, and a fixed end positioned the center of the nylon joint. Sensors 2014, 14 Sensors 2014, 14 6681 Two pneumatic muscles (SPCU-S-1, The Shadow Robot Company Ltd., London) connecting to the
nylon axis could drive the rotation of the joint, as shown in Figure 2. The main parameters of the
pneumatic muscle were as follows: the initial diameter was 20 mm; the maximum contraction rate
was 25%; the initial braiding angle of the fiber was 25◦; and the highest pressure was 0.6 MPa. The
relationship among contraction force, air pressure and contraction rate was defined as [27]: F = P[a(1 −ε)2 −b]
(1) (1) where a =
3πD2
0
4tan2θ0
, b =
πD2
0
4sin2θ0
, D
ε was the contraction rate, F was th where a =
3πD2
0
4tan2θ0
, b =
πD2
0
4sin2θ0
, D0 was the initial diameter, θ0 was the initial braiding angle of fiber,
ε was the contraction rate, F was the contraction force and P was the air pressure. 0
0
ε was the contraction rate, F was the contraction force and P was the air pressure. As shown in Figure 2a, in order to make the joint move in the maximum range, two pneumatic
muscles’ initial air pressure was set as P0, the contraction rate was ε0, the contraction force of both
pneumatic muscles was F0 and the length was L0. When a pressure signal, ∆P, was input to the
pneumatic muscles, the air pressure became P0 + ∆P and P0 −∆P, the contraction rate became εa
and εb, the contraction force became Fa and Fb and the length became L0 −∆L and L0 + ∆L (∆L was
the changes of the length), as shown in Figure 2b. The joint would move because of the unbalance of the
torques, and then, a new balancing of the torques occurred [28]. The changes of length ∆L and the contraction rates, εa, εb, were calculated by: ∆L = θ2πR
180◦
(2) ∆L = θ2πR
180◦ (2) where R is the radius of the nylon joint. where R is the radius of the nylon joint. where R is the radius of the nylon joint. 2.2. Hardware The rotation axis connected to
the nylon joint, and the fixed end connected to the upper arm link. Figure 2. The elbow-joint model. Figure 2. The elbow-joint model. 2.3. Experimental Protocol Upon arrival, subjects were asked to have anthropometric measurements taken (age, height, weight),
and then, the procedures and equipment used for these experiments were introduced to them. Before
each experiment, the subject was requested to wear no shirt. There were two movements of the elbow
joint: flexion and extension. The agonistic muscle and the synergistic muscle of the flexion movement
were the biceps brachii and the brachioradialis [29]. The agonistic muscle and the synergistic muscle
of the extension movement were the triceps brachii and the anconeus [29]. The four pairs of electrodes
were attached to the four muscles of the right arm to collect the EMG signals in the experiments. The
inter-electrode distance was 2 cm. The placement of electrodes was in the direction of the muscle fibers
on the midline of the muscle belly and avoided the innervation zone of the muscles [30]. The position
was marked with a pen to ensure the same positions on every experiment. Before electrode attachment,
alcohol was used to clean the skin, and conductive gel was used to improve the contact of the electrode
with the skin [31]. In each experiment, new electrodes were attached again on the pen mark. After the surface electrodes were attached and the signal was normal, the upper-limb power-assist
exoskeleton was attached to the subject’s right arm. The angle of the shoulder (θ1) was 90◦, and the
angle of the elbow (θ2) was from 0◦to 90◦. There were a total of four experiments. In experiment
one, the subject held a 1-kg load wearing the exoskeleton, but with no actuation, and performed the
elbow flexion-extension movement under three different motion periods (a 2-s, 4-s and 8-s period). The
EMG signals of four muscles and the angle of the elbow were collected. Every subject performed sixty
sets of movement under every motion period. Three models (EMG-angle) corresponding to the three
different motion periods were built. The reliability of the control strategy was evaluated. The conditions
of the other three experiments were as follows: (1) in experiment two, the subject held a 1-kg load by
hand without wearing the exoskeleton; (2) in experiment three, the subject held a 1-kg load wearing
the exoskeleton, but with no actuation; (3) in experiment four, the subject held a 1-kg load wearing
the exoskeleton under direct proportional myoelectric control. 2.3. Experimental Protocol The model (EMG-angle) that had the
best prediction performance in experiment one was used to predict the elbow angle and to control the
exoskeleton. The contrast of the three experiments could evaluate the power-assist effectiveness. Sensors 2014, 14 Sensors 2014, 14 Sensors 2014, 14 6682 Sensors 2014, 14 εa = ε0 + θ2πR
180◦L0
(3)
εb = ε0 −θ2πR
180◦L0
(4) εa = ε0 + θ2πR
180◦L0
εb = ε0 −θ2πR
180◦L0 (3) (4) The contraction forces, Fa and Fb, were calculated by: The contraction forces, Fa and Fb, were calculated by: The contraction forces, Fa and Fb, were calculated by: Fa = (P0 + ∆P)[a(1 −εa)2 −b]
(5)
Fb = (P0 −∆P)[a(1 −εb)2 −b]
(6) (5) (6) According to Equations (3)–(6), the pressure signal, ∆P, was calculated by: ∆P =
Fa
2[a(1 −ε0 −
θ2πR
180◦L0)2 −b] −
Fb
2[a(1 −ε0 +
θ2πR
180◦L0)2 −b]
(7) (7) When the angle, θ2, was measured, the pressure signal, ∆P, could be calculated; as we can see from
Equation (7), the relationship between them was nonlinear. There was a nonlinear relationship between
the pressure signal and the control signal of the pneumatic muscles (voltage), and then, the control signal
was gained according to the related conversion equations. 2.4. Control Scheme As shown in Figure 3, a control scheme based on the proportional myoelectric control was
constructed. After the procedures of the preamplifier, the analog-to-digital converter and the filter, the raw EMG
signal was transferred to the filtered EMG signal. However, it was not suitable as the input signals for
the classifier (back-propagation neural network (BPN)); the feature of the signal should be extracted. As
the time-domain indicator of EMG, the root mean square (RMS) was calculated and used as the feature. The RMS was calculated as [32]:
v RMS =
v
u
u
t 1
N
N
X
i=1
v2
i
(8) (8) where vi is the voltage at the i-th sampling and N is the number of sampling points. where vi is the voltage at the i-th sampling and N is the number of sampling points. 6683 Sensors 2014, 14 Figure 3. The control scheme based on the proportional myoelectric control. BPN,
back-propagation neural network; EMG, electromyogram; RMS, root mean square. The back-propagation neural network (BPN) as the classifier was used for the pattern recognition. A three-layer BPN (input layer, hidden layer and output layer) was created using MATLAB. There were
four nodes in the input layer corresponding to the four features of the EMG (RMS of four muscles), and
there was one node in the output layer corresponding to the angle. The number of the hidden layer’s
nodes should not be too large or small, or it would affect the learning speed and the generalization ability
of the network [33]. At the same time, the hidden layer’s nodes had to be smaller than the input layer
to prevent over train [34]. The nodes of the hidden layer were set to three. BPN had the ability to map
the nonlinear properties through training. During training, some sets of data and classes were selected
to adjust the weights in order to get the proper input-output relation. All sets of data were randomly
selected to separate into the training (80%) sets and the testing (20%) sets. A sigmoid function was used
for the transfer function when training the network. The output is calculated by: The back-propagation neural network (BPN) as the classifier was used for the pattern recognition. A three-layer BPN (input layer, hidden layer and output layer) was created using MATLAB. 2.4. Control Scheme There were
four nodes in the input layer corresponding to the four features of the EMG (RMS of four muscles), and
there was one node in the output layer corresponding to the angle. The number of the hidden layer’s
nodes should not be too large or small, or it would affect the learning speed and the generalization ability
of the network [33]. At the same time, the hidden layer’s nodes had to be smaller than the input layer
to prevent over train [34]. The nodes of the hidden layer were set to three. BPN had the ability to map
the nonlinear properties through training. During training, some sets of data and classes were selected
to adjust the weights in order to get the proper input-output relation. All sets of data were randomly
selected to separate into the training (80%) sets and the testing (20%) sets. A sigmoid function was used
for the transfer function when training the network. The output is calculated by: y = f
X
wixi
=
1
1 + e−(P wixi)
(9) (9) where y is the output, xi is the input, wi is the weighting factor attached to that input, e is the error
function and f
is the transfer function. When the training ended, an EMG-angle model was built, namely the mapping from the four muscles’
EMG signals to the elbow angle was found. An elbow angle (output) could be predicted from the EMG
signals (input) using the model in real time. Then, the value of angle was input to the computer interface. According to Equation (7), the angle could transfer to the air pressure of pneumatic muscles (the control
signals of actuators). There was a nonlinear relationship between the air pressure and the control signal
of pneumatic muscles (voltage), and then, the control signal was gained. The mapping from the input
signals (EMG signal) to the output signal (voltage) was set up. A threshold was applied to eliminate the
noise of the EMG signal. When the EMG signal was below the threshold, no signal (voltage = 0 V) was
output. When the EMG signal was above the threshold, it indicated the start of the movement. A direct 6684 Sensors 2014, 14 relation between the nervous system and the motion of the exoskeleton was founded. Subjects would
adapt to the exoskeleton easily by using simple and smooth (proportional) control. 2.5. Data Collection Four pairs of surface electrodes were used to collect the EMG data from the biceps brachii, the
brachioradialis, the triceps brachii and the anconeus. The sensors (MyoScan sensor), which connected
with electrodes, could record the EMG signals up to 1,600 micro-volts (µV) and an active range from
20 to 500 Hz. The EMG signals during four experiments were collected, amplified and transmitted by
a ten-channel digital EMG system (FlexComp Infiniti System, Thought Technology Ltd., Canada). All
EMG data were collected at 1,024 Hz. Then, the raw EMG signals were high-pass filtered (4th-order
Butterworth; the cutoff frequency was 50 Hz), rectified and low-pass filtered (4th-order Butterworth; the
cutoff frequency was 10 Hz). According to Equation (8), the filtered EMG signals were transferred to
the RMS (time-domain signals of EMG). We applied the overlapped windowing technique to process
data windowing [35]. We set the length of the window as 200 ms (feature extraction), and the processing
time (classification) was often less than 50 ms. Therefore, the window length and the processing time
should be less than 300 ms in total in order to allow that subjects do not feel any delay, which made the
exoskeleton user friendly. The potentiometer (RV30YN30S, TOCOS, Japan) positioned on the center of the nylon joint was
used to measure the angle. The data were collected at 1,024 Hz. When the joint of exoskeleton moved in
angle, the rotation axis of the potentiometer moved in the same angle, and the corresponding voltage was
output at the same time [36]. According to an analog-to-digital converter, the voltage was transferred to
the corresponding angle. The data of EMG and angle were collected and processed synchronously using
MATLAB (MathWorks, Inc., Natick, MA, USA). 3.1. The Evaluation of the Control Scheme All data were collected and processed from the same subject (one of the six subjects), who had
better experimental performance (a better rhythm in the elbow flexion-extension movement). The data
(EMG, RMS and angle) of one flexion-extension movement under three motion periods are shown in
Figures 4–6. The subject performed sixty flexion-extension movements under every motion period. Forty eight sets of data were randomly selected to form the training data, and the other twelve were
selected to form the testing data. A total of three EMG-angle models (a 2-s, 4-s and 8-s period)
were constructed. 6685 Sensors 2014, 14 Figure 4. The muscles’ EMG and RMS and the elbow angle under 2-s period (1–3 s). Figure 4. The muscles’ EMG and RMS and the elbow angle under 2-s period (1–3 s). Figure 4. The muscles’ EMG and RMS and the elbow angle under 2-s period (1–3 s). Figure 5. The muscles’ EMG and RMS and the elbow angle under 4-s period (1–5 s). Figure 5. The muscles’ EMG and RMS and the elbow angle under 4-s period (1–5 s). Sensors 2014, 14 6686 Figure 6. The muscles’ electromyogram (EMG) and root mean square (RMS) and the elbow
angle under the 8-s period (1–9 s). Figure 6. The muscles’ electromyogram (EMG) and root mean square (RMS) and the elbow
angle under the 8-s period (1–9 s). The RMSE (root-mean-square error) and R2 were used to evaluate the prediction performance. The
RMSE showed the error between the actual angle and the predicted angle, which could be calculated by: The RMSE (root-mean-square error) and R2 were used to evaluate the prediction performance. The
RMSE showed the error between the actual angle and the predicted angle, which could be calculated by: The RMSE (root-mean-square error) and R2 were used to evaluate the prediction performance. The
RMSE showed the error between the actual angle and the predicted angle, which could be calculated by: RMSE =
v
u
u
t 1
N
N
X
n=1
(An −A′
n)2
(10) (10) where An is the predicted angle and A′
n is the actual angle. Normally, a smaller RMSE value (close to
zero) and a larger R2 value (close to one) indicates that the prediction performance of the network is
better. Linear regression was used to analyze the difference between the actual angle and the predicted
angle (deviation rate). k and j were the slope and the intercept of the optimal regression line. The value
of k was closer to one, and the value of j was closer to zero, meaning that there was a smaller error
between the actual angle and the predicted angle. The values of RMSE, R2, k and j for each network are shown in Table 1. For the different motion
periods, the elbow movement under the four-second period had a lower RMSE (9.67) and greater
R2 (0.87), when compared with the two-second period (RMSE: 10.70; R2: 0.83) and the eight-second
period (RMSE: 12.42; R2: 0.79). Similar results were obtained from linear regression. Figure 4. The muscles’ EMG and RMS and the elbow angle under 2-s period (1–3 s). Sensors 2014, 14 Sensors 2014, 14 6688 Figure 4. The muscles’ EMG and RMS and the elbow angle under 2-s period (1–3 s). The value of
k (0.9107) was closer to one, and the value of j (4.4201) was closer to zero under the four-second
period, when compared with the two-second period (k: 1.1985; j: −5.0340) and eight-second period
(k: 0.8799; j: 10.6008). The prediction performance of the network under the four-second period was
better than the other two, and the predicted angle was much more closely related to the actual angle. 6687 Sensors 2014, 14 Table 1. The performance of three networks (2-s, 4-s and 8-s period). Table 1. The performance of three networks (2-s, 4-s and 8-s period). Table 1. The performance of three networks (2-s, 4-s and 8-s period). 2-s Period
4-s Period
8-s Period
RMSE
10.70
9.67
12.42
R2
0.83
0.87
0.79
k
1.1985
0.9107
0.8799
j(◦)
−5.0340
4.4201
10.6008 The relation between the actual angle and the predicted angle can be seen in Figures 7 and 8. In the
movement under the four-second period, the curve of the predicted angle was capable of reflecting the
curve of the actual angle well. The error between the actual angle and the predicted angle was smaller,
but there was a little offset. In the movement under the two-second period, there was a larger error
between the actual angle and the predicted angle. From 0◦to 70◦, the predicted angle was larger than
the actual angle. From 70◦to 90◦and 90◦to 0◦, the predicted angle was lower than the actual angle. Especially from 80◦to 90◦, there was the largest error. In the movement under the eight-second period,
there was a larger error between the actual angle and the predicted angle, as well. From 0◦to 90◦, the
predicted angle was lower than the actual angle. From 90◦to 0◦, the predicted angle was larger than the
actual angle. Especially from 90◦to 40◦, there was the largest error. However, the curve of the predicted
angle could roughly reflect the direction and trend of the curve of the actual angle in all movements (2-s,
4-s and 8-s period). Figure 7. The curve of the actual angle and the curve of the predicted angle under three
motion periods during one flexion-extension movement. Figure 8. The optimal regression line of the actual angle and the predicted angle under three
motion periods during flexion movement. Figure 8. The optimal regression line of the actual angle and the predicted angle under three
motion periods during flexion movement. 3.2. The Evaluation of Power-Assist Effectiveness All data were collected and processed from the same subject (one of the six subjects), who had
better experimental performance (a better rhythm in the elbow flexion-extension movement). The
EMG and RMS output in three experiments (experiment two, experiment three and experiment four)
were compared to verify the power-assist effectiveness, as shown in Figures 9 and 10. In the three
experiments, the subject performed the elbow flexion-extension movement under the four-second period
(this was the best prediction performance of the network). The amplitude of the four muscles’ EMG
and RMS was similar in experiment two and experiment three, which meant that the muscle activity was
similar when the subject held a 1-kg load without the exoskeleton and with the exoskeleton, but with
no actuation. The amplitude of the four muscles’ EMG and RMS were decreased while wearing the
exoskeleton under direct proportional myoelectric control (experiment four), which verified the positive
power-assist effectiveness. Figure 10. The four muscles’ RMSs of the three experiments. Figure 10. The four muscles’ RMSs of the three experiments. Figure 9. The four muscles’ EMGs of the three experiments. Figure 9. The four muscles’ EMGs of the three experiments. Figure 9. The four muscles’ EMGs of the three experiments. Sensors 2014, 14 6689 Figure 10. The four muscles’ RMSs of the three experiments. 4. Discussion
This study demonstrated that there was the best prediction performance of the network (EMG-angle)
under the four-second period. When one flexion-extension motion period was short (two-second period),
the motion acceleration would affect the EMG amplitude. The higher speed would lead to the use
of greater force, so the larger EMG amplitude was tested. Because muscle force reached the maximum
value and the minimum value when the acceleration reached the maximum value and the minimum value,
the functional state and the motion state of the nervous system would be in an unstable region, which led
to a worse prediction performance. When one flexion-extension motion period was long (eight-second
period), the speed, which was not constant during the motion period, would affect the EMG amplitude. Because the speed could not be constant in a long motion period, the muscle force of subjects could not
maintain an isometric level, which led to a worse prediction performance. as well. Keeping constant
motion speed and rhythm would obtain the good prediction performance. Several previous studies had
discussed the relation between acceleration, speed and prediction performance. Fougner [23] thought
that the nervous system could control the angles and positions of the many parts of the arm, and it could
control the speed and muscle force, as well. However, the nervous system could not keep the constant
motion speed and the isometric muscle force during a short or long period without external equipment. Au [37] used EMG signals to evaluate the performance of a time-delayed artificial neural network
(TDANN) to predict the shoulder and elbow motions. There was a 20◦difference between the maximum
RMSE and the minimum RMSE at the different speeds and different accelerations. Lee [38] studied
human intention during a lifting movement (up/down movement) from EMG signals with different
speeds (0.2, 0.3, 0.4 and 0.5 Hz). The movement direction was estimated with higher accuracy at speeds
of 0.3 Hz (93.6%) and 0.4 Hz (94.8%), but with lower accuracy at speeds of 0.2 Hz (80.1%) and 0.5 Hz 4. Discussion However, there
were many factors affecting the relation between the EMG amplitude and the muscle force [39], like
muscle length, muscle contraction velocity and fatigue [40]. One subject was requested to perform
elbow flexion-extension movement continuously (more than two hundred times in experiment one)
and hold a 1-kg load in experiments two, three and four, which might have led to the muscle fatigue. Muscle fatigue could induce changes in the EMG signals: the amplitude and RMS (root mean square) of
EMG would be increased [41]; the MF (median frequency) and MPF (mean power frequency) of EMG
would be decreased [42]. These changes could affect the prediction performance of the network and the
power-assist effectiveness. Muscle fatigue should be considered an important factor in our future studies. The actuator of the upper-limb power-assist exoskeleton in this study was the artificial pneumatic
muscle. Due to the greater range of motion at the elbow (from 0◦to 145◦), the contraction rate of the
pneumatic muscle needed to be considered. However, pneumatic muscle could only shorten or lengthen
about one third of the initial length. It could not be stretched beyond its limit length, which led to a limit
on the range of motion. We reduced the diameter of the nylon joint and made the range of motion be from
0◦to 90◦, which was still less than the average human anthropometric boundaries. The portability and
flexibility of the exoskeleton was another limitation. Due to the heavy air supply equipment (air pump),
the subject could not move around wearing the exoskeleton. Future designs could choose different
actuators (servo motors or hydraulic cylinders) to alleviate these limitations. According to the above results of our study and previous studies, proportional myoelectric control
had some advantages and disadvantages for the control of an upper-limb power-assist exoskeleton
compared to the other control methods. The advantages were: (1) it provided an effective way to
control the exoskeleton in real time using biological signals [43]; (2) it led to a greater mechanical
assistance to improve the power-assist effectiveness; and (3) the nervous system could easily adapt
the exoskeleton control for different motions [40]. The disadvantages were: (1) it was hard to obtain
the same EMG signals from one motion, even with one person [1]; and (2) every subject needed to
go through the system training (BPN) to recognize the input signals, due to the individual differences
of subjects. 4. Discussion This study demonstrated that there was the best prediction performance of the network (EMG-angle)
under the four-second period. When one flexion-extension motion period was short (two-second period),
the motion acceleration would affect the EMG amplitude. The higher speed would lead to the use
of greater force, so the larger EMG amplitude was tested. Because muscle force reached the maximum
value and the minimum value when the acceleration reached the maximum value and the minimum value,
the functional state and the motion state of the nervous system would be in an unstable region, which led
to a worse prediction performance. When one flexion-extension motion period was long (eight-second
period), the speed, which was not constant during the motion period, would affect the EMG amplitude. Because the speed could not be constant in a long motion period, the muscle force of subjects could not
maintain an isometric level, which led to a worse prediction performance. as well. Keeping constant
motion speed and rhythm would obtain the good prediction performance. Several previous studies had
discussed the relation between acceleration, speed and prediction performance. Fougner [23] thought
that the nervous system could control the angles and positions of the many parts of the arm, and it could
control the speed and muscle force, as well. However, the nervous system could not keep the constant
motion speed and the isometric muscle force during a short or long period without external equipment. Au [37] used EMG signals to evaluate the performance of a time-delayed artificial neural network
(TDANN) to predict the shoulder and elbow motions. There was a 20◦difference between the maximum
RMSE and the minimum RMSE at the different speeds and different accelerations. Lee [38] studied
human intention during a lifting movement (up/down movement) from EMG signals with different
speeds (0.2, 0.3, 0.4 and 0.5 Hz). The movement direction was estimated with higher accuracy at speeds
of 0.3 Hz (93.6%) and 0.4 Hz (94.8%), but with lower accuracy at speeds of 0.2 Hz (80.1%) and 0.5 Hz Sensors 2014, 14 Sensors 2014, 14 6690 (83.0%). Motion acceleration and speed would affect the prediction performance to some extent. The
relations between the acceleration and speed and the prediction performance will be discussed in our
future studies. The results in Figures 9 and 10 demonstrated a clear reduction in four muscles’ EMG amplitude while
wearing the exoskeleton under direct proportional myoelectric control (experiment four). 5. Conclusions In this paper, we developed an upper-limb power-assist exoskeleton actuated by pneumatic muscles. The proportional myoelectric control method was proposed to control the exoskeleton according to the
user’s motion intention in real time. The elbow angle was estimated based on the EMG signals in the
proposed method. A back-propagation neural network (BPN) was applied to construct the EMG-angle
model to make the exoskeleton adaptable to every subject. There were a total of four experiments in this
study. In experiment one, the reliability of the control scheme was evaluated according to the prediction
performance of networks under different motion periods, and there was the best prediction performance
under the four-second period. In experiments two, three and four, the power-assist effectiveness was
evaluated under different conditions, and a positive effect was verified. The results indicated that the
exoskeleton could be controlled by the user’s motion intention in real time and that it was useful for
augmenting arm performance with neurological signal control, which could be applied to assist with
elbow rehabilitation after neurological injury. 4. Discussion The development of EMG electrodes and the optimization of control algorithms could
overcome these disadvantages in the near future. At the same time, according to the state-of-the-art
in this field, using EMG signals to control an upper-limb exoskeleton faces challenges for clinical use. Scheme and Englehart [44] thought that the dexterity with which one might control an exoskeleton
had progressed very little, especially when controlling multiple degrees of freedom. Using pattern
recognition to discriminate multiple degrees of freedom had shown great promise, but it had yet to
transition to a clinically viable option. Jiang [45] found that myoelectric control had a great potential
for improving the quality of life of persons with limb deficiency; however, its clinical and commercial
impact is still limited. Given the difficulty of robust control solely by using EMG, the use of other sensor
modalities seems necessary for the control of complex devices. The sensor-fusion approach would be
the research direction in our future studies. Sensors 2014, 14 6691 Acknowledgments This study is partly supported by the National Natural Science Foundation of China (61103100,
61303137, 51205059, 51305077), the National Natural Science Foundation of Zhejiang Province
(Y13F020143), the Shandong Province Outstanding Young Scientist Award Fund (BS2013ZZ014) and
the Fundamental Research Funds for the Central Universities (2014QNA5009, 2232013D3-31). This study is partly supported by the National Natural Science Foundation of China (61103100,
61303137, 51205059, 51305077), the National Natural Science Foundation of Zhejiang Province
(Y13F020143), the Shandong Province Outstanding Young Scientist Award Fund (BS2013ZZ014) and
the Fundamental Research Funds for the Central Universities (2014QNA5009, 2232013D3-31). Author Contributions Zhichuan Tang developed the control scheme, carried out the measurements of the results and took
care of the writing for the work. Kejun Zhang and Shouqian Sun worked on the development of
the experimental setup, measurements and revision of the paper. Zenggui Gao, Lekai Zhang and
Zhongliang Yang worked on the problem definition and the revision of the results. All the authors
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This article is an open access article
distributed
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and
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of
the
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terms
and
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of
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Attribution
license
(http://creativecommons.org/licenses/by/3.0/). © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
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Las experiencias de relaciones humanas como atisbos de la Gracia. Investigación dialogante con Hans Urs von Balthasar y Krzysztof Kieslowski.
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3 Las experiencias de relaciones humanas como atisbos
de la Gracia. Investigación dialogante
con Hans Urs von Balthasar y Krzysztof Kieslowski
DOI: 10.22199/S07198175.2010.0002.00003
Juan Pablo MOYANO SJ
Resumen
En nuestra vida cotidiana nos relacionamos con otras personas. Algunos son muy cercanos,
como familiares o amigos, otros pueden ser simples desconocidos. Seguramente muchas de estas relaciones no pasan de ser encuentros fugaces o sólo determinados por nuestras necesidades
prácticas. Pero algunas pueden ser mucho más que eso. Súbitamente, si miramos con más atención, podemos encontrarnos mediante ellas con la Belleza, la Verdad o el Amor. Podemos atisbar
la gracia de Dios en el mundo. El siguiente trabajo intenta ser una reflexión sobre las posibilidades
y alcances de tal encuentro gracioso. Para hacerlo dialogaremos con dos sugerentes autores. El
cuerpo de la investigación será la Teología de Hans Urs von Balthasar en su obra Epílogo y sus intuiciones acerca de la presencia de la Gracia en el mundo por medio de la comprensión de los trascendentales del ser. Posteriormente, a manera de una extensa conclusión aplicada, analizaremos
y nos dejaremos cuestionar por la película Bleu del director polaco Krzysztof Kieslowski. Nuestro
empeño es desentrañar esta presencia de la Gracia en medio de nuestra humanidad pretendiendo
el diálogo entre estas dos obras, es decir, entre la teología y el arte.
Palabras clave: Gracia – H.U. von Balthasar – K. Kieslowski – relaciones humanas.
The experiences of human relationships as glimpses of Grace.
A dialogical investigation with Hans Urs von Balthasar and
Krzysztof Kieslowski.
Abstract
In our daily life we interact with other people. Some are very close to us, like family or friends;
others may be just unknown people. Surely, many of these relationships are no more than fleeting
encounters or they are just determined by our practical needs. But some relationships may be much
more than that. Suddenly, if we look closely, through them we can find Beauty, Truth and Love. Even
more, through them we can get a glimpse of the Grace of God in the world. This paper aims to be
a reflection on the possibilities and scope of such a gracefulness encounter. In order to do it, we
are going to dialogue with two authors. The body of our research will be based on von Balthasar’
s theology, specially his work Epilogue and his intuitions about the presence of Grace in the world
through out the comprehension of the Trascendentals of being. Lately, as a very extensive applied
conclusion, we will analyze –and let us question- by the film Bleu, directed by Krzysztof Kieslowski.
Our commitment is to unravel the presence of Grace in the middle of our humanity; pretending the
dialogue between these two works: between theology and art.
Key words: Grace – H.U. von Balthasar – K. Kielowski – human relationships.
Introducción
E
n nuestra vida tenemos experiencias radicales al encontrarnos con otras
personas. Lo más común es que estas relaciones sean entre familiares, amigos
profundos o personas significativas. Pero también pueden darse encuentros más
inesperados con otros que son desconocidos. Lo que acontece en ellas es la experiencia del Amor, de una Belleza que nos configura como seres con espíritu. Lo
que quiero decir es que experimentamos en algunos momentos de la vida que ante
la realidad de otro, de un rostro distinto, se ilumina el propio rostro y se atisban
características del Misterio. De esa manera algo tiene el ser humano que con su
llamada puede generar en otro ser humano la experiencia de la Gracia, que no es
otra cosa que la experiencia de Dios mismo, del Rostro, del Amor. Y esta experiencia si bien, no es de la misma profundidad y radicalidad que la del hombre con Dios,
devela el ser de cada hombre y mujer en su realidad más veraz.
Por tanto cabe preguntarnos, ante esta realidad de las experiencias humanas,
¿de qué manera podemos iluminar teológicamente estos acontecimientos? La
experiencia nos abre al milagro del ser que está en los entes, nos preguntamos
¿cómo puede acontecer esto? Podemos tomar como punto de partida y presupuesto, varias características de una metafísica del Amor que nos ayudarán en la
investigación. Primeramente, el Ser se-muestra, se da a conocer, se presenta en el
mundo; la pregunta es por qué medios ¿será por el hombre?, ¿por cuáles seres humanos concretos?, ¿por cuáles actuaciones de esos seres humanos? En segundo
lugar el Ser se-da, sólo puede existir en relación, recordando acá la realidad perichorética en la Trinidad, esta entrega tiene una doble injerencia en el que se da: se
vacía pero al mismo tiempo se llena con la respuesta del otro; pero ¿qué pasa con
los hombres y las mujeres que nos parecen feos, sin importancia, repugnantes,
malos, injustos, sin nada que entregar?, ¿se nos dará algo en el pobre, el que aparece como un no-otro, excluido, con vacíos internos y externos, que al parecer es
pura necesidad?, ¿No fue así Jesús para sus contemporáneos? Y la última de las
características: el Ser se-dice, se expresa en palabras y le acontece al hombre, en
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gran medida porque lo oído es lo único que el ser humano no procesa sino que le
viene tal cual es, desde acá nos cuestionamos: ¿cuánto hay de la Gracia del Logos
eterno en el ser que se devela en la comunicación?
La pregunta que está a la base de nuestra investigación será acerca de la condición de posibilidad del hombre de acceder a la Gracia que es Dios mismo. Pero
al ser el hombre y la mujer un ente concreto histórico, no podemos olvidar que ese
acceso es primariamente mediante lo que puede conocer desde su naturaleza. Y
por medio del llamado de un otro u otra como él que en la experiencia le abre la
posibilidad de abrirse al ser. El desafío será reconocer qué es lo que posibilita este
atisbo de la Gracia. ¿Qué responsabilidad le cabe al Espíritu Santo en esta relación entre seres humanos?, ¿Cómo la realidad del rostro de Cristo fijado en cada
ser humano transforma para ese y para otros la experiencia humana?, ¿Cuánto
de la Gracia se develará por medio de la relación con otros?, ¿De qué manera el
acontecimiento de la Encarnación, Cruz y Resurrección de Cristo, posibilita una
Metafísica nueva que hace de las relaciones entre los entes finitos una forma
de explicitación del infinito?, ¿No será que el mundo se puso “patas para arriba”
después de Cristo y nos supuso una nueva relación entre los hombres y mujeres,
entre Dios y los hombres, en que somos algo que Dios es?
Tomaremos como texto base para la investigación la síntesis que hace Hans
Urs von Balthasar de su Trilogía en la obra Epílogo. Desarrollaremos el planteamiento de este autor acerca de nuestro tema. En primer lugar lo referente a la
centralidad de Jesucristo y la manera en que es la clave para entender al hombre.
Y luego, a modo de una gran conclusión, haremos dialogar lo que hemos recogido
de nuestro autor con la película Bleu del director polaco Krzysztof Kieslowski; todo
esto como explicitación desde el cine de lo reflexionado, acogiendo que muchas
veces las palabras no alcanzan a expresar el misterio, y el arte viene en nuestro
auxilio.
I.- LA CENTRALIDAD DE JESUCRISTO
Nuestra pregunta se posiciona radicalmente dentro de una antropología profunda. Incluso nuestra motivación más primigenia es sobre la pregunta del hombre, sus características y capacidades. Para el hombre de Fe la realidad de la
Gracia actuante en el mundo es profundamente transformante. La actuación de
Dios en la realidad, sea ésta individual o comunitaria, se nos aparece por muchos
medios. Todos ellos son modos en que la gracia se presenta, trata de llevar a cabo
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su misión y en definitiva “interactúa” con el mundo. Esta presencia del Espíritu de
Jesús, la gracia, en la realidad de hombres y mujeres es lo que hace finalmente
posible toda religiosidad personal y comunitaria en esta vida. Decimos esto precisamente porque toda la historia del cristianismo ha sido un gran devenir dentro de
un desafío: relacionarse de buena manera con Dios.
Ahora bien, esto podríamos afirmarlo para cualquiera de las religiones de la humanidad. Todas han querido relacionarse con la divinidad y encontrar así su paso
por este mundo de hombres y mujeres. Esta es la razón por la cual Balthasar comienza su libro con un capítulo dedicado a relacionar lo específico cristiano con la
esencia y afanes de otras religiones, monoteístas o no. La conclusión es que solamente el cristianismo va más allá y por eso “ve más”. Lo específico del cristianismo
es que ese Dios se ha encarnado, y este acontecimiento sí que ha transformado
radicalmente la realidad. Pero no es solo la realidad que se ha transformado sino
también la vida de cada uno de los seres humanos que habitan esta tierra. La dinámica de un Dios que se encarna nos lleva a pensar profundamente en la humanidad
nueva que de ahí emerge.
La pregunta por el ser del hombre puede ser respondida por el mismo, esta ha
sido la pretensión eterna del ser humano en la historia. Y lo han tratado de responder por tres caminos: el escepticismo, una reducción de la filosofía a la antropología y una mirada desde la filosofía-religiosa. Esta última en la historia ha sido o
monista o dualista. Y los dos han fracasado, tanto los dualistas: antropología científica; como los monistas: todos los caminos de las religiones orientales. El monismo
quiso superar el dualismo (filosofía) pero se encontró con que salvando a Dios se
olvidó del hombre. A continuación un ejemplo de esto: Si la apariencia mundana se
entiende, pues, como lugar de aparición de lo divino en el mundo humano (avatara),
entonces será visible una posibilidad, la de experimentar epifanías de lo divino bajo
las formas transitorias del mundo, ya sea en el ser singular que manifiesta lo divino,
ya sea en una determinada categoría de hombres, que, como los gnósticos, encuentran en sí un núcleo divinal y procuran liberarlo de su envoltura de lo material
aparente. Si se radicaliza esta perspectiva, entonces todo el mundo fenoménico
puede convertirse en una especie de organismo de lo divino, como en la estoa y
sus múltiples derivaciones; el hombre debe reconocer entonces la identidad de su
“chispa anímica” con el gran fuego central divino del ser del mundo y procurar vivir
prácticamente lo que pide de él: que nivele como insignificantes para su realidad
íntima las diferencias de lo que le afecta en su situación mundana.1
1 BALTHASAR H. U. von, Epílogo. Ediciones Encuentro. Madrid. 1998. Pp. 24-25.
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La pregunta es ¿qué tiene el acontecimiento de Jesucristo que hace que veamos al hombre de distinta manera? Y por otra parte, siendo la exhalación de la
gracia una de las consecuencias de ese acontecimiento, ¿de qué manera ese Otro
es el que posibilita todo acercamiento a la gracia desde las relaciones humanas?
Estas son preguntas capitales pues nos llevan al centro del misterio del cristianismo y por tanto también del hombre. Y nos abren a la verdadera posibilidad de
hablar de una experiencia de la gracia en la realidad. Para esto debemos de tomar
en serio la centralidad de Jesucristo, sobre todo de su Encarnación y su Pasión.
Es esto lo que precisamente desarrolla Balthasar en todo el tercer capítulo de su
libro, el cual ha llamado: Catedral.
Hubo Dios de hacerse uno de nosotros para que pudiéramos acercarnos a
contemplar la verdad, la belleza y la bondad. Pues en Cristo se mantiene, y debe
permanecer siempre, la máxima de la similitudo in major dissimilitudo. La major
dissimilitudo deberá revelarse siempre dentro de la similitudo, sino el hombre no
podrá relacionarse con lo divino como ha pretendido en toda su historia la humanidad. Para nuestro autor, Jesucristo es la verdadera clave para entender al hombre
y a Dios, es por esto que debe ocuparse en desarrollar esa segunda persona de la
Trinidad que se ha encarnado dándole mucha relevancia.
Jesús LA analogia entis
Un presupuesto importante es que Jesucristo es la analogia entis por excelencia. Cuando Jesús se iguala al Padre lo que está haciendo es decir que en él, ser
finito, se da la adecuada afirmación de Dios. Para adentrarnos en esta realidad
de Dios que es una realidad paradojal necesitamos de la analogia entis. Analogía
dice relación con “proporción” por eso se puede relacionar con el término “analogía proporcional” de Santo Tomás. Pero la analogia entis significa que el ser de
un ente es aclarado por otro ente. Por tanto se infiere que hay coincidencia y diversidad entre estos dos entes. Para nosotros en la analogía la matriz es el Espíritu
Santo: que es la unidad de la diversidad (Padre-Hijo), no una semejanza imperfecta de dos cosas, sino una perfecta semejanza proporcional entre cosas totalmente
desemejantes. Y también es el que posibilita la relación analógica entre otros dos
entes: Dios y el Hombre. Tiene que haber acá semejanza y diferencia, de ahí que
haya analogía entis entre ellos. Así como se da dentro de la Trinidad. Queda claro
que esta identidad de Jesús hace posible para cualquier ser humano entrar en
una nueva relación con Dios: “Su persona se revela en su aparición sensible (en
los tres trascendentales) tan convincentemente que puede decir: “Quien me ve a
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mí, ve al Padre” (Jn. 14, 9)… En la demanda de un tal mirar a través de lo sensible
hay, por cierto, una pretensión exagerada de la naturaleza humana; por esto, para
el logro de esta demanda, le es prometido el Espíritu Santo, que “introducirá en
toda (mi) verdad (Jn 16, 13). Pero como Jesús en cuanto Hijo de Dios se ha hecho
verdaderamente hombre, así el Espíritu no quedará flotando por encima de la comprensión humana, sino que entrará en esta, para, en unión con ella, capacitándola,
posibilitar la demandada perspectiva del hombre Jesús para lo divino”2
Podemos notar acá la presencia de la Trinidad en esta nueva creación que se
ha ido conformando. El Padre envía al Hijo que no toma nada de sí mismo sino de
su relación con El y luego el Espíritu Santo posibilita que el hombre Jesús se vincule estrechamente con lo divino. Incluso podemos ver en este texto que para Jesús
también es necesario (igual que para cada uno de nosotros) que su comprensión
humana sea inhabitada por el Espíritu para entrar desde su humanidad en relación
con su Padre.
Es importante dejar claro junto con Balthasar que la afirmación acerca de
lo analógico de Jesús no debe suponer que se suprima el axioma fundamental
que explica en el comienzo de sus reflexiones: “De ahí la afirmación fundamental
“esse significat aliquid complutum et simples, sed non subsistens”: “ser real significa algo completo y simple, pero sin existencia en sí” (sino sólo en entes particulares) (Thomas, de pot. 1,1) El todo de la realidad sólo existe en el fragmento de
un ente finito, pero el fragmento no existe más que por el ser real”3.
Dios es el único subsistente en sí, lo cual complica aún más su encarnación,
pues todo ente es completo y simple pero no subsistente. Dios está más bien, por
encima del ser y por eso su esencia no es que tenga o manifieste las procesiones
divinas sino que las es en sí mismo. Por tanto pulcrum, bonum y verum en Dios se
manifiestan idénticos a las personas. Por tanto la analogía entis todavía nos es
insuficiente para decir cómo Jesús es la imagen del Padre que se aparece en el
mundo. Por esto el ser del Padre es el que siempre se dona hacia el Hijo, entre los
dos solo puede haber relación de total generación-recepción-devolución; por eso
la centralidad del amor en Dios. En este amor de Dios se halla toda posible verdad
y bondad, se manifiesta lo que es más propio del ser. Y esto se entrega al mundo
mediante el ente particular que es Jesucristo. Pero de una manera bastante particular tal como lo enuncia San Pablo en su carta a los Corintios: 1 Co 1, 26-31.
2 Ibíd., p. 85
3 Ibíd., p. 47
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Al entrar la segunda persona de la Trinidad en el mundo lo que acontece es
que la Palabra de Dios (que está más allá del ser) se revela (diría Balthasar: mostrando, diciendo, dando). Acontece la revelación del Dios uno y trino entero en el
devenir humano. Pero el problema principal que se mantiene, y que según nuestro
autor es uno de las dos grandes novedades del cristianismo, es el de la posibilidad
que el absoluto se haga presente en lo finito: “¿cómo puede hacerse presente
el Absoluto –de manera definitiva- en una efímera forma finita de vida? Desde el
mundo parece esto imposible; pero ¿quién puede decir que tal forma de vida es
desde Dios aprióricamente imposible porque encierra en sí una contradicción?”4
A esta pregunta Balthasar propone dos acercamientos para que sea posible
esto. Por una parte, apoyándose en la carta a los Hebreos (1, 1-2), que Dios ya
ha compatibilizado en tiempos pasados con el pueblo de Israel lo que parecía incompatible. Y en segundo lugar reconociendo que la fuerza divina compatibiliza elementos que parecen desde el mundo como contrarias, y lo hace desde su
amor siempre nuevo, servicio a Dios/servicio al prójimo, espada y enemistad/paz
y reconciliación, ha vencido al mundo/sigue la lucha contra los poderes rebeldes.
Estas tensiones precisamente en Dios y sobre todo con Jesucristo, se mantienen
en El sin violencia y sin esfuerzo. Aquí está presente en toda su profundidad la
paradoja de la realidad del Amor de Dios en el mundo, o más precisamente con
el tema de nuestro trabajo, la Gracia que actúa en una realidad mundana permanentemente quebrada. Y toda esta síntesis se da sólo en Jesucristo, como Palabra
verdadera y definitiva, mientras los hombres han querido buscarla desde ellos han
fracasado, el único capaz de la unidad de lo plenamente humano es Dios mismo.
La verdadera revolución de la frase de San Juan “Dios es amor” (1 Jn. 4, 8) es que
en Cristo eso se hace carne y por tanto opera la síntesis (que incluye toda paradoja) y que muestra al verdadero hombre.
La pregunta que nos queda ahora es: ¿De qué manera esto que ha acontecido
en Jesucristo y que sigue proclamándose hasta el fin de los tiempos por medio de
Cristo resucitado presente entre nosotros, se puede atisbar en los seres humanos
y las relaciones de unos con otros? Antes de pasar a ese tema, que es el central
de nuestro asunto, enunciemos el comentario que Balthasar hace a raíz de esa
manifestación del amor de Dios en Jesucristo. Y es que sólo los de ojos cándidos
pueden reconocer la verdadera forma de Jesús, pues ellos no pierden la mirada
holística.
4 Ibíd., p. 88
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“Pues sólo el ojo cándido puede ver juntamente en su unidad las aparentes
contradicciones en la forma de Jesús, sólo los “nepioi”, los pequeños, pobres,
incultos, no son inducidos, por el amontonamiento de sus tesoros del saber, a considerar por sí los rasgos particulares y a perder de vista la forma ante puros análisis… El poder ver el misterio del amor de Dios mediante el ojo sencillo no está,
por consiguiente, sólo en la complacencia trinitaria como tal, sino absolutamente
también en una disposición del ojo, que, mediante la gracia divina, se ha dejado
dócilmente empobrecer y hacer más cándido”5
Probablemente por esto Jesús declara bienaventurados a los pobres y los limpios de corazón (cándidos), claramente está diciendo que los pobres reales por
naturaleza y los que se hacen pobres y limpios de mirada, conocerán al verdadero
Dios, podrán vislumbrar esa verdad de que Dios es Amor.
La novedad de la Encarnación
Veremos cómo, apoyándonos en Balthasar, lo que posibilita el acercamiento
al misterio por medio de los seres humanos concretos es la dinámica de la encarnación. Esto se fundamentará en nuestro autor de tres maneras, que tendrán
como base común la humanidad asumida radicalmente. En primer lugar, el Logos
encarnado no es simple hombre sino que toma en serio la carne, es un Logos-sarx.
Esta teología del logos-sarx del primer cristianismo afirma que Cristo se hizo cargo
de la carne del mundo pues él mismo se hace carne. La dinámica alejandrina del
movimiento de asumir la carne por parte de la segunda persona de la Trinidad
posibilita hablar de dos naturalezas como lo definirá más tarde el Concilio de Calcedonia. El sujeto Jesús que padece en la cruz es la divinidad que padece, ésta
afirmación es sólo posible hacerla por medio de una precisa teología de la unión
hipostática. En ella la única persona de Jesucristo conserva las propiedades de
las dos naturalezas. Por tanto podemos decir que lo predicado de cada naturaleza
se dice de la persona entera, aunque no todo lo que se diga de la persona se puede achacar a cada naturaleza. Esto significa que pone en el centro de su encarnación a la mortalidad y la caducidad del ser humano. Los padres griegos vieron
claramente que la encarnación tenía que ver con la cruz, que es ahí donde la Palabra termina de asumir todo lo humano: “El sufrimiento salvador del mundo sólo
fue posible dentro del tipo de comunidad con los demás hombres fundamentado
por medio de la materialidad. La encarnación del Logos afecta a toda la naturale5 Idíd., pp. 91-92
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za humana sobre el fundamento del conjunto de los individuos que se basa en la
unidad material”6 Podemos decir entonces, siguiendo la lógica de los polos que se
unen, que toda síntesis en lo humano tiene su base en esta dinámica encarnatoria.
Y más específicamente en nuestro tema, aquí radica la posibilidad de la salvación,
pero no sólo eso sino de la relación con la gracia que es mediada por el Espíritu y
prefigurada en Cristo. Que esto acontezca tiene especial repercusión para nuestro
tema, pues de aquí radica toda posibilidad de hablar de una cristificación de todo
ser humano.
El segundo tema para poder hablar de un atisbo de la gracia en los entes finitos
está en el proyecto “crístico” del mundo. La historia del mundo que comienza con
la creación no tiene más que un proyecto profundamente divino. La humanidad
está “cristificada” desde su comienzo, prológica y escatológicamente el modelo
siempre es Cristo. Por eso San Pablo puede decir que la creación está en “dolores
de parto” (Rm 8, 22). Nosotros estamos también esperando nuestra plenitud pues
ya hemos sido injertados en el cuerpo de Cristo. Tomarse en serio la teología del
cuerpo místico es radicalizar el cuerpo en toda su plenitud y también la afirmación
de que en cada ser humano habita Jesucristo, no ya en su caducidad y mortalidad
sino resucitado. Al unirse resurrección (de Cristo) y mortalidad (nuestra) en cada
ser finito lo que acontece es la posibilidad de rasguñar eso que verdaderamente
somos, pero que está en ciernes en cada uno. ¡Cuánta verdad tiene San Juan al
afirmar lo que somos y que todavía no se ha manifestado! (1 Jn. 3, 1-2). Por eso es
tan importante para nuestra reflexión tomar en cuenta esta cristificación, y Balthasar también le da una centralidad absoluta: “En ambas direcciones, tanto desde
el comienzo de la creación como desde la salvación final, está en el centro la corporalización del Logos, y, en tanto todo el cosmos, en desarrollo ascendente hacia
el hombre, destinado a reinar sobre él como sobre su gran cuerpo, es inseparable
del hombre, la soberanía del Hijo encarnado se hace necesaria para la soberanía
sobre el universo”7
El tercero de los puntos fundamentales es que con esta encarnación que asume la carne y la hace propia en la única persona de Cristo, lo que se transforma es
nuestro propio modo de entender lo carnal en cada uno y el encuentro interpersonal. Además de tomar en serio a cada uno y que no caigamos en un disolverse en
la divinidad que nos sobreviene. Dice Balthasar “mi cuerpo es una inconcebible
6 Ibíd., p. 95
7 Ibíd., p. 96
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zona intermedia entre mí y el mundo”8. Mi cuerpo es el lugar y el medio para el
encuentro con los otros cuerpos y todo el mundo externo. Y es precisamente en el
encuentro con otros donde se me aparece esa doble característica corporal: “Si
se trata en este choque de un prójimo, entonces descubro especialmente ambos
aspectos: el límite de mi libertad y la realidad de la suya, que se me hace realidad
mediante el encuentro de los cuerpos”.9 Pero incluso nuestro autor va más allá y
concede a esta realidad de intersubjetividad la responsabilidad de crear todo conjunto de seres humanos. “Pero donde el choque de los cuerpos se convierten en
percibir mutuo –y esto sucede porque los sentidos corporales devienen ocasión
para el conocimiento y el reconocimiento-, allí y sólo allí se origina la “entre-palabra”, en griego el dia-logo”10. Mediante estos diálogos entre dos sentidos corporales se puede hablar de la posibilidad de la comunidad humana. Ahora bien, según
Balthasar es el verdadero sentido de la encarnación el que nos hará entrar en una
verdadera comprensión del misterio que estamos tratando de entender. Solamente en Jesucristo Dios ha mostrado su palabra profética en que se da un definitivo
mostrarse, decirse y regalarse. Pero nuestra humana mirada nos hace incapaces
de mirar esto que hemos llamado la unidad de lo divino en Jesucristo. Al toparse
con el ser humano que es Jesús de Nazaret, lo que tienen que hacer los discípulos
es mirar de otra forma, por eso es tan importante la fe. San Juan comienza su primera carta haciendo alusión a esto que tanto ha traído controversia y escándalo
(y que seguirá trayendo a lo largo de la historia), y que en definitiva llevó a la cruz
a Jesús: “Lo que existía desde el principio, lo que hemos oído, lo que hemos visto
con nuestros ojos, lo que contemplamos y tocaron nuestras manos acerca de la
Palabra de vida” (1 Jn. 1,1). Probablemente por esto mismo el Evangelista Juan de
tanta importancia a que el creyente tiene que abrirse a un ver distinto, uno que lo
haga tener una mirada de Fe (cfr. Jn. 9, 1-41).
Para Balthasar los que vivimos en el tiempo post-pascual tenemos una ayuda
adecuada para abrirnos a esa realidad de Cristo que ya no es carnal-terreno sino
que pneumatológico. La única manera de que podamos hablar de los atisbos de la
gracia en cada uno es afirmar que el Cristo que sigue presente en el mundo es el
resucitado que actúa permanentemente. Por esto nuestro autor se preocupa tanto
de dejar en claro que toda la sacramentalidad de la Iglesia (recordemos acá que
8 Ibíd., p. 97
9 Idem.
10 Ibíd., p. 98
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para Balthasar Iglesia no es solamente la visible e institucional) nace desde este
modo de ser pneumatológico.
“Y al ser Cristo (corporal por supuesto) sacramento original como aparición,
entrega y afirmación del amor de Dios para el mundo, la Iglesia obtiene parte,
por medio de la universalización eucarística de este cuerpo personalmente entregado, en esta sacramentalidad original, tanto por la inclusión de los creyentes
como miembro de su “cuerpo espiritual creador de vida” (1 Cor 15, 45; o “cuerpo
místico”), como en tanto es fruto de su cuerpo entregado, en cuanto “esposa” que
procede de él, que es “una carne con él” (Ef 5, 31)”11
De aquí podemos afirmar que la presencia de Cristo en cada ser humano está
plenamente asegurada, por medio de este doble movimiento en que la Iglesia se
hace parte del amor de Dios para el mundo: por la inclusión de los creyentes en un
cuerpo místico y como fruto de ese cuerpo de Jesús entregado en la cruz. Ahora
bien, no es necesario ser creyente para poder participar de esta gracia, hay que
recordar la universalidad de la salvación. Basta entonces con abrirse a la gracia
que ya está actuando en el propio cuerpo y que al mismo tiempo se nos presenta
en el cuerpo de los otros con los cuales convivimos.
Como hemos visto, Jesucristo es la analogía por excelencia y donde se da
el definitivo mostrase, decirse y regalarse. Estos tres verbos hacen relación con
los trascendentales del ser los cuales desarrolla Balthasar para dar con eso que
es el ser y que lo constituye como tal. “Lo que se llama las propiedades del ser
(los “trascendentales”), que traspasan todo ente particular, pareció ofrecer el más
apropiado acceso a los misterios de la teología cristiana. De estas propiedades se
resaltaron tres: “bello”, “bueno”, “verdadero”.12 Es con estas categorías con que
podemos vislumbrar el misterio y, por lo tanto más específicamente en nuestro
tema, atisbar la gracia que hay en cada cual. Son ellas las que se nos han mostrado en Jesucristo en toda su plenitud, las que permanecen en su cuerpo pneumatológico y que, por la creación, la encarnación y del cuerpo místico, las podemos
atisbar en la historicidad. Faltará por tanto, para nuestro propósito, explayarnos
sobre estas tres propiedades y hacernos la pregunta sobre la posibilidad de atisbarlas en cada ser humano que se presenta ante el mundo.
11 Ibíd., p. 108
12 Ibíd., p. 46
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II.- LOS TRASCENDENTALES DEL SER
La complejidad en el Ser
La trilogía de nuestro autor: “Estética”, “Dramática” y “Lógica”, está trabajada íntegramente desde estas propiedades del ser que hemos nombrado. Pero
no seríamos lo suficientemente justos con Balthasar si mostráramos cada uno
de los trascendentales independientes y por tanto simplemente presentes cada
cual por sus medios en la realidad humana. Si hay algo que ha dejado en claro el
Epílogo es que los trascendentales se inter- penetran, se relacionan, se jalonan
unos a otros y finalmente son parte simultáneamente de todo el ser. Ahora bien,
en la realidad mundana los entes son distintos entre sí pero contienen la totalidad
del ser, es lo que Balthasar llama el ser real, el cual no tiene subsistencia en sí
pero es completo y simple. Aunque existen entes que tienen más conciencia de
sí y son más transparentes de su esencia que otros. El ser humano, con su autoconciencia como sujeto libre, no puede alcanzar la unidad de esencia y existencia
(ningún ente lo puede hacer) pero si puede darse cuenta que tiene su fundamento
en una realidad superesencial que lo convierte en imagen de Dios. Este prototipo
le asegura la posibilidad de encontrar la manera en que más plenamente puede
ser él mismo. Esta parece ser la grandeza del ser humano, siempre y cuando se
mantenga dentro de la subsistencia en otro y no pretenda darse existencia en sí
mismo (esto es acaso lo que remite el mito de Adán y Eva en el pecado original,
como camino originante de toda perdición del hombre).
Aún más grandeza le cabe al ser humano, la de ser un ente en relación. Balthasar plantea, refiriéndose al ser humano, que tiene una paradoja en sí mismo, la
cual puede parecer una maldición pero que en realidad es una bendición, puede
fundamentarse en sí mismo y además salir de sí mismo para manifestándose se
realice. Es la paradoja principal del hombre y la mujer que se auto-realiza pero que
no lo puede hacer sin los otros.
“La realidad proporciona a todo ente su ser-en-sí (su ser-para-sí en el ente
espiritual), pero también, puesto que todos los entes reales lo son por la única
realidad, su ser-con (su ser-para-un-otro en el ente espiritual). Por eso todo ente
tiene el don de poder “expresarse” frente a otros, lo que presupone una “capacidad interior” de poder comunicarse, que significa un misterioso “partir” “con” los
otros, pues lo que se comunica se da a la vez y –para poder darse- se conserva. El
ser real, que ha sido regalado al ente, entraña en sí, por esto, una dualidad que por
de pronto puede aparecer contradictoria: fundamentarse en sí mismo (lo cual el
simple ente no lo podría realizar desde sí mismo, de lo contrario sería Dios) y salir
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de sí, por una dinámica dada a él, para realizarse también a sí mismo (su interior)
en esta manifestación.”13
Pero en este comunicarse se realiza el darse por excelencia ya que en la manifestación hay un darse, pero no completo sino que una parte. Debe guardarse algo
de sí mismo en el misterio, sino en su manifestación terminaría vaciándose en su
interlocutor y dejaría de ser él mismo. De esta manera se mantiene la singularidad
de cada uno y al mismo tiempo cada cual se regala para afirmarse en su ser en la
relación. No se trata de vaciarse en el otro, como hemos dicho más arriba, sino de
mantener la paradoja y encontrar que en la relación se mantiene la singularidad
(¿Acaso no pasa eso mismo en la Trinidad?).
Precisamente el hecho de aparecer frente a otro es lo que posibilita hablar de
una expresión de los trascendentales. “Pues si la primera propiedad omnirreinante
del ser no se ha de reducir a ningún concepto univoco, así deberá valer necesariamente lo mismo también en el caso de todos los siguientes trascendentales: de
lo verdadero, bueno y bello, que sólo pueden tener su sitio dentro del ser real.”14
Por lo tanto habría que mantener la univocidad de la realidad y el ser real que se
muestra en los entes, para poder seguir hablando de trascendentales que no traspasan los entes sino que se encuentran en ellos. Veremos ahora como cada uno
de los trascendentales del ser son la expresión de lo más humano y lo más divino,
y que por tanto, es nuestro modo de afirmar que la gracia está actuando en cada
ser humano.
Pulcrum: mostrar-se
La afirmación principal es que todo ente mundano es epifánico, se muestra
ante otros y, específicamente el ser humano, con ese aparecer se constituye en
sujeto. Aunque esto ya es mucho decir, Balthasar va un poco más allá, y es que
“La forma de aparición del ente es el modo como éste se expresa, una especie de
lenguaje átono, pero no desarticulado, en el que las cosas no sólo se expresan a sí
mismas, sino siempre también la realidad total presente en ellas, que (como “non
subsistens”) remite a lo real subsistente”15. Esto es importante porque podemos
decir que todo ente que se presenta ante el mundo lo hace de una manera com13 Ibíd., p. 50
14 Ibíd., p. 53
15 Ibíd., p. 55
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pleta, el ser no se puede subdividir, por lo tanto lo que se nos presenta en otra
persona es siempre la totalidad de la realidad presente en ella. Ahora bien, para
Balthasar no toda manifestación puede llegar a ser bella, necesita remitirse a la
profundidad que la origina para que pueda expresarse trascendentalmente. Hace
uso de los conceptos de apariencia y aparición para expresar estas dos posibilidades. Una aparición puede ser bella siempre y cuando este unida a los otros
trascendentales (verdadero y bueno), porque de esa manera alcanzará la profundidad de manifestación y auto-donación. Además solo de esa manera alcanzará el
verdadero grado estético, que no se quede solamente en un aparecer que no lleve
al asombro. Si todo esto no se da la manifestación puede quedar (como muchas
veces en nuestra experiencia mundana) en una simple apariencia. Aquí siguiendo
nuestra tesis, podemos decir que hay algunas relaciones (con ciertas características) que posibilitan esta aparición que es epifanía. La pregunta será entonces,
desde el pensamiento de Balthasar: ¿Cuáles serían esas condiciones para que el
mostrarse se convierta en Bello?.
La primera de la condiciones se puede inferir fácilmente, y nace de la capacidad autoconsciente del ser humano. Claramente esta capacidad de ofrecerse al
otro con una claridad de que la luz que ofrece no le pertenece (recordemos acá lo
que hemos dicho acerca de la paradoja del ser humano), pues reconoce que en el
mero hecho de apropiársela pierde la luz que puede haber en ella. Lo segundo es
que la imagen pase a ser aparición de aquello a lo que remite, y por tanto tenga
la capacidad de no quedarse en la mera presentación de sí misma sino que tenga
una profundidad tal que mueva a hacerse cargo de lo que está subyacente a lo
que se presenta simplemente. Lo tercero es considerar que toda epifanía trascendental puede remitir o llevarnos a la revelación de la realidad absoluta que tiene
como centro a Jesucristo. Y este ejercicio nos puede llevar a tener un profundo
acercamiento a la realidad del misterio. El mismo Jesús se ha revelado y presentado frente a los demás hombres en un hombre particular, con una apariencia que
ha llegado a ser epifanía. Su aparición está desplegada en varias imágenes: representación de Dios el Padre, reconocible en el Espíritu Santo, muerto en cruz, cuerpo eucarístico. Estas son algunos aspectos de la “cosa en si” que es Jesucristo,
aunque aquí ya se presente otra paradoja: Jesús en la cruz, como deformación
de la imagen del Padre. Acoger esa paradoja es la forma precisa de abrirse a la
univocidad de Jesucristo, sin olvidar que es la clave para entender toda epifanía
en nuestra, también paradojal, existencia. Si la existencia paradójica de Cristo es
la condición de posibilidad de hablar de la trascendencia y la gracia en nuestras
propias paradojas. Y esto nos lleva a la última condición, toda aparición que es
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verdadera epifanía debe acogerse desde el regalo intrínseco que contiene, “En
toda belleza hay un momento de la gracia: se me muestra más de lo que tenía
derecho a esperar, por eso se produce el asombro y la admiración de que “haya”
ser en una abundancia que fluye inmensurable, pero que se vierte en entes y ahí
llega a la realidad perfecta; también en mí, que no me debo a mí, sino a él (para mi
eterno asombro). La gracia entitativa que actúa en todo esto es peraltada cualitativamente allí donde el Absoluto se ilumina y se forma acabadamente en los seres
finitos; ante esta gracia por antonomasia, que ya no manifiesta belleza, sino gloria,
ya no se requiere sólo admiración y encanto, sino adoración”16
Tenemos derecho a esperar, por tanto, que se ilumine el Absoluto en cada una
de las verdaderas epifanías, donde lo bello es puerta de entrada a la gloria debido
a la acción de la gracia. Esto es lo que hemos llamado “atisbos de la gracia” que
son posibles en el encuentro con los demás hombres y mujeres.
Bonum: donar-se
Una afirmación importante de los trascendentales es que no pueden existir
sin relación unos con otros. Por tanto podemos decir que lo que se comunica, que
como vimos es siempre la persona en su totalidad, se da a un otro. Y la estructura
polar que hemos reseñado se da también acá, cuando alguien se me regala estoy recibiendo el ser ya que solo ahí puedo darme cuenta que soy un “otro”, y al
mismo tiempo estoy siendo obligado a donarme yo también, para desarrollar esto
que hay dentro de cada uno que es ser auto-donador (no olvidemos que estamos
creados a imagen y semejanza de un Dios que es siempre donación). Por lo tanto
ésta es la base de que la relación de amor entre dos se pueda llevar a cabo en la
libre donación mutua. Para Balthasar es muy importante dejar en claro que este
amor es un derecho de toda persona. Amar y ser amado aparecen acá como una
paradoja, pues el derecho a ser amado por otro no puede ser violento, el amor solo
puede darse gratuitamente. Esto es lo que Balthasar ha llamado la Dramática que
se da siempre en el mundo, todo bonum se necesita para ser persona pero no puede exigirse a otro que me lo de. El ser humano convive en sociedad y por tanto se
presenta ante las demás personas, exigiendo lo inexigible por naturaleza, el amor.
Pero la paradoja existencial no deja sólo estos problemas, está también presente en la incapacidad del amor de obligar a ser acogido. ¿Hasta dónde lo bueno
que regala un ser humano exige ser acogido (no digamos ya correspondido)? Se16 Ibíd., p. 62
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gún nuestro autor claramente el amor está llamado a no ser violento tanto en su
génesis como en la respuesta que puede suscitar. Se trata de mantener incólume
la libertad de todo hombre, es lo que quiere mantener Jesucristo en su predicación, la persuasión excluye toda obligación a acoger. Por eso la donación de Jesús en la cruz, que es un donarse radicalmente y además con una persuasión que
confunde (recordamos acá eso de San Pablo “Cristo en la cruz: locura para los
gentiles y escándalo para los judíos”), es la posibilidad de ofrecer el amor absoluto
y dejar libre al hombre para acoger. De hecho la imagen de Jesucristo es de donación absoluta pero también de libertad infinita, Cristo en la cruz (aunque desfigurado en su belleza) aparece como el que puede dar respuesta a la imperfección de la
libertad amorosa del hombre. En ese camino kenótico de Jesús aparece el fracaso
del darse y mediante este la libertad queda resguardada. La libertad infinita, de
esta manera, va a ofrecer la perfección de la finita. Es lo mismo que expresó el
profeta Jeremías de otra manera, preparando el camino al Señor: “Ya no tendrán
que adoctrinar más el uno a su prójimo y el otro a su hermano, diciendo: “conoced
al Señor”, pues todos me conocerán del más chico al más grande, oráculo del
Señor” (Jr. 31, 34). El conocimiento del Señor acá es reconocer su amor y además
de asombrarse ante él, aunque venga de un mero hombre, abrirse a acoger el don
que se regala que es desde ya una acogida libre.
Verum: decir-se
Dice Balthasar “La autodeclaración en la palabra es más que un mero expresarse en el aparecer o hacer”.17 El sujeto espiritual puede dar a conocer su interioridad con una libertad que solo la da el mero hecho de hacerse consciente, ésta
es más que un mero hablar, por la misma elaboración de lo dicho se está dando a
entender a la persona misma y por lo tanto lo más profundamente humano. Nuestro autor considera que el decirse es el remate del mostrarse y el donarse, pues
es lo que hace al hombre un ser autoconsciente. Si no tuviera la capacidad de
elaborar y manifestar su verdad el hombre y la mujer podría mostrarse y donarse, pero no llegaría a ser una verdadera epifanía que mostrara el trascendental.
Para tener ésta capacidad de elaboración es necesaria la gracia, por de pronto
porque toda capacidad autocomprensiva es al hombre dada gratuitamente. Ahora bien, se puede ir mucho más profundo si nos hacemos cargo de las palabras
de Balthasar en que “Por otra parte, el mostrarse y el darse deben ser también
17 Ibíd., p. 71
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ya, prehumanamente, formas incoativas del decirse, lo que sólo es pensable si
(como continuamente ha inculcado J. Pieper) las cosas mismas son “palabras”
dichas por un entendimiento libre infinito (dicho teológicamente: entes creados en
la Palabra eterna), entes (inconscientes, conscientes o autoconscientes) que solo
pueden decirse perfectamente en el hombre, que es un ente apto para la palabra,
donde su autonomía y su autoentrega entran también en momentos indispensables en su devenir lingüístico”18
Aquí queda clara la supremacía del hombre frente a toda la creación, como
único capaz de palabra y por lo tanto único que puede cerrar el círculo de sentido
que conforman el bonum (bueno), pulcrum (bello) y verum (verdadero). Además el
modo en que los hombres se entienden es el lenguaje, es como nos comunicamos
y cómo podemos formular quienes somos frente a los otros. Estamos acá hablando
de todo medio de comunicación entre los hombres, por lo tanto entra acá el lenguaje expresado por los labios, también el gestual y el de las emociones.
Ahora bien, el signo visible, y a esto apunta claramente las consideraciones
teológicas anteriores, puede ser transportador de mucho más que lo que es capaz
de expresar y entenderse. En el dialogo interpersonal siempre hay un “quedarse
corto” de lo que quiero expresar con toda su profundidad. Es por tanto imprescindible, para que se devele la gracia en un ser humano, que pueda dar cuenta de esa
angostura de sus palabras y gestos, y que se mantenga en su “pobreza” constitutiva. De esta manera es posible no tratar de agotar el misterio y caer en la trampa
de querer simplemente amarrar el ser para “expresarse más adecuadamente”.
El bonum de Cristo como la clave de comprensión
Queda aún precisar la interrelación que tienen los tres trascendentales que
Balthasar ha desarrollado, en su Epílogo le ha dado cierto orden y sistematización
para poder encontrar el mejor modo de explicarlos. Igualmente nos aventuraremos a lo que me parece dos puntos claves desde los cuales es posible construir
toda argumentación de nuestra tesis.
El primero de ellos ya lo hemos ido desarrollando en los puntos anteriores y lo
podríamos formular de la siguiente manera: Jesucristo, como persona histórica,
en su esencia y en su proyecto de mundo, es la clave para entrar a lo que Balthasar llama en su obra la Catedral. Hemos visto como los trascendentales se presen18 Idíd., p. 72
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tan en su plenitud en la segunda persona de la Trinidad encarnada. Hay una verdadera manifestación de lo divino, una imagen que reclama ser reconocida como tal
y que el mismo Jesús dilucida a quién se refiere, en sus palabras al apóstol Felipe
“el que me ha visto a mí, ha visto al Padre” (Jn.14, 9). Es la manifestación por excelencia que cambia el curso de la historia pues ahí lo que se aparece ya no es sólo
un modo trascendental del ser sino que el más allá del ser, el Padre se muestra
en la más radical de las Epifanías. Y lo hace en dos momentos en que la paradoja
del Dios mayor y menor al mismo tiempo se hace más patente: en un pesebre naciendo en Belén y muriendo en una cruz. Ahora bien, debemos hacernos cargo del
hecho de la cristificación de todo hombre, y podemos afirmarlo por dos medios:
la creación de cada uno teniendo como prototipo a Jesús y la conformación del
misterio del cuerpo místico por medio de la Eucaristía (que actualiza la salvación
universal acontecida en la cruz). Estos dos modos hacen que todo hombre y mujer
pueda acercarse a participar de la salvación ofrecida por Jesús y participar de
las gracias que emanan de El. Esta participación de la vida divina que nos ofrece
Jesucristo, hace que el aparecer de los trascendentales de cada uno puedan ser
una verdadera epifanía del misterio.
Mientras que el segundo punto clave, nos aleja de lo presentado por nuestro
autor, a mi juicio, siguiendo a Jesucristo por modelo, el bonum es la clave de toda
formulación del ser y de toda gracia en el mundo. Es cierto lo que hemos dicho del
mostrar-se de Jesucristo, El que es la verdadera imagen del Padre. Por otra parte,
podemos decir que el verum está plenamente expuesto en la Encarnación. Jesús
es la palabra definitiva del Padre en su comunicación con los hombres, es lo que
trata de exponer la introducción de la carta a los Hebreos (Hb. 1, 1-4) y es la fuerza
que tiene el prologo de Juan (“La Palabra se hizo carne”). Pero podemos decir que
todo alcanza sentido en el Amor, desde ahí puede comprenderse más claramente
quien es Dios y quien es el hombre. La palabra definitiva de Dios es Jesús en la
cruz, es el amor hecho carne y la actualización del trascendental que le da sentido
al ser. El hombre sin darse no puede vivir, podrá manifestarse y decirse, pero hemos dejado claro que todo puede quedar en simple aparición si no hay donación
de sí que pueda entablar verdadera relación entre dos seres humanos.
A modo de conclusión: un diálogo con el cine
Quizás una de las cosas que podemos afirmar cada vez que nos acercamos a
lo esencial de la vida es que nos enfrentamos con el misterio. Este misterio se nos
presenta en todo momento como algo inabarcable, cada vez que intentamos de-
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jarlo circunscrito a nuestros propias maneras de ver, nuestros esquemas o ideas,
se nos escapa por sí mismo. En definitiva hay que mantener la realidad del hombre
y de Dios con la necesaria cuota de desconocimiento e inadecuación a nuestra inteligencia. Es esta convicción acerca de nuestro precario conocimiento del hombre y de Dios que nos serviremos de una película al tratar de dar con nuestra propuesta definitiva sobre la tesis que nos mueve esta investigación. Hemos afirmado
que al haber encuentros verdaderamente profundos entre dos seres humanos,
puede acontecer un reconocimiento de la gracia que está en cada uno. Para ello
nos hemos hecho cargo de la presentación que hace Balthasar sobre la centralidad de Jesucristo en el misterio del ser humano y el recurso a los trascendentales
del ser que son expresión de la gracia que ayuda a expresar lo fundamental del
ser humano.
El cine es una expresión artística junto con muchas otras: la música, la poesía,
la pintura y tantas otras. Creemos que toda obra de arte puede ser una buena
manera de decir y elaborar elementos para los cuales las palabras se nos quedan
cortas. Quizás el cine con mayor razón puede dar cuenta del misterio que se nos
escapa, es una expresión artística que utiliza la imagen, la palabra, la música y los
gestos humanos. De esta manera, parece ser adecuado que terminemos nuestra investigación en un diálogo entre el pensamiento de Balthasar (que hemos
presentado) y la historia que nos presenta la película Bleu del director polaco Krzysztof Kieslowski19. Intentando hallar en este filme los ejemplos que nos permitan
vislumbrar de mejor manera lo que Balthasar presenta del Misterio; y porque no…
encontrar algo nuevo.
La película es parte de una trilogía que creó éste director polaco basándose
en los ideales de la revolución francesa. Utilizando como nombre de cada una
de ellas los colores de la bandera francesa: Bleu, Blanc y Rouge; el director nos
entrega una reflexión sobre la libertad, la fraternidad y la igualdad. En el caso de
nuestra película el ideal desarrollado es la libertad. Aunque, quizás haciendo una
analogía con los trascendentales, en las películas de la trilogía estén presentes
los otros tres ideales. La historia trata de Julie, una mujer de unos 35 años, que
en un accidente pierde a su marido y a su hija. La película comienza con el accidente y toda ella será la narración de lo que sucede con Julie, con su duelo y sus
búsquedas al quedar sola. Julie intenta escapar de su dolor, del pasado y de todo
lo que la ate a la realidad. Para eso se cambia de casa, es distante con un amigo
19 Trois couleurs: Blue. Director: Krzysztof Kieslowski. Protagonizada por Juliette Binoche. Polonia.
1993.
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que intenta acercarse amorosamente a ella, deja la música, en definitiva intenta
sólo sobrevivir. Julie y su marido eran músicos y él estaba componiendo una obra
de solo una canción inspirada en la unificación europea (no hay que olvidar que
el director es polaco y que está filmada poco tiempo después de la caída del muro
de Berlín). El título del himno es elocuente: Song for the unification of Europe. Se
trataba de un proyecto que debía ser interpretado sólo una vez en doce lugares de
Europa simultáneamente. Pero el tema queda inconcluso con la trágica muerte.
Pero será la música y algunas personas con las cuales Julie se va topando que la
ayudan a salir de su ensimismamiento y a ir reconociendo algunas verdades de lo
que le ha acontecido.
En primer lugar, tenemos que referirnos a la obra que el marido de Julie está
componiendo cuando muere y que ella completa. La letra de la canción son extractos del himno del amor de San Pablo en Corintios. Y lo decimos en primer lugar
puesto que al parecer es la clave para apreciar la película. Con esto en ella se
expresa la centralidad del amor en la vida humana y de qué manera el hecho de
donar-se es la llave que puede abrir el sentido de la persona humana. En el fondo
la vida de Julie se va desplegando a medida que se va dando su apertura a lo que
los versos de la canción le propone. La clave está en que ella se cierra al amor
durante gran parte de la película y todo se trata de las maneras en las que el amor
intenta irrumpir en su vida y como ella se va dejando seducir. Es un tira y afloja
permanente que incluso no queda concluido ni siquiera al final del filme. Es el
bonum, expresado en la canción de la unificación, pero que está presente también
en la sensibilidad de la mujer prostituta, en el cariño desinteresado de Olivier o
en la justicia del niño que le intenta entregar la cadena que tomó del lugar del
accidente. Hay varios ejemplos de esta centralidad del amor en la película, que los
podemos usar también como elementos que confirmen nuestra posición de que
finalmente el bonum es la clave. A lo largo de toda la película cada vez que a Julie
le preguntan por sí misma, por algo que la haga salir de su ensimismamiento, dejarse interpelar y abrirse ante otro, hay un silencio inmediato y la pantalla se pone
en negro y suenan los acordes del himno central que estamos aludiendo. Esta
escena repetida durante el largo camino de la protagonista remite a la “vida” que
intenta entrar en la existencia de Julie y a la que ella cierra la puerta constantemente. Podemos recordar acá todo lo que dijimos acerca de la libertad de acoger
el bien que se ofrece a cada uno por el prójimo. No se puede “obligar” a otro ni a
acoger ni a responder al amor que se dona, esto no sería profundamente humano
ni lo constituiría en su verdad fundamental. Claramente en la película ella tiene la
199
ANTROPOLOGÍA TEOLÓGICA
capacidad y la decisión de poder negarse a ese otro que intenta entrar a su vida y
parece que a ratos le funciona, podemos verlo en el diálogo con su madre:
Julie: “Ahora sé que solo haré una cosa. Nada. No quiero posesiones, ni recuerdos, ni amigos, ni ataduras. Son todas trampas”
Madre: “Tienes dinero para vivir?, es importante, no podemos renunciar a
todo”
Este es el proyecto de Julie, no tener ataduras, es por esto que trata de alejarse de todos y de pasada de las necesidades de los demás: no toma en cuenta al
hombre que golpean en la calle, no quiere tener relación de amistad con Lucille (la
prostituta), incluso no quiere el cariño que le ofrece el joven único testigo del accidente o de la gente que trabajaba en su casa. Al parecer la idea es ser “nadie”,
lo que le dice al que le arrienda el departamento es elocuente:
Arrendatario: “¿Cuál es su ocupación?”
Julie: “Ninguna”
Arrendatario: “¿En qué trabaja?”
Julie: “En nada”
Arrendatario: “¿Absolutamente nada?”
Julie: “Absolutamente nada”
Finalmente sabemos que desde la mitad de la película ella empieza a dar pasos
para abrirse y reconocerse nuevamente. Las voces del coro del himno suenan
solo dos veces en la película: cuando rompe las partituras en el camión de la basura y al final cuando va con Olivier y al parecer comienza a vivir. ¡Cuánto camino
recorrido hay en lo vivido por Julie! Desde la cerrazón a la bondad en ella y en los
demás hasta la apertura (aunque todavía incipiente) a la donación. No sólo es la
vida que ella abre para sí sino que para los demás en la obra de arte, en la música, que particularmente como hemos visto tiene un significado especial al utilizar
esos versos de la carta a los Corintios.
Pero hay algo más acerca del pulcrum en la película. Y es que al parecer lo
bello no tiene que ver con las apariencias, la manifestación del ser profundo no
se presenta por lo sensiblemente agradable a los ojos o los demás sentidos. Esto
queda muy bien reflejado en elementos simbólicos y personales de la película.
Todo lo relacionado con el ratón que Julie encuentra en su departamento es bien
central en este sentido. Luego tendrá sus crías y para ella eso no importará, buscará la forma de eliminarlos, corre a buscar un gato. Incluso le tiene miedo y no
duerme muy bien por la presencia de la “vida” que está al otro lado de la puerta.
200
CUADERNOS DE TEOLOGÍA - Vol. II, Nº2
La verdad, que es la vida misma que irrumpe, no aparece como algo manifiestamente bonito, incluso es un poco repulsivo. Pero el director de la película se
esfuerza para hacernos caer en la cuenta que ahí donde hay vida las apariencias
no importan. O sea, la gracia puede manifestarse por medio de lo que nos parece
repugnante. No olvidemos que la primera vez que Olivier toca los acordes del himno en el piano la imagen que acompaña a la música es el ratón con sus crías. Por
otro lado la persona que la ayuda a salir de su ensimismamiento, con sus preocupaciones y conflictos, es la prostituta: Lucille. Ella acude a Julie en búsqueda de
amistad y termina ayudándola con lo de los ratones gratuitamente. Esta gratuidad
nos recuerda la capacidad de donarse pero también la verdad que se va revelando
y que motiva a Julie a encontrarse también con la suya. Hay que remarcar que el
momento en que ella comienza a rearmar la realidad de su vida y la verdad de su
marido, que terminará con el darse cuenta de la infidelidad, es en el club nocturno
en que acoge la verdad de Lucille. Incluso es decisivo el diálogo que ahí se da:
Lucille: “Me has salvado la vida”
Julie: “No he hecho nada”
Lucille: “Si, te he llamado y has venido. Es lo mismo”
Se nota acá la interrelación de los tres trascendentales. El bonum en la donación que casi a contrapelo emerge de Julie frente a la situación de Lucille. El
pulcrum que no es sino la manifestación de la persona del otro en toda su complejidad y belleza, aunque aquello nos parezca contradictorio al venir de una prostituta. Y el verum que aparece reflejado en la verdad tanto de Lucille como de Julie
que se da cuenta ahí, en el lugar menos imaginable, de la verdad de su vida. La
autoconsciencia irá desplegándose de a poco, pero ya hay un inicio de apertura.
Acá se da finalmente lo que hemos dicho, Julie empieza a comprender que en un
otro (y también en sí misma, aunque no se dé cuenta fácilmente) hay atisbos de la
gracia que emergen en situaciones de mayor cercanía y diálogo. Para nosotros los
espectadores la gracia emerge por lo inesperado, por los personajes que creímos
no iban a darnos nada o eran sólo carencia. Quizás acá se nos está otorgando
otra mirada nueva a lo que nos sale naturalmente. ¿Acaso no es así también con
la experiencia de contacto con Jesucristo? Y ¿no será ésta la gran paradoja que
nos plantea Jesús en el Evangelio de Mateo, capítulo 25?
Por último, la película también por medio de muchas imágenes y reflexiones
nos ilustra acerca de la relación de no-apropiación que debe tener el ser humano
con lo bello y lo que se dona. Parece ser que en el momento de donarse, de salir
fuera de sí, ya no me pertenece. Y, si una de las cosas que desarrollamos anterior-
201
ANTROPOLOGÍA TEOLÓGICA
mente es que el ser del ente no es divisible, por tanto lo que entregamos ya somos
todo nosotros lo que no nos pertenece. Esto se ve claramente en nuestra película
en dos escenas de Julie, a mi entender claves: los encuentros con el flautista y
el diálogo final con Olivier. El flautista al parecer está tocando música que es de
Julie o su marido, no queda claro, lo cierto es que le reprocha que está tocando
sin derecho pero éste le dice que son de él. Y antes le había dicho a Julie, cuando
le ayuda: “siempre debemos guardarnos algo”. En esto se aprecia la paradoja
existencial que ya hemos visto, tanto en la vida de todo ser humano como de Jesús, tenemos que entregar de lo nuestro para vivir y constituirnos como sujetos
pero debemos guardarnos algo para poder ser singulares y dignos de diálogo y no
terminar por diluirnos en el otro. Y también el dialogo final, cuando ella le ofrece ir
a buscar la partitura que han arreglado ya para ser interpretada:
Olivier: “No iré a buscar esa partitura”
Julie: “¿Cómo?”
Olivier: “No iré a buscarla, lo he pensado bien. Esta música puede ser mi música. Algo torpe y pesada, pero mi música. O la suya, pero habría que decírselo a
todos”
Julie: “Tiene razón”
Parece ser que Olivier ha entendido una de las condiciones para poder atisbar
la gracia que hay en el otro, y que coincide muy estrechamente con lo que hemos dicho de Jesucristo y la salvación que ofrece, que lo necesario es donar-se,
mostrar-se, decir-se a sí mismos, no ofrecer lo que no le pertenece. Aunque lo
propio sea “torpe o pesado”, será “mi música”. Y por eso mismo hay que trasparentarlo. Quizás esa sea la única forma en que Julie comienza a comprender que
la verdad lleva a darse cuenta que el pulcrum y el bonum emergen igualmente de
las personas. A veces sin quererlo, como ha acontecido en el bien hecho a Lucille
al no firmar la lista para echarla del edificio o la forma en que trata al amante de la
esposa sin darse cuenta de su bondad:
Chica: “Patrice me habló mucho de usted”
Julie: “Y que le dijo”
Chica: “Que usted es buena. Que usted es buena y generosa. Que así quiere
ser. Y que se puede contar con usted. Incluso yo.”
Hemos visto en este diálogo entre la película Bleu y toda la reflexión balthasariana dos verdades fundamentales. La primera, es en la mundaniedad y en la
202
CUADERNOS DE TEOLOGÍA - Vol. II, Nº2
relación entre seres humanos donde aparece la gracia y por lo tanto donde podemos atisbar el misterio de esa gracia actuando en el hombre. En la película se
nos muestra a Julie, una persona en total desamparo que pretende la no-relación
y que al parecer no tiene nada que entregar. Finalmente reconoceremos que mediante su aparición en el mundo, y mientras deja espacio a que se manifieste la
gracia, puede abrirse a dar y recibir vida. Es en la mundaniedad, diríamos que
con la Encarnación de Jesucristo se inaugura radicalmente esta verdad, donde se
manifiesta la gracia. Si no es en la vida misma, que siempre se las arregla para ser
relacional, donde tendríamos que ir a buscar la gracia actuando que nos permite
desplegar el rostro verdadero de cada uno. Mientras que la segunda verdad es
que en el hombre y la mujer los trascendentales se topan, se mueven, se atraen
entre sí. Con esto afirmamos que el ser no es divisible en los entes y que se presenta siempre desbordando nuestras expectativas. La historia de Julie, que como
hemos visto refleja la batalla interna que libra la gracia, nos ayuda a comprender
al hombre como un ser nunca acabado y por sobre todo en una unidad profunda
en que todos sus pliegues se relacionan unos con otros. Por último, no olvidemos
nuestra tesis, hemos dicho que en las relaciones humanas se atisba la gracia. Y
ha quedado demostrado que no podemos dejar de unir la realidad humana con la
centralidad de Cristo. Con la Encarnación y la Pasión la humanidad se cristificó,
por tanto hizo posible lo que para los no cristianos parece imposible: que el hombre con su vida hable de lo profundamente divino. Con el análisis de la película nos
damos cuenta que en una historia, completamente plausible para la vida real, se
ejemplifica ésta verdad cristológica y antropológica. La irrupción de los trascendentales en cada cual nos entrega una forma de acercarnos a dialogar con todos
nuestros prójimos con la reverencia debida ya que como citamos anteriormente:
“ante esta gracia por antonomasia, que ya no manifiesta belleza, sino gloria, ya no
se requiere sólo admiración y encanto, sino adoración”20
Juan Pablo MOYANO SJ
Pontificia Universidad Católica de Chile
jpmoyanosj@gmail.com
20 BALTHASAR, H. U. von. Op.cit., p. 62
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Peer Review #2 of "Genome annotation across species using deep convolutional neural networks (v0.2)"
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cc-by
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Computer Science Computer Science Computer Science Computer Science Manuscript to be reviewed Genome Annotation across Species using
1
Deep Convolutional Neural Networks
2
Ghazaleh Khodabandelou1,2*, Etienne Routhier1, and Julien
3
Mozziconacci1,3,4*
4 Genome Annotation across Species using
1
Deep Convolutional Neural Networks
2
Ghazaleh Khodabandelou1,2*, Etienne Routhier1, and Julien
3
Mozziconacci1,3,4*
4
1Sorbonne Universit´e, CNRS, Laboratoire de Physique Th´eorique de la Mati`ere
5
Condens`ee, LPTMC, 75005, Paris, France. 6
2University of Val-de-Marne, Laboratoire Images, Signaux et Syst`emes Intelligents
7
(LISSI), Paris, France
8
3Structure et Instabilit´e des G´enomes MNHN - CNRS UMR 7196 / INSERM U1154 -
9
Sorbonne Universit´es, Paris, France. 10
4Institut Universitaire de France. 11
*ghazaleh.khodabandelou@u-pec.fr, julien.mozziconacci@mnhn.fr
12 1Sorbonne Universit´e, CNRS, Laboratoire de Physique Th´eorique de la Mati`ere
Condens`ee, LPTMC, 75005, Paris, France. 2University of Val-de-Marne, Laboratoire Images, Signaux et Syst`emes Intelligents
7
(LISSI), Paris, France
8 3Structure et Instabilit´e des G´enomes MNHN - CNRS UMR 7196 / INSERM U1154 -
9
Sorbonne Universit´es, Paris, France. 10 *ghazaleh.khodabandelou@u-pec.fr, julien.mozziconacci@mnhn.fr
12 ABSTRACT
13 Application of deep neural networks is a rapidly expanding field that has now reach many disciplines
including genomics. In particular, convolutional neural networks have been exploited for identifying
the functional role of short genomic sequences. These approaches rely on gathering large sets of
sequences with known functional role, extracting those sequences from whole genomes. The sets are
then split into training, test and validation sets in order to train the networks. While the obtained networks
perform well on validation sets, they often perform poorly when applied on whole genomes in which
the ratio of positive over negative examples can be very different than in the training set. We here
address this issue by assessing the genome-wide performance of networks trained with sets exhibiting
different ratios of positive to negative examples. As a case study, we use sequences encompassing gene
starts from the RefGene database as positive examples, and random genomic sequences as negative
examples. We than demonstrate that models trained using data from one organism can be used to predict
gene-start sites in a related species, when using training sets providing good genome-wide performance. This cross-species application of convolutional neural networks provides a new way to annotate any
genome from existing high-quality annotations in a related reference species. It also provides a way to
determine whether the sequence motifs recognised by chromatin-associated proteins in different species
are conserved or not. Ghazaleh Khodabandelou 1 Laboratoire de Physique Théorique de la Matière Condensée (LPTMC), Sorbonne Université, Paris, France
2 Laboratoire Images, Signaux et Systèmes Intelligents (LISSI), Université Val-de-Marne (Paris XII), Paris, France
3 CNRS UMR 7196 / INSERM U1154 - Sorbonne Université, Museum national d'Histoire naturelle (MNHN), Paris, France
4 Institut Universitaire de France, Paris, France 1 Laboratoire de Physique Théorique de la Matière Condensée (LPTMC), Sorbonne Université, Paris, France
2 Laboratoire Images, Signaux et Systèmes Intelligents (LISSI), Université Val-de-Marne (Paris XII), Paris, France
3 CNRS UMR 7196 / INSERM U1154 - Sorbonne Université, Museum national d'Histoire naturelle (MNHN), Paris, France
4 Institut Universitaire de France, Paris, France Corresponding Authors: Ghazaleh Khodabandelou, Julien Mozziconacci
Email address: ghazaleh.khodabandelou@u-pec.fr, julien.mozziconacci@mnhn.fr Application of deep neural network is a rapidly expanding field now reaching many
disciplines including genomics. In particular, convolutional neural networks have been
exploited for identifying the functional role of short genomic sequences. These approaches
rely on gathering large sets of sequences with known functional role, extracting those
sequences from whole-genome-annotations. These sets are then split into learning, test
and validation sets in order to train the networks. While the obtained networks perform
well on validation sets, they often perform poorly when applied on whole genomes in
which the ratio of positive over negative examples can be very different than in the
training set. We here address this issue by assessing the genome-wide performance of
networks trained with sets exhibiting different ratios of positive to negative examples. As a
case study, we use sequences encompassing gene starts from the RefGene database as
positive examples and random genomic sequences as negative examples. We than
demonstrate that models trained using data from one organism can be used to predict
gene-start sites in a related species, when using training sets providing good genome-wide
performance. This cross-species application of convolutional neural networks provides a
new way to annotate any genome from existing high-quality annotations in a related
reference species. It also provides a way to determine whether the sequence motifs
recognised by chromatin-associated proteins in different species are conserved or not. PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) Computer Science Computer Science Manuscript to be reviewed Manuscript to be reviewed We show that a CNN trained on
94
GSS containing regions in human is able to recover regions containing GSS in the mouse genome and
95
vice versa. We also assess the generalisation of the approach to more distant species, taking as examples
96
Gallus gallus and Danio rerio. 97
METHODS
98
Input Generation
99 features since they rely on the optimisation of convolution filters that can be directly matched to DNA
48
motifs [7]. Stacking several of these convolution layers together can lead to the detection of nested motifs
49
at larger scales. Pioneering studies illustrated this ability of CNNs to reliably grasp complex combinations
50
of DNA motifs and their relationship with functional regions of the genome [25, 34, 2, 39, 19, 26]. 51 Min et al. [25] used a CNN to predict enhancers, which are specific sequences that regulate gene
52
expression at a distance. This method performed very well and ranked above state-of-the-art support
53
vector machine based methods. Similar tools were used in different contexts, aiming at identifying
54
promoters [34, 26] or detecting splice sites [24, 17]. In these approaches, a sample set is first created
55
by taking all positive class sequences (e.g. enhancers) and adding the same amount of randomly picked
56
negative class examples (e.g. non-enhancers). This sample set is then divided into training, validation
57
and test sets. Balancing the data ensures that the model will be trained on the same number of positive
58
and negative examples, thus giving the same importance to both classes. While these approaches are
59
very successful when assessed on test sets derived from the sample set, we show here that they tend
60
to perform poorly when applied on entire chromosome sequences as required for the task of complete
61
genome annotation. This is due to the fact that the networks are optimised on a similar number of positive
62
and negative examples during training, but that they will usually face very different ratios of negative over
63
positive classes when used on a full chromosome sequence [16]. 64 Alternative approaches [2] [19] used unbalanced datasets for training (i.e., with more negative than
65
positive examples) to predict DNA-binding sites for proteins and genome accessibility. In these two
66
studies, however, the prediction performance of the model is also assessed on test sets derived from training
67
sets, not on full genomic sequences. Manuscript to be reviewed The task of genome-wide prediction has been assessed in a more
68
recent study aiming at identifying cell type specific regulatory elements [18]. In order to infer long range
69
relationships between these elements, Kelley et al. used very long (131 kb) non-overlapping windows
70
covering the whole genome. This approach has proven efficient but requires a lot of computational
71
memory. 72 As our goal is to provide genome-wide predictions, the methodology we used is inspired from this last
73
study. Since we do not aim here at predicting cell type specific features, we could use shorter sequences
74
as input and a simpler network architecture. We also present two novelties for the development and
75
for performance assessment of genome-wide predictions. Firstly, we do not use as a quality measure
76
the classical prediction scores computed on test sets obtained by dividing the sample data into training,
77
validation and test sets as commonly done in machine learning. We rather compute prediction scores
78
that assess the ability of our model to annotate a full chromosome sequence by designing a specific
79
metric (described in Material and Methods). Secondly, we change the ratio between positive and negative
80
examples in order to obtain the highest prediction scores and show that this tuning is has an important
81
effect on the outcome. As a proof of principle, we use in this work gene start sites (GSS) as features. DNA
82
motifs around GSS are recognised by the transcription machinery and indicate the location of the initiation
83
of transcription [22]. The DNA sequence surrounding GSS therefore contains the information that could
84
in principle be used by an algorithm to identify in silico the GSS locations. These DNA sequence
85
motifs are different for different classes to genes. For instance, protein coding genes can have either CG
86
di-nucleotide (CpG) rich or poor sequences upstream their GSS [10]. We show that using training sets
87
with a higher ratio of negative over positive examples, we can faithfully retrieve GSS positions, with
88
performances varying for different classes of genes such as coding or non coding genes. 89 We then propose a new application of CNNs in genomics that leverages the fact that similar organisms
90
tend to have similar regulatory mechanisms, i.e. rely on homologous molecular machinery and on
91
homologous DNA regulatory motifs. INTRODUCTION
30 The improvement of DNA sequencing techniques lead to an explosion in the number and completeness of
31
fully sequenced genomes. One of the major goals in the field is to annotate these DNA sequences, which
32
is to associate a biological function with sequence motifs located at different positions along the genome
33
[33]. In the human genome for instance, while some DNA sequences encode proteins, most sequences
34
do not code for any protein. Many of these non-coding sequences are nevertheless conserved in related
35
species and are necessary for the correct regulation of gene expression. Deciphering the function of these
36
non-coding sequences has been increasingly achieved through improvements in the throughput of next
37
generation sequencing [28]. The 3.2 Billion base pair (bp) long human genome is now annotated with
38
many functional and bio-chemical cues [23, 9], among which are the initiation sites of gene transcription
39
[5, 13]. While these annotations are becoming more numerous and precise, they cannot be determined
40
experimentally for every organism and every cell type, as the experiments needed to produce these
41
annotations are often difficult to carry and costly. Computational methods are therefore widely used to
42
extract sequence information from known annotations and extrapolate the results to different genomes or
43
conditions, e.g. [23, 12]. 44 g
An related question is to understand the link between these annotations and the underlying DNA
45
sequence. To this end, supervised machine learning algorithms [14] have been particularly successful
46
[40, 3]. Among those, deep Convolution Neural Networks (CNNs) are very efficient at detecting sequence
47 PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) Manuscript to be reviewed Exploiting these homologies, we first train a model on a dataset
92
corresponding to a given organism and use it to predict the annotation on the genome of a related organism,
93
opening new opportunities for the task of de-novo genome annotation. We show that a CNN trained on
94
GSS containing regions in human is able to recover regions containing GSS in the mouse genome and
95
vice versa. We also assess the generalisation of the approach to more distant species, taking as examples
96
Gallus gallus and Danio rerio. 97 Manuscript to be reviewed features since they rely on the optimisation of convolution filters that can be directly matched to DNA
48
motifs [7]. Stacking several of these convolution layers together can lead to the detection of nested motifs
49
at larger scales. Pioneering studies illustrated this ability of CNNs to reliably grasp complex combinations
50
of DNA motifs and their relationship with functional regions of the genome [25, 34, 2, 39, 19, 26]. 51
Min et al. [25] used a CNN to predict enhancers, which are specific sequences that regulate gene
52
expression at a distance. This method performed very well and ranked above state-of-the-art support
53
vector machine based methods. Similar tools were used in different contexts, aiming at identifying
54
promoters [34, 26] or detecting splice sites [24, 17]. In these approaches, a sample set is first created
55
by taking all positive class sequences (e.g. enhancers) and adding the same amount of randomly picked
56
negative class examples (e.g. non-enhancers). This sample set is then divided into training, validation
57
and test sets. Balancing the data ensures that the model will be trained on the same number of positive
58
and negative examples, thus giving the same importance to both classes. While these approaches are
59
very successful when assessed on test sets derived from the sample set, we show here that they tend
60
to perform poorly when applied on entire chromosome sequences as required for the task of complete
61
genome annotation. This is due to the fact that the networks are optimised on a similar number of positive
62
and negative examples during training, but that they will usually face very different ratios of negative over
63
positive classes when used on a full chromosome sequence [16]. 64
Alternative approaches [2] [19] used unbalanced datasets for training (i.e., with more negative than
65
positive examples) to predict DNA-binding sites for proteins and genome accessibility. In these two
66
studies, however, the prediction performance of the model is also assessed on test sets derived from training
67
sets, not on full genomic sequences. The task of genome-wide prediction has been assessed in a more
68
recent study aiming at identifying cell type specific regulatory elements [18]. In order to infer long range
69
relationships between these elements, Kelley et al. used very long (131 kb) non-overlapping windows
70
covering the whole genome. Manuscript to be reviewed This approach has proven efficient but requires a lot of computational
71
memory. 72
As our goal is to provide genome-wide predictions, the methodology we used is inspired from this last
73
study. Since we do not aim here at predicting cell type specific features, we could use shorter sequences
74
as input and a simpler network architecture. We also present two novelties for the development and
75
for performance assessment of genome-wide predictions. Firstly, we do not use as a quality measure
76
the classical prediction scores computed on test sets obtained by dividing the sample data into training,
77
validation and test sets as commonly done in machine learning. We rather compute prediction scores
78
that assess the ability of our model to annotate a full chromosome sequence by designing a specific
79
metric (described in Material and Methods). Secondly, we change the ratio between positive and negative
80
examples in order to obtain the highest prediction scores and show that this tuning is has an important
81
effect on the outcome. As a proof of principle, we use in this work gene start sites (GSS) as features. DNA
82
motifs around GSS are recognised by the transcription machinery and indicate the location of the initiation
83
of transcription [22]. The DNA sequence surrounding GSS therefore contains the information that could
84
in principle be used by an algorithm to identify in silico the GSS locations. These DNA sequence
85
motifs are different for different classes to genes. For instance, protein coding genes can have either CG
86
di-nucleotide (CpG) rich or poor sequences upstream their GSS [10]. We show that using training sets
87
with a higher ratio of negative over positive examples, we can faithfully retrieve GSS positions, with
88
performances varying for different classes of genes such as coding or non coding genes. 89
We then propose a new application of CNNs in genomics that leverages the fact that similar organisms
90
tend to have similar regulatory mechanisms, i.e. rely on homologous molecular machinery and on
91
homologous DNA regulatory motifs. Exploiting these homologies, we first train a model on a dataset
92
corresponding to a given organism and use it to predict the annotation on the genome of a related organism,
93
opening new opportunities for the task of de-novo genome annotation. METHODS
98 genome were extracted from their respective NCBI RefSeq Reference Gene annotations (RefGene) genome were extracted from their respective NCBI RefSeq Reference Gene annotations (RefGene). 102
Table 1. URL of the data used in the present work. Genomes
human
https://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/hg38.fa.gz
mouse
https://hgdownload.soe.ucsc.edu/goldenPath/mm10/bigZips/chromFa.tar.gzcell4
chicken
https://hgdownload.soe.ucsc.edu/goldenPath/galGal4/bigZips/galGal4.fa.gz
zebrafish
https://egg.wustl.edu/d/danRer10/refGene.gz
Reference Gene annotations
human
https://egg.wustl.edu/d/hg38/refGene.gz
mouse
https://egg.wustl.edu/d/mm10/refGene.gz
chicken
https://egg.wustl.edu/d/galGal4/refGene.gz
zebrafish
https://egg.wustl.edu/d/danRer10/refGene.gz 102 Table 1. URL of the data used in the present work. As a positive input class, we use regions of 299 bp flanking GSS (i.e., ±149 bp around the GSS) which
103
are supposed to contain multiple sequence signals indicating the presence of a GSS to the transcription
104
machinery of the cell. For instance in the human genome, 31,037 GSS positions are extracted on both
105
DNA strands (15,798 for the positive strand and 15,239 for the negative strand). In order to generate
106
the negative class, we select 31,037×Q sequences of 299 bp at random positions on a random strand,
107
rejecting regions that do contain a GSS. The odds of getting at random a genomic region containing a
108
GSS are close to 0.28%. For Q = 1, there is an equal number of negative and positive class examples. 109
Unbalanced datasets are produced using different values of Q ranging from 1 to 100. For Q = 100, the
110
negative class encompasses 100×299×31k ≈1Gb, which represents one third of the human genome. 111
For the other genomes a similar procedure was implemented. The total number of GSS used was 25,698
112
for the mouse, 6876 for the chicken and 14805 for the zebrafish. 113 Convolution Neural Network (CNN)
114 METHODS
98 Input Generation
99 p
Genomic sequences were downloaded for the reference genomes for Human (hg38), Mouse (mm10),
100
Chicken (gg4) and Zebrafish (dr10) via the URLs shown in Table 1. Similarly, GSS positions for each
101 2/13 PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) Computer Science Computer Science Manuscript to be reviewed Figure 1. Overview of the CNN model. 299 bp-long sequences are one hot encoded into a 4×299
input matrix. The first CNN layer performs a convolution on each input matrix to recognise relevant
motifs. The next convolutional layers models the interplay among these motifs to grasp higher-level
features. Max-pooling layers reduce the dimensions of the layers. The model is trained to correctly label
input sequences as GSS or non-GSS. The output layer of the trained network then gives a probability for
any 299 bp region to contain a GSS. It can be applied along a full chromosome, i.e. on all 299 bp-long
sequences with a 1 bp shift. Figure 1. Overview of the CNN model. 299 bp-long sequences are one hot encoded into a 4×299
input matrix. The first CNN layer performs a convolution on each input matrix to recognise relevant
motifs. The next convolutional layers models the interplay among these motifs to grasp higher-level
features. Max-pooling layers reduce the dimensions of the layers. The model is trained to correctly label
input sequences as GSS or non-GSS. The output layer of the trained network then gives a probability for
any 299 bp region to contain a GSS. It can be applied along a full chromosome, i.e. on all 299 bp-long
sequences with a 1 bp shift. Figure 1. Overview of the CNN model. 299 bp-long sequences are one hot encoded into a 4×29 Figure 1. Overview of the CNN model. 299 bp-long sequences are one hot encoded into a 4×299
input matrix. The first CNN layer performs a convolution on each input matrix to recognise relevant
motifs. The next convolutional layers models the interplay among these motifs to grasp higher-level
features. Max-pooling layers reduce the dimensions of the layers. The model is trained to correctly label
input sequences as GSS or non-GSS. The output layer of the trained network then gives a probability for
any 299 bp region to contain a GSS. It can be applied along a full chromosome, i.e. on all 299 bp-long
sequences with a 1 bp shift. Implementation
150 We implement CNN using Keras library [8] and Tensorflow [1] as back-end. Training on a GPU is
151
typically faster than on a CPU. We use here a GTX 1070 Ti GPU. We use Adaptive Moment Estimation
152
(Adam) to compute adaptive learning rates for each parameter [20]. Adam optimiser is an algorithm for
153
first-order stochastic gradient-based optimisation of functions, based on adaptive estimates of lower-order
154
moments. The network architecture (see figure 1) is detailed in Table 2. The models are trained for
155
150 epochs and they mostly converge rapidly (around 30-35 epochs, we use early stopping to prevent
156
overfitting). Hyper-parameters tuning is detailed in the supplementary materials. 157 Source codes are available at https://github.com/StudyTSS/DeepTSS/. Source codes are available at https://github.com/StudyTSS/DeepTSS/. 158 Convolution Neural Network (CNN)
114 A CNN (see figure 1) is trained in order to predict the presence of a GSS in a DNA sequence of size
115
299 bp. The shape of the input layer is c × b in which c = 4 is the number of different nucleotides
116
and b = 299 is the length of the input sequence. The nucleotide sequences are one hot encoded so that
117
A=(1,0,0,0), T=(0,1,0,0), C=(0,0,1,0), and G=(0,0,0,1). The training set contains N samples of labelled
118 3/13 PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) Manuscript to be reviewed pairs (X(n),y(n)), for n ∈{1,··· ,N}, where X(n) are matrices of size c×b and y(n) ∈{0,1}. Each X(n) is
119
associated with y(n) = 1 when it corresponds to a region containing a GSS and y(n) = 0 otherwise. The
120
first convolution layer consists of k kernels of length s which are applied on b−s+1 successive sequences
121
at positions p ∈{1,··· ,(b−s+1)} to recognise relevant DNA motifs of size s. This operation generates
122
an output feature map of size k ×(b−s+1) for an input X(n) of size c×b. The feature map M resulting
123
from the convolution operation is computed as follows:
124 pairs (X(n),y(n)), for n ∈{1,··· ,N}, where X(n) are matrices of size c×b and y(n) ∈{0,1}. Each X(n) is
119
associated with y(n) = 1 when it corresponds to a region containing a GSS and y(n) = 0 otherwise. The
120
first convolution layer consists of k kernels of length s which are applied on b−s+1 successive sequences
121
at positions p ∈{1,··· ,(b−s+1)} to recognise relevant DNA motifs of size s. This operation generates
122
an output feature map of size k ×(b−s+1) for an input X(n) of size c×b. The feature map M resulting
123
from the convolution operation is computed as follows:
124 Mp,i =
c
∑
j=1
s
∑
r=1
Wi, j,rXp+r−1, j +Bi,
i ∈{1,··· ,k}
(1 (1) where W denotes the network weights with size (k × c × s) and B denotes the biases with size
125
(k ×1) (see e.g. [14]). After the convolution layer a non-linear function is applied to the output, here a
126
Rectified Linear Unit (ReLU). This activation function computes fReLU(M ) = max(0,M ) to incorporate
127
non-linearity by transforming all negative values to zero. In order to reduce the input dimension we
128
apply a max-pooling process with a pool size m over the output of fReLU(M ). Similar convolution layers
129
followed by ReLu and max-pooling are added sequentially on the input of the first layer to grasp higher
130
order motifs. The output of the last max-pooling layer is then fed into a fully connected layer which output
131
x is transformed by a softmax layer, i.e. a sigmoid function (φ =
1
1+e−x ), in order to give the final output
132
of the CNN. Manuscript to be reviewed This final score of the input sequence is ideally 0 for non-GSS and 1 for GSS containing
133
sequences. When we need to perform a classification we use a threshold of 0.5 to discriminate between
134
the two classes. 135 In the training phase, the weights and biases of the convolution layers and the fully connected layer
136
are updated via back-propagation [29] in a way which decreases the loss, which measures the discrepancy
137
between the network predictions and the reality averaged over individual examples. We use here the
138
binary cross-entropy computed as:
139 L = −1/N
N
∑
i=1
[y(n)log(ˆy(n))+(1−y(n))×log(1−ˆy(n))]
(2) (2) where ˆy(n) is the estimated score for the input sample X(n). 140 As data are imbalanced for Q > 1, the model may reach a local optimum when predicting the non-GSS
141
class for all input sequences. In order to deal with this issue, we attribute different weights to the positive
142
and negative classes. We assign a greater importance to the less represented GSS class by multiplying the
143
associated term in the loss by a weight CW = number of non-GSS
number of GSS
= Q. 144 One of the important issues of any learning algorithm is overfitting. Overfitting occurs when one
145
achieves a good fit of the model on the training and validation data, while it does not generalise well
146
on new, unseen data. To deal with this issue, a regularisation procedure called dropout is usually used
147
[32]. In the training step, some outputs of the pooling layers are randomly masked while the remaining
148
information is fed as inputs for the next layer. 149 Training models for genome annotation of GSS
177 Training models for genome annotation of GSS
177
The problem of detecting human GSS using deep neural networks has been tackled in [34]. We first follow
178
a similar approach and use a balanced dataset (see Methods for details). The model is trained/validated on
179
an equal number of 299 bp long positive and negative examples and is evaluated on a test set composed of
180
15% of the original data that was left aside prior to training. The specificity (Sp), the sensitivity (Sn) and
181
the Matthews Correlation Coefficient (MCC) (see Supplementary materials for definition) were found to
182
be similar to the ones found in [34] which used a similar approach albeit separating the sample data into
183
TATA-containing GSS and non-TATA GSS (Sp = 0.94, Sn = 0.92 and MCC = 0.86). 184
In order to assess how this model would perform as a practical tool for detecting GSS on a genome-
185
wide scale, we apply it on all the sequences along chromosome 21 (which has been withdrawn from the
186
training phase, i.e. from the training and validation sets) obtained using a 299 bp long window sliding
187
with an offset of 1 bp. Figure 2A illustrates the predictions of the CNN model over a typical region of 300
188
kbp containing 7 out of the 480 GSS of chromosome 21. Although the predictions yield higher scores over
189
GSS positions, they also yield high scores over many non-GSS positions reflecting a low signal-to-noise
190
ratio. This is due to the fact that the reality is biased in the training phase during which the CNN model
191
learns an equal number of examples from the positive and the negative classes [16]. Applied over all the
192
299-bp sequences of chromosome 21, the model encounters many more examples of the negative class
193 The problem of detecting human GSS using deep neural networks has been tackled in [34]. We first follow
178
a similar approach and use a balanced dataset (see Methods for details). The model is trained/validated on
179
an equal number of 299 bp long positive and negative examples and is evaluated on a test set composed of
180
15% of the original data that was left aside prior to training. Manuscript to be reviewed Table 2. Network architecture of the CNN model. The first column depicts the different layers used
consecutively in the network. The ”layer shape” column reports the shape of the convolutional kernels,
the max-pooling windows and the fully connected layers. The ”output shape” column reports the
variation of layer shapes at each step. Layer name
Layer shape
Output shape
Input
-
4×299×1
Conv2D
32×4×(4×1)
32×296×1
Max-pooling
2×1
32×148×1
Dropout
-
32×148×1
Conv2D
64×32×(4×1)
64×145×1
Max-pooling
2×1
64×72×1
Dropout
-
64×72×1
Conv2D
128×64×(4×1)
128×69×1
Max-pooling
2×1
128×34×1
Dropout
-
128×34×1
Dense
128
128
Dropout
-
128
Dense (sigmoid)
1
1 on all the 299 bp windows spanning a full chromosome and eventually chromosomes from other species. 163
Specifically, the model was tested on chromosome 21 which was withdrawn from the training set. We
164
therefore developed a measure to evaluate the performance of the trained models in this case. This
165
metric, called λ, measures the enhancement of the predicted signal specifically in the regions surrounding
166
the known GSS. We use in the present papers regions of length r = 400 bp. To compute λ, we first
167
compute the genome-wide Z-score [21] Zg = yg−¯µ
σ
from the predictions yg where g denotes positions on
168
the genome, and ¯µ and σ stand for the prediction mean and standard deviation, respectively. We extract
169
ZGSS, the Zg signal over 10 kb windows centred on each GSS of the test region, e.g. a full chromosome. 170
Zg is a 2D-array whose rows correspond to different genes and columns to different distances to the GSS. 171
We then average element-wise ZGSS over all GSS, i.e. along all rows. This gives us S, the average of the
172
Z-transformed prediction score in a 10 kb window around all GSS. In order to measure the signal increase
173
close to the GSS, that we call λ, we compute the average of the curve S on a region of r bp centred on the
174
GSS. A higher value of λ corresponds to a higher signal-to-noise ratio around the GSS. 175 on all the 299 bp windows spanning a full chromosome and eventually chromosomes from other species. 163
Specifically, the model was tested on chromosome 21 which was withdrawn from the training set. We
164
therefore developed a measure to evaluate the performance of the trained models in this case. Manuscript to be reviewed This
165
metric, called λ, measures the enhancement of the predicted signal specifically in the regions surrounding
166
the known GSS. We use in the present papers regions of length r = 400 bp. To compute λ, we first
167
compute the genome-wide Z-score [21] Zg = yg−¯µ
σ
from the predictions yg where g denotes positions on
168
the genome, and ¯µ and σ stand for the prediction mean and standard deviation, respectively. We extract
169
ZGSS, the Zg signal over 10 kb windows centred on each GSS of the test region, e.g. a full chromosome. 170
Zg is a 2D-array whose rows correspond to different genes and columns to different distances to the GSS. 171
We then average element-wise ZGSS over all GSS, i.e. along all rows. This gives us S, the average of the
172
Z-transformed prediction score in a 10 kb window around all GSS. In order to measure the signal increase
173
close to the GSS, that we call λ, we compute the average of the curve S on a region of r bp centred on the
174
GSS. A higher value of λ corresponds to a higher signal-to-noise ratio around the GSS. 175 Genome wide performance measure
159 Different measures have been developed in order to assess the performance of different models on
160
conventional test sets, i.e. test sets derived from a subset of the initial data. Such measures are described
161
in details in the corresponding supplementary materials section. In our case, we want to apply our model
162 4/13 PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) Training models for genome annotation of GSS
177 The specificity (Sp), the sensitivity (Sn) and
181
the Matthews Correlation Coefficient (MCC) (see Supplementary materials for definition) were found to
182
be similar to the ones found in [34] which used a similar approach albeit separating the sample data into
183
TATA-containing GSS and non-TATA GSS (Sp = 0.94, Sn = 0.92 and MCC = 0.86). 184 In order to assess how this model would perform as a practical tool for detecting GSS on a genome-
185
wide scale, we apply it on all the sequences along chromosome 21 (which has been withdrawn from the
186
training phase, i.e. from the training and validation sets) obtained using a 299 bp long window sliding
187
with an offset of 1 bp. Figure 2A illustrates the predictions of the CNN model over a typical region of 300
188
kbp containing 7 out of the 480 GSS of chromosome 21. Although the predictions yield higher scores over
189
GSS positions, they also yield high scores over many non-GSS positions reflecting a low signal-to-noise
190
ratio. This is due to the fact that the reality is biased in the training phase during which the CNN model
191
learns an equal number of examples from the positive and the negative classes [16]. Applied over all the
192
299-bp sequences of chromosome 21, the model encounters many more examples of the negative class
193 5/13 PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) Computer Science Manuscript to be reviewed Figure 2. CNN predictions for two regions of chromosome 21. (A) Prediction scores for balanced 1*
model (Q = 1) and unbalanced 100* model (Q = 100), respectively in blue and red on a 300 kb region. The position of genes is indicated below. The annotation track was done using the UWash Epigenome
browser (https://epigenomegateway.wustl.edu/). Both models detect 7 GSS positions, but the 1* model
returns a higher background signal at non GSS positions. Adding negative examples using the 100* model
mitigates the noise while preserving the high scores over GSS. (B) Application of 30 1* models, trained
on different datasets, over a 3.2 kb region of chromosome 21. At each site, the maximum and minimum
prediction scores are respectively displayed in black and red. Other prediction scores are plotted in grey. p
pute Sc e ce Figure 2. CNN predictions for two regions of chromosome 21. Training models for genome annotation of GSS
177 (A) Prediction scores for balanced 1*
model (Q = 1) and unbalanced 100* model (Q = 100), respectively in blue and red on a 300 kb region. The position of genes is indicated below. The annotation track was done using the UWash Epigenome
browser (https://epigenomegateway.wustl.edu/). Both models detect 7 GSS positions, but the 1* model
returns a higher background signal at non GSS positions. Adding negative examples using the 100* model
mitigates the noise while preserving the high scores over GSS. (B) Application of 30 1* models, trained
on different datasets, over a 3.2 kb region of chromosome 21. At each site, the maximum and minimum
prediction scores are respectively displayed in black and red. Other prediction scores are plotted in grey. To address this issue and train a network for genome annotation, we propose a heuristic where more
195
negative examples are added into the balanced dataset to reduce the importance of the positive class
196
during training and to allocate more weight to the negative class. We call these augmented datasets limited
197
unbalanced datasets. The parameter Q is the ratio between negative and positive training examples and
198
denote as Q∗models trained with the corresponding ratio. For instance, on Figure 2A the model trained
199
on the balanced data yielding to blue signal predictions is denoted as 1∗. We train our CNN model on
200
a 100* dataset (Q = 100) and assess the efficiency of the trained model. As depicted on Figure 2A by
201
a red signal, the predictions for this model display a much higher signal-to-noise ratio, with significant
202
peaks over each of the 7 GSS (C21orf54, IFNAR2, IL10RB, IFNAR1, IFNGR2, TMEM50B, DNAJC28)
203
and a much weaker signal between these sites. Predicting GSS using the 100* model is thus expected to
204
generate fewer false positives than the 1* model, regardless the value of the threshold used to identify
205
GSS-containing regions. In order to assess how changing the value of Q affects GSS classification, we
206
apply a threshold on the prediction and compute the precision and the recall obtained for both models
207
(i.e. 1* and 100*) at 600 bp resolution on a full chromosome. The precision recall curves confirmed the
208
compromising effect of a lower signal-to-noise ratio on the accuracy of the classification (Supplementary
209
Figure 1). Comparing 1* and 100* models over a full chromosome
224 Models trained on 1* and 100* sets are applied to the full chromosome 21 and the Z-normalized prediction
225
scores around GSS are presented as heat-maps. While the 1* model (Figure 3A) presents a noisy signal
226
around GSS positions, the 100* model (Figure 3B) presents a higher signal-to-noise ratio. To investigate
227
the performance of different models on a genome-wide scale we devised a custom metric λ which
228
measures the average signal-to-noise ratio around GSS (see Methods for the definition of λ). 229 Figure 3C,D illustrate the average of the Z-score over all the GSS of chromosome 21 for the models
230
1* and 100*, respectively, and λ denotes the average of this average over a r=400 bp region centred on
231
the GSS. A larger λ score corresponds to a higher signal-to-noise ratio. In this particular case, we find a
232
λ score of 2.21 and 5.81 for the 1* and 100* model, respectively. 233 Figure 3C,D illustrate the average of the Z-score over all the GSS of chromosome 21 for the models
230
1* and 100*, respectively, and λ denotes the average of this average over a r=400 bp region centred on
231
the GSS. A larger λ score corresponds to a higher signal-to-noise ratio. In this particular case, we find a
232
λ score of 2.21 and 5.81 for the 1* and 100* model, respectively. 233 To illustrate the variability of prediction scores achieved around different GSS, we randomly selected
234
four GSS within the chromosome. The first GSS corresponds to the gene CXADR, shown in Figure
235
3E. While the prediction of 1* model results in a low averaged Z-scores over all positions, the averaged
236
Z-score of 100* model strongly peaks around the GSS position and shows low variations over non-GSS
237
positions. Figure 3F depicts the second selected GSS corresponding to the KRTAP19-2 gene. This gene is
238
part of a cluster of similar genes belonging to the family of Keratin Associated Proteins (highlighted by a
239
yellow rectangle on Figure 3A,B). For this particular cluster, the predictions are poor for both 1* and 100*,
240
probably reflecting a specific GSS signature that has not been grasped by the model. Another example of
241
gene cluster with a poor prediction score for GSS is the t-RNA cluster, highlighted in green in Figure
242
3A,B. Investigating the effect of random selection of the negative examples on predictions While positive examples are always the same in different sample sets, the negative examples are randomly
214
picked out of the genome. The performance of the model in different regions of chromosome 21 can
215
thus vary for different training sets [38]. To investigate this variation, we set up 30 balanced 1∗datasets
216
and train 30 CNNs separately. The 30 models are then applied over human chromosome 21 to study the
217
fluctuations of the predictions. The variation of 30 predictions is depicted in Fig 2B. The first observation
218
is that almost all predictions present a peak over the DIP2A GSS. However, the large gap between the
219
minimum and maximum predictions underlines the variability of predictions obtained with different
220
training datasets. This variability illustrates the uncertainty of the predictions obtained from a single CNN
221
trained on a balanced dataset and highlights the need to use limited unbalanced datasets for the task of
222
genome annotation. 223 Training models for genome annotation of GSS
177 For the sake of completeness, the performance of more models (1*, 10*, 20*, 30*, 50*, 100*)
210 6/13 PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) Computer Science Computer Science Manuscript to be reviewed Figure 3. Comparison of the 1* and 100* model predictions on chromosome 21. (A) and (B) Heat
maps depict the Z-score of the prediction for the 1* and 100* models respectively on 5000 bp flanking
each GSS of chromosome 21. (C) and (D) Averaged Z-score of the predictions over each GSS of
chromosome 21. (E-H) Zoom on regions around randomly selected GSS. Genes are indicated at the
bottom of each plot. (I-K) Averaged Z-score of the predictions over each GSS of mouse chromosome X
(I) and for networks trained on mouse/human chromosomes (except X) and applied on human/mouse
chromosome X (J,K). Figure 3. Comparison of the 1* and 100* model predictions on chromosome 21. (A) and (B) Heat
maps depict the Z-score of the prediction for the 1* and 100* models respectively on 5000 bp flanking
each GSS of chromosome 21. (C) and (D) Averaged Z-score of the predictions over each GSS of
chromosome 21. (E-H) Zoom on regions around randomly selected GSS. Genes are indicated at the
bottom of each plot. (I-K) Averaged Z-score of the predictions over each GSS of mouse chromosome X
(I) and for networks trained on mouse/human chromosomes (except X) and applied on human/mouse
chromosome X (J,K). 7/13 PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) Comparing 1* and 100* models over a full chromosome
224 Figure 3G,H displays the predictions around the GSS of the SCAF4 and, PCNT and C21ORF58
243
genes, respectively. On these more typical GSS the 100* model shows a higher signal-to-noise ratio than
244
the 1* and regions containing GSS are detected. These regions often stretch over 1 kb while our training
245
sequence centred on each GSS is only 299bp long. This could indicate the presence either of alternative
246
GSS close to the annotated GSS or of similar sequence patterns in broader regions surrounding the GSS
247
[5, 30]. 248 Manuscript to be reviewed evaluated using conventional metrics on test sets derived from the initial sample sets can be found in
211
Supplementary materials. 212 Learning and predicting in human and mouse
249 ea
g a d p ed ct
g
u
a
a d
ouse
9
To show the potential of our annotation method in a different context, we replicate a similar GSS analysis
250
in mouse. Models with values of Q ranging from 1 to 100 trained on mouse chromosomes (except
251
X) are applied over the mouse chromosome X to assess the model performance (see Figure 3I, and
252
Supplementary Figure 2 a,d,g). The averaged Z-score of λ reaches values of 2.24 and 4.90 respectively
253
for the 1* and 100* models in quantitative agreement with the model performance in human. 254
Mammals show a substantial degree of homology in the DNA sequence found at GSS [37], and earlier
255
computational models were trained to recognise transcription start site in any mammalian species [11]. 256
This study focused on 313 sequences, of which 50 were kept aside for test purposes and we want here
257
to extend the validity of this initial study at the genome wide level. Following this line, we determine
258
the possibility of predicting GSS in one organism with a network trained on a related organisms. This
259
possibility has previously been shown to be effective for sequence variants calling [27] To this end, the
260
mouse trained model is applied on human chromosome X and the human trained model is applied on
261
mouse chromosome X. The two chromosomes carry homologous genes [37], the number of annotated
262
GSS varies with a total of 4,968 GSS in human and 2,005 GSS in mouse. While the model trained and
263 To show the potential of our annotation method in a different context, we replicate a similar GSS analysis
250
in mouse. Models with values of Q ranging from 1 to 100 trained on mouse chromosomes (except
251
X) are applied over the mouse chromosome X to assess the model performance (see Figure 3I, and
252
Supplementary Figure 2 a,d,g). The averaged Z-score of λ reaches values of 2.24 and 4.90 respectively
253
for the 1* and 100* models in quantitative agreement with the model performance in human. 254 Mammals show a substantial degree of homology in the DNA sequence found at GSS [37], and earlier
255
computational models were trained to recognise transcription start site in any mammalian species [11]. Learning and predicting in human and mouse
249 256
This study focused on 313 sequences, of which 50 were kept aside for test purposes and we want here
257
to extend the validity of this initial study at the genome wide level. Following this line, we determine
258
the possibility of predicting GSS in one organism with a network trained on a related organisms. This
259
possibility has previously been shown to be effective for sequence variants calling [27] To this end, the
260
mouse trained model is applied on human chromosome X and the human trained model is applied on
261
mouse chromosome X. The two chromosomes carry homologous genes [37], the number of annotated
262
GSS varies with a total of 4,968 GSS in human and 2,005 GSS in mouse. While the model trained and
263 8/13 PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) Computer Science Manuscript to be reviewed Figure 4. Evaluation of the model performance for different classes of genes. (A) and (B) CpG
number in 299bp regions centred on mRNA-GSS in X chromosomes for human (A) and mouse (B). These regions were divided in three groups of similar size according to their CpG number into low,
medium and high groups (the bounds are 35% and 60% for human and 30% and 60% for mouse). The
proportion of genes in each class is similar on the X chromosome (test set) than on other chromosomes
(training and validation sets). (C) λ values computed for networks trained on each species non-X
chromosome GSS (t) and predicted on either species’ X-chromosome GSS (p). λ values for each
mRNA-CpG sub-group and ncRNA genes are also shown to highlight different levels of performance. Figure 4. Evaluation of the model performance for different classes of genes. (A) and (B) Cp
number in 299bp regions centred on mRNA-GSS in X chromosomes for human (A) and mouse (B
These regions were divided in three groups of similar size according to their CpG number into low
medium and high groups (the bounds are 35% and 60% for human and 30% and 60% for mouse). proportion of genes in each class is similar on the X chromosome (test set) than on other chromoso
(training and validation sets). (C) λ values computed for networks trained on each species non-X
chromosome GSS (t) and predicted on either species’ X-chromosome GSS (p). Manuscript to be reviewed applied on mouse shows a better signal-to-noise ratio, the same model applied to human chromosome
264
X still captures most of the GSS and gives a λ score of 5.18 for the 100* model (see Figure 3J and
265
Supplementary Figure 2b,e,h). Similarly, the models trained on human capture most of GSS on the mouse
266
X chromosome as shown in Figure 3K and Supplementary Figure 2c,f,i and reaches a λ score of 4.32 for
267
the 100* model. In all cases, the signal-to-noise ratio is improved in the 100* models with respect to the
268
1* models. The human model applied on human provides the highest scores for both 1* and 100* models
269
probably a signature of an overall better GSS annotation. 270 Evaluation of the prediction for different GSS classes
271 The potential of our trained networks to recover GSS containing regions along the human and mouse
272
genomes is assessed in the previous parts without any distinction between different GSS classes. Since
273
we find that some GSS are better predicted than others (Figure 3), we compute the λ score independently
274
for the two main classes of GSS: mRNA-GSS and ncRNA-GSS. While λ is higher for the mRNA-GSS
275
class, the model is versatile and is also able to predict the ncRNA-GSS (Figure 4B). In human and
276
mouse, mRNA-GSS are found in different classes, that can be derived from the CpG content of the region
277
flanking the GSS. High CpG regions, also called ”CpG island” can be methylated and play an important
278
role in gene regulation [10]. Figure 4A displays the distribution of the CpG number in 299 bp regions
279
surrounding the all mRNA-GSS for the mouse and human X chromosome. From this distribution, we
280
identify three classes of mRNA-GSS with respectively a high, medium and low CpG content. High CpG
281
GSS correspond to genes regulated by DNA methylation and have been shown to exhibit a different
282
pattern of chromatin modifications [35]. Assessing the performance of the model for the three different
283
classes, we find that better scores are obtained for CpG richer GSS (Figure 4B). The worst performing
284
GSS are low CpG content GSS which are hardly recovered by our model. In order to test whether CpG
285
content alone could be used to predict GSS we computed the λ score over all GSS using the Z-normalized
286
CpG content as predictor. We get values of 1.30 and 0.92 respectively for the human and mouse GSS
287
indicating that the CpG content is a strong indicator of the present of GSS but that our models use as well
288
other features which allow them to reach much higher scores. 289 Application of the approach to other vertebrates
290 The performance of a CNN trained on human GSS to recover mouse GSS is not surprising given the
291
similarity between their genomes [37]. We next set out to apply the same methodology on more diverse
292
species, including chicken and zebrafish (Figure 5). Four CNNs were trained on all the GSS from
293
the genomes of Homo Sapiens (human), Mouse musculus (mouse), Gallus gallus (chicken) and Danio
294
rerio (zebrafish). G.g. and D.r. are model organisms, and together with H.s. and M.m. provide the
295
most comprehensive GSS annotations for mammals, birds and fishes. These four CNNs were then
296
applied genome wide on each of the four species and the λ metric is computed for each chromosome
297
independently, using a r value of 400 bp (see Methods). 298 The results for the human and mouse genomes are very similar, with only a slightly better performance
299
when the model trained on a species is applied on the same species. The model trained on the chicken
300
genome performs less well when applied on the mammalian genomes and the model trained on the
301
zebrafish genome is not able recover the mammalian GSS as shown by a λ value of 0. 302 When applied on the chicken genome, the mouse and human models surprisingly outperform the
303
chicken model, probably because the GSS annotation is better in the two mammals so that the training
304
phase is more efficient. This result highlights the potential of the method when used across different
305
species when the genome of one species is more precisely annotated. 306 When applied on the zebrafish genome on the other hand, the human, mouse and chicken models
307
all show poor performances while the zebrafish model performs well. This is in line with the fact that
308
the CpG composition of zebrafish regions around GSS if very different than in chicken and mammals. 309
CpG islands, which are high density CpG regions, are found upstream of many GSS for coding genes in
310
chicken and mammals while they are less abundant in the zebrafish’s genome which has a low GC content
311
[15]. All together, these results suggest that the molecular machinery that interprets the genome sequence
312
in order to find start sites of genes has a similar specificity in human, mouse and chicken, but a different
313
specificity in zebrafish. Learning and predicting in human and mouse
249 λ values for each
mRNA-CpG sub-group and ncRNA genes are also shown to highlight different levels of performan Figure 4. Evaluation of the model performance for different classes of genes. (A) and (B) CpG Figure 4. Evaluation of the model performance for different classes of genes. (A) and (B) CpG
number in 299bp regions centred on mRNA-GSS in X chromosomes for human (A) and mouse (B). These regions were divided in three groups of similar size according to their CpG number into low,
medium and high groups (the bounds are 35% and 60% for human and 30% and 60% for mouse). The
proportion of genes in each class is similar on the X chromosome (test set) than on other chromosomes
(training and validation sets). (C) λ values computed for networks trained on each species non-X
chromosome GSS (t) and predicted on either species’ X-chromosome GSS (p). λ values for each
mRNA-CpG sub-group and ncRNA genes are also shown to highlight different levels of performance. Figure 4. Evaluation of the model performance for different classes of genes. (A) and (B) CpG
number in 299bp regions centred on mRNA-GSS in X chromosomes for human (A) and mouse (B). gu e
p
g
( )
( )
p
number in 299bp regions centred on mRNA-GSS in X chromosomes for human (A) and mouse (B). These regions were divided in three groups of similar size according to their CpG number into low,
medium and high groups (the bounds are 35% and 60% for human and 30% and 60% for mouse). The
proportion of genes in each class is similar on the X chromosome (test set) than on other chromosomes
(training and validation sets). (C) λ values computed for networks trained on each species non-X
chromosome GSS (t) and predicted on either species’ X-chromosome GSS (p). λ values for each
mRNA-CpG sub-group and ncRNA genes are also shown to highlight different levels of performance. 9/13 PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) CONCLUSIONS ith the surge of DNA sequencing technologies, over a million genome datasets are now available and
316
petabases of transcripts are sequenced every year to annotate these datasets with functional marks [36]. 317
It has not escaped the notice of many computational biologists that deep neural networks are a key tool
318
to deal with this exponentially increasing amount of data [36]. One possible application is to leverage
319
datasets with good annotations in order to train neural networks and to predict annotations on other
320
datasets. One of the practical issues when applying neural networks on genomic sequences is unbalanced
321
data, a well-known issue in the machine learning literature [16, 6, 4]. In the present paper, we address this
322
problem using GSS as a case study. Indeed, GSS occupy only a few locations on the genome (31,037 GSS
323
for human) leading to extreme unbalances in datasets (i.e., the ratio of GSS-containing 299 bp windows
324
to non-GSS in the human genome is 1/400). In this case, the lack of examples of the minority class (i.e.,
325
true GSS) impacts the learning process as conventional machine learning algorithms usually measure the
326
model performance on the majority class (i.e., non-GSS) leading to biased or inaccurate prediction of the
327
minority class. To deal with this disparity, we adopt a weighting strategy to decrease the importance of
328
the majority class samples (non-GSS) during the learning process thereby improving identification of the
329
rare minority class samples (GSS). Using this approach, which we call ”limited unbalanced datasets”, we
330
show that learning on imbalanced datasets can be performed effectively, and that for GSS recognition, a
331
ratio of 1 to 100 positive over negative examples is usually sufficient to achieve a good signal to noise
332
ratio in the prediction. This approach can be easily extended to identify other functional regions in any
333
annotated genome. 334 We also show that our method can be efficiently used across genomes of different species, i.e. training
335
the model on one genome and applying it to another genome. We use the X chromosomes of human and
336
mouse GSS as a case study, and apply models trained on each one’s other chromosomes to its own and
337
the other one’s X chromosome. While the sequence of this chromosome has evolved differently in both
338
species, many genes are homologous [31]. CONCLUSIONS The fact that we are able to recover GSS in mouse/human
339
with a model trained on the other organism suggests that the machinery capable of recognising GSS in
340
each organism is overall conserved. We also show that this methodology can be applied to more distant
341
species, and use as examples chicken and a zebrafish. Our results point toward a higher similarity between
342
mammal and chicken while zebrafish GSS cannot cannot be reliably predicted with models trained on
343
mammal and chicken sequences While the genome sequence conservation can be computed directly from
344
DNA sequences, further developments of our method may provide a new tool to quantify more complex
345
patterns of similarity between different organism’s nuclear machinery that interprets DNA sequences in
346
vivo. 347 Computer Science Manuscript to be reviewed Figure 5. λ scores obtained with CNN trained on four different species: human, mouse, chicken
and zebrafish. λ scores are computed from predictions done on GSS of (A) human, (B) mouse, (C)
chicken and (D) zebrafish chromosomes. Manuscript to be reviewed
mputer Science Figure 5. λ scores obtained with CNN trained on four different species: human, mouse, chicken
and zebrafish. λ scores are computed from predictions done on GSS of (A) human, (B) mouse, (C)
chicken and (D) zebrafish chromosomes. Application of the approach to other vertebrates
290 314 10/13 PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) Computer Science REFERENCES
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is work was supported by the Agence Nationale pour la Recherche [HiResBac ANR-15-CE11-0023-03 This work was supported by the Agence Nationale pour la Recherche [HiResBac ANR-15-CE1
359 ADDITIONAL INFORMATION
352 Data availability statement
353 Data availability statement
353 y
Genome sequences data and gene annotation are available at http://hgdownload.soe.ucsc.edu/ and
354
http://egg.wustl.edu/. 355 Genome sequences data and gene annotation are available at http://hgdownload.soe.ucsc.edu/ and
354
http://egg.wustl.edu/. 355 Conflict of interest statement
356 Conflict of interest statement
356 The authors declare no competing interests. 357 ACKNOWLEDGEMENTS
348 We would like to thank L´eopold Carron for helping us with datasets, Hugues Roest Croeluis for discussions,
349
Michel Quaggetto for technical support and Annick Lesne for comments on the manuscript. We also wish
350
to thank our editor James Procter and the two anonymous referees for their invaluable work. 351 We would like to thank Leopold Carron for helping us with datasets, Hugues Roest Croeluis for discussions,
349
Michel Quaggetto for technical support and Annick Lesne for comments on the manuscript. We also wish
350
to thank our editor James Procter and the two anonymous referees for their invaluable work. 351 Michel Quaggetto for technical support and Annick Lesne for comments on the manuscript. We also wish
350
to thank our editor James Procter and the two anonymous referees for their invaluable work 11/13 PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) REFERENCES
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A primer on deep learning in genomics. Nature genetics, page 1, 2018. 454 13/13 PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) Computer Science Computer Science Manuscript to be reviewed Overview of the CNN model. 299 bp-long sequences are one hot encoded into a 4 x 299 input matrix. The first CNN layer
performs a convolution on each input matrix to recognise relevant motifs. The next
convolutional layers models the interplay among these motifs to grasp higher-level features. Max-pooling layers reduce the dimensions of the layers. The model is trained to correctly
label input sequences as GSS or non-GSS. The output layer of the trained network then gives
a probability for any 299 bp region to contain a GSS. It can be applied along a full
chromosome, i.e. on all 299 bp-long sequences with a 1 bp shift. PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) CNN predictions for two regions of chromosome 21 ((A) Prediction scores for balanced 1* model (Q=1) and unbalanced 100* model (Q=100),
respectively in blue and red on a 300 kb region. The position of genes is indicated below. The
annotation track was done using the UWash Epigenome browser ((A) Prediction scores for balanced 1* model (Q=1) and unbalanced 100* model (Q=100),
respectively in blue and red on a 300 kb region. The position of genes is indicated below. The
annotation track was done using the UWash Epigenome browser
(https://epigenomegateway.wustl.edu/). Both models detect 7 GSS positions, but the 1*
model returns a higher background signal at non GSS positions. Adding negative examples
using the 100* model mitigates the noise while preserving the high scores over GSS. (B)
Application of 30 1* models, trained on different datasets, over a 3.2 kb region of
chromosome 21. At each site, the maximum and minimum prediction scores are respectively
displayed in black and red. Other prediction scores are plotted in grey. (https://epigenomegateway.wustl.edu/). Both models detect 7 GSS positions, but the 1*
model returns a higher background signal at non GSS positions. Adding negative examples
using the 100* model mitigates the noise while preserving the high scores over GSS. (B)
Application of 30 1* models, trained on different datasets, over a 3.2 kb region of
chromosome 21. At each site, the maximum and minimum prediction scores are respectively
displayed in black and red. Other prediction scores are plotted in grey. PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) Computer Science Computer Science Manuscript to be reviewed Computer Science Computer Science Manuscript to be reviewed PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) Computer Science Computer Science Manuscript to be reviewed Comparison of the 1* and 100* models predictions over chromosome 21 Comparison of the 1* and 100* models predictions over chromosome 21 (A) and (B) Heat maps depict the Z-score of the prediction for the 1* and 100* models
respectively on 5000 bp flanking each GSS of chromosome 21. (C) and (D) Averaged Z-score
of the predictions over each GSS of chromosome 21. (E-H) Zoom on regions around randomly
selected GSS. Genes are indicated at the bottom of each plot. (I-K) Averaged Z-score of the
predictions over each GSS of mouse chromosome X (I) and for networks trained on
mouse/human chromosomes (except X) and applied on human/mouse chromosome X (J,K). PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) Computer Science Computer Science Manuscript to be reviewed Evaluation of the model performance for different classes of genes Evaluation of the model performance for different classes of genes (A) and (B) CpG number in 299bp regions centred on mRNA-GSS in X chromosomes for
human (A) and mouse (B). These regions were divided in three groups of similar size
according to their CpG number into low, medium and high groups (the bounds are 35% and
60% for human and 30% and 60% for mouse). The proportion of genes in each class is similar
on the X chromosome (test set) than on other chromosomes (training and validation sets). (C) Lambda values computed for networks trained on each species non-X chromosome GSS
(t) and predicted on either species' X-chromosome GSS (p). Lambda values for each mRNA-
CpG sub-group and ncRNA genes are also shown to highlight different levels of performance. PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) Manuscript to be reviewed
Computer Science Manuscript to be reviewed PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020) Figure 5 Lambda scores obtained with CNN trained on four different species: human, mouse,
chicken and zebrafish Lambda scores are computed from predictions done on GSS of (A) human, (B) mouse, (C)
chicken and (D) zebrafish chromosomes. Lambda scores are computed from predictions done on GSS of (A) human, (B) mouse, (C)
chicken and (D) zebrafish chromosomes. PeerJ Comput. Sci. reviewing PDF | (CS-2019:10:42134:3:0:NEW 13 May 2020)
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http://www.scielo.br/pdf/po/v8n3/8307.pdf, http://pdfs.semanticscholar.org/b5c9/d6b82cc6b21fdf24142ce734fd5d4cae1689.pdf
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Tela de largura de temperatura do LCD de tela TFT para vendaof polyester resin
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cc-by
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Reaproveitamento de Resíduos de Laminados de
Fibra de Vidro na Confecção de Placas
Reforçadas de Resina Poliéster.
Patrícia Risson, G. A. Carvalho, S. L. Vieira, Mara Zeni e Ademir J. Zattera
Resumo: Visando o aproveitamento de resíduos de laminados de poliéster insaturado com fibra de vidro, foi
realizado o estudo da incorporação do resíduo moído em placas confeccionadas à base de resina poliéster
insaturado pelo processo de moldagem por compressão de preformas (BMC). Foram testadas cinco formulações com diferentes quantidades de resina poliéster, resíduo moído, aditivos e cargas. As placas foram confeccionadas em prensa hidráulica com molde plano e pressões da ordem de 18,1 MPa à temperatura de
115 ºC. Os corpos de prova, preparados segundo normas ASTM, foram submetidos a ensaios de tração,
impacto Charpy e dureza do material. Os resultados dos ensaios mecânicos indicam que a substituição de
fibra de vidro pelo resíduo moído no compósito polimérico promove perda de resistência à flexão, ao impacto Charpy e a dureza do compósito; a resistência à tração mostra que a incorporação do resíduo pode ter um
aumento de 23% quando a quantidade de resíduo adicionada é de 20% p/p.
Palavras-chave: Resíduos, laminados de poliéster insaturado , reaproveitamento, reciclagem, fibra de vidro.
Introdução
Os materiais poliméricos sintéticos são utilizados para a produção de uma variada gama de artigos,
suprindo os mais diversos requisitos funcionais de
forma econômica e eficaz. Após a segunda guerra
mundial intensificou-se a criação e a utilização de
novos tipos de polímeros, surgindo, de maneira lenta
e gradual, o uso de termofixos e fibras de vidro como
reforço de peças moldadas em resina poliéster
insaturado, resultando num compósito.
Os compósitos são uma classe dos materiais de
engenharia em que um dos componentes (fase
descontínua) confere resistência ao compósito quan-
do submetido a algum tipo de esforço, enquanto que
o outro componente (fase contínua) é responsável
pela transferência deste esforço (componente
matricial). O compósito polímero reforçado com fibra de vidro é usualmente denominado de
“fiberglass” e possui as seguintes características:
leveza; facilidade de transporte; rapidez de instalação; custo inferior ao de equipamentos construídos
com ligas especiais; aço inoxidável ou até mesmo
materiais menos nobres; propriedades mecânicas
satisfatórias para aplicações estruturais, resistência
química a diversos ambientes agressivos; dispensa
pintura, revestimento ou qualquer proteção
anticorrosiva; manutenção simples e barata; pode
Patrícia Risson, G. A. Carvalho, S. L. Vieira e Ademir J. Zattera., Departamento de Engenharia Química (CCET), Universidade de Caxias do Sul, Mara
Zeni, Departamento de Física e Quimica (CCET), Universidade de Caxias do Sul, CEP 95001-970, Caxias do Sul, RS.
Polímeros: Ciência e Tecnologia - Jul/Set - 98
89
C
O
M
U
N
I
C
A
Ç
Ã
O
T
É
C
N
I
C
O
C
I
E
N
T
Í
F
I
C
A
ser translúcido ou opaco, na cor desejada; possibilidade de uso em materiais que estarão em contato
com alimentos; pode ser construído com detalhes
complexos, sem emendas podendo ser modificado
no campo com ferramentas simples[1].
São vários os processos de fabricação de
compósitos resina poliéster insaturado e fibra de vidro passíveis de desenvolvimento. Todos partem do
princípio básico de processamento no qual a resina
termofixa é dissolvida num solvente apropriado que
faz a impregnação das fibras de reforço. A cura é
realizada tanto à temperatura ambiente como em
prensas ou moldes pré-aquecidos (dependendo do
processo de fabricação: BMC, SMC), sendo que o
tempo de cura deste último pode variar com a espessura e com o tipo de material, utilizando temperatura e pressões adequadas[2]. Figueiredo apresentou
um estudo bastante completo sobre a reciclagem de
termofixos à base de resina poliéster insaturado e
fibra de vidro obtidos pelos processos BMC e
SMC[3].
A crescente utilização de termofixos reforçados
com fibra de vidro no setor automobilístico, trouxe
consigo a preocupação com a produção de resíduos
sólidos no seu processamento[4,5]. Na região de Caxias
do Sul (RS), são geradas cerca de 80 toneladas por
mês de resíduos de resina poliéster insaturado reforçada com fibra de vidro sem utilização que comumente são lançadas em aterros[6].
A estabilidade química, física e térmica dos compostos termorrígidos são um desafio para seu reaproveitamento, pois, ao contrário dos termoplásticos e
metais, não podem ser refundidos[3,7]. Muitas técnicas têm sido apresentadas como forma de reutilizar
ou degradar estes materiais, sendo que algumas têm
sido criadas apenas para a utilização da porção orgânica que representa somente 20 a 25% do conteúdo[8]. Dentre elas estão a incineração, a pirólise, a
degradação química e a moagem e reutilização do
material polimérico[9].
Neste trabalho é avaliada a possibilidade de
reutilização de resíduos sólidos gerados no processo
de laminação à pistola (spray-up) como cargas
reforçantes em compósitos termofixos fabricados por
BMC. O processo utilizado foi o de moagem em virtude de sua facilidade de implementação e do fator
econômico[10]. Estudos preliminares mostraram que
o custo da moagem somado ao custo do refugo, proporcionado pelo processo, correspondem a 10% do
valor da resina virgem[11].
90
Tabela 1. Porcentagem de cada componente na formulação do BMC (p/p)
Amostra
I
II
III
IV
V
resina
25,08
30,63
35,44
35,44
35,19
aditivos
0,97
0,19
0,17
0,17
0,83
carga mineral
8,05
8,33
7,79
7,79
7,79
fibra de vidro
49,64
22,92
—
—
—
outros*
16,26
15,02
13,97
4,97
—
resíduos
0,00
22,92
42,63
51,63
56,19
*agentes tixotrópicos , de compatibilização e desmoldantes1,3
Parte Experimental
O material utilizado (resíduo de laminados de resina poliéster insaturado reforçada com fibra de vidro) era proveniente do processo de laminação à
pistola e do setor de corte das rebarbas. O resíduo foi
moído e analisada sua granulometria. Escolheu-se a
faixa de diâmetros maior que 6 e menor que 14 mm
(economicamente mais viável), e uma composição
média de 68% de fibra de vidro e 32% de matéria
orgânica (p/p).
Com a finalidade de reutilizar resíduos de
laminados poliéster insaturado com fibra de vidro no
processamento BMC, foram feitas cinco misturas com
diferentes composições desses resíduos, conforme a
Tabela 1. Depois de prontas, as misturas foram acondicionadas em um filme de polietileno e conservadas
à temperatura de 25oC, durante 10 dias. As massas de
cada amostra foram então prensadas em uma prensa
hidráulica com molde de chapa plana (previamente
aquecido) para a confecção de placas. A pressão utilizada foi de aproximadamente 1,87 kgf/cm2 (18,1
MPa) e a temperatura média de 115°C.
Depois de prontas as placas, foi determinada a
massa específica de cada amostra através do deslocamento do volume de água em uma proveta. A seguir, os corpos de prova foram submetidos a ensaios
de tração (ASTM D-638), impacto (ASTM D-256),
flexão (ASTM D-790) e dureza Barcol (ASTM D2583). Para cada medida foram utilizados três corpos de prova, de cada amostra.
Resultados e Discussão
Na tabela 2 são apresentados os resultados dos ensaios mecânicos realizados e a massa específica de cada
uma das amostras feitas pelo processamento BMC.
Polímeros: Ciência e Tecnologia - Jul/Set - 98
Tabela 2. Resultados dos testes realizados para cada formulação do BMC
Amostra
Resistência à
tração (MPa)
Resistência à
flexão (MPa)
Dureza Barcol
(Barcol)
Resistência ao
impacto Charpy
(J/cm2)
Massa Específica
(g/cm3)
I
22,20
58,59
53,5
1,20
1,83
II
27,73
29,85
52,0
0,80
1,57
III
27,35
32,71
47,0
0,78
1,43
IV
22,37
32,71
43,4
0,31
1,39
V
21,70
30,44
39,5
0,21
1,27
Os resultados obtidos por meio dos ensaios de tração mostraram que a incorporação do resíduo juntamente
com o aumento da quantidade de resina (amostras I e
II), conferiram ao compósito uma maior resistência à
tração (24,9%). Neste caso, o resíduo de laminado de
fibra de vidro atua como reforço, melhorando esta pro60
60
Resistência à tração
55
Resistência à flexão
Dureza Barcol
50
50
45
45
40
40
35
35
30
30
25
25
20
20
15
Dureza B
barcol (Barcol)
Resistências à tração e flexão (MPa)
55
15
II
I
III
V
IV
Amostra
Figura 1. Resistência à tração, resistência à flexão e dureza Barcol para
os compósitos estudados.
2
i
Resistência ao Impacto
Charpy (J/cm )
1.5
1.0
priedade mecânica. Entretanto, aumentando-se ainda
mais a concentração de resíduos e de resina sem adicionar fibra de vidro à mistura (amostra III), pouco altera a
resistência à tração. Porém, se uma maior quantidade
de resíduo for acrescentada (amostras IV e V), a mistura se torna menos resistente à tração.
Com relação aos ensaios de flexão, os resultados
mostraram que a substituição da fibra de vidro pelo
resíduo (amostras I e II) reduziu a resistência à flexão
em 49%. Para os compósitos que não continham fibra de vidro (amostras II, IV e V), o aumento da quantidade de resíduo diminui a resistência à flexão das
amostras.
Nos ensaios de dureza Barcol, observou-se que,
da amostra I para a II, a dureza teve redução de menos de 3%. Porém, a ausência da fibra de vidro nas
amostras e o aumento da concentração de resíduos
(amostras III. IV e V) provocou uma diminuição de
mais de 12% na dureza.
Na figura 1 são apresentados os resultados dos
ensaios de tração, flexão e dureza Barcol para cada
amostra ensaiada.
Quanto à resistência ao impacto Charpy, todas as
amostras que continham uma certa quantidade de resíduo de laminado de fibra de vidro apresentaram uma
diminuição desta propriedade mecânica. A figura 2
ilustra o comportamento dos diferentes compósitos
com respeito à resistência ao impacto Charpy.
Conclusões
0.5
0.0
0
1I
2II
III
3
IV
4
V
5
6
Amostra
Figura 2. Resistência ao impacto Charpy para os compósitos estudados.
Polímeros: Ciência e Tecnologia - Jul/Set - 98
A proposta de misturar resina poliéster com resíduos de laminados de resina poliéster insaturado reforçada com fibra de vidro propiciou a obtenção de
placas de excelente aspecto e com boas propriedades
mecânicas, permitindo sua utilização em painéis, pisos, divisórias entre outras.
91
Os resultados deste trabalho resumem-se em:
1. a substituição da fibra de vidro pelo resíduo diminui a resistência à flexão, a resistência ao impacto Charpy e a dureza do material;
2. dependendo da quantidade de resíduo adicionado, a resistência à tração pode ter um aumento da ordem de 23%;
Agradecimentos
Os autores agradecem ao CNPq, à UCS, à
FAPERGS e à empresa São Marcos Fibras Ltda.
Referências Bibliográficas
1. Biryukovich, K.L. - “Glassfibre Reinforced
Cement”, Budivelnik, Kiev (1994)
2. Carvalho, A. - “Manual Técnico da Fiberglass do
Brasil”, São Paulo (1991)
3. Figueiredo, E.M. - “Reciclagem de Plásticos
Termofixos à Base de Resina Poliéster Reforçados com Fibra de Vidro”, I Congresso Brasileiro de Polímeros, 460-464 (1991)
4. Anom - “El Reciclado como Fuente Rentable de
Materias Primas”, Revista de Plásticos Modernos, 27(327), 386-390, Madrid (1976)
5. Ferraro Jr., D. - “Reciclagem Transforma Problema em Solução”, Plástico Moderno, 21(208),
20-23, São Paulo (1990)
6. CSD - GEOLOCK - Geologia e Engenharia Ambiental
Ltda.; “Inventário de Resíduos Industriais e Hospitalares”, Caxias do Sul, RS, p.15 (1992)
92
7. Bilwastsch, D. - “Garantia de Calidad en los Plásticos Reciclados”, Plásticos Universalles (16),
70-74, Barcelona (1992)
8. Rubin, I.I. - “Handbook of Plastics Materials and
Technology”, John Wiley & Sons, 799-1039,
New York (1990)
9. Bonelli, C.M.C. - “Recuperação Secundária de
Plásticos Provenientes de Resíduos Sólidos Urbanos do Rio de Janeiro”, tese de mestrado
IMA/UFRJ, Rio de Janeiro, julho (1993)
10. Almeida, M.G., Zeni, M. e Zattera, A.J. - “Estudo sobre o Reaproveitamento do EVA (Etileno
Acetato de Vinila)”, anais do XI CBECIMAT,
Águas de São Pedro, dezembro (1994)
11. Zattera, A.J., Cormelato, E., Bianchi, F., De Bortoli,
V. e Zeni, M. - “Reciclagem de Materiais
Poliméricos” anais do II Congresso Brasileiro de
Polímeros, São Paulo, 718-722, outubro (1993)
12. Maczko, J. - “An Alternative to Landfills for Mixed
Plastic Waste”, Plastics Engineering, 46(4), 5153, Roselle (1990).
13. Majumdar, A.J. and Ryder, J.F. - “Glassfibre
Reinforcement for Cement Products”, Glass
Technology, 9, 78-84 (1986).
14. Majumdar, A.J. - “Glassfibre Reinforced Cement
and Gypsum Products”, Proc. Roy. Soc. Lond.
A., (319), 69-78 (1970).
Recebido: 09/02/98
Aprovado: 30/07/98
Polímeros: Ciência e Tecnologia - Jul/Set - 98
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https://openalex.org/W2013700732
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https://europepmc.org/articles/pmc3463344?pdf=render
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English
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Porphyromonas gingivalis Mediates Inflammasome Repression in Polymicrobial Cultures through a Novel Mechanism Involving Reduced Endocytosis
|
Journal of biological chemistry/The Journal of biological chemistry
| 2,012
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cc-by
| 9,274
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g
y
Received for publication,July 17, 2012 Published, JBC Papers in Press,July 26, 2012, DOI 10.1074/jbc.M112.401737 Debra J. Taxman‡§1, Karen V. Swanson‡§, Peter M. Broglie¶2, Haitao Wen‡§, Elizabeth Holley-Guthrie‡§,
Max Tze-Han Huang§, Justin B. Callaway‡§, Tim K. Eitas‡§, Joseph A. Duncan§¶3, and Jenny P. Y. Ting‡§
From the ‡Department of Microbiology and Immunology, School of Medicine, the §Lineberger Comprehensive Cancer Center, the
¶Division of Infectious Diseases, School of Medicine, and the Department of Pharmacology, School of Medicine, University of North
Carolina, Chapel Hill, North Carolina 27599 Background: Porphyromonas gingivalis has low immunogenicity and synergizes with other periodontal pathogens, includ-
ing Fusobacterium nucleatum. g
Results: Porphyromonas gingivalis selectively represses the activation of the IL-1-processing inflammasome by Fusobacterium
nucleatum and inducers that are endocytosed. Conclusion: Porphyromonas gingivalis suppresses inflammasome activity through a novel mechanism involving modulation of
endocytosis. y
Significance: Inflammasome suppression may contribute to periodontitis and other chronic diseases. y
Significance: Inflammasome suppression may contribute to periodontitis and other chronic diseases. The
interleukin
(IL)-1-processing
inflammasome
has
recently been identified as a target for pathogenic evasion of the
inflammatory response by a number of bacteria and viruses. We
postulated that the periodontal pathogen, Porphyromonas gin-
givalis may suppress the inflammasome as a mechanism for its
low immunogenicity and pathogenic synergy with other, more
highly immunogenic periodontal bacteria. Our results show
that P. gingivalis lacks signaling capability for the activation of
the inflammasome in mouse macrophages. Furthermore, P. gin-
givalis can suppress inflammasome activation by another peri-
odontal bacterium, Fusobacterium nucleatum. This repression
affects IL-1 processing, as well as other inflammasome-medi-
ated processes, including IL-18 processing and cell death, in
both human and mouse macrophages. F. nucleatum activates
IL-1 processing through the Nlrp3 inflammasome; however,
P. gingivalis repression is not mediated through reduced levels
of inflammasome components. P. gingivalis can repress Nlrp3
inflammasome activation by Escherichia coli, and by danger-
associated molecular patterns and pattern-associated molecular
patterns that mediate activation through endocytosis. However,
P. gingivalis does not suppress Nlrp3 inflammasome activation
by ATP or nigericin. This suggests that P. gingivalis may prefer-
entially suppress endocytic pathways toward inflammasome
activation. To directly test whether P. gingivalis infection affects
endocytosis, we assessed the uptake of fluorescent particles in
the presence or absence of P. gingivalis. Our results show that P. gingivalis limits both the number of cells taking up beads and
the number of beads taken up for bead-positive cells. These
results provide a novel mechanism of pathogen-mediated
inflammasome
inhibition
through
the
suppression
of
endocytosis. 4 The abbreviations used are: NLR, nucleotide-binding domain and leucine-
rich repeat; ASC, apoptosis-associated speck-like protein containing a CARD;
PGN, peptidoglycan; BMDM, bone marrow-derived mouse macrophages;
m.o.i., multiplicity of infection; MSU, monosodium urate. 1 To whom correspondence should be addressed: Department of Microbiol-
ogy and Immunology, University of North Carolina, Chapel Hill, NC 27599.
Tel.: 919-966-2662; Fax: 919-966-8212; E-mail: debra_taxman@med.
unc.edu. * This study was supported, in whole or in part, by National Institutes of
Health Grants DE016326, AI029564, and AI057157 (to J. T.) and AI088255
(to J. A. D). THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 39, pp. 32791–32799, September 21, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 39, pp. 32791–32799, September 21, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 39, pp. 32791–32799, September 21, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. * This study was supported, in whole or in part, by National Institutes of
Health Grants DE016326, AI029564, and AI057157 (to J. T.) and AI088255
(to J. A. D).
1 To whom correspondence should be addressed: Department of Microbiol-
ogy and Immunology, University of North Carolina, Chapel Hill, NC 27599.
Tel.: 919-966-2662; Fax: 919-966-8212; E-mail: debra_taxman@med.
unc.edu.
2 Awardee of the UNC STD and HIV Training Program (National Institutes of
Health Grant T32AI007001).
3 Supported by the Burroughs Wellcome Fund through the Career Award for
Medical Scientists. 2 Awardee of the UNC STD and HIV Training Program (National Institutes of
Health Grant T32AI007001). 3 Supported by the Burroughs Wellcome Fund through the Career Award for
Medical Scientists. Porphyromonas gingivalis Mediates Inflammasome
Repression in Polymicrobial Cultures through a Novel
Mechanism Involving Reduced Endocytosis* g
y
Received for publication,July 17, 2012 Published, JBC Papers in Press,July 26, 2012, DOI 10.1074/jbc.M112.401737 P. gingivalis Inflammasome Repression via Reduced Endocytosis target for pathogenic inhibition by specific bacteria and viruses. At least two pathogenic viruses have evolved strategies for
suppressing the inflammasome by producing inactivating
homologs of apoptosis-associated speck-like protein contain-
ing a CARD (ASC) (6, 7). Other viruses have been shown to
mediate inflammasome inhibition through their production of
serpin family proteins (8–10) and others (11) that can directly
interact with caspase-1. Additionally, a viral NLR homolog was
recently described that can repress inflammasome activation by
Kaposi sarcoma-associated herpes virus (12). An alternative
strategy for pathogenic inflammasome inhibition involves the
modulation of the upstream signals that lead to its activation. Several bacteria minimize the inflammasome response through
the production of Type III secretion system effector molecules
that either regulate caspase-1 activity (13–15) or mask inflam-
masome detection (16). Staphylococcus aureus suppresses the
inflammasome through enzymatic modification of its cell wall
peptidoglycan (PGN) to make it resistant to lysosomal degra-
dation following endocytosis (17). A variety of additional bac-
terial proteins suppress the inflammasome by unknown mech-
anisms (18, 19). lenium Inc.; Asc/ mice from Dr. Vishva Dixit at Genentech;
and Casp1/ mice from Dr. Richard Flavell, Yale University. All the mice were backcrossed for a minimum of nine genera-
tions to C57BL/6 mice. Bone marrow-derived macrophages
were harvested from WT or gene-deletion mice and cultured in
DMEM 10% fetal calf serum, M-CSF for 6–7 days. Cells were
plated without M-CSF 16 h prior to infection. Peripheral blood
macrophages were isolated on buffy coats from healthy donors
(American Red Cross). Primary human macrophages were har-
vested by Ficoll-Hypaque gradients and seeded in 10 ml of
RPMI 1640 medium containing 10% heat-inactivated FBS. Nonadherent cells were removed prior to infection. p
Bacterial Strains and Bacterial Infection—P. gingivalis strain
A7436 was isolated from a refractory case of periodontitis and
has been described previously (40). P. gingivalis strain 381,
F. nucleatum strain PK 1594, and Eschericha coli strain LF82
were obtained from American Type Culture Collection
(Manassas, VA). P. gingivalis and F. nucleatum were cultured
anaerobically, and E. coli aerobically until late exponential
phase (0.8–1.2 optical density units at 660 nm). Aliquots were
stored in media containing 20% glycerol at 80 °C and used
within 3–4 months of preparation. Bacterial counts were con-
firmed to within 2–3-fold by replating of frozen cultures. Infec-
tions were performed by adding the desired multiplicity of
infection (m.o.i.) of P. gingivalis either alone or together with
F. P. gingivalis Inflammasome Repression via Reduced Endocytosis nucleatum or E. coli to macrophages for 16–18 h. 10 g/ml
gentamycin was added to cell cultures 2 h following infection. Porphyromonas gingivalis is one of the most common patho-
gens in chronic periodontitis, and its presence in the oral cavity
is associated with a variety of related systemic diseases (20). In
addition to its immunostimulatory properties, evidence is
emerging to suggest that P. gingivalis has evolved several dis-
tinct mechanisms for evasion of the immune response that may
contribute to its ability to perpetuate the chronic state of peri-
odontal diseases. Like other oral pathogens, P. gingivalis pro-
duces proteases and toxins that directly attack host tissue, as
well as lipopolysaccharide (LPS) and other microbial products
that induce inflammation. However, cell wall extracts and puri-
fied cellular components from P. gingivalis have comparatively
weak host immunostimulatory activity (21–24). Additionally,
P. gingivalis lipid A has a unique structure that can either stim-
ulate or antagonize Toll-like receptor 4 (TLR4) activation
depending on environmental growth conditions and its phos-
phorylation and acylation states (25–29). Furthermore, P. gin-
givalis actively limits the immune response by producing pro-
teases that degrade complement component C3 and IgG (30)
and promotes intrinsic signaling that inhibits E-selectin expres-
sion and secretion of the neutrophil chemoattractant IL-8 (31,
32). Additionally, P. gingivalis confers resistance to apoptosis
induced by pharmacologic agents (33–35). It is likely that these
evasion mechanisms may explain the pathogenic synergism
that is observed between P. gingivalis and other periodontal
bacteria in animal models of infection (36–39). Treatment with Signal 1 and Signal 2 Inducers—To induce
signal 1 activation in macrophages, 1 g/ml Ultrapure E. coli
LPS (InvivoGen) was added to macrophage cultures for 3 h
prior to the addition of the signal 2 activator. Signal 2 activation
was induced by the addition of 2 mM ATP (Sigma) or 20 M
nigericin (InvivoGen) for 30 min; 200 g/ml monosodium
urate (MSU) or 400 g/ml alum crystals (InvivoGen) for 6 h; or
20 g/ml PGN from S. aureus (InvivoGen) for 14–16 h. Inhibi-
tion of second signal activation by P. gingivalis was assessed by
adding bacteria immediately prior to the second signal inducer. ELISA Analysis—Supernatants were collected 18–24 h fol-
lowing infection unless otherwise indicated. Secreted cytokine
levels were assessed using the human ELISA set for IL-1 (BD
Biosciences) or the human ELISA kit for IL-18 (R&D Systems). Samples were assayed within linear range. P. gingivalis Inflammasome Repression via Reduced Endocytosis RNA Isolation and Real-time PCR—RNA was isolated using
RNeasy kits (Qiagen). Real-time PCR was performed using
TaqMan Assays on Demand (Applied Biosystems). Values
represent averages S.D. of biological triplicates. All values
were standardized to 18 S rRNA expression. Western Analysis—For assessment of secreted IL-1 levels,
supernatants were clarified by centrifugation for 10 min and
then boiled for 5 min in 1/3 volume of 3 SDS sample buffer
(187.5 mM Tris, pH 6.8, 6% SDS, 30% glycerol, 150 mM DTT,
0.03% bromphenol blue). Cell lysates were prepared by washing
cells in 1 PBS and then lysing for 20 min in ice-cold 1 lysis
buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM
EGTA, 1% Triton X-100) supplemented with Complete EDTA-
free Protease Inhibitor (Roche Applied Science). Lysates were
centrifuged for 10 min and boiled for 5 min in 1/3 volume of 3
SDS sample buffer. Immunoblots were processed using 3ZD Given the knowledge that P. gingivalis has evolved a unique
set of mechanisms for subverting inflammatory activity, we
hypothesized that P. gingivalis may function to suppress the
inflammasome. Our results demonstrate that P. gingivalis sup-
presses inflammasome activation by other pathogenic bacteria,
and that this suppression occurs by a novel mechanism involv-
ing the blockade of endocytosis. g
y
Received for publication,July 17, 2012 Published, JBC Papers in Press,July 26, 2012, DOI 10.1074/jbc.M112.401737 The innate immune inflammatory cytokine, interleukin
(IL)-1 is critical in the host defense against infection, and con-
sequently, pathogens have evolved an array of mechanisms for
inhibiting its production (1). This includes the inhibition of the
transcription of the pro-Il-1 precursor mRNA, the production
of IL-1 decoy receptors, and most recently, the inhibition of
the IL-1 processing inflammasome (see Ref. (2) for a review). The primary functions of the inflammasome are to cleave the
precursor pro-IL-1 protein to its active secreted form and to
promote caspase-dependent signaling pathways leading to cell
death in response to foreign or intrinsic “danger signals,”
including bacterial-encoded “pattern-associated molecular
patterns” and host-derived “danger-associated molecular pat-
terns.” In addition to its role in combating pathogenic infection,
recent evidence suggests a role for the inflammasome in medi-
ating host metabolic responses (3, 4). The dysregulation of
inflammasome components has been linked to a number of
inherited inflammatory and immune disorders, further under-
scoring its relevance in human disease (5). Classically, the inflammasome is composed of a nucleotide-
binding domain and leucine-rich repeat (NLR)4 family protein,
the adaptor molecule PYCARD/ASC, and pro-caspase-1,
which provides the core enzymatic activity of the complex. Each of these components has been postulated to serve as a JOURNAL OF BIOLOGICAL CHEMISTRY 32791 32791 SEPTEMBER 21, 2012•VOLUME 287•NUMBER 39 EXPERIMENTAL PROCEDURES Mouse Strains and Macrophage Culture—MyD88/ mice
were obtained from Dr. Shizuo Akira; Nlrp3/ mice from Mil- 32792
JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 39•SEPTEMBER 21, 2012 FIGURE 1. P. gingivalis lacks second signal activation ability for IL-1 production and can repress second signal activation by F. nucleatum. ELISAs of
IL-1incellsupernatantswereperformedfollowing12–16-hinfection.A,mouseBMDMwereinfectedwith20m.o.i.P.gingivalis(Pg),F.nucleatum(Fn),orE. coli
(Ec). Where indicated, cells were treated for 30 min with 2 mM ATP immediately prior to collection of supernatants. B, BMDM were infected with Fn and Pg alone
or in combination as indicated. C, BMDM were infected with Fn and Ec alone or in combination as indicated. D, BMDM were treated with a range of doses of Fn
and Pg. E, BMDM were treated with Fn and Pg strains A7436 or 381. Results represent the averages and standard deviation of duplicates and are representative
of at least three independent experiments. P. gingivalis Inflammasome Repression via Reduced Endocytosis P. gingivalis Inflammasome Repression via Reduced Endocytosis P. gingivalis Inflammasome Repression via Reduced Endocytosis FIGURE 1. P. gingivalis lacks second signal activation ability for IL-1 production and can repress second signal activation by F. nucleatum. ELISAs of
IL-1incellsupernatantswereperformedfollowing12–16-hinfection.A,mouseBMDMwereinfectedwith20m.o.i.P.gingivalis(Pg),F.nucleatum(Fn),orE. coli
(Ec). Where indicated, cells were treated for 30 min with 2 mM ATP immediately prior to collection of supernatants. B, BMDM were infected with Fn and Pg alone
or in combination as indicated. C, BMDM were infected with Fn and Ec alone or in combination as indicated. D, BMDM were treated with a range of doses of Fn
and Pg. E, BMDM were treated with Fn and Pg strains A7436 or 381. Results represent the averages and standard deviation of duplicates and are representative
of at least three independent experiments. monoclonal antibody against Pro-IL-1 (Frederick National
Laboratory for Cancer Research), H-153 against IL-1 (Santa
Cruz Biotechnology), IMG-5028 against caspase-1 (Imgenex),
AL177 against ASC (Enzo Life Sciences), Clone Cryo-2 against
NLRP3 (Enzo Life Sciences), sc-1615 against actin (Santa Cruz
Biotechnology), and MAB374 against GAPDH (Millipore). ond, inflammasome-activating signal (21). To test whether
P. gingivalis lacks “signal 2” activation capability in macro-
phages, we measured IL-1 production in bone marrow-de-
rived mouse macrophages (BMDM) following infection. Our
results showed that overnight infection with P. gingivalis was
not sufficient for the stimulation of significant levels of IL-1,
but that IL-1 secretion could be induced by the subsequent
addition of ATP, a well characterized second signal activator
(42). EXPERIMENTAL PROCEDURES This was in contrast to the high levels of IL-1 activation
elicited in the absence of ATP for E. coli and for F. nucleatum,
another periodontal pathogen that is commonly isolated from
periodontal disease sites together with P. gingivalis (20) (Fig. 1A). This suggests that the lack of a signal 2 activation capability
is a property of P. gingivalis that is not conserved among all
bacteria. Cell Death Measurement—ToxiLight bioassays were per-
formed according to the manufacturer’s instructions (Lonza). Propidium iodide staining was performed as a measure of cell
leakage upon death and Hoechst staining as a general nuclear
stain as described previously (41). Endocytosis
Assays—FluoresbriteTN
Carboxyl
YG
2.0
Micron Microspheres latex beads (Polysciences, Inc.) were
added to macrophages at a concentration of 10 beads/cell either
alone or in combination with 100 moi P. gingivalis. 1 h follow-
ing exposure, cells were washed three times with 1 PBS and
then fixed with 4% paraformaldehyde. Cells and beads were
visualized using a Zeiss 710 confocal microscope. P. gingivalis has been shown to promote synergistic pathoge-
nicity in polymicrobial infections with F. nucleatum (36–39). Given the emerging role of inflammasome repression as a
mechanism for pathogenic stealth (2), we postulated that P. gin-
givalis may contribute to the synergistic effects of these two
bacteria in vivo by repressing F. nucleatum-mediated activation
of IL-1 processing. To assess this as a possible mechanism,
IL-1 levels were measured following infection with 20 m.o.i. F. nucleatum and increasing doses of P. gingivalis. Results ver-
ified that P. gingivalis repressed IL-1 activation by F. nuclea-
tum (Fig. 1B). This mechanism of P. gingivalis in repressing
F. nucleatum is not a common feature shared with other bacte- P. gingivalis Inflammasome Repression via Reduced Endocytosis A, real-time PCR of Il1b mRNA in mouse BMDM was performed 2 or 5 h following infection with a
range of doses of Fn and Pg. Expression is normalized to the expression of 18 S rRNA and standardized to 1 in uninfected cells. Results represent the average
S.D. (error bars) of biological triplicates and are representative of three independent experiments. B, Western analysis is shown for pro-IL-1 and pro-
caspase-1 protein in cell lysates and cleaved (activated) IL-1 and caspase-1 p10 in cell supernatants 14 h following infection with 20 m.o.i. Fn and 100 m.o.i. Pg. GAPDH is shown as a loading control. Results are representative of three independent experiments. C, ELISA of IL-18 expression in BMDM is shown following
14-h infection with Fn and Pg at indicated m.o.i. Results represent the averages S.D. of duplicates and are representative of at least three independent
experiments. D, real-time PCR of Il18 mRNA in BMDM was performed 2 or 5 h following infection with a range of dose of Fn and Pg. Expression is normalized to
the expression of 18 S rRNA and standardized to 1 in uninfected cells. Results represent the averages S.D. of biological triplicates and are representative of
three independent experiments. E, percentage of cell death in BMDM was determined by ToxiLight assay 16 h following infection with 20 m.o.i. Fn alone or
together with () 12.5, () 50, or () 200 m.o.i. Pg. Results are representative of three independent experiments. F, propidium iodide (P. I.) stain is shown
as an indicator of cell death in BMDM 16 h following infection with 20 m.o.i. Fn and 100 m.o.i. Pg. Hoechst staining is shown as a control. Results are
representative of two independent experiments. G, ELISA of IL-1 secretion in human macrophages is shown 14 h following infection with Fn and Pg at a range
of m.o.i. Results represent the averages S.D. of duplicates and are representative of two independent experiments. H, percentage of cell death in human
macrophages was determined by ToxiLight assay 16 h following infection with Fn and Pg at a range of m.o.i. or in combination with P. gingivalis. Real-time PCR analysis of
IL1b levels showed that this gene was transcriptionally induced
by F. nucleatum, but that its induction was not repressed upon
co-infection with P. gingivalis (Fig. 2A). These results indicate
that P. P. gingivalis Inflammasome Repression via Reduced Endocytosis P. gingivalis Inflammasome Repression via Reduced Endocytosis i
i
th t E
li
h
dd d t
F
l
t
h
d
i
bi
ti
ith P
i
i
li
R
l ti
PCR
FIGURE 2. Pg represses inflammasome activation by Fn. A, real-time PCR of Il1b mRNA in mouse BMDM was performed 2 or 5 h following infect
range of doses of Fn and Pg. Expression is normalized to the expression of 18 S rRNA and standardized to 1 in uninfected cells. Results represent th
S.D. (error bars) of biological triplicates and are representative of three independent experiments. B, Western analysis is shown for pro-IL-1
caspase-1 protein in cell lysates and cleaved (activated) IL-1 and caspase-1 p10 in cell supernatants 14 h following infection with 20 m.o.i. Fn and 100
GAPDH is shown as a loading control. Results are representative of three independent experiments. C, ELISA of IL-18 expression in BMDM is shown
14-h infection with Fn and Pg at indicated m.o.i. Results represent the averages S.D. of duplicates and are representative of at least three ind
experiments. D, real-time PCR of Il18 mRNA in BMDM was performed 2 or 5 h following infection with a range of dose of Fn and Pg. Expression is norm
the expression of 18 S rRNA and standardized to 1 in uninfected cells. Results represent the averages S.D. of biological triplicates and are represe
three independent experiments. E, percentage of cell death in BMDM was determined by ToxiLight assay 16 h following infection with 20 m.o.i. Fn
together with () 12.5, () 50, or () 200 m.o.i. Pg. Results are representative of three independent experiments. F, propidium iodide (P. I.) stain
as an indicator of cell death in BMDM 16 h following infection with 20 m.o.i. Fn and 100 m.o.i. Pg. Hoechst staining is shown as a control. R
representative of two independent experiments. G, ELISA of IL-1 secretion in human macrophages is shown 14 h following infection with Fn and Pg
of m.o.i. Results represent the averages S.D. of duplicates and are representative of two independent experiments. H, percentage of cell death
macrophages was determined by ToxiLight assay 16 h following infection with Fn and Pg at a range of m.o.i. FIGURE 2. Pg represses inflammasome activation by Fn. RESULTS IL-1 is produced in many circumstances via a two-step
process involving the activation of transcription and translation
of pro-IL-1 (signal 1) followed by its proteolysis to an active
secreted form by the inflammasome (signal 2). Most bacteria
have the ability to provide both signals in macrophages, which
are the most active physiologic producers of this potent inflam-
matory cytokine. By contrast, recent studies in epithelial cells
suggest that P. gingivalis may lack the ability to produce a sec- SEPTEMBER 21, 2012•VOLUME 287•NUMBER 39 32793 JOURNAL OF BIOLOGICAL CHEMISTRY 32 SEPTEMBER 21, 2012•VOLUME 287•NUMBER 39 P. gingivalis Inflammasome Repression via Reduced Endocytosis gingivalis repression was not at the level of the activation
of transcription (signal 1), implying a role in the IL-1 process-
ing pathway. To determine whether P. gingivalis affects inflam-
masome activation by F. nucleatum (signal 2), levels of pro-
IL-1 and pro-caspase-1 in cell lysates and levels of mature
IL-1 and caspase-1 p10 in supernatants were examined by ria, given that E. coli, when added to F. nucleatum, enhanced,
rather than repressed IL-1 release (Fig. 1C). The repressive
effect of P. gingivalis is dose dependent at a range of m.o.i. of
F. nucleatum (Fig. 1D). Furthermore, this activity does not
appear to be strain-specific, given that both the A7436 and 381
strains of P. gingivalis were able to repress F. nucleatum-medi-
ated IL-1 activation (Fig. 1E). To distinguish the level at which P. gingivalis represses
F. nucleatum-mediated IL-1 activation, RNA was isolated
from macrophages following infection with F. nucleatum alone VOLUME 287•NUMBER 39•SEPTEMBER 21, 2012 P. gingivalis Inflammasome Repression via Reduced Endocytosis FIGURE 3. Pg does not repress the inflammasome by ablating the expression of its protein components. A, ELISA of IL-1 secretion is shown for BMDM
isolated from wild type (WT) and gene-deletion mice 16 h following infection with Fn. B, ELISA of IL-1 secretion in wild type mouse BMDM is shown following
a time course of infection with 20 m.o.i. Fn and 100 m.o.i. Pg alone or in combination. C, real-time PCR of Nlrp3, Asc, and caspase-1 mRNA in BMDM following a
time course of infection with 20 m.o.i. Fn and 100 m.o.i. Pg is shown. Expression is normalized to the expression of 18 S rRNA and standardized to 1 in uninfected
cells. Results represent the average S.D. (error bars) of biological triplicates and are representative of three independent experiments. D, Western analysis was
performedforNlrp3,Asc,andcaspase-1proteinlevelsinBMDM8,11,and14hfollowinginfectionwith20m.o.i.Fnand100m.o.i.Pg.Actinisshownasaloading
control, and cleaved caspase-1 in cell supernatants is provided for reference. Results are representative of three independent experiments. P. gingivalis Inflammasome Repression via Reduced Endocytosis FIGURE 3. Pg does not repress the inflammasome by ablating the expression of its protein components. A, ELISA of IL-1 secretion is shown for BMDM
isolated from wild type (WT) and gene-deletion mice 16 h following infection with Fn. B, ELISA of IL-1 secretion in wild type mouse BMDM is shown following
a time course of infection with 20 m.o.i. Fn and 100 m.o.i. Pg alone or in combination. C, real-time PCR of Nlrp3, Asc, and caspase-1 mRNA in BMDM following a
time course of infection with 20 m.o.i. Fn and 100 m.o.i. Pg is shown. Expression is normalized to the expression of 18 S rRNA and standardized to 1 in uninfected
cells. Results represent the average S.D. (error bars) of biological triplicates and are representative of three independent experiments. D, Western analysis was
performedforNlrp3,Asc,andcaspase-1proteinlevelsinBMDM8,11,and14hfollowinginfectionwith20m.o.i.Fnand100m.o.i.Pg.Actinisshownasaloading
control, and cleaved caspase-1 in cell supernatants is provided for reference. Results are representative of three independent experiments. Western analysis. Whereas the pro-forms of each protein were
expressed in F. nucleatum-infected cells both without and with Western analysis. Whereas the pro-forms of each protein were
expressed in F. nucleatum-infected cells both without and with
P. gingivalis co-infection, the mature forms were only detected
in cells infected with F. nucleatum alone (Fig. 2B). These results
verify that P. gingivalis blocks IL-1 secretion at the level of
inflammasome activation. 32794
JOURNAL OF BIOLOGICAL CHEMISTRY 32794
JOURNAL OF BIOLOGICAL CHEMISTRY P. gingivalis Inflammasome Repression via Reduced Endocytosis P. gingivalis Inflammasome Repression via Reduced Endocytosis masome activation requires an initial activation through the
MyD88 adaptor molecule, followed by the assembly of a protein
complex composed of NLRP3, ASC, and caspase-1 (43). To
determine whether MyD88 and these three inflammasome
components are required for F. nucleatum activation, we
infected macrophages from gene-deletion mice with F. nuclea-
tum and measured IL-1 release. Our results showed that each
of these genes was required for F. nucleatum-mediated IL-1
release (Fig. 3A). We next tested whether transcript levels of the
three inflammasome components were regulated by F. nuclea-
tum and P. gingivalis. RNA was isolated over a time course of
infection that preceded and spanned the 11-h peak of IL-1
induction following F. nucleatum infection (Fig. 3B). Nlrp3
transcription was activated by both P. gingivalis and F. nuclea-
tum, and levels persisted following infection with the two bac-
teria in combination at each time point (Fig. 3C, top panel). By
contrast, levels of Asc and caspase-1 were not significantly
modulated following infection with F. nucleatum either alone
or in combination with P. gingivalis throughout the time course
(Fig. 3C, bottom two panels). To determine whether repression
of these inflammasome components occurs at the level of pro-
tein expression, Western blotting was performed 8, 11, or 14 h
post-infection. Levels of Nlrp3, Asc, and pro-caspase-1 were
similar in lysates from cells infected with F. nucleatum alone or
in combination with P. gingivalis at each earlier time point. At
the 14-h time point, levels of Nlrp3, Asc, and pro-caspase-1 in masome activation requires an initial activation through the
MyD88 adaptor molecule, followed by the assembly of a protein
complex composed of NLRP3, ASC, and caspase-1 (43). To
determine whether MyD88 and these three inflammasome
components are required for F. nucleatum activation, we
infected macrophages from gene-deletion mice with F. nuclea-
tum and measured IL-1 release. Our results showed that each
of these genes was required for F. nucleatum-mediated IL-1
release (Fig. 3A). We next tested whether transcript levels of the
three inflammasome components were regulated by F. nuclea-
tum and P. gingivalis. RNA was isolated over a time course of
infection that preceded and spanned the 11-h peak of IL-1
induction following F. nucleatum infection (Fig. 3B). Nlrp3
transcription was activated by both P. gingivalis and F. nuclea-
tum, and levels persisted following infection with the two bac-
teria in combination at each time point (Fig. 3C, top panel). P. gingivalis Inflammasome Repression via Reduced Endocytosis By
contrast, levels of Asc and caspase-1 were not significantly
modulated following infection with F. nucleatum either alone
or in combination with P. gingivalis throughout the time course
(Fig. 3C, bottom two panels). To determine whether repression
of these inflammasome components occurs at the level of pro-
tein expression, Western blotting was performed 8, 11, or 14 h
post-infection. Levels of Nlrp3, Asc, and pro-caspase-1 were
similar in lysates from cells infected with F. nucleatum alone or
in combination with P. gingivalis at each earlier time point. At
the 14-h time point, levels of Nlrp3, Asc, and pro-caspase-1 in Western analysis. Whereas the pro-forms of each protein were
expressed in F. nucleatum-infected cells both without and with
P. gingivalis co-infection, the mature forms were only detected
in cells infected with F. nucleatum alone (Fig. 2B). These results
verify that P. gingivalis blocks IL-1 secretion at the level of
inflammasome activation. The inflammasome is responsible for the processing and
secretion of other inflammatory cytokines besides IL-1,
including IL-18, as well as for the promotion of cell death. Our
results showed that, consistent with its role in inflammasome
suppression, P. gingivalis repressed the secretion of IL-18 (Fig. 2C), and that this repression, like that of IL-1, did not appear
to be transcriptional (Fig. 2D). P. gingivalis also suppressed the
induction of cell death by F. nucleatum as measured by Toxi-
Light assay (Fig. 2E) or propidium iodide staining (Fig. 2F). These findings rule out the possibility that reduced cytokine
secretion was caused by P. gingivalis-induced cell death in
F. nucleatum- and P. gingivalis-infected cells and further sup-
port a role for P. gingivalis in inhibiting multiple signaling path-
ways downstream of the inflammasome. P. gingivalis was also
able to repress IL-1 levels and cell death in human macro-
phages, demonstrating that these findings are not limited to
murine cells (Fig. 2, G and H). One potential mechanism of inflammasome repression
involves the modulation of the expression of inflammasome
components. The most commonly utilized pathway of inflam- SEPTEMBER 21, 2012•VOLUME 287•NUMBER 39 SEPTEMBER 21, 2012•VOLUME 287•NUMBER 39 32795 JOURNAL OF BIOLOGICAL CHEMISTRY P. gingivalis Inflammasome Repression via Reduced Endocytosis P. gingivalis Inflammasome Repression via Reduced Endocytosis FIGURE 4. Pg inflammasome repression is mediated through blockade of
endocytosis. ELISA of mouse BMDM demonstrates that Pg can repress the
activationofIL-1by(A)E. coli;by(B)LPSplusMSUorPGN;andby(C)LPSplus
alum, but not LPS plus ATP or nigericin (Nig). P. gingivalis Inflammasome Repression via Reduced Endocytosis Results are representative of
three independent experiments. D, IL-1 ELISA of BMDM treated with LPS
plus ATP is shown. Pg was added over a time course preceding the ATP treat-
ment. Representative of two independent experiments. E, percentages of
BMDM uptaking beads following 2-h exposure are shown. Cells were pre-
treated with 100 m.o.i. Pg where indicated. F, numbers of beads in the bead-
containing cell population for uninfected cells and cells infected with
100 m.o.i. Pg are shown. Numbers are expressed as a percentage of the total
bead-containing cells. G, a diagrammatic representation of the number of
beads per cell for uninfected cells and cells infected with 100 m.o.i. Pg is
shown. Each cell is represented as a dot. H, a representative confocal micros-
copy image for bead uptake in uninfected cells and cells infected with
100 m.o.i. Pg is provided. The number of internalized beads was counted in
100 cells per each of three independent experiments. P. gingivalis Inflammasome Repression via Reduced between ATP/nigericin and the other NLRP3 inflammasome
inducers is the shortened time required for IL-1 stimulation. For inflammasome stimulation by ATP and nigericin, cells are
treated with LPS for 3 h and then with ATP for 30 min. By
contrast, to achieve optimal inflammasome activation, other
inducers are applied for 6 h to overnight. To determine whether
a critical time of P. gingivalis exposure is required for its inhi-
bition of ATP-mediated inflammasome activation, P. gingivalis
was applied at increasing times prior to the activation of the
inflammasome by ATP. However, prolonged P. gingivalis expo-
sure did not restore its ability to repress inflammasome activa-
tion, suggesting that its failure to repress ATP-mediated
inflammasome activation is not explained by differential timing
(Fig. 4D). An additional difference between ATP and nigericin and the
other NLRP3 inflammasome inducers is the requirement for
endocytosis to elicit IL-1 production. The inducers that were
blocked by P. gingivalis all require endocytosis to activate the
signal; however, ATP and nigericin are thought to activate
the inflammasome directly (43). One possible explanation for
the selective ability of P. gingivalis to block IL-1 production
could be that P. gingivalis has the ability to regulate endocyto-
sis. To determine whether this might be the case, we exposed
macrophages to nonimmunogenic fluorescent latex beads and
examined uptake by confocal microscopy 1 h following incuba-
tion. P. gingivalis Inflammasome Repression via Reduced Endocytosis Our data showed that 80% of the uninfected cells took
up one or more beads, with 20% of the cells having no bead
uptake; however, cells that were infected with P. gingivalis prior
to exposure to beads had a distribution of bead-positive and
-negative cells closer to 50:50 (Fig. 4E). Furthermore, among
the bead-positive cells, P. gingivalis-infected cells were more
likely to contain one or two beads, whereas the uninfected cells
were more likely to contain three or more beads (Fig. 4, F–H). These findings suggest that P. gingivalis infection causes reduc-
tion in levels of endocytosis. FIGURE 4. Pg inflammasome repression is mediated through blockade of
endocytosis. ELISA of mouse BMDM demonstrates that Pg can repress the
activationofIL-1by(A)E. coli;by(B)LPSplusMSUorPGN;andby(C)LPSplus
alum, but not LPS plus ATP or nigericin (Nig). Results are representative of
three independent experiments. D, IL-1 ELISA of BMDM treated with LPS
plus ATP is shown. Pg was added over a time course preceding the ATP treat-
ment. Representative of two independent experiments. E, percentages of
BMDM uptaking beads following 2-h exposure are shown. Cells were pre-
treated with 100 m.o.i. Pg where indicated. F, numbers of beads in the bead-
containing cell population for uninfected cells and cells infected with
100 m.o.i. Pg are shown. Numbers are expressed as a percentage of the total
bead-containing cells. G, a diagrammatic representation of the number of
beads per cell for uninfected cells and cells infected with 100 m.o.i. Pg is
shown. Each cell is represented as a dot. H, a representative confocal micros-
copy image for bead uptake in uninfected cells and cells infected with
100 m.o.i. Pg is provided. The number of internalized beads was counted in
100 cells per each of three independent experiments. P. gingivalis Inflammasome Repression via Reduced Endocytosis determine whether P. gingivalis has the ability to actively sup-
press inflammasome induction by F. nucleatum and other
stimulatory agents. pro-caspase-1 levels. Therefore, the ability of P. gingivalis to
suppress IL-1 in our model system is not likely to involve
reduction in components of the core Nlrp3 inflammasome, but
rather its ability to suppress endocytosis. pro-caspase-1 levels. Therefore, the ability of P. gingivalis to
suppress IL-1 in our model system is not likely to involve
reduction in components of the core Nlrp3 inflammasome, but
rather its ability to suppress endocytosis. Inflammasome activation is a two-step process, and general-
ized low stimulatory responses to P. gingivalis may be explained
in part by our findings that P. gingivalis can induce the tran-
scription of the IL-1 gene (signal 1) but that it lacks the ability
to activate the processing of mature IL-1 (signal 2) in primary
mouse macrophages. The ability of P. gingivalis to stimulate
first signal activation without second signal activation is unique
among pathogenic bacteria (Ref. 5 and Fig. 1). Additionally, this
selective first signal activation could explain why P. gingivalis
has the ability to stimulate IL-1 and cell death in monocytic
cells (45–47), but not macrophages (Figs. 1 and 2). Immature
monocytes are able to provide an endogenous signal that
negates the requirement for second signal activation, but this
endogenous second signal is lost upon maturation (48, 49). Studies to determine which components of F. nucleatum
require phagocytosis to activate the inflammasome may help to
further characterize mechanisms of P. gingivalis-mediated
repression of endocytosis. There are multiple steps in the cel-
lular pathway toward endocytosis, and it would be of interest to
identify the step or steps that are affected. Given that P. gingi-
valis has been shown to cause actin cytoskeletal rearrange-
ments (52), it is intriguing to postulate that P. gingivalis might
suppress endocytosis by modulating the function of a core cyto-
skeletal protein. P. gingivalis has been shown to evade the endo-
cytic pathway to lysosomes in favor of trafficking to autopha-
gosomes (53), where it activates autophagy to provide a
replicative niche (54); thus, there is a basis for its role in direct-
ing cellular trafficking. Our results show that P. gingivalis can actively suppress the
secretion of IL-1 induced by F. nucleatum. Given that co-in-
fection with P. gingivalis blocks the F. nucleatum-mediated
activation of caspase-1 without reducing F. REFERENCES 1. Dinarello, C. A. (1998) Interleukin-1, interleukin-18, and the interleu-
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kin-1-converting enzyme. Ann. N.Y. Acad. Sci. 856, 1–11 We propose a novel mechanism for P. gingivalis-mediated
inflammasome inhibition through the suppression of endocy-
tosis, as is indicated by the selectivity of P. gingivalis in its ability
to inhibit Nlrp3 inflammasome inducers that are endocytosed. As a direct measure of effects on endocytosis, P. gingivalis
infection was shown to modulate the levels of endocytosis of
nonimmunogenic fluorescent beads. Additionally, our results
rule out the possibility that P. gingivalis suppresses F. nuclea-
tum-induced inflammasome activation through the reduction
of endogenous levels of Nlrp3 or the other core components of
its inflammasome. A recent study suggested that Nlrp3 is
down-regulated by 33% in gingival fibroblast cultures colonized
with a nine-species biofilm upon the inclusion of P. gingivalis
(50). However, Il1b transcription was also reduced 35%, and it is
therefore unclear whether reduced IL1b, rather than Nlrp3,
may contribute to the reduced IL-1 secretion in that model. P. gingivalis can activate Nlrp3 transcription both in primary
macrophages (Fig. 3) and in primary human gingival cells (51). Additionally, P. gingivalis does not significantly alter ASC or 2. Taxman, D. J., Huang, M. T., and Ting, J. P. (2010) Inflammasome inhibi-
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and Ting, J. P. (2011) Fatty acid-induced NLRP3-ASC inflammasome ac-
tivation interferes with insulin signaling. Nat. Immunol. 12, 408–415 4. Wen, H., Ting, J. P., and O’Neill, L. A. (2012) A role for the NLRP3 inflam-
masome in metabolic diseases: did Warburg miss inflammation? Nat. Im-
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Il-1 transcript levels, the inflammasome suppression by P. gin-
givalis occurs at the level of the second signal. These results are
supported by the finding that P. gingivalis also can repress the
secretion of IL-18, another cytokine that is known to be pro-
cessed by the inflammasome, without affecting its transcript
levels. Suppression is not explained by death induction by
P. gingivalis. Instead, P. gingivalis is shown to suppress
F. nucleatum-induced cell death. Cell death is an additional
physiological process that is associated with inflammasome
activation, and its suppression, therefore, is consistent with the
notion that P. gingivalis IL-1 suppression is an inflam-
masome-mediated event. The induction of IL-1 and cell death
also is suppressed in primary human macrophages, further sup-
porting the findings that P. gingivalis acts at the level of the
second signal. The suppression of the innate immune response
by P. gingivalis in human macrophages could facilitate the
maintenance of the chronic state of infection during periodon-
tal diseases. An additional important future direction would include the
identification of a P. gingivalis component responsible for
inflammasome inhibition through the suppression of endocy-
tosis. Interestingly, S. aureus produces an enzyme that sup-
presses its own inflammasome activation by making the cell
wall PGN-resistant to degradation during phagocytosis (17). Although P. gingivalis produces abundant gingival proteases, a
similar mechanism for enzymatic modulation of bacterial
immunostimulatory components has not been determined. Our unpublished results suggest that P. gingivalis LPS is not
sufficient for inflammasome inhibition, but that another
secreted component may be involved.5 The identification and
purification of this component could lead to the development
of a therapeutic agent for the treatment of periodontitis, and
considering that P. gingivalis can suppress the activation of the
inflammasome by MSU and other danger-associated molecular
patterns and pattern-associated molecular patterns, its thera-
peutic value could potentially extend to the treatment of gout
and other inflammatory disorders. 5 D. J. Taxman, unpublished data. DISCUSSION P. gingivalis resides within periodontal tissue in combination
with a wide variety of other pathogenic bacteria that can medi-
ate a robust immune response, chief among which is F. nuclea-
tum. In vivo studies have suggested that P. gingivalis and
F. nucleatum can work synergistically to promote infection and
pathogenesis (36–39). The mechanism for F. nucleatum-medi-
ated synergy is postulated to involve its abundant production of
adhesins that facilitate the co-aggregation of bacteria and
attachment to host cells (44). However, the contribution of
P. gingivalis to this synergy has remained largely unknown. We
postulated that P. gingivalis may contribute to the synergy
between the two bacteria through the evasion of F. nucleatum-
mediated immune responses by inflammasome suppression. Inflammasome suppression is employed as a stealth mecha-
nism by a variety of other pathogenic viruses and bacteria (2)
and would be a likely avenue for promoting the chronic state of
periodontitis through immune evasion. Furthermore, P. gingi-
valis has intrinsically low ability to stimulate immune responses
(21–24), which could contribute to its stealth potential. Given
these considerations, we sought to specify the effects of P. gin-
givalis on inflammasome activation in macrophages and to P. gingivalis-infected
cells
were
similar
or
higher
than
F. nucleatum- and P. gingivalisF. nucleatum-infected cells,
though the processing of pro-caspase-1 to caspase-1 only
occurred in F. nucleatum-infected cells and not the other
groups. These findings clearly demonstrate that P. gingivalis
does not suppress inflammasome activation by reducing the
expression of the inflammasome components (Fig. 3D). To determine whether P. gingivalis-mediated inhibition is
limited to F. nucleatum activation of the inflammasome, we
tested whether P. gingivalis inhibits E. coli as an alternate bac-
terial NLRP3 inflammasome inducer. Results showed that
P. gingivalis also can inhibit E. coli-mediated IL-1 release (Fig. 4A). To further delineate the mechanism of P. gingivalis inflam-
masome inhibition, we tested the effect of P. gingivalis infection
upon stimulation with LPS plus a panel of signal 2 inducers that
are known to activate the NLRP3 inflammasome, including
MSU, PGN, alum, ATP, and nigericin. Interestingly, P. gingiva-
lis inhibited activation by MSU PGN, and alum but did not
inhibit activation by ATP and nigericin under the same condi-
tions of P. gingivalis addition (Fig. 4, B and C). One difference VOLUME 287•NUMBER 39•SEPTEMBER 21, 2012 32796 32796
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Studien �ber Adsorption in L�sungen
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751
Studien tiber Adsorption in LSsungen.
V. Abhandlung:
Lsung, Adsorption,claemischeYerbindung,Adhiisi0n
von
G. v. G e o r g i e v i c s .
A a s dem Laboratorium fiir chemische T e c b n o l o g i e o r g a n i s c h e r Stoffe an der
k. k. D c u t s c h e n t c c h n i s c h e n H o c h s c h u l e in Prag.
(Mit 2 Textfiguren.)
( V o r g e l e g t in der S i t z u n g a m 6. F e b r u a r 1913.)
Das Studium der Sorption von Siiuren durch Wolte hat zu
einer Erkltirung dieses Vorganges geftihrt, gegen welche verschiedene Einwtinde gemacht werden kSnnten. Man k6nnte
einerseits die physikalische Auffassung der Sorption als nicht
genfigend begrtindet betrachten u n d andrerseits den Einwurf machen, dab die gegebene Erkl/irung in anderen F/illen,
namentlich bei manchen Fttrbungen oder bei Sorptionen mit
Kohle als Adsorbens, nicht befriedigend w~ire. Es sollen daher im
folgenden die Grtinde, welche zu einer physikalischen Auffassung der Sorption gedr/ingt haben, n/iher erSrtert und eine
prtizise Definition ftir den Vorgang der Sorption und Adsorption
gegeben werden.
Was zuntichst den Vorgang der ,,LOsung<< eines Stoffes im
Adsorbens anbelangt, so kann man denselben entweder rein
physikalisch auffassen oder man kann die Annahme machen,
.dab sich zun/ichst eine chemische Verbindung des sorbierten
Stoffes mit einem kleinen Anteil des Adsorbens bildet, die sich
in dem letzteren vollkommen gleichmiil3ig ver~eilt. Es wiirde
dann im letzteren Falle bei weiter gehender Sorption auch eine
752
G.v. G e o r g i e v i c s ,
weitere Bildung dieser chemischen Verbindung stattfinden
mfissen und es wS.re daher such die Adsorption und mithin die
ganze Sorption im wesentlichen ein chemischer Vorgang.
Gegen diese Auffassung lassen sich folgende Grtinde anffihren:
Zun/ichst sei daran erinnert, daft die Bildung einer chemischen Verbindung einen anderen Verlauf zeigt als eine Sorption,
wie aus den Figuren 1 und 2 deutlich ersehen werden kann.
Als Beispiet hierfCtr sei die Bildung vor~ Diphenylaminpikrat 1
und die von Naphthalinpikrat ~ angeftihrt. Hieraus allein ergibt:
sich schon, daf3 eine Sorption nicht ohneweiters als ein chemischer Vorgang aufgefal3t werden kann; denn die chemische
AffinitS.t kSnnte es nicht bewirken, dab selbst so schwache
/
i~ Lb'Su~ g~lieberu
Fig. I. $orption.
9.~, L&.Tu~jybtieben~
Fig. 2. Chemischer Vorgang.
S~turen, wie Butters/iure, schon aus den verdfmntesten LSsunge n
von Wolle aufgenommen werden. Die chemische Auffassung
der Sorption ist aber such nicht imstande, die Tatsache zu erklg.ren, dab die Euttersg.ure sta.rker sis EssigsS.ure und Phosphors~.ure stti.rker als die anderen starken MineralsS.uren sorbiert
wird, w/ihrend die physikalische Auffassung dieses Vorgangs eine
Erkl~rung hierf~r zu geben vermag, a
Die meisten Anhgmger der chemischen Auffassung der
Sorption stellen sich fibrigens diese Reaktion etwas anders vor,
indem sie annehmen, dab hierbei mehr oder weniger leicht
hydrolysierbare salzartige Verbindungen gebildet werden. Nach
dieser Auffassung k/Snnte beim LSsungsvorgalag, also im Anfange
1 J. W a l k e r und J. A p p l e y a r d , Journ. of the Chem. Soc., 1896, p. 1334.
2 G e o r g i e v i c s , Monatshefte ffir Chemie, 1911, p. 323.
3 Siehe die vorige Abhar, dlung.
L6sung, Adsorption etc.
753
d e r Sorption, eine chemische Verbindung tiberhaupt nicht entstehen, sie wtirde hydrolysiert w e r d e n mtissen und es wfirde
d e r sorbierte Stoff als solcher zun/ichst eine starre L/3sung im
A d s o r b e n s bilden und erst sp/iter, beim Eintritt der Adsorption,
k~Snnte die Bildung der chemischen Verbindung stattfinden. Bei
steigender Is
der zur Sorption a n g e w e n d e t e n Sfiure16sungen sollte d e m n a c h die H y d r o l y s e der gedachten cl~emi:schen V e r b i n d u n g zurtickgedr/ingt werden; die Bildung dieser
Verbindung mtil3te also begtinstigt, nicht abet g e h e m m t werden,
,,vie es tats/ichlich geschieht!
Auch hier versagt d e m n a c h die chemische Theorie, w~ihrend
n a c h der physikalischen Auffassung der Sorption die genannte
H e m m u n g , welche das charakteristischeste Merkmal der Adsorption bildet, notwendigerweise eintreten muB.
Ein weiteres Argument, das gegen die chemische Auff a s s u n g der Sorption spricht, ist folgendes: Der basische Charakter der W o l l s u b s t a n z ist jedenfalls recht s c h w a c h und es
w t i r d e n daher auch die Verbindungen derselben mit den
s c h w a c h e n einbasischen Fetts~turen recht lose sein m~ssen.
Aceton, das keinen S/iurecharakter m e h r besitzt, sotlte daher
von Wolle nicht m e h r sorbiert werden k/3nnen! Es geschieht
dies aber doch und die graphische Darstellung des Vorgangs
zeigt, dab derselbe , v o n d e r Sorption der Sfiuren durch ~Volle
nicht wesentlich verschieden ist.
Gegen die A n n a h m e einer in diesem speziellen Falle (VVolle
und verdtinnte S/iuren) stattfindenden unmittelbaren Salzbi/dung
spricht auch, wie P. D. Z a c h a r i a s
wiederholt betont hat, der
c h e m i s c h e C h a r a k t e r der Proteinsubstanzen. Sie sind recht
reaktionstr/ige; .wenn sie aber einmal zu reagieren anfangen,
d a n n tritt auch schon ein Zerfall ihres komplizierten Molektils
ein. Der Beweis, daft Proteinsubstanzen Salze zu geben verm/3gen,
ist auch bisher noch nicht erbracht worden. Es miJl3te also einer
S a l z b i l d u n g die Z e r s e t z u n g der W o l l s u b s t a n z v o r a u s g e h e n . 1
DaB tibrigens auch bei Sorptionen a u s verdtinnten L/3s u n g e n chemische Reaktionen stattfinden k/3nnen, habe ich
i Daft eine solche bei den gemachten Versuehen nicht eintritt, ist frgher
(Monatshefte f/Jr Chemie, 1911, p. 671) gezeigt worden.
754
G.v. Georgi evics,
selbst in dem Falle Pikrins~ure--Seide 1 nachgewiesen. Gerade
dieser Fall hat abet auch gezeigt, dab eine solche A b w e i c h u n g
von dem gewShnlichen Verlauf der Sorptionen durch die Form
der Sorptionskurve leicht zu erkennen ist. W e n n diese aber
normal verl&uft, dann ist man nicht berechtigt, y o n dem Stattfinden einer chemischen Reaktion zu sprechen.
Die Vorg~nge der LSsung eines Stoffes im Adsorbens und
seine Adsorption dutch dasselbe aus verdtinnten L S s u n g e n
werden daher physikalisch aufzufassen sein. Die chemische
Auffassung v e r m a g keine Erkl/irung der Sorption zu geben u n d
sie ist auch tats~chlich bisher nieht imstande gewesen, die Erforschung dieses Problems zu f6rdern. DaB bei A n w e n d u n g
konzentrierter L S s u n g e n eine chemische Reaktion stattfinden
kann und in dem Falle: Wolle--S~.uren auch tats~chlich stattfindet, ist schon in der vorhergehenden A b h a n d l u n g g e s a g t
worden.
Die heute herrschende A n s c h a u u n g tiber die Adsorptio~
besteht in der Annahme einer Verdichtung des adsorbierten
Stoffes an der Oberfl~.che des als Adsorbens wirkenden KSrpers,
wobei unentschieden gelassen wird, ob man sich diese Verdichtung nur an der inneren Seite des Adsorbens oder a u f
beiden Seiten seiner Oberfl/iche zu denken hat. Eine Prtifung
dieser Annahme auf ihre Zul~ssigkeit konnte bisher nicht gemacht werden, weil die ganze E r s c h e i n u n g der Sorption w e d e r
in ihrem Verlauf noch in b e z u g auf ihre Ursachen gentigend
studiert war. Dieselbe ist abet, wie wir sehen werden, nicht befriedigend und sie ftihrt auch hS.ufig zu einer V e r w e c h s l u n g
von Adsorption und Adhiision, die man doch als verschiedene
Vorg~nge zu betrachten hat.
Unklar ist hierbei zun~chst, w a s man sich unter ,>Oberfl&che<< zu denken hat. Der F~rbereitechniker versteht darunter
die Fl~.chen, von welchen die einzelnen Gespinstfasern b e g r e n z t
sind, und es ist auch sicher, dab diese beim F/irbeprozel3 eine
w'ichtige Rolle spielen. Betrachtet man n~.mlich den Querschnitt yon
gef/irbten Wollfasern, so sieht man in m a n c h e n FSllen eine starke
1 Monatshefte f/Jr Chemie, 1911, p. 1075. Es ist sehr wahrscheinlich, dab
dieser Fall nur bei einer durch den Waschprozeg mit S5uren angegriffenen Seide
vorkommt.
L6sung, Adsorption etc.
755
F/irbung der genannten Oberfl/iche, die sich gegen das Innere
der Faser zu allmS.hlich abstuff. In anderen F~illen erscheint nur
diese Oberfl~iche gef/irbt, das Innere der Fasern ist ganz farblos.
Und doch sind auch im Innern der Wollfasern Elementarteilchen,
die Fibrillen enthalten, welche Oberfl/ichen besitzen! Manchmal
werden nicht einmal die Oberfl/ichen aller Fasern, sondern nur
jene gef/~rbt, welche an der Oberfl~iche des Gewebes liegen.
Dies kommt z. B. bei T u c h e n vor, die mit Cochenille und Zinnbeize ,>auf einem Wasser~ gefS.rbt worden sin& Durchschneidet
man ein solches T u c h , so zeigt es den ,,weil3en Schnitt% d. h.
es erscheint n u t seine Oberfl/tche gefg,rbt.
In allen diesen F~illen handelt es sich um eine mehr oder
weniger starke D i f f u s i o n s h e m m u n g ;
die F/irbung ist in
s o l c h e n F ~ l l e n im w e s e n t l i c h e n e i n e A d h / i s i o n s e r s c h e i nungund derVorgang, welcherSorptiongenanntwird,
t r i t t i n d e n H i n t e r g r u n d . Hierher gehSren auch die F/irbungen mit Suspensionen u n d wahrscheinlich auch die mit
kolloidalen L6sungen yon Indigo (R. M 6 h l a u ) und Metallsulf i d e n ( W . Biltz). M a n w i r d d e m n a c h s o l c h e V o r g ~ i n g e ,
diesichnuroderfastnur
a n d e r O b e r f l ~ t c h e e i n e s Adsorbensabspielen,
n i c h t m e h r a l s S o r p t i o n o d e r Adsorption, sondernalsAdhg.sion
zu bezeichnen
haben.
Wie nahe verwandt diese Vorg/inge sind und wie leicht sie miteinander kombiniert auffreten kSnnen, mSge aus dem folgenden
Beispiel ersehen werden: Es ist bekannt, dab manche Farbstoffe
bei langsamem vorsichtigem F/irben die Fasern gut durchf/irben;
f/irbt man aber rasch, so sitzt die entstandene Fiirbung nur
oberflg.chlich; sie zeigt die E r s c h e i n u n g des Abf/irbens oder
Abrui3ens. Dieses Beispiel zeigt uns auch recht deutlich, wie
unbefriedigend die Annahme yon Oberfl~ichenkr/iften als Ursache der Adsorption ist. Diese Annahme wfirde, auf den geschilderten Fall angewendet, zu dem Schlusse nStigen, dal3 die
angenommenen Oberfl/ichenkr~,ffe nut bei raschem, nicht aber
abet bei langsamem F~.rben zur W i r k u n g kommen!
Auch bei A n w e n d u n g von Kohle als Adsorbens wird die
GrSl3e der freiliegenden Oberfl/ichen die Sorption im gfinstigen
Sinne beeinflussen u n d e s wird daher auch eine Zerkleinerung
tier Kohle eine grSI3ere Sorptionsf/ihigkeit derselben zur Folge
756
G.v. Georgievics,
haben. Freiliegende Oberfliichen wirken eben sicher, w/ihrend
die im Innern des Adsorbens liegenden Molektile durch eine
Diffussionshemmung in ihrer Sorptionsf/ihigkeit gehindert
werden kOnnen.
Man wird daher in allen diesen F/illen, wo die M~3glichkeit
einer S o r p t i o n s h e m m u n g vorhanden ist, aus der Bef/Srderung
der Sorption durch Vergr613erung der Oberflg.che des Adsorbens
nicht ohneweiters auf eine st/irkere Anziehungskraft der an
der Oberfl~.che des Adsorbens liegenden Molektile schliel3en
dtirfen.
W e n n man aber gut genetzte Fasern im losem Zustand
oder in Form eines lose gedrehten G a m e s und molekulardispers gel6ste Stoffe, wie Minerals~iuren und Fetts/iuren, aufeinander wirken ltil3t, dann wird wohl eine vollst/indige Durchdringung des Adsorbens eintreten und man wird annehmen
diirfen, dal3 alle Molektile des letzteren in die Lage kommen
werden, ihre anziehende W i r k u n g auf den sorbierten Stoff auszuflben.
Das ist also ein Fall, wo die E r s c h e i n u n g der Sorption
recht vollkommen stattfinden kann, und hier werden wir zu
prfifen haben, ob die gegenw/irtig t~bliche Annahme, dab die
an den Oberflg.chen des Adsorbens liegenden Molekiile eine
grbl3ere Anziehungskraft besitzen als die im Innern liegenden,
berechtigt ist.
Betrachten wit nun den in diesem Fall stattfindenden Vorgang, so sehen wir, dab am Anfange der Sorption eine st~irkere
Anziehung der S/iuremolekiile von den an den Oberfl/ichen der
Wollfasern gelegenen Keratinmolektilen n i c h t vorhanden sein
kann, da ja zuerst eine, starre L6sung, eine homogene Verteilung der Sfi.ure in der Wollfaser stattfindet. W e n n man demnach an der /_iblichen Auffassung der Adsorption festhalten will,
so mug man annehmen, daf3 erst spg.ter, beim Eintritt der
eigentlichen Adsorption, die grOf~ere Anziehungskraft der Oberfl~.chenmolek~le des Adsorbens zur Oeltung kommt. Da aber,
wie in der letzten Abhandlung gezeigt w o r d e n ist, die Adsorption durch die c h e m i s c h e Affinitg.t bewirkt wird, so wt'lrde
man die Annahme zu machen haben, dab die hier zur Wirkung
L6sung, Adsorption etc.
787
kommende chemische Affinit/it der Oberfl/ichenmolekfile gr/3f3er
ist als jene der im Innern des Adsorbens liegenden Molekfile!
Ffir eine solche Annahme liegt abet wohl kein Grund vor
und man gelangt daher zu der Vorstellung, d a b s ~ . m t l i c h e
MolekfiledesAdsorbens
diegleiche Anziehungskraft
b e s i t z e n . Der Vorgang der Sorption wtirde sich demnach in
folgender Weise abspielen: Die in Wasser gel6ste S/iure diffundiert in die Wollfaser und verteilt sich in derselben zun/i.chst
ganz gleichm/il3ig. Sp/iter treten weitere Anteile an, die Konzentration des sorbierten Stoffes im Adsorbems wird gr613er, es
macht sich die chemische Anziehungskraft der Molekfile des
Adsorbens geltend und die A d s o r p t i o n , b e s t e h e n d in e i n e r
allmg.hlich w a c h s e n d e n V e r d i c h t u n g des s o r b i e r t e n
S t 0 f f e s u m d i e e i n z e l n e n M o l e k f i l e (oder Moiekfilaggregate) d e s A d s o r b e n s , a l s o a u c h um die, w e l c h e im I n n e r n
l i e g e n , t r i t t in E r s c h e i n u n g . In d e m MaI3e, als d i e s
stattfindet, wirddievon den MolekfilendesAdsorbens
aufdiein der Sorptionsl/3sung enthaltenen Molekfile
a u s g e f i b t e A n z i e h u n g g e r i n g e r w e r d e n u n d es m u g
dahereine Sorptionshemmung, das charakteristische
Kennzeichen derAdsorption, eintreten.
g s sei nochmals betont, dab die Sorption in der hier deftnierten Form in erster Linie bei Anwendung eines quellbaren
Adsorbens und bei m6glichst vollstgndigem AusschluB einer
Diffusionshemmung einerseits, bei Anwendung yon molekulardispers gel/Ssten Stoffen andrerseits stattfinden wird. In anderen
F/illen, wie bei den oben angeffihrten Beispielen, wird man mit
einer mehr oder weniger starken Diffusionshemmung zu rechnen
haben, wodurch die Sorption zurttckgedr/ingt und ein zweiter
Vorgang, die Adh/ision, zur Geltung gelangt.
Die Erscheinungen, die man bisher unter dem Namen Adsorption zusammengefal3t hat, k/3nnen daher sehr verschieden
voneinander sein; man hat es eben hier mit Vorg/ingen zu
tun, bei welchen Sorption und Adh/ision in sehr wechselndem
Umfange miteinander kombiniert oder auch fftr sich allein auftreten k/Snnen.
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W4388041167.txt
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https://www.qeios.com/read/2VFEF2/pdf
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en
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Review of: "Empowering Minds: The Evolution of Higher Education in Tamil Nadu towards Innovation and Excellence"
| null | 2,023
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cc-by
| 138
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Qeios, CC-BY 4.0 · Review, October 31, 2023
Review of: "Empowering Minds: The Evolution of Higher
Education in Tamil Nadu towards Innovation and Excellence"
Pavel Krpálek1
1 University of Economics Prague
Potential competing interests: No potential competing interests to declare.
The article has the character of an overview study on the state of higher education and development perspectives, it is not
a scientific probe supported by exact research based on quantitative methods with verified hypotheses, the authors do not
even have such ambitions. However, a comprehensive philosophy of educational development and an analysis of
paradigmatic questions is in place here and represents a necessary and well-understood topic. Undoubtedly, it will be
interesting and worth publishing. I consider them beneficial, at least for the deeper development of the discussion on the
current topic.
Qeios ID: 2VFEF2 · https://doi.org/10.32388/2VFEF2
1/1
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https://openalex.org/W2013419897
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https://jcmr-online.biomedcentral.com/track/pdf/10.1186/1532-429X-12-S1-P241
|
English
| null |
Regional matrix metalloproteinase activation correlates with microstructure diffusion tensor indices post myocardial infarction
|
Journal of cardiovascular magnetic resonance
| 2,010
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cc-by
| 1,669
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Published: 21 January 2010 Published: 21 January 2010
Journal of Cardiovascular Magnetic Resonance 2010, 12(Suppl 1):P241
doi:10.1186/1532-429X-12-S1-P241 This abstract is available from: http://jcmr-online.com/content/12/S1/P241
© 2010 Mekkaoui et al; licensee BioMed Central Ltd. Introduction
di l 7). Following MR imaging hearts were sliced (5 mm), cut
in 8 radial pies and divided into endocardial and epicar-
dial segments for gamma-well-counting for determina-
tion of RP805 activity, expressed as percent of injected
dose/gram of tissue (%ID/g). Similarly, T2-weighted
images were segmented using the same anatomical land-
marks and used to classify tissue as infarcted (I) or non-
infarcted (NI). TC is defined as the maximum Gaussian
curvature of the toroid-based representation of the DT. Figure 1 displays an example of the, FA and TC maps used
in quantification compared with RP805 and morphology. 7). Following MR imaging hearts were sliced (5 mm), cut
in 8 radial pies and divided into endocardial and epicar-
dial segments for gamma-well-counting for determina-
tion of RP805 activity, expressed as percent of injected
dose/gram of tissue (%ID/g). Similarly, T2-weighted
images were segmented using the same anatomical land-
marks and used to classify tissue as infarcted (I) or non-
infarcted (NI). TC is defined as the maximum Gaussian
curvature of the toroid-based representation of the DT. Figure 1 displays an example of the, FA and TC maps used
in quantification compared with RP805 and morphology. Post myocardial infarction (MI) activated matrix metallo-
proteinases (MMPs) degrade the extracellular matrix
(ECM) and alter the tissue microstructure (TMS). Diffu-
sion Tensor MRI (DT-MRI) yields diffusivity and anisot-
ropy indices that characterize the TMS. Our previous
studies in pigs post-MI have showed that gross spatial and
temporal changes in mean diffusivity () and fractional
anisotropy (FA) correlate with MMP activation assessed
with a 99 mTc-labeled radiotracer (RP805) targeted at
activated MMPs. Purpose To determine the correlation between regional MMP acti-
vation defined by RP805 with alterations in, FA and a new
coefficient of anisotropy, the toroidal curvature (TC). Mid-ventricular cross-section of MD (A), FA (B), TC (C), and
RP805 (D) maps, as well as the relative post mortem slice (E)
for a 2-week post-MI porcine heart
Figure 1
Mid-ventricular cross-section of MD (A), FA (B), TC
(C), and RP805 (D) maps, as well as the relative post
mortem slice (E) for a 2-week post-MI porcine heart. Figure shows the spatial correlation between the infacted
area (red arrow) and the increse in MD and RP805 and the
decrease in FA and TC. BioMed Central
Journal of Cardiovascular Magnetic
Resonance
Open Access
Poster presentation
Regional matrix metalloproteinase activation correlates
with microstructure diffusion tensor indices post myocardial
infarction
Choukri Mekkaoui*1, Marcel P Jackowski2, Roberto Martuzzi1,
Donald P Dione1, Francis G Spinale3 and Albert J Sinusas1
Address: 1Yale University, New Haven, CT, USA, 2University of São Paulo, São Paulo, Brazil and 3Medical University of South Carolina, Charleston,
SC, USA
* Corresponding author BioMed Central
Journal of Cardiovascular Magnetic
Resonance
Open Access
Poster presentation
Regional matrix metalloproteinase activation correlates
with microstructure diffusion tensor indices post myocardial
infarction
Choukri Mekkaoui*1, Marcel P Jackowski2, Roberto Martuzzi1,
Donald P Dione1, Francis G Spinale3 and Albert J Sinusas1
Address: 1Yale University, New Haven, CT, USA, 2University of São Paulo, São Paulo, Brazil and 3Medical University of South Carolina, Charleston,
SC, USA
* Corresponding author Open Ac
Poster presentation
Regional matrix metalloproteinase activation correlates
with microstructure diffusion tensor indices post myocardial
infarction
Choukri Mekkaoui*1, Marcel P Jackowski2, Roberto Martuzzi1,
Donald P Dione1, Francis G Spinale3 and Albert J Sinusas1 Address: 1Yale University, New Haven, CT, USA, 2University of São Paulo, São Paulo, Brazil and 3Medical University of South Carolina, Charleston,
SC, USA * Corresponding author from 13th Annual SCMR Scientific Sessions
Phoenix, AZ, USA. 21-24 January 2010 from 13th Annual SCMR Scientific Sessions
Phoenix, AZ, USA. 21-24 January 2010 Published: 21 January 2010 Journal of Cardiovascular Magnetic
Resonance BioMed Central Open Access Methods
l Conclusion
Therefore, MMP-mediated degradation of the ECM post-
MI was associated with increased water diffusivity as
reflected by and reduced anisotropy by a decrease in FA
and TC. Hence, evaluation of regional DT-MRI indices of
microstructure in combination with evaluation of MMP
activation may provide new insight in the remodeling
process post-MI. Scatter diagrams showing the correlation between RP805
and MD (A), FA (B), and TC (C) respectively with I (red
dots) and NI (black dots) for all animales
Figure 2
Scatter diagrams showing the correlation between
RP805 and MD (A), FA (B), and TC (C) respectively
with I (red dots) and NI (black dots) for all animales. The correlation coefficient r and the relative p values are
indicated in the upper right corner of each panel. Conclusion Therefore, MMP-mediated degradation of the ECM post-
MI was associated with increased water diffusivity as
reflected by and reduced anisotropy by a decrease in FA
and TC. Hence, evaluation of regional DT-MRI indices of
microstructure in combination with evaluation of MMP
activation may provide new insight in the remodeling
process post-MI. Scatter dia
and MD (A
dots) and
Figure 2 g
g
( ),
( ),
( )
p
y
(
)
(
)
g
Scatter diagrams showing the correlation between
RP805 and MD (A), FA (B), and TC (C) respectively
with I (red dots) and NI (black dots) for all animales. The correlation coefficient r and the relative p values are
indicated in the upper right corner of each panel. g
g
( )
( )
( )
p
y
(
)
(
)
g
Scatter diagrams showing the correlation between
RP805 and MD (A), FA (B), and TC (C) respectively
with I (red dots) and NI (black dots) for all animales. The correlation coefficient r and the relative p values are
indicated in the upper right corner of each panel. Methods
l Correlation between regional MMP activation using
RP805 and DT-MRI was assessed in 3 infarcted porcine
hearts at 2- and 4-week post-MI. Two hours prior to eutha-
nasia, RP805 (28 ± 3 mCi) was injected. Each heart was
then excised and placed in a container and filled with
Fomblin. DT-MRI was performed on a 3.0 T scanner (Sie-
mens, Erlangen, Germany) using a segmented EPI
sequence, 6 gradient directions; b-values = 0 (T2-
weighted) and 600 s/mm2; voxel-size = 2 × 2 × 2 mm3;
slices = 50; TR/TE = 5400/84 ms; 40 averages (EPI-factor = Mid-ventricular cross-section of MD (A), FA (B), TC (C), and
RP805 (D) maps, as well as the relative post mortem slice (E)
for a 2-week post-MI porcine heart
Figure 1
Mid-ventricular cross-section of MD (A), FA (B), TC
(C), and RP805 (D) maps, as well as the relative post
mortem slice (E) for a 2-week post-MI porcine heart. Figure shows the spatial correlation between the infacted
area (red arrow) and the increse in MD and RP805 and the
decrease in FA and TC. Page 1 of 2
(page number not for citation purposes) Page 1 of 2
(page number not for citation purposes) Journal of Cardiovascular Magnetic Resonance 2010, 12(Suppl 1):P241 http://jcmr-online.com/content/12/S1/P241 http://jcmr-online.com/content/12/S1/P241 http://jcmr-online.com/content/12/S1/P241 http://jcmr-online.com/content/12/S1/P241 Scatter diagrams showing the correlation between RP805
and MD (A), FA (B), and TC (C) respectively with I (red
dots) and NI (black dots) for all animales
Figure 2
Scatter diagrams showing the correlation between
RP805 and MD (A), FA (B), and TC (C) respectively
with I (red dots) and NI (black dots) for all animales. The correlation coefficient r and the relative p values are
indicated in the upper right corner of each panel. Conclusion
Therefore, MMP-mediat
MI was associated wit
reflected by and reduce
and TC. Hence, evaluati
microstructure in comb
activation may provide
process post-MI. Scatter diagrams showing the correlation between RP805
and MD (A), FA (B), and TC (C) respectively with I (red
dots) and NI (black dots) for all animales
Figure 2
Scatter diagrams showing the correlation between
RP805 and MD (A), FA (B), and TC (C) respectively
with I (red dots) and NI (black dots) for all animales. The correlation coefficient r and the relative p values are
indicated in the upper right corner of each panel. Results Figure 2 illustrates the positive correlation between
regional RP805 activity and for both I (r = 0.563, p <
0.001) and NI (r = 0.628, p < 0.001). There was an inverse
correlation between RP805 activity and FA (r = -0.426, p <
0.001) within NI regions, although no correlation within
I regions (r = -0.096, p = 0.3). There were inverse correla-
tions between RP805 activity and TC for both NI (r = -
0.532, p < 0.001) and I (r = -0.329, p < 0.05) regions. The
average (I = 0.52 ± 0.10 mm2/s; NI = 0.43 ± 0.06 mm2/s),
FA (I = 0.44 ± 0.06; NI = 0.47 ± 0.06), TC (I = 113.55 ±
39.64, NI = 152.44 ± 45.98) and RP805 activity (I = 0.75
± 0.34; NI = 0.46 ± 0.19) were all significantly (p < 0.001)
different between I and NI regions. Publish with BioMed Central and every
scientist can read your work free of charge
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(page number not for citation purposes) Publish with BioMed Central and every
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Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
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BioMedcentral Publish with BioMed Central and every
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|
https://openalex.org/W4253122018
|
https://www.degruyter.com/document/doi/10.2390/biecoll-jib-2010-113/pdf
|
English
| null |
A New Approach for Modelling Gene Regulatory Networks Using Fuzzy Petri Nets
|
Berichte aus der medizinischen Informatik und Bioinformatik/Journal of integrative bioinformatics
| 2,010
|
cc-by
| 7,422
|
2 Bioinformatics and Computational Biology, Patna University - 800005, India 2 Bioinformatics and Computational Biology, Patna University - 800005, India Summary Gene Regulatory Networks are models of genes and gene interactions at the expression
level. The advent of microarray technology has challenged computer scientists to develop
better algorithms for modeling the underlying regulatory relationship in between the
genes. Fuzzy system has an ability to search microarray datasets for activator/repressor
regulatory relationship. In this paper, we present a fuzzy reasoning model based on the
Fuzzy Petri Net. The model considers the regulatory triplets by means of predicting
changes in expression level of the target based on input expression level. This method
eliminates possible false predictions from the classical fuzzy model thereby allowing a
wider search space for inferring regulatory relationship. Through formalization of fuzzy
reasoning, we propose an approach to construct a rule-based reasoning system. The
experimental results show the proposed approach is feasible and acceptable to predict
changes in expression level of the target gene. doi:10.2390/biecoll-jib-2010-113
* To whom correspondence should be addressed. Email: raed.inf@gmail.com Journal of Integrative Bioinformatics, 7(1):113, 2010 Journal of Integrative Bioinformatics, 7(1):113, 2010 http://journal.imbio.de A New Approach for Modelling Gene Regulatory Networks
Using Fuzzy Petri Nets
Raed I. Hamed1*, S. I. Ahson2, R. Parveen1 Raed I. Hamed1*, S. I. Ahson2, R. Parveen1 1 Department of Computer Science, JMI University, New Delhi-110025, India Journal of Integrative Bioinformatics, 7(1):113, 2010 http://journal.imbio.de http://journal.imbio.de The rule-based system has played an important role in such an expert system. Especially in an
uncertain information environment, the rule-based system must have the capability of
performing fuzzy reasoning which is based on the fuzzy sets foundation [9]. Several existing
work puts emphasis on details of actual implementation of fuzzy system, and lacks formal
specifications of fuzzy reasoning. Modeling and simulation methods provided by systems theory can help improving the level of
understanding of biological phenomena, [10]. In particular, Petri nets are becoming the
reference modeling formalism for GRNs (see, e.g., [11, 12, 13, 14]): activation and inhibition
of gene activity is intrinsically an on/off mechanism, and the dynamics governing proteins
concentration are described by hybrid Petri nets (HPNs), while the activation and the
deactivation of these dynamics are triggered by discrete switches encoding protein
concentration reaching some threshold. Fuzzy Petri net (FPN), which combines fuzzy logic
with Petri net, is useful tool in dealing with uncertain and incomplete information. In this paper, we introduce and motivate a new modeling approach fuzzy Petri net which
provides a powerful and intuitive tool for investigating biological processes and systems. We
then apply this method to predict changes in expression level of the target gene. Since every
triplet of genes (one as the activator, one as an repressor, and one as the target gene) is
checked, in our model. With difference from those existing system, we formalize the fuzzy
reasoning mechanism with Fuzzy Petri Net [15]. With the FPN’s graphical nature and
mathematical foundation, we visualize the structure of a rule-based fuzzy reasoning system,
and make the model relatively simple and legible. In an attempt to find new applications and stimulate new research topics, researchers such as
[16] and [17] combined fuzzy theory and the basic Petri net to form a new model and define
the associated operations of fuzzy Petri net in modeling biological processes. We present a
more complete and efficient model for rule-based reasoning system modeled as FPN. The
model is developed to simulate the inference process from the antecedent to the consequent
propositions. Also, we modify fuzzy Petri nets to model gene regulatory network with fuzzy
logic. Our ultimate goal is to develop a model that is similar to, but simpler than, classical
fuzzy models (see [1, 7] for instance), and that has the power to perform predicate logic. The paper is structured as follows. Journal of Integrative Bioinformatics, 7(1):113, 2010 Section 2 introduces a gene regulatory network. Section 3
introduces the definition of Petri nets and fuzzy Petri nets. Section 4 describes the process of
changes in expression values. Section 5 describes the FPN model based specifications of
fuzzy reasoning rules and procedure of reasoning. Finally, Section 6 concludes the paper and
points out directions for future work. Introduction 1 Gene regulatory networks control biological functions by regulating the level of gene
expression. Discovering and understanding the complex causal relationships within gene
regulatory networks has become a major issue in systems biology, computational biology and
bioinformatics. The benefits of characterizing gene interaction are many; for example, the
effects of drugs on a regulatory pathway can be found, the development of cancer in a cell can
be tracked, etc. DNA microarray experiments today allow to monitor the output of gene
regulatory networks by measuring the gene expression levels of thousands of genes [1]. Several methods have been proposed to develop maps of gene interaction, including Bayesian
networks [2], dynamic Bayesian networks with hidden Markov model [3], and Boolean
networks [4]. More recently, neural networks have also been applied to the problem of gene
expression data analysis (eg. [5], [6]). Woolf and Wang [7] introduced a fuzzy logic approach
to analyse the activator/repressor relationship of gene interaction using a normalized subset of
Saccharomyces cerevisiae data [8]. They applied every possible combination of activators and repressors for each gene and the
output from the model was compared to the expression levels of the remaining genes. Since
gene expression levels are qualitatively classified into low, medium and high states to a
varying degree based on a set of membership functions. Genes are then paired into an
activator and repressor, and this gene pair determines the predicted target gene expression
values based on a set of heuristic rules. Since a fuzzy logic algorithm [7] searches a
microarray dataset for regulatory triplets consisting of activator, repressor and target gene. 1 1 Gene regulatory network 2 Generally, a genetic network can be expressed by a set of nonlinear differential equations with
each gene expression level as variables [18]. The accuracy of this design depends on the
accuracy of the data in terms of concentrations, rate constants, and expression levels [19]. The
expression level of gene i at time instant t+1 is given by ))
(
(
)1
(
t
x
f
t
x
i
i
=
+ where
)1
( +
t
xi
is the expression level (mRNA concentration) of gene i at time instant t + 1,
x(t) is the vector of expression levels of all genes at time instant t ,fi is the function that
determines the expression level of genei from the previous expression values of all genes. Note that the function fi is static, without altering during simulation. Although the vector that
holds values of all genes is the parameter of the function fi, it takes in practice only the genes doi:10.2390/biecoll-jib-2010-113 2 Journal of Integrative Bioinformatics, 7(1):113, 2010
http://journal.imbio.de Journal of Integrative Bioinformatics, 7(1):113, 2010 Journal of Integrative Bioinformatics, 7(1):113, 2010 http://journal.imbio.de that control the genei . In most cases, the expression function fi uses only a couple of genes’
values. For instance, the expression function of the gene 3 in Fig. 1 is that control the genei . In most cases, the expression function fi uses only a couple of genes’
values. For instance, the expression function of the gene 3 in Fig. 1 is
))
(
(
3
t
x
f
≡
))
(
),
(
(
1
2
3
t
x
t
x
f that control the genei . In most cases, the expression function fi uses only a couple of genes’
values. For instance, the expression function of the gene 3 in Fig. 1 is as the gene 3 receives regulation from gene 1, and gene 2. However, this model indicates that
the expression level of gene 3 is directly influenced be expression level of gene 1 and gene
2. Fig. 1: Generalized GRN model Fig. 1: Generalized GRN model We can obtain mRNA concentrations of gene xi(t) at time t, but do not know the expression of
fi(x(t)) ,i.e., we do not know the interaction relation between gene xi(t). To study the genetic
regulatory network, we should obtain the expression of fi(x(t)), according to the microarray
data. 3
A Petri Nets and Fuzzy Petri Nets Review Stage 3 Gene regulatory network In fact, it is impossible to find the exact fi(x(t)). However, many people have become
aware that the real world is not linear quadratic and that many situations can not be modeled
accurately by mathematically tractable equations [20, 21]. Combining with Petri net and
knowledge representation, a Fuzzy Petri Nets can be used to depict fuzzy generating rules that
can be taken as rules of fuzzy relationships between two propositions [22, 23]. So we will
construct a fuzzy system according to the microarray data in different time points, and make
the fuzzy system universal approximator of fi(x(t)). FPN = (P, T, D, I, O, f, α, β, λ) where: P = {pl, p2 , ..., pn} is a finite set of places, corresponding to the propositions of FPRs; P = {pl, p2 , ..., pn} is a finite set of places, corresponding to the propositions of FPRs;
T = {tl, t2 , ..., tn} is a finite set of transitions, P ∩T = Ø , corresponding to the execution of
FPRs; D = {d1, d2 , ..., dn} is a finite set of propositions of FPRs. P ∩ T ∩D = Ø, |P | =| D |, di (i=
1,2,..., n) denotes the proposition that interprets fuzzy linguistic variables, such as: “low”
“medium” “high”, as in our model; D = {d1, d2 , ..., dn} is a finite set of propositions of FPRs. P ∩ T ∩D = Ø, |P | =| D |, di (i=
1,2,..., n) denotes the proposition that interprets fuzzy linguistic variables, such as: “low”
“medium” “high”, as in our model; O : is an output incidence matrix; f = {µ1, µ 2 ,..., µm} where µi denotes the certainty factor (CF) of Ri , which indicates the
reliability of the rule Ri , and µi∈[0,1]; f = {µ1, µ 2 ,..., µm} where µi denotes the certainty factor (CF) of Ri , which indicates the
reliability of the rule Ri , and µi∈[0,1]; α : P → [0,1] is the function which assigns a token value between zero and one to each place;
β : P → D is an association function, a bijective mapping from places to propositions. β : P → D is an association function, a bijective mapping from places to propositions. λ : T→ [0, 1] is the function which assigns a threshold λi between zero and one to a transition
ti; By carefully connecting related place and assigning reasonable values of certainty factors to
transitions, we can come up with a fuzzy Petri net that can make decisions based on the
expertise we gave it during its construction. By carefully connecting related place and assigning reasonable values of certainty factors to
transitions, we can come up with a fuzzy Petri net that can make decisions based on the
expertise we gave it during its construction. There are a significant number of generalizations of Petri nets into the domain of fuzzy sets;
most of these approaches are focused on modeling the mechanisms of approximate reasoning. 3.1
Petri Nets A Petri net is a directed, weighted, bipartite graph consisting of two kinds of nodes, called
places (Pi) and transitions (Tj), where arcs are either from a place to a transition or from a
transition to a place [24]. Murata has formally defined Petri nets as a 5-tuple [25]: PN = (P, T,
F, W, M0), where P = {P1, P2, …, Pm} is a finite set of places, T = {t1, t2,… , tn} is a finite set
of transitions, F ⊆ (P ×T )∪(T ×P) is a set of arcs, W : F → {1, 2, 3, …,} is a weight function,
and M0 : P → {1, 2, 3, …,} is the initial marking. A marking M is an m-vector,
{M(P1),…,M(Pm)}, where M(Pi) denotes the number of the tokens in place Pi. The incidence
matrix A = [aij] is a n×m matrix of integers and its typical entry is defined by aij = aij
+ - a ij
−,
where aij
+ is the weight of the arc from a transition ti to its output place Pj, and a ij
− is the
weight of the arc to a transition ti from its input place Pj .The reachability set R(M0) of a Petri
net is defined as the set of all possible markings reachable from M0. Some notations are
introduced as follows: •tj denotes the input places of tj, tj• denotes the output places of tj, •Pi
denotes the input transitions of Pi, and Pi• denotes the output transitions of Pi. Because PNs 3 doi:10.2390/biecoll-jib-2010-113 Journal of Integrative Bioinformatics, 7(1):113, 2010 http://journal.imbio.de http://journal.imbio.de (ordinary Petri nets)cannot deal with vague or fuzzy information such as “very good“ and
“healthy” several kinds of Fuzzy Petri Nets (FPNs) have been introduced [15]. They are used
for fuzzy knowledge representation and reasoning. A FPN differs from a PN only in
markings, the firing rule, and possible-token locations. In this paper, we use the FPNs defined
in [26]. (ordinary Petri nets)cannot deal with vague or fuzzy information such as “very good“ and
“healthy” several kinds of Fuzzy Petri Nets (FPNs) have been introduced [15]. They are used
for fuzzy knowledge representation and reasoning. A FPN differs from a PN only in
markings, the firing rule, and possible-token locations. In this paper, we use the FPNs defined
in [26]. 3.2
Definition of Fuzzy Petri nets FPN expanded from a Petri net is a bipartite graph that has place and transition nodes like the
Petri net. However, in FPN a token incorporated with a place is associated with a real value
between 0 and 1; a transition is associated with a certain factor (CF) between 0 and 1. Fuzzy
Petri net is a promising modeling tool for expert systems, and it has shown itself to be suitable
for fuzzy knowledge representation and reasoning. In order to capture more information of
modeling gene regulatory networks, many authors have developed FPNs, for example, 8-tuple
[15], 13-tuple [20], and 9-tuple [26]. As shown in [26], a fuzzy Petri net structure is defined as
9-tuple: 3.4
Construction of Input Incidence Matrix I and Output Incidence Mat Assuming that there are m places, n transitions in the fuzzy Petri net, we have Input incidence matrix Im×n : Input incidence matrix Im×n : I= (aij) m×n I= (aij) m×n
( i= 1,2,…..,m; j=1,2,….,n ) ( i= 1,2,…..,m; j=1,2,….,n ) where where
1 pi ∈•tj & pi ∉ t•
j
aij =
0 others where
1 pi ∈•tj & pi ∉ t•
j
aij =
0 others 1 pi ∈•tj & pi ∉ t•
j
aij =
0 others aij = 1 means that there is an arc connecting place pi to transition tj; aij = 1 means that there is an arc connecting place pi to transition tj;
aij = 0 means that there is not an arc connecting place pi to transition tj; aij = 0 means that there is not an arc connecting place pi to transition tj; Output incidence matrix Om×n : Output incidence matrix Om×n : O = (bij) m×n O = (bij) m×n ( i= 1,2,…..,m; j=1,2,….,n ) ( i= 1,2,…..,m; j=1,2,….,n ) where
1 pi ∈t•
j & pi ∉•tj
bij =
0 others where
1 pi ∈t•
j & pi ∉•tj
bij =
0 others where others bij = 1 represents that there is an arc connecting transition tj to place pi;
bij = 0 means that there is not an arc connecting transition tj to place pi; bij = 1 represents that there is an arc connecting transition tj to place pi;
bij = 0 means that there is not an arc connecting transition tj to place pi; bij = 1 represents that there is an arc connecting transition tj to place pi;
bij = 0 means that there is not an arc connecting transition tj to place pi; bij = 0 means that there is not an arc connecting transition tj to place pi; FPN = (P, T, D, I, O, f, α, β, λ) where: The primary thrust of these attempts is in a proper representation of the semantics of the
underlying reasoning mechanisms. For the limitations, fuzzy Petri net may be not suitable to
parallel reasoning, such as in [16]. doi:10.2390/biecoll-jib-2010-113 4 4 Journal of Integrative Bioinformatics, 7(1):113, 2010 http://journal.imbio.de 3.3
Definition of pre-set and post-set 2) post-set: ∀x∈ T ∪P, x• ={z| (x,z) ∈ F} is called the post-set of x ; 3.4
Construction of Input Incidence Matrix I and Output Incidence Matrix O 3.4
Construction of Input Incidence Matrix I and Output Incidence Matrix O (Excitatory component) Observe that for wi = 0 and λi= 1, the inhibitory effect z¯ equals directly
)
(
ip
α
hence
)
(
ip
α
=
1 completely prohibits the transition from firing. For lower level of marking
)
(
ip
α
this
prohibition effect becomes limited. So to understand the inhibitory mechanism, and make that
possible to implement our model to predict changes in target expression level, we set wi =0
and λi=0. 3.5
The concept of inhibition arc Pedrycz and Gomide [27] provides a novel scheme for machine learning using fuzzy Petri
nets. Their formulation is based on the usual definition of t-and s-norms. A transition ti fires if
its degree of firing exceeds its threshold λi. ]
))
(
[(
j
j
j
n
i
j
sw
p
T
z
α
λ →
=
≠ doi:10.2390/biecoll-jib-2010-113 5 doi:10.2390/biecoll-jib-2010-113 Journal of Integrative Bioinformatics, 7(1):113, 2010 http://journal.imbio.de http://journal.imbio.de http://journal.imbio.de where, “→” denoted a fuzzy implication. An important knowledge representation addition
pertains to inhibitory arcs (connections) occurring within the net [27]. The role of this arc is to
model an inhibitory action coming from a certain input place. In the two-valued version of the
net these places while carrying a nonzero number of tokens prevent the associated transitions
from firing. In the framework of the fuzzy Petri net the inhibitory action is completed by
considering a complement of the marking of the inhibitory place see Fig 2. complement
ix , contributes to the following expression describing the level of firing z. This complement
ix , contributes to the following expression describing the level of firing z. Fig. 2: Illustrating inhibition in a fuzzy Petri net Fig. 2: Illustrating inhibition in a fuzzy Petri net −
+
≠
=
→
→
=
tz
z
sw
p
t
sw
p
T
z
i
i
j
j
j
j
n
i
j
]
))
(
[(
]}
))
(
{[(
α
λ
α
λ
where −
+
≠
=
→
→
=
tz
z
sw
p
t
sw
p
T
z
i
i
j
j
j
j
n
i
j
]
))
(
[(
]}
))
(
{[(
α
λ
α
λ
where −
+
≠
=
→
→
=
tz
z
sw
p
t
sw
p
T
z
i
i
j
j
j
j
n
i
j
]
))
(
[(
]}
))
(
{[(
α
λ
α
λ
where where where (Inhibitory component) ]
))
(
[(
i
i
i
sw
p
z
α
λ →
=
− and ]
))
(
[(
j
j
j
n
i
j
sw
p
T
z
α
λ →
=
≠
+ Firing fuzzy production rules can be considered as firing transitions. Firing fuzzy production rules can be considered as firing transitions. Firing fuzzy production rules can be considered as firing transitions. 3.7
Rule representation for fuzzy reasoning Production rules (PRs) are suitable to express expert knowledge. In most cases, collecting
data in a precise way is difficult; FPRs are thus adopted, which have the ability of process
uncertain or incomplete knowledge [16, 26, 29]. For these reasons, inference rules are
obtained in the form of FPRs, enhancing reasoning capacity. FPNs are built on the basis of
FPRs. 3.7.1
Fuzzy Production Rules (FPRs) Let R be a set of fuzzy production rules: 3.6
Firing Principles of Transitions 3.6 A transition can be fired under the condition that the degrees of the truth of all its input places
are not null and greater than certain threshold values. We follow the common firing principle
in [28]. The degree of truth of an output place is equal to the minimum of the degrees of the
input places multiplying the certainty factor (CF) of the transition. Once transition tj meets its
firing conditions, the degrees of truth of the places under the state marking M (k) are computed
by: Min { M (k) (•tj) } × uj pi ∈t•
j & pi ∉•tj
M (k) (pi) =
M (k) (pi) others others doi:10.2390/biecoll-jib-2010-113
6 doi:10.2390/biecoll-jib-2010-113 doi:10.2390/biecoll-jib-2010-113 6 6 Journal of Integrative Bioinformatics, 7(1):113, 2010 http://journal.imbio.de Journal of Integrative Bioinformatics, 7(1):113, 2010 http://journal.imbio.de where tj ∈ T, j = 1,2,...,n ;
pi∈ P, i= 1,2,..., m ; tj ∈ T, j = 1,2,...,n ;
P i
1 2 tj ∈ T, j = 1,2,...,n ; pi∈ P, i= 1,2,..., m ; M (k) (pi) denoted the degree of truth of the pi under the state marking M (k); M (k) (pi) denoted the degree of truth of the pi under the state marking M (k); k denoted the times of iteration; k denoted the times of iteration; uj denoted the certainty factor (CF) of the jth rule. uj denoted the certainty factor (CF) of the jth rule. Let R be a set of fuzzy production rules: Let R be a set of fuzzy production rules: R = {R1, R2,..., Rm}, and a fuzzy production rule Ri is shown as follows [30 R = {R1, R2,..., Rm}, and a fuzzy production rule Ri is shown as follows [30]: Ri : If dj then dk , (CF = µi ) Ri : If dj then dk , (CF = µi ) IF all propositions in the antecedent dj have value true THEN the propositions in the
consequent dk are true. di = { dj1, dj2,..., dj n}, represents the antecedent part which comprises of one or more
propositions connected by either “AND” or “OR” in the rule; di = { dj1, dj2,..., dj n}, represents the antecedent part which comprises of one or more
propositions connected by either “AND” or “OR” in the rule; dk = { dk1, dk2,..., dkn } represents the consequent part which comprises of one or more
propositions connected by “AND” in the rule; µi denotes the certainty factor (CFi ) of the rule Ri . Generally, FPRs are classified into four
types as follows: Type 1: If dj, then dk, (CF = µ),
Type 2: IF djl and dj2 and … and djn THEN dk (CF = µ),
Type 3: IF dj1 or dj2 or… or djn, THEN dk (CF = µ),
Type 4: IF dj THEN dk1 and dk2 and … and dkn (CF = µ), Type 1: If dj, then dk, (CF = µ),
Type 2: IF djl and dj2 and … and djn THEN dk (CF = µ),
Type 3: IF dj1 or dj2 or… or djn, THEN dk (CF = µ), Type 4: IF dj THEN dk1 and dk2 and … and dkn (CF = µ), doi:10.2390/biecoll-jib-2010-113 7 7 Journal of Integrative Bioinformatics, 7(1):113, 2010 Journal of Integrative Bioinformatics, 7(1):113, 2010 http://journal.imbio.de FPN models of the 4 types of composite fuzzy production rules are shown in Fig. 3. Places
(drawn as circles) represent entities or concentrations of a protein, mRNA, complex of
proteins, metabolites, etc. Transitions (drawn as bars) represent biological processes like
enzymatic reactions, transport, etc. Arcs represent dependencies of places and transitions or
define which and how places are affected whenever a transition fires. Fig. 3: Fuzzy Petri net models of composite fuzzy production rules. Firing of Transitions in FPN. (a) Before firing transitions. (b) After firing transitions. Fig. Let R be a set of fuzzy production rules: 3: Fuzzy Petri net models of composite fuzzy production rules. Firing of Transitions in FPN. (a) Before firing transitions. (b) After firing transitions. doi:10.2390/biecoll-jib-2010-113 8 8 Journal of Integrative Bioinformatics, 7(1):113, 2010 Journal of Integrative Bioinformatics, 7(1):113, 2010 http://journal.imbio.de 4
Process of changes in expression values 4 The fuzzy predict changes in expression values of the target gene are based on fuzzy logic
control theory. It consists of the following four steps: 4.1
Defining the membership functions for the input and output Fuzzification consists of defining the membership function for the input and output as well as
mapping from crisp data to fuzzy membership. In the process of changes in expression values
we search a microarray dataset for regulatory triplets consisting of activator, repressor and
target gene [7, 31]. Gene expression levels are first qualitatively classified into low, medium,
and high states to a varying degree based on a set of fuzzy membership functions. Genes are
then paired into an activator and repressor, and this gene pair determines the predicted target
gene expression profile based on a set of heuristic rules. In this process we set the activator
and repressor as input, with changes in target gene expression level as output. The fuzzy
membership functions of activator and repressor are described in Fig.4 .In order to measure
these input and output metadata universally, we normalize them into the same standard scale
of [0, 1]. The activator and repressor gene are classified into three sets, respectively. The
value of input data may belong to 1, 2 or 3 sets with corresponding membership degree. For
example, µactivator_expression=low (0.375) = 0.25, µactivator_expression=medium (0.375) = 0.75, means the activator
expression, 0.375 belongs to medium with confidence value (truth degree) of 75% while 25%
belongs to low. Fig. 4: The fuzzy input membership functions as a function of a normalized gene expression
level. Fig. 4: The fuzzy input membership functions as a function of a normalized gene expression
level. The fuzzy membership function of the output, i.e. changes in target gene expression level is
defined in Fig.5. It is represented with five levels or five sets with respect to fuzzy theory,
namely Very low, Low, Medium, High, and Very high. 9 doi:10.2390/biecoll-jib-2010-113 doi:10.2390/biecoll-jib-2010-113 9 doi:10.2390/biecoll-jib-2010-113 Journal of Integrative Bioinformatics, 7(1):113, 2010
http://journal.imbio.de Journal of Integrative Bioinformatics, 7(1):113, 2010 http://journal.imbio.de Fig. 5: The fuzzy membership function of output. Fig. 5: The fuzzy membership function of output. 4.2
Fuzzification Fuzzification consists of defining the membership function for the input and output as well as
mapping from crisp values to fuzzy membership. Mapping a particular activator and repressor
expression to the fuzzy membership correspondingly. By using the membership functions
defined above, we translate the input crisp values of activator and repressor expression into a
set of linguistic values and assign a membership degree for each linguistic value. 4.4
Defuzzification In order to represent the global output variable in fuzzy Petri nets (for example, the change in
target gene expression level has high, medium, or low) a defuzzification method is required to
produce a non-fuzzy output (crisp value). In Fig. 8, firing transition T1, T2,…,T9 deposits token
in places P7, P8, and P9 which can be defined as a defuzzification token and can be expressed
mathematically by the “centre of gravity” method [32]. We adopt the “center of gravity”
method as the defuzzification of the output predict change in target gene expression level Copyright 2010 The Author(s). Published by Journal of Integrative Bi
This article is licensed under a Creative Commons Attribution-NonCo Change_ target_ expression_level =
[ ]
[ ]
∑
∑
=
=
×
n
i
n
i
i
i
y
i
1
1
μ
μ where: where: • µ[i] is the height of output area from the i-th rule, • µ[i] is the height of output area from the i-th rule, • µ[i] is the height of output area from the i-th rule, • yi is the gravity’s horizontal coordinate of output area from the i-th rule, • n is the total number of matching rules for given values of each input dimension. After get the crisp value of the output, we map it into its fuzzy membership and get the
linguistic value whose membership degree has the highest level for change in target gene
expression level. Journal of Integrative Bioinformatics, 7(1):113, 2010 http://journal.imbio.de The reasoning engine performs decision-making based on the fuzzy logic reasoning rules with
first order predicate logic. Each rule can be defined as an If-Then clause, which determines
the linguistic value of output according to the linguistic values of input. Those fuzzy
reasoning rules are shown in Fig. 7. Fig 7: Rules for reasoning
1. If activator is “Low” and repressor is “Low” then target shift is “Medium”
2. If activator is “Low” and repressor is “Medium” then target shift is “Low”;
3. If activator is “Low” and repressor is “High” then target shift is “Low”
4. If activator is “Medium” and repressor is “Low” then target shift is “High”;
5. If activator is “Medium” and repressor is “Medium” then target shift is “Medium”
6. If activator is “Medium” and repressor is “High” then target shift is “Low”;
7. If activator is “High” and repressor is “Low” then target shift is “High”
8. If activator is “High” and repressor is “Medium” then target shift is “High”;
9. If activator is “High” and repressor is “High” then target shift is “Medium” 4.3
Reasoning with fuzzy reasoning rules In our algorithm, input genes are drivers. The heuristic rules are constructed accordingly
based on an activator/repressor regulatory logic. For example, if the activator and the
repressor genes are qualitatively classified low, then the predicted change in the target
expression level is considered Medium. In another case, if the activator is high and repressor
is low, then the predicted change at target is a High equivalent to the activation input as the
repressor is below threshold expression level. Similar heuristics are applied to construct the
decision matrix shown in Fig. 6. Fig. 6: Fuzzy decision matrix for predicting change in expression level of the target gene in an activator/
repressor regulatory relationship. Target
Medium
Target
Low
Target
Low
Target
High
Target
Medium
Target
Low
Target
High
Target
High
Target
Medium
Repressor
Low Medium High Fig. 6: Fuzzy decision matrix for predicting change in expression level of the target gene in an activator/
repressor regulatory relationship. Fig. 6: Fuzzy decision matrix for predicting change in expression level of the target gene in an activator/
repressor regulatory relationship. doi:10.2390/biecoll-jib-2010-113
10 doi:10.2390/biecoll-jib-2010-113 doi:10.2390/biecoll-jib-2010-113 10 Journal of Integrative Bioinformatics, 7(1):113, 2010 Journal of Integrative Bioinformatics, 7(1):113, 2010 http://journal.imbio.de http://journal.imbio.de Suppose the available activator expression value is 0.78, and the repressor expression value is
0.3. For these values we will map the normalized expression values to the fuzzy input
membership function defined in Fig. 4, the activator expression value, 0.78 is between
“Medium” and “High” µActivator_expression=medium (0.78)=0.44, µActivator_expression=high (0.78)=0.56. The repressor expression value, 0.3 is between “Low” and “Medium”, µRepressor_expression=low
(0.3) =0.38, µRepressor_expression=medium (0.3) =0.62. Suppose the available activator expression value is 0.78, and the repressor expression value is
0.3. For these values we will map the normalized expression values to the fuzzy input
membership function defined in Fig. 4, the activator expression value, 0.78 is between
“Medium” and “High” µActivator_expression=medium (0.78)=0.44, µActivator_expression=high (0.78)=0.56. The repressor expression value, 0.3 is between “Low” and “Medium”, µRepressor_expression=low
(0.3) =0.38, µRepressor_expression=medium (0.3) =0.62. FPN model to predict changes in target expression values 5 In this paper, we introduce a novel fuzzy Petri net model to predict changes in expression
values and infer causal relationship between genes. 11 doi:10.2390/biecoll-jib-2010-113 11 doi:10.2390/biecoll-jib-2010-113 doi:10.2390/biecoll-jib-2010-113 5.1
Constructing Petri Net model Fig. 8 shows how we realize the steps of fuzzy inference using the FPN structure as FPN = (P,
T, D, I, O, f, α, β, λ), where P= {p1, p2 , p3 , p4 , p5 , p6 , p7 , p8 , p9},
T= { t1 , t2 , t3 , t4 , t5 , t6 , t7 , t8 , t9 }, P= {p1, p2 , p3 , p4 , p5 , p6 , p7 , p8 , p9},
T= { t1 , t2 , t3 , t4 , t5 , t6 , t7 , t8 , t9 }, P= {p1, p2 , p3 , p4 , p5 , p6 , p7 , p8 , p9},
T= { t1 , t2 , t3 , t4 , t5 , t6 , t7 , t8 , t9 }, 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
0 1 1 0 0 1 0 0 0
1 0 0 0 1 0 0 0 1
0 0 0 1 0 0 1 1 0
0 0 0 0 0 0 0 0 0
O =
1 1 1 0 0 0 0 0 0
0 0 0 1 1 1 0 0 0
0 0 0 0 0 0 1 1 1
1 0 0 1 0 0 1 0 0
0 1 0 0 1 0 0 1 0
0 0 1 0 0 1 0 0 1
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
I = The linguistic meaning for propositions together with certainty factor (i.e. CF=µi) are listed in
the table 1. Because we used our algorithm to search for Activator_Repressor_Target triplets,
we expected to find the confidence degree of each initial rule usually depends on those
experiences of experts. The linguistic meaning for propositions together with certainty factor (i.e. CF=µi) are listed in
the table 1. Because we used our algorithm to search for Activator_Repressor_Target triplets,
we expected to find the confidence degree of each initial rule usually depends on those
experiences of experts. doi:10.2390/biecoll-jib-2010-113 5.1
Constructing Petri Net model The linguistic meaning for propositions together with certainty factor (i.e. CF=µi) are listed in
the table 1. Because we used our algorithm to search for Activator_Repressor_Target triplets,
we expected to find the confidence degree of each initial rule usually depends on those
experiences of experts. Therefore, these initial confidence degrees have been defined before the reasoning begins. Besides, the linguistic meaning of each proposition may have an effect on the confidence
degree. We set the confidence degree of each rule as shown in Fig. 8. Therefore, the confidence
degree vector CF=µi is We set the confidence degree of each rule as shown in Fig. 8. Therefore, the confidence
degree vector CF=µi is µi = {0.8, 0.6, 0.8, 0.7, 0.9, 0.95, 0.8, 0.99, 0.9}. As we computed the initial truth degree for each place (i.e. α(pi) =yi), the truth degree vector
is As we computed the initial truth degree for each place (i.e. α(pi) =yi), the truth degree vector
is α = {0, 0.44, 0.56, 0.38, 0.62, 0, 0, 0, 0}T. The initial marking vector is The initial marking vector is M0 ={1,1,1,1,1,1,0,0,0}T . M0 ={1,1,1,1,1,1,0,0,0}T . doi:10.2390/biecoll-jib-2010-113 12 Journal of Integrative Bioinformatics, 7(1):113, 2010
http://journal.imbio.de Journal of Integrative Bioinformatics, 7(1):113, 2010 http://journal.imbio.de Fig. 8: A FPN model Fig. 8: A FPN model Fig. 8: A FPN model Tab. 1: propositions and certainty factor for FPN Model 6
Conclusions 6 In this work, a fuzzy Petri nets GRN model is proposed for searching activator/ repressor
regulatory relationship between gene triplets in the microarray data. The model predicts
changes in expression levels in the target gene caused due to possible regulation based on
input expression levels. The genes that fit the model are more likely to exhibit activator/
repressor relationship. We propose a novel approach of fuzzy reasoning to predict changes in
expression levels. The major features of our approach are: (1) Since the FPN is a graphical
tool, we are able to give a description of the typical procedure of fuzzy reasoning; (2)
visualize the structure of a rule-based fuzzy reasoning system; (3) with the mathematical
foundation of FPN, we construct the reasoning steps for FPN reasoning; and (4) Finally, we
describe the FPN based modeling to predict changes in expression levels in the target gene to
validates the feasibility of FPN model. With the definition of FPN and the procedure of FPN
reasoning, some flaws of FPN based reasoning should be pointed out. In a FPN model, both
the truth degree of a proposition and the confidence degree of a rule should be determined
beforehand. The determination of these two degrees usually relies on experiences of experts,
which induce some uncertainty in the reasoning. Further biological experiments are needed to
determine the validity of the genetic interactions suggested by the model. For future work, we plan to integrate the proposed approach with neural network for modeling
gene regulatory network. Tab. 1: propositions and certainty factor for FPN Model Tab. 1: propositions and certainty factor for FPN Model
Propositions
Places
Initial
truth
degree
Initial
marking
Certainty
factor
Activator _Low
P1
0
1
0.8
Activator_Medium
P2
0.44
1
0.6
Activator _High
P3
0.56
1
0.8
Repressor_Low
P4
0.38
1
0.7
Repressor_Medium
P5
0.62
1
0.9
Repressor_High
P6
0
1
0.95
Target_Low
P7
0
0
0.8
Target_Medium
P8
0
0
0.99
Target_High
P9
0
0
0.9 doi:10.2390/biecoll-jib-2010-113 doi:10.2390/biecoll-jib-2010-113 13 Journal of Integrative Bioinformatics, 7(1):113, 2010 Journal of Integrative Bioinformatics, 7(1):113, 2010 http://journal.imbio.de http://journal.imbio.de 5.2
FPN reasoning The execution of FPN will change the truth degree vector (i.e. α(pi) =yi) and the marking
vector M0. The procedure of execution of FPN is shown as follows. α(pi) = {0, 0.44, 0.56, 0.38, 0.62, 0, 0, 0.396, 0.392}T . After the execution mentioned above, the change in target is got. Transfer the linguistic values
into a crisp value according to “center of gravity”, see section 4. Change_Target_Expression_Level=
392
. 0
396
. 0
0
392
. 0
396
. 0
0
+
+
×
+
×
+
×
high
medium
low Change_Target_Expression_Level=
392
. 0
396
. 0
0
392
. 0
396
. 0
0
+
+
×
+
×
+
×
high
medium
low
Change_Target_Expression_Level=
392
. 0
396
. 0
0
75
. 0
392
. 0
5
. 0
396
. 0
25
. 0
0
+
+
×
+
×
+
×
≈ 0.62 392
. 0
396
. 0
0
+
+
Change_Target_Expression_Level=
392
. 0
396
. 0
0
75
. 0
392
. 0
5
. 0
396
. 0
25
. 0
0
+
+
×
+
×
+
×
≈ 0.62 According to Fig. 5, the change target expression level, 0.62 is between “Medium” and
“High”. Apparently, the change in target gene expression level belongs to “Medium”, because
µChange_target_expression_level =medium is much larger than µChange_target_expression_level =high . Hence, we
decide the final change in target expression level as “Medium”. Acknowledgements The authors would like to thank the reviewers for their best comments that help improve the
manuscript. Many thanks for my professor Syed I. Ahson for improving this paper. And I also
give my thanks to my colleagues for their good advices to this paper. The work is partially
supported by the national nature foundation under Contact Number 5576. 14 doi:10.2390/biecoll-jib-2010-113 Journal of Integrative Bioinformatics, 7(1):113, 2010 http://journal.imbio.de References [1] R. Ram, M. Chetty and T. I. Dix. Fuzzy Model for Gene Regulatory Network. IEEE
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English
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Silicone v1.0.0: an open-source Python package for inferring missing emissions data for climate change research
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cc-by
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Correspondence: Robin Lamboll (r.lamboll@imperial.ac.uk) Correspondence: Robin Lamboll (r.lamboll@imperial.ac.uk) Received: 13 May 2020 – Discussion started: 28 May 2020
Revised: 14 September 2020 – Accepted: 23 September 2020 – Published: 4 November 2020 Received: 13 May 2020 – Discussion started: 28 May 2020
Revised: 14 September 2020 – Accepted: 23 September 2020 – Published: 4 November 2020 Abstract. Integrated assessment models (IAMs) project fu-
ture anthropogenic emissions which can be used as input
for climate models. However, the full list of climate-relevant
emissions is lengthy and most IAMs do not model all of
them. Here we present Silicone, an open-source Python pack-
age which infers anthropogenic emissions of unmodelled
species based on other reported emissions projections. For
example, it can infer nitrous oxide emissions in one sce-
nario based on carbon dioxide emissions from that scenario
plus the relationship between nitrous oxide and carbon diox-
ide emissions found in other scenarios. Infilling broadens the
range of IAMs available for exploring projections of future
climate change, and hence Silicone forms part of the open-
source pipeline for assessments of the climate implications
of IAM scenarios, led by the Integrated Assessment Mod-
elling Consortium (IAMC). This paper presents a variety of
infilling options and outlines their suitability for different
cases. We recommend certain infilling techniques as good
defaults but emphasise that considering the specifics of the
model being infilled will produce better results. We demon-
strate the package’s utility with three examples: infilling all
required gases for a pathway with data for only one emis-
sion species, splitting up a Kyoto emissions total into sepa-
rate gases, and complementing a set of idealised emissions
curves to provide a complete, consistent emissions portfolio. The code and notebooks explaining details of the package
and how to use it are available on GitHub (https://github.com/
GranthamImperial/silicone, last access: 2 November 2020). The repository with this paper’s examples and uses of the
code to complement existing research is available at https: //github.com/GranthamImperial/silicone_examples (last ac-
cess: 2 November 2020). 1.1
General context and problem setting Integrated assessment models (IAMs) are scientific mod-
elling tools that integrate knowledge from different academic
disciplines with the aim of exploring and informing policy
decisions (Clarke et al., 2014; Rogelj et al., 2018a; Weyant,
2017). They are widely used in climate change research to
combine insights from energy, economy, agricultural, and
natural sciences, with the aim of creating scenarios that ex-
plore how societal decisions can affect projected greenhouse
gases and other emissions, as well as their related climate
outcomes (Clarke et al., 2014; Huppmann et al., 2018; Riahi
et al., 2017; Rogelj et al., 2018b). However, IAMs do not always exhaustively represent all
possible processes or sources of climate-relevant emissions. Thus, many IAM scenarios lack projections for some climate
forcers, be it specific greenhouse gas emissions or aerosol
precursors. A complete set of these climate forcers is re-
quired to accurately estimate the overall climatic effects of a
given scenario (Meinshausen et al., 2011; Smith et al., 2018),
as a large number of supposedly minor emissions may col-
lectively exert a significant radiative forcing (Meinshausen
et al., 2017; O’Neill et al., 2016). Geosci. Model Dev., 13, 5259–5275, 2020
https://doi.org/10.5194/gmd-13-5259-2020
© Author(s) 2020. This work is distributed under
the Creative Commons Attribution 4.0 License. Geosci. Model Dev., 13, 5259–5275, 2020
https://doi.org/10.5194/gmd-13-5259-2020
© Author(s) 2020. This work is distributed under
the Creative Commons Attribution 4.0 License. R. D. Lamboll et al.: Silicone v1.0.0 However, the former clearly
does not scale easily to larger databases (because making
ad hoc decisions for a thousand scenarios requires a signif-
icant time input), and the latter approach, termed the “equal
quantile walk” (EQW), ignores trade-offs and specific rela-
tionships between emission species resulting from how com-
peting technologies, behaviours, and industrial practices re-
sult in different emissions. A few alternative approaches have
been used recently: for instance, using the pathway with the
smallest mean squared distance over time was used in Ro-
biou du Pont and Meinshausen (2018). This works well for
large databases containing similar paths but is less reliable
for smaller databases or for paths with an unusual behaviour
over time. A more sophisticated “generalised quantile walk”
technique can capture the effect of trade-offs and was re-
cently introduced in Sect. 3.8.1 in Teske et al. (2019), in-
volving quantile regression between a lead variable (fossil
CO2 emissions) and other gases for every individual year. Unfortunately, the implementation there did not consistently
guarantee that higher quantiles resulted in higher emissions,
and has not been followed up with any peer-reviewed work
that does so. A tool for infilling was provided by Rogelj et al. (2014) using a cubic spline between specific points in a small
database; however, this type of infiller behaves chaotically
when applied to large databases incorporating many differ-
ent models. It was also coded in Excel, limiting the ease of
open-source development. H
lb
f
h d
dd
h p
Silicone is compatible with a suite of Python tools that
make up the IAM climate assessment pipeline developed un-
der the umbrella of the Integrated Assessment Modelling
Consortium (IAMC). Compatibility with these tools allows
us to load, manipulate, and save files using a common file
format. The pipeline is based around the pyam package (Gid-
den and Huppmann, 2019), specifically its IamDataFrame
class, which Silicone makes extensive use of; pyam data
frames easily convert to and from widely used pandas data
frames, which pyam and Silicone also use internally (McK-
inney, 2011). The pipeline also includes tools to harmonise
(i.e. correct projection made in the past to match now-known
emissions) (called aneris; Gidden et al., 2018) before infilling
and to pass the complete projections to climate simulators. The estimation of climatic impact is performed by Open-
SCM (Nicholls et al., 2020), which is compatible with the
data structure of the pipeline. R. D. Lamboll et al.: Silicone v1.0.0 5260 R. D. Lamboll et al.: Silicone v1.0.0 to a broader set required for a sensible climate assessment. In
essence, its methods are grounded in a comparison of the co-
evolution of anthropogenic emissions in scenarios that are
readily available in the literature (Huppmann et al., 2018;
Riahi et al., 2017; Rogelj et al., 2018b). Silicone aims to
provide IAM teams that do not represent all individual cli-
mate forcers with robust methods to complement their model
output and facilitate a climatic assessment of their work. Furthermore, Silicone also aims to provide geoscience re-
searchers with a tool to easily develop stylised, yet inter-
nally consistent future emission pathways of the most impor-
tant climate forcers. It can also estimate or calculate missing
emissions from particular sectors. Notebooks describing how
to use these tools are available on the accompanying GitHub
repository (Lamboll et al., 2020b) and the formal documen-
tation is available in Lamboll et al. (2020a). Additional ex-
amples of using Silicone for the specific situations outlined
below are included in a separate GitHub repository (Lam-
boll, 2020). The package is open-source and intended to al-
low groups to write their own infilling methods if desired. Users and collaborators are encouraged to add any such de-
velopments to the code base via GitHub. Scenarios that only report a limited set of greenhouse
gases or climate forcers must thus be complemented by es-
timated evolutions of missing emissions derived without fur-
ther economic analysis. We term this estimation “infilling”. If no infilling is attempted, the unevaluated emissions would
effectively be considered zero, which would clearly create
systematic biases and potential artefacts in the projected tem-
peratures. Depending on the radiative forcing of the species
in question, this bias may be positive or negative, so infilling
with zeros would not necessarily be a conservative choice. Most earlier studies overcame this problem in one of two
ways: with expert-based ad hoc decisions on how to ade-
quately fill in missing species (Schaeffer et al., 2015) or by
assuming that a pathway will occur at the same quantile for
each set of emissions in a particular year, although the quan-
tile can vary over time (Gütschow et al., 2018; Meinshausen
et al., 2006; Nabel et al., 2011). R. D. Lamboll et al.: Silicone v1.0.0 This pipeline is being devel-
oped in support of the IAM community and the IAM scenario
assessment for the forthcoming Sixth Assessment Report of
the Intergovernmental Panel on Climate Change (IPCC AR6)
in particular. This paper is structured as follows: Sect. 2 presents an
overview of the different infiller methods, then goes through
the infiller techniques in precise and mathematical detail. In
Sect. 3, we present our analysis of emissions projections in
the SR1.5 database. This includes correlation statistics for
the database and how well Silicone reproduces one entry in
the database given the other entries. We use this to draw con-
clusions on the implications for using Silicone on unknown
data. In Sect. 4, we present three examples of using Silicone
for infilling a pathway with limited information, splitting up
an aggregate basket of emissions, and infilling stylised emis-
sions trajectories. We end with a summary of our paper. Here we present a new toolbox of methods to address these
recurring infilling challenges in the climatic assessment of
socioeconomic emissions scenarios. The toolbox introduces
new approaches as well as building on and combining previ-
ous approaches. The code base is a significant improvement
compared to existing options in terms of flexibility, applica-
bility, reproducibility, and versatility. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. https://doi.org/10.5194/gmd-13-5259-2020 1.2
The aim of Silicone Silicone is a Python package designed to enable users to ex-
pand scenario projections of a limited set of climate forcers Geosci. Model Dev., 13, 5259–5275, 2020 https://doi.org/10.5194/gmd-13-5259-2020 5261 R. D. Lamboll et al.: Silicone v1.0.0 Figure 1. Flow chart suggesting how to choose the cruncher (peach oblongs) or multiple infiller (yellow oblongs) to use when infilling. sting how to choose the cruncher (peach oblongs) or multiple infiller (yellow oblongs) to use when infilling. Figure 1. Flow chart suggesting how to choose the cruncher (peach oblongs) or multiple infiller (yellow oblo Figure 1. Flow chart suggesting how to choose the cruncher (peach oblongs) or multiple infiller (yellow oblongs) to use when infilling. https://doi.org/10.5194/gmd-13-5259-2020 Geosci. Model Dev., 13, 5259–5275, 2020 Pitfalls Has no basis in the data – only used as
a last resort in cases of complete uncer-
tainty. No reason to assume that the rela-
tionship between emissions holds for
all times. No restriction on signs of
follower gas, so potential sign errors
when the lead (but not follower) emis-
sions may become negative. Sensitive
to emissions trajectories with a high co-
efficient of variation. Used when no data are available
for most times; this generalises
from the latest information we
have, e.g. if only historic data
are available. Allows
arbitrarily
high
emissions. Can behave unexpectedly if emissions
change sign, and an error is produced if
emissions with this sign are not seen at
the same time in the infiller database. Used when two emissions
should track each other or one
represents a portion of the other. Multiplies the lead variable by
the ratio of the averages of the
lead and follower data in the in-
filler database. (Note: this ratio
is not the same as the average
of the ratios and is more stable
to inclusion of extreme ratios.)
Optionally calculates this using
only values with the same sign
of lead emissions. Multiplies the lead variable by
the ratio of the averages of the
lead and follower data in the in-
filler database. (Note: this ratio
is not the same as the average
of the ratios and is more stable
to inclusion of extreme ratios.)
Optionally calculates this using
only values with the same sign
of lead emissions. Used when behaviour at one
time should strongly determine
behaviour at another and conti-
nuity is needed between times. The only cruncher that does not
treat each time separately. A small change in the target data at
a single time step can result in large
changes in output at every time step. All
the results returned are found exactly in
the infiller database, so if that database
is small, the same values are returned in
many cases. Results more extreme than
those found in the infiller database all
return the same value. Finds the most similar path-
way in the infiller database and
uses those values. Most simi-
lar means smallest root mean
squared difference between the
lead values of infiller pathways
and target pathway averaged
over all times. Linear interpolation;
interpolate specified
scenarios and models R. D. Lamboll et al.: Silicone v1.0.0 Table 1. A guide to crunchers. Names followed by asterisks use a ratio-based approach; i.e. they find a multiplicative factor and then multiply
the target lead by this value. These crunchers do not preserve harmonisation. If the asterisk is in brackets, a ratio-based approach is optional. Otherwise, techniques all return linear combinations of values seen in the infiller database. Otherwise, techniques all return linear combinations of values seen in the infiller database. Name
Description
Use case
Pitfalls
Constant ratio∗
Multiplies the lead variable by a
constant (not fitted to any data). Used
when
no
information
about
the
follower
variable
is available in any database. Mainly used for infilling with
zeros. Has no basis in the data – only used as
a last resort in cases of complete uncer-
tainty. Latest time ratio∗
Multiplies the lead variable by
a constant fitted to a single
(latest) time point in the infiller
data. Used when no data are available
for most times; this generalises
from the latest information we
have, e.g. if only historic data
are available. No reason to assume that the rela-
tionship between emissions holds for
all times. No restriction on signs of
follower gas, so potential sign errors
when the lead (but not follower) emis-
sions may become negative Sensitive Used for infilling where we
have a small number of com-
parable models and/or scenar-
ios. The required filtering gives
control over the narrative used
for infilling. 2
Methods like population number before infilling, provided that com-
parable emissions statistics can be found in the remaining
database. Silicone takes a database that contains data for at least two
emissions species (this database is referred to as the “infiller”
database) and derives a relationship between these time se-
ries. It then applies that relationship to a second database
(the “target” database), which does not have any data for
one of the emissions species in the infiller database. For ex-
ample, based on an infiller database of CO2 and N2O emis-
sions, Silicone could then derive N2O emissions compatible
with the CO2 emissions in a less complete target database. In all cases, the infillers will perform best if the target data
come from a scenario that is socioeconomically similar to
scenarios found in the infiller database. The performance of
most crunchers can be improved by filtering out scenarios
that are known to assume radically different characteristics Silicone offers a range of tools that apply methods for do-
ing this infilling which are appropriate in different circum-
stances, depending on the amount of complete data and how
much we know about the narrative behind our emissions. These tools are referred to as “crunchers”. Each of these
crunchers takes a “lead variable”, found in both the infiller
and target databases, and uses it to infer the value of a “fol-
lower variable”, found only in the infiller database (hence
missing in the target database). There are also several tools
for easily infilling multiple variables, called “multiple in-
fillers”. These may have multiple follower or lead variables. https://doi.org/10.5194/gmd-13-5259-2020 Geosci. Model Dev., 13, 5259–5275, 2020 5262 R. D. Lamboll et al.: Silicone v1.0.0 R. D. Lamboll et al.: Silicone v1.0.0 Used when two emissions
should track each other or one
represents a portion of the other. Used for infilling where we
have a small number of com-
parable models and/or scenar-
ios. The required filtering gives
control over the narrative used
for infilling. Conceptually simple, used by
previous work. 2.1.1
Cruncher guide There are currently seven types of cruncher. These are out-
lined in Table 1 below. A flow chart to guide the choice is
shown in Fig. 1. There is also a series of notebooks with ex-
amples of how to use them all in the main GitHub repository
(Lamboll et al., 2020b). The ratio-based approaches are better for cases in which
the lead values to be infilled are outside the range in the in-
filler database and we expect the emissions to scale with each
other, for instance if we are infilling one incomplete com-
bustion product based on another or splitting up aggregated
emissions into their components. However, care needs to be
taken when infilling emissions that are non-negative using a
lead value that may be of any sign, for example CO2 emis-
sions. In that case, the ratio method might produce values for
the target emissions that are unsupported by any available
evidence. Singular behaviour may also be encountered when
the lead data are close to zero in the infiller database. The dif-
ferent crunchers present different ways to estimate the ratio
to use. 2.1
Methods overview results are to be harmonised, then harmonising both the in-
filler and target data before infilling is required for improved
consistency (otherwise, infilling depends on outdated data). Absolute value infilling techniques preserve harmonisation;
however, ratio-based approaches do not necessarily do this
and may need harmonisation again afterwards. 2.1.2
Multiple infiller and aggregation tool guide Multiple infillers are for cases in which there are relation-
ships between multiple lead or follower values that need to
be considered at the same time. They allow less tailored ap-
proaches to infilling but can ensure that the infilling is faster
or more consistent than infilling each of the variables sepa-
rately. These are outlined in Table 2. R. D. Lamboll et al.: Silicone v1.0.0 R. D. Lamboll et al.: Silicone v1.0.0
Table 1. Continued. Name
Description
Quantile rolling
windows (QRW), time-
dependent quantile
rolling windows (∗)
At
each
time,
applies
a
1 / (1 + (lead variable
difference)2) weighting to data
points at equally spaced points
across the infiller lead. Then
calculates a specified quantile
(usually the median) for the
infiller follower value at these
points. Can also be used in ratio
mode, in which case the ratio
between lead and follower in
the infiller database is treated
as
above. Time-dependent
QRW allows the quantile to be
different at different times (but
is computationally slower). Equal quantile walk
(EQW)
Calculates the quantile of the
infiller database corresponding
to the lead value in each indi-
vidual year. Returns that quan-
tile in that year of the follower
value from the same database. Table 1. Continued. Use case Pitfalls Pitfalls Using with any quantile larger than 0.5
will result in all emissions being higher,
even if the lead and follower emis-
sions anticorrelate. Results more ex-
treme than found in the infiller database
all return the same value, unless in ra-
tio mode. In ratio mode, sign changes
in the lead variable can result in fol-
lower emissions being assigned unde-
sired negative values. Quantile rolling
windows (QRW), time-
dependent quantile
rolling windows (∗) Can choose options to give
more smoothing (less noise)
or more localised behaviour
(shows trends better). Allows
the option to generate a distri-
bution of outputs, not just a sin-
gle optimum. Can add to the
narrative through time depen-
dence. Ratio mode allows better
infilling outside the range of the
infiller data. Equal quantile walk
(EQW) Assumes all variables are monotoni-
cally increasing together. Results more
extreme than those found in the infiller
database all return the same value. Calculates the quantile of the
infiller database corresponding
to the lead value in each indi-
vidual year. Returns that quan-
tile in that year of the follower
value from the same database. Equal quantile walk
(EQW) Pitfalls At each time, linearly interpo-
lates between the follower val-
ues at the two nearest lead
values, taking averages where
multiple points have identical
lead values. Interpolates speci-
fied scenarios and models filters
in the infiller database before
applying the same technique. A small change in the target data can
result in a large change in the output
at the same time step because pathways
in the infiller database can be very dif-
ferent in follower variables for nearly
identical values of the lead variable. For similar reasons, results can vary er-
ratically between time steps for large
infiller datasets. Results more extreme
than those found in the infiller database
all return the same value. Geosci. Model Dev., 13, 5259–5275, 2020 https://doi.org/10.5194/gmd-13-5259-2020 R. D. Lamboll et al.: Silicone v1.0.0
Table 1. Continued. Name
Description
Quantile rolling
windows (QRW), time-
dependent quantile
rolling windows (∗)
At
each
time,
applies
a
1 / (1 + (lead variable
difference)2) weighting to data
points at equally spaced points
across the infiller lead. Then
calculates a specified quantile
(usually the median) for the
infiller follower value at these
points. Can also be used in ratio
mode, in which case the ratio
between lead and follower in
the infiller database is treated
as
above. Time-dependent
QRW allows the quantile to be
different at different times (but
is computationally slower). Equal quantile walk
(EQW)
Calculates the quantile of the
infiller database corresponding
to the lead value in each indi-
vidual year. Returns that quan-
tile in that year of the follower
value from the same database. 5263 R. D. Lamboll et al.: Silicone v1.0.0 Pitfalls Use case Requires all information to be
known already – no statistical
inference, just adding. Requires all information to be
known already – no statistical
inference, just adding. Requires only the target
database. Adds together known
values to construct a consistent
output (with optional weight-
ing). Infilling aggregate values (e.g. Kyoto gas totals) or finding re-
mainders given aggregates and
values for the other compo-
nents. Multiple infillers
Decompose collection
with time-dependent
ratio∗ Infiller scenarios which do not
have values for all compo-
nents at all times are ignored. Ignores the aggregate if the
infiller database has inconsis-
tency between that and the sum
of reported components. As-
sumes direct proportionality be-
tween components and sum,
which is problematic around
sign changes. Breaking down aggregate val-
ues into their components, as-
suming all should be treated
similarly. Constructs a consistent version
of the aggregate in the in-
filler database. Breaks a known
quantity
down
into
compo-
nents, estimated by the time-
dependent ratio method. Breaking down aggregate val-
ues into their components when
one emission type is much
larger than the others or may be
either positive or negative The remainder emission is not
constrained nor as precisely es-
timated as the other values. The remainder emission is not
constrained nor as precisely es-
timated as the other values. Low confidence in the results
being accurate as the method
does not consider the specific
characteristics of the data. For infilling scattered,
minor gaps in a largely sound
database. Pyam data frames assign values to variables as a function
of different models, scenarios, regions, and times. All meth-
ods work on databases with only a single region at a time,
although the region can be different between the infiller and
target databases. than increasing in line with other variables. They may be
considered more stable and more conservative. The quantile-
rolling-windows (QRW) cruncher can be used in either ratio
or absolute (non-ratio) mode, the absolute mode being the
default. As one final detail, we discuss the data model which is
assumed by Silicone. Silicone is built around the pyam pack-
age (Gidden and Huppmann, 2019). As a result, it assumes
that all input data are provided in a particular structure. The
structure includes the model which created the time series,
the scenario with which the time series is associated (e.g. 2.2.1
Ratio infilling methods These methods all firstly estimate the ratio of the lead vari-
able to the follower variable. In all cases, we first determine
the ratios, written as R(t) at time t. Once these have been
calculated, the follower value in the target database, Ef (t),
is valued as Ef (t) = R(t)El(t),
(1) (1) Ef (t) = R(t)El(t), where El(t) is the lead value in the target database. Pitfalls a
high BECS 1.5◦scenario), the region the emissions occurs
in, and the unit of the data (full details available at https:
//pyam-iamc.readthedocs.io/en/stable/data.html, last access:
2 November 2020). Accordingly, Silicone is able to work on
specific subsets of models (e.g. only the MESSAGE model)
or subsets of scenarios (e.g. all SSP1-like scenarios). We
therefore follow the pyam convention and refer to a “model–
scenario combination” to mean a single projected world that
in some contexts might be called a “scenario”. 2.2
Mathematical detail The absolute-value-based techniques infill with values de-
rived from the absolute data found in the infiller database or
linear combinations of them. This means that they will al-
ways return values within the range spanned by the infiller
database. This is most appropriate for processes whereby we
have a greater number of cases, preferably with both larger
and smaller lead emissions in the infiller database or when we
expect the follower emissions to be strongly bounded rather Notebooks presenting the benefits and risks of each cruncher
type can be found in the Silicone GitHub (Lamboll et
al., 2020b) and may be useful to have as examples when
analysing the work below, as well as demonstrating how to
use them. There are two main classes of infillers: those based on the
ratios between two emission pathways and those based on
the absolute emission values in the infiller database. If the https://doi.org/10.5194/gmd-13-5259-2020 Geosci. Model Dev., 13, 5259–5275, 2020 5264 R. D. Lamboll et al.: Silicone v1.0.0 Table 2. Guide to aggregation tools and multiple infillers. Names followed by asterisks use a ratio-based approach; i.e. they find a multi-
plicative factor and then multiply the target lead by this value. If the asterisk is in brackets they are ratio-based. Table 2. Guide to aggregation tools and multiple infillers. Names followed by asterisks use a ratio-based approach; i.e. they find a multi-
plicative factor and then multiply the target lead by this value. If the asterisk is in brackets they are ratio-based. Quantile-rolling-window cruncher The quantile-rolling-window method may be applied in ratio
mode, in which case we calculate R(t) by first calculating
the ratio for each scenario, R = ⟨ef (tlast)⟩
El (tlast) ,
(2) (2) rs(t) = ef (t)
el(t) ,
(6) rs(t) = ef (t)
el(t) , (6) where the angular brackets mean taking the (algebraic) mean
with equal weighting for all estimates (typically historical es-
timates) at that time and with a lower-case ef (t) representing
the follower values in the database at time t. This ensures that
at tlast, all infilled data will fulfil then following the calculation in the absolute value section
using this instead of el. This method finds quantiles of the
ratio in the infiller database at set points along the range of
lead values in the infiller database. Ef (tlast) = R × El (tlast) = ⟨ef (tlast)⟩. (3) (3) (3) Constant-ratio and latest-time-ratio crunchers Constant-ratio and latest-time-ratio methods both use the
same ratio for all infill times, R(t) = R. With the constant-
ratio method, the ratio must be given as an input parameter. Geosci. Model Dev., 13, 5259–5275, 2020 https://doi.org/10.5194/gmd-13-5259-2020 5265 RMS-closest cruncher The time-dependent ratio is appropriate when there are some
data in the infiller database for all times and allows the ratio
to vary with time. The ratio used is The RMS-closest cruncher filters the infiller database for
models with data at all the times found in the target data. It then ranks models and scenarios by the root mean squared
(RMS) difference between the lead data in the infiller and
target database, with the average being taken over all time
slices. It returns the follower data from the scenario–model
combination with the smallest RMS difference: the formula
is Ef (t) = ef,i(t), where the subscript i refers to the model–
scenario case that minimises R(t) = ⟨ef (t)⟩
⟨el(t)⟩. (4) R(t) = ⟨ef (t)⟩
⟨el(t)⟩. (4) Optionally, the averaging can be taken only over model–
scenario cases in which the sign of the lead variable is the
same in both the infiller and target case – this will guarantee
that the infilled value takes the same sign as that of follower
values in the database. It will produce an error if there are no
data with the required sign. This cruncher has a useful con-
servativity property (with or without the sign restriction): if
in every scenario averaged over, the emissions of several sub-
stances sum to another substance, e.g. if e1 = e2 + e3, then
⟨e1⟩= ⟨e2⟩+ ⟨e3⟩. It then follows that X
t
El(t) −ef,i(t)
2. (7) (7) (7) In the case of a draw, the value that occurs earlier in the in-
filler database will be used. This is the only infiller that is not
time-independent; i.e. changing the value of the lead at one
time may result in different outputs at other times. 1 = ⟨e2⟩
⟨e1⟩+ ⟨e3⟩
⟨e1⟩,
(5) 1 = ⟨e2⟩
⟨e1⟩+ ⟨e3⟩
⟨e1⟩, (5) R. D. Lamboll et al.: Silicone v1.0.0 The latest-time-ratio method uses the ratio between the mean
follower data in the infiller database (we denote this database
with lower-case ef ) and the value of the lead variable in the
target data (El), with both values evaluated at the latest time
for which there are follower data in the infiller database, tlast. The mean is taken over all infiller data at that time. This is
designed for the case in which we have estimates only until a
certain time, after which they stop – for instance, if we have
no projections for some new hydrofluorocarbon (HFC) emis-
sions, but we have historic measurements for recent years. This gives us the equation on all scenarios having values for all of these variables, so
misses it cases which do not have one of the constituents or
only reports at some of the required times, unless the over-
ride option only_consistent_cases is set to false. It always
constructs a new, consistent version of the aggregate variable
in case different modellers used different conversion factors
in the infiller database. , last
The mean is taken over all infiller data at that time. This is
designed for the case in which we have estimates only until a
certain time, after which they stop – for instance, if we have
no projections for some new hydrofluorocarbon (HFC) emis-
sions, but we have historic measurements for recent years. This gives us the equation Quantile-rolling-windows cruncher Note that we sum over smaller follower values, but the
weighting is determined by the lead values: where dl is the decay length, which defaults to half the
separation between ep values, and i is the label for which
model–scenario we are investigating. Increasing the decay
length will reduce the weight difference between points, so
the rolling window becomes wider and more even, with the
limit case of calculating quantile q of all data for large dl. Amongst other things, this is a clear improvement over the
generalised quantile walk approach, as the latter uses equal
weights within a fixed window of a certain fraction of the
infiller database’s lead values in a certain year. These values
are then normalised so that Pwp = 1 and sorted into ascend-
ing order by ef . The follower value at quantile q, evaluated
at lead point el(j), is where the quantile equals the sum of
weights of all smaller ef plus half the weight of ef (j) it-
self. Note that we sum over smaller follower values, but the
weighting is determined by the lead values: q (el(j)) =
X
ef (i) < ef (j)wp (el(i)) + wp (el(j))
2
. (10) (10) Quantiles between these are evaluated by linearly interpolat- Quantiles between these are evaluated by linearly interpolat-
ing this relationship. We are usually interested in the case
in which q = 0.5. To infill a point at El, we interpolate be-
tween the known points ep. Quantile crossing is not possi-
ble in this framework because at any given evaluation point
higher quantiles cannot have lower values, and only linear
fits between these points are used. Figure 2. Schematic of how the quantile-rolling-windows cruncher
determines the follower value to use. (a) Example relationships be-
tween lead (CO2) and follower (CH4) variables over time. (b) A
number of rolling window centres (here 5, by default 10) are drawn,
and a weighting function is constructed for each window. It has
a continuous distribution rather than a discrete cutoff, hence the
name. (c) A relationship between the sum of the weights and the
follower value is established, and the follower value at the desired
quantile is returned. R. D. Lamboll et al.: Silicone v1.0.0 5266 The follower value returned is then the interpolated value for
the target lead. The “interpolate specified scenarios and mod-
els” cruncher filters for scenarios and models that match a
given text string before performing the same action of the
linear interpolation cruncher. Figure 2. Schematic of how the quantile-rolling-windows cruncher
determines the follower value to use. (a) Example relationships be-
tween lead (CO2) and follower (CH4) variables over time. (b) A
number of rolling window centres (here 5, by default 10) are drawn,
and a weighting function is constructed for each window. It has
a continuous distribution rather than a discrete cutoff, hence the
name. (c) A relationship between the sum of the weights and the
follower value is established, and the follower value at the desired
quantile is returned. Linear interpolation The linear interpolation method constructs a linear (un-
smoothed) interpolator function between all lead and fol-
lower points in the infiller database at a given point in time. It is similar in concept to the cubic spline interpolator used
in Rogelj et al. (2014). The equation for our case is the right-hand side of which we can identify as the two R(t)
values of using Eq. (3) twice for different followers. This
means when the aggregate is the lead and the components
are followers, the sum of the two ratios is 1, so we can use
this infiller to break an aggregate value into components and
know that the total is conserved. This relationship generalises
to any number of components, still holds when emissions can
be negative, and is irrespective of whether the averaging in-
cludes all values or only those for which the lead has a par-
ticular sign. Ef (t) = ef <(t) + (El(t) −el <(t)) ef >(t) −ef <(t)
el >(t) −el <(t)′ ,
(8) (8) where subscript < or > signs indicate the model–scenario
combination with lead values immediately below or above
the target lead value at that time. If multiple points have ex-
actly the same lead value, the average follower value is used. This cruncher is the foundation for the “decompose collec-
tion with time-dependent ratio” multiple infiller. This relies https://doi.org/10.5194/gmd-13-5259-2020 Geosci. Model Dev., 13, 5259–5275, 2020 Geosci. Model Dev., 13, 5259–5275, 2020 Quantile-rolling-windows cruncher The quantile-rolling-windows cruncher, applied with the de-
fault option use_ratio=false, infills the values based on inter-
polating between the required quantile of the follower vari-
able. This is calculated at fixed points across the range of
lead values in the infiller database for each time. The pro-
cess is identical to the above discussion wherein use_ratio is
true, except using the actual follower values instead of the
ratios between lead and follow. It is inspired by the gener-
alised quantile walk approach in Sect. 3.8.1 of Meinshausen
and Dooley (2019). An illustration of the idea behind this
cruncher is shown in Fig. 2. For each time in the infiller
database, it splits the range of lead values into nwindows points
(defaults to 10) with values ep, including the highest and low-
est values. For each window, the weightings of each point are
given as Figure 2. Schematic of how the quantile-rolling-windows cruncher
determines the follower value to use. (a) Example relationships be-
tween lead (CO2) and follower (CH4) variables over time. (b) A
number of rolling window centres (here 5, by default 10) are drawn,
and a weighting function is constructed for each window. It has
a continuous distribution rather than a discrete cutoff, hence the
name. (c) A relationship between the sum of the weights and the
follower value is established, and the follower value at the desired
quantile is returned. wp(el(i)) = 1/
1 +
ep −el(i)
/dl
2
,
(9) (9) where dl is the decay length, which defaults to half the
separation between ep values, and i is the label for which
model–scenario we are investigating. Increasing the decay
length will reduce the weight difference between points, so
the rolling window becomes wider and more even, with the
limit case of calculating quantile q of all data for large dl. Amongst other things, this is a clear improvement over the
generalised quantile walk approach, as the latter uses equal
weights within a fixed window of a certain fraction of the
infiller database’s lead values in a certain year. These values
are then normalised so that Pwp = 1 and sorted into ascend-
ing order by ef . The follower value at quantile q, evaluated
at lead point el(j), is where the quantile equals the sum of
weights of all smaller ef plus half the weight of ef (j) it-
self. Equal quantile walk The equal quantile walk calculates the quantile of the lead
value at each time (Meinshausen et al., 2006). This is zero https://doi.org/10.5194/gmd-13-5259-2020 Geosci. Model Dev., 13, 5259–5275, 2020 5267 2.3
General limitations Note that all of the methods listed above are purely statistical
in nature: if the scenarios in the infiller database are funda-
mentally different from those in the target database, different
relationships are likely and the validity of the results is poor. The adequate use of Silicone requires users to select an in-
filler database most appropriate for each respective applica-
tion. Using Silicone with an infiller database that has itself
been infilled may distort the model democracy of the results. Note also that in version 1.0.0 of Silicone, all methods take
only a single lead value, although forthcoming work will add
the capacity to use multiple lead values to some crunchers. This will improve the ability to resolve more complex rela-
tionships, since it is possible for very different worlds to have
similar emission trends in one emission without being similar
in other emissions. The immediate observation from the study of absolute
rank correlations is that there is no clear, overall best infiller
gas. CH4 has a slightly higher average than other emissions
and is reported by most models. CO2 is reported by all mod-
els and has the second-highest correlation; however, this is
somewhat inflated by having two of its constituents listed
separately (agriculture, forestry, and other land use – AFOLU
– and energy and industrial processes; a similar concern can
be raised about F gases). Generally, CO2 and CH4 are there-
fore the best choices for a default lead variable. However,
there are some specific cases in which the correlations are
low, and much better choices could be made. There is a cluster of emissions species, specifically black
carbon, organic carbon, and carbon monoxide, that correlate
well with each other but less well with other emission path-
ways. Physically, these relate to incomplete burning and are
best infilled using each other. The F gases, SF6, hydroflu-
orocarbons (HFCs), and perfluorinated compounds (PFCs)
also primarily relate to each other. Many models report F-
gas emissions as a basket. Infilling these should best be done
by splitting the F-gas basket into its constituents. Otherwise,
the default infillers, CO2 and CH4, should do reasonably. 3.1
Rank correlations The infilling method is important. However, equally impor-
tant is the choice of lead variable. The best choice is where
there is a causal link between the lead and follower vari-
able, particularly if there is a clear understanding of the im-
plications of this link for the relative behaviour of the two
variables; for instance, black carbon and carbon monoxide
are both produced by incomplete combustion. In most cases,
there is no such certainty, and the best choice is then to
find the lead variable with the best predictive power. We es-
timate this by the Spearman’s rank correlation coefficient,
a measurement of the monotonicity of the relationship be-
tween the two variables. In cases in which this value is low,
we anticipate the need for higher effort to select relevant
cases from the infilling database. We use the data from the
IPCC Special Report on Global Warming of 1.5 ◦C (Hupp-
mann et al., 2018) as our database of scenarios and com-
pare the correlations between the different variables. The Sil-
icone package has a function in the statistics section called
calc_all_emissions_correlations, which will produce tables
of both the Spearman (rank) and Pearson correlation coeffi-
cients, calculated separately for each year requested, and the
time-averaged magnitude of the correlations. Since there is
no reason to expect the relationships between variables to be
linear, we will focus on the rank correlation in this analysis. We also plotted the relationships between CO2 and all other R. D. Lamboll et al.: Silicone v1.0.0 variables (using the plotting function in the Silicone exam-
ples on GitHub) to check that there were no obvious cases of
a non-monotonic relationship. All the crunchers work just as
well with negative trends as with positive, so the sign of the
correlations is not relevant for considering goodness of fit. Using this tool, we can calculate the decadal-averaged mag-
nitude of the rank correlation coefficient, found in Table 3. We also calculate the variation of this value with time, and
in cases in which this exceeds 0.03 (chosen to highlight only
extreme cases) we write the values with asterisks. This is to
indicate cases in which more care needs to be taken to ensure
that values are representative for the times of interest. for values below the database minimum, 1 for those above the
database maximum, and the fraction of infiller data smaller or
equal to this value otherwise. We interpolate between neigh-
bouring values in the infiller data to find the fraction that
would match the target value exactly. We then apply the same
logic to calculate the appropriate value for the derived quan-
tile of the follower data. R. D. Lamboll et al.: Silicone v1.0.0 Table 3. Absolute values of Spearman’s rank correlation between emissions, averaged over the start of decades from 2020 to 2100. We use
the following abbreviations: BC as black carbon; VOCs as volatile organic compounds; AFOLU as agriculture, forestry, and other land use;
and En & IP as energy and industrial processes. “CO2|” represents subtypes of CO2. We also calculate the average of these rows, with or
without the CO2 and subtypes. Cells are bold if the value in them is > 0.7 and have asterisks if the variance of the rank correlation between
years exceeds 0.03. There is no overlap between these categories. Table 3. Absolute values of Spearman’s rank correlation between emissions, averaged over the start of decades from 2020 to 2100. We use
the following abbreviations: BC as black carbon; VOCs as volatile organic compounds; AFOLU as agriculture, forestry, and other land use;
and En & IP as energy and industrial processes. “CO2|” represents subtypes of CO2. We also calculate the average of these rows, with or
without the CO2 and subtypes. Cells are bold if the value in them is > 0.7 and have asterisks if the variance of the rank correlation between
years exceeds 0.03. There is no overlap between these categories. R. D. Lamboll et al.: Silicone v1.0.0 Variable
BC
CH4
CO
CO2
CO2| AFOLU
CO2| En & IP
F gases
HFC
N2O
NH3
NOx
OC
PFC
SF6
Sulf
VOCs
BC
0.47
0.75
0.46
0.37
0.42
0.23
0.10
0.40
0.40
0.58
0.73
0.41
0.20
0.48
0.45∗
CH4
0.32
0.74
0.49
0.73
0.64∗
0.58
0.86
0.34
0.58
0.30
0.66
0.41
0.65
0.24
CO
0.36
0.38
0.32
0.06
0.16∗
0.29
0.35
0.48
0.78
0.05
0.17
0.36
0.68
CO2
0.54
0.96
0.60
0.57
0.54
0.30
0.61
0.24
0.35
0.22∗
0.69
0.37
CO2| AFOLU
0.36
0.27
0.40∗
0.53
0.36
0.33
0.34
0.23∗
0.21∗
0.31
0.20
CO2| En & IP
0.58∗
0.51
0.50
0.25
0.61
0.17
0.32∗
0.18∗
0.69
0.36
F gases
0.91
0.57
0.19
0.50
0.10
0.90
0.77
0.60
0.12
HFC
0.46
0.11
0.30
0.14
0.71
0.68
0.36
0.23
N2O
0.44∗
0.46
0.30
0.65
0.40
0.49
0.17
NH3
0.23
0.39
0.10
0.05
0.23
0.25
NOx
0.22
0.53
0.26
0.76
0.39
OC
0.20
0.11
0.19∗
0.41
PFC
0.77
0.46
0.16
SF6
0.26∗
0.24
Sulfur
0.46∗
VOCs
Average
0.43
0.53
0.37
0.50
0.36
0.46
0.47
0.42
0.47
0.27
0.46
0.31
0.43
0.33
0.47
0.32
Average, no CO2
0.43
0.50
0.37
0.46
0.34
0.43
0.47
0.40
0.46
0.26
0.44
0.32
0.47
0.36
0.44
0.32
No. scenarios
389
412
353
414
412
414
368
108
411
345
363
363
180
191
412
345 the standard deviation in the follower value in the database at
that time (σ), i.e. *s Ef,inf−Ef,act
σ
2
i
+
decade
, with the sub-
i
“i f” i di
i
h
h
l
i i fill d “
” i di the standard deviation in the follower value in the database at
that time (σ), i.e. *s Ef,inf−Ef,act
σ
2
i
+
decade
, with the sub-
i
“i f” i di
i
h
h
l
i i fill d “
” i di Table 4. Root mean squared error in reconstructing known data us-
ing different crunchers, with CO2 as the lead variable, normalised
by the standard deviation at that time. Table 4. Root mean squared error in reconstructing known data us-
ing different crunchers, with CO2 as the lead variable, normalised
by the standard deviation at that time. decade
script text “inf” indicating that the value is infilled, “act” indi- script text “inf” indicating that the value is infilled, “act” indi-
cating actual, and i/decade indicating averaging over model–
scenario cases or decades. 3.2
Reconstructing data The choice of cruncher to use in different situations will
depend on the expectations about the specific emissions in
question. However, in cases in which there are no clear ex-
pectations, it is good to have a default. In this section we
assess to what degree the cruncher reproduces the follower
data from one model and scenario given the lead data from
that case and all data from all the other model–scenario com-
binations in the SR1.5 database. We try this with both CH4
and CO2 as our lead variables. We use the crunchers that
are designed for use on complete datasets with only default
settings: QRW (default settings mean in absolute mode and
for the 0.5 quantile), RMS closest, EQW, time-dependent ra-
tio, and linear interpolation. “Interpolate selected model” be-
haves identically to linear interpolation with default settings
and is not treated separately here. We perform the infilling
for each model–scenario combination for each decade from
2020 to 2100 and report the root mean squared difference be-
tween the original value and the infilled value, normalised by https://doi.org/10.5194/gmd-13-5259-2020 Geosci. Model Dev., 13, 5259–5275, 2020 5268 R. D. Lamboll et al.: Silicone v1.0.0 These results are found in Tables 4
and 5. Given the definition of standard deviations, values
larger than 1 would indicate that this infiller is worse than
simply using the mean value in the database. Time-
dependent
RMS
Linear
Inter-
Species
ratio
QRW
Closest
polation
EQW
BC
1.763
0.734
0.668
1.021
0.921
CH4
0.774
0.460
0.392
0.520
0.500
CO
2.236
0.804
0.764
1.049
1.006
F gases
0.576
0.537
0.485
0.619
0.603
HFC
0.618
0.559
0.512
0.606
0.581
N2O
1.566
0.645
0.535
0.797
0.786
NH3
1.681
0.781
0.676
1.076
1.060
NOx
1.538
0.662
0.606
0.826
0.771
OC
2.062
0.792
0.706
1.069
1.112
PFC
0.649
0.576
0.441
0.600
0.764
SF6
0.754
0.653
0.499
0.762
0.809
Sulfur
0.819
0.570
0.494
0.658
0.637
VOCs
2.223
0.812
0.708
1.056
1.007
Mean
1.328
0.660
0.576
0.820
0.812 p y
g
We see with this fairly large infiller database that for both
CO2 and CH4 the approach that generates follower pathways
most similar to those removed from the initial scenarios (i.e. the smallest errors) is the RMS technique, with the QRW
technique being the next smallest. Linear interpolation with-
out smoothing is expected to produce a noisy fit when given
a large infiller dataset, so its performance is unsurprisingly
worse. The equal quantile walk (EQW) performs similarly
poorly due to effectively ignoring the relationship between
the lead and follower data. The time-dependent ratio method
is worst of all – its errors are potentially unbounded and for
CO2 the average error far exceeds 1. To determine the ap-
propriate statistics to apply on the errors, we first perform a
Shapiro–Wilk test to detect any non-Gaussian aspect for the
error distribution (details can be found in the statistics_for_
paper notebook of the examples on the GitHub repository). This indicated that the distributions are statistically signif-
icantly non-Gaussian for several crunchers when analysed
separately and most clearly as an aggregate. We will there-
fore use non-parametric tests where possible. The small dif-
ferences in rank between CH4 and CO2 manifest in slightly
lower values for CH4. https://doi.org/10.5194/gmd-13-5259-2020 4
Use cases Data in the Silicone examples package rely on the IAMC
pyam open-source software data structure (Gidden and
Huppmann, 2019) and fit into the IAMC scenario assessment
pipeline prepared in support of the IPCC AR6 literature as-
sessment. Time-
Linear
dependent
RMS
Inter-
Species
ratio
QRW
Closest
polation
EQW
BC
1.082
0.729
0.657
0.971
0.875
CO
1.410
0.798
0.642
1.017
1.018
CO2
0.626
0.468
0.448
0.541
0.483
F gases
0.659
0.565
0.506
0.657
0.664
HFC
0.697
0.593
0.471
0.669
0.649
N2O
0.719
0.457
0.364
0.497
0.441
NH3
1.134
0.756
0.533
0.958
1.048
NOx
0.919
0.680
0.625
0.823
0.758
OC
1.318
0.777
0.584
0.972
0.989
PFC
0.592
0.546
0.312
0.550
0.702
SF6
0.703
0.633
0.502
0.768
0.799
Sulfur
0.610
0.580
0.508
0.627
0.644
VOCs
1.398
0.802
0.618
0.972
1.038
Mean
0.913
0.645
0.521
0.771
0.778 As part of the pipeline, emissions projections are also har-
monised, i.e. modified to be consistent with known historical
emissions in a smooth way (Gidden et al., 2018). The Sili-
cone process is assumed to be part of the IAMC pipeline after
harmonisation, as the harmonisation process will potentially
differently affect the target and infiller data, resulting in in-
consistencies. All infiller options except the latest time ratio
are designed such that if both the data being infilled (the “tar-
get data”) and the data drawn on for infilling (“infiller data”)
are harmonised, the result must also be harmonised, so there
is no need for harmonisation again after infilling. (Latest time
ratio only preserves the harmonisation of the last time point
in the infiller database.) The infilled results can then be run
via climate models, most easily via the OpenSCM package
(Nicholls et al., 2020). We now demonstrate several uses of the package for spe-
cific purposes. The notebooks demonstrating the steps for
these calculations can be found in the Silicone_examples
GitHub repository (Lamboll, 2020), along with several other
use cases. ing one infiller species, with CH4 performing slightly better
while also generally having a slightly lower availability of
data. We perform similar pairwise Wilcoxon t tests on the re-
sults of different crunchers and find that the ordering of mean
errors (RMS closest < QRW < linear interpolation ≈EQW
< time-dependent ratio) is statistically robust. 4
Use cases The p values
are < 0.01 for almost all pairs except linear interpolation and
EQW, which are much greater than 0.1 whether the compari-
son uses CO2 lead data, CH4 lead data, or all data combined. The one pairwise exception to this is time-dependent ratio
and EQW for CH4, which has only p = 0.028, though the
values for other combinations still have p < 0.01. R. D. Lamboll et al.: Silicone v1.0.0 Table 5. Root mean squared error in reconstructing known data us-
ing different crunchers, with CH4 as the lead variable, normalised
by the standard deviation at that time. Table 5. Root mean squared error in reconstructing known data us-
ing different crunchers, with CH4 as the lead variable, normalised
by the standard deviation at that time. R. D. Lamboll et al.: Silicone v1.0.0 Performing a Wilcoxon’s t test on the results indicates that this result is statistically signifi-
cant for the data as a whole (relative t-test statistic 376, p =
0.00007), although when considering each of the crunchers
individually, only the RMS-closest and time-dependent ra-
tio crunchers are significantly better with CH4 than CO2 (p
values for time-dependent ratio = 0.012, QRW = 0.48, RMS
closest = 0.041, linear interpolation = 0.060, EQW = 0.39). We therefore conclude that using either CO2 or CH4 as the
default will produce the most reasonable results when us- the results indicates that this result is statistically signifi-
cant for the data as a whole (relative t-test statistic 376, p =
0.00007), although when considering each of the crunchers
individually, only the RMS-closest and time-dependent ra-
tio crunchers are significantly better with CH4 than CO2 (p
values for time-dependent ratio = 0.012, QRW = 0.48, RMS
closest = 0.041, linear interpolation = 0.060, EQW = 0.39). We therefore conclude that using either CO2 or CH4 as the
default will produce the most reasonable results when us- Geosci. Model Dev., 13, 5259–5275, 2020 https://doi.org/10.5194/gmd-13-5259-2020 5269 Geosci. Model Dev., 13, 5259–5275, 2020 R. D. Lamboll et al.: Silicone v1.0.0 5270 Figure 3. (a) The POEM scenario B projection for CO2 from energy and industrial applications data. The fine lines represent the different
time series in the SR1.5 database used to perform the infilling and are not included in the legend for clarity. (b) The results of interpolating
these data using five different crunchers. The “interpolate specified model” approach used the MESSAGE model and only chose scenarios
based on SSP2. Figure 3. (a) The POEM scenario B projection for CO2 from energy and industrial applications data. The fine lines represent the different
time series in the SR1.5 database used to perform the infilling and are not included in the legend for clarity. (b) The results of interpolating
these data using five different crunchers. The “interpolate specified model” approach used the MESSAGE model and only chose scenarios
based on SSP2. infillers designed for this use is preferable. The symmetric
way to divide the basket into its constituent parts (CO2, CH4,
N2O, and F gases) is using the “decompose collection with
time-dependent ratio” multiple infiller, which uses a ratio-
based technique to ensure conservation of the total amounts. Alternatively, the “split collection with remainder” multiple
infiller can estimate the fractions of CH4, N2O, and F gases,
then assign the remainder to CO2. F gases could be further
subdivided using similar methods. strong, direct relationship between CO2 and CH4 emissions,
which the other crunchers do not uphold at early times, al-
though this would disappear if the data were harmonised. The other cruncher results are all fairly similar and look con-
sistent. The RMS-closest pathway is consistent by construc-
tion (and precisely overlines a point in the original database). The quantile-rolling-windows result also looks consistent
and tends to move closer to dense clouds of values in the
infiller database. In deciding which is the best infiller to use,
the RMS-closest result is more consistent over time but more
arbitrary in its selection of the pathway, while quantile rolling
windows is more conservative in the sense of giving results
closer to the median behaviour of the whole dataset. As can be seen in Fig. 5, the curves that result from “de-
compose collection” are generally smooth, in spite of being
separately calculated at each time point. It is important to
ensure that the number of scenarios reported at each time are
consistent. R. D. Lamboll et al.: Silicone v1.0.0 In the SR1.5 database, some scenarios only report
values at decadal intervals, whereas others use 5-year inter-
vals. We interpolated all models to 5-year intervals to give
consistent representation. In the CH4 and F gases, the low-
est orange line is clearly seen to rise discontinuously after
2060. This is the last point before the Kyoto total goes nega-
tive. To ensure that the sign of the constituents is correct, the
formula only considers data from SR1.5 paths for which the
Kyoto total has the same sign as in the data being infilled. In
this way, emissions that are unlikely to go negative like CH4
are ensured positive; however, their magnitude increases the
more negative the aggregate is. 4.1
Infilling the IMAGE model POEM scenario B To demonstrate the uses of this package alone, we will ap-
ply the methods directly using unharmonised data in the
SR1.5 repository (Huppmann et al., 2019) to infill the emis-
sion pathways of the POEM scenario B from the AR5
database (Clarke et al., 2014). The POEM scenarios only
report CO2 from certain sources and are thus an excellent
use case. The crunchers are all used via the multiple infiller,
infill_all_required_emissions_for_openscm. No active deci-
sions are taken except to use the SSP2 scenarios from the
MESSAGE model for the specified model interpolation. The
choice of SSP2 in this case is ultimately arbitrary but sup-
ported by POEM scenario B in being fairly middle of the
road and usually fitting in the SSP2 range. The choice of
MESSAGE model is because this is the marker model for
SSP2 (Riahi et al., 2017). Other POEM scenarios would need
different ranges of scenarios for infilling. We stress that this does not always mean that the RMS-
closest technique is the best default, as it makes the assump-
tion that the pathway being infilled is similar to a whole path-
way found in the database. The advantage of the quantile-
rolling-windows technique is its choice of conservativity –
for example, it tends to produce values more towards the me-
dian value if the default 0.5 quantile is used – and time in-
dependence, whereas RMS closest is better at reconstructing
the data and has better consistency over time. Linear interpo-
lation, EQW, and the time-dependent ratio are best used in
cases in which there is a large degree of knowledge about the
expected relationship between variables. We see from Fig. 3 that the linear interpolation model
(without filtering the database) provides a chaotic pathway
due to its value being determined only by the two points ei-
ther side of it in the database, which changes semi-randomly
with time and should not be used here. Although the “in-
terpolate specified model” approach is also determined by
only a few model–scenario pairs because there are only data
from a small number of related scenarios, the pathway is
smoother and more consistent. The EQW pathway assumes a https://doi.org/10.5194/gmd-13-5259-2020 Geosci. Model Dev., 13, 5259–5275, 2020 4.2
Splitting up a Kyoto greenhouse gas path The Silicone package has features that can split a basket
of gases into its constituents. In this example we take data
from the Climate Action Tracker (CAT) website (https://
climateactiontracker.org/, last access: 9 July 2020; Climate
Action Tracker, 2020), which reports projected global emis-
sions in terms of Kyoto gas totals, shown in Fig. 4. While
it is possible to use this to infill all other values directly as
above, the subcategories of Kyoto gas will not necessarily
add up to the Kyoto gas total. Therefore, one of the multiple Geosci. Model Dev., 13, 5259–5275, 2020 https://doi.org/10.5194/gmd-13-5259-2020 R. D. Lamboll et al.: Silicone v1.0.0 R. D. Lamboll et al.: Silicone v1.0.0 5271 Figure 4. The Climate Action Tracker (CAT) Kyoto gas totals (thick lines) compared with the portfolio of values in the SR1.5 database (thin
lines). Figure 4. The Climate Action Tracker (CAT) Kyoto gas totals (thick lines) compared with the portfolio of values in the SR1.5 database (thin
lines). Figure 4. The Climate Action Tracker (CAT) Kyoto gas totals (thick lines) compared with the portfolio of values in the SR1.5 database (thin
lines). Figure 4. The Climate Action Tracker (CAT) Kyoto gas totals (thick lines) compared with the portfolio of val
lines). Figure 5. The CAT Kyoto gas baskets decomposed into their components using the decompose collection multiple infiller. Figure 5. The CAT Kyoto gas baskets decomposed into their components using the decompose collection multiple infiller. https://doi.org/10.5194/gmd-13-5259-2020 Geosci. Model Dev., 13, 5259–5275, 2020 https://doi.org/10.5194/gmd-13-5259-2020 5272 R. D. Lamboll et al.: Silicone v1.0.0 Figure 6. Kyoto gases decomposed by first infilling the non-negative emissions using the (non-ratio) quantile rolling windows, then infilling
the CO2 using infill composite values. Figure 6. Kyoto gases decomposed by first infilling the non-negative emissions using the (non-ratio) quantile rolling windows, then infilling
the CO2 using infill composite values. Figure 6. Kyoto gases decomposed by first infilling the non-negative emissions using the (non-ratio) quantile
the CO2 using infill composite values. mposed by first infilling the non-negative emissions using the (non-ratio) quantile rolling windows, then infillin
ite values. For this reason, the “split collection with remainder”
method produces more robust results with sign changes in the
lead variable. This technique can use any cruncher, usually
RMS closest or (probably non-ratio) quantile rolling win-
dows, to infill the positive values and then allow the value
that may be negative (CO2) to make up the rest. This pro-
duces the results seen in Fig. 6. Here the behaviour of all
curves is fairly smooth, with no obvious features around
zero-crossing points and no negative values except in CO2,
as expected. tailing the calculations and demonstrating this usage, titled
Infill_stylised_path.ipynb (Lamboll, 2020), using data from
Riahi et al. (2011) and van Vuuren et al. (2011). It shows
that curves with different values in some of the parameters,
termed E∞and τ, can be complemented using a number of
techniques. Here we highlight the method of interpolating re-
sults from any of the SSP scenarios as implemented by vari-
ants of the MESSAGE model. 4.3
Stylised trajectories Another use of this software is to infill simple, stylised tra-
jectories generated to explore a wide range of possibilities
without detailed economic modelling. For example, Sander-
son et al. (2016) suggest simple formulae whereby one may
construct emissions trajectories characterised by a few free
variables – in this case, based on rates of transition between
the RCPs and a long-term emissions value. They present gen-
eral formulae for generating plausible total CO2 pathways
with several free variables. Silicone provides an alternative
means of complementing such results – instead of specify-
ing the functional forms of all emissions, you can have a
few key emissions prescribed and infill the remainder using
scenarios with similarities to the desired narrative. A note-
book can be found in the Silicone examples on GitHub de- R. D. Lamboll et al.: Silicone v1.0.0 As the different SSPs have dif-
ferent narratives, this allows the user to decide what narrative
is relevant to the infilling, rather than adding more arbitrary
values (Gidden et al., 2019). An example of this output can
be found in Fig. 7. R. D. Lamboll et al.: Silicone v1.0.0 R. D. Lamboll et al.: Silicone v1.0.0 R. D. Lamboll et al.: Silicone v1.0.0
527
Figure 7. Illustration of using the “interpolate specified scenario” cruncher to infill a series of stylised trajectories (solid lines) characteris
by two different parameters (τ and E∞), as defined in (Sanderson et al., 2016). The first column compares the total CO2 calculated for t
tylised trajectories to the values of the MESSAGE model for a given group of SSP scenarios (dotted lines). These are our lead values
ach case. The second column shows the range of follower values for that SSP. The third column shows the resultant AFOLU (agricultur
orestry, and other land use) trajectories that emerge from using the “interpolate specified scenario” infiller. Figure 7. Illustration of using the “interpolate specified scenario” cruncher to infill a series of stylised trajectories (solid lines) characterised
by two different parameters (τ and E∞), as defined in (Sanderson et al., 2016). The first column compares the total CO2 calculated for the
stylised trajectories to the values of the MESSAGE model for a given group of SSP scenarios (dotted lines). These are our lead values in
each case. The second column shows the range of follower values for that SSP. The third column shows the resultant AFOLU (agriculture,
forestry, and other land use) trajectories that emerge from using the “interpolate specified scenario” infiller. 2 November 2020), which also contains full documentation. In addition, a flow chart to guide the choice of cruncher
for a given situation is included in the text. The results of
Spearman’s rank correlations and applying the crunchers to
the SR1.5 database implied that the best default lead vari- ables are CH4 and CO2 and that the best default cruncher
is the root-mean-squared-closest cruncher, followed by the
quantile-rolling-windows cruncher. Both of these crunchers
perform significantly better at reconstructing known path-
ways compared to the commonly used equal quantile walk https://doi.org/10.5194/gmd-13-5259-2020 5
Summary In this paper we have outlined the features of the open-source
Silicone package. This provides tools for complementing
emissions pathways with other climate-relevant emissions
through relationships found in the scenario literature. The
package features several scripts for analysing data to es-
tablish the relationships between the variables in the com-
plete infiller database to establish the best variables to use
when infilling. The values of the follower data are estimated
using objects called crunchers. Notebooks describing the
use of the crunchers are included in a GitHub repository
(https://github.com/GranthamImperial/silicone, last access: https://doi.org/10.5194/gmd-13-5259-2020 Geosci. Model Dev., 13, 5259–5275, 2020 5273 Competing interests. The authors declare that they have no conflict
of interest. Competing interests. The authors declare that they have no conflict
of interest. Huppmann, D., Kriegler, E., Krey, V., Riahi, K., Rogelj, J., Calvin,
K., Humpenoeder, F., Popp, A., Rose, S. K., Weyant, J., Bauer,
N., Bertram, C., Bosetti, V., Doelman, J., Drouet, L., Emmer-
ling, J., Frank, S., Fujimori, S., Gernaat, D., Grubler, A., Guiv-
arch, C., Haigh, M., Holz, C., Iyer, G., Kato, E., Keramidas,
K., Kitous, A., Leblanc, F., Liu, J.-Y., Löffler, K., Luderer, G.,
Marcucci, A., McCollum, D., Mima, S., Sands, R. D., Sano,
F., Strefler, J., Tsutsui, J., Van Vuuren, D., Vrontisi, Z., Wise,
M., and Zhang, R.: IAMC 1.5 ◦C Scenario Explorer and Data,
IIASA, Integr. Assess. Model. Consort. Int. Inst. Appl. Syst. Anal., https://doi.org/10.5281/zenodo.3363345, 2019. Acknowledgements. We thank Nicholai Meinshausen for useful
statistics discussions. F., Strefler, J., Tsutsui, J., Van Vuuren, D., Vrontisi, Z., Wise, Financial support. This project has received funding from the Eu-
ropean Union’s Horizon 2020 research and innovation programme
under grant agreement no. 820829 (CONSTRAIN). Lamboll, R. D.: GranthamImperial/silicone_examples (Version
v1.0.0).,
Zenodo,
https://doi.org/10.5281/zenodo.4020372,
2020. Review statement. This paper was edited by Rolf Sander and re-
viewed by two anonymous referees. Lamboll, R. D., Nicholls, Z., and Kikstra, J.: Silicone documenta-
tion, available at: https://silicone.readthedocs.io/en/latest/index. html, last access: 11 May 2020a. Lamboll, R. D., Nicholls, Z., and Kikstra, J.: Silicone (Ver-
sion v1.0.0), Zenodo, https://doi.org/10.5281/zenodo.3822259,
2020b. https://doi.org/10.5194/gmd-13-5259-2020 Geosci. Model Dev., 13, 5259–5275, 2020 R. D. Lamboll et al.: Silicone v1.0.0 5274 Climate, Human, and Environmental Systems, J. Open Source
Softw., 4, 1095, https://doi.org/10.21105/joss.01095, 2019. technique, although this and many other crunchers are in-
cluded in the package for specific situations in which they
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Outcomes of wildlife translocations in protected areas: what is the type and extent of existing evidence? A systematic map protocol
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Environmental evidence
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To cite this version: Joseph Langridge, Romain Sordello, Yorick Reyjol. Outcomes of wildlife translocations in protected
areas: what is the type and extent of existing evidence? A systematic map protocol. Environmental
Evidence, 2020, 9 (1), pp.16. 10.1186/s13750-020-00199-4. hal-03988150 Distributed under a Creative Commons Attribution 4.0 International License SYSTEMATIC MAP PROTOCOL
Open Access Open Access Abstract Background: Conversion, fragmentation, and loss of natural habitats are among the main causes of declining spe-
cies’ populations worldwide. Protected areas are therefore crucial for biodiversity as they provide refuge and ensure
key ecological processes. Wildlife translocations, defined as “the deliberate movement of organisms from one site for
release in another”, have been used in conjunction as a conservation tool for a number of decades as wild popula-
tions become increasingly fragmented and endangered. Not only are translocations used to bolster the viability of
imperiled species but are also recommended for improving population resilience and adapting species’ ranges in
response to climate change. Despite translocation being a recognised conservation tool, it remains complex with
variable results due to the different factors that can determine its success. Accordingly, the Map will investigate the
existing evidence on the links between different types of wildlife translocation interventions and factors that may be
important to consider for planning. This will provide an overview of relevant studies for possible future syntheses, and
may help to inform management decisions. Method: We will perform a thorough search of peer-reviewed journal articles and grey literature sources document-
ing the occurrence of translocations in the context of protected areas. Two databases will be used: Web of science
core collection and Scopus, with a supplementary search in Google Scholar. Multiple key specialized websites will also
be used. All bibliographic data will be extracted, managed, and screened in Microsoft excel. Three screening stages
will be undertaken (title, then abstract, then full texts) against predefined inclusion criteria. The retained relevant
literature will be subjected to coding and meta-data extraction. No formal validity appraisal will be undertaken. The
Map will particularly highlight translocation operations in terms of origin and destination (i.e. translocating from one
protected area to another, within the same area, and from and to non-protected areas) by taxonomic group, among
other important factors (e.g. number of individuals, age class, release strategy, distance between capture and release
sites etc.). Finally, a database will be provided along with a Map narratively describing the evidence with summary
figures and tables of pertinent study characteristics. Keywords: Managed relocations, Reintroduction, Supplementation, Introduction, Conservation areas © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material
in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material
is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the
permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco
mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/
zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Outcomes of wildlife translocations
in protected areas: what is the type and extent
of existing evidence? A systematic map protocol Joseph Langridge* , Romain Sordello and Yorick Reyjol Backgroundi Modification, fragmentation, and loss of natural habitats
are among the main causes of declining species’ popula-
tions worldwide [1–4]. In the face of such threats, extinc-
tion rates have been accelerating and biological diversity
diminishing for the last several decades [5–7]. Accord-
ingly, protected areas such as national parks, nature and *Correspondence: joseph.langridge1@mnhn.fr
UMS Patrimoine Naturel (PatriNat), OFB-CNRS-MNHN, 36 Rue
Geoffroy‑Saint‑Hilaire CP41, 75005 Paris, France *Correspondence: joseph.langridge1@mnhn.fr
UMS Patrimoine Naturel (PatriNat), OFB-CNRS-MNHN, 36 Rue
Geoffroy‑Saint‑Hilaire CP41, 75005 Paris, France HAL Id: hal-03988150
https://hal.science/hal-03988150v1
Submitted on 26 Jul 2023 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Langridge et al. Environ Evid (2020) 9:16
https://doi.org/10.1186/s13750-020-00199-4 Environmental Evidence © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material
in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material
is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the
permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco
mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/
zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Langridge et al. Environ Evid (2020) 9:16 Page 2 of 11 Page 2 of 11 Langridge et al. Environ Evid (2020) 9:16 conjunction with conservation areas more regularly as
populations become progressively more fragmented
and endangered [21]. Even though past efforts have
not been entirely uniform with a notably marked tax-
onomic bias towards birds and mammals (e.g. [5, 22])
and an apparent prioritization for larger more charis-
matic species [15, 22, 23], attention being paid to other
groups has rapidly increased since the early 2000s (e.g. English Nature’s Species Recovery Programme involv-
ing 62 species, of which only 11 were birds or mam-
mals) [22]. Thus, for management purposes, the need to
synthesize this profuse information is apparent. More-
over, concerning success, studies regularly identify the
value of habitat quality at recipient sites and the impor-
tance of species being relocated to non-degraded habi-
tats [5, 20, 24, 25]. Indeed, in a previous review on plant
reintroductions, Godefroid et al. [26] confirmed that
reintroducing species to protected areas significantly
increased survival rate. Equally, regarding vertebrate
translocations, several papers highlight the positive
effects of protected habitat (e.g. [27]), and in a number
of canid translocations protected areas were regularly
chosen as release sites [28–30]. With the overarching
pressure of climate change, several authors have con-
tinued to propose translocations as a viable means to
enhance the resilience of threatened species, improve
ecosystem integrity, and assist migration to favourable
habitats [9, 14, 24, 31, 32]. biosphere reserves play vital roles in maintaining refuge
and ensuring fundamental ecological mechanisms such
as dispersal and gene exchange [5, 8–11]. Further, not
only do they provide key habitat and conserve biodiver-
sity from various human pressures [8, 10] but they are
able to maintain higher species population levels, includ-
ing threatened species, better than other management
approaches [10, 11]. biosphere reserves play vital roles in maintaining refuge
and ensuring fundamental ecological mechanisms such
as dispersal and gene exchange [5, 8–11]. Further, not
only do they provide key habitat and conserve biodiver-
sity from various human pressures [8, 10] but they are
able to maintain higher species population levels, includ-
ing threatened species, better than other management
approaches [10, 11]. Translocation is an umbrella term referring to the
“deliberate movement of organisms from one site for
release in another” [12]. Indeed, it may occur in different
contexts such as reintroduction in which organisms are
transported and released into their historical native range
but from which they have become extirpated or extinct,
or supplementation (also known as reinforcement),
which refers to the addition of individuals to an existing
population of conspecifics [12]. Thirdly, introduction,
which from a conservation perspective is often referred
to as assisted colonization [12–14], attempts to establish
a species outside of its recorded historical distribution
but within appropriate habitat and biogeographical area. Each strategy sharing the ultimate goal of population per-
sistence [14]. Historically, the intentional movement and release of
species has occurred for millennia [12, 15], but the use
of translocations to address species-focused conserva-
tion objectives is more recent [15]. For example, between
1973 and 1989 more than 700 translocations were esti-
mated to have taken place per year across the USA, Aus-
tralia, and New Zealand in order to restore and enhance
populations [5]. Latterly, interest in assisted colonization
has been driven by predicted habitat and climate changes
[13, 15]. In 1985, Peters and Darling [16] suggested that
climate change might alter habitat suitability for species
confined within protected areas, effectively stranding
them as habitat becomes increasingly unfavourable [15]. They proposed the translocation of individuals into new
reserves encompassing habitat that was or would become
appropriate [16], therefore potentially compensating for
the fixed nature of perimeters [17, 18]. More recently,
the concept of rewilding has emerged [15, 19]. Origi-
nally based on the keystone role played by wide-ranging
predators and their ability to maintain ecosystem equi-
librium through top-down trophic interactions [15], the
concept has since harmonized with the current conserva-
tion translocation framework to include the role of spe-
cies reintroduction to restore ecological processes [15,
19], and to a broader extent, the restoration of ecosystem
functions by means of introducing ecological replace-
ments [15]. Despite the number of translocations rapidly growing
and it increasingly being recognized as a key conserva-
tion measure, implementation is often complex and
different programs have had varying results. From a bio-
logical perspective, this is notably due to the numerous
different factors that influence its success [33, 34] such as
the number of translocated individuals [35], the distances
involved [36], whether acclimatisation strategies (e.g. protective enclosures or supplemental feeding) are used
[37], and what levels of habitat quality individuals are
faced with at release sites [25, 26]. From a social perspec-
tive, interventions are still considered controversial: cost,
feasibility, and political acceptability remain the principal
influencing factors [38]. l
Although previous overviews exist (e.g. [23, 34]), and
while others have explored the effectiveness of anti-pred-
ator training and conditioning interventions (e.g. [37]),
there appears to be a deficit in terms of systematic litera-
ture assessments on the role of protected areas. Hence,
our aim is to map evidence of translocation operations
carried out in the context of protected areas detailing the
distribution and abundance of relevant studies in relation
to key factors that influence success. This will provide
an evidence base for possible future reviews, and should In the current context of the biodiversity crisis, trans-
locations and particularly reintroductions of threatened
species are more numerous [20]. They are also used in Page 3 of 11 Langridge et al. Environ Evid (2020) 9:16 Page 3 of 11 help to inform eventual management and stakeholder
decisions. motive is to restore natural ecosystem functions will be
included. In accordance with Seddon et al. [15], translo-
cation rewilding will only entail (i) population restoration
through reintroduction, where release within the indig-
enous range aims at reestablishing some ecological func-
tion, or (ii) in the form of a conservation introduction
through ecological replacement [15, 19]. Neither invasive
species nor historical introductions for hunting purposes
will be included. We will aim to provide a comprehen-
sive overview of the distribution of studies by taxonomic
group and type of translocation, in conjunction with
other key drivers (e.g. age class, release strategy, distance
between capture and release sites, number of individu-
als initially translocated etc.) that may influence various
biological outcomes i.e. success of wildlife translocation
operations.h Search strings
Fi
l Firstly, a scoping exercise was conducted in the Web of
Science Core Collection database to explore the effi-
ciency of chosen words and the number of articles
returned. In accordance with our main objective, we
combined all search terms relating to protected areas and
wildlife translocations. Concerning protected areas, the
chosen key words represent synonyms of the different
types of reserves and management categories that exist.hfi Searching for articles Our search strategy has been designed in order to
retrieve a broad range of articles covering the topic of
wildlife translocations in protected areas. Indeed, the
systematic map will follow the Environmental Evidence
Guidelines and conforms to the ROSES standards (see
Additional file 1 for our declaration and checklist of
adherence to the ROSES guidelines). Search terms and languages All searches will be performed using English terms only. Hence, all relevant studies published in English will be
included in this systematic map. This will include diverse
bibliographic documents (e.g. books conference proceed-
ings, journal articles, theses, technical reports etc.) Stakeholder engagementh The current systematic map will be conducted as part
of a wider European LIFE programme (the EU’s fund-
ing instrument for environment and climate action). The
LIFE project, entitled “Natur’Adapt”, is coordinated by the
French Nature Reserves Network (Les Réserves Naturelles
de France (RNF)), and co-financed by the French Min-
istry of Ecology and the French Office of Biodiversity
(OFB). RNF is accompanied by nine other beneficiaries,
including The French National Natural History Museum
(Muséum National d’Histoire Naturelle (MNHN)), who
will be responsible for the mapping process.h The project’s principal aim is to align conservation
efforts in protected areas to the challenges associated
with climate change, in France and across Europe. Sub-
sequently, the progressive development of an adaptation
plan will be undertaken based, firstly, on six “experimen-
tal” nature reserves then progressively made adaptable
to all protected areas in France and Europe. The MNHN
is responsible for a key LIFE action: to provide evidence
syntheses. This will help reserve managers build their
adaptation plan by transferring scientific knowledge to
them in an accessible and summarized form. As a first
step, several working groups were conducted between
RNF, MNHN, and reserve managers. This was an oppor-
tunity for reserve managers to define all relevant con-
servation strategies, in the context of climate change,
of which they were most in need of scientific evidence
to support decision-making. At the end of this process,
translocation, among other measures, was retained as it
was considered a necessary conservation action plan. As
a result of numerous discussions, a systematic map was
chosen as a central reference tool. Further workshops
were held to specifically learn the stakeholders’ needs
and involve them in the definition of the Map’s meta-data
variables. Therefore, the primary question for this Map protocol
is as follows: What type, extent, and distribution of evi-
dence exists on the outcomes of wildlife translocations
carried out in protected areas? Components of the primary question in Table 1. Objectives of the reviewh The main objective is to systematically map transloca-
tion operations within the context of protected areas
(i.e. operations from, to or within a protected area). The IUCN protected area management categories will
be used for this as they represent a global standard for
defining conservation areas. In agreement with the spe-
cific aims of the LIFE project, this Map will consider
translocations for species conservation—where the pri-
mary goal is to improve the status of the focal species
through supplementation, reintroduction, or assisted
migration. Translocation for rewilding—where the initial Thus, the search string that produced the highest effi-
ciency is presented below (see Additional file 2 for test
list details and Additional file 3 for information of the
building process of the search string). Page 4 of 11 Langridge et al. Objectives of the reviewh Environ Evid (2020) 9:16 p
y
p
a Concerning context, this will equate to all interventions from, to, or within these types of protected areas
Population (P)
All plant and animal species of wild or captive source
Intervention (I)
Type of interventions
Definition of interventions
Conservation aim of intervention
Introduction
This refers to the intentional manual
transfer/movement and release of an
organism outside of its indigenous range/
historical distribution [12]
(i) Assisted migration: this refers to the inten-
tional manual transfer/movement and
release outside of the indigenous range,
to primarily avoid extinction of popula-
tions of the focal species [12]
This occurs if the persistence of a species in
its indigenous range is threatened from
current or future impacts than at alterna-
tive sites [12]
(ii) Ecological replacement:
This refers to the intentional manual
transfer/movement and release of an
organism outside its indigenous range/
historical distribution, to perform a spe-
cific ecological function [12]
This is used to re-establish an ecological
function lost through extinction; involving
the most suitable existing sub-species, or a
close relative of the extinct species [12]
Reintroduction
This refers to the intentional manual transfer/movement and release of an organism
inside its indigenous range/historical distribution but from which it has disappeared or
become extinct locally, regionally, or otherwise (No conspecifics are present in situ) [12]
The conservation aim is to re-establish a
viable population of the focal species
within its historical range [12]
Supplementation
This refers to the intentional manual transfer/movement and release of an organism into
the existing distribution of a population of conspecifics [12]
The aim is to enhance and reinforce popula-
tion viability e.g. by increasing population
size, or by increasing genetic diversity [12]
Comparator (C)
Studies will not be required stricto sensu to have a comparator. Although in certain cases the study design may translate as a time series comparison (before and after translocation)
Outcomes (O)
All relevant outcomes related to the translocated population, including space use, demography, survival, reproduction, feeding, behaviour, genetics, and physiology (cf. Objectives of the reviewh Table 3 for
full typology)
Context (C)a
Type of protected areas
Definitions of protected areas
Strict reserves for the protection of nature
(Ia)
Areas set aside to strictly protect biodiversity where human visitation, use, and impacts are strongly limited [39]
Wilderness areas (Ib)
Areas that are largely unmodified, retaining their natural character, and free of inappropriate or excessive human use or presence [39]
National Parks (II)
Protected areas of large natural or near natural areas set aside to protect large-scale ecological processes [39]. Natural monuments (III)
Protected areas set aside to protect a specific natural feature in the landscape [39]. Management areas (IV)
Specific protected areas that aim to safeguard a particular species or habitat. Consequently, the management reflects this priority [39]
Protected landscapes (V)
A protected area where humans and nature together over time have produced an area of significant ecological, biological, cultural and
scenic value [39]
Protected areas with sustainable use of
natural resources (VI)
Protected areas which conserve ecosystems and habitats together with associated cultural values and traditional natural resource
management and use [39] Langridge et al. Environ Evid (2020) 9:16 Langridge et al. Environ Evid (2020) 9:16 Page 5 of 11 • Google Scholar (https://scholar.google.com/). We
used the same key words in the software programme
Publish or perish (version 6) to retrieve all academic
citations. The software’s use of Boolean characters
differs from WOS and Scopus. As a result, the search
string was broken down into eight separate searches,
in order to achieve a similar comprehensiveness, as
only a single term can be included in the field “all of
the words”. Consequently, each sub-search was lim-
ited to the first 200 search hits, in line with recom-
mendations [40]. (Refer to Additional file 5.) • Google Scholar (https://scholar.google.com/). We
used the same key words in the software programme
Publish or perish (version 6) to retrieve all academic
citations. The software’s use of Boolean characters
differs from WOS and Scopus. As a result, the search
string was broken down into eight separate searches,
in order to achieve a similar comprehensiveness, as
only a single term can be included in the field “all of
the words”. Consequently, each sub-search was lim-
ited to the first 200 search hits, in line with recom-
mendations [40]. Specialist searchesh The following specialist organisations will be searched
for reports which contain translocations to, from and
within protected areas. • Scopus. We will equally search for all published doc-
uments. We will use the field tag “TITLE-ABS-KEY”,
which operates in the same way as the “TS” tag in
WOS. • US Federal Science database (https://www.scien
ce.gov/). • US Fish and wildlife service (https://www.fws.gov/).fi • Office National de la Chasse et de la Faune Sauvage
(http://www.oncfs.gouv.fr/). These databases were chosen as they provide compre-
hensive citation data for numerous different academic
disciplines. The English search string detailed above will
be used for both literature sources. The search string will
be adapted as necessary to account for the differences in
the use of field tags and Boolean characters [an additional
file provides details on number of search hits and dates of
searches (see Additional file 5)]. • IUCN Conservation Planning Specialist Group’s
document library (http://www.cpsg.org/document-
repository). • Association of Zoos & Aquariums (https://www. aza.org/). Including the European Association
(https://www.eaza.net/). • Rewilding Europe (https://rewildingeurope.com/). Internet searches to be conducted A supplementary retrieval of publications will be under-
taken using web-based search engines. Publication databases to be searched All published material will be collected from the follow-
ing databases (and managed in excel). • Web of Science (WOS) core collection. The entire
database i.e. all citation indexes will be searched by
Topic i.e. using the “TS” field tag, which searches for
key words in the title, abstract and key-words of pub-
lished documents (see Additional file 4 for Web of
Science subscription details). Estimating the comprehensiveness of the searchi A test list of 40 scientific articles was established and
used to assess the comprehensiveness of the search
string. The test list was composed of relevant scientific
articles identified by the review team prior to the map-
ping process. The overall comprehensiveness was 100%. [Two additional files provide further details (see Addi-
tional files 2 and 3)]. • Conservation
Evidence
(https://www.conse
rvationevidence.com/)—we will collect primary
research using the Journal’s “Advanced search”. Use
of five key words: “reintroduction”, “reinforcement”,
“introduction”, “translocation”, or “rewilding” will
be used for collecting individual studies. We will
extract the first 40 hits per keyword search (total
hits: 200). Objectives of the reviewh (Refer to Additional file 5.) TS = (“protected area$” OR “protected landscape$”
OR “protected site$” OR “receptor site$” OR “reintro-
duction site$” OR “natur* reserve$” OR “national park$”
OR “regional park$” OR “national reserve$” OR “bio-
logical reserve$” OR “biosphere reserve$” OR “regional
reserve$” OR “wilderness area$” OR “natural monu-
ment$” OR “management area$” OR sanctuar*) AND
TS = (“assisted colonization” OR “assisted population
migration” OR “assisted migration” OR “assisted gene
flow” OR “managed relocation$” OR transloc* OR rein-
troduc* OR reinforc* OR “assisted range expansion$” OR
“assisted long-distance migration$” OR rewilding OR
“wild release”). i
• A retrieval of theses will also be done using UK
Theses and Dissertations (https://ethos.bl.uk). We
will search for theses using the intervention key
words only. We will search using five key words:
“reintroduction” OR “reinforcement” OR “introduc-
tion” OR “translocation” OR “rewilding”. Hits lim-
ited to 200. Title Inclusion criteria Firstly, all titles will be retained if pres-
ence of the terms reintroduction, supplementation (and
its common synonyms i.e. reinforcement, augmenta-
tion, re-stocking, enhancement) and introduction (and
its common synonyms i.e. assisted migration, managed
relocation etc.). Secondly, any title containing compat-
ible synonyms such as, re-wilding, release, range-shifts,
transfer, restoration etc., will also be retained. In cases
where none of the above words are present, a title would
still meet eligibility if it strongly implies a translocation
event (i.e. reference to captive of wild stock) or meta-
population management. Nb. At title screening stage, all
types of literature (including review, meta-analyses and
relevant discussion and opinion articles) will be retained. Supplementary searches A call for literature will be made through the pro-
fessional networks of Les Réserves Naturelles de
France (RNF) and EuroParc. An advert will be Langridge et al. Environ Evid (2020) 9:16 Langridge et al. Environ Evid (2020) 9:16 Page 6 of 11 Page 6 of 11 Article screening and study eligibility criteria
Screening processi In accordance with the pre-defined screening and study
eligibility criteria (detailed in “Eligibility criteria” section),
study selection will follow a three-stage filtering process
carried out by two members of the mapping team. Firstly,
all titles will be screened, followed by abstracts and
thirdly full texts. During screening, we will choose to take
a conservative approach. Hence, if the qualifying infor-
mation is not detailed sufficiently to reject or to retain
with certainty, then the article in question will be kept for
assessment at the next eligibility stage in the overall fil-
tering process. In addition, articles or grey literature that
qualify after title screening but do not contain an abstract
will pass by default to the full-text screening stage. Lastly,
should our search string retrieve, in addition, any rel-
evant published material in French it will also be incor-
porated into the mapping process because these are the
two languages spoken and understood by all members of
the map team. Exclusion criteria clear absence of the above key words. Translocation in a genetic context, e.g. chromosomal
translocation, will also be excluded. Abstract Inclusion criteria Presence of words related to survival,
mortality, space use, genetics and all other relevant bio-
logical outcomes (cf. Table 3). The abstract will also be
retained if it contains words confirming a transloca-
tion event to, from, within or away from protected area
perimeters. Additionally, for the purpose of the Map, if
the translocation event has occurred to solve human-
wildlife conflicts then this will also satisfy the inclusion
criteria. Eligibility criteriaf published in the monthly newsletter of RNF. Euro-
Parc, who act as a federation of protected areas at the
European continental scale will also solicit their net-
work. Since translocations programs are often carried
out without being published in the form of scientific
articles this will provide further opportunity to gather
additional grey literature such as PhD and MSc theses,
various technical reports, and other documentation. AirTable, which works like a database will be the spe-
cific software used to acquire the documents sent via
the stakeholders’ contacts. Different eligibility criteria will be applied at each filter-
ing stage. Table 2 describes a summary description of the
eligibility criterion. Consistency checking To fully assess whether both reviewers adhere to the
eligibility criteria, a Kappa test will be performed at the
start of each filtering stage. Accordingly, 10% of retained
titles, 10% of retained abstracts, and 10% of retained full
texts will be pre-screened to check for agreement. Kappa
scores should be equal to or greater than 0.6. If differ-
ences of opinion occur, the process will be repeated with
new samples until a score of 0.6 or greater is reached. Even if statistical agreement is reached, all (if any)
remaining disagreements will be discussed before begin-
ning the screening process. A consistency check for
meta-data extraction will also be undertaken based on
training articles representing 10% of the retained corpus. All eventual disagreements will be discussed between the
reviewers. Exclusion criteria If no obvious description of interven-
tion exists. Full text Environ Evid (2020) 9:16 Page 8 of 11 Table 3 Outcome categories and corresponding descriptions
Outcome category
Description
Example references
(from test list or scoping
exercise)
Space use
Studies measuring all movement/dispersal of translocated individuals. This will include notably
home range measurements, or euclidean distance travelled
[43]
Demography
Studies outlining the changes in number of individuals, males/females, of the translocated popula-
tion i.e. population growth overtime
[44, 45]
Survival
Studies illustrating precisely the proportion of individuals alive or level of mortality since transloca-
tion
[46]
Reproduction
Any impacts on reproduction, expressed by number of young born since translocation, or specifi-
cally the survival rate of offspring
[47]
Feeding
All impacts specifically on diet and feeding of translocated individuals. (Nb. cascade effects will not
be included as an outcome)
[48, 49]
Behaviour
Studies measuring changes in terms of communication (e.g. vocal), social structure, or anti-preda-
tor behaviour i.e. stress/vigilance levels, of translocated individuals
[50, 51]
Genetics
Studies relating to the genetic structure of the translocated species
[52]
Physiology
All biological or physiological impacts measured at the molecular, cellular or organic level (e.g. hormone activity)
[53] Table 3 Outcome categories and corresponding descriptions
Outcome category
Description Table 3 Outcome categories and corresponding descriptions • Publication source (name of journal). for hunting purposes. We will provide a list of articles
excluded at full text with reasons for exclusion. for hunting purposes. We will provide a list of articles
excluded at full text with reasons for exclusion. • Full-text language (English, French or other). • Document type (journal article, book, conference
object, thesis (Phd, or Msc), technical documenta-
tion, or other). Study validity assessment No formal validity appraisal of included studies will be
performed. All studies that are deemed eligible at the
full text stage based on the Population, Intervention,
Outcomes, and Context criteria/screening stages will be
included in the Map. Thus, this Systematic Map will be
considered a thorough narrative synthesis ahead of any
review providing a comprehensive and robust overview
of the existing evidence. )
• Study content (study, review, meta-analysis, discus-
sion paper, modelling, or other). Full text Inclusion criteria Primarily but not exclusively, if the
outcome has been obtained from field studies (e.g. indi-
viduals equipped with radio-collars at time of release,
reported number of individuals surviving after a pre-
determined time-scale). However, discussion and review
articles will be retained if presence of PICO elements is
sufficiently described. Also, if the article presents evi-
dence of comparison of release strategies. All articles that
clearly state that population/individuals (plants or ani-
mals) are of captive or wild stock and have been trans-
ferred to, from, or within protected areas. Exclusion criteria Similar to those applied for title
or abstract screening, or information informing that
the translocated population is invasive or introduced Page 7 of 11 Langridge et al. Environ Evid (2020) 9:16 Table 2 Systematic map inclusion and exclusion criteria, and PICO definitions for the three-stage screening process
Criteria
Description
PICO definition(s)
Inclusion criteria
Eligible population
Any terrestrial, marine, or aquatic plant or animal species of wild or captive source
IN: Wild—free-ranging species from natural environments; not domesticated or
cultivated
Captive—species born, bred, or cultivated in captive setting (e.g. zoos, nurseries)
Eligible intervention
Conservation-based translocation operations (i.e. reintroduction, supplementation
and introduction) (see Table 1 for definitions)
IN: Conservation-based interventions—where the aim is improving viability and
persistence of translocated population. Regarding introduction, this will also include
ecological replacement and assisted migration [14]
Eligible comparator
Studies will not be required stricto sensu to have a comparator present
NA
Eligible outcomes
All relevant biological outcomes related to the subject population, including space
use, demography, survival, reproduction, trophic, behaviour, genetics, and physiol-
ogy
IN: For full description and definitions (cf. Table 3)
Exclusion criteria
Ineligible population
Invasive species or population historically introduced for hunting purposes
OUT: Invasive alien species outside of its natural distribution area and that threatens
biological function and diversity of native populations
Game/Hunting purposes: if transfer of species is for hunting purposes
Ineligible intervention
Non conservation-based introductions (e.g. historically introduced animals for hunt-
ing purposes or accidental introductions)
OUT: Non-conservation based transfer of species—where persistence or conservation
of individuals is not the motivation (e.g. sport/game hunting)
Accidental introductions: any documentation reporting on accidental invasions,
pathways, and risks etc. Ineligible outcomes
Sources not studying one of either Space use, Demography, Survival, Reproduction,
Feeding, Behaviour, Genetics, Physiology
OUT: Non-relevant outcomes that would not enable any interpretation of success. Trophic cascades will not be retained Langridge et al. Data coding strategy • Capture and release site locality coded as two sepa-
rate fields (name and geographic coordinates will be
recorded if given). A thorough meta-data extraction for the Map will be per-
formed by the same two members of the mapping team. Each selected article will be double coded. If, due to
resource limitations, true double coding is not possible,
a posteriori cross-check will be carried out and poten-
tial disagreements will be discussed until a consensus
is reached. Concerning missing data, if data is not suf-
ficiently detailed or simply unknown, it will be coded as
such. The following meta-data will be extracted from all
articles retained after completion of the screening pro-
cess [an additional file is provided with full explanations
(see Additional file 6)]: • Capture and release site climate types coded as two
separate fields (under the Köppen-Geiger Climate
Classification). i
• IUCN protected area management categories coded
for each protected area. (This will be achieved by
accessing the IUCN PAs database via http://www. protectedplanet.net, and then matching each PA with
the PAs in the database based on NAME). • Protected area (this will be coded in order to deci-
pher if individuals are translocated from-to different
protected areas, to, from, or within same protected
area). Study characteristics • Study country. • Study country. Outcome characteristics • Source and destination (wild-to-wild, captive-to-
wild, breeding-to-wild). If transferred individuals are
bred at specific sites, then released to wild this will be
coded as “breeding-to-wild”. • The following biological outcomes will be recorded:
space use, demography, survival, reproduction, feed-
ing, behaviour, genetics, and physiology (cf. Table 3
for full descriptions). • Study sample size (number of individuals initially
translocated, as stated by article authors). • Study age class at release (adult, juveniles or both, as
stated by authors). However, concerning plant trans-
locations it will be appropriate to detail life stage at
translocation e.g. seed, seedling, and adult plant. Intervention characteristics Intervention characteristics • Study interventions (supplementation, reintroduc-
tion, or introduction). 5 possible intervention catego-
ries will be coded as follows: iv. Within same: transfer occurring within the
same PA perimeter. • Study area biome (as stated by article authors). But
regrouped into 6 broad categories (Additional file 6
gives explanations on chosen habitat classes): i. Introduction—if a study is based on a single
one-off intervention i.e. assisting the migration
of a given species to suitable habitat outside of
its historical distribution. i. Forest/wooded. ii. Intro + Suppl—an introduction intervention
followed for the supplementation of the same
introduced population.f ii. Savannah. iii. Open habitats. iv. Wetland/humid. iii. Reintroduction—a single one-off reintroduc-
tion event (not followed by supplementation). v. Marine. vi. Aquatic. iv. Reintro + Suppl—a reintroduction followed by
the supplementation of the same reintroduced
population. • Study release strategy. Two release strategies will be
coded as follows: • Study release strategy. Two release strategies will be
coded as follows: v. Supplementation—where a given study only
reports on the supplementation of an already
threatened species. i. Soft
release:
studies
having
sufficiently
described methods to acclimatize individu-
als at the recipient site. Two key methods will
define soft release: use of protective enclo-
sures, and use of supplemental feeding [41, 42]. • Duration of intervention i.e. “translocation period”
(number of years). This will be relevant for cases
where an initial reintroduction event is followed by
several supplementations. g
ii. Hard release: immediate release (no acclimati-
zation and no supplementary food) [41]. • If translocation is climate motivated or not. • Study cost (in the rare case that such information
is reported, we will record figures stated by article
authors). • Programme motivation (this will outline the overall
motive of the manual transfer/movement of the spe-
cies in question). • Distance between capture and release site (coded à
posteriori with recorded geographic coordinates and
use of geographic software). i. Conservation (improving status of focal spe-
cies). ii. Rewilding (restoring natural functions). iii. Experimental or trial translocations.l iv. Human-wildlife conflict. Population characteristics l
v. Wildlife rescue operations (from human devel-
opment projects/urbanisation). • The lowest taxonomic rank will be recorded if suffi-
ciently detailed i.e. species name. Otherwise, higher
taxonomic classification will be used e.g. genus, fam-
ily or Order. vi. Metapopulation management. vi. Metapopulation management. Bibliographic information i. From-to: transfer from one protected area (PA)
to another. • Authors of article. ii. To: transfer from a non-protected habitat i.e. outside of PA perimeter to a PA. • Title and abstract. • Year of publication. Langridge et al. Environ Evid (2020) 9:16 Page 9 of 11 Page 9 of 11 iii. From: transfer from a PA to a non-protected
habitat i.e. outside of PA perimeter. Funding g
This work will be undertaken within the framework of the LIFE program
entitled “Natur’Adapt”. The project is co-financed by the LIFE programme
(European commission), The French Ministry of Ecology and the French Office
of Biodiversity. Acknowledgements g
We would like to thank the reserve managers of Les Réserves Naturelles de
France for their contribution during the round-tables. We thank François Sar-
razin of the Sorbonne University and Bruno Colas of the University of Paris-Sud
for their expertise on the subject. We would also like to thank Dakis-Yaoba
Ouédraogo for her invaluable feedback. 12. IUCN. Guidelines for reintroductions and other conservation transloca-
tions. IUCN. 2013. https://www.iucn.org/content/guidelines-reintroduc
tions-and-other-conservation-translocations. Accessed 31 Oct 2013. 13. Hoegh-Guldberg O, Hughes L, McIntyre S, Lindenmayer DB, Parmesan
C, Possingham HP, et al. Assisted colonization and rapid climate change. Science. 2008;321:345–6. Study mapping and presentation A systematic map database will be provided, detailing all
included articles from the full text screening stage. The Langridge et al. Environ Evid (2020) 9:16 Langridge et al. Environ Evid (2020) 9:16 Page 10 of 11 provided overall scientific expertise and proof-reading. All authors read and
approved the final manuscript. provided overall scientific expertise and proof-reading. All authors read and
approved the final manuscript. systematic map will include all the metadata coded for
each article. For the cases where more than one study is
reported in the same article, each study will be recorded
as a unique entry in the excel database with its corre-
sponding geographical coordinates, if given, and a unique
study ID. This database will be available as an open access
excel spreadsheet and included as an appendix to the sys-
tematic map publication.h Supplementary information A Codebook outlining the meta-data extraction
methods. Additional file 6. A Codebook outlining the meta-data extraction
methods. 10. Watson JEM, Dudley N, Segan DB, Hockings M. The performance and
potential of protected areas. Nature. 2014;515:67–73. 11. Gray CL, Hill SLL, Newbold T, Hudson LN, Börger L, Contu S, et al. Local
biodiversity is higher inside than outside terrestrial protected areas
worldwide. Nat Commun. 2016;7:1–7. Competing interests Competing interests
Authors declare having no competing interests. Received: 29 January 2020 Accepted: 17 July 2020 Received: 29 January 2020 Accepted: 17 July 2020 References 1. Murphy SE, Greenaway F, Hill DA. Patterns of habitat use by female brown
long-eared bats presage negative impacts of woodland conservation
management. J Zool. 2012;288:177–83. 2. Ceballos G, Ehrlich PR, Barnosky AD, García A, Pringle RM, Palmer TM. Accelerated modern human–induced species losses: entering the sixth
mass extinction. Sci Adv. 2015;1:e1400253. 3. Nowakowski AJ, Thompson ME, Donnelly MA, Todd BD. Amphibian sen-
sitivity to habitat modification is associated with population trends and
species traits. Glob Ecol Biogeogr. 2017;26:700–12. Competing interests
Authors declare having no competing interests. Competing interests
Authors declare having no competing interests. Ethics approval and consent to participate Ethics approval and consent to participate
No ethics agreement was required. Supplementary information 4. Newbold T, Hudson LN, Hill SLL, Contu S, Lysenko I, Senior RA, et al. Global effects of land use on local terrestrial biodiversity. Nature. 2015;520:45–50. Supplementary information accompanies this paper at https://doi. org/10.1186/s13750-020-00199-4. Supplementary information accompanies this paper at https://doi. org/10.1186/s13750-020-00199-4. Supplementary information acco
org/10.1186/s13750-020-00199-4. Supplementary information accompanies this paper at https://doi. org/10.1186/s13750-020-00199-4. 5. Griffith B, Scott J, Carpenter J, Reed C. Translocation as a species conser-
vation tool: status and strategy. Science. 1989;245:477–80. Additional file 1. Our declaration and checklist of adherence to the
ROSES guidelines. Additional file 2. Test list and overall comprehensiveness of search string. Additional file 3. Illustrating the building process of the search string. Additional file 4. Web of Science Core Collection database subscription
details. Additional file 5. Corresponding number of Search hits from Web of Sci-
ence core collection, Scopus, and supplementary search in google scholar
and organisation websites. Additional file 6. A Codebook outlining the meta-data extraction
methods. Additional file 1. Our declaration and checklist of adherence to the
ROSES guidelines. 6. Caizergues A, Rätti O, Helle P, Rotelli L, Ellison L, Rasplus J-Y. Population
genetic structure of male black grouse (Tetrao tetrix L.) in fragmented vs. continuous landscapes. Mol Ecol. 2003;12:2297–305. Additional file 2. Test list and overall comprehensiveness of search string. Additional file 3. Illustrating the building process of the search string. 7. Rivera-Ortíz FA, Aguilar R, Arizmendi MDC, Quesada M, Oyama K. Habitat
fragmentation and genetic variability of tetrapod populations. Anim
Conserv. 2015;18:249–58. 7. Rivera-Ortíz FA, Aguilar R, Arizmendi MDC, Quesada M, Oyama K. Habitat
fragmentation and genetic variability of tetrapod populations. Anim
Conserv. 2015;18:249–58. 8. Cantú-Salazar L, Gaston KJ. Very large protected areas and their contribu-
tion to terrestrial biological conservation. Bioscience. 2010;60:808–18. 8. Cantú-Salazar L, Gaston KJ. Very large protected areas and their contribu-
tion to terrestrial biological conservation. Bioscience. 2010;60:808–18. Additional file 5. Corresponding number of Search hits from Web of Sci-
ence core collection, Scopus, and supplementary search in google scholar
and organisation websites. 9. Lunt ID, Byrne M, Hellmann JJ, Mitchell NJ, Garnett ST, Hayward MW, et al. Using assisted colonisation to conserve biodiversity and restore ecosys-
tem function under climate change. Biol Cons. 2013;157:172–7. 9. Lunt ID, Byrne M, Hellmann JJ, Mitchell NJ, Garnett ST, Hayward MW, et al. Using assisted colonisation to conserve biodiversity and restore ecosys-
tem function under climate change. Biol Cons. 2013;157:172–7. Additional file 6. 14. Seddon PJ. From reintroduction to assisted colonization: moving along
the conservation translocation spectrum. Restor Ecol. 2010;18:796–802. Availability of data and materials y
Data sharing is not applicable to the publication of the review protocol. All
datasets associated with the Systematic Map will be made available as open
access files (Additional file 6). The map database will be described in the map publi-
cation with summary figures and tables of the relevant
study characteristics. A geographic map will present
the location of each translocation event/study. Possible
knowledge gaps (under-represented subtopics that war-
rant further primary research) and knowledge clusters
(well-represented subtopics for full synthesis by a sys-
tematic review) will be identified by cross-tabulating
key meta-data variables (e.g. biological groups and out-
comes). Based on these results, recommendations will be
made on priorities for future research concerning trans-
location and protected areas. Recommendations will also
be made to inform management. To this end, regarding
the specific objectives of the LIFE project, all Map results
will be transferred to reserve managers. In addition, a
practitioner brief will be provided to reserve managers
with the aim of summarizing key results in an operational
manner in order to aid decision making. Workshops are
already planned for this. Authors’ contributions The social and ecological integration of
captive-raised adolescent male African elephants (Loxodonta africana)
into a wild population. PLoS ONE. 2013;8:e55933. 43. Fernando P, Leimgruber P, Prasad T, Pastorini J. Problem-elephant
translocation: translocating the problem and the elephant? PLoS ONE. 2012;7:e50917. 22. Hodder KH, Bullock JM. Translocations of native species in the UK: impli-
cations for biodiversity. J Appl Ecol. 1997;34:547–65. 44. Hochkirch A, Agnes W, Anje T, Friedhelm N. Translocation of an endan-
gered insect species, the field cricket (Gryllus campestris Linnaeus, 1758)
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Effects of Hand Configuration on the Grasping, Holding, and Placement of an Instrumented Object in Patients With Hemiparesis
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Frontiers in neurology
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To cite this version: Ross Parry, Sandra Macias Soria, Pascale Pradat-Diehl, Veronique Marchand-Pauvert, Nathanael
Jarrasse, et al.. Effects of Hand Configuration on the Grasping, Holding, and Placement of an
Instrumented Object in Patients With Hemiparesis. Frontiers in Neurology, 2019, 10, pp.240. 10.3389/fneur.2019.00240. hal-02086396 Effects of Hand Configuration on the Grasping, Holding,
and Placement of an Instrumented Object in Patients
With Hemiparesis
Ross Parry, Sandra Macias Soria, Pascale Pradat-Diehl, Veronique
Marchand-Pauvert, Nathanael Jarrasse, Agnès Roby-Brami Ross Parry, Sandra Macias Soria, Pascale Pradat-Diehl, Veronique
Marchand-Pauvert, Nathanael Jarrasse, Agnès Roby-Brami HAL Id: hal-02086396
https://hal.sorbonne-universite.fr/hal-02086396v1
Submitted on 1 Apr 2019 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
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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. ORIGINAL RESEARCH
published: 19 March 2019
doi: 10.3389/fneur.2019.00240 Ross Parry 1,2*, Sandra Macias Soria 1, Pascale Pradat-Diehl 3,4,5,
Véronique Marchand-Pauvert 5, Nathanaël Jarrassé 1 and Agnès Roby-Brami 1 1 Institut des Systèmes Intelligents et de Robotique, Sorbonne Université, Paris, France, 2 Centre de Recherche sur le Sport
et le Mouvement, EA 2931, Université Paris Nanterre, Nanterre, France, 3 Service de Médecine Physique et de Réadaptation,
Assistance Publique-Hôpitaux de Paris, Hôpitaux Universitaires Pitié Salpêtrière-Charles Foix, Paris, France, 4 AP-HP, GRC
n◦18 Handicap cognitif et réadaptation (HanCRe), Sorbonne Université, Hôpitaux Universitaires Pitié Salpêtrière-Charles
Foix, Paris, France, 5 Laboratoire d’Imagerie Biomédicale, Sorbonne Université, Paris, France Objective: Limitations with manual dexterity are an important problem for patients
suffering from hemiparesis post stroke. Sensorimotor deficits, compensatory strategies
and the use of alternative grasping configurations may influence the efficiency of
prehensile motor behavior. The aim of the present study is to examine how different grasp
configurations affect patient ability to regulate both grip forces and object orientation
when lifting, holding and placing an object. Edited by:
Martin Lotze,
University of Greifswald, Germany Edited by:
Martin Lotze,
University of Greifswald, Germany Methods: Twelve stroke patients with mild to moderate hemiparesis were recruited. Each was required to lift, hold and replace an instrumented object. Four different
grasp configurations were tested on both the hemiparetic and less affected arms. Load
cells from each of the 6 faces of the instrumented object and an integrated inertial
measurement unit were used to extract data regarding the timing of unloading/loading
phases, regulation of grip forces, and object orientation throughout the task. Reviewed by:
Margit Alt Murphy,
University of Gothenburg, Sweden
Eric Wolbrecht,
University of Idaho, United States
*Correspondence:
Ross Parry
rparry@parisnanterre.fr Reviewed by:
Margit Alt Murphy,
University of Gothenburg, Sweden
Eric Wolbrecht,
University of Idaho, United States Results: Grip forces were greatest when using a palmar-digital grasp and lowest when
using a top grasp. The time delay between peak acceleration and maximum grip force
was also greatest for palmar-digital grasp and lowest for the top grasp. Use of the
hemiparetic arm was associated with increased duration of the unloading phase and
greater difficulty with maintaining the vertical orientation of the object at the transitions to
object lifting and object placement. The occurrence of touch and push errors at the onset
of grasp varied according to both grasp configuration and use of the hemiparetic arm. Specialty section:
This article was submitted to
Stroke,
a section of the journal
Frontiers in Neurology Received: 10 September 2018
Accepted: 22 February 2019
Published: 19 March 2019 Conclusion: Stroke patients exhibit impairments in the scale and temporal precision
of grip force adjustments and reduced ability to maintain object orientation with various
grasp configurations using the hemiparetic arm. Nonetheless, the timing and magnitude
of grip force adjustments may be facilitated using a top grasp configuration. Conversely,
whole hand prehension strategies compound difficulties with grip force scaling and inhibit
the synchrony of grasp onset and object release. Keywords: hand function, grasp, stroke, assessment, instrumented objects for rehabilitation Effects of Hand Configuration on the
Grasping, Holding, and Placement of
an Instrumented Object in Patients
With Hemiparesis Ross Parry 1,2*, Sandra Macias Soria 1, Pascale Pradat-Diehl 3,4,5,
Véronique Marchand-Pauvert 5, Nathanaël Jarrassé 1 and Agnès Roby-Brami 1 Edited by:
Martin Lotze,
University of Greifswald, Germany INTRODUCTION The cerebral hemisphere implicated
in the CVA (13, 47) and the extent of the resulting sensory deficits
(48, 49) have also been observed to influence anticipatory grip
force scaling. This body of work highlights the potential interest
of using instrumented objects for the diagnosis and evaluation of
the impairments associated with hemiparesis (45, 46, 48, 50–53). These impairments in patient hand function manifest in
multiple different aspects of motor performance. This may
include reduced strength (3), loss of individuated finger control
(12), and abnormal force control at the level of the fingers (13). Increased muscle tone and spasticity though the flexors of the
wrist and hand may further compound these difficulties and
inhibit the ability to open the hand in preparation for grasping
(14). Atypical reaching and grasping patterns are often seen to
emerge both as a consequence of and as a response to the motor
dysfunction (15, 16). As it stands, these objective studies of hand function post
stroke have focused primarily upon either the lifting or the
vertical movement components in object handling. To a certain
extent, this limitation has been related to technical restrictions. Other than a handful of studies by Hermsdorfer et al. (8, 49),
research in this field has predominantly used manipulanda
designed for the study of precision grip, where strain gauge
force transducers are attached to a separate base unit [e.g., (23–
25, 29, 33, 35, 37)]. These devices cannot be freely handled
by subjects, much less a person with an upper-limb movement
disorder. Indeed, patients with hemiparesis often experience
specific impairments with precision grip (53) and regularly
use alternative grasping strategies such as whole hand grasping
(15, 16, 54). Previous researchers have hypothesized that these
alternative grasp strategies may impact grip force scaling (55) and
compromise patient ability to manage hand-object-environment
relationships during object manipulation (56). Unfortunately, rehabilitation of upper limb impairments
proves
to
be
challenging. Whilst
numerous
therapeutic
modalities (e.g., bilateral training, constraint-induced therapy,
electrical stimulation, task-oriented, high intensity programs)
have been evaluated in clinical trials, none have demonstrated
consistent effects upon hand function (17–19). Indeed, previous
research papers have described therapy outcomes in upper
limb rehabilitation post stroke as “unacceptably poor” (20). Ideally, the design of neurorehabilitation programs should
be grounded upon an understanding of basic mechanisms
involved in neural plasticity and motor learning (21, 22). INTRODUCTION underlying challenge involves analyzing sensorimotor function
of the hand with respect to its interaction with objects in the
environment (30). Cerebrovascular accidents (stroke) are a frequent cause of
disability (1) and the recovery of upper-limb function in
particular, is a key determinant of independence in activities
of daily living (2). Broadly speaking, the physical impairment
experienced by patients is characterized by loss of strength,
abnormal movement patterns (pathological synergies), and
changes in muscle tone to the side of the body contralateral to
the stroke (3, 4). This presentation is commonly referred to as
hemiparesis and its severity tends to reflect the extent of the
lesion to the corticospinal tract (5). Subtle changes in movement
kinematics and hand function on the ipsilesional upper-limb
have also been documented and may be the consequence
of direct impairment of ipsilateral motor pathways (6, 7),
as well as reorganization of the non-lesioned hemisphere to
support recovery of motor-function in the hemiparetic limb
(8–10). Above all though, patients living with stroke find that
limitations with manual dexterity of the hemiparetic arm have
the most significant effect upon their ability to carry out activities
involving hand use in daily life (11). Successfully managing grasping and object handling tasks
requires skilled control of prehensile finger forces. In healthy
adults, grip forces are regulated to be marginally greater than
the minimum required to prevent the object from slipping
(31). This safety margin is calibrated according to the shape,
surface friction and weight distribution of the object (32, 33). As the hand moves through space (lifting, transporting, object
placement), grip force is continually modulated, proportional to
the load forces associated with the mass and acceleration of that
object (34). This temporal coupling between grip and load forces
is considered a hallmark of anticipatory sensorimotor control
(35). Disruption to motor planning, volitional motor control or
somatosensory feedback may lead to a breakdown in the timing
and magnitude of grip force adjustments. Numerous studies have examined grip force regulation in
neurological pathologies including cerebellar dysfunction (36),
peripheral sensory neuropathy (37, 38), Parkinson’s disease (36,
37, 39, 40) as well as congenital and acquired brain lesions (13,
36, 41–45). For patients suffering from hemiparesis post stroke,
difficulty with coordinating the grasping and lifting action are
frequently associated with temporal discrepancies between grip
forces and load forces (46). Citation: Parry R, Macias Soria S,
Pradat-Diehl P, Marchand-Pauvert V,
Jarrassé N and Roby-Brami A (2019)
Effects of Hand Configuration on the
Grasping, Holding, and Placement of
an Instrumented Object in Patients
With Hemiparesis. Front. Neurol. 10:240. doi: 10.3389/fneur.2019.00240 Front. Neurol. 10:240. doi: 10.3389/fneur.2019.00240 March 2019 | Volume 10 | Article 240 Frontiers in Neurology | www.frontiersin.org 1 Grasp Regulation Post Stroke Parry et al. Frontiers in Neurology | www.frontiersin.org Clinical Measures of Upper-Limb Function Clinical Measures of Upper-Limb Function
Prior to completing the experimental phase of this study, an
upper-limb motor-function assessment was carried out. The
Fugl-Meyer upper-limb evaluation (FME) and Frenchay Arm
Test (FAT) was conducted for each patient and, in addition to
this, 8 of the 12 patients completed the Jebsen Taylor Hand
Function test (JTT). The FME evaluation provides an overall
score of upper limb function (max of 126), which may then be
broken down into its sensory function component (max of 60)
motor function component (max of 66) (60). The FAT assesses
patient ability to carry out five different actions providing a score
on a scale of 1 to 5 (61). The JTT provides an overall score
in seconds, representing the time taken to complete a series of
functional task with each arm. Finally, hand strength for both
arms was measured using a grip-strength dynamometer (DGS). Pinch grasp: opposition between the pads of the thumb and
palmar aspect of the four fingers (Figure 2C). Palmar-digital grasp: opposition of fingers and palm, with the
thumb in abduction as for a power grip (Figure 2D). Palmar-digital grasp: opposition of fingers and palm, with the
thumb in abduction as for a power grip (Figure 2D). This combination of grasps was selected to represent common
hand configurations which may support functionally different
tasks in daily activities. For example, pinch grasps are a versatile
hand configuration that can support an object whilst enabling
transition to in-hand manipulation if necessary, while precision
grasps are important for handling smaller objects. By contrast,
a palmar digital grasp serves to fix an object in the hand
while the arm is in motion (i.e., scrubbing a surface with a
sponge) whereas the top grasp configuration may assist with
tasks such as repositioning objects on a table’s surface [see (62)
for greater detail on the frequency of grasp configuration in
household tasks]. MATERIALS AND METHODS Subjects were seated at a horizontal table throughout the
experiment. In the starting posture, both hands were positioned
at each corner of the proximal edge of the table. The iBox
was placed vertically before the patient. It was positioned in
the parasagittal plane, 20 cm in front of the hand used for the
pinch, precision and top grasps. For the palmar digital grasp, the
iBox was placed in front of the opposite hand so as to ensure a
comfortable grasp (15, 57). In all cases the iBox was rotated 30◦
around the vertical axis, in the direction of the patient’s midline. This reference orientation was calibrated at the beginning of the
experiment and repeated prior to each trial. The experimental
setup is illustrated in Figure 1B. Participants Twelve adult patients (6 males, 6 females) with a diagnosis
of a unique stroke and a mean age of 58 years (range 48–70
years) participated in this study. Of these patients, 8 suffered
from hemiparesis on their dominant right hand side; 4 right
handed patients and 1 ambidextrous patient suffered from left
sided hemiparesis [hand preference verified using the Edinburgh
Handedness Inventory, see (59)]. Each patient was in a subacute
or chronic phase of recovery and was assessed between 1 and
13 months following the neurological event. The ability to grasp
and hold an object was a requirement for inclusion to this study. Patients with additional neurological or orthopedic conditions,
important cognitive deficits or aphasia were not eligible for this
study. A summary of clinical characteristics of the patient group
is provided in Table 1. This study was approved by the local ethics
committee at University Paris Descartes and all subjects provided
written consent prior to commencement of the evaluation. Experimental Apparatus p
pp
An instrumented object (iBox) with 6 integrated load cells and
an inertial measurement unit (IMU) was used for the purposes
of this study (see Figure 1A). This device measures 108 × 70
× 40mm and has a mass of 0.370 kg. It enables recording of
acceleration, rotational velocity, orientation of the unit as well
as the forces applied normally to each of its six faces. The force
of the load cell on the bottom face was calibrated so that the
weight of the device, equivalent to 3.63 N, was subtracted (i.e.,
the reference force signal was zero when the object lay on the
table and decreased to −3.63 N when the object was lifted from
the supporting surface). All data was sampled at a frequency
of 100 Hz and transmitted wirelessly to a local computer via
Bluetooth. Overall acceleration was measured as a combination
of gravity and kinematic acceleration (39). Object orientation
was calculated from IMU data and expressed as the alpha angle, INTRODUCTION Part
of this process implies coming to terms with the factors which
characterize the disorganization in voluntary motor output
(21). However, the majority of clinical tools currently used
for evaluating hand function distinguish motor performance
according to ordinal rating scales or task completion time (e.g.,
Frenchay Arm Test, Jebson-Taylor Hand Function Test) (23, 24). These kinds of assessments lack sensitivity and may prove
insufficient for detecting the presence of mild motor deficits or
subtle, yet clinically important changes in hand coordination
(25, 26). Evidence based frameworks for hand rehabilitation
have specifically called for the integration of new technology
to support patient assessment and treatment planning (27). Despite this, the transposition of technology for upper limb
rehabilitation from the research domain into clinical practice has
been limited (28, 29). In the assessment of manual dexterity, the In a recent study with healthy adult subjects, (57) we
demonstrated how an instrumented object with multiple load
cells and an integrated inertial measurement unit (58) may
be used to examine relationships between different grasp
configurations, grip force regulation and object orientation. The purpose of the present investigation was to extend this
work to the study of patients with hemiparesis post stroke. The first objective was to compare how four alternative grasp
configurations commonly used in daily tasks affect grip force
regulation in this population. The second objective was to March 2019 | Volume 10 | Article 240 2 Parry et al. Grasp Regulation Post Stroke explore the timing and coordination of the whole task sequence
(grasping, lifting, holding, placement and object release). The
third and final objective was to evaluate the stability of the hand-
held object’s orientation across the different phases of the task. indicating the deviation of the longitudinal axis of the iBox from
the vertical axis. Further technical details regarding the iBox are
provided in (58). Frontiers in Neurology | www.frontiersin.org Grasp Configurations Used The experimental procedure involved grasping and holding the
iBox using 4 different hand configurations. Each of these grasps,
described below is illustrated in Figure 2. Precision grip: opposition between the pads of the thumb and
index (Figure 2A). Top Grasp: opposition using a pinch grip, the object is
approached and grasped from above (Figure 2B). Experimental Procedure Each patient was given a brief period of time to handle the
iBox with both hands prior to beginning the experimental
tasks in order to become familiar with the weight and surface
characteristics of the object. During the experimental task,
patients were asked to lift and hold the iBox approximately
10 cm above the table. For the pinch, precision and palmar-digital
hand configurations, patients were instructed to hold the iBox
for between 2 and 5 s before replacing it in an approximately
similar position. For the top grasp configuration, patients were
asked to place the iBox in the frontal plane, 10 cm distal to
the initial position (deposit area indicated in Figure 1) (57). A
demonstration was provided prior to commencement of each March 2019 | Volume 10 | Article 240 Frontiers in Neurology | www.frontiersin.org 3 Grasp Regulation Post Stroke Parry et al. TABLE 1 | Results from the functional upper limb evaluations for stroke patients. Experimental Procedure The Jebsen Taylor provides a score in seconds,
being the total time required to complete a series of manual handling tasks; the score in brackets provides the reference time for the less affected arm. FIGURE 1 | Illustration of the iBox device and the experimental setup. (A) The iBox instrumented object. (B) Setup for the experimental procedure. Initial positions of
the iBox and hand start area are indicated by the dotted lines. The gray shaded rectangle indicates the deposit area for the top grasp task. FIGURE 1 | Illustration of the iBox device and the experimental setup. (A) The iBox instrumented object. (B) Setup for the experimental procedure. Initial positions of
the iBox and hand start area are indicated by the dotted lines. The gray shaded rectangle indicates the deposit area for the top grasp task. FIGURE 2 | Grasp configurations used during the iBox protocol. (A) Precision grip. (B) Top grasp. (C) Pinch grip. (D) Palmar-digital grasp. Image adapted from
Martin-Brevet et al. (57). FIGURE 2 | Grasp configurations used during the iBox protocol. (A) Precision grip. (B) Top grasp. (C) Pinch grip. (D) Palmar-digital grasp. Image adapted from
Martin-Brevet et al (57) s used during the iBox protocol. (A) Precision grip. (B) Top grasp. (C) Pinch grip. (D) Palmar-digital grasp. Image adapted from FIGURE 2 | Grasp configurations used during the iBox protocol. (A) Precision grip. (B) Top grasp. (C) Pinch grip. (D) Palmar-digital grasp. Image adapted from
Martin-Brevet et al. (57). Visual inspection of all force, acceleration and orientation
signals was carried out immediately following data acquisition. Events where signals were compromised or patients were unable
to complete the set task were excluded. All patients were able
to perform the palmar and top grasp tasks with both limbs. Using the hemiparetic arm, one patient (patient 9) was unable to task. Patients were asked to perform each grasp and place task
3 times to the best of their ability. The ensemble of grasping
and holding tasks were performed first with the less affected arm
and then with the hemiparetic arm. The experimenter verified
the patient’s initial posture and repositioned the iBox between
movements as required. task. Patients were asked to perform each grasp and place task
3 times to the best of their ability. The ensemble of grasping
and holding tasks were performed first with the less affected arm
and then with the hemiparetic arm. Experimental Procedure Patient
ID
Hemiparetic
arm
Time since
stroke
Dynamometer grip
strength (reference from
less-affected side)
Fugl-Meyer upper limb
evaluation (sensory/motor
subscores)
Jebsen Taylor hand function
test (reference from
less-affected side)
Frenchay
arm test
P1
Right (d)
5 months
361.6 N (353.1 N)
124 (58/66)
79 s (80 s)
5
P2
Right (d)
13 months
156.8 N (473.3 N)
95 (39/56)
303 s (95 s)
3
P3
Right (d)
11 months
215.6 N (363.6 N)
105 (56/49)
89 s (84 s)
5
P4
Right (d)
2 months
38.2 N (197.0 N)
84 (42/42)
337 s (110 s)
5
P5
Right (d)
18 months
245.9 N (382.8 N)
105 (56/49)
261 s (163 s)
5
P6
Left (n)
1 months
107.8 N (367.2 N)
109 (53/56)
308 s (52 s)
4
P7
Left (n)
2 months
52.9 N (235.9 N)
78 (41/37)
362 s (45 s)
3
P8
Right (d)
19 months
146.0 N (189.4 N)
124 (59/65)
61 s (65 s)
5
P9
Left (n)
5 months
26.5 N (156.8 N)
104 (38/66)
NA
3
P10
Left (a)
13 months
266.6 N (275.4 N)
120 (60/60)
NA
5
P11
Right (d)
2 months
332.2 N (381.2 N)
125 (65/60)
NA
5
P12
Right (d)
14 months
16.7 N (124.5 N)
96 (48/48)
NA
5
n = 12
8 right/4 left
9 months
163.7 N (291.7 N)
106 (51/55)
225 s (87 s)
5
For the hemiparetic arm, (d), (n), (a) signify if this is the dominant, non-dominant or ambidextrous hand. Grip strength scores are provided in newton with values for the less affected side
in brackets. Fugl-Meyer provides the total score on the upper limb evaluation with sensory and motor subscores indicated in brackets. The Jebsen Taylor provides a score in seconds,
being the total time required to complete a series of manual handling tasks; the score in brackets provides the reference time for the less affected arm. TABLE 1 | Results from the functional upper limb evaluations for stroke patients. For the hemiparetic arm, (d), (n), (a) signify if this is the dominant, non-dominant or ambidextrous hand. Grip strength scores are provided in newton with values for the less affected side
in brackets. Fugl-Meyer provides the total score on the upper limb evaluation with sensory and motor subscores indicated in brackets. Statistical Analysis Data for continuous variables were examined using Shapiro-
Wilk tests. As the ensemble of these variables was found to
have non-normal distributions, Kruskall-Wallis non-parametric
analysis of variance was used for statistical comparisons. Both side (hemiparetic arm/less-affected arm) and grasp
configuration (pinch/precision/palmar digital/top) factors were
included. Where indicated, post-hoc analysis was conducted
using Dunn’s method. The frequency of touch and push errors
was analyzed using Chi-Squared tests. The Bonferroni method
was used for correction of p-values when comparing across grasp
configurations. The threshold for statistical significance was set
at p = 0.05. Data Processing and Analysis Transitions between grasping, lifting, and placement phases were
identified in an automated manner with reference to load cell
data (57) (Figure 3 indicates the different phase transitions with
vertical lines). Grasp onset (tg) was defined as the moment when
the mean of the forces applied to the two lateral load cells
exceeded 0.15 N. Onset of lifting (tl), when the base load cell
value was inferior to the −3.4 N threshold. Placement time (tp)
was the moment when the base load cell then returned to the
threshold value of −3.4 N. Object release time (tr) was defined
as the moment when the mean of the forces applied to the two
lateral load cells were inferior to 0.15 N. The hold onset (ho) and
hold end (he) events were chosen subjectively to delimit a plateau
of relative stability during holding and tagged manually from
data in each trial using a graphic interface. From these events,
five separate phases were identified: (1) unloading of the bottom
face between tg and tl1, (2) lifting between tl and ho, (3) holding
between ho and he, (4) descent between he and tp, and (5) release
between tp and te. In
order
to
evaluate
relationships
between
clinical
characteristics and task performance, test results from the
DGS, FME, JTT, and FAT were transformed into z-scores prior
to testing with Spearman correlation coefficients against the
hemiparetic upper-limb variables assessed using the iBox. Values
>0.7 or <-0.7 were considered to represent strong correlation
between clinical motor-function tests and iBox variables. In order
to control for multiple correlation analysis, a resampling method
with 10,000 randomized permutations of each iBox variable was
used. Percentile values (2.5 and 97.5%) from the distribution
of the resulting coefficient matrix served as a symmetric two-
sided 95% confidence interval (63). Correlations of clinical
motor tests and iBox variables outside of this confidence
interval were considered as statistically significant. All statistical
analyses were conducted using Matlab and the JASP software
package (https://jasp-stats.org). Further to this, the occurrence of push and touch errors (57)
were identified. Touches were identified where extraneous forces
were applied to the object prior to grasp onset or following object
release. A touch was defined as an event where the sum of forces
on the exposed (front, back, top, and lateral) load cells exceeded
0.7 N before tg or after tr for any given trial. Data Processing and Analysis The first face of the
object touched was identified and noted. A push was detected
as increased force (>0.4 N) on the base load cell during the
unloading or release phases. Examples of touch and push events
are illustrated in the load cell signals provided in Figure 3C2. Experimental Procedure The experimenter verified
the patient’s initial posture and repositioned the iBox between
movements as required. March 2019 | Volume 10 | Article 240 Frontiers in Neurology | www.frontiersin.org 4 Grasp Regulation Post Stroke Parry et al. perform the pinch grasp task and four patients (patients 3,6,7,9)
were unable to complete the precision grip task. perform the pinch grasp task and four patients (patients 3,6,7,9)
were unable to complete the precision grip task. RESULTS Clinical Measures of Upper-Limb Function
Average grip-strength for the affected arm was 163.7 N (s.d. 120.5 N; range 16.7–361.6 N) compared to 292.0 N (s.d. 109.8 N;
range 124.46–473.3 N) for the less affected arm. The patient
group was assessed as having mild to moderate upper-limb
impairment using the FME motor assessment (median = 56;
range 37–66) with variable levels of sensory deficits (range 38–
60 on the sensory function subscore). The median score on the
Frenchay Arm Test was 5 (range 3–5), indicating that patients
were able to carry out basic functional tasks with their affected
upper-limb. The median time for completion of the JTT with the
hemiparetic arm was 282 s (range 61–362 s), vastly superior to
that of average times for similarly aged individuals (average 30 s,
(64, 65). Clinical measures of upper-limb function are displayed
in Table 1. Based upon the time-tagged data sequences, the following
series of variables were extracted for analysis: • Duration and rate of grip force change for unloading and
release phases • Grip force at tg, tl, tp, te (mean of the front and back load cells) • Maximal grip force and peak acceleration during the
lifting phase • Time difference between maximal grip force and peak
acceleration during the lifting phase • Grip force during holding (median and standard deviation of
the front and back load cells during the whole period) • iBox orientation at times tg, tl, td, te (alpha angle) • iBox orientation at times tg, tl, td, te (alpha angle) • iBox orientation during holding (alpha angle median and
standard deviation) • Frequency of touch events before grasping and after object
release and of push events during the unloading and
release phases Time Courses for iBox Data Signals From top to bottom: (A1,2) angle measuring the deviation of the iBox from the vertical (B1,2) vertical acceleration of
the iBox (C1,2) force signals: grasping force is indicated with plain (thumb) and dashed lines (digits), the unloading of the bottom face of the object is indicated with
(dotted lines); inset in (C1) shows a larger scale. Vertical lines indicate the times of transitions between phases tg = onset of grip; tl = onset of lifting; ho = hold onset;
he = hold end; tp = placement time; tr = release time. Time = 0 s at tg. In (C2), arrows indicate touch and push errors upon establishing and releasing grasp. (0.49 s on average) (Kruskal Wallis, p = 0.002; Figure 5A) and
grip force rate was accordingly diminished on the hemiparetic
side (Kruskal Wallis, p = 0.003; Figure 5B). speaking, those patients who experienced a better recovery had
regular acceleration and orientation profiles. For these patients,
maximal grip force occurred during lifting and a smooth decrease
of force was observed before placement while the holding phase
was characterized by relative stability of grip forces. Patients with
more severe motor deficits demonstrated greater variability in
the acceleration and object orientation profiles (see examples in
Figures 3A1,2,B1,2). In the following section, the main results
of this experiment are presented according to the five phases
(unloading, lifting, holding, descent, release) which characterize
the task. The mean orientation of the iBox at tl was 5.4◦on the
hemiparetic arm, significantly greater than that of the 1.8◦for the
less affected arm (Kruskall-Wallis p = 0.001; Figure 6A). The occurrence of touch and push errors varied with both the
grasp configuration and the arm used (Chi-Squared p < 0.001;
per Figure 7). Touch errors were most frequent when using the
palmar (48% of trials) and pinch grasps (23% of trials). This type
of error was also twice as frequent in the hemiparetic arm (35%
of trials) than in the less-affected arm (17% of trials). When using
the hemiparetic arm, these errors were associated predominantly
with sub-threshold touches on the load cell corresponding to
finger contact (18%) than for the load cell corresponding to the
thumb (8%). On the unaffected arm, this trend was reversed with
many more errors attributed to sub-threshold contact from the
thumb (10%) than for the fingers (2%). Push errors occurred
more systematically than touch errors. Time Courses for iBox Data Signals Time Courses for iBox Data Signals
Time courses of force, acceleration and object orientation signals
were generally consistent across the different grasp patterns used. Changes in grip forces reflected the phase progression in the
grasping, lifting, holding and placement of the iBox, although the
regularity and magnitude of these signals were less consistent. Figure 3 provides typical examples of these signals for two
patients with contrasting functional abilities (patient 1 had a
FME motor score of 66 compared to 37 for patient 7). Broadly All data analysis was performed using customized Matlab scripts. 1Probably due to the design of the iBox, we could not distinguish a first phase of
increasing grasping force without change in vertical force (see inset of Figure 3). The unloading period in this work corresponds to the sum of pre-loading and
loading periods commonly identified in previous studies where the vertical force
sensor is fitted between the handle of the manipulandum and its main mass. March 2019 | Volume 10 | Article 240 Frontiers in Neurology | www.frontiersin.org 5 Parry et al. Grasp Regulation Post Stroke FIGURE 3 | Examples of recording of a lifting task carried out with the hemiparetic arm using the pinch grip in two patients with contrasting functional abilities (P1,
FME 66 and P7 FME 37; see Table 1 for details). From top to bottom: (A1,2) angle measuring the deviation of the iBox from the vertical (B1,2) vertical acceleration of
the iBox (C1,2) force signals: grasping force is indicated with plain (thumb) and dashed lines (digits), the unloading of the bottom face of the object is indicated with
(dotted lines); inset in (C1) shows a larger scale. Vertical lines indicate the times of transitions between phases tg = onset of grip; tl = onset of lifting; ho = hold onset;
he = hold end; tp = placement time; tr = release time. Time = 0 s at tg. In (C2), arrows indicate touch and push errors upon establishing and releasing grasp. FIGURE 3 | Examples of recording of a lifting task carried out with the hemiparetic arm using the pinch grip in two patients with contrasting functional abilities (P1,
FME 66 and P7 FME 37; see Table 1 for details). Unloading Phase Grip force at tg was found to vary with grasp configuration
(Kruskal Wallis, p = 0.011) and post-hoc testing showed that
force in the palmar-digital grasp was greater than in the precision
(p = 0.009) and top grasps (p = 0.018). The subsequent unloading phase was characterized by a
progressive increase in grasp forces and a corresponding
decreased load on the base of the instrumented object until tl
when it reached −3.63 N (see examples in Figures 3C1,2). At tl,
grip force was found to vary with grasp configuration (Kruskall-
Wallis p = 0.038, Figure 4A). Grip forces were significantly lower
when using the top grasp (average of 12.85 N) than when using a
palmar-digital grasp (average of 19.03 N; p = 0.013). The overall
duration of the unloading phase was greater when using the
hemiparetic arm (0.85 s on average) than the less-affected arm Time Courses for iBox Data Signals They occurred most
frequently with the top grasp (91% of trials) and pinch grasps
(68% of trials). Again, these errors were more common for the
hemiparetic arm (75% of trials) than for the less-affected arm
(64% of trials). Frontiers in Neurology | www.frontiersin.org Lifting Phase During the lifting phase, grip forces were generally observed to
continue to increase in accordance with the vertical acceleration March 2019 | Volume 10 | Article 240 Frontiers in Neurology | www.frontiersin.org 6 Parry et al. Grasp Regulation Post Stroke FIGURE 4 | Grip forces for the hemiparetic (red symbols) and less affected arms (black symbols) for the different grasp configurations (in abscissa). (A) Grip force at
the time of lifting (tl). (B) Maximum grip force during the lifting phase (C) Average force during the holding phase (D) Grip force at the time of release.*Dunn’s post-hoc
p < 0.05; **Dunn’s post-hoc p < 0.01. FIGURE 4 | Grip forces for the hemiparetic (red symbols) and less affected arms (black symbols) for the different grasp configurations (in abscissa). (A) Grip force at
the time of lifting (tl). (B) Maximum grip force during the lifting phase (C) Average force during the holding phase (D) Grip force at the time of release.*Dunn’s post-hoc
p < 0.05; **Dunn’s post-hoc p < 0.01. FIGURE 5 | Temporal data for unloading and lifting phases in the hemiparetic (red symbols) and less affected arms (black symbols) using different grasp
configurations (in abscissa). (A) Duration of the unloading phase. (B) Time difference between maximal grip force and peak acceleration during the lifting phase. (C)
Rate of grip force change during the unloading phase. *Dunn’s post-hoc p < 0.05. FIGURE 5 | Temporal data for unloading and lifting phases in the hemiparetic (red symbols) and less affected arms (black symbols) using different grasp
configurations (in abscissa). (A) Duration of the unloading phase. (B) Time difference between maximal grip force and peak acceleration during the lifting phase. (C)
Rate of grip force change during the unloading phase. *Dunn’s post-hoc p < 0.05. FIGURE 6 | Object orientation for the hemiparetic (red symbols) and less affected arms (black symbols) at: (A) Time of lift and, (B) Time of placement. *Dunn’s
post-hoc p < 0.05; ***Dunn’s post-hoc p < 0.001. FIGURE 6 | Object orientation for the hemiparetic (red symbols) and less affected arms (black symbols) at: (A) Time of lift and, (B) Time of placement. *Dunn’s
post-hoc p < 0.05; ***Dunn’s post-hoc p < 0.001. March 2019 | Volume 10 | Article 240 7 Frontiers in Neurology | www.frontiersin.org Parry et al. Grasp Regulation Post Stroke FIGURE 7 | Frequency of touch and push errors. Holding Phase Grip forces during the holding phase were observed to be
particularly variable from one individual to another (s.d. 9.70 N;
range 3.92–40 N). In the examples provided in Figure 3, the
grip force during holding for patient 1 (panel C1) is more
than twice as great as the grip force for patient 7 (panel C2)
for the same grasp and place task using the pinch grip. Three
patients (10–12) were again observed to saturate load cells during
this phase. Figure 8A provides a comparison of average grip
force during holding when using the pinch grasp. Overall, grip
force during holding was found to vary in relation to grasp
configuration (Kruskall-Wallis p = 0.027; see Figure 4C). On
average, grip force when holding with the top grasp was 12.75 N, Lifting Phase (A) Frequency of touch and push errors made at grasp onset by the hemiparetic (red) and less affected (black) arms. (B) Same data distributed according to the different types of grasps used. (C) Frequency of touch and push errors made at grasp release by the hemiparetic (red) and
less affected (black) arms. (D) Same data distributed according to the different types of grasps used. ***Chi-squared test p < 0.001. FIGURE 7 | Frequency of touch and push errors. (A) Frequency of touch and push errors made at grasp onset by the hemiparetic (red) and less affected (black) arms. (B) Same data distributed according to the different types of grasps used. (C) Frequency of touch and push errors made at grasp release by the hemiparetic (red) and
less affected (black) arms. (D) Same data distributed according to the different types of grasps used. ***Chi-squared test p < 0.001. significantly lower than holding with a palmar-digital grasp at
19.77 N (p = 0.022). of the iBox (examples in Figures 3B1–C1, B2–C2). Several
patients (1, 10–12) were found to have particularly high
maximal grip forces in the lifting phase, to the point where
the load cells were saturated (limit of 40 N) on several trials. While no differences were observed for peak acceleration,
the maximal grip force through the lifting phase varied with
the grasp used (Kruskall-Wallis p = 0.009, Figure 4B) and
post-hoc testing showed that the maximal grip forces were
significantly greater for the palmar-digital than for the top
grasp (p = 0.003). Descent and Placement In the descent phase, average object orientation and standard
deviation were observed to vary with grasp configuration
(Kruskall-Wallis p < 0.001; p = 0.007), post-hoc testing
confirmed that these variables were greater for top grasp than for
pinch (p = 0.011; p = 0.037), precision (p = 0.001; p = 0.047) and
palmar-digital grasps (p = 0.003; p = 0.004). Upon placement of the iBox, certain patients appeared
to control downward acceleration smoothly, whereas others
exhibited important variations in acceleration around the time
of placement, tp, suggesting vibrations due to the impact of the
object on the table (see examples in Figures 3B1,2). Despite this,
no significant differences in grip force at tp were found. Time difference between maximal grip force and peak
acceleration varied with grasp configuration (Kruskall-Wallis
p = 0.02) and the arm used (Kruskall-Wallis p = 0.03; see
Figures 5C, 8B). For example, the average lag time was 185 ms
when using a top grasp, significantly lower than that of 486 ms
when using the palmar-digital grasp (p = 0.02). The deviation of the object from the vertical was greater when
using the hemiparetic arm (alpha angle at tp of 6.38◦) than
for the less affected side (alpha angle at tp of 3.45◦) (Kruskall
Wallis p = 0.012; see Figure 6B). Grasp configuration was also
found to influence object orientation at tp (Kruskal-Wallis p =
0.003). When using top grasp, alpha angle was 8.18◦on average,
significantly greater than for the precision (p = 0.008), pinch (p
= 0.06) and palmar-digital grasps (p = 0.007). Release During the release phase, the force on the bottom face of the
object increased while the grip forces decreased. Those patients
with better functional ability appeared to perform this transition
relatively smoothly (progressive increase of force on bottom face
of iBox and progressive decrease in grip forces, see Figure 3C1). March 2019 | Volume 10 | Article 240 Frontiers in Neurology | www.frontiersin.org 8 Parry et al. Grasp Regulation Post Stroke FIGURE 8 | Examples of individual differences during pinch grasp with hemiparetic (red) and less affected arms (black). (A) Grip force during holding. Each bar
represents median grip force recorded for each patient. (B) Time delay from peak acceleration to maximum grip force. Each bar represents mean of time delay over
three trials. FIGURE 8 | Examples of individual differences during pinch grasp with hemiparetic (red) and less affected arms (black). (A) Grip force during holding. Each bar
represents median grip force recorded for each patient. (B) Time delay from peak acceleration to maximum grip force. Each bar represents mean of time delay over
three trials. A table providing all significant correlation data is provided
in Tables S1–S6. The release phase was comparatively more irregular in patients
with poorer functional ability and occasionally associated with
an impact of the object on the surface of the table in addition to
extraneous touch and push errors (see Figure 3C2). For the precision grip, FME was correlated with the temporal
parameters of the task (positive correlation with the rate of force
change during lifting and placement, inverse correlation with
the duration of unloading and placement phases,) and inversely
correlated with the angle of the object during holding and at
tp. Further to this, the FME sensory function subscore was also
positively correlated with grip force at several stages of the task
(tl, tp, maximal grip force, average grip force during holding),
while the FME motor subscore was positively correlated with
peak acceleration and negatively correlated with the angle of the
object during holding (refer to Tables S2, S3, respectively). DGS
was correlated with the grip force during holding and at tp. Grip force at tr was greater on the hemiparetic side (average
of 0.27 N) than on the less-affected side (0.12 N) (Kruskal-
Wallis p = 0.01; Figure 4D). Release At the same time, grip force at
tr was also observed to vary according to grasp configuration
(Kruskal-Wallis p = 0.032) and post-hoc testing showed that these
forces were significantly higher in top grasp than in precision
grasp (p = 0.017). The occurrence of push errors was found to vary with grasp
configuration (Chi-Squared p < 0.001), the palmar-digital grasp
being associated with the greatest frequency (82% of trials,
see Figure 7D). In the case of top grasp, FAT was inversely correlated with
touch frequency at grip onset, grip force at tg, object angle at tl
and variability of object angle during holding. It was positively
correlated with the rate of force during unloading. The FME
motor subscore was negatively correlated with the duration of
the unloading and loading phases. The JTT was correlated with
temporal parameters during the unloading phase, object angle at
tg and grip force at tl (see Table S5). Correlation of Clinical Measures for
Upper-Limb Function With iBox Variables be correlated with temporal parameters and force parameters
during object release (positive correlation with rate of force
change during release, negative correlation with release phase
duration and grip force at tr). The JTT was correlated with
several temporal parameters (positive correlation with release
phase duration and lag time from maximal grip force to peak
acceleration, negative correlation with rate of force change during
unloading and release) as well as being positively correlated with
object angle at tp. the action sequence (grasping, lifting, holding, placement, and
release) and stability of the hand-held object. Motor performance
was compared across four
different grasp configurations
commonly used in daily life activities for both the hemiparetic
and less affected arms. The results of this study confirmed
the hypothesis that grasp configuration has a significant
effect upon grip force scaling for patients suffering from
hemiparesis (55). The ability to manage object orientation
was reduced in the hemiparetic arm when compared to the
less affected arm while grasp configuration had comparably
less effect. For the palmar-digital grasp, FAT was inversely correlated
with the object angle and object angle variability during holding. FME was correlated with the maximum grip force, the rate
of force change during the unloading and loading phases,
and inversely correlated with the duration of the unloading
and loading phases as well as object angle at tg. The FME
sensory subscore was negatively correlated with object angle
at tl and average object angle during the holding phase,
while the FME motor subscore was associated with temporal
parameters (negative correlation with duration of unloading
and loading phases, negative correlation with rate of force
change during loading and unloading phases). JTT score was
positively correlated with object angle during the holding phase. DGS was correlated with the rate of grip force change during
loading, maximum grip force and average grip force during the
holding phase. Correlation of Clinical Measures for
Upper-Limb Function With iBox Variables A
summary
of
statistically
significant
correlations
of
dynamometer grip-strength (DGS), Fugl Meyer evaluation
(FME) and Frenchay Arm Tests (FAT) scores with iBox variables
for each grasp configuration is provided in Figure 9. Each
line represents a significant Spearman correlation (black) or
negative correlation (red) between a clinical variable (FAT, FMA,
and DGS, on the left) and a biomechanical behavioral variable
(grouped according force, timing and orientation variables). For the pinch grip, FAT was inversely correlated with the
object angle at tl and FME was inversely correlated with the
duration of the loading phase and the grip force at tr. Both
the sensory and motor subscores of the FME were found to March 2019 | Volume 10 | Article 240 Frontiers in Neurology | www.frontiersin.org 9 Parry et al. Grasp Regulation Post Stroke FIGURE 9 | Correlation between clinical data and behavioral variables for the different grasp types. Lines represent significant Spearman correlations (positive in
black, negative in red) between clinical measures and iBox variables. FAT, Frenchay arm test; FME, Fugl Meyer Evaluation; DGS, dynamometer grip strength; Touches
on, frequency of touches before tg; GF, grip force at different time points; D, phase duration; Time lag, time difference between maximal grip force and peak
acceleration during lifting; Alpha, deviation of the iBox from the vertical at the different time points; Alpha var, variability of alpha angle during holding. FIGURE 9 | Correlation between clinical data and behavioral variables for the different grasp types. Lines represent significant Spearman correlations (positive in
black, negative in red) between clinical measures and iBox variables. FAT, Frenchay arm test; FME, Fugl Meyer Evaluation; DGS, dynamometer grip strength; Touches
on, frequency of touches before tg; GF, grip force at different time points; D, phase duration; Time lag, time difference between maximal grip force and peak
acceleration during lifting; Alpha, deviation of the iBox from the vertical at the different time points; Alpha var, variability of alpha angle during holding. be correlated with temporal parameters and force parameters
during object release (positive correlation with rate of force
change during release, negative correlation with release phase
duration and grip force at tr). The JTT was correlated with
several temporal parameters (positive correlation with release
phase duration and lag time from maximal grip force to peak
acceleration, negative correlation with rate of force change during
unloading and release) as well as being positively correlated with
object angle at tp. Grip Force Regulation During Lifting and
Holding The results of this study are generally consistent with previous
research in demonstrating that patients with hemiparesis were
globally capable of regulating grip forces with respect to
load force variations (8, 22–25, 27, 47). Specific impairments
manifested as irregularities in the magnitude and timing of
grip force modulation through the grasping, lifting, holding and
release of the instrumented object. Broadly speaking, excessive grip force has been a notable
feature of quantitative research on object manipulation in
patients with neurological disorders (52). Hermsdorfer et al. reported particularly important grip force increases for holding,
transportation and cyclical vertical movements when using
a pinch grip for the hemiparetic arm of stroke patients
when compared to the less affected arm. This type of “grip
force overshoot” (52) has been interpreted as an increase
in the safety margin between the applied force and the
minimum force necessary to prevent the object from slipping Frontiers in Neurology | www.frontiersin.org March 2019 | Volume 10 | Article 240 Timing and Coordination Errors at Grasp
Initiation and Release Issues with the timing of grip force modulation were most
notable during the unloading and lifting phases of the task
sequence. The increased duration of the grasp time prior to
the object being raised from a flat surface is consistent with
results from prior studies (10, 13, 49, 66) and reflects the
diminished rate of change in grip force during this phase (45,
46, 67). The temporal discrepancy between peak acceleration
and maximum grip force observed for the hemiparetic arm in
this study is typical of a breakdown in the nervous system’s
ability to regulate the coupling of grip forces with load forces. McDonnell et al. (46) previously documented a disruption to
the coupling between grip and load forces in stroke patients
during lifting with a precision grip. The present study expands
upon these results, demonstrating that this effect is consistent
across the pinch, palmar-digital and top grasps. At the same
time, it should be noted that experiments by Hermsdorfer
et al. did not observe similar temporal delays when examining
cyclical vertical movements (48, 49). This suggests that deficits
with temporal coupling for the hemiparetic arm depends upon
the type of activity and supports the postulate that motor
control for rhythmic motion is relatively distinct from discrete
movements (68). Mechanisms for predictive control may be
sufficient to regulate grip force load force coupling in regular,
continuous alternating movement (48) whereas discrete actions
such as lifting would require highly efficient integration of
sensory feedback and corresponding muscular adjustments
(69). Another (non-exclusive) interpretation is that the lifting
and holding task performed by stroke patients with severe
impairment is composed of multiple segmented actions and/or
may be corrupted by irregularities in proximal control of the
arm such that that maximum grip force and acceleration do
not coincide. The specific design of the instrumented object used in this study
allowed us to identify micro errors upon grasp initiation and
object release. The rate of these touch and push errors was greater
for both the hemiparetic and the less affected side than the rates
observed in healthy young adults (57). The increased frequency
of push errors during lifting here is generally consistent with the
observations of McDonnel et al. (46). Similarly, Duque et al. (44)
observed a greater duration between the first touch by the thumb
or index and the onset of grasp forces for children with cerebral
palsy when compared to age-matched controls. Orientation and Stability of the Hand-Held
Object Increased grip force magnitude may reflect compensatory
mechanisms in order to compensate for deficits with sensory
feedback mechanisms (37, 48) or motor deficits involving
poor rate of force development (49). Generalized weakness
however may be difficult to discern during lifting and holding
as grip forces may be comparable to the grip-load force
safety margin. j
The current body of literature on hand-object orientation in
manual dexterity tasks is limited. In the previous study using
the iBox with healthy young adults performing the same tasks,
Martin-Brevet et al. reported that the object was close to
vertical (angle <0.5◦) at the times of lifting and placement
and marginally more variable during holding (<3◦). The values
obtained in the present study are considerably higher, particularly
during holding. Moreover, significant differences between the
hemiparetic and less affected sides were observed (per Figure 6). Whilst not directly measuring object orientation, García Álvarez
et al. (53) previously rated object stability for stroke patients
when grasping daily objects. They found that object stability
was correlated with upper-limb strength (Medical Research
Council Scale) and spasticity (Modified Ashworth Scale). Here,
quantitative data on iBox orientation resulted in multiple
correlations with the Frenchay Arm Test (FAT), although
the limited range of scores means caution should be taken
with interpretation. Nonetheless, these combined observations
suggest that global upper-limb strength is a key factor in
regulating the vertical object orientation during lifting, holding
and placement tasks. DISCUSSION This study investigated the hand function of stroke patients. Using an instrumented object, several aspects of dexterity were
examined: grip force regulation, timing and coordination of March 2019 | Volume 10 | Article 240 Frontiers in Neurology | www.frontiersin.org 10 Parry et al. Grasp Regulation Post Stroke Orientation and Stability of the Hand-Held
Object (49). Large security margins used by stroke patients have
previously been associated with the level of somatosensory
impairment (37, 48). Nonetheless, Wenzelburger et al. also
observed moderate increases in grip force during holding
in patients with purely motor capsular stroke (45). In the
present study, we observed limited correlation between grip
force magnitude with either the FME sensory or motor
subscores obtained on the hemiparetic upper limb. Furthermore,
consistent with the observation of Nowak et al. (8), a
number of patients in the present study also presented with
excessive grip forces in their less affected arm (e.g., Figure 8A,
patients 1,10,11,12;). Perhaps most striking though was the
important variability between patients, with grip force during
holding in the range of 4 N−40 N. These vast differences
in grip forces underscore the fact that stroke patients are a
heterogeneous population and that a clinical presentation of
hemiparesis alone is not sufficient for one to presume the
magnitude, nor the laterality of changes in grip force scaling. Increased grip force magnitude may reflect compensatory
mechanisms in order to compensate for deficits with sensory
feedback mechanisms (37, 48) or motor deficits involving
poor rate of force development (49). Generalized weakness
however may be difficult to discern during lifting and holding
as grip forces may be comparable to the grip-load force
safety margin. (49). Large security margins used by stroke patients have
previously been associated with the level of somatosensory
impairment (37, 48). Nonetheless, Wenzelburger et al. also
observed moderate increases in grip force during holding
in patients with purely motor capsular stroke (45). In the
present study, we observed limited correlation between grip
force magnitude with either the FME sensory or motor
subscores obtained on the hemiparetic upper limb. Furthermore,
consistent with the observation of Nowak et al. (8), a
number of patients in the present study also presented with
excessive grip forces in their less affected arm (e.g., Figure 8A,
patients 1,10,11,12;). Perhaps most striking though was the
important variability between patients, with grip force during
holding in the range of 4 N−40 N. These vast differences
in grip forces underscore the fact that stroke patients are a
heterogeneous population and that a clinical presentation of
hemiparesis alone is not sufficient for one to presume the
magnitude, nor the laterality of changes in grip force scaling. Timing and Coordination Errors at Grasp
Initiation and Release These kinds of
touch errors may be seen as evidence of an impairment in the
transition between reach and grasp. We would suggest that the
apparent lack of synchrony between thumb and finger movement
as they close upon or withdraw from an object may be associated
with the hand and palmar arch pre-shaping deficits previously
documented by Sangole et al. (70). Effect of Grasp Configuration p
g
The effects of hand configuration upon grasp regulation during
lifting, holding and object placement represents the central
finding of the present study. As hypothesized, the use of
the different grasps (precision, top, pinch, palmar-digital) had
important effects upon the magnitude and timing of grip force
adjustments, object orientation as well as the frequency of
errors. Most notably, grip forces were greatest when using
the palmar-digital grasp. This observation is consistent with
prior results in healthy adult subjects (57). Whilst coupling
between grip forces and load forces was apparent across all
the grasp combinations, the time delay between maximum grip
and peak acceleration was greater in the palmar-digital grasp March 2019 | Volume 10 | Article 240 Frontiers in Neurology | www.frontiersin.org 11 Grasp Regulation Post Stroke Parry et al. than the top grasp. In an experimental paradigm involving
cyclic vertical movements, Flanagan and Tresilian similarly
observed temporal delays in the coordination between grip forces
and load forces when using a palmar-digital grasp (34). They
suggested that these differences may reflect diminished tactile
information in certain parts of the hand. A lower density of
glabrous skin receptors through the palm than in the thumb
and fingers may limit the precision of fine tuning abilities (32). The increased grip force observed in palmar-digital grasp would
thus represent an increased safety margin to account for this
limitation. In the present study, we found that the frequency
of touch errors was greatest when initiating a palmar-digital
grasp and that this grasp configuration was associated with
variable object orientation at tl. Importantly, stroke patients
with more important impairments tend to use palmar-digital
grasp configurations more consistently than less impaired stroke
patients or healthy adults (53). Therefore, whilst this behavior
may assist stroke patients to compensate for reduced dexterity
or muscle strength (53), the results presented here indicate that
this preferential use of the palmar-digital grasping strategies may
impact upon task execution in terms of grip force economy,
temporal precision of grip force adjustments, and stability of the
hand-held object. previously described kinematic patterns where patients move
their hand around an object in the approach phase, a strategy
which may serve to compensate for weakness in the wrist
extensors and/or finger flexors (54, 76). Effect of Grasp Configuration In other terms, this
could be thought of as “leading with the fingers” in preparing
for object handling with the hemiparetic arm as opposed to
“leading with the thumb” when preparing for object handling
with the less affected arm. Release phase transitions were also
characterized by asynchrony between the thumb and fingers on
the hemiparetic side. Certain studies have suggested that this
type of issue is linked to a distinct impairment of the grasp
release mechanisms (77, 78). At the same time, such an error
could also conceivably be hindered by limitations with proximal
control as the patient attempts to withdraw their hand. Future
studies should seek to combine kinematic analysis of upper-limb
movement with measures from instrumented objects in order
to understand patterns of coordination across the arm, hand
and object as an ensemble. Finally, as evoked above (section
Orientation and Stability of the Hand-Held Object), it is likely
that upper-limb strength is important for maintaining vertical
object orientation. The specific increases in the variability of
object orientation at tl and tp seen in the hemiparetic arm (per
Figure 6) further suggest that patients have the greatest difficulty
maintaining object stability in the transition of the object to and
from the working surface. In contrast to this, the top grasp configuration was
associated with lower grip forces and comparably lower temporal
discrepancies between peak acceleration and maximal grip
force. The increased levels of wrist flexion when using the
top grasp configuration may contribute to these differences. In
healthy subjects, maximum grip-strength varies according to
wrist position (71–73) and influences grip force regulation (74). Of course, when in an extended position, extrinsic flexors of
the wrist and fingers are stretched, and conversely, a flexed
position brings about passive finger extension (tenodesis effect). Increased flexor tone is common following a stroke, hence
this effect may be exaggerated (75). Additionally, it has been
proposed that the modification of afferent input associated
with the changes in muscle length across the wrist could
affect cortical and spinal excitability (74). Allowing a stroke
patient to use a top grasp may thus limit these passive
increases in muscle tension and further inhibit (excessive)
neurological drive. Regardless of the precise mechanisms
involved, the increased temporal precision of grip force
adjustments when using a top grasp may be informative
in clinical practice. Effect of Grasp Configuration It would suggest that use of top grasp
hand configurations may be an adaptive strategy to assist
stroke patients with tasks specifically requiring responsive grip
force adjustments. LIMITATIONS OF THE STUDY The principal limitation in the design of this study is the lack
of control group. Whilst one of our previous studies involved
a similar protocol, data was obtained only for young adults. In the absence of an age matched control group we have
limited our analysis to differences in grasp regulation between
the hemiparetic and less affected arms of patients following
a stroke. Secondly, whilst the iBox affords certain advantages
(ease of manipulation, multiple integrated sensors), it measures
exlusively those forces normal to the surface of each face—it is
unable to estimate tangential forces or torque. The choice for
linear load cells was motivated by the possibility of an affordable
object which could be used in the clinic or at home (58). Finally, the design of this study allows for considerable
variation in surface contact. The coefficient of friction between
a hand and an object varies according to the properties
of a subject’s skin (79) and the texture of the object (31). Increasing surface contact increases the coefficient of friction
(80), a factor which was not controlled for in this experiment
from one grasp configuration to the next. Consequently, the
analysis of force exchanges with the iBox has certain limits for
comparison across the grasping strategies. It is interesting to
note however, that the subjects employed greater grasp forces
when using the palmar-digital grasp despite having a greater
coefficient of friction. This underscores that grip force regulation
is contingent upon numerous biomechanical and neurological
variables. In the present study, we consider the measurable
behaviors as representative of the strategies associated with each
grasp configuration. Frontiers in Neurology | www.frontiersin.org REFERENCES 9. Graziadio S, Tomasevic L, Assenza G, Tecchio F, Eyre JA. The myth of the
‘unaffected’ side after unilateral stroke: Is reorganisation of the non-infarcted
corticospinal system to re-establish balance the price for recovery? Exp Neurol. (2012) 238:168–75. doi: 10.1016/j.expneurol.2012.08.031 1. Miller EL, Murray L, Richards L, Zorowitz RD, Bakas T, Clark P, et al. Comprehensive overview of nursing and interdisciplinary rehabilitation
care of the stroke patient: a scientific statement from the American Heart
Association. Stroke. (2010) 41:2402–48. doi: 10.1161/STR.0b013e3181e7512b 10. Quaney BM, Perera S, Maletsky R, Luchies CW, Nudo RJ. Impaired
grip force modulation in the ipsilesional hand after unilateral middle
cerebral artery stroke. Neurorehabil Neural Repair. (2005) 19:338–49. doi: 10.1177/1545968305282269 2. Veerbeek JM, Kwakkel G, van Wegen EEH, Ket JCF, Heymans MW. Early
prediction of outcome of activities of daily living after stroke: a systematic
review. Stroke. (2011) 42:1482–8. doi: 10.1161/STROKEAHA.110.604090 2. Veerbeek JM, Kwakkel G, van Wegen EEH, Ket JCF, Heymans MW. Early
prediction of outcome of activities of daily living after stroke: a systematic
review. Stroke. (2011) 42:1482–8. doi: 10.1161/STROKEAHA.110.604090 11. Ekstrand E, Rylander L, Lexell J, Brogårdh C. Perceived ability to perform daily
hand activities after stroke and associated factors: a cross-sectional study. BMC
Neurol. (2016) 16:208. doi: 10.1186/s12883-016-0733-x 3. Twitchell TE. The restoration of motor function following hemiplegia in man. Brain. (1951) 74:443–80. doi: 10.1093/brain/74.4.443 12. Xu J, Ejaz N, Hertler B, Branscheidt M, Widmer M, Faria AV, et al. Separable
systems for recovery of finger strength and control after stroke. J Neurophysiol. (2017) 118:1151–63. doi: 10.1152/jn.00123.2017 4. Buma F, Kwakkel G, Ramsey N. Understanding upper limb recovery after
stroke. Restor Neurol Neurosci. (2013) 31:707–22. doi: 10.3233/RNN-130332 5. Byblow WD, Stinear CM, Barber PA, Petoe MA, Ackerley SJ. Proportional
recovery after stroke depends on corticomotor integrity. Ann Neurol. (2015)
78:848–59. doi: 10.1002/ana.24472 5. Byblow WD, Stinear CM, Barber PA, Petoe MA, Ackerley SJ. Proportional
recovery after stroke depends on corticomotor integrity. Ann Neurol. (2015)
78:848–59. doi: 10.1002/ana.24472 13. Raghavan P, Krakauer JW, Gordon AM. Impaired anticipatory control of
fingertip forces in patients with a pure motor or sensorimotor lacunar
syndrome. Brain. (2006) 129:1415–25. doi: 10.1093/brain/awl070 6. Schwerin S, Dewald JPA, Haztl M, Jovanovich S, Nickeas M, MacKinnon
C. Ipsilateral versus contralateral cortical motor projections to a shoulder
adductor in chronic hemiparetic stroke: implications for the expression of arm
synergies. Exp Brain Res. (2008) 185:509–19. doi: 10.1007/s00221-007-1169-8 14. ACKNOWLEDGMENTS We would like to extend a special thank you to Sara Panera,
Ariane Bozon-Verduraz and Marie-Laure Descamps of the
physiotherapy department of the Salpêtrière Hospital, as well
as to Claire Kemlin, Eric Moulton and Charlotte Rosso of the
Institut du Cerveau et de la Moelle Epiniere for their assistance
in the recruitment of patients for this study. These observations may assist in understanding the functional
implications of compensatory grasp strategies in patients with
hemiparesis and assist with facilitating adaptive prehension
patterns in the context of rehabilitation. That is to say, whilst
patients suffering from stroke may have exhibit preferences for
taking objects with palmar-digital grasp configurations (53), this
strategy may have negative effects upon grip force economy and
temporal precision of grip force adjustments. The use of top
grasp may thus be indicated in order to facilitate more responsive
control in day to day object handling for this population. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fneur. 2019.00240/full#supplementary-material CONCLUSION AND PERSPECTIVES To surmise, the magnitude and temporal precision of grip
force adjustments varied according to the different grasp
configurations
employed
by
hemiparetic
patients. More
specifically, grip forces were consistently greatest when patients
used a palmar-digital grasp and lowest when using a top
grasp. Similarly, the time delay between peak acceleration and
maximum grip during lifting were highest in palmar-digital
grasp and lowest top grasp. Use of the hemiparetic arm resulted
in greater variability in the vertical orientation of the object, in
particular upon lifting the object from and placing the object
upon the working surface. Both grasp configuration and use of
the hemiparetic arm were found to contribute to the occurrence
of touch and push errors when establishing grasp or releasing the
object. Our interpretation of this is that structural aspects of hand
configuration contribute considerably to the grip force scaling
while the effects of hemiparesis on upper-limb coordination
more globally bring about deficits with object control and
orientation at transitions in task sequence such as grasp onset,
lifting, object placement and release. RP, SM, and PP-D were responsible for patient recruitment. Data
collection
and
patient
evaluations
were
carried
out
by
RP
and
SM. The
iBox
device
and
software
was
designed
and
adapted
by
NJ. Data
analysis
was
performed by RP, NJ, and AR-B while VM-P participated
in the discussions. The manuscript was drafted by RP
and
AR-B. All
authors
participated
in
the
revision
of
the manuscript. RP, SM, and PP-D were responsible for patient recruitment. Data
collection
and
patient
evaluations
were
carried
out
by
RP
and
SM. The
iBox
device
and
software
was
designed
and
adapted
by
NJ. Data
analysis
was
performed by RP, NJ, and AR-B while VM-P participated
in the discussions. The manuscript was drafted by RP
and
AR-B. All
authors
participated
in
the
revision
of
the manuscript. Effects of Side Differences in grasp regulation between the hemiparetic and
less affected arms were observed most notably in the frequency
of errors at grasp onset, the duration of the unloading phase
and object angle at lifting and placement. Interestingly, the
frequency of touch errors on grasping with the hemiparetic side
was associated with sub-threshold finger contact, whereas in the
less affected arm, touches they were more frequently associated
with sub-threshold thumb contact. This appears consistent with March 2019 | Volume 10 | Article 240 Frontiers in Neurology | www.frontiersin.org 12 Grasp Regulation Post Stroke Parry et al. FUNDING This work was made possible by funding from the Sorbonne
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DA. Sensorimotor
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flexion/extension on force scaling. Clin Neurophysiol. (2009) 120:1588–95. doi: 10.1016/j.clinph.2009.05.007 61. Wade DT, Langton-Hewer R, Wood VA, Skilbeck CE, Ismail HM. The
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usage in daily household and machine shop tasks. IEEE Trans Haptics. (2013)
6:296–308. doi: 10.1109/TOH.2013.6 75. Gillen G. Stroke Rehabilitation: A Function-based Approach. Mosby, MO; East
Lansing, MI: Michigan State University; Elsevier Health Sciences (2015). 76. Roby-Brami A, Jacobs S, Bennis N, Levin MF. Hand orientation for
grasping and arm joint rotation patterns in healthy subjects and hemiparetic
stroke patients. Brain Res. (2003) 969:217–29. doi: 10.1016/S0006-8993(03)
02334-5 63. Westfall PH, Young SS. Resampling-Based Multiple Testing: Examples
and Methods for p-Value Adjustment, 1st ed. REFERENCES New York, NY: Wiley-
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the aging adult as determined by the Jebsen Test of Hand Function. Phys Ther. (1992) 72:373–7. doi: 10.1093/ptj/72.5.373 77. Lindberg PG, Roche N, Robertson J, Roby-Brami A, Bussel B, Maier
MA. Affected and unaffected quantitative aspects of grip force control
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in chronic stroke: grip formation and release phases. Exp Brain Res. (2011)
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pinch grip lifts under somatosensory guidance: comparison between people
with stroke and healthy controls. Arch Phys Med Rehabil. (2006) 87:418–29. doi: 10.1016/j.apmr.2005.11.018 79. Adams
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12.0467 67. Avarello M, Bolsinger P, Mai N. Fast repetitive force changes in hemiparetic
and cerebellar patients. Eur Arch Psychiatry Neurol Sci. (1988) 237:135–8. doi: 10.1007/BF00451280 80. Han H-Y, Shimada A, Kawamura S. Analysis of friction on human fingers and
design of artificial fingers. In: Proceedings of IEEE International Conference
on Robotics and Automation. Vol. 4. Minneapolis, MN (1996). p. 3061–6. doi: 10.1109/ROBOT.1996.509177 68. Hogan N, Sternad D. On rhythmic and discrete movements: reflections,
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Humphrey DR, Freund H-J, editors. Motor Control Concepts and Issues. Chichester, NY: John Wiley & Sons Ltd (1991). p. 331–55. Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
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199:59–70. doi: 10.1007/s00221-009-1972-5 Copyright © 2019 Parry, Macias Soria, Pradat-Diehl, Marchand-Pauvert, Jarrassé
and Roby-Brami. This is an open-access article distributed under the terms of
the Creative Commons Attribution License (CC BY). Frontiers in Neurology | www.frontiersin.org March 2019 | Volume 10 | Article 240 REFERENCES The use, distribution or
reproduction in other forums is permitted, provided the original author(s) and the
copyright owner(s) are credited and that the original publication in this journal
is cited, in accordance with accepted academic practice. No use, distribution or
reproduction is permitted which does not comply with these terms. Copyright © 2019 Parry, Macias Soria, Pradat-Diehl, Marchand-Pauvert, Jarrassé
and Roby-Brami. 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. 71. Kattel BP, Fredericks TK, Fernandez JE, Lee DC. The effect of upper-extremity
posture on maximum grip strength. Int J Ind Ergon. (1996) 18:423–9. doi: 10.1016/0169-8141(95)00105-0 72. O’Driscoll SW, Horii E, Ness R, Cahalan TD, Richards RR, An KN. The
relationship between wrist position, grasp size, and grip strength. J Hand Surg. (1992) 17:169–77. March 2019 | Volume 10 | Article 240 Frontiers in Neurology | www.frontiersin.org 15
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Effects of gold mining on the general population
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International journal of recent scientific research
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cc-by
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INTRODUCTION The local demand for gold in India has always been ona rise. According to national survey data there are only three
functioning gold mines in India namely Hirabuddini mines in
Jharkhand, and Hatti, Uti gold minesinKarnataka.1 Arsenic also serves as an important constituent of gold mining. It has a low abundance in the earth’s crust so it exists in the
form of sulfide bearing minerals. The mining of gold and base
metals is associated with sulfide mineralization thus ending up
releasing arsenic in the environment.6 The village of Hutti is located in Raichur District in the Indian
state of Karnataka. This mine is probably one of the most
ancient metal mines in the world. There was an extensive
increase in gold mining in the area between 1890 and 1920. The industry closed down in 1920 due to technical difficulties
and lack of funds. After the end of the second world war, the
production started in September 1948, at the rate of 130 tonns
of ore per day. By 1972 this rate had progressively increased to
600 tonnes of ore per day.2 The Company was originally
formed in 1947 as the Hyderabad Gold mines Company Ltd.,
with the Hyderabad State Government holding a majority of
the shares. With the re-organisation of the States in 1956, the
Company was transferred to Mysore State (now Karnataka
State) and became Hutti Gold Mines Limited.2,3 Hg and As are known toxicants that are hazardous to humans,
wildlife and domestic animals and may accumulate in the
environment causing serious damage to ecosystems and human
health. Studies conducted throughout the world where gold
mining occurs have reported the presence of high mercury and
arsenic concentrations in human urine, breast milk, blood,hair,
and nails, and in plant and fish samples.7 High levels of Hg and As have been linked to detrimental
effects on humans, such as skin problems, cancer, high blood
pressure, cardiovascular diseases, and neurological and
reproductive disorders among others.7 The potential harm of
these toxicants to pregnant women, their fetuses and young
children is an area of special concern. Women in these gold
mining regions may engage in geophagy, or earth-eating,
behaviors, which are common in artisanalmining areas due to
poor nutritional status and cultural acceptance of this practice.7 Mercury, being an active constituent of the Gold mining
industry dates back to the 16th century. Key Words: Gold mining, Mercury, Arsenic, Gold mining, Mercury, Arsenic,
pigmentation, human health, Hutti village pigmentation, human health, Hutti village Copyright © Amitha Hegde et al, 2017, 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 work is
properly cited. Copyright © Amitha Hegde et al, 2017, 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 work is
properly cited. brought about due to gold mining procedures (Lacerda 1997)5. There was an exceptional hike in the gold prices experienced in
the country in the 1970s which further led to extensive gold
mining for a very long period. brought about due to gold mining procedures (Lacerda 1997)5. There was an exceptional hike in the gold prices experienced in
the country in the 1970s which further led to extensive gold
mining for a very long period. *Corresponding author: Ankit Varun
Department of Pedodontics, A.B. Shetty Memorial Institute of Dental Sciences, NITTE University, Mangalore ABSTRACT Hutti, one of the three functioning gold mines in India, is located in Raichur District of Karnataka. Mercury and arsenic, being active constituents of the Gold mining industry are known toxicants that
are hazardous to humans, wildlife and domestic animals and may accumulate in the environment
causing serious damage to ecosystems and human health. Hence a questionnaire based survey was
conducted amongst the local population of the Hatti village. Most of the people who participated in
the survey were aware of the ill effects of mining. In growing age these signs and symptoms are
more pronounced than elders. Some of the most common health problems noticed were dark brown
pigmentation of the skin (47%), loose stool, breathing difficulties and fatigue. Female population
reported 3% abortions and miscarriages. The poor accessibility and unavailability of health care
centers was the main reason for the increased incidence of health issues. Article History:
Received 17th December, 2016
Received in revised form 21st
January, 2017
Accepted 05th February, 2017
Published online 28th March, 2017 Research Article DOI: http://dx.doi.org/10.24327/ijrsr.2017.0803.0115 ARTICLE INFO
Article History:
Received 17th December, 2016
Received in revised form 21st
January, 2017
Accepted 05th February, 2017
Published online 28th March, 2017
Key Words:
Gold mining, Mercury, Arsenic,
pigmentation, human health, Hutti village ARTICLE INFO ISSN: 0976-3031 Available Online at http://www.recentscientific.com
CODEN: IJRSFP (USA) International Journal of
Recent Scientific
Research International Journal of Recent Scientific Research
Vol. 8, Issue, 3, pp. 16266-16269, March, 2017 DOI: 10.24327/IJRSR MATERIALS AND METHOD
MATERIALS AND METHOD
MATERIALS AND METHOD 4)
The poor accessibility and unavailability of health care centers
was the main reason for the increased incidence of health
Fig 1
Fig 2
22
11
No
Don’t Know
Knowledge about the ill effects of mining
39
50
Once a month
More than once a
month
Frequency of child's leave from school Fig 1
Fig 1
67
0
20
40
60
80
Yes
Percentage
Knowledge about the ill effects of mining
Fig 1
22
11
No
Don’t Know
Knowledge about the ill effects of mining Knowledge about the ill effects of mining
Knowledge about the ill effects of mining The study was conducted at the village of Hutti located in
Raichur District in the Indian state of Karnataka. The estimated
population of the area of Hutti Gold Mines, as of
2001 India census is 14,716 out of which 9% of the population
is under six years of age. Males constitute 51% of the
population and females 49%. Hutti Gold Mines has an average
literacy rate of 24%. Most people in the village depend on
water from rivers, natural wells, and wells constructed for
human and livestock consumption and for mining. The study was conducted at the village of Hutti located in
Raichur District in the Indian state of Karnataka. The estimated
population of the area of Hutti Gold Mines, as of
2001 India census is 14,716 out of which 9% of the population
is under six years of age. Males constitute 51% of the
population and females 49%. Hutti Gold Mines has an average
literacy rate of 24%. Most people in the village depend on
water from rivers, natural wells, and wells constructed for
human and livestock consumption and for mining. The study was conducted at the village of Hutti located in
Raichur District in the Indian state of Karnataka. The estimated
population of the area of Hutti Gold Mines, as of
2001 India census is 14,716 out of which 9% of the population
is under six years of age. Males constitute 51% of the
population and females 49%. Hutti Gold Mines has an average
literacy rate of 24%. Most people in the village depend on
water from rivers, natural wells, and wells constructed for
human and livestock consumption and for mining. Using a cross sectional design with a simple sampling
technique, 100 people were recruited into the study and
participated in the structured questionnaire survey. MATERIALS AND METHOD
MATERIALS AND METHOD
MATERIALS AND METHOD All of the
individuals approached agreed to participate, (response rate
was 100%). These participants included the teachers and
attenders of the Primary school of Hutti Village and also the
general village people residing and working at the adjoining
streets. Written informed consent was obtained from each
participant. Since mining occurs in close proximity to housing
and other economic activities, all people over the age of 18
were considered eligible to participate. The individuals who
were recent immigrants of the place were excluded from the
survey. A single researcher carried out the questionnaire
survey, structured in English and provided to the participants. Those who needed it to be translated to the native language of
Kannada, were assisted by the researchers. Using a cross sectional design with a simple sampling
technique, 100 people were recruited into the study and
participated in the structured questionnaire survey. All of the
individuals approached agreed to participate, (response rate
was 100%). These participants included the teachers and
attenders of the Primary school of Hutti Village and also the
general village people residing and working at the adjoining
streets. Written informed consent was obtained from each
participant. Since mining occurs in close proximity to housing
and other economic activities, all people over the age of 18
were considered eligible to participate. The individuals who
were recent immigrants of the place were excluded from the
survey. A single researcher carried out the questionnaire
survey, structured in English and provided to the participants. Those who needed it to be translated to the native language of
Kannada, were assisted by the researchers. Using a cross sectional design with a simple sampling
technique, 100 people were recruited into the study and
participated in the structured questionnaire survey. All of the
individuals approached agreed to participate, (response rate
was 100%). These participants included the teachers and
attenders of the Primary school of Hutti Village and also the
general village people residing and working at the adjoining
streets. Written informed consent was obtained from each
participant. Since mining occurs in close proximity to housing
and other economic activities, all people over the age of 18
were considered eligible to participate. The individuals who
were recent immigrants of the place were excluded from the
survey. A single researcher carried out the questionnaire
survey, structured in English and provided to the participants. INTRODUCTION Mercury is used in
amalgamation and concentration of gold and thus is a major
source of contamination in developing countries (Rojas et al
2001).4 The release of mercury in the biosphere is said to have
exceeded 260,000 tons between the year 1550 and 1930, *Corresponding author: Ankit Varun
Department of Pedodontics, A.B. Shetty Memorial Institute of Dental Sciences, NITTE University, Mangalore Amitha Hegde et al., Effects of Gold Mining on The General Population
Amitha Hegde et al., Effects of Gold Mining on The General Population
Amitha Hegde et al., Effects of Gold Mining on The General Population More alarming is the fact that even low Hg exposure in
mothers due to the contaminants released by gold mining could
result in the fetus being exposed to high levels of Hg as it is
concentrated by a factor of ten in the fetus relative to the
mother. Thus, even a woman with a low level of mercury
exposure could give birth to a child with significant birth
defects.7 In addition, Hg exposure due to transmission through
breast milk could have an effect on the healthy development of
infants.8 Finally, infants and children are sometimes directly
exposed to the mining processes themselves, since mothers
often have their children at the mine site and processing areas
while they are working and sometime young children even
participate in mining as workers.9
More alarming is the fact that even low Hg exposure in
mothers due to the contaminants released by gold mining could
result in the fetus being exposed to high levels of Hg as it is
concentrated by a factor of ten in the fetus relative to the
mother. RESULTS
RESULTS
RESULTS The age of the participants ranged from 18 years to 58 years;
however, most were between 25 and 38 years of age. Of the
hundred participants who participated in the survey, 39% lived
in Hutti for more than ten years, and 34% lived in the area for
five to ten years, remaining 27% lived for less than five years. Hutti village was the main center for mining activities in
Raichur. Amongst the study population, the proportion of
participants who were aware of the gold mining were 81% and
rest were unaware of the activity. The age of the participants ranged from 18 years to 58 years;
however, most were between 25 and 38 years of age. Of the
hundred participants who participated in the survey, 39% lived
in Hutti for more than ten years, and 34% lived in the area for
five to ten years, remaining 27% lived for less than five years. Hutti village was the main center for mining activities in
Raichur. Amongst the study population, the proportion of
participants who were aware of the gold mining were 81% and
rest were unaware of the activity. The age of the participants ranged from 18 years to 58 years;
however, most were between 25 and 38 years of age. Of the
hundred participants who participated in the survey, 39% lived
in Hutti for more than ten years, and 34% lived in the area for
five to ten years, remaining 27% lived for less than five years. Hutti village was the main center for mining activities in
Raichur. Amongst the study population, the proportion of
participants who were aware of the gold mining were 81% and
rest were unaware of the activity. Most of the people who participated (67%) in the survey were
aware of the ill effects of mining. (Fig. 1) Around 30% people
said that the quality of drinking water has improved. Gold
mining and its byproducts are known to cause lots of health
issues. In growing age these signs and symptoms are more
pronounced than elders. So the next question was to see the
frequency of children taking leave from school due to health
issues. 50% and 39% children took leave from school once a
month and more than once a month respectively. (Fig. 2)
Most of the people who participated (67%) in the survey were
aware of the ill effects of mining. (Fig. MATERIALS AND METHOD
MATERIALS AND METHOD
MATERIALS AND METHOD Those who needed it to be translated to the native language of
Kannada, were assisted by the researchers. Fig 1
Fig 1
Fig 1 Fig 2
Fig 2
11
0
10
20
30
40
50
60
Once a week
Percentage
Frequency of child's leave from school
Fig 2
39
50
Once a month
More than once a
month
Frequency of child's leave from school Frequency of child's leave from school
Frequency of child's leave from school Fig 2
Fig 2
Fig 2 The survey consisted of a total of 20 questions which included
categorical questions (“Yes”, “No”, “Don’t know”) and open-
ended questions. Questions pertaining to their quality of life
and the source, quality of drinking water were included so as to
identify the cause for the prevailing health issues. Participant’s
knowledge of the ill effects of gold mining was determined
from basic questions regarding their awareness of associated
health impacts. Questions were also directed at the effects on
the general health of their children and prevalence of any dental
abnormaltites observed with the sudden increase of gold
mining in the region. The survey consisted of a total of 20 questions which included
categorical questions (“Yes”, “No”, “Don’t know”) and open-
ended questions. Questions pertaining to their quality of life
and the source, quality of drinking water were included so as to
identify the cause for the prevailing health issues. Participant’s
knowledge of the ill effects of gold mining was determined
from basic questions regarding their awareness of associated
health impacts. Questions were also directed at the effects on
the general health of their children and prevalence of any dental
abnormaltites observed with the sudden increase of gold
mining in the region. The survey consisted of a total of 20 questions which included
categorical questions (“Yes”, “No”, “Don’t know”) and open-
ended questions. Questions pertaining to their quality of life
and the source, quality of drinking water were included so as to
identify the cause for the prevailing health issues. Participant’s
knowledge of the ill effects of gold mining was determined
from basic questions regarding their awareness of associated
health impacts. Questions were also directed at the effects on
the general health of their children and prevalence of any dental
abnormaltites observed with the sudden increase of gold
mining in the region. Some of the most common health problems noticed were dark
brown pigmentation of the skin (47%), (Fig. RESULTS
RESULTS
RESULTS 1) Around 30% people
said that the quality of drinking water has improved. Gold
mining and its byproducts are known to cause lots of health
issues. In growing age these signs and symptoms are more
pronounced than elders. So the next question was to see the
frequency of children taking leave from school due to health
issues. 50% and 39% children took leave from school once a
month and more than once a month respectively. (Fig. 2)
Most of the people who participated (67%) in the survey were
aware of the ill effects of mining. (Fig. 1) Around 30% people
said that the quality of drinking water has improved. Gold
mining and its byproducts are known to cause lots of health
issues. In growing age these signs and symptoms are more
pronounced than elders. So the next question was to see the
frequency of children taking leave from school due to health
issues. 50% and 39% children took leave from school once a
month and more than once a month respectively. (Fig. 2) Hence a questionnaire based survey was conducted amongst
the local population of the Hatti village. The primary objective
of this survey was to assess community risk knowledge and
perception of potential Mercury and Arsenic toxicity and/or
exposure from artisanal gold mining in the Hatti village
locality. Hence a questionnaire based survey was conducted amongst
the local population of the Hatti village. The primary objective
of this survey was to assess community risk knowledge and
perception of potential Mercury and Arsenic toxicity and/or
exposure from artisanal gold mining in the Hatti village
locality. Hence a questionnaire based survey was conducted amongst
the local population of the Hatti village. The primary objective
of this survey was to assess community risk knowledge and
perception of potential Mercury and Arsenic toxicity and/or
exposure from artisanal gold mining in the Hatti village
locality. INTRODUCTION Thus, even a woman with a low level of mercury
exposure could give birth to a child with significant birth
defects.7 In addition, Hg exposure due to transmission through
breast milk could have an effect on the healthy development of
infants.8 Finally, infants and children are sometimes directly
exposed to the mining processes themselves, since mothers
often have their children at the mine site and processing areas
while they are working and sometime young children even
participate in mining as workers.9
More alarming is the fact that even low Hg exposure in
mothers due to the contaminants released by gold mining could
result in the fetus being exposed to high levels of Hg as it is
concentrated by a factor of ten in the fetus relative to the
mother. Thus, even a woman with a low level of mercury
exposure could give birth to a child with significant birth
defects.7 In addition, Hg exposure due to transmission through
breast milk could have an effect on the healthy development of
infants.8 Finally, infants and children are sometimes directly
exposed to the mining processes themselves, since mothers
often have their children at the mine site and processing areas
while they are working and sometime young children even
participate in mining as workers.9 MATERIALS AND METHOD
MATERIALS AND METHOD
MATERIALS AND METHOD Some of the most common health problems noticed were dark
brown pigmentation of the skin (47%), (Fig. 5) loose stool
(38%), breathing difficulties (49%) and fatigue
(47%). Around 11% people noticed loss of sensation of feet and hands
especially towards the toes and fingers. When dental health was
considered around 55%people reported with white spot lesions
on their tooth surface. (Fig. 3) When the female population was
surveyed, around 3% reported abortions and miscarriages. (Fig. 4)
The poor accessibility and unavailability of health care centers
was the main reason for the increased incidence of health
Fig 1
Fig 2
Some of the most common health problems noticed were dark
brown pigmentation of the skin (47%), (Fig. 5) loose stool
(38%), breathing difficulties (49%) and fatigue
(47%). Around 11% people noticed loss of sensation of feet and hands
especially towards the toes and fingers. When dental health was
considered around 55%people reported with white spot lesions
on their tooth surface. (Fig. 3) When the female population was
surveyed, around 3% reported abortions and miscarriages. (Fig. 4)
The poor accessibility and unavailability of health care centers
was the main reason for the increased incidence of health
Fig 1
Fig 2
67
0
20
40
60
80
Yes
Percentage
Knowledge about the ill effects of mining
11
0
10
20
30
40
50
60
Once a week
Percentage
Frequency of child's leave from school
Some of the most common health problems noticed were dark
brown pigmentation of the skin (47%), (Fig. 5) loose stool
(38%), breathing difficulties (49%) and fatigue
(47%). Around 11% people noticed loss of sensation of feet and hands
especially towards the toes and fingers. When dental health was
considered around 55%people reported with white spot lesions
on their tooth surface. (Fig. 3) When the female population was
surveyed, around 3% reported abortions and miscarriages. (Fig. MATERIALS AND METHOD
MATERIALS AND METHOD
MATERIALS AND METHOD Mining activities are largely concentrated in rura
areas that have very little infrastructure, and the individual
undertaking informal mining generally lack education, training
management skills and essential equipment for safe mining
Fig 3
Fig 4
Fig 5
Fig 6 Fig 4
3
40
0
10
20
30
40
50
60
Yes
No
Percentage
Frequency of abortions and miscarriages
Fig 4
57
Don’t know
Frequency of abortions and miscarriages
Fig 4 In the present study area in a northeastern region of Karnataka
(Hutti), India, factors impacting the natural secondary
geochemical dispersion of Arsenic and mercury in groundwater
and soil can depend upon factors such as topography, water
table depth, drainage, soil type, and underlying geology. This
area also has several historic gold mines with some presently
active and many abandoned.7,9
In the present study area in a northeastern region of Karnataka
(Hutti), India, factors impacting the natural secondary
geochemical dispersion of Arsenic and mercury in groundwater
and soil can depend upon factors such as topography, water
table depth, drainage, soil type, and underlying geology. This
area also has several historic gold mines with some presently
active and many abandoned.7,9
In the present study area in a northeastern region of Karnataka
(Hutti), India, factors impacting the natural secondary
geochemical dispersion of Arsenic and mercury in groundwater
and soil can depend upon factors such as topography, water
table depth, drainage, soil type, and underlying geology. This
area also has several historic gold mines with some presently
active and many abandoned.7,9 Frequency of abortions and miscarriages
Frequency of abortions and miscarriages In the current study 100 inhabitants of the village participated
in the questionnaire survey of which majority were in the age
group of 25-38 years. It was observed that 81% of the
participants were aware of the gold mining in Hutti. In the current study 100 inhabitants of the village participated
in the questionnaire survey of which majority were in the age
group of 25-38 years. It was observed that 81% of the
participants were aware of the gold mining in Hutti. In the current study 100 inhabitants of the village participated
in the questionnaire survey of which majority were in the age
group of 25-38 years. It was observed that 81% of the
participants were aware of the gold mining in Hutti. MATERIALS AND METHOD
MATERIALS AND METHOD
MATERIALS AND METHOD Fig 4
Fig 4
Fig 4 The most common ill effects of arsenic and mercury poisoning
could be skin problems, malignancies, cardiovascular diseases,
gastro intestinal problems, neurological and reproductive
disorders as seen in other similar studies across the world. Similar defects were seen in our present study amongst the
local population which were in accordance with the global
trend of mining related health disorders.12Although more toxic
mercury species (e.g., dimethylmercury) are present in trace
levels in seafood, the primary risk of toxicity arises from
MeHg. MeHg is the predominant form of mercury in seafood,
is readily absorbed from the digestive tract, and can rapidly
cross the blood-brain barrier to exert its neurological
effects. Long-lived and predatory fish (e.g., sharks) absorb
MeHg from their diets, leading to higher MeHg levels than
those present in species lower on the food chain.12, 13
The most common ill effects of arsenic and mercury poisoning
could be skin problems, malignancies, cardiovascular diseases,
gastro intestinal problems, neurological and reproductive
disorders as seen in other similar studies across the world. Similar defects were seen in our present study amongst the
local population which were in accordance with the global
trend of mining related health disorders.12Although more toxic
mercury species (e.g., dimethylmercury) are present in trace
levels in seafood, the primary risk of toxicity arises from
MeHg. MeHg is the predominant form of mercury in seafood,
is readily absorbed from the digestive tract, and can rapidly
cross the blood-brain barrier to exert its neurological
effects. Long-lived and predatory fish (e.g., sharks) absorb
MeHg from their diets, leading to higher MeHg levels than
those present in species lower on the food chain.12, 13
The most common ill effects of arsenic and mercury poisoning
could be skin problems, malignancies, cardiovascular diseases,
gastro intestinal problems, neurological and reproductive
disorders as seen in other similar studies across the world. Similar defects were seen in our present study amongst the
local population which were in accordance with the global
trend of mining related health disorders.12Although more toxic
mercury species (e.g., dimethylmercury) are present in trace
levels in seafood, the primary risk of toxicity arises from
MeHg. MeHg is the predominant form of mercury in seafood,
is readily absorbed from the digestive tract, and can rapidly
cross the blood-brain barrier to exert its neurological
effects. MATERIALS AND METHOD
MATERIALS AND METHOD
MATERIALS AND METHOD Epidemiological and experimental evidence11 is furthermore
mounting that in utero or early life exposure to As and Hg may
affect fetal development or increase rates of several malignant
and non-malignant diseases. Chronic arsenic (As) and mercury (Hg) exposure through
ingestion can cause severe adverse effects on human health,
especially via the consumption of contaminated groundwater. Epidemiological and experimental evidence11 is furthermore
mounting that in utero or early life exposure to As and Hg may
affect fetal development or increase rates of several malignant
and non-malignant diseases. Chronic arsenic (As) and mercury (Hg) exposure through
ingestion can cause severe adverse effects on human health,
especially via the consumption of contaminated groundwater. Epidemiological and experimental evidence11 is furthermore
mounting that in utero or early life exposure to As and Hg may
affect fetal development or increase rates of several malignant
and non-malignant diseases. Fig 3
Fig 3
Fig 3 DISCUSSION
Traditional mining is increasingly common in many parts o
the world with more than 30 million active miners in more than
55 countries. Mining activities are largely concentrated in rura
areas that have very little infrastructure, and the individual
undertaking informal mining generally lack education, training
management skills and essential equipment for safe mining
Fig 3
Fig 4
Fig 5
Fig 6
3
40
0
10
20
30
40
50
60
Yes
No
Percentage
Frequency of abortions and miscarriages
47
33
0
10
20
30
40
50
Yes
No
Percentage
Dark brown pigmentation on skin
49
29
0
10
20
30
40
50
60
Yes
No
Don’t know
Percentage
Breathing difficulties in family
DISCUSSION
Traditional mining is increasingly common in many parts o
the world with more than 30 million active miners in more than
55 countries. Mining activities are largely concentrated in rura
areas that have very little infrastructure, and the individual
undertaking informal mining generally lack education, training
management skills and essential equipment for safe mining
Fig 3
Fig 4
Fig 5
Fig 6
57
Don’t know
Frequency of abortions and miscarriages
20
Don’t know
Dark brown pigmentation on skin
2
Don’t know
Breathing difficulties in family
DISCUSSION
Traditional mining is increasingly common in many parts o
the world with more than 30 million active miners in more than
55 countries. MATERIALS AND METHOD
MATERIALS AND METHOD
MATERIALS AND METHOD reside in the same area because of the lack of awareness about
the ill effects of gold mining. practices. Mercury (Hg), used in the processing of gold ore,
and arsenic (As), which is a constituent of some gold ores, are
common occupational exposures that can result in widespread
environmental contamination. Hg and As are known toxicants
that are hazardous to humans, wildlife and domestic animals
and may accumulate in the environment causing serious
damage to ecosystems and human health.10
practices. Mercury (Hg), used in the processing of gold ore,
and arsenic (As), which is a constituent of some gold ores, are
common occupational exposures that can result in widespread
environmental contamination. Hg and As are known toxicants
that are hazardous to humans, wildlife and domestic animals
and may accumulate in the environment causing serious
damage to ecosystems and human health.10
practices. Mercury (Hg), used in the processing of gold ore,
and arsenic (As), which is a constituent of some gold ores, are
common occupational exposures that can result in widespread
environmental contamination. Hg and As are known toxicants
that are hazardous to humans, wildlife and domestic animals
and may accumulate in the environment causing serious
damage to ecosystems and human health.10 he ill effects of gold mining. Fig 3
Fig 4
Fig 5
55
43
0
10
20
30
40
50
60
Yes
No
Percentage
White spot lesions in teeth
3
40
0
10
20
30
40
50
60
Yes
No
Percentage
Frequency of abortions and miscarriages
47
33
0
10
20
30
40
50
Yes
No
Percentage
Dark brown pigmentation on skin
60
Breathing difficulties in family
he ill effects of gold mining. Fig 3
Fig 4
Fig 5
2
Don’t know
White spot lesions in teeth
57
Don’t know
Frequency of abortions and miscarriages
20
Don’t know
Dark brown pigmentation on skin
Breathing difficulties in family
he ill effects of gold mining. Fig 3
Fig 4
Fig 5 Fig 3
55
43
0
10
20
30
40
50
60
Yes
No
Percentage
White spot lesions in teeth
Fig 3
2
Don’t know
White spot lesions in teeth
Fig 3 Chronic arsenic (As) and mercury (Hg) exposure through
ingestion can cause severe adverse effects on human health,
especially via the consumption of contaminated groundwater. MATERIALS AND METHOD
MATERIALS AND METHOD
MATERIALS AND METHOD 5) loose stool
(38%), breathing difficulties (49%) and fatigue
(47%). Around 11% people noticed loss of sensation of feet and hands
especially towards the toes and fingers. When dental health was
considered around 55%people reported with white spot lesions
on their tooth surface. (Fig. 3) When the female population was
surveyed, around 3% reported abortions and miscarriages. (Fig. 4)
Some of the most common health problems noticed were dark
brown pigmentation of the skin (47%), (Fig. 5) loose stool
(38%), breathing difficulties (49%) and fatigue
(47%). Around 11% people noticed loss of sensation of feet and hands
especially towards the toes and fingers. When dental health was
considered around 55%people reported with white spot lesions
on their tooth surface. (Fig. 3) When the female population was
surveyed, around 3% reported abortions and miscarriages. (Fig. 4)
Some of the most common health problems noticed were dark
brown pigmentation of the skin (47%), (Fig. 5) loose stool
(38%), breathing difficulties (49%) and fatigue
(47%). Around 11% people noticed loss of sensation of feet and hands
especially towards the toes and fingers. When dental health was
considered around 55%people reported with white spot lesions
on their tooth surface. (Fig. 3) When the female population was
surveyed, around 3% reported abortions and miscarriages. (Fig. 4) The poor accessibility and unavailability of health care centers
was the main reason for the increased incidence of health
issues. The majority of inhabitants of the village still choose to
The poor accessibility and unavailability of health care centers
was the main reason for the increased incidence of health
issues. The majority of inhabitants of the village still choose to
The poor accessibility and unavailability of health care centers
was the main reason for the increased incidence of health
issues. The majority of inhabitants of the village still choose to 16267 | P a g e
16267 | P a g e
16267 | P a g e International Journal of Recent Scientific Research Vol. 8, Issue, 3, pp. 16266-16269, March, 2017
International Journal of Recent Scientific Research Vol. 8, Issue, 3, pp. 16266-16269, March, 2017
International Journal of Recent Scientific Research Vol. 8, Issue, 3, pp. 16266-16269, March, 2017 reside in the same area because of the lack of awareness about
the ill effects of gold mining. reside in the same area because of the lack of awareness about
the ill effects of gold mining. MATERIALS AND METHOD
MATERIALS AND METHOD
MATERIALS AND METHOD Long-lived and predatory fish (e.g., sharks) absorb
MeHg from their diets, leading to higher MeHg levels than
those present in species lower on the food chain.12, 13 47
33
0
10
20
30
40
50
Yes
No
Percentage
Dark brown pigmentation on skin
20
Don’t know
Dark brown pigmentation on skin Fig 5
Fig 5
Fig 5 Fig 6
49
29
0
10
20
30
40
50
60
Yes
No
Don’t know
Percentage
Breathing difficulties in family
Fig 6
2
Don’t know
Breathing difficulties in family
Fig 6 Breathing difficulties in family
Breathing difficulties in family Inorganic arsenic is highly toxic: high levels can cause
neurological damage, anemia, leucopenia, and vascular disease,
while low-level chronic exposure increases an individual’s risk
of developing cancer. MMA and DMA were previously
believed to be non-toxic, but have recently been linked to
arsenic-induced toxicity.1,5 Humans can metabolize inorganic
arsenic to MMA and DMA, and it is believed that these
metabolites contribute significantly to carcinogenicity and
overall toxicity, particularly in their trivalent forms. Arsenic
toxicity due to gold mining is notable, but the metal is present
in the form of non-toxic arsenobetaine and arsenosugars. Inorganic arsenic is highly toxic: high levels can cause
neurological damage, anemia, leucopenia, and vascular disease,
while low-level chronic exposure increases an individual’s risk
of developing cancer. MMA and DMA were previously
believed to be non-toxic, but have recently been linked to
arsenic-induced toxicity.1,5 Humans can metabolize inorganic
arsenic to MMA and DMA, and it is believed that these
metabolites contribute significantly to carcinogenicity and
overall toxicity, particularly in their trivalent forms. Arsenic
toxicity due to gold mining is notable, but the metal is present
in the form of non-toxic arsenobetaine and arsenosugars. Inorganic arsenic is highly toxic: high levels can cause
neurological damage, anemia, leucopenia, and vascular disease,
while low-level chronic exposure increases an individual’s risk
of developing cancer. MMA and DMA were previously
believed to be non-toxic, but have recently been linked to
arsenic-induced toxicity.1,5 Humans can metabolize inorganic
arsenic to MMA and DMA, and it is believed that these
metabolites contribute significantly to carcinogenicity and
overall toxicity, particularly in their trivalent forms. Arsenic
toxicity due to gold mining is notable, but the metal is present
in the form of non-toxic arsenobetaine and arsenosugars. Fig 6
Fig 6
Fig 6 DISCUSSION
DISCUSSION
DISCUSSION The major heavy metal risk posed by gold mining is exposure
of a developing fetus to MeHg. Even low Hg exposure in
mothers due to the contaminants released by gold mining could
result in the fetus being exposed to high levels of Hg as it is
concentrated by a factor of ten in the fetus relative to the
mother.7,12 Thus, even a woman with a low level of mercury
exposure could give birth to a child with significant birth
The major heavy metal risk posed by gold mining is exposure
of a developing fetus to MeHg. Even low Hg exposure in
mothers due to the contaminants released by gold mining could
result in the fetus being exposed to high levels of Hg as it is
concentrated by a factor of ten in the fetus relative to the
mother.7,12 Thus, even a woman with a low level of mercury
exposure could give birth to a child with significant birth
The major heavy metal risk posed by gold mining is exposure
of a developing fetus to MeHg. Even low Hg exposure in
mothers due to the contaminants released by gold mining could
result in the fetus being exposed to high levels of Hg as it is
concentrated by a factor of ten in the fetus relative to the
mother.7,12 Thus, even a woman with a low level of mercury
exposure could give birth to a child with significant birth Traditional mining is increasingly common in many parts of
the world with more than 30 million active miners in more than
55 countries. Mining activities are largely concentrated in rural
areas that have very little infrastructure, and the individuals
undertaking informal mining generally lack education, training,
management skills and essential equipment for safe mining
Traditional mining is increasingly common in many parts of
the world with more than 30 million active miners in more than
55 countries. Mining activities are largely concentrated in rural
areas that have very little infrastructure, and the individuals
undertaking informal mining generally lack education, training,
management skills and essential equipment for safe mining
Traditional mining is increasingly common in many parts of
the world with more than 30 million active miners in more than
55 countries. DISCUSSION
DISCUSSION
DISCUSSION Mining activities are largely concentrated in rural
areas that have very little infrastructure, and the individuals
undertaking informal mining generally lack education, training,
management skills and essential equipment for safe mining 8 | P a g e
8 | P a g e
8 | P a g e 16268 | P a g e
16268 | P a g e
16268 | P a g e 16268 |
16268
16268 Amitha Hegde et al., Effects of Gold Mining on The General Population defects. In addition, Hg exposure due to transmission through
breast milk could have an effect on the healthy development of
infants. 4. Drake PL, Rojas M, Reh CM, Mueller CA, Jenkins FM. Occupational exposure to airborne mercury during gold
mining
operations
near
El
Callao,
Venezuela. International archives of occupational and environmental
health. 2001 Apr 1; 74(3):206-12. This is in accordance with the results of our survey which
reported an abortion and miscarriage rate of 3%. Also, the
percentage of children taking leave from school due to health
problems were between 40% - 50%. 5. Van Straaten P. Mercury contamination associated with
small-scale gold mining in Tanzania and Zimbabwe. Science of the Total Environment. 2000 Oct 2;
259(1):105-13. Another hurdle which contributed in worsening gold mining
related health problems was absence of a proper primary health
care or referral centre. Thus, there was lack of awareness as
well as failure to intervene the disease in the early phase
amongst the population. 6. Wickre JB, Folt CL, Sturup S, Karagas MR. Environmental exposure and fingernail analysis of
arsenic and mercury in children and adults in a
Nicaraguan gold mining community. Archives of
Environmental Health: An International Journal. 2004
Aug 1; 59(8):400-9. While groundwater contamination is hypothesized to be the
main route of As and Hg exposure, there could also be presence
of highly elevated As and Hg levels in soil samples in the area. Investigation of other possible significant routes of exposure as
well as their relative contributions to toxicity should be
studied.8 The historic gold mine workings of the Hutti Gold
Mining company may be a significant contributor to localized
pollution in Hutti and other neighbouring regions. 7. Charles E, Thomas DS, Dewey D, Davey M, Ngallaba
SE, Konje E. A cross-sectional survey on knowledge
and perceptions of health risks associated with arsenic
and mercury contamination from artisanal gold mining
in Tanzania. BMC public health. DISCUSSION
DISCUSSION
DISCUSSION 2013 Jan 25; 13(1):74. 8. Skerfving
S. Mercury
in
women
exposed
to
methylmercury through fish consumption, and in their
newborn
babies
and
breast
milk. Bulletin
of
environmental contamination and toxicology. 1988 Oct
1; 41(4):475-82. CONCLUSION Additional studies to determine, temporal variation, the nature
of As and Hgmobilization, particularly whether previous and
ongoing mining activities are playing a role, are crucial in
guiding efforts to identify at-risk communities and remedy the
effects of this contamination. If historic gold mining has played
a role in contamination, increasing interest in there opening of
previously closed mines and the prospecting of new regions in
the and around Karnataka should be conducted along with
sound monitoring and assessment of the environment and the
health effects resulting from its degradation. As various
surveys have identified many additional communities with
elevated groundwater arsenic and mercury, environmental
contamination appears to be more widespread than the present
study area. Both medical screening and regular water quality
analysis programs should be continued or organized in the
region immediately.8 9. Chakraborti D, Rahman MM, Murrill M, Das R, Patil SG,
Sarkar A, Dadapeer HJ, Yendigeri S, Ahmed R, Das KK. Environmental arsenic contamination and its health
effects in a historic gold mining area of the Mangalur
greenstone belt of Northeastern Karnataka, India. Journal
of hazardous materials. 2013 Nov 15; 262:1048-55. 10. Kolb J, Rogers A, Meyer FM. Relative timing of
deformation and two-stage gold mineralization at the
Hutti Mine, Dharwar Craton, India. Mineralium
Deposita. 2005 Mar 1; 40(2):156-74. 11. Hoedoafia MA, Cheabu BS, Korang V. The effects of
small scale gold mining on living conditions: A case
study of the west gonja district of Ghana. International
Journal of Social Science Research. 2014 Mar 11;
2(1):151-64. 12. Bose-O’Reilly S, Lettmeier B, Gothe RM, Beinhoff C,
Siebert U, Drasch G. Mercury as a serious health hazard
for children in gold mining areas. Environmental
research. 2008 May 31; 107(1):89-97. How to cite this article:
Amitha Hegde et al.2017, Effects of Gold Mining on The General Population. Int J Recent Sci Res. 8(3), pp. 16266-16269.
DOI: http://dx.doi.org/10.24327/ijrsr.2017.0803.0115 Amitha Hegde et al.2017, Effects of Gold Mining on The General Population. Int J Recent Sci Res. 8(3), pp. 16266-16269.
DOI: http://dx.doi.org/10.24327/ijrsr.2017.0803.0115 References 1. Naganna C. Gold mineralization in the Hutti mining
area, Karnataka, India. Economic Geology. 1987 Dec 1;
82(8):2008-16. 1. Naganna C. Gold mineralization in the Hutti mining
area, Karnataka, India. Economic Geology. 1987 Dec 1;
82(8):2008-16. 13. Grandjean P, White RF, Nielsen A, Cleary D, de Oliveira
Santos EC. Methylmercury neurotoxicity in Amazonian
children downstream from gold mining. Environmental
health perspectives. 1999 Jul; 107 (7): 587. 2. Curtis LC, Radhakrishna BP. Hutti-Gold Mine into 21st
Century. GSI Publications. 1995 May 7; 3(1). 2. Curtis LC, Radhakrishna BP. Hutti-Gold Mine into 21st
Century. GSI Publications. 1995 May 7; 3(1). 3. Sarma DS, Mcnaughton NJ, Fletcher IR, Groves DI,
Mohan MR, Balaram V. Timing of gold mineralization
in the Hutti gold deposit, Dharwar Craton, South India. Economic Geology. 2008 Dec 1; 103(8):1715-27. 14. Smedley PL, Edmunds WM, Pelig-Ba KB. Mobility of
arsenic in groundwater in the Obuasi gold-mining area
of Ghana: some implications for human health. Geological Society, London, Special Publications. 1996
Jan 1; 113(1):163-81. ******* ******* 16269 | P a g e 16269 | P a g e
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Self-Replicating RNA Viruses for Vaccine Development Against Infectious Diseases and Cancer
| null | 2,021
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cc-by
| 21,008
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Citation: Lundstrom, K. Self-Replicating RNA Viruses for
Vaccine Development against
Infectious Diseases and Cancer. Vaccines 2021, 9, 1187. https://
doi.org/10.3390/vaccines9101187 Keywords: self-replicating RNA viruses; vaccines; infectious diseases; cancer; immune response;
tumor regression; protection; approval Academic Editors: Ângela
Maria Almeida de Sousa, Christiane
Pienna Soares, Aldo Venuti and
François Meurens Academic Editors: Ângela
Maria Almeida de Sousa, Christiane
Pienna Soares, Aldo Venuti and
François Meurens 1. Introduction Vaccine development has always had a central position in prevention of infectious
diseases, but with the onset of the COVID-19 pandemic it has reached unprecedented
levels. Similarly, the area of cancer vaccines has drawn plenty of attention. Obviously, the
development of vaccines against SARS-CoV-2 has been approached from every possible
angle including inactivated and attenuated viruses, protein and peptide subunit-based
vaccines, nucleic acid-based vaccines, and viral vectors [1]. In this review the focus will be
on viral vectors. Although the strongest progress has been achieved for adenovirus vectors
with Emergency Use Authorization (EUA) for the ChAdOx1 nCoV-19 [2], Ad26.COV2.S [3],
and rAd26-S/rAd5-S [4], only vaccine candidates based on self-replicating RNA viruses
will be described here. In addition to SARS-CoV-2, other viral pathogens such as influenza
virus, human immunodeficiency virus (HIV), hepatitis B virus (HBV), human papilloma
virus (HPV), Ebola virus (EBOV) and Lassa virus (LASV) have been targeted [5]. Self-
replicating RNA viruses have also been used for cancer vaccine development. In this review
multiple examples of immunization with self-amplifying RNA viral vectors expressing
various antigens against infectious agents and tumors are presented. The advantages and
disadvantages of using self-replicating RNA viral vectors, especially RNA-based delivery,
are also discussed. Received: 16 August 2021
Accepted: 12 October 2021
Published: 15 October 2021 Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Copyright: © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). Review
Self-Replicating RNA Viruses for Vaccine Development against
Infectious Diseases and Cancer Kenneth Lundstrom PanTherapeutics, 1095 Lutry, Switzerland; lundstromkenneth@gmail.com PanTherapeutics, 1095 Lutry, Switzerland; lundstromkenneth@gmail.com Abstract: Alphaviruses, flaviviruses, measles viruses and rhabdoviruses are enveloped single-
stranded RNA viruses, which have been engineered for recombinant protein expression and vaccine
development. Due to the presence of RNA-dependent RNA polymerase activity, subgenomic RNA
can replicate close to 106 copies per cell for translation in the cytoplasm providing extreme transgene
expression levels, which is why they are named self-replicating RNA viruses. Expression of surface
proteins of pathogens causing infectious disease and tumor antigens provide the basis for vaccine
development against infectious diseases and cancer. Self-replicating RNA viral vectors can be admin-
istered as replicon RNA at significantly lower doses than conventional mRNA, recombinant particles,
or DNA plasmids. Self-replicating RNA viral vectors have been applied for vaccine development
against influenza virus, HIV, hepatitis B virus, human papilloma virus, Ebola virus, etc., showing
robust immune response and protection in animal models. Recently, paramyxovirus and rhabdovirus
vector-based SARS-CoV-2 vaccines as well as RNA vaccines based on self-amplifying alphaviruses
have been evaluated in clinical settings. Vaccines against various cancers such as brain, breast, lung,
ovarian, prostate cancer and melanoma have also been developed. Clinical trials have shown good
safety and target-specific immune responses. Ervebo, the VSV-based vaccine against Ebola virus
disease has been approved for human use. Citation: Lundstrom, K.
Self-Replicating RNA Viruses for
Vaccine Development against
Infectious Diseases and Cancer.
Vaccines 2021, 9, 1187. https://
doi.org/10.3390/vaccines9101187 2. Self-Replicating RNA Viruses Application of self-replicating RNA viruses for vaccines against infectious diseases
and cancer has clear advantages compared to other viral vectors and non-viral delivery https://www.mdpi.com/journal/vaccines Vaccines 2021, 9, 1187. https://doi.org/10.3390/vaccines9101187 Vaccines 2021, 9, 1187 2 of 28 systems. Self-replicating RNA viruses deposit their RNA directly into the cytoplasm of
infected host cells [6], which requires no delivery to the nucleus as is the case for some
other RNA viruses such as influenza virus, and also for DNA-based delivery. In the case
of positive strand RNA viruses such as alphaviruses, the most significant feature relates
to the efficient self-replication/amplification of delivered RNA by the established RNA
replication complex, which can accumulate close to 106 copies of subgenomic RNA per
cell in the host cell cytoplasm [7]. It will generate high levels of antigen expression, which
can potentially elicit superior immune responses and may also allow immunizations with
smaller doses resulting in reduced adverse events. It can also provide extreme expression
of toxic, anti-tumor and immunostimulatory genes for cancer vaccination and therapy. Additionally, self-replicating RNA viral vectors can be utilized as recombinant replication-
deficient viral particles, replicon RNA, or layered DNA/RNA vectors (Figure 1). Another
feature of interest is the transient nature of high levels of transgene expression provided by
self-replicating RNA viruses due to the degradation of RNA transcribed from recombinant
particles and RNA replicons within 5–7 days post-immunization. It is advantageous for
vaccine development against both infectious diseases and cancers. Furthermore, in contrast
to for instance retroviruses, alphavirus RNA is not subjected to reverse transcription and
integration into the host genome. Figure 1. Schematic illustration of self-replicating RNA alphavirus-based expression systems. Alphavirus-based deliv-
ery and expression systems comprise of infection of recombinant viral particles, electroporation/lipid-based transfection
of in vitro transcribed RNA or transfection of plasmid DNA. Recombinant protein expression can be obtained as follows. In vitro transcribed RNA carrying the replicase gene and the gene of interest is electroporated/transfected into mammalian
host cells (A). Alternatively, the replicon RNA can be delivered to host cells by infection with recombinant alphavirus
particles (B). The third option is to transfect alphavirus DNA replicons (C), which after DNA delivery to the nucleus RNA is
in vivo transcribed. The replicase complex will amplify RNA molecules (self-replication) and recombinant protein will be
expressed from the 26S subgenomic promoter. 2. Self-Replicating RNA Viruses To develop a vaccinia virus-free system, the VSV N, P and L genes were
introduced downstream of both the T7 promoter and an internal ribosomal entry site (IRES)
and the T7 polymerase was provided by the BSR-T7/5 stable cell line. The elimination of
the vaccinia virus from the reverse genetics system presented an attractive alternative for
generation of infectious VSV from DNA Self-replicating RNA viruses can be divided into two groups based on the polarity
of their RNA genome. All self-replicating RNA viruses possess a single-stranded non-
fragmented RNA (ssRNA) genome. However, alphaviruses [6] and flaviviruses [8] have
a positive-sense RNA genome, whereas the genome of paramyxoviruses [9] and rhab-
doviruses [10] is of negative polarity. The difference in polarity has consequences for
their applications as the positive sense ssRNA is immediately after infection translated
in the cytoplasm. In the case of alphaviruses, expression systems are based on delivery
of recombinant viral particles, RNA replicons or plasmid DNA replicons. Infection with
recombinant particles and electroporation or lipid-based transfection of in vitro transcribed
replicon RNA deliver positive sense ssRNA to the cytoplasm of host cells. Utilization of
plasmid DNA transfection requires initial delivery of DNA to the nucleus followed by
in vivo transcription of RNA. The recombinant RNA containing the non-structural replicase
genes and the gene of interest (GoI) is efficiently amplified (self-replication) from a minus
strand RNA template and translation of recombinant protein coding for the GoI occurs in
the cytoplasm. A schematic illustration of alphavirus self-replicating expression systems is
presented in Figure 1. The most prominent alphavirus expression systems are based on
Semliki Forest virus (SFV) [11], Sindbis virus (SIN) [12] and Venezuelan equine encephalitis
virus (VEEV) [13]. Flavivirus expression systems have been engineered for Kunjin virus
(KUN), where the gene of interest is introduced between the first 60 nucleotides of the C20
core protein and the last 22 codons of the E22 envelope protein [14]. The GoI is expressed as
part of a larger polyprotein from which the flanking regions are cleaved off by the FMDV-
2A protease sequence in the KUN vector [15]. KUN production has been facilitated by the
engineering of a packaging cell line [16]. In addition to KUN, expression vectors have been
engineered for West Nile virus (WNV) [17], yellow fever virus (YFV) [18], Dengue virus
(DENV) [19], and tick-borne encephalitis virus (TBEV) [20]. 2. Self-Replicating RNA Viruses Furthermore, the bovine viral
diarrhea virus (BVDV) has been engineered as an expression vector by introducing the
GFP reporter gene between the N(pro) and C genes of the non-cytopathic type-1 BVDV
strain SD1 [21]. Similarly, expression of GFP from a bicistronic classical swine fever virus
(CSFV) in infected host cells confirmed the potential of CSFV as an expression vector [22]. I
h
f RNA
i
i h
i
RNA
l
i
h
i
l
i i (
)
p
p
[
]
In the case of RNA viruses with negative ssRNA polarity such as vesicular stomatitis
virus (VSV), the RNA-dependent RNA polymerase (RdRp) responsible for self-replication
is encoded in the L gene and the phosphoprotein (P) is an essential cofactor for the RdRp
activity. In the case of VSV expression systems, the VSV glycoprotein (G) gene is generally
replaced by the GoI or the GoI is inserted between the G and L genes for the generation
of either pseudotype or recombinant VSV particles (Figure 2A) [23,24]. Pseudotype VSV
can be produced in mammalian cells by transfection of plasmid DNA containing foreign
envelope genes followed by infection with the VSV G-complemented G-VSV∆G pseudo-
type virus. The generated pseudotype VSV can infect target cells, but do not produce
infectious viral progeny. In contrast, infection of mammalian producer cells with VSV
G-complemented recombinant virus replacing the VSV G with a foreign envelope generates
fully infectious viral progeny [24]. Originally, application of reverse genetics for expression
vector engineering was based on recombinant vaccinia virus vectors. In the case of VSV,
the nucleoprotein (N), phosphoprotein (P), polymerase (L) and the full-length antigenomic
RNA were expressed from four plasmids under the control of the T7 promoter from a
vaccinia virus [25]. To develop a vaccinia virus-free system, the VSV N, P and L genes were
introduced downstream of both the T7 promoter and an internal ribosomal entry site (IRES)
and the T7 polymerase was provided by the BSR-T7/5 stable cell line. The elimination of
the vaccinia virus from the reverse genetics system presented an attractive alternative for
generation of infectious VSV from DNA. In the case of rabies virus (RABV), the GoI can be inserted between the N and P genes
and G and L (Figure 2B) [26,27]. A vaccinia virus-free reverse genetics system has also been
engineered for RABV [28]. 2. Self-Replicating RNA Viruses 5′ cap, 5′ end cap analogue; 26S, alphavirus subgenomic promoter; CMV,
cytomegalovirus promoter; GoI, gene of interest; pA, poly A signal; SP6, bacteriophage SP6 RNA polymerase promoter. Figure 1. Schematic illustration of self-replicating RNA alphavirus-based expression systems. Alphavirus-based deliv-
ery and expression systems comprise of infection of recombinant viral particles, electroporation/lipid-based transfection
of in vitro transcribed RNA or transfection of plasmid DNA. Recombinant protein expression can be obtained as follows. In vitro transcribed RNA carrying the replicase gene and the gene of interest is electroporated/transfected into mammalian
host cells (A). Alternatively, the replicon RNA can be delivered to host cells by infection with recombinant alphavirus
particles (B). The third option is to transfect alphavirus DNA replicons (C), which after DNA delivery to the nucleus RNA is
in vivo transcribed. The replicase complex will amplify RNA molecules (self-replication) and recombinant protein will be
expressed from the 26S subgenomic promoter. 5′ cap, 5′ end cap analogue; 26S, alphavirus subgenomic promoter; CMV,
cytomegalovirus promoter; GoI, gene of interest; pA, poly A signal; SP6, bacteriophage SP6 RNA polymerase promoter. Vaccines 2021, 9, 1187 3 of 28 3 of 28 Self-replicating RNA viruses can be divided into two groups based on the polarity
of their RNA genome. All self-replicating RNA viruses possess a single-stranded non-
fragmented RNA (ssRNA) genome. However, alphaviruses [6] and flaviviruses [8] have
a positive-sense RNA genome, whereas the genome of paramyxoviruses [9] and rhab-
doviruses [10] is of negative polarity. The difference in polarity has consequences for
their applications as the positive sense ssRNA is immediately after infection translated
in the cytoplasm. In the case of alphaviruses, expression systems are based on delivery
of recombinant viral particles, RNA replicons or plasmid DNA replicons. Infection with
recombinant particles and electroporation or lipid-based transfection of in vitro transcribed
replicon RNA deliver positive sense ssRNA to the cytoplasm of host cells. Utilization of
plasmid DNA transfection requires initial delivery of DNA to the nucleus followed by
in vivo transcription of RNA. The recombinant RNA containing the non-structural replicase
genes and the gene of interest (GoI) is efficiently amplified (self-replication) from a minus
strand RNA template and translation of recombinant protein coding for the GoI occurs in
the cytoplasm. A schematic illustration of alphavirus self-replicating expression systems is
presented in Figure 1. 2. Self-Replicating RNA Viruses The most prominent alphavirus expression systems are based on
Semliki Forest virus (SFV) [11], Sindbis virus (SIN) [12] and Venezuelan equine encephalitis
virus (VEEV) [13]. Flavivirus expression systems have been engineered for Kunjin virus
(KUN), where the gene of interest is introduced between the first 60 nucleotides of the C20
core protein and the last 22 codons of the E22 envelope protein [14]. The GoI is expressed as
part of a larger polyprotein from which the flanking regions are cleaved off by the FMDV-
2A protease sequence in the KUN vector [15]. KUN production has been facilitated by the
engineering of a packaging cell line [16]. In addition to KUN, expression vectors have been
engineered for West Nile virus (WNV) [17], yellow fever virus (YFV) [18], Dengue virus
(DENV) [19], and tick-borne encephalitis virus (TBEV) [20]. Furthermore, the bovine viral
diarrhea virus (BVDV) has been engineered as an expression vector by introducing the
GFP reporter gene between the N(pro) and C genes of the non-cytopathic type-1 BVDV
strain SD1 [21]. Similarly, expression of GFP from a bicistronic classical swine fever virus
(CSFV) in infected host cells confirmed the potential of CSFV as an expression vector [22]. In the case of RNA viruses with negative ssRNA polarity such as vesicular stomatitis
virus (VSV), the RNA-dependent RNA polymerase (RdRp) responsible for self-replication
is encoded in the L gene and the phosphoprotein (P) is an essential cofactor for the RdRp
activity. In the case of VSV expression systems, the VSV glycoprotein (G) gene is generally
replaced by the GoI or the GoI is inserted between the G and L genes for the generation
of either pseudotype or recombinant VSV particles (Figure 2A) [23,24]. Pseudotype VSV
can be produced in mammalian cells by transfection of plasmid DNA containing foreign
envelope genes followed by infection with the VSV G-complemented G-VSV∆G pseudo-
type virus. The generated pseudotype VSV can infect target cells, but do not produce
infectious viral progeny. In contrast, infection of mammalian producer cells with VSV
G-complemented recombinant virus replacing the VSV G with a foreign envelope generates
fully infectious viral progeny [24]. Originally, application of reverse genetics for expression
vector engineering was based on recombinant vaccinia virus vectors. In the case of VSV,
the nucleoprotein (N), phosphoprotein (P), polymerase (L) and the full-length antigenomic
RNA were expressed from four plasmids under the control of the T7 promoter from a
vaccinia virus [25]. 3. Infectious Diseases The classic approach for targeting of infectious diseases for vaccine development
has comprised overexpression of immunogenic surface epitopes or proteins as antigens,
which elicit immune responses leading to protection against challenges with lethal doses
of pathogens [31]. A large number of preclinical and some clinical studies have been
conducted for vaccine candidates based on self-replicating RNA viral vectors and it is only
possible to provide some examples below and in Tables 1 and 2. The most common targets
have been viral infections, but additionally vaccine candidates against bacterial infections
and tropical diseases have been developed. Table 1. Examples of preclinical studies on self-replicating RNA viral vector vaccines against infectious diseases. Virus/Disease
Antigen
Vector
Findings
Ref. 2. Self-Replicating RNA Viruses For paramyxoviruses, measles virus (MV) vectors, packaging
systems and helper cell lines have been engineered to allow rescue of replicating MV from
plasmid DNA vectors [29,30]. Generally, the GoI is inserted between the phosphoprotein Vaccines 2021, 9, 1187 4 of 28 (P) and the matrix protein (M) or alternatively between the hemagglutinin (HA) gene a
the large protein (L) (Figure 2C). As a comparison of positive and negative strand RN
viruses, their replication strategies are illustrated in Figure 3. Figure 2. Self-replicating RNA viral vectors of negative polarity. (A) VSV vector for replacement of VSV G protein. (B) Rabies virus and (C). Measles virus expression vectors. CMV, cytomegalovirus promoter; Fu, fusion protein, G,
glycoprotein; GoI, gene of interest; H, hemagglutinin; L, large protein; M, matrix protein; P. phosphoprotein; T7, T7 RNA
polymerase promoter; T7T, T7 terminal sequence. (P) and the matrix protein (M) or alternatively between the hemagglutinin (HA) gene and
the large protein (L) (Figure 2C). As a comparison of positive and negative strand RNA
viruses, their replication strategies are illustrated in Figure 3. Figure 2. Self-replicating RNA viral vectors of negative polarity. (A) VSV vector for replacement of VSV G protein. (B) Rabies virus and (C). Measles virus expression vectors. CMV, cytomegalovirus promoter; Fu, fusion protein, G,
glycoprotein; GoI, gene of interest; H, hemagglutinin; L, large protein; M, matrix protein; P. phosphoprotein; T7, T7 RNA
polymerase promoter; T7T, T7 terminal sequence. Figure 3. Replication strategy of positive- and negative-strand self-replicating RNA viruses. For positive-strand RNA
viruses, the viral RNA is directly translated in the cytoplasm and replication of new positive-strand RNA copies require
transcription of a negative-strand RNA template. In contrast, negative-strand RNA viruses rely on mRNA transcription
before translation can take place. Figure 3. Replication strategy of positive- and negative-strand self-replicating RNA viruses. For positive-strand RNA
viruses, the viral RNA is directly translated in the cytoplasm and replication of new positive-strand RNA copies require
transcription of a negative-strand RNA template. In contrast, negative-strand RNA viruses rely on mRNA transcription
before translation can take place. Vaccines 2021, 9, 1187 5 of 28 3. Infectious Diseases Alphaviruses
CHIKV/CHIK
E3-E2-6K-E1
Chimeric VSV-Env
Protection against CHIKV in mice
[32]
VEEV/VEE
E3-E2-6K-E1
VEEV-Env
Protection against VEE in mice, macaques
[33]
WEEV/WEE
E3-E2-6K-E1
WEEV-Env
Partial protection in macaques, strong in mice
[33]
EEEV/EEE
E3-E2-6K-E1
EEEV-Env
Protection against EEE in mice, macaques
[33]
VEEV/VEE
V4020 strain
VEEV DNA
Protection against VEE in mice
[34]
VEEV/VEE
V4020 strain
VEEV DNA
Protection against VEE in macaques
[35]
CHIKV/CHIK
C, Env
MV-CHIKV VLPs
Protection against CHIKV in macaques
[36]
Arenaviruses
LASV/LHF
GPC
VSV-GPC
Protection in guinea pigs and macaques
[37]
LASV/LHF
GPC
MV-LASV-GPC
Protection against LASV in macaques
[38]
LASV/LHF
GPC
YFV-LASV GPC
80% protection in guinea pigs, vector instability
[39]
LASV/LHF
GPC G1/G2
YFV-LASV G1 + G2
83% protection in guinea pigs, stable vector
[40]
LASV/LHF
GPC G1/G2
YFV-LASV G1 + G2
No protection in marmosets
[41]
LASV/LHF
GPC or NP
VEEV-GPC/NP
Protection in guinea pigs after 3 immunizations
[42]
LASV/LHF
GPC
Multivalent VEEV
Protection in inbred CBA/J mice
[43]
JUNV/AHF
GPC
VEEV-GPC
Protection against JUNV in mice
[44]
MACV/BHF
GPC
VEEV-GPC
Protection against MACV in mice
[44]
Filoviruses
EBOV/EVD
GP D637L
KUN-GP D637L
Protection in 75% of nonhuman primates
[45]
EBOV/EVD
GP
VSV-GP
Protection against two EBOV strain in macaques
[46,47]
MARV/MHF
GP
VSV-GP
Protection against MARV in macaques
[48]
SUDV/EVD
GP
VEEV-GP
Protection against SUDV and EBOV in macaques
[48]
Flaviviruses
DENV/DF
E85
VEEV-E85
Protection against DENV in mice
[49]
DENV/DF
ED3
MV-ED3
Strong immunogenicity, partial protection in mice
[50]
DENV/DF
Tetravalent DENV
YFV (CYD-TDV)
Good safety, immunogenicity in rodents, primates
[51,52]
DENV/DF
Tetravalent DENV
YFV (CYD-TDV)
Approved vaccine for endemic populations
[53]
ZIKV/ZVD
prME
VEEV-NLC RNA
Protection in mice with 10 ng of RNA
[54]
ZIKV/ZVD
prME
Chimeric
VSV-prME
Protection against ZIKV in mice
[32]
ZIKV/ZVD
E-NS1
VSV-E-NS1
Protection against ZIKV in mice
[55]
Hepatotropic
HBV/Hepatitis
HBsAg
MV-HBsAg
Protection against HBV in 50% of rhesus monkeys
[56]
HBV/Hepatitis
MHB
SFV-MHB
Protection against HBV in mice
[57]
HBV/Hepatitis
HBcAg
SFV-HBcAg
No protection against HBV in mice
[57] Examples of preclinical studies on self-replicating RNA viral vector vaccines against infectious diseases. Antigen
Vector
Findings Table 1. Examples of preclinical studies on self-replicating RNA viral vector vaccines against infe reclinical studies on self-replicating RNA viral vector vaccines against infectious diseases. mples of preclinical studies on self-replicating RNA viral vector vaccines against infectious diseases. Vaccines 2021, 9, 1187 6 of 28 Table 1. Cont. Table 1. Cont. Virus/Disease
Antigen
Vector
Findings
Ref. 3. Infectious Diseases Lentiviruses
HIV/AIDS
HIV gp160 Env
MV-gp160 Env
Humoral and cellular immune responses in mice
[58]
HIV/AIDS
HIV Env
SFV-Env
Superior immunogenicity compared to immunization
with DNA and Env protein
[59]
HIV/AIDS
HIV Env
SFV-Env RNA
Immune response in 75% of mice
[60]
HIV/AIDS
HIV
Env/Gag/PolRT
SFV RPs/RNA
VLPs superior immunogenicity to RNA in mice
[61]
HIV/AIDS
HIV Env,
Gag/Pol/Nef
SFV DNA
Robust immune responses in mice
[62]
HIV/AIDS
HIV gp140
VEEV-RNA-CNE
Superior Ab response compared to VLPS in primates
[63]
Influenza Viruses
IFVA/Influenza
HA
MV AIK-C-HA
Protection against influenza virus in cotton rats
[64]
IFVA/Influenza
HA, NA
VSV∆G-HA/NA
Protection against influenza virus in mice
[65]
IFVA/Influenza
HAfl
VSV-HAfl
Protection against influenza virus in mice
[66]
IFVA/Influenza
HA
VEEV-HA
Protection in chickens
[67]
IFVA/Influenza
HA
SFV-HA RNA
Protection in 90% of mice
[68]
IFVA/Influenza
HA
VEEV-HA RNA
Protection in mice with 64-fold less RNA *
[69]
IFVA/Influenza
M2e
SIN E2S1-M2e
Protection in mice
[70]
IFVA/Influenza
HA, NP
CSFV-HA/NP
VRPs
Strong humoral and cellular response in pigs
[71]
Coronaviruses
SARS-CoV/SARS
S
VEEV-S
Protection against SARS-CoV in mice
[72]
MERSCoV/MERS
S
VSV∆G-S
Neutralizing Abs and T cell responses in monkeys
[73]
SARS-CoV-2/COV
S
MV-S
Th1-biased Ab and T cell responses in mice
[74]
SARS-CoV-2/COV
S
MV (TMV-083)
Phase I: weak immunogenicity, trial discontinued
[75,
76]
SARS-CoV-2/COV
S
VSV-S
Neutralizing Abs, protection in mice
[77]
SARS-CoV-2/COV
S
VSV (V590)
Phase I: weak immunogenicity, trial discontinued
[78,
79]
SARS-CoV-2/COV
S
VSV∆G-S
Protection against SARS-CoV-2 in hamsters
[80]
SARS-CoV-2/COV
S
VSV∆G-S
Phase I/II: study in progress
[81,
82]
PEDV/PED
S fragment
BVDV
Neutralization of BVDV and PEDV in mice
[83]
SARS-CoV-2/COV
S
LNP-VEEV-S RNA
Robust Ab responses in mice
[84]
SARS-CoV-2/COV
S
LNP-VEEV-S RNA
Phase I/II: study in progress
[85]
SARS-CoV-2/COV
S
LUNAR-VEEV
RNA
Protection in mice after single dose
[86]
SARS-CoV-2/COV
S
SIN-S + αOX40
Protection against SARS-CoV-2 in mice
[87]
Bacterial
B. anthracis/Anthrax
PA
SIN-PA
Immune responses, some protection in mice
[88]
B. abortus/Brucellosis
B. abortus IF3
SFV-CS
Immune responses, protection in mice
[89] Vaccines 2021, 9, 1187 7 of 28 Table 1. Cont. Virus/Disease
Antigen
Vector
Findings
Ref. Parasitic
Plasmodium/Malaria
Pf332 antigen
SFV-Pf332
Robust Th1-type immune response in mice
[90]
Plasmodium/Malaria
P. 3. Infectious Diseases yoelii CS
epitope
SIN-CS
Protection against malaria in mice
[91]
Leishmania/Leishmaniasis
PpSP15-
LmSTI1
SFV-PpSP15-
LmSTI1
Superior expression from replicon RNA
[92]
αOX40, immunostimulatory antibody; Ab, antibody; AHF, Argentine hemorrhagic fever; BHF, Bolivian hemorrhagic fever; BVDV, bovine
viral diarrhea virus; C, capsid, CHIKV, Chikungunya virus; CNE, cationic nanoemulsion; COV, COVID-19 CS, circumsporozoite; DENV,
Dengue virus; DF, Dengue fever; EBOV, Ebola virus; EEEV, eastern encephalitis virus; E85, DENV envelope ectodomain; ED3, DENV
envelope protein domain III; Env, envelope proteins; EVD, Ebola virus disease; G1/G2; Glycoprotein subunit of GPC; GPC, glycoprotein
complex; HA, hemagglutinin; HAfl, full-length HA; HBcAg, HBV core antigen; HBsAg, HBV surface antigen; HBV, hepatitis B virus;
IFVA, influenza virus A; JUNV, Junin virus; LASV, Lassa virus; LHF, Lassa hemorrhagic fever; MACV, Machupo virus; MARV, Marburg
virus; MERS-CoV, Middle East respiratory syndrome-coronavirus; MHB, middle HBV surface envelope glycoprotein; MHF, Marburg
hemorrhagic fever; NA, neuraminidase; NP, nucleoprotein; NS1, nonstructural protein 1; PA, protective antigen; PEDV, porcine epidemic
diarrhea virus; prME, membrane-envelope protein; RPs, recombinant particles; RSP, recombinant subviral particle; S, spike protein; SFV,
Semliki Forest virus; VEEV, Venezuelan equine encephalitis virus; VLPs, virus-like particles; VSV, vesicular stomatitis virus; WEEV, western
equine encephalitis virus; YFV, yellow fever virus; ZIKV, Zika virus; ZVD, Zika virus disease.* Compared to synthetic mRNA. Table 1. Cont. Among alphaviruses, Chikungunya virus (CHIKV) [93,94] and VEEV [95] have been
responsible for epidemics in Africa, Polynesia, and South America. Vaccine development
has included expression of the CHIKV envelope polyprotein E3-E2-6K-E1 from a chimeric
VSV vector, which elicited neutralizing antibody responses and provided protection against
CHIKV in mice after a single administration of 1 × 107 pfu of VSV particles [32]. In another
approach, expression vectors for VEEV, western equine encephalitis virus (WEEV), and
eastern equine encephalitis (EEEV) were engineered by removing the furin cleavage site
at the junction between the E2 and E3 envelope proteins [33]. It prevents the cleavage of
the precursor p62 into E2 and E3 to produce infectious particles but generates replication-
deficient recombinant virus particles [33]. The combination of 1 × 107 IU of VEEV, WEEV,
and EEEV or individual viral recombinant particles induced strong neutralizing antibody
responses and protected mice from subcutaneous or aerosol challenges with VEEV, WEEV,
and EEEV [33]. Similarly, immunization of cynomolgus macaques with 2 × 108 IU of the
VEEV-WEEV-EEEV combination elicited strong immune responses and protected against
challenges with VEEV and EEEV. 3. Infectious Diseases Furthermore, a multivalent VEEV vaccine encoding GPC from
the distantly related LP and Josiah strains showed protection in inbred CBA/J mice [43]. VEE vectors have also been used for targeting other arenaviruses such as Junin virus (JUNV)
and Machupo virus (MACV) [44]. VEEV-based expression of JUNV- and MACV-GPC,
respectively, elicited humoral immune responses and provided protection in guinea pigs
after immunization with 1 × 107 pfu. Table 2. Examples of clinical studies on self-replicating RNA viral vector vaccines against infectious diseases. Virus/Disease
Antigen
Vector
Findings
Ref. Alphaviruses
CHIKV/CHIK
C, Env
MV-CHIKV VLPs
Phase I: 100% seroconversion after two doses
[96]
CHIKV/CHIK
C, Env
MV-CHIKV VLPs
Phase II: good safety, strong immunogenicity
[97]
Filoviruses
EBOV/EVD
GP (Zaire strain)
VSV-ZEBOV
Phase III: high vaccine efficacy, protection
[99,100]
EBOV/EVD
GP (Zaire strain)
VSV-ZEBOV
Phase III: high vaccine efficacy
[100]
EBOV/EVD
GP (Zaire strain)
VSV-ZEBOV
Ervebo approval by the FDA, EMA
[101]
Flaviviruses
ZIKV/ZVD
E
MV-ZIKA-E
Phase I: study completed; no results available
[102]
ZIKV/ZVD
E
MV-ZIKA-RSP-E
Phase I: study in progress
[103]
Lentiviruses
HIV/AIDS
HIV Gag
VEEV-Gag
Phase I: trials halted, stability & documentation
[104]
Coronaviruses
SARS-CoV-2/COV
S
MV (TMV-083)
Phase I: weak immunogenicity, trial discontinued
[75,76]
SARS-CoV-2/COV
S
VSV (V590)
Phase I: weak immunogenicity, trial discontinued
[78,79]
SARS-CoV-2/COV
S
VSV∆G-S
Phase I/II: study in progress
[81,82]
SARS-CoV-2/COV
S
LNP-VEEV-S RNA
Phase I/II: study in progress
[85]
C, capsid, CHIKV, Chikungunya virus; COV, COVID-19; EBOV, Ebola virus; E & Env, envelope proteins; EVD, Ebola virus disease; GP;
Glycoprotein; S, spike protein; SARS, severe acute respiratory syndrome; VEEV, Venezuelan equine encephalitis virus; VLPs, virus-like
particles; VSV, vesicular stomatitis virus; ZIKV, Zika virus; ZVD, Zika virus disease. C, capsid, CHIKV, Chikungunya virus; COV, COVID-19; EBOV, Ebola virus; E & Env, envelope proteins; EVD, Ebola virus disease; GP;
Glycoprotein; S, spike protein; SARS, severe acute respiratory syndrome; VEEV, Venezuelan equine encephalitis virus; VLPs, virus-like
particles; VSV, vesicular stomatitis virus; ZIKV, Zika virus; ZVD, Zika virus disease. Among filoviruses, especially EBOV has been a common target for vaccine devel-
opment due to its transmissibility and the severity of Ebola virus disease (EVD) during
outbreaks in 2014–2016 [105]. In one approach, the EBOV glycoprotein (GP) D637L mu-
tant, which displays superior cleavability and shedding compared to wildtype GP, was
expressed from a KUN vector [45]. Two subcutaneous doses of 1 × 109 KUN-GP/D637L
VLPs was sufficient to provide protection in three out of four immunized nonhuman pri-
mates. 3. Infectious Diseases In contrast, the immune response against WEEV was
weak and the protection against challenges with WEEV was only partial [33]. In the
context of DNA-based delivery, the attenuated VEEV V4020 strain was administered to
BALB/c mice as a DNA/RNA layered replicon vector, which elicited robust neutralizing
antibodies and protected mice from challenges with wildtype VEEV [34]. Protection against
aerosol challenges with wildtype VEEV was also demonstrated in vaccinated cynomolgus
macaques [35]. Furthermore, an MV-based vector expressing CHIKV capsid and envelope
proteins showed strong immunogenicity and protection from viremia in macaques [36]. The
MV-CHIKV VLP vaccine candidate was evaluated for safety and efficacy in a randomized,
double-blind phase I clinical trial showing a seroconversion rate of 44–92% after a single
dose, which reached 100% after a second immunization [96]. It was followed by a phase II
study, which elicited strong neutralizing antibodies without causing any serious adverse
events making it a promising CHIKV vaccine candidate [97]. Arenaviruses including such pathogens as LASV have also been targeted for vaccine
development. In this context, VSV-based expression of the LASV glycoprotein complex
(GPC) provided protection against LASV strains from Liberia, Mali, and Nigeria in guinea
pigs and macaques immunized with 1 × 106 and 6 × 107 pfu, respectively [37]. MV-based
GPC expression has also demonstrated protection in macaques after a single immunization
with 6 × 106 pfu of MV-GPC particles [38]. A randomized, placebo-controlled, dose-finding
phase I trial is in progress in healthy volunteers receiving two doses of MV-LASV [98]. In
another approach, the LASV GPC gene was introduced into the YFV vector between the
envelope (E) and non-structural protein 1 (NS1) [39]. Immunization of guinea pigs was 80% Vaccines 2021, 9, 1187 8 of 28 protective, but due to instability of the full-length GPC, GP1 and GP2 subunit constructs
were engineered in individual YFV vectors [40]. Combined immunization with YFV-LASV
GP1 and -GP2 showed 83% protection in guinea pigs with no stability issues. However,
prime-boost vaccination of marmosets failed to provide protection confirming previous
findings that robust immune responses and protection seen in rodents is not necessarily
reproducible in non-human primates [41]. Expression of either LASV GPC or nucleoprotein
(NP) from VEEV replicons protected guinea pigs from challenges with the LASV Josiah
strain [42]. However, protection was only established after three immunizations with
recombinant VEEV particles. 3. Infectious Diseases In another study, when macaques vaccinated with 5 × 107 VSV-EBOV GP particles
were challenged with the EBOV-Makona [46] and Zaire (ZEBOV) [47] strains, they were
resistant to EVD. The VSV-based EBOV-GP vaccine (VSV-ZEBOV) has been subjected to
phase III clinical evaluation in 7651 individuals with suspected EVD, of which 4123 were
vaccinated immediately and 3528 received a delayed vaccination [99]. No EVD cases were
detected in individuals immediately vaccinated while 16 EVD cases were observed among
those receiving delayed vaccination, which indicated that the vaccination was successful. Another phase III trial confirmed the efficacy, where no new EVD cases were detected in Vaccines 2021, 9, 1187 9 of 28 9 of 28 neither the 2119 individuals who received immediate vaccination nor in the 2041 subjected
to a 21-day delay in vaccine administration [100]. The VSV-ZEBOV vaccine has been ap-
proved by both the FDA and the EMA [101]. In the context of other filoviruses, VEEV-based
expression of the Marburg virus (MARV) GP provided protection in macaques against
MARV challenges after immunization with 1 × 107 pfu of VEEV-MARV-GP particles [100]. A VEEV vector expressing the Sudan virus (SUDV) GP was subjected to intramuscular
administration of 1 × 1010 particles in cynomolgus macaques, which protected the vac-
cinated animals against SUDV challenges [48]. Moreover, macaques immunized with
VEE-SUDV GP particles provided partial protection against EBOV challenges. Macaques
co-administrated with VEEV-SUDV-GP and VEEV-EBOV-GP were protected against both
SUDV and EBOV challenges. In the context of flaviviruses, BALB/c mice immunized with a single dose of VEEV
particles expressing the ectodomain of the DENV envelope resulted in protective immunity
against challenges with DENV [49]. Moreover, immunization of mice with 2 × 106 pfu of an
MV vector expressing the DENV envelope protein domain III (ED3) elicited robust immune
responses and resulted in partial protection against DENV [50]. A live-attenuated chimeric
YFV-DENV tetravalent vaccine (CYD-TDV) has been engineered and tested in rodent and
primate models, showing no toxicity, good safety, and robust immune responses [51,52]. The CYD-TDV vaccine has been tested in endemic populations [53]. In another approach, VEEV replicon RNA was engineered to express the codon
optimized Zika virus (ZIKV) membrane-envelope protein (prME) [54]. The delivery of
the VEEV-ZIKV-prME replicon RNA was facilitated by formulation of nanocarrier lipids
(NLCs). A single intramuscular dose of 10 ng RNA provided complete protection against
ZIKV challenge. 3. Infectious Diseases Furthermore, intramuscular Vaccines 2021, 9, 1187 10 of 28 10 of 28 administration of SFV-HIV-Env replicon RNA induced Env-specific immune responses in
four out of five mice [60]. In another approach, immunization of mice with SFV particles
expressing the Indian HIV-1C Env-Gag-Pol-RT genes elicited significant T-cell responses
with higher antibody titers compared to replicon RNA immunization [61]. SFV DNA
replicon delivery of HIV Env and a Gag-Pol-Nef fusion protein generated strong immune
responses in immunized BALB/c mice [62]. In attempts to improve stability and delivery
of VEE-HIV-1 gp140 RNA replicons, cationic nanoemulsion (CNE) formulations consisting
of squalene, 1,2-dioleoyl-3-tri-methylammonium-propane (DOTAP) and sorbitan trioleate
were developed [63]. In a comparative study, intramuscular injection of 50 µg of VEEV
RNA-CNE elicited stronger immune responses in rhesus macaques than what was obtained
for VEEV particles or MF59 adjuvanted HIV gp140 protein [110]. In the case of clinical eval-
uations for self-replicating RNA virus-based HIV vaccines, the safety and immunogenicity
of an alphavirus replicon HIV-1 Gag vaccine (AVX101) was subjected to a double-blind,
randomized, placebo-controlled trial in healthy adults [104]. The study was conducted
in the US and South Africa, but it was halted due to vaccine stability issues. Another
phase I trial was initiated, but it was prematurely terminated because of documentation
issues encountered by the contract manufacturer. However, the study results indicated that
in contrast to preclinical findings, only low levels of immune responses were elicited in
humans. Measurement of anti-vector antibodies showed only modest local reactogenicity. y
g
y
The importance of vaccine development against influenza virus relates to the occur-
rence of seasonal global outbreaks. In the context of MV, a recombinant MV AIK-C vaccine
expressing the hemagglutinin (HA) protein from the influenza A/Sapporo/107/2013
(H1N1pdm) strain elicited strong immune responses in cotton rats and provided protection
against challenges with influenza virus [64]. In the case of VSV, the VSV∆G vector lacking
the VSV G protein was engineered to express the HA protein of the highly pathogenic avian
influenza virus (HPAIV) A/Vietnam/1203/04 (VN1203) strain and the neuraminidase (NA
protein) of the mouse-adapted H1N1 influenza virus A/Puerto Rico/8/34 (PR8) [65]. A single immunization of mice with VSV∆G-H5N1 provided protection against lethal
H5N1 infection. In another study, a VSV-based H5N1 influenza virus vector containing
the full-length hemagglutinin (HAfl) was administered as a single dose or a prime-boost
regimen in mice, generating protection against lethal challenges with various H5 clade 2
viruses [66]. 3. Infectious Diseases Moreover, a single immunization of mice with 1 × 107 pfu of the chimeric
VSV vector expressing the ZIKA-prME resulted in robust antibody responses and rendered
mice resistant to ZIKV challenges [32]. A live MV-based vaccine expressing the ZIKV E
gene (MV-E2) provided protection against the nonlethal ZIKV Asian strain PRVABC59
and the lethal African strain MR766 in mice [55]. Despite 100% survival complete viral
clearance in the brain and reproductive tract did not occur. However, co-administration of
an MV vector expressing the ZIKV NS1 gene (MV-NS1 [2]) led to complete clearance of
ZIKV from the female reproductive tract and fetal protection was achieved. Although most clinical trials conducted on ZIKV vaccines are based on inactivated or
attenuated viruses and nucleic acids [106], a dose-finding phase I study with MV-ZIKA
(MV-E2) was carried out in 48 individuals, however, no results have been published
yet [102]. Furthermore, another phase I trial to validate the safety and immunogenicity of
MV-ZIKA is in progress in healthy 18–55-year-old volunteers in Austria [103]. Although HBV vaccines have been approved [107] there is still need for new vaccine
development due to the discovery of breakthrough infections, for instance. In attempts
to target HBV, MV vectors have been used for the expression of the surface antigen (HB-
sAg) [56]. Immunization with HBV-HBsAg resulted in 50% protection of rhesus monkeys. In another approach, the SFV RNA replicon expressing the HBV middle surface enve-
lope glycoprotein (MHB) and the core antigen (HBcAg) were packaged into a VSV G
envelope [57]. Mice immunized with 1 × 107 pfu of SFV-G-MHB were protected against
HBV challenges, while SFV-HBcAg immunizations did not provide protection. So far
clinical trials on HBV vaccines have focused mainly on DNA, live virus, and peptide-based
approaches [108]. Only one adenovirus-based phase I trial has been described [109]. No
published data is available for clinical applications of self-replicating RNA vector-based
HBV vaccines. The HIV/AIDS epidemic has obviously accelerated the development of vaccines
against HIV. Live attenuated MV vectors expressing HIV-1 Gag like particles with a gp160
Env protein envelope elicited robust cellular and humoral immune responses in mice [58]. Much attention has been dedicated to alphavirus-based HIV vaccine development. For
example, mice immunized with SFV-HIV-Env particles showed superior antibody titers
compared to plasmid DNA and recombinant Env protein [59]. 3. Infectious Diseases Prior to COVID-19
vaccines, both SARS-CoV and Middle East respiratory syndrome-coronavirus (MERS-CoV) Vaccines 2021, 9, 1187 11 of 28 11 of 28 have been targeted. For example, mice immunized with a VEEV vector expressing the
SARS-CoV Spike (S) protein resulted in protection against SARS-CoV challenges [72]. In
the context of MERS-CoV, the VSV G protein was replaced by the MERS-CoV S protein [73]. A single intramuscular or intranasal immunization with VSV∆G-MERS-CoV S elicited
neutralizing antibodies and T cell responses in rhesus macaques. g
p
q
Obviously due to the COVID-19 pandemic, SARS-CoV-2 has received major attention
as a vaccine target. MV-based expression of the SARS-CoV-2 S protein elicited robust Th1-
biased antibody and T cell responses in mice [74]. The MV-SARS-CoV-2 S vaccine candidate
TMV-083 was subjected to a randomized, placebo-controlled phase I clinical trial, which
based on disappointing weak immune responses in vaccinated volunteers was discontin-
ued [75,76]. VSV vectors have also been applied for overexpression of the SARS-CoV-2 S
protein [77]. Immunization of mice with VSV-SARS-CoV-2 S particles elicited neutralizing
antibody responses and protected against SARS-CoV-2 related pathogenesis. In the context
of clinical trials, the VSV-SARS-CoV-2 S vaccine candidate V590 was evaluated in a phase
I clinical trial in 252 volunteers [78]. The immunization proved safe and showed good
tolerability, but the immune responses were weaker than seen in COVID-19 patients, which
justified the termination of the trial [79]. In another approach the replication competent
VSV∆G vector was engineered by replacing the VSV G protein with the SARS-CoV-2 S
protein [80]. A single immunization with 5 × 106 pfu of VSV∆G-SARS-CoV-2 S elicited
potent neutralizing antibodies and protected Syrian golden hamsters against challenges
with SARS-CoV-2. In the case of clinical trials, the VSV∆G-SARS-CoV-2 S vaccine candidate
is subjected to a phase I/II clinical study, where volunteers will receive a single dose of
5 × 105, 5 × 106, or 5 × 107 pfu of VSV∆G-SARS-CoV-2 in the first part of the study [81]. As the trial is still in progress the interim experience has caught attention as other vaccines
have received EUA and the ethical question has been raised whether individuals in the
placebo group should be entitled to other vaccines during the study period [82]. The
decision was to balance the individual risk with the common good of participants without
compromising the quality of randomized studies. 3. Infectious Diseases In the context of alphaviruses, a single dose of 1 × 107 pfu of VEE-HA resulted
in protection against influenza A virus isolate A/HK/156/97 challenges in chickens [67]. In another study, 10 µg of SFV-HA replicon RNA provided protection in 90% of vaccinated
BALB/c mice [68]. The superiority of self-replicating replicon RNA was confirmed by
demonstrating that only 1.25 µg was required to provide protection in mice compared
to 80 µg needed for synthetic mRNA [69]. In a novel approach, the external domain of
the influenza virus M2 protein (M2e) was introduced into the E2 membrane protein in a
SIN vector, resulting in SIN particles (E2S1-M2e) with M2e expressed on its surface [70]. Mice intranasally immunized with SIN E2S1-M2e were protected from challenges with a
virulent influenza A virus strain. As CSFV targets monocytes and dendritic cells (DCs)
the nucleoprotein (NP) and HA genes of influenza virus were inserted into the CSFV
replicon RNA (RepRNA) vector [71]. Packaging of a Rep-HA/Rep-NP mix in viral replicon
particles (VRPs) was compared with polyethylenimine (PEI)-based RNA complexes and
naked RepRNA in pigs. Both VRPs and PEI-RepRNA complexes elicited strong HA and
NP specific humoral and cellular immune responses, whereas naked RNA induced only
low-level immunogenicity. Overall, CSFV VRPs showed superior immunogenicity in pigs. The current COVID-19 pandemic has promoted vaccine development to a new level. The breath and intensity of global activities related to vaccines have been unprecedented
leading to EUA of both nucleic acid- [111,112] and viral vector-based [2–4] COVID-19
vaccines in approximately a year from the onset of the outbreak. As the authorized viral
vector based COVID vaccines are based on adenoviruses they are not discussed here, and
the focus of the current review will be on self replicating RNA viruses Prior to COVID 19 g
y
p
g
y
p g
The current COVID-19 pandemic has promoted vaccine development to a new level. The breath and intensity of global activities related to vaccines have been unprecedented
leading to EUA of both nucleic acid- [111,112] and viral vector-based [2–4] COVID-19
vaccines in approximately a year from the onset of the outbreak. As the authorized viral
vector based COVID vaccines are based on adenoviruses they are not discussed here, and
the focus of the current review will be on self-replicating RNA viruses. 3. Infectious Diseases In the context of animal coronaviruses,
the chimeric flavivirus BVDV vector was applied for the expression of a spike antigen of
the porcine epidemic diarrhea virus (PEDV) [83]. Intramuscular injection of BALB/c mice
elicited BVDV- and PEDV-specific antibodies and neutralized both BVDV and PEDV. p
Self-amplifying RNA viruses have also been used in COVID-19 vaccine development
as liposome nanoparticle (LNP) encapsulated RNA replicons. In this context, VEEV-based
RNA replicons were engineered to express the prefusion-stabilized SARS-CoV-2 S RNA [84]. Intramuscular administration of LNP SARS-CoV-2 S RNA in BALB/c mice elicited robust
and dose-dependent SARS-CoV-2 specific antibody responses and SARS-CoV-2 neutral-
ization. The antibody titers were superior to those seen in recovered COVID-19 patients. The LNP SARS-CoV-2 S RNA vaccine candidate has been subjected to a randomized,
placebo-controlled, dose-finding phase I/II study in healthy volunteers [85]. No results
from the study are available yet. In another study, the VEEV-based self-replicating RNA
(STARR)-based vaccine (LUNAR-COV19) expressing the full-length SARS-CoV-2 S protein
has been evaluated in BALB/c mice [86]. A single immunization elicited strong neutral-
izing antibody responses and both 2 µg and 10 µg doses protected humanized ACE2
transgenic mice from mortality and measurable infection after challenges with wildtype
SARS-CoV-2. Recently, SIN particles expressing the SARS-CoV-2 S protein were combined
with the OX40 immunostimulatory antibody (αOX40) for intraperitoneal immunization
of C57BL/6J mice [87]. A prime-boost vaccination strategy with 14 days between the two
doses elicited long-lasting neutralizing antibodies and robust T-cell responses and sera
from vaccinated mice inhibited the function of the SARS-CoV-2 S protein. Furthermore,
immunized mice were protected against challenges with SARS-CoV-2. In addition to viral targets, bacterial and parasite infections have also been studied. For example, SIN vectors have been applied for the expression of the Bacillus anthracis
protective antigen (PA) [88]. Immunization of Swiss Webster mice elicited PA-specific
IgG and neutralizing antibody responses and provided some protection against a lethal Vaccines 2021, 9, 1187 12 of 28 12 of 28 bacterial strain. SFV particles expressing the Brucella abortus translation initiation factor 3
(IF3) elicited IF3-specific IgM antibodies and T cell proliferative responses and protected
immunized BALB/c mice [89]. In the context of parasites and malaria vaccines, SFV particles expressing the Plasmod-
ium falciparum Pf332 antigen were subjected to immunization studies in BALB/c mice [90]. A single immunization elicited Th1-type immune responses, which were further enhanced
by a second immunization. 3. Infectious Diseases In another approach, the Plasmodium yoelii circumsporozoite
(CS) protein class I major histocompatibility complex-restricted-9-mer epitope SYVpSAEQI
was expressed from a SIN vector [91]. Subcutaneous immunization of BALB/c mice with
SIN-Mal particles induced robust epitope-specific CD8+ T cell responses and provided
protection against malaria. Recently, the Phlebotomus papatasi SP15-Leishmania major stress
inducible protein 1 (PpSP15-LmSTI1) fusion protein was compared for expression in BHK-
21 cells after transfection of SFV replicon RNA, SFV DNA replicons and a conventional
DNA plasmid [92]. The relative expression was significantly higher for the SFV replicon
RNA than both SFV and conventional DNA vectors, making it an attractive alternative for
vaccine development against leishmaniasis. 4. Cancer Self-replicating RNA viruses have also been applied for cancer immunotherapy and
cancer vaccine development [5]. The general approach has been to introduce a tumor
antigen into the self-replicating RNA viral vector for immunization studies, which have
demonstrated both prophylactic and therapeutic efficacy in preclinical animal models. Other approaches have involved expression of immunostimulatory genes such as cytokines
and even reporter genes. Obviously, the application of reporter genes such as GFP and
luciferase allows efficient monitoring of expression. The therapeutic effect seen after
administration of alphavirus vectors expressing reporter genes relates to the induction
of apoptosis, but the efficacy has been inferior compared to treatment with alphavirus
vectors expressing cytokines like interleukin-12 (IL-12) [113]. While cancer vaccines aim at
providing protection against tumor development, oncolytic viruses possess therapeutic
activity, also named virotherapy, for the treatment of existing tumors [114]. Oncolytic
viruses are characterized by efficient replication in and killing of tumor cells without
causing harm to normal cells, which make them attractive for cancer therapy. There are
different types of engineered oncolytic viruses such as herpes simplex virus, adenovirus,
vaccinia virus and reovirus. Moreover, naturally oncolytic viruses have been identified
for Newcastle disease virus [115]. Among self-replicating RNA viruses, attenuated MV
strains [116], engineered VSV vectors [117], and the naturally oncolytic M1 alphavirus [118]
exist. Although the focus is on prophylactic and therapeutic cancer vaccines, examples
of virotherapy are also included here. So far, a limited number of clinical trials have also
been conducted. Examples of preclinical studies and clinical trials are presented below and
summarized in Tables 3 and 4. Table 3. Examples of preclinical studies on self-replicating RNA viral vector vaccines against cancers. Cancer
Antigen/Therapeutic
Vector
Findings
Ref. Brain
Glioblastoma
Endostatin
SFV
Complete tumor regression in mice
[119]
Glioblastoma
IL-18
DC-SFV + IL-12
Enhanced Th1-type response, anti-tumor immunity
[120]
Glioblastoma
gp100, IL-18
SIN DNA
Therapeutic effect, prolonged survival in mice
[121]
Glioblastoma
CHIKV E3-E2-6K-E1
VSV∆G-CHIKV
Selective infection, elimination of tumor cells
[122]
Glioblastoma
GFP, CEA, NIS
GSC-MV
Anti-tumor effect, prolonged survival in mice
[123]
Glioblastoma
EGFP
SFV VA
Tumor inhibition, prolonged survival in mice
[124]
CT-2A glioma
miRT124
SFV4
Replication in tumor cells, prolonged survival
[125] Table 3. Examples of preclinical studies on self-replicating RNA viral vector vaccines against cance 13 of 28 13 of 28 Vaccines 2021, 9, 1187 Vaccines 2021, 9, 1187
13 of 28
Table 3. Cont. Cancer
Antigen/Therapeutic
Vector
Findings
Ref. 4. Cancer Breast
A2L2
HER2/neu
Ad/SIN DNA
Prolonged survival in mice
[126]
A2L2
HER2/neu
Ad + SIN DNA
Tumor protection in mice with 80% less DNA
[127]
HER2
HER2 ECD, TMs
VEEV (VRP-HER2)
Preventive, therapeutic tumor growth control in mice
[128]
4T1
IL-12
SFV + S. 4. Cancer typhimurium
Inhibition of metastasis, long-term survival in mice
[129]
TNBC
M1
M1 + Doxorubicin
Synergistic effect of M1 and Doxorubicin
[118]
MCF7
SLAMblind
MV
Targeting and killing of breast cancer cells
[130]
Cervical
HPV-16
Capsid
MV
Humoral immune responses in mice
[131]
HPV-16
Capsid
MV + HPV protein
IgG and neutralizing antibody responses
[132]
CRPV
E1, E2, E6, E7
VSV
Reduced papilloma volumes, elimination of disease
[133]
HPV-16
E7
VSV
Tumor regression in mice
[134]
HPV-16
E7
VEEV
Immune response, protection against tumors in mice
[135]
HPV-16
E6/E7 fusion
SFVEnh
Tumor regression, complete eradication
[136]
HPV
E6-E7
SFV DNA + EP
85% of immunized mice became tumor-free
[137]
Colon
CT26
GM-CSF
KUN
Tumor regression, cure in 50% mice
[138]
CT26
VEGFR-2
SFV
Inhibition of tumor growth, metastasis
[139]
CT26
VEGFR-2 + IL-4
SFV
Super immunogenicity, prolonged survival
[139]
CT26
LacZ
SFV RNA
Tumor regression, protection against tumor cells
[140]
MC28cea
GM-CSF
MV
Tumor regression, prevention of re-engraftment
[141]
Lung
H358cea
EGFP
SFV
Protection against HBV in 50% of rhesus monkeys
[142]
A549
EGFP
SFV VA
Superior survival compared to adenovirus delivery
[143]
CT26
LacZ
SIN
Complete tumor remission, prolonged survival
[144]
CL25
oMV
MV Hu-191
Suppressed tumor growth, prolonged survival
[145]
LLC
oMV
MV Schwarz
Suppression of uncontrollable tumor growth
[146]
Adenocarcinoma
CEA
MV
Tumor regression in mice
[147]
H2009, A549
IFNβ
VSV
Tumor regression in mice
[148]
LM2
IFNβ
VSV
Prolonged survival, cure in 30% of mice
[148]
Melanoma
B16-OVA
GM-CSF
KUN
Tumor regression, cure of more than 50% of mice
[138]
B16-OVA,
B16F0
SIIINFEKL epitope
YFV
Immune response, protection in mice
[149]
B16
TRP-2
VEEV
Immune response, prolonged survival in mice
[150]
B16
TRP-2
VEEV + GITR mAb
Complete tumor regression in 90% of mice
[151]
B16
TRP-2
VEEV + CTLA-4 mAb
Complete tumor regression in 50% of mice
[151]
B16
VEGFR-2/IL-12 +
survivin/β-hCG
SFV DNA
Superior tumor growth inhibition, prolonged
survival after combination therapy
[152]
mel Z
oMV
MV L-16
Tumor cell killing, inhibition of tumor growth
[153]
B16-OVA
LCMV GP
VSV
Efficacy in subcutaneous tumor models
[154]
Ovarian
A2780
LCMV GP
VSV + ruxolitinib
Tumor regression in mice
[155]
SKOV3ip.1
αFR scFV
MV
Tumor volume reduction, prolonged survival
[156]
SKOV3ip.1
CEA, NIS
MV
Dual therapy superior in mice
[157]
ES2
IL-12
SIN + irinotecan
Long-term survival in mice
[158]
MOSEC
OVA
SFV + VV
Immune response, enhanced anti-tumor activity
[159]
Pancreatic
PDAC
GFP
VSV
Superior oncolytic activity compared to Sendai, RSV
[160]
PDAC
GFP
VSV-∆M51
Anti-tumor activity enhanced by gemcitabine
[161]
KLM1
SLAMBlind
MV
Suppression of tumor growth in mice
[162]
Capan-2
SLAMBlind
MV
Suppression of tumor growth in mice
[162] Table 3. 4. Cancer Cont. Vaccines 2021, 9, 1187 14 of 28 14 of 28 Table 3. Cont. Table 3. Cont. Cancer
Antigen/Therapeutic
Vector
Findings
Ref. Prostate
PC-3
CEA
MV
Delay in tumor growth, prolonged survival in mice
[163]
PC-3
oMv, oMuV
MV + MuV
Immune responses, protection in mice
[164]
DU145, PC-3
GFP
VSV-∆M51
Apoptosis in tumor cells, prolonged survival
[165]
DU-145,
22Rv1
LCMV GP
VSV
Long-term remission in mice
[166]
TRAMP-C
PSMA
VEEV
PSMA-specific immune response in mice
[167]
TRAMP
STEAP
VEEV
STEAP-specific immune response, prolonged
survival
[168]
TRAMP
PSCA
VEEV
Long-term survival for 12 months in 90% of mice
[169]
Ad, adenovirus; CEA, carcinoembryonic antigen; CRPV, cottontail rabbit papilloma virus; CSC, glioma stem cell; CTLA-4, CTL antigen-4;
DC, dendritic cell; GFP, green fluorescent protein; GITR, glucocorticoid-induced tumor necrosis factor receptor; GM-CSF, granulocyte
macrophage-colony stimulating factor; IL. interleukin; KUN, Kunjin virus; LCMV, lymphocytic choriomeningitis virus; Lewis lung
carcinoma; MOSEC, murine ovarian surface epithelial carcinoma; MV, measles virus; NIS, sodium iodine symporter; PDAC, pancreatic
ductal adenocarcinoma; RROC, refractory recurrent ovary cancer; RSV, respiratory syncytial virus; SFV, Semliki Forest virus; TNBC, triple-
negative breast cancer; SIN, Sindbis virus; STEAP, six-transmembrane epithelial antigen of the protein; TRAMP, transgenic adenocarcinoma
mouse prostate; VEEV, Venezuelan equine encephalitis virus; VEGFR-2, vascular endothelial growth factor receptor-2; VSV, vesicular
stomatitis virus; YFV, yellow fever virus. Table 4. Examples of clinical studies on self-replicating RNA viral vector vaccines against cancers. Cancer
Antigen/Therapeutic
Vector
Findings
Ref. Breast
HER2
HER2 ECD TMs
VEEV (VRP-HER2)
Phase I: immune response, PR and SD
[128]
HER2
HER2 ECD TMs
VEEV (VRP-HER2)
Phase II: study in progress
[170]
Cervical
HPV-16
E6/E7 fusion
SFVEnh (Vvax001)
Phase I: safe, immune responses in all patients
[171]
Colon
Stage III-IV
CEA
VEEV
Phase I: immune responses, prolonged survival
[172]
Ovarian
RROC
CEA
MV
Phase I: well-tolerated, dose-dependent activity
[173]
Pancreatic
Metastatic
CEA
VEEV
Phase I: immunogenicity, prolonged overall survival
[174]
CEA, carcinoembryonic antigen; MV, measles virus; RROC, refractory recurrent ovary cancer; VEEV, Venezuelan equine encephalitis virus. e 4. Examples of clinical studies on self-replicating RNA viral vector vaccines against cancers. Brain tumors have been the target of several studies due to the severity of disease
and very few alternative options of successful treatment. For instance, SFV particles
expressing endostatin were compared to a retrovirus vector expressing endostatin, and
SFV-LacZ in a B16 brain tumor mouse model [119]. 4. Cancer In another approach, neuron-specific micro-
RNA miRT124 sequences were introduced into the replication-competent SFV4 vector,
which modified its tropism [125]. A single intraperitoneal administration of SFV4-miRT124
to C57BL/6 mice with implanted CT-2A orthotopic gliomas demonstrated significant tumor
growth inhibition and provided prolonged survival. g
p
p
g
Related to breast cancer, immunization of BALB/c mice with adenovirus particles and
SIN DNA replicons expressing the HER2/neu gene inhibited A2L2 tumor growth [126]. However, if the tumor challenges took place prior to immunization, no inhibition was
observed. A strategy of prime immunization with SIN DNA followed by a boost with
adenovirus particles significantly prolonged the survival of mice. In another study, intra-
dermal administration of BALB/c mice with SIN-HER2/neu DNA replicons generated
robust antibody responses and required 80% less replicon DNA than conventional plasmid
DNA to achieve tumor protection [127]. In another study, a novel VEEV vector expressing
the extracellular domain (ECD) and transmembrane (TM) domains of HER2 (VRP-HER2)
showed robust immunogenicity, both preventive and therapeutic efficacy. and control of
tumor growth in a HER2 transgenic mouse model [128]. Moreover, VRP-HER2 showed
good tolerance in a phase I trial in stage IV HER2 overexpressing breast cancer patients
and generated partial response (PR) in one patient and continued stable disease (SD) in
two other patients [170]. Additionally, a phase II trial on VRP-HER2 and pembrolizumab
in 39 HER2-positive breast cancer patients is in progress [170]. In another study, 2 × 108
SFV-IL12 particles and 2 × 107 units of an aroC- Salmonella typhimurium strain (LVR01)
were administered to mice with 4T1 tumor nodules, which provided complete inhibition of
lethal lung metastases and long-term survival in 90% of immunized mice [129]. Compared
to administration of either SFV-IL12 or LVR01 alone, the synergistic effect of combination
therapy presents a promising alternative for prevention and eradication of metastases
in advanced breast cancer. In the case of triple-negative breast cancer (TNBC), the most
aggressive breast cancer molecular subtype, Doxorubicin was demonstrated to increase
the oncolytic effect of the oncolytic M1 alphavirus by 100-fold, specifically in TNBC cells
in vitro and significantly inhibited tumor growth in vivo [118]. In the context of MV, reverse
genetics was applied to engineer a recombinant MV named rMV-SLAMblind, which is se-
lectively unable to use the signalling lymphocyte activation molecule (SLAM) [130]. 4. Cancer Intratumoral administration of SFV-
Endostatin showed significant tumor growth inhibition and reduction of intratumoral
vascularization, which was superior to both retrovirus and SFV-LacZ treatments. In
addition, intravenous SFV-Endostatin administration generated 3-fold increase in serum
levels of endostatin. In another approach, intratumoral injection of DCs transduced with
SFV particles expressing interleukin-18 (IL-18) in combination with recombinant IL-12
protein therapy, elicited Th1-type immune responses and provided anti-tumor immunity
in a B16 brain tumor mouse model [120]. Furthermore, a SIN DNA replicon encoding the
human gp100 and mouse IL-18 was evaluated in B16-gp100 implanted brain tumor models
in mice [121]. Immunization with SIN-gp100-IL-18 DNA resulted in therapeutic effects,
enhanced protection of malignant brain tumors, and significantly prolonged survival
rates. A chimeric VSV vector, where the VSV G protein was replaced by the CHIKV
E3-E2-6K-E1 polyprotein (VSV∆G-CHIKV), showed selective infection and elimination Vaccines 2021, 9, 1187 15 of 28 of brain tumor cells [122]. Moreover, tumor-bearing mice showed extended survival for
more than 100 days. VSV∆G-CHIKV also showed targeting in intracranial melanoma
xenografts derived from patients with only minor detectable infection of normal cells. Moreover, oncolytic MV vectors expressing GFP, carcinoembryonic antigen (CEA) and
sodium iodide symporter (NIS) have been shown to replicate and providing cytotoxic
effects in glioma stem cells (GSCs) [123]. When GSCs transduced with MV-GFP were
implanted into the right caudate nucleus of nude mice a significant prolongation of survival
was obtained. Application of oncolytic replication competent SFV VA-EGFP particles was
evaluated in BALB/c mice in a subcutaneous orthotopic tumor model stably expressing
firefly luciferase [124]. A single intravenous administration of SFV VA-EGFP completely
inhibited intracranial firefly bioluminescence and provided long-term survival in 16 out
of 17 mice. The treatment was well tolerated causing no damage to heart, liver, spleen,
or brain. Applications of alphavirus vectors for cancer immunotherapy and gene therapy of
brain tumors have raised some concerns due to their neurotropic nature [175]. In one
approach, distribution of recombinant SFV particles (recSFV) and RNA replicons (recRNA)
expressing firefly luciferase was compared in tumor-free and 4T1 mammary tumor-bearing
mice [176]. Intravenous administration of recRNA resulted in primary brain targeting in
both tumor-free and tumor-bearing mice. However, local intratumoral injection led to high
levels of luciferase expression in tumors. Interestingly, predominant tumor targeting of
recSFV was observed after low intravenous or intraperitoneal viral doses, whereas higher
doses led to a broader luciferase distribution. 4. Cancer Unlike
the MV-Edmonston vaccine strain, rMV-SLAMblind used the polio virus receptor-related
4 (PVRL4) as a receptor to infect breast cancer cells showing superior oncolytic activity. Vaccines 2021, 9, 1187 16 of 28 16 of 28 In vivo studies of rMV-SLAMblind in monkeys showed no clinical symptoms, suggesting
that the vector could be a promising oncolytic candidate for breast cancer therapy. In vivo studies of rMV-SLAMblind in monkeys showed no clinical symptoms, suggesting
that the vector could be a promising oncolytic candidate for breast cancer therapy. Although the recombinant protein-based human papilloma virus (HPV) vaccine Gar-
dasil was approved by the FDA in 2006 against cervical cancer [177], there is a continuous
development in this area. Recombinant MV expressing the HPV-16 L1 capsid protein was
subjected to immunization studies in transgenic mice, which resulted in strong humoral
immune responses [131]. In another study, the MV-HPV16 L1 capsid vaccine was com-
pared to recombinant HPV16L1 and 18L1 protein vaccines produced in Pichia pastoris in
immunized non-human primates [132]. Both MV- and P. pastoris-based vaccines induced
immune responses. Prime-boosting combination immunization elicited HPV-specific total
IgG and neutralizing antibodies, which was not affected by pre-existing antibodies against
MV. Moreover, recombinant VSV vectors have been utilized for the expression of the
cottontail rabbit papillomavirus (CRPV) E1, E2, E6, and E7 proteins and immunization
of rabbits [133]. VSV-E1, E2, E6, and E7 immunizations significantly reduced papilloma
volumes, the VSV-E7 being the most efficient reducing the papilloma volumes by 96.9%,
which ultimately eradicated the disease. In another approach, mice bearing TC-1 syngeneic
tumors were immunized with VSV-HPV E7 [134]. A single intramuscular injection of
C57BL/6 mice with 5 × 106 pfu of VSV-HPV E7 elicited HPV16 E7 specific T cells and dis-
played anti-tumor activity resulting in a 10-fold reduction in tumor volume and regression
of pre-existing tumors. p
g
Among alphaviruses, VEEV vectors have been utilized for the expression of the HPV-
16 E7 protein [135]. Immunization of C57BL/6 mice elicited CD8+ T cell responses and
protected mice from tumor challenges. In another study, an SFV vector containing the
translation enhancer signal from the SFV capsid gene was engineered to express the HPV
E6-E7 fusion [136]. Tumor regression and complete eradication of established tumors
were observed in immunized C57BL/6 mice. The SFVenh-HPV E6/E7 vaccine candidate
Vvax001 has been subjected to a phase I clinical trial in 12 individuals with a history of
cervical intraepithelial neoplasia [171]. 4. Cancer Complete tumor remission was observed in one third of immunized mice
and tumor re-engraftment was rejected. g
j
Another area of opportunity is lung cancer. Human H358a non-small cell lung cancer
(NSCLC) cells transduced by SFV-EGFP particles were efficiently killed and the growth
of H358a spheroids was inhibited [142]. Moreover, nu/nu mice with H358a xenografts
were injected with SFV-EGFP particles, which resulted in complete tumor regression in
three out of seven mice. In comparison to a conditionally replicating adenovirus vector
(Ad5-Delta24TK-GFP), the replication-competent SFV (VA7)-EGFP particles were locally
administered to nude mice with A549 lung adenocarcinoma xenografts [143]. Mice immu-
nized with SFV-EGFP showed superior survival compared to adenovirus-based vaccination. Systemic administration, however, did not elicit significant immune responses with either
vector. In another approach, SIN-LacZ particles were intravenously administered to mice
with implanted CT26.CL25 colon tumors [144]. SIN-LacZ particles induced complete tumor
remission and provided long-term survival. MV vectors have also been subjected to lung
cancer treatment. In this context, the oncolytic MV Hu-191 strain effectively suppressed
tumor growth and significantly prolonged the survival of C57BL/6 mice implanted with
Lewis lung carcinoma (LLC) cells [145]. It was demonstrated in another study that the
live-attenuated oncolytic MV Schwarz strain prevented uncontrollable growth of estab-
lished lung and colorectal adenocarcinomas in nude mice with xenografts [146]. Similarly,
the expression of CEA from the MV Edmonston strain resulted in potent killing of lung
cancer cell lines and tumor regression in mice [147]. Additionally, a VSV vector expressing
interferon-β (VSV-IFNβ) reduced tumor growth in intratumorally immunized mice with
H2009 and A549 lung tumors [148]. Superior efficacy was achieved by intratumoral admin-
istration of VSV-IFNβ leading to tumor regression, prolonged survival, and cure in 30% of
immunized syngeneic LM2 lung tumors [148]. y g
g
Melanoma is an indication that has been frequently visited for vaccine development ap-
plying viral vectors [178]. For example, immunization of C57BL/6 mice carrying B16-OVA
melanoma tumors with KUN-GM-CSF particles resulted in substantial tumor regression
and cure of more than 50% of treated animals [138]. Moreover, expression of the chicken
ovalbumin (OVA) cytotoxic T cell lymphocyte (CTL) epitope SIINFEKL from an YFV vector
induced SIINFEKL-specific CD8+ lymphocytes and provided protection against challenges
with B16-OVA or B16F0 melanoma cells in immunized C57BL/6 mice [149]. Alphaviruses
have also been used for melanoma therapy. 4. Cancer Patients received three immunizations of 5 × 105,
5 × 106, 5 × 107, or 2.5 × 108 infectious SFVenh-HPV E6/E7 particles at a three-week
interval. The vaccination showed high safety and tolerability in patients with HPV-induced
cancers. HPV-specific immune responses were detected in all 12 patients. SFV DNA
replicons have also been employed for HPV vaccine development [137]. Intradermal
immunization of mice with SFV-HPV E6-E7 DNA replicons accompanied by electroporation
eradicated 85% of tumors [135]. The efficacy of DNA replicon immunization compared
to conventional plasmid DNA demonstrated that a 200-fold lower dose of only 0.05 µg of
SFVDNA was sufficient for therapeutic efficacy. p
y
Colon cancers have also been targeted by self-replicating RNA viral vectors. For in-
stance, a noncytopathic KUN vector was engineered to express the granulocyte macrophage-
colony stimulating factor (GM-CSF) [138]. Intratumoral immunization of BALB/c mice
implanted with CT26 colon xenografts induced CD8+ T cell responses, resulted in tumor
regression, and in cure of 50% of vaccinated mice. SFV particles expressing the vascular
endothelial growth factor receptor-2 (VEGFR-2) inhibited tumor growth, reduced tumor
angiogenesis, and prevented metastatic spread in immunized BALB/c mice [139]. Addi-
tionally, combination therapy of SFV-VEGFR2 and SFV-IL-4 elicited stronger VEGFR-2
antibody responses and provided prolonged survival of vaccinated mice. Immunization
with RNA replicons has also been successful, the classic example being the immunization
of mice with SFV-LacZ RNA, which elicited antigen-specific CD8+ T cell responses [140]. A single immunization with 0.1 µg SFV-LacZ RNA provided protection against tumor
challenges and therapeutic immunization prolonged survival of mice with pre-existing
tumors. In a phase I clinical trial, patients with stage IV colorectal cancer received VEEV
particles expressing the CEA every three weeks for four immunizations [172]. Later the
study was expanded to include stage III patients. Antigen-specific effector T cells were
elicited, and long-term survivors were identified suggesting prolonged overall survival. In the case of oncolytic MV vectors the expression of GM-CSF resulted in therapeutic Vaccines 2021, 9, 1187 17 of 28 17 of 28 efficacy and adaptive immune responses in a colon adenocarcinoma MC38cea model [141]. Intratumoral administration of MV-GM-CSF delayed tumor progression and prolonged
survival time. Complete tumor remission was observed in one third of immunized mice
and tumor re-engraftment was rejected. efficacy and adaptive immune responses in a colon adenocarcinoma MC38cea model [141]. Intratumoral administration of MV-GM-CSF delayed tumor progression and prolonged
survival time. 4. Cancer For instance, humoral immune responses,
anti-tumor activity, and prolonged survival were obtained in a B16 mouse melanoma
model after immunization with VEEV particles expressing the tyrosine-related protein-2
(TRP-2) [150]. In another approach, VEEV-TRP-2 particles were combined with antagonist
anti-CTL antigen-4 (CTLA-4) or agonist anti-glucocorticoid-induced tumor necrosis factor
receptor (GITR) monoclonal antibodies (mAbs) [151]. Immunization with VEEV-TRP-2 and
anti-GITR mAbs induced complete tumor regression in 90% of mice, whereas VEEV-TRP-2
and anti-CTLA-4 treatment resulted in tumor shrinkage in 50% of animals. In the context of
DNA-based delivery, SFV DNA replicons expressing VEGFR-2 and IL-12 from one plasmid
vector and survivin and β-hCG antigens from another plasmid were co-administered
to mice with implanted B16 melanoma tumors [152]. The combination immunization
provided superior tumor growth inhibition and prolonged survival compared to adminis-
tration of either SFV DNA replicon alone. In the case of MV vectors, the oncolytic potential
of the MV Leningrad-16 (L-16) strain was demonstrated to provide efficient killing of tumor
cells and inhibition of tumor growth in the mel Z mouse melanoma model [153]. Related to
VSV, a pseudotyped vector, where the VSV-G protein was replaced by the non-neurotropic
lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP), showed efficacy in subcu-
taneous A375 xenograft and B16-OVA syngeneic mouse tumor models, and also reduced
the size of lung metastasis after systemic treatment [154]. Vaccines 2021, 9, 1187 18 of 28 18 of 28 In the context of ovarian cancer, the pseudotyped VSV-LCMV-GP vector showed
oncolytic activity against A2780, HOC7, SKOV6 and other ovarian cancer cell lines and
in vivo in the A2780 ovarian mouse tumor model [155]. Tumor regression was further en-
hanced by combination treatment with the JAK1/2 inhibitor ruxolitinib. MV vectors have
also been evaluated for ovarian cancer therapy. Tumor-specific targeting has been achieved
by engineering of the MV-αFR vector with a single-chain antibody (scFV) sequence for
the alpha-folate receptor (αFR) [156]. Intratumoral injection of MV-αFR into mice with
ovarian SKOV3ip.1 xenografts demonstrated reduced tumor volumes and prolonged over-
all survival. Moreover, MV-CEA and MV-NIS have been applied alone or in combination
for immunization of mice implanted with SKOV3ip.1 xenografts [157]. The dual therapy
was superior to either MV-CEA or MV-NIS treatment alone. The MV-CEA vector has been
evaluated in a phase I clinical trial in patients with taxol and platinum-refractory recurrent
ovarian cancer (RROC) [173]. 4. Cancer The study demonstrated that intraperitoneal administration
of MV-CEA was well tolerated and provided dose-dependent biological activity in heavily
pre-treated patients, of which SD was achieved in 14 out of 21 patients. Alphavirus vectors
have also been evaluated for ovarian cancer therapy. Combination therapy of SIN-IL-12
particles and the CPT-11 topoisomerase inhibitor irinotecan provided long-term survival
in SCID mice with grafted highly aggressive ES2 human ovarian tumors [158]. In another
study, C57BL/6 mice with murine ovarian surface epithelial carcinoma (MOSEC) received
a prime immunization of SFV-OVA followed by boost vaccination with vaccinia virus
expressing OVA (VV-OVA), which elicited OVA-specific CD8+ T cell immune responses
and enhanced anti-tumor activity [159]. Due to the poor prognosis of pancreatic cancer patients plenty of efforts have been
dedicated to the development of vaccines. The oncolytic potential of VSV vectors has
been verified in highly aggressive pancreatic ductal adenocarcinoma (PDAC) [160]. In
comparison to Sendai virus and respiratory syncytial virus (RSV), VSV showed superior
oncolytic activity although PDAC cells were shown to be highly heterogenous to VSV sus-
ceptibility reducing the therapeutic efficacy. In another study, wildtype VSV, VSV-GFP and
the oncolytic VSV-∆M51-GFP were tested in five PDAC cell lines with (+MUC1) or without
(MUC1 null) MUC1 expression [161], showing oncolytic activity independent of MUC1
expression. The VSV-∆M51-GFP vector generated significant reduction in tumor growth
in mice with implanted PDAC xenografts. The anti-tumor activity was improved when
gemcitabine was co-administered with VSV. Related to MV vectors, SCID mice with KLM1
and Capan-2 pancreatic tumor xenografts were immunized with MV-SLAMBlind, which
resulted in significant suppression of tumor growth [162]. In the case of alphaviruses, a
phase I clinical study in pancreatic cancer patients was conducted with VEEV-CEA particles
efficiently infecting DCs [174]. Repeated intramuscular injection of VEEV-CEA induced
clinically relevant T cell and antibody responses, which mediated cellular cytotoxicity
against tumor cells and prolonged overall survival in patients. g
p
g
p
In the context of prostate cancer, a significant delay in tumor growth and prolonged
survival was seen in a prostate PC-3 mouse model after intratumoral immunization with
MV-CEA [163]. In another application, co-administration of oncolytic MV and mumps virus
(MuV) vectors generated superior anti-tumor activity and prolonged survival in the PC-3
prostate cancer model compared to individual administration of MV or MuV [164]. 5. Conclusions In summary, numerous examples of applications of self-replicating RNA viral vectors
have been presented for targeting both infectious diseases (Tables 1 and 2) and various
cancers (Tables 3 and 4). In many cases, target-specific humoral and cellular immune
responses have been obtained. In the context of cancer therapy and cancer vaccinations,
inhibition of tumor growth, tumor regression and even tumor eradication have been
observed. Moreover, immunized animals including mice, guinea pigs and non-human
primates were protected against challenges with lethal doses of infectious agents and tumor
cells. One attractive characteristic of self-amplifying RNA viruses, especially alphaviruses,
is the flexibility of applying them as recombinant viral particles, RNA replicons or lay-
ered DNA/RNA vectors (Figure 1). The main feature of RNA replication/amplification
has allowed similar immune responses and challenge protection to be achieved for self-
replicating RNA viruses with significantly lower doses compared to conventional viral
particles, synthetic RNA, or plasmid DNA. Alternatively, higher doses could potentially in-
duce stronger immune responses. Additionally, the prolonged release of antigens expressed
from self-replicating RNA contributes to B cell stimulation and immune stimulation is also
enhanced by generation of double-stranded RNA intermediates in transfected cells [69]. Moreover, the rapid RNA degradation renders the heterologous gene expression transient,
which is an advantage for vaccine development and cancer therapy, where high-level short-
term expression is preferable. On the other hand, although not the topic of this review,
self-replicating RNA virus vectors are not suitable for the treatment of chronic diseases,
where long-term gene expression is required. Self-replicating RNA viruses do not possess
reverse transcriptase activity and therefore do not integrate into the host genome. However,
application of self-replicating RNA viral vectors also presents some disadvantages. In the
case of replicon RNA, the ssRNA structure is sensitive to degradation, which demands
careful handling and has required RNA encapsulation in LNPs for improved stability and
delivery [84,85]. RNA-based vaccines have also stricter demands on storage and transporta-
tion temperatures. In the case of recombinant self-replicating RNA virus particles, safety
concerns have been raised, requiring engineering of helper vectors for conditionally infec-
tious particles [180] and split helper systems [181]. The use of replication-proficient and
oncolytic viruses for cancer therapy also needs special attention to ensure that no damage
is caused to normal tissue. 4. Cancer In the
context of VSV vectors, the VSV-∆M51-GFP showed efficient replication in human DU145,
and PC-3 cell lines, which induced apoptosis and killing of tumor cells [165]. In vivo,
malignant cells were eradicated while normal tissue was relatively unaffected in nude mice
immunized with VSV-∆M51-GFP. The survival of immunized mice was also significantly
prolonged. In another study, the oncolytic VSV-LCMV-GP efficiently infected 6 different
prostate cancer cell lines [166]. Intratumoral and intravenous immunization generated
long-term remission of subcutaneous tumors and bone metastases in the DU145 and 22Rv1
prostate tumor mouse models. In the case of alphaviruses, a VEEV vector expressing
the prostate-specific membrane antigen (PSMA) elicited strong PSMA-specific immune
responses in immunized BALB/c and C57BL/6 mice [167]. Immunization studies with Vaccines 2021, 9, 1187 19 of 28 19 of 28 VEEV expressing the six-transmembrane epithelial antigen of the protein (STEAP) has been
evaluated in prophylactic and therapeutic mouse models [168]. The study demonstrated
CD8+ T cell responses against a newly defined mouse STEAP epitope, which prolonged
the overall survival of mice. Moreover, TRAMP mice immunized with VEEV particles
expressing the prostate stem cell antigen (PSCA) provided long-term survival in 90% of
mice at 12 months post-vaccination [169]. In the context of clinical applications, a phase I
study was conducted in patients with castration resistant metastatic prostate cancer (CRPC)
by immunization with either 0.9 × 107 or 3.6 × 107 IU of VEEV-PSMA particles [179]. Although the vaccination was well tolerated the PSMA-specific immune response was
weak. To address this issue, thorough dose optimization and vector engineering should
be considered. 5. Conclusions For instance, alphavirus vectors showing strong neurotropism,
engineering of neuron-specific miRT124 sequences restricted replication to tumor cells only,
allowing efficient treatment of CT-2A gliomas in mice [125]. Self-amplifying RNA viruses have been applied for some clinical trials. So far, the
numbers are significantly lower than what have been seen for adenoviruses, AAV, retro-
viruses and lentiviruses. However, the positive results obtained so far has encouraged
further engineering of improved vectors and delivery systems and optimization of dosing
and prime-boost strategies. Reflecting on the success and failure of vaccine development
based on self-replicating RNA viral vectors, it is difficult to point out any vector system
showing superiority over other systems. Clearly, the choice of target plays a role, especially
for vaccines targeting infectious diseases. It might also be good to underline the differ- Vaccines 2021, 9, 1187 20 of 28 20 of 28 ences between cancer vaccines and vaccines against infectious pathogens. In the case of
cancer vaccines, the approach is to provide both prophylactic and therapeutic efficacy and
it therefore includes in a broader meaning cancer immunotherapy. For that reason, the
repertoire of expressible genes of interest is much larger and in addition to tumor antigens,
anti-tumor genes, cytotoxic genes and immunostimulatory genes can be overexpressed to
provide preventive or therapeutic effects. Another issue relates to the differences in cancer
development and infectious diseases. While viral and bacterial outbreaks can quickly
develop into epidemics and even pandemics as familiarly experienced with COVID-19,
although many cancers have a high mortality rate, there is no risk of causing epidemics. For this reason, the urgency for cancer vaccines seems to be less prominent compared to
infectious diseases, particularly when they have reached pandemic levels. p
y
y
p
Related to the efficacy of vaccine development, various self-replicating RNA viral
vectors have elicited high neutralizing antibody titers in immunized rodents and non-
human primates. Moreover, protection has been achieved in rodents and primates against
challenges with lethal doses of infectious pathogens. Similarly, immunization of rodents
with cancer vaccine candidates has elicited strong immune responses and in certain cases
inhibition of tumor growth and/or tumor regression have been observed. Moreover,
immunized animals were protected against challenges with tumor cells. Administration of
oncolytic viruses has also resulted in tumor regression and in some favorable situations
total tumor eradication and cure of treated mice. Conflicts of Interest: The author declares no conflict of interest. Conflicts of Interest: The author declares no conflict of interest. References Kunjin virus replicons: An RNA-based non-cytopathic viral vector system for protein
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particularly studies in mice, has often struggled to generate the same efficacy in clinical
trials. This phenomenon has been attributed to different delivery demands in larger animals
and humans and suboptimal dosing. For this reason, we should not be discouraged by
these setbacks, but continue the engineering of more efficacious delivery vectors and
continue dose optimization studies. It might also be advantageous to consider canine
tumor models for the following reasons. First, the bigger size of dogs compared to rodents
might provide a more similar situation to establish delivery in humans. Second, naturally
occurring tumors in canine models resemble more closely human cancers than induced
tumors in rodent models. Third, prophylactic and therapeutic evaluation in dogs might
lead to veterinary applications in support of human use although it should not be seen
as a shortcut to applications in humans. In the context of MV-based vaccines, potential
pre-existing immunity against MV, which cannot be assessed in studies in rodents, can
compromise the efficacy of vaccines in humans. The approval by the FDA and the EMA of Ervebo, the VSV-based vaccine against EVD,
has given a glimpse of hope for this strategy and further strengthens the position of self-
replicating RNA viral vectors as attractive vehicles for vaccine development. One might
ask why it took so long to obtain the approval for the first vaccine based on self-replicating
RNA viral vectors? There is obviously no simple answer, and although breakthrough in
vector and technology development since the 1980s has been remarkable, especially the
combination of omics initiatives including bioinformatics, genomics, proteomics, immu-
nomics, and vaccinomics, all pieces had to come together to guarantee the highest possible
safety level of therapeutics and vaccines. Although efficient vaccines based on adenovirus vectors against COVID-19 have
received EUA, issues related to vaccine-induced immune thrombotic thrombocytopenia
(VITT) and the circulation of novel more transmissible SARS-CoV-2 variants demonstrate
the need for re-engineering of novel vaccines to which self-replicating RNA viral vectors
may provide a substantial contribution. Funding: The author has received no external funding for this review. Institutional Review Board Statement: No approval needed as no studies involving humans
are conducted. Informed Consent Statement: No studies including humans were conducted, only results have from
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https://openalex.org/W2555471252
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https://asp-eurasipjournals.springeropen.com/track/pdf/10.1186/s13634-016-0416-1
|
English
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Range cell migration correction analysis of one-step and two-step motion compensation for millimeter-wave airborne SAR imaging
|
EURASIP Journal on Advances in Signal Processing
| 2,016
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cc-by
| 7,028
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RESEARCH Open Access © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made. Abstract Conventional two-step motion compensation (MOCO) method is widely adapted for airborne synthetic aperture
radar (SAR) imaging due to its conciseness combining with the SAR focusing procedure. For two-step MOCO, range-
independent compensation is processed before range cell migration correction (RCMC), and the range-dependent
phase correction is implemented after RCMC. However, the accuracy of RCMC would be seriously decreased by the
residual range-dependent phase, which is a fatal problem for high-resolution millimeter-wave (MMW) SAR imaging. In
this paper, an extensive investigation on the RCMC accuracy is provided by establishing an accurate formula
expression between the range cell migration error and the residual range-dependent phase error. One-step MOCO-
based SAR imaging algorithm is investigated by compensating the range-dependent motion error before RCMC, so
the presence of range cell migration error would be significantly suppressed. What is more, a modified azimuth match
filtering (AMF) function is given by precise topography and aperture-dependent motion compensation (PTA) method
to overcome the residual azimuth-dependent phase error in the azimuth compression stage. Both simulated and
real-measured MMW SAR data sets are used to validate the analysis for high-resolution airborne SAR imaging. Keywords: Synthetic aperture radar (SAR), Motion compensation (MOCO), Millimeter-wave (MMW), One-step MOCO rds: Synthetic aperture radar (SAR), Motion compensation (MOCO), Millimeter-wave (MMW), One-st while the first step is processed to the range compressed
data and the second step is processed after range cell
migration correction (RCMC). The problem of con-
ventional two-step MOCO processing is also obvious. The residual range-dependent motion error remained
after the first step seriously decreases the accuracy
of RCMC in two-dimensional wavenumber domain,
which presents as a curving range cell migration (RCM)
range profile in two-dimensional time domain, and
destroys the performance of azimuth pulse compres-
sion. Reference [11] describes the problem above for
Omega-k algorithm [12, 13]. In their work, a one-step
MOCO method is proposed, but the detailed analysis
of the RCMC error is not given. Besides, the original
MOCO methods only take range-dependent motion
error into account, and the residual azimuth-dependent
motion error should also be considered, which is non-
ignorable
for
high-resolution
airborne
MMW
SAR
imaging with wide swath. The existed azimuth-dependent
MOCO algorithms [14–18] could precisely compensate Range cell migration correction analysis of
one-step and two-step motion compensation
for millimeter-wave airborne SAR imaging Guanyong Wang1,2, Lei Zhang1*, Jun Li2 and Qingrong Hu2 Guanyong Wang1,2, Lei Zhang1*, Jun Li2 and Qingrong Hu2 EURASIP Journal on Advances
in Signal Processing EURASIP Journal on Advances
in Signal Processing Wang et al. EURASIP Journal on Advances in Signal
Processing (2016) 2016:115
DOI 10.1186/s13634-016-0416-1 *Correspondence: leizhang@xidian.edu.cn
1National Lab of Radar Signal Processing and the Collaborative Innovation
Center of Information Sensing and Understanding, Xidian University, Xi’an
710071, People’s Republic of China
Full list of author information is available at the end of the article 1
Introduction The
expression of echo signal from target P is given by: S (τ, X) = εp · rect
τ −t
Tp
· rect
X −x0 −x
L
× exp
j2π
−fct + α(τ −t)2
2
(2) (2) where α is the chirp rate, Tp is the pulse duration width,
fc is the center frequency, τ denotes the range fast-time,
t = 2Rn (X)/c stands for the round-way propagation
time of electromagnetic wave between target P and radar,
c is the speed of light. εp corresponds to the complex-
valued scattering amplitude of the point target, symbol
rect (·) denotes the rectangular window function. Accord-
ing to [7], the expression of range compressed signal is
given by: S (τ, X) = εp · sinc
Tpα
τ −2Rn (X)
c
· rect
X −x0 −x
L
× exp
−j4π
λ Rn (X)
(3) The whole paper is organized as follows: Section 2
gives the signal and geometry model of the SAR imaging,
RCMC accuracy respect to the residual range-dependent
error is analyzed as well, and flowcharts of one-step and
two-step MOCO-based SAR imaging algorithms are then
given for comparison. In Section 3, RCMC error compar-
ison between one-step and two-step MOCO is discussed,
and computational burden of both methods is also ana-
lyzed in detail. In Section 4, extensive experimental results
are given with both simulated and real measured MMW
airborne SAR data. Conclusions are given in the last
section. (3) where symbol sinc (·) is expressed as sinc (a)
=
sin (πa)/πa. However, the actual motion of platform is
not ideal, so the range compressed signal in (3) is blurred
with motion error R = A′P −AP. Motion compensation
is then processed to the blurred range compressed sig-
nal in order to remove the envelope and phase of motion
errors, so that most of the errors would be compensated. Due to our early works in [3], the signal model as well
as the residual phase after deramp processing are calcu-
lated for a refined phase gradient autofocus. Based on
the former accumulation, in this paper, we try to inves-
tigate the mathematical explanation of RMC error with
respect to the residual motion errors remained by the
range-independent MOCO. 1
Introduction Motion compensation (MOCO) [1–4] is a crucial operat-
ing step for airborne synthetic aperture radar (SAR) [5–7]
imaging because the non-ideal movement deviates the
radar platform from the predetermined flight trajectory. More importantly, for high-resolution millimeter-wave
(MMW) [8] SAR systems, imaging performance is more
sensitive to the envelope and phase of motion errors,
so a precise MOCO is essential with the availability of
high-precision inertial navigation system (INS) mea-
surement. An efficient two-step MOCO algorithm [4] is
proposed by Moreira and Huang, combining with chirp
scaling algorithm (CSA) [9, 10] for airborne SAR imaging. This method is divided into range-independent com-
pensation step and range-dependent compensation step, *Correspondence: leizhang@xidian.edu.cn
1National Lab of Radar Signal Processing and the Collaborative Innovation
Center of Information Sensing and Understanding, Xidian University, Xi’an
710071, People’s Republic of China
Full list of author information is available at the end of the article Wang et al. EURASIP Journal on Advances in Signal Processing (2016) 2016:115 Page 2 of 11 Page 2 of 11 from target P to the radar in the conical coordinate system
[19] is given by: the
azimuth-dependent
motion
error
and
modify
the azimuth matched filtering function in order to
eliminate
the
influence
of
azimuth-variant
motion
error. Rn (X) =
(r cos ϕ)2 + (X −x −r sin ϕ)2, −L
2 ≤X ≤L
2
(1) Based on the signal model in [3], we investigate the
cause of RCMC error as well as its definite expres-
sion deduction for MMW SAR system in this paper. A
background assumption is confirmed that the trajectory
information is accurately recorded by the INS and the
whole motion compensation procedure is processed with-
out autofocus step. The one-step MOCO-based imaging
algorithm is investigated, which compensates range-
dependent motion error before RCMC in order to sup-
press the residual envelope and phase error of RCM range
profile. Moreover, according to the analytical expression
of the residual spatial variant error, an accurate azimuth
match filtering (AMF) function is modified by precise
topography- and aperture-dependent motion compensa-
tion (PTA) [17], which compensates the residual azimuth-
dependent motion errors remained by RCMC. In this
paper, the conventional two-step MOCO-based imag-
ing algorithm is introduced for comparison, theoretical
analysis to the superiority of one-step MOCO would be
adequately verified by simulated and real-measured data
experiments. (1) where X = vtm denotes the instantaneous azimuth coor-
dinate of antenna phase center at slow time tm. 1
Introduction Order RE to represent the
residual motion error remained by the range-independent
MOCO before RCMC, which would induce serious enve-
lope and phase error to RCMC, and decrease the azimuth
pulse compression accuracy. The envelope part of RE is
usually limited within a range bin in actual processing, so
we ignore the small envelope of RE. Applying the range
Fourier transform (FT) with respect to τ, the signal is
transferred to the range wavenumber domain as shown in
(4). The constant and envelope terms of signal expression
could be omitted for analysis without adverse effect. 2
One-step MOCO-based SAR imaging algorithm
2.1
SAR imaging and RCMC error analysis 2.1
SAR imaging and RCMC error analysis
The squinted SAR geometry is given in Fig. 1. In the model
above, the SAR sensor travels along a straight-line flight
path with a constant velocity v in ideal conditions, and the
synthetic aperture length is L. Symbol O stands for the
original point of SAR sensor. The ideal linear trajectory is
defined as X-axis corresponding to the azimuth direction,
point A denotes the ideal position of platform. During
the data acquisition, the radar beam illuminates at squint
angle ϕ, symbol C denotes the scene center, and r repre-
sents the range from C to radar at squint angle ϕ. Symbol P
stands for the target located on the scene center line O′C,
which is parallel to the trajectory. The distance between
target P and scene center C is given by x, and the distance
between O′ and C is given by x0. The instantaneous range Wang et al. EURASIP Journal on Advances in Signal Processing (2016) 2016:115 Page 3 of 11 Fig. 1 Geometric model of squint SAR imaging Fig. 1 Geometric model of squint SAR imaging Fig. 1 Geometric model of squint SAR imaging S (Kr, X) =
S (τ, X) exp
−jKr
τc
2
· d τc
2
= exp
−jKr·
(r cos ϕ)2+(X−x −r sin ϕ)2+RE (X)
(4) where the second term of (7) represents the range cell
migration and the third term represents the second-order
coupling phase. One may multiply both sides of Eq. (7) by
HRCMC (Kx, Kr) and HSORC (Kx, Kr) to perform the RCMC
and second-order range compression (SORC). where the second term of (7) represents the range cell
migration and the third term represents the second-order
coupling phase. One may multiply both sides of Eq. (7) by
HRCMC (Kx, Kr) and HSORC (Kx, Kr) to perform the RCMC
and second-order range compression (SORC). (4) HRCMC (Kx, Kr) = exp
jKr
Krcr cos ϕ
K2rc −K2x
−r
(8)
HSORC (Kx, Kr) = exp
⎡
⎣−j
K2
x r cos ϕ
2
K2rc −K2x
3
2
K2
r
⎤
⎦
(9) (8) where Kr represents the range wavenumber spectrum
with Kr = Krc + Kr and Krc = 4πfc/c is the range
wavenumber center with Kr ∈
−2παTp/c, 2παTp/c
. 2.2
Flowcharts of one-step and two-step MOCO-based
imaging algorithms One-step MOCO has compensated most of the range-
dependent motion errors, but the residual azimuth-
dependent motion error is remained after RCMC, which
decreases the accuracy of azimuth pulse compression. D
h
id
l
i
h d
d
i
ϑ The compensated data is then transformed into
two-dimensional wavenumber domain, and SORC and
RCMC are processed by multiplying HSORC (Kx, Kr)
and HRCMC (Kx, Kr), shown in (8) and (9), respectively. One-step MOCO has compensated most of the range-
dependent motion errors, but the residual azimuth-
dependent motion error is remained after RCMC, which
decreases the accuracy of azimuth pulse compression. Due to the residual azimuth-dependent motion error, ϑ0
is calculated in (18), and a method of PTA is introduced to
compensate ϑ0 by modifying the AMF function. Accord-
ing to [17], the modified AMF function HAMF (Kx, r) is
expressed as: Substituting (15) into (12) with replacement of Kx by K∗
x
and ordering X −x = 0 for simply analyzing, the resid-
ual motion error in azimuth time and range wavenumber
domain is given by: RE (X) = RE +
r sin ϕ
Krccos2ϕ · ∂RE
∂X
· Kr
(16) (16) Substituting (16) into (14), the expression of signal is
translated to the following equation: S (Kr, X) = exp
−jKrc
(r cos ϕ)2 + (X −x −r sin ϕ)2
× exp
−j
ϑ0 + ϑ1Kr + ϑ2K2
r
(17) (17) where the second term of (17) is the RCMC error with
respect to the residual motion error RE. This phase term
is expressed as a series of Kr, so the phase and envelope
component would be distinguished where HAMF (Kx, r) = exp
jKrcRE
X∗
e
· exp
jKrcRn
r, X∗
e
+jKx
X∗
e −x
(23) (23) ϑ0 = KrcRE
(18)
ϑ1 = RE + r sin ϕ
cos2ϕ · ∂RE
∂X
(19)
ϑ2 =
r sin ϕ
Krccos2ϕ · ∂RE
∂X
(20) (18) where X∗
e is calculated by ∂Rn (Xe∗)
∂Xe∗
+ ∂RE (Xe∗)
∂Xe∗
+ Kx
Krc
= 0
(24) (24) (20) As the one-step MOCO-based imaging algorithm is
described in detail, we briefly describe the procedure
of the two-step MOCO-based imaging algorithm for
comparison. 2
One-step MOCO-based SAR imaging algorithm
2.1
SAR imaging and RCMC error analysis Then, applying the azimuth FT to (4) with respect to X,
the impact of RE to the stationary phase point could be
ignored for simplifying the analysis formula. The expres-
sion of signal in two-dimensional wavenumber domain is
given by [3]: (9) The expression of signal in two-dimensional wavenum-
ber domain processed by RCMC and SORC is given as
follows: S (Kr, Kx) = exp
−j
K2r −Kx2 cos ϕ + Kx sin ϕ
· r + Kx · x
× exp
−Kr · RE
X∗
(5)
X∗= −r cos ϕ · Kx
K2r −Kx2
+ r sin ϕ + x
(6) S (Kr, Kx) = exp
−j
Kx (x + r sin ϕ) + Krr
+ r cos ϕ
K2rc −K2x
× exp
−jKr · RE
X∗
(10) (5) (10) (6) × exp
−jKr · RE
X∗ where Kx represents the azimuth wavenumber spectrum
and X∗denotes the ideal stationary phase point. The first
phase term of (5) is expressed by a Taylor expansion with
respect to Kr = Kr −Krc around Kr = 0, so (5) is
expressed as: where the second term of (10) is shown in (11), which
is considered the residual motion error phase in two-
dimension wavenumber domain, decreasing the accuracy
of RCMC seriously. (Kr, Kx) = exp
−jKrRE
X∗
(11) (11) S (Kr, Kx) ≈exp
−j
K2rc −Kx2r cos ϕ + Kx (r sin ϕ + x)
× exp
−j Krcr cos ϕ
√
K2rc−K2x Kr
. exp
j
K2
x r cos ϕ
2
K2rc−K2x
3
2 K2
r
× exp [−Kr · RE (X∗)] Then, the relationship between (Kr, Kx) and RCMC
error would be theoretically analyzed. It could be
observed in (6) that the residual motion error RE (X∗)
is a function of range wavenumber spectrum Kr, so we
may expand the second phase term in (10) by a Taylor
expansion with respect to Kr around Kr = 0, which (7) Wang et al. EURASIP Journal on Advances in Signal Processing (2016) 2016:115 Page 4 of 11 Page 4 of 11 is expressed as follows, remaining the constant term and
first-order term: is expressed as follows, remaining the constant term and
first-order term: 2.2
Flowcharts of one-step and two-step MOCO-based
imaging algorithms As shown in Fig. 2, a flowchart of the one-step MOCO-
based imaging algorithm is given in this subsection, while
the conventional two-step MOCO-based imaging algo-
rithm is also given for comparison. It is here needed
to explain that the flowchart is displayed in such form
for the purpose of distinguishing the procedure of the
one-step MOCO and two-step MOCO more legible. We
introduce PTA for azimuth-variant MOCO in order to
eliminate the influence caused by other residual special-
dependent errors. In a one-step procedure, bulk envelope
compensation is processed in range wavenumber domain
by multiplying HMOCO (Kr, X) to the range compressed
data. The envelope compensation function is expressed as: RE
X∗
≈RE
X∗
Kr=0+ ∂RE (X∗)
∂Kr
Kr=0
Kr
(12) where ∂RE(X∗)
∂Kr
=
∂X∗
∂Kr · ∂RE(X∗)
∂X∗
= Kx·(Krc+Kr)r cos ϕ
(Krc+Kr)2−K2x
3
2 · ∂RE(X∗)
∂X∗
(13) (13) Ignoring the impact to the stationary phase point made
by RE (X∗), we apply azimuth inverse Fourier transform
(IFT) to (10), and we have: S (Kr, X) =
S (Kr, Kx) · exp
jKxX
· dKx
= exp
−jKrc
(r cos ϕ)2 + (X −x −r sin ϕ)2
× exp
RE
K∗
x
· (Krc + Kr)
( HMOCO (Kr, X) = exp
−jRr (rs) Kr
(21) (21) where Rr denotes the slant range projection of motion
error, which is with respect to slant range r and would
be expressed in (26). Then, the range-dependent motion
error is compensated in range time domain by multiplying
HMOCO (r, X), where where Rr denotes the slant range projection of motion
error, which is with respect to slant range r and would
be expressed in (26). Then, the range-dependent motion
error is compensated in range time domain by multiplying
HMOCO (r, X), where (14) where the stationary phase point K∗
x is given by: K∗
x ≈−Krc
X −r sin ϕ −x
(cos ϕ · r)2 + (X −x −r sin ϕ)2
(15) HMOCO (r, X) = exp
−jKrcRr (r, X)
(22) (15) (22) The compensated data is then transformed into
two-dimensional wavenumber domain, and SORC and
RCMC are processed by multiplying HSORC (Kx, Kr)
and HRCMC (Kx, Kr), shown in (8) and (9), respectively. 2.2
Flowcharts of one-step and two-step MOCO-based
imaging algorithms For two-step MOCO, the envelope com-
pensation and the first-phase compensation are pro-
cessed with respect to the reference slant range rs before
RCMC, and the second range-dependent MOCO step is It is worthy to note that ϑ0 represents the phase com-
ponent of RCMC error and ϑ1 denotes the envelope error
of RCMC, which seriously destroys the imaging perfor-
mance. ϑ2 is the second-order term, which slightly reflects
the focusing performance in range, and the effect could be
ignored in most cases. Wang et al. EURASIP Journal on Advances in Signal Processing (2016) 2016:115 Page 5 of 11 Fig. 2 Flowchart of one-step MOCO-based imaging algorithm compared with conventional two-step MOCO-based imaging algorithm azimuth-independent motion error respect to range r is
expressed as: processed after RCMC, but serious RCM envelope error
is remained at this step. In order to make the comparison
more equitable, azimuth-dependent motion error is then
compensated by method of PTA. Rr (r) = Xt
r x + Yt
r y + H
r z
(26) (26) 3
Comparative analysis of one-step and two-step
MOCO For
the
two-step
MOCO
algorithm,
the
range-
independent component of motion error is compensated
in the first step. The residual motion error ˜RE contains
range-dependent component and azimuth-dependent
component, which is given by: 3.1
RCMC error comparison between one-step and
two-step MOCO In the previous section, we analyze the RCMC error with
respect to the residual motion error RE, but RE is
different for the one-step and two-step MOCO. In this
subsection, we focus on calculating RE and compar-
ing the RCMC error between the one-step and two-step
MOCO. ˜RE (r, X) = ˜R (r, X) −Rn (r, X) −Rr (rs)
= rs−r
r2s
·
−
H2
√
r2s cos2ϕ−H2 y + Hz
+ δRa (X)
(27) (27) The actual range history at slant range r is expressed as: where r denotes the range bin of target, rs denotes the
slant range from the radar to the beam center, δRa is the
azimuth-dependent motion error. It is obvious in (27) that
the residual motion error is in proportion to the range
between target and scene center, so the error is diffused
along range direction. ˜R (r, X) =
(X −x −r sin ϕ −x)2 + (Yt −y)2 + (H −z)2
(25) (25) where x, y, and z represent the along track error,
cross track error, and height error, respectively. Xt
=
r sin ϕ denotes the projection of r on x direction,
Yt
=
r2cos2ϕ −H2 denotes the projection of r on
y direction, and H is the height of the platform. The For one-step MOCO, range-dependent motion error is
compensated before RCMC. However, a new error δRo
is introduced for squint SAR, the residual motion error
before RCMC is given by: Wang et al. EURASIP Journal on Advances in Signal Processing (2016) 2016:115 Page 6 of 11 Fig. 3 Real measured motion error and corresponding residual RCMC envelope error. a Real-measured motion error. b Corresponding residual
RCMC envelope error Fig. 3 Real measured motion error and corresponding residual RCMC envelope error. a Real-measured motion error. 3.1
RCMC error comparison between one-step and
two-step MOCO b Corresponding residual
RCMC envelope error RE (X) = δRo (X) + ˜R (r, X) −Rn (r, X) −Rr (r)
= δRo (X) + δRa (X)
≈−(X −x) sin ϕ
r2s
·
−
H2
r2s cos2ϕ −H2 y + Hz
+ δRa (X)
(28) Similarly, substituting (30) into (19), the RCMC enve-
lope error for the one-step MOCO is given by: T2 ≈(X −x) sin ϕ
r2
u (X) + r sin ϕ
cos2ϕ · u′ (X)
(31) (31) where the azimuth-dependent motion error δRa has little
impact on the RCMC envelope, so it is not considered in
this paper. The ratio between T1 and T2 is expressed
as: (28) where x denotes the azimuth position of the target. In this
section, we mainly focus on providing the comparison of
RCMC envelope error between the two-step and one-step
MOCO, which is expressed as ϑ1 in (19). Order T1
T2
≈
rs −r
(X −x) sin ϕ
(32) (32) u (X) = −
H2
r2s cos2ϕ −H2 y + Hz
(29) where the numerator of (32) represents the range differ-
ence between target and scene center and the denomina-
tor represents the length of range walk. It’s clear that the
RCMC envelope error is in proportion to the range dif-
ference for two-step MOCO, so the imaging performance
would be seriously destroyed at the points far from scene
center line. However, in some cases, the effect of resid-
ual range-dependent motion error could be neglected if
the maximum of RCMC envelope is within 1/4 rang bin,
which is shown as: where the numerator of (32) represents the range differ-
ence between target and scene center and the denomina-
tor represents the length of range walk. It’s clear that the
RCMC envelope error is in proportion to the range dif-
ference for two-step MOCO, so the imaging performance
would be seriously destroyed at the points far from scene
center line. However, in some cases, the effect of resid-
ual range-dependent motion error could be neglected if
the maximum of RCMC envelope is within 1/4 rang bin,
which is shown as: (29) Substituting (29) into (19), the RCMC envelope error for
the two-step MOCO is given by: T1 ≈rs −r
r2
u (X) + r sin ϕ
cos2ϕ · u′ (X)
(30) (30) Table 1 Simulation parameters Carrier frequency
35 GHz
Pulse repetition frequency
2000 Hz
Velocity
70 m/s
Pulse width
2 μs
Center closest slant range
4000 m
Squint angle
5◦
Grazing angle
45◦
Range resolution
0.15 m
Azimuth resolution
0.15 m
Point A coordinate
(0, 400)
Point B coordinate
(0, 0)
Point C coordinate
(0, −400) max
RE + r sin ϕ
cos2ϕ · ∂RE
∂X
≤1
4r
(33) (33) Velocity where max |·| denotes the maximum of the absolute value. For the two-step MOCO, the inequality becomes: where max |·| denotes the maximum of the absolute value. For the two-step MOCO, the inequality becomes: max
rs −r
r2
u (X) + r sin ϕ
cos2ϕ · u′ (X)
≤1
4r
(34) (34) And for the one-step MOCO, the inequality becomes: And for the one-step MOCO, the inequality becomes: max
(X −x) sin ϕ
r2
u (X) + r sin ϕ
cos2ϕ u′ (X)
≤1
4r max
(X −x) sin ϕ
r2
u (X) + r sin ϕ
cos2ϕ u′ (X)
≤1
4r
(35) (35) Wang et al. EURASIP Journal on Advances in Signal Processing (2016) 2016:115 Page 7 of 11 Fig. 4 Comparison of the two-step MOCO and one-step MOCO in RCMC results of points A, B and C. a Two-step MOCO results. b One-step MOCO
results Fig. 4 Comparison of the two-step MOCO and one-step MOCO in RCMC results of points A, B and C. a Two-step MOCO results. b One-step MOCO
results Fig. 4 Comparison of the two-step MOCO and one-step MOCO in RCMC results of points A, B and C. a Two-step MOCO results. b One-step MOCO
results Fig. 5 Comparison of the two-step MOCO and one-step MOCO in imaging results of points A, B, and C. a Two-step MOCO results. b One-step
MOCO results Fig. 5 Comparison of the two-step MOCO and one-step MOCO in imaging results of points A, B, and C. a Two-step MOCO results. b One-step
MOCO results Wang et al. EURASIP Journal on Advances in Signal Processing (2016) 2016:115 Page 8 of 11 Fig. 6 Comparison of the two-step MOCO and one-step MOCO in azimuth pulse response curves of points A, B, and C Fig. Table 1 Simulation parameters 6 Comparison of the two-step MOCO and one-step MOCO in azimuth pulse response curves of points A, B, and C It could be found that, for SAR imaging with wide swath,
the requirement of inequation (34) is hard to satisfy at
points far from the scene center line, while inequation
(35) is easier to meet. A simulation result of the RCM
error comparison with real-measured trajectory devia-
tions between the two-step and one-step MOCO is shown
in Fig. 3. The trajectory deviations are extracted from a
real position and orientation system shown in Fig. 3a. The
residual RCMC envelope error is calculated with a squint
angle of 5◦and a pitch angle of 45◦, the center closest
slant range is 4000 m, and the range difference between
target and scene center is 100 m. The corresponding resid-
ual RCMC envelope errors are shown in Fig. 3b. It is clear
that the envelope error of the two-step MOCO is up to
several meters, while the one-step MOCO significantly
overcomes this problem. the computational burden and exchanges for a better
focused imagery. 4
Simulated and real data experiments
4.1
Experiments with simulated data In this subsection, a group of point target simulation
experiments are set to demonstrate the effectiveness of
the one-step MOCO-based imaging algorithm compared
with the two-step MOCO-based imaging algorithm. Sim-
ulation parameters and three-point target coordinates are
shown in Table 1. It needs to be emphasized that points A
and C are far from the scene center line, while point B lies
on the scene center line. In this simulation experiment, the
performance of the one-step MOCO imaging algorithm
is validated by simulated data mixed with motion errors
which are extracted from airborne INS, which are shown
in Fig. 3a. At first, we present the comparison of RCMC error
with respect to the residual range-dependent motion error
between the two-step MOCO and one-step MOCO. In
order to illustrate the RCMC error explicitly, the range
profile of three points are shown in two-dimensional
time domain. It could be easily found in Fig. 4a that
the residual RCMs of points A and C are obvious,
except for point B because it is lying on the scene cen-
ter line without range-dependent motion error before
RCMC. These RCMC errors are deemed to coincide well 3.2
Computational burden analysis In this subsection, the computational burden of the
one-step and two-step MOCO-based SAR imaging algo-
rithms is respectively measured by operating number of
fast Fourier transform (FFT), inverse fast Fourier trans-
form (IFFT), and complex multiplication for compari-
son. As shown in Fig. 2, suppose the azimuth and range
point numbers are denoted by Na and Nr. It needs
to be noticed that we analyze the operand by merg-
ing adjoining phase terms and without regard of the
calculation of PTA operation. For the one-step MOCO-
based imaging algorithm, there are 4Na times Nr-point
FFT/IFFT operators, 2Nr times Na-point FFT/IFFT oper-
ators, and 5 times Nr × Na-point complex multiplications
to obtain a focused imaging. Comparing with the conven-
tional two-step based imaging algorithm, there are 2Na
times Nr-point FFT/IFFT operators, 2Nr times Na-point
FFT/IFFT operators, and 5 times Nr × Na-point com-
plex multiplications. It could be found that the one-step
MOCO-based imaging algorithm adds 2Nr more times
Na-point FFT/IFFT operators, which slightly increases Table 2 Focusing performance comparison between two
focusing algorithms
Target point
Method
PSLR (dB)
ISLR (dB)
IRW (m)
A
One-step MOCO
−13.0416
−10.4187
0.1444
Two-step MOCO
−4.1975
−4.4064
0.3631
B
One-step MOCO
−13.3593
−11.3825
0.1531
Two-step MOCO
−11.5402
−11.1442
0.1575
C
One-step MOCO
−13.4320
−11.4704
0.1663
Two-step MOCO
−4.9471
−5.0134
0.3894 Table 2 Focusing performance comparison between two
focusing algorithms Page 9 of 11 Wang et al. EURASIP Journal on Advances in Signal Processing (2016) 2016:115 Table 3 Experiment parameters
Carrier frequency
35 GHz
Pulse repetition frequency
5000 Hz
Velocity
70 m/s
Pulse width
20 μs
Center closest slant range
4000 m
Squint angle
5◦
Grazing angle
45◦
Range resolution
0.15 m
Azimuth resolution
0.15 m Table 3 Experiment parameters
Carrier frequency
35 GHz
Pulse repetition frequency
5000 Hz
Velocity
70 m/s
Pulse width
20 μs
Center closest slant range
4000 m
Squint angle
5◦
Grazing angle
45◦
Range resolution
0.15 m
Azimuth resolution
0.15 m Table 3 Experiment parameters spreading response of points A, B, and C. In order to eval-
uate the focused improvement of the proposed algorithm
comparing with the conventional one, three quantita-
tive metrics are introduced to measure the point impulse
responses of points A, B, and C, which are shown in
Table 2. The quantitative metrics are peak side-lobe ratio,
(PSLR), integrated side-lobe ratio (ISLR), and impulse
response width (IRW). 3.2
Computational burden analysis It is obvious that the focusing
degradation of the conventional two-step MOCO-based
imaging algorithm is overcame by the one-step MOCO-
based imaging algorithm, especially for points A and C,
which are far from the scene center line. 4.2
Experiments with real-measured data with the expression of ϑ1 in (19). It is also shown in
Fig. 4a that the residual RCMC errors of points A and
C processed by two-step MOCO are spanning across
several range cells, so the focusing performance would
be seriously damaged. The RCM corrected range pro-
files of points A, B, and C processed by the one-step
MOCO are shown in Fig. 4b for comparison, while
all the RCMC errors of points A, B, and C are well
removed. p
In this subsection, a set of comparison experiment is pro-
vided based on the processing of measured data recorded
by an experimental airborne MMW SAR system. The
instantaneous position and motion parameters of plat-
form are measured by a high-accuracy INS equipped
on the platform. Detailed radar parameters are shown
in Table 3. An imaging result processed by the one-
step MOCO-based imaging algorithm is shown in Fig. 7. In order to compare the imaging performance between
the conventional two-step MOCO-based imaging algo-
rithm and one-step MOCO-based imaging algorithm
more vividly, two typical areas named scene 1 and scene
2 with obvious point-like targets are highlighted by the
yellow rectangles in the scene. The picked two scenes are
respectively magnified in Fig. 8a, b for imaging perfor-
mance comparison, where the two-step MOCO imaging
results are lied on the left, while the one-step MOCO
imaging results are lied on the right. It is shown that
defocusing of targets in the images is distinct for the two-
step MOCO-based imaging algorithm, and the one-step Then, azimuth compression performance of the one-
step MOCO-based imaging algorithm is investigated. The
two-dimensional images of points A, B, and C processed
by the conventional two-step MOCO-based imaging algo-
rithm and one-step MOCO-based imaging algorithm are
shown in Fig. 5. Because of the existence of residual
RCMC errors, the imaging algorithm based on the con-
ventional two-step MOCO fails to focus in azimuth for
points A and C, as shown in Fig. 5a. While the well-
focused imaging results of points A, B, and C processed by
the one-step MOCO-based imaging algorithm are shown
in Fig. 5b. Figure 6 shows the comparison of azimuth point Fig. 7 Imaging result processed by the one-step MOCO-based imaging algorithm Fig. 7 Imaging result processed by the one-step MOCO-based imaging algorithm Wang et al. EURASIP Journal on Advances in Signal Processing (2016) 2016:115 Page 10 of 11 Fig. 4.2
Experiments with real-measured data 8 Comparison of the two-step MOCO-based imaging algorithm and one-step MOCO-based imaging algorithm in local amplification results of
scene 1 and scene 2. a Comparison of scene 1 (the two-step MOCO-based imaging result is on the left, the one-step MOCO-based imaging result is
on the right). b Comparison of scene 2 (two-step MOCO-based imaging result is on the left, the one-step MOCO-based imaging result is on the right) Fig. 8 Comparison of the two-step MOCO-based imaging algorithm and one-step MOCO-based imaging algorithm in local amplification results of
scene 1 and scene 2. a Comparison of scene 1 (the two-step MOCO-based imaging result is on the left, the one-step MOCO-based imaging result is
on the right). b Comparison of scene 2 (two-step MOCO-based imaging result is on the left, the one-step MOCO-based imaging result is on the right) by the one-step MOCO-based imaging algorithm, com-
pared with the two-step. The quantitative analysis results
of the azimuth point spreading response functions of
Fig. 9 are listed in Table 4. It could be found in the exper-
iments above that, focusing performance is sensitive to
the residual RCMC error especially for high-resolution
MMW SAR imaging, so conventional two-step MOCO-
based imaging algorithm is not suitable for this case. The
one-step MOCO removes the range-dependent motion MOCO significantly removes the RCMC error, so the tar-
gets are well focused. Moreover, in order to check the
azimuth point spreading response improvement of the
one-step MOCO-based imaging algorithm, two isolated
point-like targets named point A and point B are extracted
from Fig. 7 by yellow circle for azimuth point spreading
response function comparison. The comparison results of
point A and point B are shown in Fig. 9a, b, respectively. A
better azimuth pulse response function could be obtained Fig. 9 Azimuth point spreading response function comparison of A and B. a Point A. b Point B . a Point A. b Point B ig. 9 Azimuth point spreading response function comparison of A and B. a Point A. b Point B Fig. 9 Azimuth point spreading response function comparison of A and B. a Point A. b Point B Wang et al. The Second Academy of China Aerospace Science and Industry Corporation
(CASIC), Beijing 100854, People’s Republic of China. The Second Academy of China Aerospace Science and Industry Corporation
(CASIC), Beijing 100854, People’s Republic of China. The Second Academy of China Aerospace Science and Industry Corporation
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Th
h
h
k h 19. G Fornaro, E Sansosti, R Lanari, et al, Role of processing geometry in SAR
raw data focusing. IEEE Trans. Aerospace Electron. Syst. 38(2), 441–454
(2002) The authors thank the anonymous reviewers for their valuable comments to
improve the paper quality. This work was supported by the National Natural
Science Foundation of China under grant numbers 61301280 and 61301293
and the High-Resolution Earth Observation System Major Special Project
Youth Innovation Foundation of China under grant number GFZX04060103. 5
Conclusions 12. R Bamler, A comparison of range-doppler and wavenumber domain SAR
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(1992) The conventional two-step MOCO algorithm remains
the range-dependent motion error before RCMC, which
decreases the accuracy of RCMC in two-dimensional
wavenumber domain, inducing serious envelope and
phase error to the range profile. In this paper, analytical
expressions of these errors are deduced in detail. The one-
step MOCO-based imaging algorithm is also investigated
to compare with the conventional two-step MOCO-based
imaging algorithm, which removes the range-dependent
motion error before RCMC, so the RCMC error is sig-
nificantly suppressed. Simulations and measured MMW
data experiments illustrate the outperforms of the one-
step MOCO-based SAR imaging algorithm, which verify
the analysis in this paper. 13. A Reigber, E Alivizatos, A Potsis, et al, Extended wavenumber-domain
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aperture-dependent motion compensation algorithms for airborne SAR. IEEE Geosci. Remote Sensing Lett. 4(3), 349–353 (2007) 16. P Prats, A Reigber, JJ Mallorqui, Topography-dependent motion
compensation for repeat-pass interferometric SAR systems. IEEE Geosci. Remote Sensing Lett. 2(2), 206–210 (2005) 17. S Perna, V Zamparelli, A Pauciullo, et al, Azimuth-to-frequency mapping in
airborne SAR data corrupted by uncompensated motion errors. IEEE
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motion compensation improvement in wide-beam SAR data processing. Electron. Lett. 37(23), 1405–1407 (2001) References 1. MD Xing, XW Jiang, RB Wu, et al, Motion compensation for UAV SAR
based on raw radar data. IEEE Trans. Geosci. Remote Sensing. 47(8),
2870–2883 (2009) 1. MD Xing, XW Jiang, RB Wu, et al, Motion compensation for UAV SAR
based on raw radar data. IEEE Trans. Geosci. Remote Sensing. 47(8),
2870–2883 (2009) 2. AE Azouz, ZF Li, Improved phase gradient autofocus algorithm based on
segments of variable lengths and minimum-entropy phase correction. IET
Radar Sonar Navigation. 9(4), 467–479 (2015) 2. AE Azouz, ZF Li, Improved phase gradient autofocus algorithm based on
segments of variable lengths and minimum-entropy phase correction. IET
Radar Sonar Navigation. 9(4), 467–479 (2015) error before RCMC, so that the range profile after RCMC
is accurate. 3. L Zhang, MD Xing, ZQ Qiao, Wavenumber-domain autofocusing for
highly squinted UAV SAR imagery. IEEE Sensors J. 12(5), 1574–1588 (2012) 3. L Zhang, MD Xing, ZQ Qiao, Wavenumber domain autofocusing for
highly squinted UAV SAR imagery. IEEE Sensors J. 12(5), 1574–1588 (2012)
4. A Moreira, YH Huang, Airborne SAR processing of highly squinted data
using a chirp scaling approach with integrated motion compensation. IEEE Trans. Geosci. Remote Sensing. 32(5), 1029–1040 (1994)
5. WG Carrara, RM Majewshi, RS Goodman, Spotlight Synthetic Aperture Radar
Signal Processing Algorithm. (Artech House, Boston, 1995), pp. 15–27
6. CV Jakowatz, DE Wahl, PH Eichel, et al, Spotlight Mode Synthetic Aperture 4. A Moreira, YH Huang, Airborne SAR processing of highly squinted data
using a chirp scaling approach with integrated motion compensation. IEEE Trans. Geosci. Remote Sensing. 32(5), 1029–1040 (1994) In order to verify the calculation analysis in Section 2,
we record the calculation time for both two-step and
one-step MOCO-based SAR imaging algorithms. The
computer platform is installed with Windows10 64-bit
operating system, Core i7-4720HQ@2.6GHz CPU, 16-GB
memory and Matlab with version of R2015a. A block of
16, 384 × 8192 (range×azimuth) points SAR data is used
for test, the whole data is divided into four sub-blocks
in azimuth, and the calculation time of the two-step and
one-step MOCO-based imaging algorithms are 855.13s
and 909.49s, respectively. With the nearly equal computa-
tion complexity compared with the two-step MOCO, the
one-step MOCO is applicative for practical MMW SAR
imaging application. 5. WG Carrara, RM Majewshi, RS Goodman, Spotlight Synthetic Aperture Radar
Signal Processing Algorithm. (Artech House, Boston, 1995), pp. 15–27 6. Received: 13 May 2016 Accepted: 28 October 2016 Received: 13 May 2016 Accepted: 28 October 2016 4.2
Experiments with real-measured data EURASIP Journal on Advances in Signal Processing (2016) 2016:115 Page 11 of 11 Table 4 Focusing performance comparison of points A and B
between two focusing algorithms
Target point
Method
PSLR (dB)
ISLR (dB)
IRW (m)
A
One-step MOCO
−16.5387
−10.7167
0.1575
Two-step MOCO
−15.5048
−8.6454
0.2975
B
One-step MOCO
−14.6624
−10.6820
0.1702
Two-step MOCO
−0.6995
2.3087
0.3755 Table 4 Focusing performance comparison of points A and B
between two focusing algorithms Author details
1N i
l L b f Author details
1National Lab of Radar Signal Processing and the Collaborative Innovation
Center of Information Sensing and Understanding, Xidian University, Xi’an
710071, People’s Republic of China. 2Beijing Institute of Radio Measurement,
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https://openalex.org/W2162150568
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https://europepmc.org/articles/pmc517939?pdf=render
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English
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Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants.
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BMC biotechnology
| 2,004
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cc-by
| 5,410
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BioMed Central BioMed Central Op
Methodology article
Rapid and reliable extraction of genomic DNA from various
wild-type and transgenic plants
Tae-Jin Kang1 and Moon-Sik Yang*2 Open Access ddress: 1Institute of Basic Science, Chonbuk National University, Jeonju 561-756, South Korea and 2Division of Biolo
olecular Biology and Genetics, Chonbuk National University, Jeonju 561-756, South Korea Email: Tae-Jin Kang - tjkang@chonbuk.ac.kr; Moon-Sik Yang* - mskyang@chonbuk.ac.kr * Corresponding author Received: 15 July 2004
Accepted: 02 September 2004 This article is available from: http://www.biomedcentral.com/1472-6750/4/20 © 2004 Kang and Yang; 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. Page 1 of 12
(page number not for citation purposes) Abstract Background: DNA extraction methods for PCR-quality DNA from calluses and plants are not
time efficient, since they require that the tissues be ground in liquid nitrogen, followed by
precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid
and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here,
we describe a simple and efficient method of isolating high-quality genomic DNA for PCR
amplification and enzyme digestion from calluses, various wild-type and transgenic plants. Results: We developed new rapid and reliable genomic DNA extraction method. With our
developed method, plant genomic DNA extraction could be performed within 30 min. The method
was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated
homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1). After centrifugation,
the supernatant was directly used for DNA template for PCR, resulting in successful amplification
for RAPD from various sources of plants and specific foreign genes from transgenic plants. After
precipitating the supernatant, the DNA was completely digested by restriction enzymes. Conclusion: This DNA extraction procedure promises simplicity, speed, and efficiency, both in
terms of time and the amount of plant sample required. In addition, this method does not require
expensive facilities for plant genomic DNA extraction. Most methods require the use of liquid nitrogen [4] or
freeze-drying (lyophilization) [5,6] of tissue for the initial
grinding, and these processes are unavailable in many
regions of the world. After grinding the tissues in various
extraction buffers, DNA is extracted with phenol-chloro-
form, or the extract is dialyzed against EDTA and a buff-
ered Tris-HCl solution [7]. After extraction, the aqueous
phase is concentrated, either by ethanol or isopropanol
precipitation [8,9], or with microconcentrators (e.g., the
Wizard genomic DNA purification system; Promega,
USA). However, these methods are not time efficient for Background g
Molecular biological studies of plants require high-quality
DNA. Several DNA extraction procedures for isolating
genomic DNA from various plant sources have been
described, including the salt extraction method and the
cetyltrimethyl ammonium bromide (CTAB) method [1]
and its modifications [2,3]. The need for a rapid and sim-
ple procedure is urgent, especially when hundreds of sam-
ples need to be analyzed. Page 1 of 12
(page number not for citation purposes) Page 1 of 12
(page number not for citation purposes) BMC Biotechnology 2004, 4:20 http://www.biomedcentral.com/1472-6750/4/20 RAPD fingerprints of all the DNA samples with primers (A) RAPD-1 (5'-CCACAGCAGT-3') and (B) RAPD-2 (5'-
AAGCCCGAGG-3')
Figure 1
RAPD fingerprints of all the DNA samples with primers (A) RAPD-1 (5'-CCACAGCAGT-3') and (B) RAPD-2
(5'-AAGCCCGAGG-3'). (A) Lane 1, the DNA template was the supernatant from the first phenol:chloroform:isoamyl alco-
hol extraction (protocol 1); lane 2, the DNA template was the supernatant after two phenol:chloroform:isoamyl alcohol
extractions and one chloroform extraction (protocol 2); lane 3, the DNA template was prepared with an additional ethanol
precipitation (protocol 3). (B) PCR products amplified using only the DNA template from protocol 1. 1 kb, DNA molecular
weight ladder. RAPD fingerprints of all the DNA samples with primers (A) RAPD 1 (5 CCACAGCAGT 3 ) and (B) RAPD 2 (5
AAGCCCGAGG 3 )
Figure 1
RAPD fingerprints of all the DNA samples with primers (A) RAPD-1 (5'-CCACAGCAGT-3') and (B) RAPD-2
(5'-AAGCCCGAGG-3'). (A) Lane 1, the DNA template was the supernatant from the first phenol:chloroform:isoamyl alco-
hol extraction (protocol 1); lane 2, the DNA template was the supernatant after two phenol:chloroform:isoamyl alcohol
extractions and one chloroform extraction (protocol 2); lane 3, the DNA template was prepared with an additional ethanol
precipitation (protocol 3). (B) PCR products amplified using only the DNA template from protocol 1. 1 kb, DNA molecular
weight ladder. Southern blot analysis to confirm homologous recombi-
nation in chloroplast transformation [10]. For our pur-
poses, we desire a simple and fast procedure for obtaining
plant genomic DNA for PCR, and good-quality DNA for
complete enzyme digestion for Southern blot analysis. Therefore, we present a protocol for extracting genomic
DNA from fresh calluses and plant leaves that is applica-
ble to a variety of organisms, regardless of the complexity
of their genomes. Page 2 of 12
(page number not for citation purposes) Background In addition, we present a rapid and reli- consistently obtaining PCR-quality DNA from calluses
and plants, since they require that the tissues be ground in
liquid nitrogen, followed by precipitation of the DNA pel-
let in ethanol, washing and drying the pellet, etc. In our laboratory, we investigate the stability of transgenes
expressed in calluses or plants transformed by nuclear or
chloroplast transformation in tobacco, lettuce, potato, etc. In addition, we need high-quality genomic DNA for Page 2 of 12
(page number not for citation purposes) Page 2 of 12
(page number not for citation purposes) http://www.biomedcentral.com/1472-6750/4/20 BMC Biotechnology 2004, 4:20 Table 1: Primers used in this study
Primer Name
Target Amplified
Size of Product
Primers (5'-3')
RAPD-1
Random
CCACAGCAGT
RAPD-2
Random
AAGCCCGAGG
1-F
Chloroplast
1700 bp
AAAACCCGTCCTCAGTTCGGATTGC
1-R
CCGCGTTGTTTCATCAAGCCTTACG
2-F
Chloroplast
1900 bp(LTB)
CTGTAGAAGTCACCATTGTTGTGC
2-R
2200 bp (bar)
TGACTGCCCACCTGAGAGCGGACA
Bar-F
Bar
550 bp
CGAGACAAGCACGGTCAACTTC
Bar-R
AAACCCACGTCATGCCAGTTC
LTB-F
B subunit of E. coli heat-labile enterotoxin
380 bp
ATGGCTCCCCAGTCTATTACAG
LTB-R
CTAGTTTTCCATACTGATTGC
PEDV-F
Porcine epidemic diarrhea virus
420 bp
TCTATGGTTACTTTGCCATC
PEDV-R
AATTAAACGTCTGTGATACC
Ure-F
A and B subunits of Helicobacter pylori urease
2450 bp
GCCACCATGAAACTCACCCCAAAAG
Ure-R
GGTACCCTAGAAAATGCTAAAGAGTTG
IFN-F
α-Interferon
580 bp
ATGGCCTCGCCCTTTGCTTTAC
IFN-R
CTCTTATTCCTTCCTCCTTAATC
F, forward primer; R, reverse primer Table 1: Primers used in this study F, forward primer; R, reverse primer DNA samples prepared using the three different extraction
procedures (lanes 1, 2, and 3 in Figure 1) were subjected
to PCR amplification using two 10-mer random primers:
RAPD-1 and RAPD-2 (Genotech, Korea) (Figure 1). All
the genomic DNA samples produced a clear, sharp, and
reproducible PCR product when primer RAPD-1 was used
for PCR amplification (Figure 1A). Although three varia-
tions of the DNA extraction procedure were used, there
was little difference between lanes 1, 2, and 3. Only a dif-
ference in the intensity of the band was observed, which
may be due to the different template concentrations used
for the PCR reaction. This result suggests that the superna-
tant after the first phenol treatment (protocol 1) was suf-
ficiently pure to be used as the DNA template for PCR
amplification. Therefore, PCR amplification with another
random primer, RAPD-2, was performed using the DNA
template extracted using the simplest protocol (Figure
1B). The PCR amplification was successful, and the same
banding pattern was seen when we repeated the PCR
amplification. Background Therefore, we confirmed that the DNA
template extracted using the simplest method was suffi-
cient for RAPD, and it was used as the DNA template to
amplify specific DNA or transgenes from transgenic cal-
luses or plants. able procedure for extracting genomic DNA for PCR or
Southern blot analysis from a small amount (~0.5 cm2) of
leaf tissue. Page 3 of 12
(page number not for citation purposes) Results and discussion (B) The PCR
products of transplastomic plants. Lane 1, wild-type plant with primers Bar-F/Bar-R; lane 2, primers Bar-F/Bar-R produce a
550-bp fragment; lanes 3 and 6, primers 1-F/1-R produce a 1700-bp fragment; lane 4, primers 2-F/2-R produce a 2200-bp frag
ment containing the bar gene; lane 5, primers LTB-F/LTB-R produce a 380-bp fragment; lane 7, primers 2-F/2-R produce a
1900-bp fragment containing the LTB gene. 1 kb, DNA molecular weight ladder. Schematic diagram of the chloroplast genome transformed with the bar or LTB gene and PCR analysis of wild-type and chloro-
plast transformants
Figure 2
Schematic diagram of the chloroplast genome transformed with the bar or LTB gene and PCR analysis of wild-
type and chloroplast transformants. (A) Map of the chloroplast targeting region in transplastomic plants. Arrows indicate
the direction of transcription. Primer 1F is located in the native chloroplast DNA; 1R, aadA; 2F, aadA; 2R, trnA. (B) The PCR
products of transplastomic plants. Lane 1, wild-type plant with primers Bar-F/Bar-R; lane 2, primers Bar-F/Bar-R produce a
550-bp fragment; lanes 3 and 6, primers 1-F/1-R produce a 1700-bp fragment; lane 4, primers 2-F/2-R produce a 2200-bp frag-
ment containing the bar gene; lane 5, primers LTB-F/LTB-R produce a 380-bp fragment; lane 7, primers 2-F/2-R produce a Schematic diagram of the chloroplast genome transformed with the bar or LTB gene and PCR analysis of wild-type and ch
plast transformants
Figure 2
Schematic diagram of the chloroplast genome transformed with the bar or LTB gene and PCR analysis of
type and chloroplast transformants. (A) Map of the chloroplast targeting region in transplastomic plants. Arrows ind
the direction of transcription Primer 1F is located in the native chloroplast DNA; 1R, aadA; 2F, aadA; 2R, trnA (B) The g
p
g
g
y
yp
p g
Schematic diagram of the chloroplast genome transformed with the bar or LTB gene and PCR analysis of wild-
type and chloroplast transformants. (A) Map of the chloroplast targeting region in transplastomic plants. Arrows indicate
the direction of transcription. Primer 1F is located in the native chloroplast DNA; 1R, aadA; 2F, aadA; 2R, trnA. (B) The PCR
products of transplastomic plants. Results and discussion We describe a simple and reproducible procedure for
RAPD or PCR amplification of transgenes from various
plant sources. Three different variations of the genomic
DNA extraction protocol for RAPD analysis were com-
pared. After simple plant leaf and callus tissue homogeni-
zation with DNA extraction buffer using a hand-operated
homogenizer, the leaf and callus cells were lysed with
20% SDS. Then, genomic DNA was extracted with the
same volume of phenol/chloroform/isoamyl alcohol
(25:24:1). An aliquot of the supernatant (~5 µl) was
diluted 5 fold with sterile dH2O, and PCR was performed
using 1 µl of the diluted supernatant as a template (Figure
1, lane 1). Alternatively, after phenol/chloroform/isoamyl
alcohol (25:24:1) extraction, the supernatant was trans-
ferred to a fresh tube for a second phenol/chloroform/iso-
amyl alcohol (25:24:1) extraction followed by a
chloroform extraction. An aliquot of the supernatant (~5
µl) was diluted 5 fold with sterile dH2O, and PCR was per-
formed using 1 µl of the diluted supernatant as the DNA
template (Figure 1, lane 2). In the third variation, after
chloroform extraction the supernatant was transferred to
a fresh tube and precipitated with two volumes of ethanol. After washing the DNA pellet with 70% ethanol, the DNA
pellet was dissolved in 50 µl of sterile dH2O containing 20
µg ml-1 DNase-free RNase A. For PCR, 50 ng of the DNA
were used as the template (Figure 1, lane 3). To examine the presence of bar [11,12] or the LTB gene
[13] at a directed site in the chloroplast DNA after homol-
ogous recombination in transplastomic tobacco plants,
putative transformants were screened by PCR analysis
(Figure 2). PCR amplification using primer combinations
Bar-F/Bar-R, 1-F/1-R, and LTB-F/LTB-R resulted in 550-,
1700-, and 380-bp fragments, respectively. Primers 2-F/2-
R produced 2200- or 1900-bp fragments containing bar Page 3 of 12
(page number not for citation purposes) Page 3 of 12
(page number not for citation purposes) BMC Biotechnology 2004, 4:20 http://www.biomedcentral.com/1472-6750/4/20 Schematic diagram of the chloroplast genome transformed with the bar or LTB gene and PCR analysis of wild-type and chlor
plast transformants
Figure 2
Schematic diagram of the chloroplast genome transformed with the bar or LTB gene and PCR analysis of wild
type and chloroplast transformants. (A) Map of the chloroplast targeting region in transplastomic plants. Arrows indicat
the direction of transcription. Primer 1F is located in the native chloroplast DNA; 1R, aadA; 2F, aadA; 2R, trnA. http://www.biomedcentral.com/1472-6750/4/20 After homogenization in DNA extraction buffer
using a hand-operated homogenizer, the template DNA
for PCR could be extracted by phenol/chloroform/isopro-
pyl alcohol treatment. Since this method does not require
liquid nitrogen, expensive commercial DNA extraction
kits, or ethanol precipitation to produce DNA template
for PCR, we can save considerable time and expense. The
time required for our DNA extraction method is less than
30 min, which is extraordinary compared with other
genomic DNA extraction methods. With our procedure,
leaf tissue (~0.5 cm2) is put in a 1.5-ml microfuge tube
and homogenized directly; consequently, a very small
sample is required for DNA extraction. There is no sample
waste with our method, whereas much larger samples are
required when plant samples are ground in a mortar and
pestle with liquid nitrogen and transferred to a tube. Pre-
viously reported techniques require several steps [19], use
of expensive enzymes such as proteinase K [20], or beads
and shakers [21]. Although the protocol for one-step
plant DNA isolation was developed by Burr et al. [22], if
plant material more than 1 mm2 was used in the extrac-
tion, co-extracts (e.g., chlorophyll) were extracted along-
side the DNA and inhibited the PCR. On the contrary, our
protocol does not require appropriate sample size to
extract DNA. Warner et al. [23] also reported a rapid DNA
extraction method in barley, which requires NaOH. How-
ever, the extracted DNA samples were easily degraded. The
DNA samples extracted by our protocol were very stable
and could be stored for a long time without degradation. bp) [18] (Figure 3). Specific PCR amplification was also
conducted using transgenic calluses as well as transgenic
plants. In transgenic calluses derived from Siberian gin-
seng plants, α-interferon was successfully amplified,
showing a 580-bp fragment in 1% agarose gels. PCR amplification products from transgenic plants and
calluses
Figure 3
PCR amplification products from transgenic plants
and calluses. Lane 1, α-interferon in transgenic calluses
from Siberian ginseng; lane 2, Core epitope of PEDV; lane 3,
LTB; lane 4, A plus B subunits of Helicobacter pylori urease. 1
kb, DNA molecular weight ladder. PCR amplification products from transgenic plants and
calluses
Figure 3
PCR amplification products from transgenic plants
and calluses. Lane 1, α-interferon in transgenic calluses
from Siberian ginseng; lane 2, Core epitope of PEDV; lane 3,
LTB; lane 4, A plus B subunits of Helicobacter pylori urease. 1
kb, DNA molecular weight ladder. http://www.biomedcentral.com/1472-6750/4/20 Using the third protocol, the DNA concentrations
obtained were between 20 and 30 µg/0.5 cm2 plant leaf,
and the absorbance ratios (A260/A280) were between 1.7
and 2.0. However, the DNA concentrations from rice,
maize, and poplar were relatively low (< 3 µg). This may
be because homogenization using a hand-operated
homogenizer with a plastic tip is incomplete, since the
leaves of these plants are stronger than the leaves of
tobacco, potato, cabbage, lettuce, and Siberian ginseng. Genomic DNA from various plant sources was electro-
phoresed on 1% agarose gels, and high-molecular-weight
DNA was obtained (Figure 4A). When the genomic DNA
was digested with EcoRI and HindIII, the DNA was com-
pletely digested, and could be used for Southern blot anal-
ysis. Therefore, we concluded that the purity and quality
of the genomic DNA was sufficient for enzyme digestion. We find the new method very useful in our laboratory,
since limited transgenic plant tissue or callus is sometimes
available in a culture bottle. Therefore, the simplicity, effi-
ciency, speed, and lack of a requirement for expensive
facilities make our method an attractive alternative to
existing methods of genomic DNA extraction. http://www.biomedcentral.com/1472-6750/4/20 http://www.biomedcentral.com/1472-6750/4/20 BMC Biotechnology 2004, 4:20 There are many advantages in using our genomic DNA
extraction method to obtain template for PCR amplifica-
tion. Many different plants could be amplified using the
same DNA extraction method and the same PCR protocol. Using this protocol, we successfully amplified DNA
repeatedly from all eight plant sources examined. Our
procedure is not only very simple, but is also time and cost
effective. After homogenization in DNA extraction buffer
using a hand-operated homogenizer, the template DNA
for PCR could be extracted by phenol/chloroform/isopro-
pyl alcohol treatment. Since this method does not require
liquid nitrogen, expensive commercial DNA extraction
kits, or ethanol precipitation to produce DNA template
for PCR, we can save considerable time and expense. The
time required for our DNA extraction method is less than
30 min, which is extraordinary compared with other
genomic DNA extraction methods. With our procedure,
leaf tissue (~0.5 cm2) is put in a 1.5-ml microfuge tube
and homogenized directly; consequently, a very small
sample is required for DNA extraction. There is no sample
waste with our method, whereas much larger samples are
required when plant samples are ground in a mortar and
pestle with liquid nitrogen and transferred to a tube. Pre-
viously reported techniques require several steps [19], use
of expensive enzymes such as proteinase K [20], or beads
and shakers [21]. Although the protocol for one-step
plant DNA isolation was developed by Burr et al. [22], if
plant material more than 1 mm2 was used in the extrac-
tion, co-extracts (e.g., chlorophyll) were extracted along-
side the DNA and inhibited the PCR. On the contrary, our
protocol does not require appropriate sample size to
extract DNA. Warner et al. [23] also reported a rapid DNA
extraction method in barley, which requires NaOH. How-
ever, the extracted DNA samples were easily degraded. The
DNA samples extracted by our protocol were very stable
and could be stored for a long time without degradation. There are many advantages in using our genomic DNA
extraction method to obtain template for PCR amplifica-
tion. Many different plants could be amplified using the
same DNA extraction method and the same PCR protocol. Using this protocol, we successfully amplified DNA
repeatedly from all eight plant sources examined. Our
procedure is not only very simple, but is also time and cost
effective. Results and discussion Lane 1, wild-type plant with primers Bar-F/Bar-R; lane 2, primers Bar-F/Bar-R produce a
550-bp fragment; lanes 3 and 6, primers 1-F/1-R produce a 1700-bp fragment; lane 4, primers 2-F/2-R produce a 2200-bp frag-
ment containing the bar gene; lane 5, primers LTB-F/LTB-R produce a 380-bp fragment; lane 7, primers 2-F/2-R produce a
1900-bp fragment containing the LTB gene. 1 kb, DNA molecular weight ladder. and LTB, respectively, which confirmed the site-specific
integration in the chloroplast genome (Table 1). No
detectable product was produced using genomic DNA
from wild-type plants (Figure 2B, lane 1), demonstrating
the specificity of these primers and genomic DNA extracts. Therefore, we concluded that chloroplast DNA was also
amplified, although we did not use liquid nitrogen, but simply used a hand-operated homogenizer with a plastic
tip. We also successfully amplified specific foreign genes
from transgenic tobacco plants transformed using the
nuclear transformation method, including the α-inter-
feron (550 bp) [14], the core epitope of the PEDV gene
(420 bp) [15,16], the LTB gene (380 bp) [17], and the A
plus B subunit of the Helicobacter pylori urease gene (2450 Page 4 of 12
(page number not for citation purposes) Page 4 of 12
(page number not for citation purposes) Page 5 of 12
(page number not for citation purposes) Conclusions Our objective was to extract genomic DNA with a simple
and fast procedure for PCR and enzyme digestion. The
present protocol is for extracting genomic DNA from fresh
calluses or plant leaf tissues that is applicable to a variety
of organisms, regardless of the complexity of their
genomes. Our procedure is not only very simple, but is
also time and cost effective. Since this method does not
require liquid nitrogen, expensive commercial DNA
extraction kits, or ethanol precipitation to produce DNA
template for PCR, we can save considerable time and Page 5 of 12
(page number not for citation purposes) Page 5 of 12
(page number not for citation purposes) http://www.biomedcentral.com/1472-6750/4/20 BMC Biotechnology 2004, 4:20 Agarose gel electrophoresis of undigested and digested genomic DNA
Figure 4
Agarose gel electrophoresis of undigested and digested genomic DNA. (A) Genomic DNA from five different plants
with 5 µg of genomic DNA loaded from each sample. (B) Genomic DNA digested with the restriction enzymes EcoRI and Hin-
dIII. 1 kb, DNA molecular weight ladder. Agarose ge
Figure 4 g
g
p
g
g
g
g
Agarose gel electrophoresis of undigested and digested genomic DNA. (A) Genomic DNA from five different plants
with 5 µg of genomic DNA loaded from each sample. (B) Genomic DNA digested with the restriction enzymes EcoRI and Hin-
dIII. 1 kb, DNA molecular weight ladder. DNA extraction (Figure 5) expense. In addition, a very small sample is required for
DNA extraction. ( g
)
We tested three different variations of the genomic DNA
extraction procedure. About 0.5 cm2 of culture room- or
greenhouse-grown plant leaves were put in a 1.5-ml
microfuge tube. The leaf tissue was homogenized in 50 µl
DNA extraction buffer (500 mM NaCl, 100 mM Tris-HCl
pH 7.5, and 50 mM EDTA pH 7.5), using a hand-operated
homogenizer (Sigma, Z35997-1) with a plastic pestle, for
15~20 s. After an initial homogenization, another 150 µl
of DNA extraction buffer were added and homogenized
with the same homogenizer for 15~20 s. Then, 20 µl of
20% SDS were added and vortexed for 30 s. The samples
were incubated at 65°C for 10 min for cell lysis. At this
point, three different DNA extraction protocols were used
for PCR amplification. Protocol 1: An equal volume of
phenol/chloroform/isoamyl alcohol (25:24:1) was added
to the samples, mixed by vortexing for 30 s, and then cen-
trifuged at 10,000 g for 3 min at 4°C. The supernatant was
diluted 5 fold, and 1 µl of the supernatant was used as the
DNA template for PCR analysis. Protocol 2: The superna-
tant from protocol 1 was transferred to a fresh tube and
extracted one more time with phenol/chloroform/iso-
amyl alcohol (25:24:1) and then with chloroform. The
supernatant was diluted 5 fold, and 1 µl of the
supernatant was used as the DNA template for PCR anal-
ysis. Protocol 3: The supernatant from protocol 2 was
transferred to a fresh tube, and a double volume of etha-
nol was added to each sample, mixed well, and the sam-
ples were incubated at -20°C for 30 min. The samples Plant material We examined plant material from plant collections com-
monly used for foreign gene expression: tobacco (Nico-
tiana tabacum), potato (Solanum tuberosum), cabbage
(Brassica oleracea), rice (Oryza sativa), lettuce (Lactuca
sativa), maize (Zea mays), poplar (Populus nigra), and Sibe-
rian ginseng (Eleutherococcus senticosus). The plants used
for genomic DNA extraction were grown in a culture room
or greenhouse. Tobacco, potato, cabbage, lettuce, and
Siberian ginseng were grown in a culture room. Seeds
were surface-sterilized with 70% ethanol for 3 min, and
then with 10% sodium hypochlorite for 15 min. The
seeds were washed five times in sterile water and placed in
Petri dishes containing 4.6 g l-1 MS salts [24], 30 g l-1
sucrose, and 7.5 g l-1 bactoagar at pH 5.7. The seeds were
grown in a controlled environment at 25°C on a 16-h
continuous light and 8-h dark daily cycle. Rice, maize, and
poplar plants were grown in a greenhouse for genomic
DNA extraction. Transgenic tobacco plants and Siberian
ginseng calluses were also used to extract genomic DNA
and to confirm foreign gene insertion by PCR
amplification. Page 6 of 12
(page number not for citation purposes) Page 6 of 12
(page number not for citation purposes) http://www.biomedcentral.com/1472-6750/4/20 BMC Biotechnology 2004, 4:20 Three different DNA extraction protocols for calluses and plants
Figure 5
Three different DNA extraction protocols for calluses and plants Three different DNA extraction protocols for calluses and plants
Figure 5
Three different DNA extraction protocols for calluses and plants type and transgenic plants, PCR amplifications were
performed on a Perkin Elmer GeneAmp PCR System 2400
(Biorad, USA) in a total volume of 25 µl containing 1 ×
PCR buffer, 0.2 mM dNTP, 10 pmol of each primer (Table
1), 50 ng template DNA from plants, and 0.25 U Ex-Taq
DNA polymerase (Takara, Japan) using the following pro-
file: a 3-min denaturation at 94°C and 40 cycles of 1-min
denaturation at 94°C, 1-min annealing at 37°C for RAPD
or 55°C for specific transgene amplification, and a 2-min
extension at 72°C, followed by a final extension at 72°C
for 7 min. The PCR products were resolved by electro-
phoresis in 1.0% agarose gels. were then centrifuged at 10,000 g for 10 min at 4°C. The
pellet was washed with 70% ethanol, dried, and resus-
pended in sterile dH2O containing 20 µg/ml DNase-free
RNase A. The concentration and purity were determined
from the A260/A280 ratio using a spectrophotometer. Plant material Five
micrograms of each genomic DNA sample were incubated
at 37°C for 3 h for complete digestion with 20 U of EcoRI
and HindIII (Life Technologies, USA) in a total volume of
100 µl and analyzed on 1.0% agarose gels using 15 µl aliq-
uots of the reaction mixture. Analysis of DNA and PCR amplifications Five micrograms of each genomic DNA sample measured
by spectrophotometer were incubated at 37°C for 3 h for
complete digestion with 20 U of EcoRI and HindIII in a
total volume of 100 µl and analyzed on 1.0% agarose gels
using 15 µl aliquots of the reaction mixture. By using the
genomic DNA isolated from the leaves or calluses of wild- References 1. Doyle JJ, Doyle JL: A rapid DNA isolation procedure for small
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(page number not for citation purposes) http://www.biomedcentral.com/1472-6750/4/20 Authors' contributions TJK developed the method and performed majority of the
experiments. MSY provided technical assistance, funding
and supervision for the work. All authors have read and
approved the final manuscript. Page 7 of 12
(page number not for citation purposes) Page 7 of 12
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Mol Biol Rep 2000, 18:61-64. References http://www.biomedcentral.com/1472-6750/4/20 BMC Biotechnology 2004, 4:20 http://www.biomedcentral.com/1472-6750/4/20 BMC Biotechnology 2004, 4:20 Page 10 of 12
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cited in PubMed and archived on PubMed Central
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https://openalex.org/W2808560070
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http://publications.lnu.edu.ua/journals/index.php/biology/article/download/128/127
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Russian
| null |
Influence of taurolithocholate 3-sulphate on calcium content in cytosol and store of isolated mice hepatocytes
|
Bìologìčnì studìï
| 2,015
|
cc-by
| 4,984
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Keywords: hepatocytes, taurolitocholate 3-sulphate, Ca2+, nicotinic acid adenine
dinucleotide phosphate. S. V. Bychkova
Ivan Franko National University of Lviv, 4, Hrushevskyi St., Lviv 79005, Ukraine
e-mail: s.bychkova@gmail.com Momohydroxylated bile acids, including taurolitocholate (TLC) and its 3-sulphate
(TLC-S), have been shown to increase [Ca2+]i in cytosol of rat hepatocytes [1, 2]. These
bile acids mobilize Ca2+ from the internal pool which is sensitive to inositol trisphosphate
(IP3). However, bile-acid mediated Ca2+ release is independent of IP3 production. Nico
tinic acid adenine dinucleotide phosphate (NAADP) is a nucleotide which can to release
calcium from specific type of intracellular store defined as endo-lysosomal system or acid
ic store. The aim of this study was to examine influence of NED-19 (antagonist of NAADP)
on TLC-S-induced change of calcium content in cytosol of and endoplasmic reticulum of
isolated mice hepatocytes in order to elucidate the role of acidic store in bile-acid medi
ated Ca2+ release. Isolated hepatocytes of mice were loaded with fluo-4 (2.5 µM). Fluores
cent images were obtained using Leica SP2 MP dual two-photon confocal microscope. Isolated hepatocytes were permeabilized in suspension with saponine (0.1 mg/mL). Next
the permeabilized suspension of hepatocytes was loaded with Mag-Fura-2 AM (5 µM). Measurement of Ca2+ content in store of permeabilized cells was conducted using spec
trofluorimetric method. We confirmed that TLC-S (50, 100 and 200 µM) elicited cytosolic
Ca2+‑signals, which were not inhibited by the IP3-receptors (IP3Rs) antagonist 2-APB
(100 µM). In suspensions of permeabilized murine hepatocytes TLC-S (100 µM) mobi
lized 66.10 ± 8.87 % of the total stored calcium as detected by ionomycin-induced release
(10 µM). After application of TLC-S thapsigargin could release only 47.94 ± 3.05 %. Previ
ous addition of NED-19 (100 nM) decreased fraction of calcium that is released by TLC-S
and equals 33.25 ± 2.15 % of the total calcium. In this case, the following use of thapsi
gargin mobilized only 21.75 ± 10.68 %. Thus, previous application of NED-19 significantly
(n = 6; P ≤ 0.01) reduced the proportion of calcium released by TLC‑S 2-fold. It was ob
served that the rate of TLC-S-induced decrease of calcium content in the intracellular
store was 1.8 times slower than after application of NED-19 (n = 6; Р ≤ 0.05). Previous
application of NED-19 increased the rate of thapsigargin-evoked calcium content reduc
tion by a factor of 2.5 (n = 6; Р ≤ 0.01). We suggest the impact of acid store in TLC-S-
elicited cytosolic Ca2+-signals in mice hepatocytes. Thus, the mechanism of TLC-S-in
duced calcium release is also NAADP-mediated. Biol. Stud. 2015: 9(1); 49–56 • DOI: https://doi.org/10.30970/sbi.0901.385
www.http://publications.lnu.edu.ua/journals/index.php/biology ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2015 • Том 9/№1 • С. 49–56 INFLUENCE OF TAUROLITHOCHOLATE 3-SULPHATE ON CALCIUM CONTENT
IN CYTOSOL AND STORE OF ISOLATED MICE HEPATOCYTES S. V. Bychkova
Ivan Franko National University of Lviv, 4, Hrushevskyi St., Lviv 79005, Ukraine
e-mail: s.bychkova@gmail.com INTRODUCTION Bile salts are synthesized from cholesterol in liver and represent the main driving
force of the bile flow. Bile is crucial for intestinal absorption of fats and fat-soluble vita
mins, as well as the elimination of excess cholesterol and waste products from body [3]. Previous work has shown that application of bile acids can cause the increase in the
levels of cytosolic [Ca2+]i in hepatocytes [1, 4]. i Specifically, bile acids activate calcium entry into the cells and cause depletion of
internal calcium store [5]. Other effects, not linked to calcium signaling, have also been
observed, including the increase in intracellular Na+ concentration [6] and depolarization
of inner mitochondrial membrane. [7]. In acinar pancreatic cells, it was also shown that bile acids can release calcium from
both ER and acidic stores in secretory granular areas. In both stores TLC-S interacts with
both the IP3Rs and the RyRs. TLC-S opens the RyRs through activation of NAADP [8]. In
hepatocytes, it is still unclear if NAADP-sensitive acidic store is involved in TLC-S-in
duced Ca2+-signals. Therefore, the main purpose of this study was to examine such
possibility. S. V. Bychkova
Ivan Franko National University of Lviv, 4, Hrushevskyi St., Lviv 79005, Ukraine
e-mail: s.bychkova@gmail.com N 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2015 • Том 9/№1 • С. 49–56 50 S. V. Bychkova RESULTS AND DISCUSSION TLC-S-induced Responses in the Intact Hepatocytes. We have shown that
TLC-S (50, 100 and 200 µM) elicited cytosolic Ca2+-signals, consistent with the previous
findings described early [1, 2]. A typical trace with repeated application of different con
centration of TLC-S is shown in Fig.1. A and B. TLC‑S (200 µM) induced calcium eleva
tion in the cytosol of intact hepatocytes comparably half the size of 10 µM ATP effect
(Fig.1, B). After TLC-S-elicited Ca2+-signal takes place hepatocytes can answer to ATP
but this signal has smaller amplitude yet longer plateau phase (Fig.1, B). The liver expresses 2 principal intracellular, calcium-release channels: the inositol
1,4,5-trisphosphate receptor (IP3R) (types 1 and 2) [10,11] and the ryanodine receptor
type 1 (RyR), detected as a truncated but functional channel-protein [12]. Previous re
sults of Mandi et al. [13] reported about NAADP-sensitive store in liver microsome frac
tion. Additionally, Zhang’s group has shown that NAADP-sensitive Ca2+ release channel
is present in the lysosome of native liver cells [14]. We have also observed NAADP-
evoked Ca2+-release in permeabilized hepatocytes [15].l So, we investigated whether inhibitors of these channels influence the TLC-S-elici
ted Ca2+ release. We tested 2-aminoethyldiphenyl borate (2-APB) as the inhibitor of the
IP3Rs. It was revealed that TLC‑S‑induced Ca2+-signals were not inhibited by the IP3Rs
antagonist 2-APB (100 µM) (Fig. 1, C). Monitoring of TLC-S action on the Ca2+ storage organelles in suspension of
permeabilized hepatocytes. In suspensions of murine hepatocytes, TLC-S (100 µM)
mobilizes 66.10 ± 8.87 % of the total stored calcium released by ionomycin (10 µM). In
this experiment, after exposure to TLC-S thapsigargin can release 47.94 ± 13.05 % of
the total stored calcium. A typical trace showing the effect of bile acid on Mag-Fura-2
(5 µM) (F/F0) in intracellular store of hepatocytes is shown on Fig.2, A.i (
µ
) (
0)
p
y
g
,
NAADP is the most potent Ca2+-mobilizing agent identified to date that acts in va
rious cell types across phyla. It was shown to selectively target the lysosome-related
organelles rich in Ca2+ and H+ and therefore called acidic Ca2+-stores. In hepatocytes
they are presented as endo-lysosomal system of the cell [16]. There are many hypoth
eses about the mechanisms of NAADP action. Much evidence suggests that NAADP
induces small yet localized cytoplasmic Ca2+-signals subsequently amplified into regen
erative global Ca2+-signals by recruitment of endoplasmic reticulum via calcium-induced
calcium release (CICR) [17]. MATERIALS AND METHODS 49–56 51 INFLUENCE OF TAUROLITHOCHOLATE 3-SULPHATE ON CALCIUM CONTENT IN CYTOSOL AND STORE... Cellular calcium content that was mobilized by 10 µM ionomycin was accepted as
100 % and represents the total amount of Ca2+ within the internal pool. l Cellular calcium content that was mobilized by 10 µM ionomycin was accepted as
100 % and represents the total amount of Ca2+ within the internal pool. l Reagents used in this study include fluo-4/Mag-Fura-2 AM (Invitrogen), thapsigar
gin (Calbiochem), collagenase (Worthington). All the other chemicals were purchased
from Sigma. MATERIALS AND METHODS Isolation of hepatocytes. CD-1 male mice were humanely sacrificed in compli
ance with the provisions of the European Convention for the Protection of Vertebrate
Animals used for Experimental and Other Scientific Purposes (Strasbourg, 1985) and in
accordance with International Convention for the Protection of Animals. The protocol for
hepatocyte isolation was as described in [9]. Isolated liver was perfused with buffer I
without Ca2+: 140 mM NaCl; 4.7 mM KCl; 10 mM HEPES; 10 mM D-glucose; 100 µM
EGTA; pH 7.4; the rate of perfusion was 5 mL/min at 37 °C. Next the liver was perfused
with buffer I in the presence of 1.3 mM CaCl2 and collagenase I (Worthington) for 10 min
at 37 °C. Dissociated hepatocytes were centrifuged at 50 × g for 1 min and then trans
ferred into buffer I containing 1 mM MgCl2 and 1.3 mM CaCl2, pH 7.4. g
g
2
2 p
Fluorescent [Ca2+] measurement. After isolation, the cells were loaded with low
affinity Ca2+-sensitive dye fluo-4 (2.5 µM) for 30-45 minutes at 36.5 °C. Cells were at
tached to poly-L-lysine-coated coverslips in flow chamber. All experiments were per
formed at room temperature. Fluorescent images were obtained using Leica SP2 MP dual two-photon confocal
microscope with a × 63 1.2 NA objective. For fluo-4 excitation and emission wavelengths
were 488 nm (argon ion laser, 3 % power) and 510–590 nm, respectively. Fluorescent
images were collected with frequency of 0.6–1.0 frame/second. Fluorescence signals
were plotted as F/F0, with F as fluorescence during the experiment and F0 as the initial
level of fluorescence. Measurement of [Ca2+] content in store of permeabilized cells. Suspension of
permeabilized hepatocytes (2 × 106) was used to load with fluorescent dye Mag-Fura-2
AM (5 µM). The dye was washed out before permeabilization. Isolated hepatocytes were
permeabilized with saponine (0.1 mg/mL) in intracellular solution for 10 min. Cells were
later washed with an intracellular solution based on K-HEPES, containing 20 mM NaCl;
127 mM KCl; 1.13 mM MgCl2; 0.05 mM CaCl2; 0.1 mM EGTA; 10.0 mM HEPES (KOH);
5 µg/mL oligomycine; 1 µg/mL rotenone; 2.0 mM АТP; рН 7.0. 2 mL of cell suspension
were transferred to the spectrofluorometer cuvette. The fluorescence of Mag-Fura-2 AM
was monitored using excitation wavelength 340-380 nm with emission at 500 nm. ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2015 • Том 9/№1 • С. RESULTS AND DISCUSSION The actual data collected on the NAADP-receptors remain
disputable. The potential NAADP-sensitive Ca2+-channels candidates include TRPML1,
TRPM2, TPCs and even RyRs [16, 17]. In order to investigate the effects of NAADP in
the cell, there was synthesized the selective antagonist of NAADP – NED-19. This small
molecule is cell-permeable and fluorescent derivative of tryptophan. NED-19 is a power
ful noncompetitive inhibitor of NAADP-binding process. It is also able to label the
NAADP-receptors in intact cells and effectively block the NAADP-induced Ca2+-release. Thus, NED-19 is commonly used for studies of NAADP-mediated events [18]. We have
found previously that NAADP triggered changes in stored Ca2+ were completely abo
lished by NED-19 as antagonist of NAADP in permeabilized rat hepatocytes [15]. ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2015 • Том 9/№1 • С. 49–56 52 52
S. V. Bychkova
Fig. 1. TLC-S-induced increase of Ca2+ level in the cytosol of intact hepatocytes loaded with fluo-4: (A) TLC-
S-mediated [Ca2+]i elevation is dependent on the concentration of TLC-S applied (30–50 µM); (B)
TLC-S (200 µM) triggers [Ca2+]i rise about half the size of 10 µM ATP-evoked elevation; after TLC-S-
elicited Ca2+-signals hepatocytes answer to ATP but this signal has smaller amplitude yet longer pla
teau phase; (C) 2-APB does not block [Ca2+]i elevation induced by TLC-S
Рис. 1. TLC-S-індуковане збільшення вмісту Са2+ в цитозолі інтактних гепатоцитів, навантажених fluo-4:
(А) TLC-S-зумовлене збільшення [Ca2+]i залежить від концентрації TLC-S (30–50 мкмоль/л); (В)
TLC-S (200 мкмоль/л) викликає підвищення рівня [Ca2+]i приблизно наполовину менше за його
збільшення під впливом 10 мкмоль/л АТФ; після Са2+-сигналів, викликаних TLC-S, гепатоцити
реагують на АТФ сигналом із меншою амплітудою, зате подовженою фазою плато; (С) 2-АРВ не
100
0
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
ATP, 1 µM
TLC-S, 30 µM
TLC-S, 50 µM
1.6
1.7
1.8
F/Fo, fluo-4
200
300
Time, s
400
500
600
700
800
A
0
0.6
0.8
1.2
1
1.4
1.8
2.2
2.4
2
1.6
200
400
600
800
1000
1200
1400
ATP, 10 µM
ATP, 10 µM
TLC-S, 200 µM
F/Fo, fluo-4
Time, s
A
B
200
400
600
800
1000
1200
1400
0
0.6
0.7
0.8
0.9
1.1
1.2
1.3
1.4
1
ATP, 1 µM
TLC-S, 50 µM
TLC-S, 50 µM
2-APB, 100 µM
F/Fo, fluo-4
Time, s
C S. V. RESULTS AND DISCUSSION Bychkova 100
0
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
ATP, 1 µM
TLC-S, 30 µM
TLC-S, 50 µM
1.6
1.7
1.8
F/Fo, fluo-4
200
300
Time, s
400
500
600
700
800
A B B Time, s
0
0.6
0.8
1.2
1
1.4
1.8
2.2
2.4
2
1.6
200
400
600
800
1000
1200
1400
ATP, 10 µM
ATP, 10 µM
TLC-S, 200 µM
F/Fo, fluo-4
Time, s
A
B Time, s
200
400
600
800
1000
1200
1400
0
0.6
0.7
0.8
0.9
1.1
1.2
1.3
1.4
1
ATP, 1 µM
TLC-S, 50 µM
TLC-S, 50 µM
2-APB, 100 µM
F/Fo, fluo-4
Time, s
C C Fig. 1. TLC-S-induced increase of Ca2+ level in the cytosol of intact hepatocytes loaded with fluo-4: (A) TLC-
S-mediated [Ca2+]i elevation is dependent on the concentration of TLC-S applied (30–50 µM); (B)
TLC-S (200 µM) triggers [Ca2+]i rise about half the size of 10 µM ATP-evoked elevation; after TLC-S-
elicited Ca2+-signals hepatocytes answer to ATP but this signal has smaller amplitude yet longer pla
teau phase; (C) 2-APB does not block [Ca2+]i elevation induced by TLC-S Рис. 1. TLC-S-індуковане збільшення вмісту Са2+ в цитозолі інтактних гепатоцитів, навантажених fluo-4:
(А) TLC-S-зумовлене збільшення [Ca2+]i залежить від концентрації TLC-S (30–50 мкмоль/л); (В)
TLC-S (200 мкмоль/л) викликає підвищення рівня [Ca2+]i приблизно наполовину менше за його
збільшення під впливом 10 мкмоль/л АТФ; після Са2+-сигналів, викликаних TLC-S, гепатоцити
реагують на АТФ сигналом із меншою амплітудою, зате подовженою фазою плато; (С) 2-АРВ не
пригнічує TLC-S-індукованого підвищення вмісту [Ca2+]i Рис. 1. TLC-S-індуковане збільшення вмісту Са2+ в цитозолі інтактних гепатоцитів, навантажених fluo-4:
(А) TLC-S-зумовлене збільшення [Ca2+]i залежить від концентрації TLC-S (30–50 мкмоль/л); (В)
TLC-S (200 мкмоль/л) викликає підвищення рівня [Ca2+]i приблизно наполовину менше за його
збільшення під впливом 10 мкмоль/л АТФ; після Са2+-сигналів, викликаних TLC-S, гепатоцити
реагують на АТФ сигналом із меншою амплітудою, зате подовженою фазою плато; (С) 2-АРВ не
пригнічує TLC-S-індукованого підвищення вмісту [Ca2+]i ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2015 • Том 9/№1 • С. 49–56 96-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2015 • Том 9/№1 • С. 49–56 INFLUENCE OF TAUROLITHOCHOLATE 3-SULPHATE ON CALCIUM CONTENT IN CYTOSOL AND STORE... RESULTS AND DISCUSSION The effect of TLC-S on store Ca2+ content in permeabilized hepatocytes loaded with mag-fura-2: (A)
100 µM TLC-S induce Ca2+ release from intracellular store, the subsequent application of thapsigargin
leads to depletion of Ca2+-store and the next adding of ionomycin releases residual Ca2+; (B) NED-19
substantially reduces TLC-S-evoked Ca2+ release and increases the rate of store emptying
Рис. 2. Вплив TLC-S на вміст депонованого Са2+ у пермеабілізованих гепатоцитах, навантажених mag-
fura-2: (А) TLC-S у концентрації 100 мкмоль/л спричиняє вивільнення Са2+ з депо, подальше Вплив TLC-S на вміст депонованого Са2+ у пермеабілізованих гепатоцитах, навантажених mag-
fura-2: (А) TLC-S у концентрації 100 мкмоль/л спричиняє вивільнення Са2+ з депо, подальше
застосування тапсигаргіну зумовлює спустошення останнього, а подальше використання іоно
міцину вивільняє залишковий Са2+; (В) NED-19 суттєво пригнічує TLC-S-індуковане вивільнення
Са2+ з депо та підвищує швидкість його спустошення Рис. 2. Вплив TLC-S на вміст депонованого Са2+ у пермеабілізованих гепатоцитах, навантажених mag-
fura-2: (А) TLC-S у концентрації 100 мкмоль/л спричиняє вивільнення Са2+ з депо, подальше
застосування тапсигаргіну зумовлює спустошення останнього, а подальше використання іоно
міцину вивільняє залишковий Са2+; (В) NED-19 суттєво пригнічує TLC-S-індуковане вивільнення
Са2+ з депо та підвищує швидкість його спустошення Рис. 2. Вплив TLC-S на вміст депонованого Са2+ у пермеабілізованих гепатоцитах, навантажених mag-
fura-2: (А) TLC-S у концентрації 100 мкмоль/л спричиняє вивільнення Са2+ з депо, подальше
застосування тапсигаргіну зумовлює спустошення останнього, а подальше використання іоно
міцину вивільняє залишковий Са2+; (В) NED-19 суттєво пригнічує TLC-S-індуковане вивільнення
Са2+ з депо та підвищує швидкість його спустошення After application of NED-19 (100 nM) fraction of calcium that was released by TLC-
S decreases and made up only 33.25 ± 2.15 % of the total calcium released by ionomy
cin. In this case, the following use of thapsigargin mobilizes only 21.75 ± 10.68 % of the
stored calcium in suspension of mice hepatocytes (Fig. 2, B). Thus, the previous applica
tion NED-19 significantly (n = 6; Р ≤ 0.01) reduced the proportion of calcium released by
TLC‑S 2-fold. We also calculated the velocity of calcium store emptying by TLC-S and
thapsigargin in control and after previous application of NED-19. It was established that
the rate of TLC-S-induced reduction of calcium level in the intracellular stores was 2-fold
slower than after application of NED-19 (n = 6; Р ≤ 0.05). RESULTS AND DISCUSSION 53 3
2.5
2
1.5
Ratio, mag-fura-2
1
0.5
0
200
400
800
1000
Ionomycin, 10 µM
Tg, 10 µM
TLS-C, 100 µM
600
Time, s
A
Ratio, mag-fura-2
0.5
1.5
2.5
3.5
1
2
3
4
0
0
200
400
600
800
1000
1200
Ionomycin, 5 µM
Tg, 10 µM
TLS-C, 100 µM
NED-19, 1 µM
Time, s
B 3
2.5
2
1.5
Ratio, mag-fura-2
1
0.5
0
200
400
800
1000
Ionomycin, 10 µM
Tg, 10 µM
TLS-C, 100 µM
600
Time, s
A Fig. 2. The effect of TLC-S on store Ca2+ content in permeabilized hepatocytes loaded with mag-fura-2: (A)
100 µM TLC-S induce Ca2+ release from intracellular store, the subsequent application of thapsigargin
leads to depletion of Ca2+-store and the next adding of ionomycin releases residual Ca2+; (B) NED-19
substantially reduces TLC-S-evoked Ca2+ release and increases the rate of store emptying
Рис. 2. Вплив TLC-S на вміст депонованого Са2+ у пермеабілізованих гепатоцитах, навантажених mag-
fura-2: (А) TLC-S у концентрації 100 мкмоль/л спричиняє вивільнення Са2+ з депо, подальше
застосування тапсигаргіну зумовлює спустошення останнього, а подальше використання іоно
міцину вивільняє залишковий Са2+; (В) NED-19 суттєво пригнічує TLC-S-індуковане вивільнення
Са2+ з депо та підвищує швидкість його спустошення
3
2.5
2
1.5
Ratio, mag-fura-2
1
0.5
0
200
400
800
1000
Ionomycin, 10 µM
Tg, 10 µM
TLS-C, 100 µM
600
Time, s
A
Ratio, mag-fura-2
0.5
1.5
2.5
3.5
1
2
3
4
0
0
200
400
600
800
1000
1200
Ionomycin, 5 µM
Tg, 10 µM
TLS-C, 100 µM
NED-19, 1 µM
Time, s
B Ionomycin, 10 µM 0.5
0
200
400
800
1000
600
Time, s
Ratio, mag-fura-2
0.5
1.5
2.5
3.5
1
2
3
4
0
0
200
400
600
800
1000
1200
Ionomycin, 5 µM
Tg, 10 µM
TLS-C, 100 µM
NED-19, 1 µM
Time, s
B Fig. 2. The effect of TLC-S on store Ca2+ content in permeabilized hepatocytes loaded with mag-fura-2: (A)
100 µM TLC-S induce Ca2+ release from intracellular store, the subsequent application of thapsigargin
leads to depletion of Ca2+-store and the next adding of ionomycin releases residual Ca2+; (B) NED-19
substantially reduces TLC-S-evoked Ca2+ release and increases the rate of store emptying Fig. 2. RESULTS AND DISCUSSION The same results were observed
on thapsigargin-elicited calcium content decrease in endoplasmic reticulum – previous ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2015 • Том 9/№1 • С. 49–56 54 S. V. Bychkova application of NED-19 increased its rate by 2.5 fold (n = 6; Р ≤ 0.01). We speculate that
the rise in velocity of TLC-S- and thapsigargin-induced calcium release from endoplasmic
reticulum after previous application of NED-19 is caused by destruction of the contact
sites between NAADP-sensitive acidic stores and endoplasmic reticulum. We assume
that the acidic Ca2+-store is important for refilling endoplasmic reticulum with calcium. CONCLUSION We suggest that acidic Ca2+-stores are involved in TLC-S-induced cytosolic Ca2+-
signals due to reduction of calcium level in the endoplasmatic reticulum of mice hepato
cytes. Thus, the mechanism of TLC-S-induced Ca2+-release is also NAADP-mediated. 1. Combettes L., Dumont M., Berthon B. et al. Release of calcium from the endoplasmic reticulum
by bile acids in rat liver cells. J. Biol. Chem, 1988; 263(5): 2299–2303. 1. Combettes L., Dumont M., Berthon B. et al. Release of calcium from the endoplasmic reticulum
by bile acids in rat liver cells. J. Biol. Chem, 1988; 263(5): 2299–2303. 2. Combettes L., Berthon B., Doucet E. et al. Characteristics of bile acid-mediated Ca2+ release
from permeabilized liver cells and liver microsomes. J. Biol. Chem, 1989; 264(1): 157–167. p
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3. Rembacz K. P., Woudenberg J., Hoekstra M. et al. Unconjugated bile salts shuttle through
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y
p
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p
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( )
4. Bouscarel B., Fromm H., Nussbaum R. Ursodeoxycholate mobilizes intracellular Ca2+ and acti
vates phosphorylase a in isolated hepatocytes. Am. J. Physiol, 1993; 264(2 Pt 1): G243–51. p
y
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p
gy
( )
4. Bouscarel B., Fromm H., Nussbaum R. Ursodeoxycholate mobilizes intracellular Ca2+ and acti
vates phosphorylase a in isolated hepatocytes. Am. J. Physiol, 1993; 264(2 Pt 1): G243–51. 5. Lau B.W., Colella M., Ruder W.C., Ranieri M., Curci S., Hofer A.M. Deoxycholic acid activates
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( )
6. Voronina S.G., Gryshchenko O.V., Gerasimenko O.V. et al. Bile acids induce a cationic cur
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7. Voronina S.G., Barrow S.L., Gerasimenko O.V. et al. Effects of Secretagogues and Bile Acids
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,
;
(
)
8. Gerasimenko J. V., Flowerdew S. E., Voronina S. G. et al. Bile acids induce Ca2+ release from
both the endoplasmic reticulum and acidic intracellular calcium stores through activation of
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40154–40163. 9. Li W.-C., Ralphs K.L., Tosh D. Isolation and culture of adult mouse hepatocytes. Methods
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by bile acids in rat liver cells. J. Biol. Chem, 1988; 263(5): 2299–2303.
2. Combettes L., Berthon B., Doucet E. et al. Characteristics of bile acid-mediated Ca2+ release
from permeabilized liver cells and liver microsomes. J. Biol. Chem, 1989; 264(1): 157–167.
3. Rembacz K. P., Woudenberg J., Hoekstra M. et al. Unconjugated bile salts shuttle through
hepatocyte peroxisomes for taurine conjugation. Hepatology, 2010; 52(6): 2167–2176.
4. Bouscarel B., Fromm H., Nussbaum R. Ursodeoxycholate mobilizes intracellular Ca2+ and acti
vates phosphorylase a in isolated hepatocytes. Am. J. Physiol, 1993; 264(2 Pt 1): G243–51.
5. Lau B.W., Colella M., Ruder W.C., Ranieri M., Curci S., Hofer A.M. Deoxycholic acid activates
protein kinase C and phospholipase C via increased Ca2+ entry at plasma membrane. Gas
troenterology. 2005. 128(3): 695–707.
6. Voronina S.G., Gryshchenko O.V., Gerasimenko O.V. et al. Bile acids induce a cationic cur
rent, depolarizing pancreatic acinar cells and increasing the intracellular Na+ concentration.
J. Biol. Chem, 2005; 280(3): 1764–1770. 1. Combettes L., Dumont M., Berthon B. et al. Release of calcium from the endoplasmic reticulum
by bile acids in rat liver cells. J. Biol. Chem, 1988; 263(5): 2299–2303. ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2015 • Том 9/№1 • С. 49–56 CONCLUSION Hirata K., Pusl T., O’Neill A.F. et al. The type II Inositol 1,4,5-Trisphosphate Receptor can trig
ger Ca2+ waves in rat hepatocytes. Gastroenterol, 2002; 122: 1088–1100 g
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11. Nagata J., Guerra M.T., Shugrue C.A. et al. Lipid rafts establish calcium waves in hepatocytes. Gastroenterology, 2007; 133: 256–267. 12. Pierobon N., Renard-Rooney D.C., Gaspers L.D., Thomas A.P. Ryanodine receptor in the
liver. J. Biol. Chem, 2006;10: 34086–34095. 13. Mandi M., Toth B., Timar G., Bak J. Ca2+ releasetriggered by NAADP in hepatocyte micro
somes. Biochem. J. 2006; 395(2): 233-238. ( )
14. Zhang F., Li P. L. Reconstitution and characterization of a nicotinic acid adeninedinucleotide
phosphate (NAADP)-sensitive Ca2+release channel from liver lysosomes of rats. J. Biol. Chem, 2007; 282(35): 25259–25269. (
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15. Bychkova S.V., Chorna T.I. NAADP-sensitive Сa2+ stores in permeabilized rat hepatocytes
Ukr. Biochem. J, 2014; 86(5): 65–73. 16. Patel S., Ramakrishnan L., Rahman T. et al. The endo-lysosomal system as an NAADP-sen
sitive acidic Ca2+ store: role for the two-pore channels. Cell Calcium, 2011; 50(2): 157–167. 17. Morgan A.J., Platt F.M., Lloyd-Evans E. et al. Molecular mechanisms of endolysosomal Ca2+
signalling in health and disease. Biochem. J, 2011; 439(3): 349–374. 16. Patel S., Ramakrishnan L., Rahman T. et al. The endo-lysosomal system as an NAADP-sen
sitive acidic Ca2+ store: role for the two-pore channels. Cell Calcium, 2011; 50(2): 157–167. g
g
,
;
( )
18. Naylor E., Arredouani A., Vasudevan S.R. et al. Identification of a chemical probe for NAADP
by virtual screening. Nature Сhemical Biology, 2009; 5(4): 220-226. N 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2015 • Том 9/№1 • С. 49–56 55 NFLUENCE OF TAUROLITHOCHOLATE 3-SULPHATE ON CALCIUM CONTENT IN CYTOSOL AND STORE... ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2015 • Том 9/№1 • С. 49–56 Ключові слова: гепатоцити, 3-сульфо-тауролітохолева кислота, Са2+, ніко
тинацидаденіндинуклеотидфосфат. ВПЛИВ 3-СУЛЬФО-ТАУРОЛІТОХОЛЕВОЇ КИСЛОТИ НА ВМІСТ ЦИТОЗОЛЬНОГО
ТА ДЕПОНОВАНОГО КАЛЬЦІЮ В ІЗОЛЬОВАНИХ ГЕПАТОЦИТАХ МИШЕЙ С. В. Бичкова
Львівський національний університет імені Івана Франка
вул. Грушевського, 4, Львів 79005, Україна
e-mail: s.bychkova@gmail.com Моногідроксильовані жовчні кислоти, такі як тауролітохолева кислота (TLС) і її
трисульфат (TLС-S), підвищують цитозольний кальцій у суспензії гепатоцитів [1,
2]. Вважають, що таке вивільнення відбувається за рахунок ІФ3-чутливого депо,
але без утворення ІФ3. Нікотинацидаденіндинуклеотидфосфат (НААДФ) здатний
вивільнювати Са2+ із органоїдів ендолізосомальної системи, яку зараховують до
кислотного депо клітин через кислий вміст. Метою роботи було дослідити вплив
NED-19 (антагоніста НААДФ) на TLC‑S‑індуковані зміни вмісту Са2+ в ізольованих
гепатоцитах мишей для того, щоб з’ясувати роль кислотного депо у TLC-S-індуко
ваному вивільненні Са2+. Ізольовані гепатоцити мишей навантажували 2,5 мкмоль/л
fluo-4. Зміни рівня концентрації кальцію в цитозолі реєстрували за допомогою ска
нуючого двофотонного мікроскопа Leica SP2 MP. З метою пермеабілізації гепато
цитів клітини обробляли сапоніном (0,1 мг/мл) у суспензії та навантажували mag-
fura-2. Зміни депонованого кальцію реєстрували спектрофлуориметричним мето
дом. Нами встановлено, що TLС-S у діапазоні концентрацій 50, 100 і 200 мкмоль/л
здатний викликати короткочасне підвищення кальцію в цитозолі ізольованих гепа
тоцитів мишей. Попередня аплікація 2-АРВ (100 мкмоль/л) не запобігала TLС-S-
індукованим Са2+-сигналам. У суспензії ізольованих гепатоцитів мишей TLС-S ви
вільнює 66,10 ± 8,87 % депонованого кальцію від усієї його кількості, яку здатний
вивільнити іономіцин. У цьому експерименті після дії TLС-S, тапсигаргін ще може
вивільнити 47,94 ± 13,05 % депонованого кальцію. Після застосування NED-19
(100 нмоль/л) частка кальцію, що вивільнюється TLС-S зменшується і становить
лише 33,25 ± 2,15 %. При цьому подальше застосування тапсигаргіну мобілізує
тільки 21,75 ± 10,68 % депонованого кальцію суспензії гепатоцитів мишей. Отже,
попередня аплікація NED-19 статистично достовірно (n = 6; Р ≤ 0,01) зменшує
частку депонованого кальцію після подальшого додавання TLС-S у 2 рази. Вста
новлено, що швидкість TLС-S-вивільнення кальцію з депо збільшується у 2 рази
після попереднього застосування NED-19 (n = 6; Р ≤ 0,05). Також і швидкість тап
сигаргін-індукованого вивільнення кальцію з депо зростає у 2,5 разу після преінку
бації клітин з NED-19 (n = 6; Р ≤ 0,01). Ми припускаємо, що у реалізації впливу
TLС-S на вміст кальцію в гепатоцитах мишей, окрім тапсигаргін-чутливого, залуче
не ще й кислотне депо, яке представлене ендолізосомами. Отже, механізм дії
TLС-S-індуковані Са2+-сигнали досліджуваних клітин є НААДФ-опосередкованим. Ключові слова: ві слова: гепатоцити, 3-сульфо-тауролітохолева кислота, Са2+, ніко
тинацидаденіндинуклеотидфосфат. N 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2015 • Том 9/№1 • С. 49–56 56 S. V. Bychkova ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2015 • Том 9/№1 • С. 49–56 ВЛИЯНИЕ 3-СУЛЬФО-ТАУРОЛИТОХОЛЕВОЙ КИСЛОТЫ НА СОДЕРЖАНИЕ
ЦИТОЗОЛЬНОГО И ДЕПОНИРОВАННОГО КАЛЬЦИЯ В ИЗОЛИРОВАННЫХ
ГЕПАТОЦИТАХ МЫШЕЙ С. В. Бычкова
Львовский национальный университет имени Ивана Франко
ул. Грушевского, 4, Львов 79005, Украина
e-mail: s.bychkova@gmail.com Моногидроксилированные желчные кислоты, такие как тауролитохолевая ки
слота (TLC) и ее трисульфат (TLC-S), повышают цитозольный кальций в суспензии
гепатоцитов [1, 2]. Полагают, что такое высвобождение происходит за счет ИФ3-чув
ствительного депо, но без образования ИФ3. Никотинацидадениндинуклеотид
фосфат (НААДФ) способен высвобождать Са2+ из органоидов эндолизосомальной
системы, которую причисляют к кислотному депо клеток из-за кислого содержи
мого. Целью работы было исследовать влияние NED-19 (антагониста НААДФ)
на TLC-S-индуцированные изменения кальция в изолированных гепатоцитах мы
шей для того, чтобы выяснить роль кислотного депо в TLC-S-индуцированном
высвобождении Са2+. Изолированные гепатоциты мышей нагружали 2,5 мкмоль/л
fluo-4. Регистрировали изменения концентрации кальция в цитозоле с использо
ванием сканирующего двухфотонного микроскопа Leica SP2 MP. С целью пермеа
билизации гепатоцитов клетки обрабатывали сапонином (0,1 мг/мл) в суспензии
и нагружали mag-fura-2. Изменения депонированного кальция регистрировали
спектрофлуориметрическим методом. Нами установлено, что TLC-S в диапазоне
концентраций 50, 100 и 200 мкмоль/л способен вызвать кратковременное повы
шение кальция в цитозоле изолированных гепатоцитов мышей. Предыдущая ап
пликация 2-АРВ (100 мкмоль/л) не предотвратила TLC-S-индуцированного Са2+-
сигнала. В суспензии изолированных гепатоцитов мышей TLC-S высвобождает
66,10 ± 8,87% депонированного кальция от всего, что способен высвободить ионо
мицин. В этом эксперименте, после воздействия TLC-S, тапсигаргин еще спосо
бен высвободить 47,94 ± 13,05% депонированного кальция. После применения
NED-19 (100 нмоль/л) доля кальция, высвобождаемая TLC-S уменьшается и со
ставляет лишь 33,25 ± 2,15 %. При этом последующее применение тапсигаргина
мобилизует только 21,75 ± 10,68 % депонированного кальция в суспензии гепато
цитов мышей. Итак, предыдущая аппликация NED-19 статистически достоверно
(n = 6; Р ≤ 0,01) уменьшает долю депонированного кальция после следующего
добавления TLC-S в 2 раза. Установлено, что скорость TLС-S-вызванного освобо
ждения кальция с депо увеличивается в 2 раза после предыдущей инкубации
с NED-19 (n = 6; Р ≤ 0,05). Также скорость тапсигаргин-индуцированного освобо
ждения кальция с депо возрастает в 2,5 раза (n = 6; Р ≤ 0,01). Мы предполагаем,
что в реализации влияния TLC-S на содержание кальция в гепатоцитах мышей,
кроме тапсигаргин-чувствительного, привлечено еще и кислотное депо, которое
представлено эндолизосомами. Итак, механизм действия TLC-S на кальциевый
гомеостаз исследуемых клеток является НААДФ-опосредованным. Ключевые слова: гепатоциты, 3-сульфо-тауролитохолевая кислота, Са2+,
никотинацидадениндинуклеотидфосфат. Ключевые слова: гепатоциты, 3-сульфо-тауролитохолевая кислота, Са2+,
никотинацидадениндинуклеотидфосфат. Ключевые слова: гепатоциты, 3-сульфо-тауролитохолевая кислота, Са2+,
никотинацидадениндинуклеотидфосфат. Одержано: 15.10.2014 6 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2015 • Том 9/№1 • С. 49–56 ISSN 1996-4536 (print) • ISSN 2311-0783 (on-line) • Біологічні Студії / Studia Biologica • 2015 • Том 9/№1 • С. 49–56
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The remotely controlled underwater robot system
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The
remotely
controlled
underwater
robot
system Cristian Nita1, , Dumitru Deleanu2* , and Ionut Voicu3 1Doctoral School of Mechanical Engineering, Electromechanical Faculty, 900663 Mircea cel Batran
Street, Romania 2 Constanta Maritime University, Department of General Engineering Sciences, 104 Mircea cel
Batran street, ZC 900663, Constanta, Romania 3 Electromechanical Faculty, Engineering Sciences in Mechanics and Environment Department,
900663 Mircea cel Batran Street, Romania Abstract. The remotely controlled underwater robot system is used
extensively by the scientific community to study the ocean, in support of
diving operations, in military use, in educational outreach, in broadcast
use. We built this system which has three components: 1) the surface
control interface; 2) the intermediate device; 3) the robot itself. The
presence of this robot replaces human activities where access is not
allowed or life is endangered. The novelty of this device is the construction
of component 2 (intermediate), which represents a storage frame for the
underwater robot and part of the control umbilical cord. The frame
descends with the robot to the depth of surveillance from where the robot
leaves the storage frame and is further controlled to the research objective. The surveillance function is performed from the intermediary with the help
of a video camera. The video camera is the IP type and can be accessed
through the data over power network. The movement of the underwater
device is accomplished by means of 8 electric motors (4 for horizontal
control and 4 for vertical control). The robot has data acquisition and
storage systems and piloting systems. The purpose of building this robot is
educational research. The surveillance function is performed from the intermediary with the help
of a video camera. The video camera is the IP type and can be accessed
through the data over power network. The movement of the underwater
device is accomplished by means of 8 electric motors (4 for horizontal
control and 4 for vertical control). The robot has data acquisition and
storage systems and piloting systems. The purpose of building this robot is
educational research. © 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: dumitru-deleanu@yahoo.com E3S Web of Conferences 180, 04002 (2020)
TE-RE-RD 2020 E3S Web of Conferences 180, 04002 (2020)
TE-RE-RD 2020 https://doi.org/10.1051/e3sconf/202018004002 1 Introduction The remotely controlled underwater robot system is used extensively by the scientific
community to study the ocean, in support of diving operations, in military use, in
educational outreach, in broadcast use (in the filming of several documentaries, coastguard
services). The remote controlled underwater robot system (RCURS) was specially designed for
underwater activities that are generally deep [1]. It is connected to the ship by an umbilical
cord, resistant to adverse conditions and to great depths, often used in conjunction with
TMS (Tether Management System). The TMS can be a garage-type device that includes the E3S Web of Conferences 180, 04002 (2020)
TE-RE-RD 2020 https://doi.org/10.1051/e3sconf/202018004002 RCURS during its dive, or it can be a sled-like device that only carries the laying cable. The
purpose of the TMS is to extend or shorten the umbilical cord, in order to dampen the
power draw of the water currents. RCURS during its dive, or it can be a sled-like device that only carries the laying cable. The
purpose of the TMS is to extend or shorten the umbilical cord, in order to dampen the
power draw of the water currents. As a use and historian, this system has been and can be used to rescue and recover
objects at a great depth, assisting in the development of oil platforms where diving access is
impossible, in initiating underwater constructions and repairing and maintaining them later
and also they can be used extensively by the scientific community in exploring the ocean
[2]. As technological variants can be designed to different sizes [3]: Micro - very small and
very light (weigh less than 3kg) - used as an alternative for divers, especially in places
where a diver could not have access (such as a canal, pipes or other small cavities); Mini -
weigh about 15kg (they are also used as an alternative for divers); · General - usually with
less than 5 horsepower (propulsion); Light Workclass - with less than 50 HP (horsepower);
Heavy Workclass - usually with less than 220HP; Trenching & Burial - with more than 200
HPr, but often no more than 500 HP. 2.1 Main system structure The remote controlled underwater robot system is composed of three components: 1) the
surface control interface; 2) the intermediate device; 3) the robot itself. The structure of the
robot body is presented in the Fig. 1. Fig. 1. The structure of the robot body
The control of the underwater robot is made from the surface with the help of
specialized software installed on the computer (Fig. 1). The control signal is transmitted
from the surface computer through an umbilical cord to the underwater robot. Both the
electrical energy required to operate the underwater robot and the control signal are
transmitted through two electrical conductors, through the data over power network system. The 2nd component, called intermediate, represents a storage frame for the underwater
robot and part of the control umbilical cord (Fig. 2). The frame descends with the robot to
the depth of surveillance from where the robot leaves the storage frame and is further
controlled to the research objective. Component number 2 is provided with a surface
controlled winch that allows the robot to receive only the umbilical cord length required for
movement, thus avoiding the possibility of tangling the control cord in foreign bodies. Fig. 1. The structure of the robot body Fig. 1. The structure of the robot body The control of the underwater robot is made from the surface with the help of
specialized software installed on the computer (Fig. 1). The control signal is transmitted
from the surface computer through an umbilical cord to the underwater robot. Both the
electrical energy required to operate the underwater robot and the control signal are
transmitted through two electrical conductors, through the data over power network system. The 2nd component, called intermediate, represents a storage frame for the underwater
robot and part of the control umbilical cord (Fig. 2). The frame descends with the robot to
the depth of surveillance from where the robot leaves the storage frame and is further
controlled to the research objective. Component number 2 is provided with a surface
controlled winch that allows the robot to receive only the umbilical cord length required for
movement, thus avoiding the possibility of tangling the control cord in foreign bodies. The control of the underwater robot is made from the surface with the help of
specialized software installed on the computer (Fig. 1). 2.1 Main system structure The control signal is transmitted
from the surface computer through an umbilical cord to the underwater robot. Both the
electrical energy required to operate the underwater robot and the control signal are
transmitted through two electrical conductors, through the data over power network system. nd g
g
p
y
The 2nd component, called intermediate, represents a storage frame for the underwater
robot and part of the control umbilical cord (Fig. 2). The frame descends with the robot to
the depth of surveillance from where the robot leaves the storage frame and is further
controlled to the research objective. Component number 2 is provided with a surface
controlled winch that allows the robot to receive only the umbilical cord length required for
movement, thus avoiding the possibility of tangling the control cord in foreign bodies. 2 2 E3S Web of Conferences 180, 04002 (2020)
TE-RE-RD 2020 https://doi.org/10.1051/e3sconf/202018004002 Fig. 2. The control umbilical cord
The surveillance function is performed from the intermediary with the help of a video
camera. The video camera is the IP type and can be accessed through the data over power
network. Component number 3, the underwater robot, hosts all motion control and navigation
systems (Fig 3) Fig. 2. The control umbilical cord Fig. 2. The control umbilical cord The surveillance function is performed from the intermediary with the help of a video
camera. The video camera is the IP type and can be accessed through the data over power
network. The surveillance function is performed from the intermediary with the help of a video
camera. The video camera is the IP type and can be accessed through the data over power
network. Component number 3, the underwater robot, hosts all motion control and navigation
systems (Fig. 3). systems (Fig. 3). Fig. 3. The underwater robot
Motion control is carried out by means of four electric motors for horizontal control and
another 4 electric motors for vertical control. Inside the robot's watertight housing are
installed two video cameras, one front for navigation and one superior with interior and
intermediate view, having a guiding role towards it. The electronic and electrical system
installed in the underwater robot is composed of 5 power sources with an input from 220
volts AC and 12 volts DC, these have the role of supplying electric motors and auxiliary
systems (Fig. 4). y
(
g
)
Fig. 3. The underwater robot Fig. 3. 2.2 The auxiliary structure The auxiliary systems are the following: the data over network system, the communication
system formed by a computer from the raspberry pi series, and the multi-device "pixhawk"
pilot system. The on-board computer communicates with the “pixhawk” and sends the
surface controls to it (Fig. 5). (
g
)
Fig. 5. The multi-device pixhawk pilot system
Fig. 6. The analog-to-digital signal converter
The robot is provided with indication for depth, electronic compass, temperature and
inclination angle. The video signal is transmitted using an analog-to-digital signal
converter, and accessed over a network over power network over IP (Fig. 6). This system is
located inside the robot housing on a platform (Fig. 7). Fig. 5. The multi-device pixhawk pilot system Fig. 5. The multi-device pixhawk pilot system Fig. 5. The multi-device pixhawk pilot system Fig. 6. The analog-to-digital signal converter Fig. 6. The analog-to-digital signal converter Fig. 6. The analog-to-digital signal converter The robot is provided with indication for depth, electronic compass, temperature and
inclination angle. The video signal is transmitted using an analog-to-digital signal
converter, and accessed over a network over power network over IP (Fig. 6). This system is
located inside the robot housing on a platform (Fig. 7). The robot is provided with indication for depth, electronic compass, temperature and
inclination angle. The video signal is transmitted using an analog-to-digital signal
converter, and accessed over a network over power network over IP (Fig. 6). This system is
located inside the robot housing on a platform (Fig. 7). converter Fig. 7. The platform with signal converter Fig. 7. The platform with signal converter 2.1 Main system structure The underwater robot Fig. 3. The underwater robot Motion control is carried out by means of four electric motors for horizontal control and
another 4 electric motors for vertical control. Inside the robot's watertight housing are
installed two video cameras, one front for navigation and one superior with interior and
intermediate view, having a guiding role towards it. The electronic and electrical system
installed in the underwater robot is composed of 5 power sources with an input from 220
volts AC and 12 volts DC, these have the role of supplying electric motors and auxiliary
systems (Fig. 4). Fig. 4. The electronic and electrical system
The underwater robot is powered by an alternating voltage of 220 volts from the Fig. 4. The electronic and electrical system Fig. 4. The electronic and electrical system The underwater robot is powered by an alternating voltage of 220 volts from the
surface. The underwater robot is powered by an alternating voltage of 220 volts from the
surface. The underwater robot is powered by an alternating voltage of 220 volts from the
surface. 3 https://doi.org/10.1051/e3sconf/202018004002 E3S Web of Conferences 180, 04002 (2020)
TE-RE-RD 2020 E3S Web of Conferences 180, 04002 (2020) 3 Experimental researches The general structure is fixed on the frame platform. The frame platform descends with the
robot itself to a depth of surveillance from where the robot leaves the storage frame and is
further controlled to the research objective (Fig. 8). 4 4 E3S Web of Conferences 180, 04002 (2020)
TE-RE-RD 2020 https://doi.org/10.1051/e3sconf/202018004002 Fig. 8. The general structure of device Fig. 8. The general structure of device
The component number 2 is provided with a surface controlled winch that allows the robot
to receive only the length of the control cord required for movement, thus avoiding the
possibility of entanglement in foreign bodies. The robot moves in a horizontal or vertical direction with 4 electric motors on each Fig. 8. The general structure of device The component number 2 is provided with a surface controlled winch that allows the robot
to receive only the length of the control cord required for movement, thus avoiding the
possibility of entanglement in foreign bodies. The robot moves in a horizontal or vertical direction with 4 electric motors on each
direction (Fig. 9). The robot moves in a horizontal or vertical direction with 4 electric motors on each
direction (Fig. 9). Fig. 9. Front view - electric motors for moving
The technology for making this robot has been developed for educational and research
purposes. Its modular design allows configuration to meet the specific needs of a research,
just as military special mission vehicles are designed [4]. Fig. 9. Front view - electric motors for moving Fig. 9. Front view - electric motors for moving Fig. 9. Front view - electric motors for moving The technology for making this robot has been developed for educational and research
purposes. Its modular design allows configuration to meet the specific needs of a research,
just as military special mission vehicles are designed [4]. 4 Conclusions Depending on the research to be carried out, this underwater robot can be equipped with
various sensory and sampling devices. The purpose of this paper is to present the
technological way of making such a robot for teaching and research purposes. The novelty of this device is the construction of component 2 (intermediate), which
represents a storage frame for the underwater robot and part of the control umbilical cord. The frame descends with the robot to the depth of surveillance from where the robot leaves
the storage frame and is further controlled to the research objective. Learning the technological modalities of achievement teaches students to acquire the
basic skills in designing ships and submarines, and encourages them to explore the
architecture and concepts of naval engineering. 5 E3S Web of Conferences 180, 04002 (2020)
TE-RE-RD 2020 https://doi.org/10.1051/e3sconf/202018004002 This paper has financial support from the Constanta Maritime University under assistance project PN-
III-P1-1.2-PCCDI-2017-0404 /31PCCDI/2018, Holistic on the Impact of Renewable Energy Sources
on Environment and Climate-HORESEC. 4. Pascu.E., Royal Netherlands Navy- Sea-Wasp ROV, J. Defense Romania, 20 iV (2020) References 1. D. Dumitru, C. Vlad, Diving and underwater vehicles (Scientific and Encyclopedic
Edition, Bucharest,1986) 2. D.Dumitru, C. Vlad, Underwater interventions (Technical Publishing House,
Bucharest, 1982) 3. R. Sundar, M. Dheepak, P. Veera Kumar, PC based remote operated underwater
vehicle for marine surveillance, Int.J.of Civil Engineering and Technology, 8, Issue 6,
716–721 (2017) 3. R. Sundar, M. Dheepak, P. Veera Kumar, PC based remote operated underwater
vehicle for marine surveillance, Int.J.of Civil Engineering and Technology, 8, Issue 6,
716–721 (2017) 6
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Validation of Questionnaire Methods to Quantify Recreational Water Ingestion
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International Journal of
Environmental Research
and Public Health
Article
Validation of Questionnaire Methods to Quantify
Recreational Water Ingestion
Laura M. Suppes 1, * , Kacey C. Ernst 2 , Leif Abrell 3
1
2
3
*
and Kelly A. Reynolds 2
Environmental Public Health Program, The University of Wisconsin-Eau Claire, 105 Garfield Avenue,
Eau Claire, WI 54702, USA
Mel and Enid Zuckerman College of Public Health, The University of Arizona, P.O. Box 245163,
Tucson, AZ 85724, USA; Kernst@email.arizona.edu (K.C.E.); Reynolds@email.arizona.edu (K.A.R.)
Department of Soil, Water & Environmental Science, The University of Arizona, Gould-Simpson Building
Room 611, 1040 East 4th Street, Tucson, AZ 85721, USA; Abrell@email.arizona.edu
Correspondence: suppeslm@uwec.edu; Tel.: +1-715-836-5977
Received: 8 September 2018; Accepted: 19 October 2018; Published: 1 November 2018
Abstract: Swimming pool water ingestion volumes are necessary for assessing infection risk from
swimming. Pool water ingestion volumes can be estimated by questionnaire or measuring a chemical
tracer in swimmer urine. Questionnaires are often preferred to the chemical tracer method because
surveys are less time consuming, but no research exists validating questionnaires accurately quantify
pool water ingestion volumes. The objective of this study was to explore if questionnaires are
a reliable tool for collecting pool water ingestion volumes. A questionnaire was issued at four
pool sites in Tucson, Arizona to 46 swimmers who also submitted a urine sample for analyzing
cyanuric acid, a chemical tracer. Perceived ingestion volumes reported on the questionnaire were
compared with pool water ingestion volumes, quantified by analyzing cyanuric acid in swimmer
urine. Swimmers were asked if they swallowed (1) no water or only a few drops, (2) one to two
mouthfuls, (3) three to five mouthfuls, or (4) six to eight mouthfuls. One mouthful is the equivalent of
27 mL of water. The majority (81%) of swimmers ingested <27 mL of pool water but reported ingesting
>27 mL (“one mouthful”) on the questionnaire. More than half (52%) of swimmers overestimated
their ingestion volume. These findings suggest swimmers are over-estimating pool water ingestion
because they perceive one mouthful is <27 mL. The questionnaire did not reliably collect pool water
ingestion volumes and should be improved for future exposure assessment studies. Images of the
ingestion volume categories should be included on the questionnaire to help swimmers visualize the
response options.
Keywords: pool water ingestion; recreational water; swimming pool; risk assessment
1. Introduction
The annual number of Recreational Water Illness (RWI) outbreaks associated with treated
recreational water venues (“pools”) in the U.S. has increased since 1978 when reporting was initiated
(pools are defined as swimming pools, spas, interactive fountains, wading pools and dive pools) [1–3].
RWIs range from acute gastrointestinal illness (AGI), skin infection or rash to acute respiratory illness
(ARI). The majority of outbreaks are associated with AGI, which accounted for 81% of outbreaks during
summer months in 2011–2012 [4]. Most AGI outbreaks in treated recreational water are associated
with ingesting Cryptosporidium. Cryptosporidium has been detected in treated recreational water and
associated with outbreaks internationally [5–8]. From 2000–2014, Cryptosporidium caused 58% of
treated recreational water outbreaks in the U.S. [9]. The volume of pool water ingested by swimmers is
necessary to quantify infection risk from enteric pathogens like Cryptosporidium [10]. Risk assessment
Int. J. Environ. Res. Public Health 2018, 15, 2435; doi:10.3390/ijerph15112435
www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2018, 15, 2435
2 of 6
can help identify unsafe swimming behaviors, at-risk populations, and priority hazards to direct the
development of pool safety guidelines. Recognizing the need for accurate data collection tools for
swimming pool risk assessment, this study compared perceived ingestion volumes reported on a
questionnaire to pool water ingestion volumes quantified by analyzing cyanuric acid in swimmer
urine. The questionnaire merged information and survey questions collected and developed by the
Centers for Disease Control and Prevention (CDC), the U.S. Environmental Protection Agency (USEPA),
and academic researchers to assess a variety of swimmer exposures. The objective was to determine if
questionnaires are a reliable tool for collecting pool water ingestion volumes.
One primary exposure related to risk of RWI is ingestion of water. Previously, the World Health
Organization (WHO) used questionnaires to estimate swimming ingestion rates and found swimmers
reported swallowing 20–50 mL/h [11]. These self-reported values, however, are underestimated
when compared to ingestion ranges found in other studies applying quantitative measurement
techniques. Thus, the WHO questionnaire may not accurately capture pool water ingestion magnitudes
among swimmers.
Ingestion can be quantified using methods that compare cyanuric acid in urine and pool
water. Cyanuric acid is added as a chlorine stabilizer to outdoor pool water, and when ingested,
passes through the human body unmetabolized [12]. Controlled studies show 98% of cyanuric acid
ingested is excreted in a 24 h period [12]. Using this technique, researchers Dufour et al. and Suppes
et al. showed swimmers ingested between 0–154 mL/h and 0–105.5 mL/h, respectively [13,14].
Information on perceived ingestion by study participants was not collected in the Dufour study,
but was collected by Suppes et al. using the questionnaire discussed in this article (see Supplementary
Materials). The questionnaire asked swimmers how much pool water was ingested during a timed
swim. The current article is one part of the Suppes et al. study and describes how accurately swimmers
perceive pool water ingestion by comparing reported to measured volumes. Our findings demonstrate
swimmers perceive higher ingestion exposures than in reality, which explains why self-reported
ingestion estimates are different than measured estimates.
2. Materials and Methods
2.1. Questionnaire Development
The CDC and USEPA websites and peer-reviewed literature were searched for pool outbreak
survey tools, tools developed in response to outbreaks, and tools designed to capture swimmer
exposures [15,16]. The CDC National Outbreak Reporting System (NORS) is available for reporting
nationwide waterborne disease outbreaks and includes exposure questions related to recreational
water. In-depth survey tools are also available through the CDC that collect data on swimmer
activity, gastrointestinal symptoms, confounding exposures, pool operations and maintenance, and are
designed to be administered by outbreak investigators [15]. Surveys intended to collect additional
exposure information, such as potential disinfection by-product exposures, were reviewed from the
USEPA assessment tool SWIMODEL among others [15].
Exposure risk factors relative to swimmer behavior and pool maintenance from the CDC surveys,
SWIMODEL, and peer-reviewed literature were compiled and organized into a draft questionnaire.
Three panels were assembled to review the draft for comprehensiveness and to recommend formatting
and included (1) six experts from the swimming pool industry; (2) an international group of
nine microbiologists, exposure scientists, and epidemiologists; and (3) an internal University of
Arizona panel of six respiratory health, epidemiology, exposure science, and public health specialists.
Meetings with each panel were held once and lasted 1–2 h following advance reviews of the
questionnaire. Individual communication with panel members by email or phone occurred throughout
the questionnaire development process. Questions from the draft were entered into DatStat Illume
Survey Developer Gateway Version 5.1.1.17347 (Seattle, WA, USA). The questionnaire was further
Int. J. Environ. Res. Public Health 2018, 15, 2435
3 of 6
evaluated by the external review panel for errors and comprehensiveness prior to use. A modified
version of the questionnaire can be viewed in Table S1 of the Supplementary Materials.
The question used in this study to estimate pool water ingestion by “mouthfuls/swim” was
developed by Schets et al. and was selected over other surveys based on recommendations from
the expert questionnaire review committees [17]. Other surveys used specific volume classifications,
like “teaspoon”, that may have been difficult for younger participants in this study to interpret.
The Schets study quantified the average volume in one mouthful (27 mL), which allowed measured
volumes in the present study to be categorized into “mouthfuls/swim”. Swimmers were asked on our
questionnaire if they swallowed (1) no water or only a few drops, (2) one to two mouthfuls, (3) three to
five mouthfuls, or (4) six to eight mouthfuls. Using data from the Schets study indicating an average
mouthful is 27 mL, qualitative variables from our questionnaire were converted to quantitative volumes.
Despite the Schets study defining “no water to a few drops” as 0–5 mL, swimmers with measured
ingestion between 0–26 mL were categorized as: “1: no water or only a few drops”. There was
no qualitative ingestion category in the Schets study representing 6–26 mL. The other categories
were: 27–54 mL (one to two mouthfuls), 55–135 mL (three to five mouthfuls), and 136–216 mL (six to
eight mouthfuls).
2.2. Data Collection
This research was approved by the University of Arizona Human Subjects Research and
Institutional Review Board (project number: 12-0272-12). The questionnaire was issued to 46 swimmers
June–September 2013 in Tucson, Arizona, recruited at two outdoor public pools and two outdoor
private pools. Swimmers arriving at the pools on data collection days were approached by a member
of the research team, given details of the study’s objectives, and asked if they would participate by
completing a questionnaire after swimming and submitting a 24 h urine sample to quantify pool
water ingestion. Urine samples were preserved then cleaned by solid phase extraction and analyzed
using ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for
cyanuric acid. Pool water samples were collected at each pool site on the day swimmers were recruited,
transferred on ice, and preserved along with urine samples. Cyanuric acid was quantified in pool
water using UHPLC-MS/MS. Pool water ingestion volumes were calculated using cyanuric acid
concentrations in urine and pool water [13] (Equation (1)). Detailed results from the 24 h urine sample
portion of this study are published elsewhere [14].
water ingestion (L) = ([cyanuric acid]urine (
µg
L ) ÷ [cyanuric
acid]pool water (
µg
L ))
× urine volume (L)
(1)
All swimmers, regardless of age, gender, or other factors, were approached and asked to
participate. Swim duration for all participants was recorded on the questionnaire. Participants accessed
the questionnaire either on-site using tablets, electronic or smart phones, or on a personal computer
through email. Questionnaires were completed within six hours of swimming.
3. Results
Thirty-eight of 46 participants had usable water ingestion values for analysis. Four did not submit
a questionnaire, one submitted a urine sample less than the accepted volume threshold, and three urine
samples had signal-to-noise ratios <3, which indicates a measurement below the analytical equipment
limit of detection (UHPLC-MS/MS). The percent recoveries of cyanuric acid from urine and pool water
were 6% and 112%, respectively. Table 1 summarizes the study population.
Table 2 illustrates the number of swimmers who correctly and incorrectly reported the volume
range of pool water ingested during swimming. Sixteen of 38 swimmers (42%) correctly reported
their ingestion volume, 20/38 (52%) overestimated the amount of pool water ingested and 2/38
(5%) underestimated their ingestion volume. Thirty-one of 38 swimmers (81%) actually ingested
0–26 mL of water, but only 11/38 swimmers (29%) correctly reported ingesting 0–26 mL. All swimmers
Int. J. Environ. Res. Public Health 2018, 15, 2435
4 of 6
(11/11) who reported ingesting “no water to a few drops” did ingest water within the volume range
categorized as “no water to a few drops” (0–26 mL). Four of 20 swimmers who reported ingesting
“one to two mouthfuls” actually ingested pool water within the volume range “one to two mouthfuls”
(27–54 mL). Only one swimmer reported ingesting “three to five mouthfuls”, but six actually did
ingest pool water within this volume range (55–135 mL). No swimmers ingested or reported ingesting
136–216 mL.
Table 1. Age and gender distributions of study participants.
Participant Demographics n = 38 (%)
Age
≤18 years
>18 years
17 (44.7)
21 (55.2)
Gender
Male
Female
25 (65.7)
13 (34.2)
Table 2. Number of swimmers reporting and actually ingesting pool water amounts within each
volume range listed on the questionnaire (n = 38).
No water–few drops
Measured
Ingestion *
One to two mouthfuls
No Water–Few
Drops
(0–26 mL)
One to Two
Mouthfuls
(27–54 mL)
Three to Five
Mouthfuls
(55–135 mL)
Six to Eight
Mouthfuls
(136–216 mL)
11 †
14
6
0
†
0
0
0
4
Three to five mouthfuls
0
2
Six to eight mouthfuls
0
0
1
†
0
0
0
* Measured ingestion values have been categorized using mouthful volumes characterized by Schets et al. [17].
† Study participants correctly reporting ingestion volume.
4. Discussion
Developers of the question used on our survey found the average volume of one mouthful to
be 27 mL, which was used in this study to categorize measured ingestion volumes to mouthfuls.
The majority (81%) of swimmers actually ingested <27 mL of pool water but reported ingesting >27 mL
(one mouthful) on the questionnaire. More than half (52%) of swimmers overestimated their ingestion
volume across all volume categories. These findings suggest swimmers are overestimating pool water
ingestion because they perceive one mouthful to be <27 mL. The lack of accurate reporting of ingestion
volumes using a question recommended by experts suggests a need for improving questionnaire
techniques to assess recreational water ingestion. Since there is uncertainty about the volume of
water in one mouthful, the questionnaire can be improved by including images of a one-cup/250 mL
measuring glass with one to eight mouthfuls of liquid (Figure 1). Eight was the maximum number of
mouthfuls on the questionnaire. The questionnaire can also be improved by changing the “no water
to a few drops” category to “less than one mouthful” for consistency in questionnaire response
options. Including Figure 1 would help swimmers visualize the ingestion volume categories to reduce
inaccurate reporting.
Inconsistencies in method performance between this study and similar studies [13,18] and low
recoveries of cyanuric acid in urine indicate a need for improving techniques to quantify pool water
ingestion. Using comparable methods, Dorevitch et al. recovered 32.7% of cyanuric acid from swimmer
urine and 96.5–99% of cyanuric acid from pool water [18]. Dufour et al. did not report recovery
efficiencies for cyanuric acid in urine or pool water using a similar method [13]. Recovery of cyanuric
acid in urine and pool water was 6% and 112%, respectively, in the current study. Like this study,
Dorevitch et al. calculated pool water ingestion using Equation (1) and did not adjust cyanuric acid in
Int. J. Environ. Res. Public Health 2018, 15, 2435
5 of 6
pool water to account for the lower recovery in urine. Self-reported pool water ingestion quantities
from a questionnaire by Dorevitch et al. were also compared to measured pool water ingestion
quantities. To be consistent with Dorevitch and Dufour, no percent recovery adjustments were made to
cyanuric acid in urine or pool water before analyzing measured and self-reported pool water ingestion
in this study. Measured ingestion estimates could be higher than reported in all three studies, but exact
pool water ingestion quantities cannot be estimated without a method that consistently recovers 100%
of cyanuric acid in urine. Cyanuric acid extraction efficiencies are dependent on the solid phase
extraction technique and analytical instrument. A more detailed comparison and discussion of method
performance and limitations between this study and others is published elsewhere [14].
Int. J. Environ. Res. Public Health 2018, 15, x FOR PEER REVIEW
5 of 7
Figure 1.
1. The
The figure
figure illustrates
illustrates one
one to
to eight
eight mouthfuls
mouthfuls of
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in aa one-cup/250
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mL measuring
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glass,
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one mouthful
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to 27
27 mL
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of liquid
liquid [17].
[17].
assuming
5. Conclusions
Inconsistencies in method performance between this study and similar studies [13,18] and low
recoveries
of cyanuric
acidthe
in need
urinefor
indicate
a need
for improving
techniques
to quantify
pool water
water
This study
highlights
improved
questionnaire
techniques
to assess
recreational
ingestion. Our
Using
comparable
methods,
Dorevitch
et al.higher
recovered
32.7%exposures
of cyanuric
from
ingestion.
findings
demonstrate
swimmers
perceive
ingestion
thanacid
in reality,
swimmer
urine
and
96.5–99%
of
cyanuric
acid
from
pool
water
[18].
Dufour
et
al.
did
not
report
which explains why self-reported ingestion estimates are different than measured estimates from
recovery studies.
efficiencies
forthere
cyanuric
acid in urine
or the
poolvolume
water using
a similar
[13].
Recovery
previous
Since
is uncertainty
about
of water
in one method
mouthful,
researchers
of cyanuric
in urine
and pool
water
wasshould
6% and
112%,images
respectively,
in the current
Like
who
use thisacid
question
technique
in the
future
include
of a one-cup/250
mLstudy.
measuring
this
study,
Dorevitch
et
al.
calculated
pool
water
ingestion
using
Equation
(1)
and
did
not
adjust
glass with one to eight mouthfuls of liquid to help swimmers visualize the ingestion volume categories.
cyanuric
acid in pool
water
towater
account
lower
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in urine.
water
The
questionnaire
category
“no
to afor
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ingestion
quantities
from
a
questionnaire
by
Dorevitch
et
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also
compared
to
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to be consistent with other response options on the questionnaire. The altered questionnaire should
be
water
ingestion
quantities.
To
be
consistent
with
Dorevitch
and
Dufour,
no
percent
recovery
validated to ensure ingestion volumes are accurately reported.
adjustments were made to cyanuric acid in urine or pool water before analyzing measured and
Supplementary
Materials:
following
at http://www.mdpi.com/1660-4601/15/11/2435/
self-reported pool
waterThe
ingestion
in are
thisavailable
study. online
Measured
ingestion estimates could be higher than
s1.
reported in all three studies, but exact pool water ingestion quantities cannot be estimated without a
Author
Conceptualization,
K.A.R.;
K.A.R.;
K.C.E. andacid
L.A.; extraction
validation,
methodContributions:
that consistently
recovers 100%
ofmethodology,
cyanuric acid
in software,
urine. Cyanuric
L.M.S., K.C.E., L.A., and K.A.R.; formal analysis, L.M.S.; investigation, K.A.R. and L.M.S.; resources, L.M.S., K.C.E.,
efficiencies are dependent on the solid phase extraction technique and analytical instrument. A more
L.A., and K.A.R.; data curation, L.M.S.; writing—original draft preparation, L.M.S.; writing—review and editing,
detailed
and discussion
of method performance
limitationsK.A.R.;
between
this acquisition,
study and
L.M.S.
andcomparison
K.A.R.; visualization,
K.A.R.; supervision,
K.A.R.; project and
administration,
funding
K.A.R.
others is published elsewhere [14].
Funding: Funding for this research was provided by the National Swimming Pool Foundation and Research
Foundation
for Health and Environmental Effects.
5. Conclusions
Acknowledgments:
Questionnaire
was madequestionnaire
possible with assistance
fromto
Kristen
Pogreba-Brown
This study highlights
the development
need for improved
techniques
assess
recreational
from the University of Arizona’s Foodborne Illness Outbreak Investigation Team. Training in video surveillance
water ingestion.
Ourbyfindings
demonstrate
swimmers
perceive College
higher ofingestion
exposures
than
in
methods
was provided
Paloma Beamer
at the University
of Arizona’s
Public Health.
Meredith
Lisse
and
Leena
Patel explains
in the Melwhy
and Enid
Zuckerman
College of
Public Health
assisted with
sampleestimates
collection
reality,
which
self-reported
ingestion
estimates
are different
thanwater
measured
and
site studies.
and participant
and urine processing.
in the
Arizona
Laboratory
for
fromanalysis,
previous
Since recruitment,
there is uncertainty
about the Analyses
volume of
water
in one
mouthful,
Emerging Contaminants were supported by NSF CBET 0722579. The researchers would also like to thank all those
researchers
who
this question
in the future
shouldtheir
include
images
of a in
one-cup/250
who
assisted in
the use
questionnaire
reviewtechnique
and all volunteers
who donated
time to
participate
this study.
mL measuring glass with one to eight mouthfuls of liquid to help swimmers visualize the ingestion
volume categories. The questionnaire category “no water to a few drops” should be changed to “less
than one mouthful” to be consistent with other response options on the questionnaire. The altered
questionnaire should be validated to ensure ingestion volumes are accurately reported.
Int. J. Environ. Res. Public Health 2018, 15, 2435
6 of 6
Conflicts of Interest: The authors declare no conflicts of interest.
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Workload balancing in identical parallel machine scheduling using a mathematical programming method
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The International journal of computational intelligence systems/International journal of computational intelligence systems
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Abstract This paper addresses the workload balancing problem in identical parallel machines context. The problem
consists of assigning n different jobs to m identical parallel machines in order to minimize the workload
imbalance among the different machines. This problem is formulated as a linear mixed integer program
to minimize the difference between the greatest and smallest workload assigned to each machine. Based
on some numerical examples reported in the literature, we establish that the classical formulation which
consists of minimizing the greatest machine completion time does not provide the optimal workload
repartition. That is why we consider a new mathematical formulation based on the minimization of the
difference between the workload of the bottleneck machine and the workload of the fastest machine. The
proposed programming method is also used to provide optimal solutions in reasonable computational
times for different test problems presented in the literature by Raghavendra and Murthy 10 to test their
genetic algorithm. Keywords: Parallel machine, workload balancing, scheduling, mathematical programming. International Journal of Computational Intelligence Systems, Vol. 7, Supplement 1 (2014), 58-67 International Journal of Computational Intelligence Systems, Vol. 7, Supplement 1 (2014), 58-67 Yassine Ouazene, Farouk Yalaoui, Hicham Chehade and Alice Yalaoui
Institut Charles Delaunay - Laboratoire d’Optimisation des Syst`emes Industriels
UMR-STMR-CNRS 6279
Universit´e de Technologie de Troyes
12 rue Marie Curie,CS 42060
10004, Troyes, France
E-mail: {yassine.ouazene, farouk.yalaoui, hicham.chehade, alice.yalaoui}@utt.fr Received 15 December 2012
Accepted 20 July 2013 Co-published by Atlantis Press and Taylor & Francis
Copyright: the authors 1.
Introduction workload among different machines as equally as
possible. Workload balancing is important for both
service and manufacturing industries. In service
industry, human resources should have a balanced
workload in order to be equitable and provide a qual-
ity service. The aim of a manager is to assign jobs to
each worker in such a way that their workloads are
as similar as possible. In manufacturing industry,
balancing the workload among the machines is im-
portant to reduce the idle times and work-in-process
8. It helps also to remove bottlenecks in manufac-
turing systems 13. Parallel manufacturing structure is one of the so-
lutions of increasing the processing capacity of a
manufacturing system. Generally, in parallel ma-
chine scheduling two decisions are taken at once. The first one is to determine the assignment of jobs
to the machines and the second one is sequencing
of the jobs assigned to each machine in order to op-
timize different objective functions. One of these
objective functions is the workload imbalance mini-
mization which can be defined as assigning available Co-published by Atlantis Press and Taylor & Francis
Copyright: the authors 58 Y. Ouazene, F. Yalaoui, H. Chehade and A. Yalaoui In the literature, the workload balancing prob-
lem has been associated with different scheduling
criteria in different ways, even by considering the
workload imbalance as a constraint or as an objec-
tive. As examples, Ouazene et al. 8 addressed the
identical parallel machine scheduling problem to
minimize simultaneously total tardiness and work-
load imbalance. The authors proposed a mathemati-
cal formulation and a genetic algorithm based on the
aggregation of the two objective functions. Yildirim
et al. 15 have studied the scheduling of semi-related
machines with sequence dependent setups and load
balancing constraints. The authors proposed a math-
ematical formulation of the problem and an approx-
imate resolution based on some heuristics and a
genetic algorithm. Recently, Keskinturk et al. 4
presented a non linear mathematical model for a
parallel machine problem with sequence-dependent
setups with the objective of minimizing the total
relative imbalance. The authors proposed two meta-
heuristic methods for the approached resolution of
the problem. The two metaheuristics consist of an
ant colony optimization algorithm and a genetic
algorithm. Based on various randomly generated
tests, the authors have concluded that the ant colony
algorithm outperforms both heuristics and genetic
algorithm. Rajakumar et al. 1.
Introduction 13 addressed the workload bal-
ancing problem using different priority rules such
as: random, shortest processing times and longest
processing times. The authors used the relative dif-
ference of imbalance to evaluate the performances
of these different strategies. In their next publica-
tion 14, the authors proposed a genetic algorithm
that outperforms these different strategies. Based
on these classical priority rules, Raghavendra and
Murthy 9 made an effort to reduce the imbalance
in random type of parallel machines addressing the
loading problem in flexible manufacturing system. Later, Raghavendra et al. 12 proposed a genetic algo-
rithm based approach with SPT and LPT rules for re-
ducing the imbalance between the parallel machines. The authors have concluded that their genetic algo-
rithm provides better solutions than the strategies
proposed in the first paper 9. Raghavendra et al. 11
applied this genetic algorithm in a case study for ten
different part styles with different batch quantity on
two vertical machining centers. The same genetic
based heuristics algorithm was compared with other
approximate approaches proposed in the literature. Indeed, Raghavendra et al. 10 presented a compara-
tive study among different test examples to illustrate
the efficiency of their algorithm. The authors have
shown that their algorithm outperforms the different
heuristics proposed by Heinrich 2 and the genetic
and simulated annealing algorithms proposed by
Liu and Wu 5. Caragiannis 1 presented an improved
upper bound for the greedy algorithm to minimize
the Lp norm of the machine loads for the problem of
scheduling permanent jobs on unrelated machines. As mentioned above, workload balancing problem
is present in different environments. For example
Moon et al. 7 considered this problem with several
operators in semi-automatic parallel machine shop
with two types of machines. There objective was
to assign the jobs to the machines and allocate the
machines to operators in such way to minimize the
operators workload imbalance under the constraint
of machine availability and operator times. The
authors have proposed a non linear mathematical
formulation of this problem. The remainder of this paper is organized as fol-
lows: section 2 details the mathematical formulation
of the problem considered. The different notations
and decision variables are described. Also, the clas-
sical list-scheduling algorithm usually used in work-
load balancing on parallel machines problem is ex-
plained. Section 3 presents the comparative study
based on different test examples reported in the liter-
ature. 1.
Introduction Finally, section 4 summarizes the contribution
of this paper and gives some perspectives for future
possible extensions. Co-published by Atlantis Press and Taylor & Francis
Copyright: the authors 2.1.
Notations RPI = 1
M ×∑M
m=1
Cmax−Cm
Cmax RPI = 1
M ×∑M
m=1
Cmax−Cm
Cmax
=
1
M×Cmax ×∑M
m=1 (Cmax −Cm)
= ∑M
m=1Cmax
M×Cmax −∑M
m=1Cm
M×Cmax
= 1−∑M
m=1Cm
M
×
1
Cmax =
1
M×Cmax ×∑M
m=1 (Cmax −Cm) = ∑M
m=1Cmax
M×Cmax −∑M
m=1Cm
M×Cmax Known that, ∑M
m=1Cm
M
= ∑N
j=1 pj
M
= µ, µ is a constant. ∑M
m=1Cm
M
= ∑N
j=1 pj
M
= µ, µ is a constant. So, the relative percentage of imbalances RPI can
be written as a function of Cmax as follows. M
M
So, the relative percentage of imbalances RPI can
be written as a function of Cmax as follows. RPI = 1−
β
Cmax The second criterion called normalized sum of
Square for workload deviations (NSSWD) has been
introduced by Ho et al. 3. This criterion is based on
the sum of squares principle known in measuring
variability in statistics and serves as a precise mea-
surement criterion. NSSWD is defined as follows. √
M
2 NSSWD =
√
∑M
m=1 (Cm−µ)2
µ 2.
Problem formulation The problem considered in this paper can be for-
mally described as follows: a set of N independent
jobs {J1,J2,...,JN} are scheduled on M identical par-
allel machines. We assume that each job Jj has a Co-published by Atlantis Press and Taylor & Francis
Copyright: the authors 59 Workload balancing in identical parallel machine scheduling using a mathematical programming method balancing problem optimization. So, minimizing
the relative percentage of imbalances criterion is the
same thing as minimizing the maximum of comple-
tion times. deterministic processing time pj. The jobs may be
assigned to any one of the machines. A machine can
process only one job at once and no preemption is
allowed. All jobs are available at time zero. deterministic processing time pj. The jobs may be
assigned to any one of the machines. A machine can
process only one job at once and no preemption is
allowed. All jobs are available at time zero. Based on these assumptions and the following nota-
tions, the problem is formulated as a mixed integer
mathematical model in order to minimize the work-
load imbalance amon the machines. Based on these assumptions and the following nota-
tions, the problem is formulated as a mixed integer
mathematical model in order to minimize the work-
load imbalance amon the machines. Remark 1. In the case of identical parallel machine,
the relative percentage of imbalances RPI depends
solely of the maximum of completion times Cmax. Co-published by Atlantis Press and Taylor & Francis
Copyright: the authors Considering the definition of RPI criterion, we have: Considering the definition of RPI criterion, we have: 2.1. Notations
N
Total number of jobs
M
Total number of machines
i, j ∈{0,1,...,N}
Job index where job 0 is
a fictitious one which is
always sequenced at the
first position on a machine
m
Machine index
p j
Processing time of job j
Cm
Completion time of machine m
Sm
Set of jobs scheduled on
the machine m
Cm = ∑j∈Sm p j
Completion time of machine
m 2.2.
Analysis of the existing formulations The first performance measure or criterion used to
deal with workload imbalance minimization prob-
lem is the total relative percentage of workloads
imbalances (RPI) introduced by 13,14. This perfor-
mance measure is also called percentage of devia-
tion from upper bound and defined as: µ = ∑M
m=1Cm
M
= ∑N
j=1 pj
M
h
hi M
M
For each machine m the square error is given by
(Cm −µ)2. It is easy to establish that the sum of
square for workloads deviations ∑M
m=1 (Cm −µ)2
depends directly on the sum of square completion
times. RPI = 1
M ×∑M
m=1
Cmax−Cm
Cmax
. ∑M
m=1 (Cm −µ)2 = ∑M
m=1 (C2
m −2× µ ×Cm + µ2)
= ∑M
m=1C2
m −2×∑M
m=1Cm +∑M
m=1 µ2 M
m=1 (Cm −µ)2 = ∑M
m=1 (C2
m −2× µ ×Cm + µ2) In the case of identical parallel machines, this
criterion depends solely of the maximum comple-
tion time criterion which is not optimal in workload Co-published by Atlantis Press and Taylor & Francis
Copyright: the authors g
60 60 Y. Ouazene, F. Yalaoui, H. Chehade and A. 2.2.
Analysis of the existing formulations Yalaoui xijm =
⎧
⎨
⎩
1,
if job j immediately follows job i
in a sequence on machine m
0,
otherwise
y jm =
⎧
⎨
⎩
1,
if job j is assigned to machine m
0,
otherwise
Cm = ∑j∈Sm pj = ∑N
j=1 (pj ×y jm),m = 1...M
Cmax = max1⩽m⩽M{Cm}
Cmin = min1⩽m⩽M{Cm} xijm =
⎧
⎨
⎩
1,
if job j immediately follows job i
in a sequence on machine m
0,
otherwise
y jm =
⎧
⎨
⎩
1,
if job j is assigned to machine m
0,
otherwise xijm =
⎧
⎨
⎩
1,
if job j immediately follows job i
in a sequence on machine m
0,
otherwise
y jm =
⎧
⎨
⎩
1,
if job j is assigned to machine m
0,
otherwise By considering that ∑M
m=1Cm = M × µ, we obtain:
∑M
m=1 (Cm −µ)2 = ∑M
m=1C2
m −2×M × µ2 +M × µ2 So, the sum of square for workloads deviations can
be written as follows: ∑M
m=1 (Cm −µ)2 = ∑M
m=1C2
m −M × µ2 Since M × µ2 is a constant, then minimizing the
sum of square for workloads deviations is the same
as minimizing the sum of squares of completion
times C2
m. Cm = ∑j∈Sm pj = ∑N
j=1 (pj ×y jm),m = 1...M
Cmax = max1⩽m⩽M{Cm}
Cmin = min1⩽m⩽M{Cm} Cm = ∑j∈Sm pj = ∑N
j=1 (pj ×y jm),m = 1...M Cmax = max1⩽m⩽M{Cm} 2.3.2. Mathematical formulation 2.3.2. Mathematical formulation 2.3.2. Mathematical formulation (1) minZ = Cmax −Cmin
(1) minZ = Cmax −Cmin N
∑
j=1
x0 jm ⩽1,m = 1...M
(2)
N
∑
i=0,i∕= j
M
∑
m=1
xijm = 1, j = 1...N
(3) N
∑
j=1
x0 jm ⩽1,m = 1...M
(2) (2) N
∑
i=0,i∕= j
M
∑
m=1
xijm = 1, j = 1...N
(3) (3) N
∑
j=1, j∕=i
xijm ⩽y jm,i = 1...N,m = 1...M
(4) N
∑
i=0,i∕= j
x j
xijm = y jm, j = 1...N,m = 1...M
(5) M
∑
m=1
y jm = 1, j = 1...N
(6)
Cmax ⩾
N
∑
j=1
(pj ×y jm),m = 1...M
(7)
Cmin ⩽
N
∑
j=1
(pj ×y jm),m = 1...M
(8) 2.3.
A new mixed integer linear programming
model Cmin = min1⩽m⩽M{Cm} The mathematical formulations based on the mini-
mization of the maximum completion time Cmax do
not provide the optimal utilization of machines as
mentioned in Rajakumar et al. 14. In fact, minimiz-
ing the maximum workload is not sufficient to obtain
the optimal repartition of the workload. This will
be illustrated by some counters examples presented
in computational experiments section. Therefore,
the optimal formulation should considers simultane-
ously the maximum and the minimum workloads. So, workload balancing problem should be defined
as the minimization of the difference between the
maximum and the minimum workloads. In other
words, it is defined as minimizing the difference be-
tween the workload of the bottleneck machine and
the workload of the fastest machine. Based on this
definition, we propose the following mathematical
model. Co-published by Atlantis Press and Taylor & Francis
Copyright: the authors Co-published by Atlantis Press and Taylor & Francis
Copyright: the authors
61 3.
Computational experiments This section presents some numerical experiments
based on different well known test configurations. These instances have been used by Liu and Wu 5 to
compare between genetic and simulated annealing
algorithms. Recently, Raghavendra and Murthy 10
have used the same instances to compare the per-
formance of their genetic algorithm with different
heuristic methods proposed by Heinrich 2 for min-
imizing the imbalance of workload among identi-
cal parallel machines. The design of the different
numerical examples is reported in table 3. These
instances present small scale scheduling problems
with 7, 10 and 21 jobs to be processed by respec-
tively 3, 2 and 6 machines. They present also a larger
scale problem with 29, 30 and 33 jobs to scheduling
on respectively 3, 10 and 5 machines. 2.3.1.
Decision variables (6) The mathematical model detailed below includes
both assignment and precedence variables. Gener-
ally assignment variables are sufficient for comput-
ing machine workloads. The precedence variables
are used here in order to determine the schedule or
the sequence of the jobs on each machine. (8) Co-published by Atlantis Press and Taylor & Francis
Copyright: the authors
61 Co-published by Atlantis Press and Taylor & Franc
Copyright: the authors g
61 61 Workload balancing in identical parallel machine scheduling using a mathematical programming method Algorithm 1: List-scheduling algorithm
Input data
M
number of machines
N
number of jobs
(p1,..., pN)
list of jobs processing times
for m = 1 to M
{
Wm = 0
workload of machine m
Sm = Ø
set of jobs assigned to machine m
}
for i = 1 to N
{
m = argminkWk
machine with the smallest workload
Sm = Sm ∪{i}
assign job i to machine m
Wm = Wm + pi
update workload of machine m
}
Return S = [S1,...,SM]
l Algorithm 1: List-scheduling algorithm Algorithm 1: List-scheduling algorithm xijm,y jm ∈{0,1};Cmax,Cmin ⩾0
(9) (9) In the above model, Equation 1 is the objec-
tive function that minimizes the workload imbalance
among the machines. Equation 2 assures that for
each machine, only one real job follows the ficti-
tious job 0. Equation 3 states that a job must be pro-
cessed at one and only one position on a machine
and it will be immediately preceded by exactly one
job.Equation 4 states that if job i is processed on ma-
chine m, it will be immediately followed by at most
one other job on this machine. Equation 5 states that
job j should immediately follow another job on ma-
chine m if it is placed on this machine. Equation 6
guaranties that each job is assigned to exactly one
machine. Equations 7 and 8 represent workload-
balancing constraints. The first one ensures that the
maximum workload is greater than or equivalent to
individual workloads and the second one ensures
that the minimum workload is smaller or equivalent
to individual workloads. Equation 9 states the prop-
erties of the decision variables. It states also that the
completion time of the fictitious job is equal to zero. Co-published by Atlantis Press and Taylor & Francis
Copyright: the authors 4.
Conclusion We present for each instance, the numerical results
obtained by the different approximate algorithms
given in the literature and the exact solutions ob-
tained by the mathematical programming model (see
tables 2, 3, 4, 5, 6 and 7). The comparative study
presented by Raghavendra and Murthy 10 concludes
that their genetic algorithm outperforms the different
heuristics proposed by Heinrich 2 (STA, SYSR and
IE) and the genetic and simulated annealing algo-
rithms proposed by Liu and Wu 5. So in this paper,
we are not especially interested by the comparison
with these approximate methods because we provide
the optimal solutions which can be used to evalu-
ate the real performances of the best known approx-
imate resolution approach (genetic algorithm 10). In this paper, a mixed linear integer mathematical
program for workload balancing on identical par-
allel machine problem is presented. The workload
balancing problem is defined as the minimization of
the difference between the workload of the bottle-
neck machine and the workload of the fastest ma-
chine. Based on this mathematical programming
model, we provide optimal solutions for different
test instances presented in the literature in reason-
able computational times. These solutions allow the
exact evaluation of the different approximate meth-
ods presented in the literature. As a second contri-
bution, we have illustrated based on some numerical
examples that the classical formulation of workload
balancing problem using the minimization of the
maximum machine completion time does not pro-
vide the optimal workload repartition. We are also interested on comparison between the
mixed integer linear program based on the mini-
mization of the maximum of workload (MILP 1) and
the proposed mathematical program (MILP 2). p
p
p
g
We note that for the small scale instances with 7 and
10 jobs, both approximate methods and maximum
completion time minimization model obtain the op-
timal solutions. Which is predictable. In the case
of the third small instance with 21 jobs, the best ap-
proached method fails to obtain the optimal solution
with a maximum completion time of 58 time units
and a maximum workload imbalance of 2 time units. The two large instances with respectively 29 and 30
jobs (see tables 6 and 7), illustrate that the mathe-
matical programming approach based on the mini-
mization of the maximum completion time is does
not provide the optimal repartition of the workload
among the different machines. 2.4.
Scheduling algorithms Or, the numerical results reported in
tables 6 and 7 illustrate that this formulation does
not provide the optimal workload repartition. of these different approached resolutions. We compare also this mathematical formulation
with the classical formulation proposed by Rajaku-
mar et al. 14. In their model, the authors have formu-
lated the workload balancing problem among identi-
cal parallel machines as a binary mixed integer pro-
gram with the objective of minimizing the maximum
of workloads. Or, the numerical results reported in
tables 6 and 7 illustrate that this formulation does
not provide the optimal workload repartition. Table 8 presents the computational times of the two
mathematical programming methods (MILP 1 and
MILP 2). We not that the second model is more
consuming in term of computational times but it pro-
vides the optimal solutions. However, the computa-
tional times still reasonable (less than one minute). 4.
Conclusion In fact for both ex-
amples, the maximum workload imbalance obtained
by this model is equal to 2 time units while the sec-
ond formulation obtains an optimal solution with A direct extension of this research is to consider this
new formulation of the workload imbalance min-
imization problem in the definition of the fitness
functions of the approximate resolution algorithms
proposed in the literature. Another possible exten-
sion of this contribution would be the combination
of workload imbalance criterion as defined here with
other scheduling criteria for multiobjective formula-
tions and resolutions. 2.4.
Scheduling algorithms have used the same instances to compare the per-
formance of their genetic algorithm with different
heuristic methods proposed by Heinrich 2 for min-
imizing the imbalance of workload among identi-
cal parallel machines. The design of the different
numerical examples is reported in table 3. These
instances present small scale scheduling problems
with 7, 10 and 21 jobs to be processed by respec-
tively 3, 2 and 6 machines. They present also a larger
scale problem with 29, 30 and 33 jobs to scheduling
on respectively 3, 10 and 5 machines. In the workload balancing problem, the different
scheduling procedures are based on list-scheduling
algorithm described by algorithm 1. These algo-
rithms are known to be the best for solving the
parallel machine scheduling problems to optimize
utilization criteria 14, 6. The main idea consists of selecting iteratively the
machine with the smallest workload the the as-
signment of a new job from the list of unfinished
jobs. Different strategies are adopted for the selec-
tion of jobs list such as: shortest processing times,
longest processing times and random processing
times. Based on a priority queue, the implementa-
tion complexity of a list-scheduling algorithm is of
the order of O(nlog(m)). The aim of this computational study is to provide
the optimal solutions of these well known instances
previously adopted in the literature. For each in-
stance, the mathematical programming model has
been solved using ILOG CPLEX 11.0 software. These optimal solutions allow the exact evaluation Co-published by Atlantis Press and Taylor & Francis
Copyright: the authors 62 Y. Ouazene, F. Yalaoui, H. Chehade and A. Yalaoui one time unit of workload imbalance. one time unit of workload imbalance. Table 8 presents the computational times of the two
mathematical programming methods (MILP 1 and
MILP 2). We not that the second model is more
consuming in term of computational times but it pro-
vides the optimal solutions. However, the computa-
tional times still reasonable (less than one minute). of these different approached resolutions. We compare also this mathematical formulation
with the classical formulation proposed by Rajaku-
mar et al. 14. In their model, the authors have formu-
lated the workload balancing problem among identi-
cal parallel machines as a binary mixed integer pro-
gram with the objective of minimizing the maximum
of workloads. Co-published by Atlantis Press and Taylor & Francis
Copyright: the authors Acknowledgments The authors would like to thank the editors and
anonymous reviewers for their valuable remarks,
comments and suggestions that helped to improve
this paper. Acknowledgments Co-published by Atlantis Press and Taylor & Francis
Copyright: the authors 63 Workload balancing in identical parallel machine scheduling using a mathematical programming method Table 1: Design of experiment instances
(N = 7,M = 3)
(N = 10,M = 2)
(N = 21,M = 6)
(N = 29,M = 3)
(N = 30,M = 10)
(N = 33,M = 5)
job i
pi
job i
pi
job i
pi
job i
pi
job i
pi
job i
pi
1
6
1
3
1
23
1
14
1
3
1
23
2
6
2
2
2
29
2
16
2
2
2
29
3
4
3
6
3
21
3
26
3
6
3
7
4
4
4
4
4
11
4
3
4
4
4
2
5
4
5
5
5
20
5
25
5
5
5
25
6
3
6
7
6
28
6
3
6
7
6
10
7
3
7
8
7
10
7
11
7
9
7
14
-
-
8
6
8
18
8
25
8
13
8
3
-
-
9
2
9
1
9
2
9
4
9
26
-
-
10
6
10
6
10
24
10
12
10
23
-
-
-
-
11
28
11
11
11
10
11
10
-
-
-
-
12
19
12
25
12
8
12
7
-
-
-
-
13
5
13
21
13
22
13
30
-
-
-
-
14
6
14
14
14
11
14
25
-
-
-
-
15
28
15
25
15
8
15
1
-
-
-
-
16
27
16
1
16
26
16
8
-
-
-
-
17
8
17
26
17
14
17
18
-
-
-
-
18
7
18
18
18
6
18
13
-
-
-
-
19
29
19
13
19
17
19
15
-
-
-
-
20
10
20
2
20
27
20
28
-
-
-
-
21
7
21
14
21
11
21
30
-
-
-
-
-
-
22
20
22
17
22
14
-
-
-
-
-
-
23
5
23
26
23
28
-
-
-
-
-
-
24
12
24
16
24
14
-
-
-
-
-
-
25
21
25
7
25
20
-
-
-
-
-
-
26
23
26
23
26
18
-
-
-
-
-
-
27
21
27
15
27
27
-
-
-
-
-
-
28
17
28
18
28
19
-
-
-
-
-
-
29
19
29
15
29
11
-
-
-
-
-
-
-
-
30
13
30
25
-
-
-
-
-
-
-
-
-
-
31
5
-
-
-
-
-
-
-
-
-
-
32
22
-
-
-
-
-
-
-
-
-
-
33
30 Table 1: Design of experiment instances Table 2: solution for the second instance (N = 10,M = 2)
Machine load
Heinrich Kuhn 1995
GA
MILP 1
MILP 2
STA
SYSR
IE
Machine 1
11
10
10
10
10
10
Machine 2
10
10
10
10
10
10
Workload Imbalance
2
0
0
0
0
0 64 Y. Acknowledgments Ouazene, F. Yalaoui, H. Chehade and A. References 1. I. Caragiannis. Better bounds for online load balanc-
ing on unrelated machines. In Proceedings of the nine-
teenth annual ACM-SIAM symposium on Discrete al-
gorithms, SODA ’08, pages 972–981, Philadelphia,
PA, USA, 2008. Society for Industrial and Applied
Mathematics. 9. B. V. Raghavendra and A. N. N. Murthy. Some solu-
tion approaches to reduce the imbalance of workload
in parallel machines while planning in flexible manu-
facturing system. International Journal of Engineer-
ing Science and Technology, 2(5), 2010. g
gy
10. B. V. Raghavendra and A. N. N. Murthy. Work-
load balancing in identical parallel machine schedul-
ing while planning in flexible manufacturing system
using genetic algorithm. ARPN Journal of Engineer-
ing and Applied Sciences, 6(1), 2011. 2. K. Heinrich. A heuristic algorithm for the loading
problem in flexible manufacturing systems. Inter-
national Journal of Flexible Manufacturing Systems,
7:229–254, 1995. 11. B. V. Raghavendra, A. N. N. Murthy, and M. Jayaram. Job sequence to minimize the workload imbalance on
parallel machines through genetic algorithm. Inter-
national Journal of Engineering Science and Technol-
ogy, 3(1), 2011. 3. J.C. Ho, T.L. Tseng, A.J. Ruiz-Torres, and F. J. L´opez. Minimizing the normalized sum of square for work-
load deviations on m parallel processors. Computers
and Industriel Engineering., 56(1):186–192, 2009. 4. T. Keskinturk, M.B. Yildirim, and M. Barut. An
ant colony optimization algorithm for load balancing
in parallel machines with sequence-dependent setup
times. Computers & Operations Research, 39(6):1225
– 1235, 2012. 12. B. V. Raghavendra, A. N. N. Murthy, and N. B. Rao. Some solution approaches to reduce the imbalance
of workload in parallel machines while planning in
flexible manufacturing system through genetic algo-
rithm. International Journal of Engineering Science
and Technology, 2(1), 2010. 5. M. Liu and C. Wu. A genetic algorithm for minimiz-
ing the makespan in the case of scheduling identical
parallel machines. Artificial Intelligence in Engineer-
ing, 13(4):399 – 403, 1999. 13. S. Rajakumar, V.P. Arunachalam, and V. Selladurai. Workflow balancing strategies in parallel machine
scheduling. International Jornal of Advanced Man-
ufacturing, 23:366–374, 2004. g
6. E. Mokotoff. Parallel machine scheduling problems:
A survey. Asia - Pacific Journal of Operational Re-
search, 18:193 – 242, 2001. 14. S. Rajakumar, V.P. Arunachalam, and V. Selladurai. Workflow balancing in parallel machines through ge-
netic algorithm. International Journal of Advanced
Manufacturing Technology, 33:1212–1221, 2007. 7. D. H. Moon, D. K. Kim, and J. Y. Jung. Acknowledgments Ouazene, F. Yalaoui, H. Chehade and A. Yalaoui ings, pages 497–502. Springer Berlin Heidelberg,
2011. Acknowledgments Yalaoui Table 3: solution for instance (N = 7,M = 3)
Machine load
Heinrich Kuhn 1995
GA
MILP 1
MILP 2 2
STA
SYSR
IE
Machine 1
11
10
10
10
10
10
Machine 2
10
10
10
10
10
10
Machine 3
9
10
10
10
10
10
Workload Imbalance
2
0
0
0
0
0
Table 4: solution for instance (N = 33,M = 5)
Method
Heinrich Kuhn 1995
GA
MILP 1
MILP 2
Machine load
STA
SYSR
IE
Machine 1
99
115
116
116
116
116
Machine 2
151
118
116
116
116
116
Machine 3
98
110
116
116
116
116
Machine 4
89
119
116
116
116
116
Machine 5
143
118
116
116
116
116
Workload Imbalance
62
9
0
0
0
0
Table 5: solution for instance (N = 21,M = 6)
Method
Heinrich Kuhn 1995
GA
MILP 1
MILP 2
Machine load
STA
SYSR
IE
Machine 1
74
61
57
58
56
57
Machine 2
42
49
58
58
57
57
Machine 3
60
61
58
57
57
57
Machine 4
33
59
58
56
57
56
Machine 5
48
49
53
56
57
57
Machine 6
84
62
57
56
57
57
Workload Imbalance
51
13
5
2
1
1
Table 6: solution for instance (N = 29,M = 3)
Method
Heinrich Kuhn 1995
GA
MILP 1
MILP 2
Machine load
STA
SYSR
IE
Machine 1
161
150
153
152
153
152
Machine 2
148
153
153
153
153
152
Machine 3
148
154
151
152
151
153
Workload Imbalance
13
4
2
1
2
1
Co-published by Atlantis Press and Taylor & Francis Table 3: solution for instance (N = 7,M = 3)
Machine load
Heinrich Kuhn 1995
GA
MILP 1
MILP 2 2
STA
SYSR
IE
Machine 1
11
10
10
10
10
10
Machine 2
10
10
10
10
10
10
Machine 3
9
10
10
10
10
10
Workload Imbalance
2
0
0
0
0
0 Table 5: solution for instance (N = 21,M = 6)
Method
Heinrich Kuhn 1995
GA
MILP 1
MILP 2
Machine load
STA
SYSR
IE
Machine 1
74
61
57
58
56
57
Machine 2
42
49
58
58
57
57
Machine 3
60
61
58
57
57
57
Machine 4
33
59
58
56
57
56
Machine 5
48
49
53
56
57
57
Machine 6
84
62
57
56
57
57
Workload Imbalance
51
13
5
2
1
1
Table 6: solution for instance (N = 29,M = 3)
Method
Heinrich Kuhn 1995
GA
MILP 1
MILP 2
Machine load
STA
SYSR
IE
Machine 1
161
150
153
152
153
152
Machine 2
148
153
153
153
153
152 Table 5: solution for instance (N = 21,M = 6)
Method
Heinrich Kuhn 1995
GA
MILP 1
MILP 2
Machine load
STA
SYSR
IE
Machine 1
74
61
57
58
56
57
Machine 2
42
49
58
58
57
57
Machine 3
60
61
58
57
57
57
Machine 4
33
59
58
56
57
56
Machine 5
48
49
53
56
57
57
Machine 6
84
62
57
56
57
57
Workload Imbalance
51
13
5
2
1
1 Table 6: solution for instance (N = 29,M = 3)
Method
Heinrich Kuhn 1995
GA
MILP 1
MILP 2
Machine load
STA
SYSR
IE
Machine 1
161
150
153
152
153
152
Machine 2
148
153
153
153
153
152
Machine 3
148
154
151
152
151
153
Workload Imbalance
13
4
2
1
2
1 Co-published by Atlantis Press and Taylor & Fran
Copyright: the authors
65 Atlantis Press and Tay
opyright: the authors
65 65 Workload balancing in identical parallel machine scheduling using a mathematical programming method Table 7: solution for instance (N = 30,M = 10
Method
Liu and Wu 1999
GA
MILP 1
MILP 2
Machine load
Machine 1
30
37
38
37
Machine 2
40
38
37
37
Machine 3
37
39
37
37
Machine 4
40
38
38
38
Machine 5
33
39
37
38
Machine 6
39
36
38
38
Machine 7
41
37
36
37
Machine 8
34
36
38
38
Machine 9
41
39
38
38
Machine 10
40
36
38
37
Workload Imbalance
11
3
2
1 Table 8: Computational times of exact resolution
-
MILP 1 CPU time (seconds)
MILP 2 CPU time (seconds)
Instance (N = 7,M = 3)
1.23
1.31
Instance (N = 10,M = 2)
0.85
1.43
Instance (N = 21,M = 6)
1.58
3.36
Instance (N = 29,M = 3)
1.73
1.17
Instance (N = 30,M = 10)
20.43
45.05
Instance (N = 33,M = 5)
20.21
26.25 Table 8: Computational times of exact resolution Co-published by Atlantis Press and Taylor & Francis
Copyright: the authors
66 66 Y. References An operator
load-balancing problem in a semi-automatic parallel
machine shop. Computers and Industrial Engineer-
ing, 46(2):355–362, 2004. f
g
gy
15. M.B. Yildirim, E. Duman, K. Krishna, and K. Sen-
niappan. Parallel machine scheduling with load bal-
ancing and sequence dependent setups. International
Journal of Operations Research, 4(1):42–49, 2007. 8. Y. Ouazene, F. Hnaien, F. Yalaoui, and L. Amodeo. The Joint Load Balancing and Parallel Machine
Scheduling Problem. Operations Research Proceed- Co-published by Atlantis Press and Taylor & Francis
Copyright: the authors
67 Co-published by Atlantis Press and Taylor & Francis
Copyright: the authors
67 67
|
https://openalex.org/W4283786673
|
https://nottingham-repository.worktribe.com/file/12624524/1/S42234-022-00099-7
|
English
| null |
Electric Field Responsive Nanotransducers for Glioblastoma
|
bioRxiv (Cold Spring Harbor Laboratory)
| 2,022
|
cc-by
| 7,708
|
Abstract Background: Electric field therapies such as Tumor Treating Fields (TTFields) have emerged as a bioelectronic treat-
ment for isocitrate dehydrogenase wild-type and IDH mutant grade 4 astrocytoma Glioblastoma (GBM). TTFields
rely on alternating current (AC) electric fields (EF) leading to the disruption of dipole alignment and induced dielec-
trophoresis (DEP) during cytokinesis. Although TTFields have a favourable side effect profile, particularly compared
to cytotoxic chemotherapy, survival benefits remain limited (~ 4.9 months) after an extensive treatment regime (20
hours/day for 18 months). The cost of the technology also limits its clinical adoption worldwide. Therefore, the discov-
ery of new technology that can enhance both the therapeutic efficiency and efficacy of these TTFields will be of great
benefit to cancer treatment and decrease healthcare costs worldwide. Methods: In this work, we report the role of electrically conductive gold (GNPs), dielectric silica oxide (SiO2), and
semiconductor zinc oxide (ZnO) nanoparticles (NPs) as transducers for enhancing EF mediated anticancer effects
on patient derived GBM cells. Physicochemical properties of these NPs were analyzed using spectroscopic, electron
microscopy, and light-scattering techniques. Results: In vitro TTFields studies indicated an enhanced reduction in the metabolic activity of patient-derived Glioma
INvasive marginal (GIN 28) and Glioma contrast enhanced core (GCE 28) GBM As per our journal style, article titles
should not include capitalisedletters unless these are proper nouns/acronyms. We have therefore usedthe article title
“Electric field responsive nanotransducers forglioblastoma” as opposed to “Electric Field Responsive Nanotransduc-
ersfor Glioblastoma” as given in the submission system. Please check ifthis is correct.cells in groups treated with NPs
vs. control groups, irrespective of NPs dielectric properties. Our results indicate the inorganic NPs used in this work
enhance the intracellular EF effects that could be due to the virtue of bipolar dielectrophoretic and electrophoretic
effects. Conclusions: This work presents preliminary evidence which could help to improve future EF applications for bio-
electronic medicine. Furthermore, the merits of spherical morphology, excellent colloidal stability, and low toxicity,
make these NPs ideal for future studies for elucidating the detailed mechanism and efficacy upon their delivery in
GBM preclinical models. Keywords: Tumor Treating Fields, Inorganic nanoparticles, Electric fields, Glioblastoma Bioelectronic Medicine Bioelectronic Medicine Bioelectronic Medicine Jain et al. Bioelectronic Medicine (2022) 8:17
https://doi.org/10.1186/s42234-022-00099-7 Open Access © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. Background While the
full extent of how TTFields work is currently unclear,
two proposed mechanisms explain the mode of action:
dipole alignment and DEP. In the first instance, spindle
formation during mitosis is affected. Microtubules are
influenced by the dipole moment of the building blocks,
therefore cell division is limited (Kirson et al., 2004). In
the second instance, an inhomogeneous distribution of
the EF within dividing cells causes molecules to become
polarised. These polar molecules then move to regions
with higher EF intensities, notably the cleavage furrow in
mitotic cells, and thereby interfere with cytokinesis (Kir-
son et al., 2007).f Despite TTFields affecting rapidly dividing cancer cells,
there appears to be no effect on healthy cells with rela-
tively slower cell division. Even cells that exhibit rapid cell
division such as those found in bone marrow or intestine
are not affected as they are protected by high impedance
of the bone, and slower replication times compared to
cancerous cells respectively (Blatt et al., 2021). Tight junctions between epithelial cells at the blood-
brain barrier (BBB) are known to inhibit the influx
and efflux of many molecules to the brain (Brightman
& Reese, 1969; Reese & Karnovsky, 1967). This barrier
can be overcome by taking advantage of the leaky vas-
culature surrounding brain tumours which leads to the
accumulation of NPs in the tumour, via the Enhanced
Permeation and Retention (EPR) effect. NPs are fre-
quently utilised to take advantage of this EPR effect,
with studies showing that NPs of a size range of 20-100
nm are ideal to allow for maximum accumulation in
the cells, tumour and longer clearance times (Perrault
et al., 2009). Gold NPs (GNPs) are of particular interest
for biomedical and bioelectronics applications due to During last decade, there has been much experi-
ment work into the mechanism of action of TTFields,
but there has been limited efforts to enhance TTFields
using NPs. One example is biocompatible barium titan-
ate NPs (BTNPs) with a high dielectric constant, which
were investigated as breast cancer sensitisers in cells that
were resistant to TTFields (Yoon et al., 2020). This study
found that BTNPs accumulate within the cytoplasm
when exposed to TTFields where they are then polar-
ised by the inhomogeneous EF as discussed earlier, caus-
ing the BTNPs to migrate to the cleavage furrow and the
cells to undergo apoptosis. Background This significant improvement to OS is not
accompanied by any major side effects, with the only
reported effect being contact dermatitis at the site of the
electrodes. their biocompatibility and tuneable properties (Shukla
et al., 2005; Perrault & Chan, 2009; Sanjuan-Alberte
et al., 2019). In recent years there have been numerous
clinical trials utilising GNPs to treat a range of can-
cers, including glioblastoma (Libutti et al., 2010; Gad
et al., 2012; Kumthekar et al., 2021). ZnO NPs (semi-
conductors) and SiO2 NPs (insulators), have also been
well researched as a potential treatment for cancers,
with the former showing the preferential killing of can-
cer cells over normal cells, while the latter has shown
advantages of being highly tuneable, allowing for tar-
geted drug delivery (Hanley et al., 2008; Wang et al.,
2009; Murugan et al., 2017). g
Electric fields have been used as external stimuli for the
delivery of drugs from NPs for cancer and tissue regener-
ation (Kolosnjaj-Tabi et al., 2019). However, the behavior
of nanoparticles under EFs in cellular environment needs
further studies. Apart from the classic endocytosis medi-
ated uptake of NPs in cancer cells, electric fields have
shown to enhance the uptake of NPs by permeabilizing
cancer cell membranes via electroporation and by modu-
lating bioelectricity through voltage-gated ion channels
(Aguilar et al., 2021; Chang et al., 2018). This has opened
new area of research to develop new tools to study intra-
cellular interaction of NPs with EFs. Apart from the well-
known electrophoretic and dielectrophoretic movement
of NPs, EFs have been shown to induce electrostatic
induction and charge separation of nanomaterials. This
electric polarization leads to generation of numerous
bipolar nanoelectrodes which acts as transducer of EFs
(Guo et al., 2021). Using modelling approaches Tiwari
et al. demonstrated that spherically capped gold nanow-
ire enhanced EFs inside the cells. They inferred that due
to the uniform and homogenous distribution of EFs over
nanomaterials (due to charge separation is over a short
distance ≈ nm) addition source and sinks are gener-
ated. This causes a local enhancement in electric field
strengths around nanomaterials in contact with cytoplas-
mic entities leading to the rupture of plasma membrane
and eventually apoptosis (Tiwari et al., 2009). TTFields are directional, mainly influencing cell behav-
iour when the electric field and axis of cell division are
parallel to one another (Kirson et al., 2007). Background Isocitrate dehydrogenase wild-type GBM is a form of
highly aggressive brain tumour accounting for 49.1% of
primary malignant brain tumours with less than 7% of
patients surviving after 5 years post-diagnosis (Ostrom
et al., 2021). The current standard of care is known as
the ‘Stupp regimen’ and consists of surgical resection *Correspondence: Akhil.Jain@nottingham.ac.uk; Frankie. Rawson@nottingham.ac.uk 1 Bioelectronics Laboratory, Division of Regenerative Medicine and Cellular
Therapies, School of Pharmacy, Biodiscovery Institute, University
of Nottingham, Nottingham NG7 2RD, UK
Full list of author information is available at the end of the article 1 Bioelectronics Laboratory, Division of Regenerative Medicine and Cellular
Therapies, School of Pharmacy, Biodiscovery Institute, University
of Nottingham, Nottingham NG7 2RD, UK
Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine (2022) 8:17 Page 2 of 9 Jain et al. Bioelectronic Medicine followed by treatment with radiotherapy and the alkylat-
ing chemotherapeutic agent, temozolomide, increasing
overall survival (OS) to a median of 14.6 months (Stupp
et al., 2005). Since this finding in 2005, there has been
little progression in the identification of new treatments
for GBM that are United States Food and Drug Adminis-
tration (FDA) approved, except for TTFields. Indeed, no
molecular targeted therapeutics predicated on genome
biology has shown efficacy in phase III trials to date. These low intensity (<4 V/cm), intermediate frequency
(100-500 kHz) and AC EFs, have been shown to further
enhance cell death when in conjunction with the Stupp
regimen, increasing OS by a median of 4.9 months (Stupp
et al., 2017). TTFields GIN 28 and GCE 28 cells were seeded on a 22 mm cell
culture treated coverslip at a density of 3.5 × 104 and
incubated for 24 hours at 37°C and 5% CO2. Next, the
media was replaced with media containing GNPs/SiO2/
ZnO NPs at a concentration of 5 μg/mL or 25 μg/mL and
incubated at 37°C for 4 hours. Next, the coverslips were
transferred to ceramic Petri dishes of the inovitro™ sys-
tem (Novocure, Haifa, Israel). Finally, the TTFields were
applied for a duration of 48 or 72 hours using the ino-
vitro system which consists of two pairs of perpendicular
transducer arrays on the outer walls of the ceramic plate
containing the Petri dishes. A sinusoidal waveform gen-
erator was attached to the transducer arrays producing
alternating EFs set at a frequency of 300 kHz and 1V/cm
intensity. TTFields of 300 KHz was chosen for this work,
this is based on previous studies that used range of fre-
quencies from 200-300 KHz for GBM cells (Branter et al.,
1982; Linder et al., 2021). TTFields were applied bi-direc-
tionally (perpendicular to each other), which switches
between the two direction every second. The temperature
was measured to be 37°C inside the dishes by thermistors
attached to the ceramic walls. Finally, the change in met-
abolic activity/ viability of GIN 28 and GCE 28 cells in
response to TTFields or NP + TTFields, was determined
using the PrestoBlueTM assay as described above. Methods
Materials All the reagents were of analytical grade and were used
as supplied without further purification unless speci-
fied. Citrate-capped spherical GNPs, ZnO, and SiO2 NPs
of size 50 nm were purchased from Sigma Aldrich, UK. PrestoBlue cell viability reagent and cell culture treated
22 mm coverslips (Nunc™ Thermanox™ Coverslips) were
purchased from ThermoFisher Scientific, UK. In vitro toxicity In vitro toxicity
HA-COR, GIN 28 and GCE 28 cells were seeded in a
96-well plate at a density of 4.5 × 103 cells per well and
incubated for 24 hours at 37°C and 5% CO2. Culture
media was replaced with medium containing GNPs/
SiO2/ZnO NPs (concentration = 0.1, 0.5, 1, 2, 5, 10, 20, 50
or 100, 200 μg/ mL) and incubated for 4 hours. Next, the
media was replaced with fresh media and cells were incu-
bated for 48 or 72 hours. Finally, metabolic activity was
determined using PrestoBlueTM. For each well, the media
was replaced with media containing 10% PrestoBlue cell
viability reagent and incubated at 37°C for 2 hours in an
incubator before transferring the coloured metabolic
product to a black-bottom 96-well plate. Finally, the
fluorescence of the plate was read using a plate reader
(TECAN Infinite 2000) with an excitation wavelength of
570 nm and an emission wavelength of 600 nm. Mean ±
S. D values are presented relative to negative controls. Background While this study is the first
example of using NPs to enhance TTFields, BTNPs are Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine Page 3 of 9 not FDA-approved, creating a barrier to translating these
findings to a clinical setting. were maintained at 37°C in an incubator with humidi-
fied atmosphere, containing 5% CO2. Cells were routinely
tested for mycoplasma where they were grown in an
antibiotic-free medium for one week before mycoplasma
testing. All cells used were mycoplasma-free. i
g
g
Here, we investigate the underlying mechanism with
the hypothesis that conductive particles would enhance
the effects of TTFields. NP-enhanced TTFields mecha-
nism of action was investigated, which is of paramount
importance for the discovery of new approaches that
can enhance these TTFields-induced anticancer effects. The NPs chosen were gold, zinc oxide (ZnO), and silica
(SiO2), which are FDA-approved conductive, semi-
conductive, and insulating NPs respectively (Rasmus-
sen et al., 2010; Zhou et al., 2018). As the two suggested
modes of TTFields action are due to dipole alignment
and DEP, the effects of using GNPs and ZnO NPs with
different electrical conductivities were chosen to address
the first mechanism, while dielectric SiO2 NPs have
potential to enhance TTFields by the second mechanism. From a clinical application perspective, the overall toxic-
ity, pharmacokinetics, and therapeutic efficacy of the NPs
must be evaluated in an accurate in vitro model that can
reflect the cancer heterogeneity observed in clinics. Fur-
thermore, as observed in clinics, the efficacy of TTFields
varies across patients which is attributed to heterogeneity
in GBM. Therefore, we utilised patient-derived GIN 28
(isolated from the invasive margin) and GCE 28 (isolated
from the contrast-enhanced core), which reflect GBM
tumour characteristics that are observed clinically. Cell culture GIN 28 cells were isolated from the 5-aminolevulinic acid
(5ALA) fluorescing infiltrative tumour margin and GCE
28 were isolated from the core central region of a GBM
patient who underwent surgery at the Queen’s Medi-
cal Centre, University of Nottingham (Nottingham, UK)
using the method described earlier (Smith et al., 2017;
Smith et al., 2020). Low-passage patient-derived GIN 28
and GCE 28 cells were cultured in DMEM (Gibco) sup-
plemented with 10% FBS, 1% Penicillin/Streptomycin,
and 1% L-Glutamine. Human derived cortical astrocytes
(HA-COR) were obtained from ScienCell (Cat. No. 1800,
Batch No. 24490) and were cultured in astrocyte medium
(AM) containing 2% FBS, 1% astrocyte growth supple-
ment, 1% Penicillin/Streptomycin from ScienCell. Cells Page 4 of 9 Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine resonance of 45 nm GNPs (Haiss et al., 2007), while the
365 nm peak in ZnO arises from the intrinsic band-gap
absorption due to electron transitions from the valence
band to the conduction band (O → Zn ) (Khokhra et al.,
2017). Furthermore, a sharp and narrow absorption peak
is a characteristic of uniform dispersion of monodisperse
GNPs and ZnO NPs. FTIR of SiO2 NPs (Fig. 1E) showed
two broad absorption peaks cantered at 795 cm-1 and
1055 cm-1 corresponding to bending vibrational modes
of the Si-O-Si groups (Brassard et al., 2011). To under-
stand the colloidal behaviour of these NPs, we carried out
ζ (Fig. 1F) and dynamic light scattering (DLS) (Fig. 1G)
measurements to determine their hd and surface charge. Previous studies have reported that ζ and hd of NPs can
influence their interaction with cells. For instance, NPs
with smaller hd have higher diffusion constant but weak
interaction with cells and vice versa with ζ (Villanueva-
Flores et al., 2020). Furthermore, surface charge and hd
plays a key role in determining the polarization and
movement of conducting and insulating NPs under the
influence of EF via electrophoresis and DEP, respectively
(Zhao & Bau, 2009). Therefore, it is important to balance
hd and ζ of the selected NPs for optimal cellular and EF
interaction. In general, NPs with ζ values of ≥ - 30 mV
or ≥ + 30 mV are considered to have optimal colloidal
stability for biological application due to the presence of
sufficient repulsive forces (Skoglund et al., 2017). Cell culture Citrate
capped GNPs and ZnO NPs showed a mean ζ value of
– 39.2 ± 2.2 mV and -36.5 ± 1 mV which is attributed
to the charge of citrate and oxide ions, respectively, sug-
gesting good physical colloidal stability. In contrast, SiO2
NPs showed a positive zeta potential value of +12.2 ± 0.9
mV due to the presence of -NH2 groups on the surface
of these NPs indicating presence of weak repulsive forces
and moderate colloidal stability. DLS analysis indicated
a monodisperse sample of GNPs with a hd of 43.8 ± 3.2
nm. A slight increase in the size of ZnO (hd = 69.4 ± 6.7
nm ) and SiO2 (hd = 50.7 ± 5.5 nm) NPs compared to
TEM measurements, further confirmed the agglomera-
tion of these NPs in colloidal solution. Collectively, the
data indicate that these inorganic NPs have optimal phys-
icochemical properties for biological applications. Physicochemical Properties of NPs Physicochemical Properties of NPs Inorganic NPs such as GNPs, SiO2, and ZnO present
several advantages for biological application such as
excellent biocompatibility, wide surface conjugation
chemistry, and colloidal stability (Sperling et al., 2008;
Hosseinpour et al., 2020; Jiang et al., 2018). Importantly
due to a large difference in the dielectric constant (SiO2
> ZnO > GNPs) (Abdelhalim et al., 2011; Ahmad et al.,
2019; Dutta & De, 2007), these NPs are best suited to gain
further insight into the role of EF in GBM therapy. Previ-
ous literature indicates that nano-bio interaction depends
on size; therefore, we chose spherical ~50 nm NPs to
demonstrate EF effects as this NP diameter is shown to
be optimum for achieving high cell uptake (Shang et al.,
2014; Zhu et al., 2013; Chithrani et al., 2006; Mittag et al.,
2021). Characterizationh Collectively, the
data indicate that these inorganic NPs have optimal phys-
icochemical properties for biological applications. The size and morphology of the NPs were analyzed
using a transmission electron microscope (JEOL 2000
FX TEM) operating at 200 kV accelerating voltage. TEM
samples were prepared by dropping 15 mL of NP solu-
tion on a carbon-coated copper grid (400 Mesh, Agar
Scientific), where the samples were allowed to sit on
the grid for at least 15 minutes before analyses. Fourier-
transform infrared spectroscopy (FTIR) was carried out
by drying silica NPs at 37°C for 48 hours and finally plac-
ing the dry sample onto a Cary 630 FTIR spectrometer
(Agilent Technologies Ltd) for the measurement of trans-
mittance spectra. UV-Vis absorption spectrum of ZnO
and GNPs was recorded on a Cary 3500 UV-Vis (Agilent
Technologies Ltd). The hydrodynamic diameter (hd) and
Zeta potential (ζ) of the NPs were monitored on a Mal-
vern Zetasizer Nano-ZS (Malvern Instruments, UK). Statistical analysis All the statistical analyses were performed using Graph-
Pad Prism v9.2.0 software (GraphPad Software, Inc). All the data are expressed as mean ± S.D., unless speci-
fied. For responses that were affected by more than one
variable, a two-way ANOVA with a Tukey multiple com-
parison post-test was used, and a p-value of ≥ 0.05 was
considered significant. Characterizationh resonance of 45 nm GNPs (Haiss et al., 2007), while the
365 nm peak in ZnO arises from the intrinsic band-gap
absorption due to electron transitions from the valence
band to the conduction band (O → Zn ) (Khokhra et al.,
2017). Furthermore, a sharp and narrow absorption peak
is a characteristic of uniform dispersion of monodisperse
GNPs and ZnO NPs. FTIR of SiO2 NPs (Fig. 1E) showed
two broad absorption peaks cantered at 795 cm-1 and
1055 cm-1 corresponding to bending vibrational modes
of the Si-O-Si groups (Brassard et al., 2011). To under-
stand the colloidal behaviour of these NPs, we carried out
ζ (Fig. 1F) and dynamic light scattering (DLS) (Fig. 1G)
measurements to determine their hd and surface charge. Previous studies have reported that ζ and hd of NPs can
influence their interaction with cells. For instance, NPs
with smaller hd have higher diffusion constant but weak
interaction with cells and vice versa with ζ (Villanueva-
Flores et al., 2020). Furthermore, surface charge and hd
plays a key role in determining the polarization and
movement of conducting and insulating NPs under the
influence of EF via electrophoresis and DEP, respectively
(Zhao & Bau, 2009). Therefore, it is important to balance
hd and ζ of the selected NPs for optimal cellular and EF
interaction. In general, NPs with ζ values of ≥ - 30 mV
or ≥ + 30 mV are considered to have optimal colloidal
stability for biological application due to the presence of
sufficient repulsive forces (Skoglund et al., 2017). Citrate
capped GNPs and ZnO NPs showed a mean ζ value of
– 39.2 ± 2.2 mV and -36.5 ± 1 mV which is attributed
to the charge of citrate and oxide ions, respectively, sug-
gesting good physical colloidal stability. In contrast, SiO2
NPs showed a positive zeta potential value of +12.2 ± 0.9
mV due to the presence of -NH2 groups on the surface
of these NPs indicating presence of weak repulsive forces
and moderate colloidal stability. DLS analysis indicated
a monodisperse sample of GNPs with a hd of 43.8 ± 3.2
nm. A slight increase in the size of ZnO (hd = 69.4 ± 6.7
nm ) and SiO2 (hd = 50.7 ± 5.5 nm) NPs compared to
TEM measurements, further confirmed the agglomera-
tion of these NPs in colloidal solution. In vitro Toxicity of NPs TEM analysis revealed that the mean diameter of GNPs
is ~ 42 ± 4 nm (Fig. 1A) with homogenous spherical
morphology, while SiO2 (Fig. 1B) and ZnO (Fig. 1C) were
observed to agglomerate with a mean diameter of ~ 46 ±
7 nm and ~ 40 ± 11 nm, respectively. UV-Vis spectros-
copy of GNPs and ZnO NPs (Fig. 1D) dispersed in phos-
phate buffer saline (PBS) showed distinctive absorption
peaks at 529 nm and 365 nm, respectively. The absorp-
tion peak at 529 nm is attributed to surface plasmon Before investigating the effect of using NPs in conjunc-
tion with TTFields, it was important to ascertain the
toxicity of the different NPs on HA-COR (healthy cells),
GIN 28 and GCE 28 cells. An experiment was therefore
carried out to investigate the effect of increasing the NP
concentration from 0 to 50 μg/ mL, by using PrestoBlue
assay, which reports on the metabolic activity of cells as
an indicator of cell viability (Peng et al., 2020; Xu et al.,
2015). However, the limitation of this assay is that it does Jain et al. Bioelectronic Medicine (2022) 8:17 Page 5 of 9 Jain et al. Bioelectronic Medicine (2022) 8:17 Fig. 1 Physico-chemical characterization of inorganic NPs (Gold – GNPs; Zinc Oxide – ZnO; Silica oxide – SiO2). Transmission electron microscopy
images of (A) GNPs, (B) SiO2 and (C) ZnO NPs. D UV-Vis absorption spectrum of ZnO and GNPs in PBS; E FTIR spectrum of SiO2 NPs; F Zeta potential;
and G Hydrodynamic diameter obtained using DLS. Error bars represent the standard deviation of the mean n=3; N= 3 Fig. 1 Physico-chemical characterization of inorganic NPs (Gold – GNPs; Zinc Oxide – ZnO; Silica oxide – SiO2). Transmissio
images of (A) GNPs, (B) SiO2 and (C) ZnO NPs. D UV-Vis absorption spectrum of ZnO and GNPs in PBS; E FTIR spectrum of S
and G Hydrodynamic diameter obtained using DLS. Error bars represent the standard deviation of the mean n=3; N= 3 Fig. 1 Physico-chemical characterization of inorganic NPs (Gold – GNPs; Zinc Oxide – ZnO; Silica oxide – SiO2). Transmission electron microscopy
images of (A) GNPs, (B) SiO2 and (C) ZnO NPs. D UV-Vis absorption spectrum of ZnO and GNPs in PBS; E FTIR spectrum of SiO2 NPs; F Zeta potential;
and G Hydrodynamic diameter obtained using DLS. TTFields and NPs Mediated Enhanced EF Effects in GBM
Cells enhancement with conductive GNPs and semiconducting
ZnO NPs, can be further explained by the ability of these
conducting and semiconducting NPs to polarize and align
themselves with the applied EF to act as bipolar nanoelec-
trodes or transducers (Guo et al., 2021; Cao et al., 2018; Li
& Anand, 2017). Furthermore, the observed enhanced elec-
tric effects could also be due to the bipolar electrophoretic
effect, as both the GNPs and ZnO are negatively charged. Moreover, TTFields are known to induce biophysical forces
on charged entities, this could have triggered the intracellu-
lar movement of GNPs and ZnO, thus enhancing the forces
on polar intracellular structures affecting dipole alignment
and disruption of mitotic spindle. In contrast, SiO2 is a
well-known dielectric material which upon cellular uptake
increases the impedance of cells. Furthermore, under
the influence of TTFields (non-uniform EFs) these NPs
experience dielectrophoretic forces which induces their
polarization. This polarization could lead to the dielectro-
phoretic movement of these particles towards the pole with
higher EF intensity thus enhancing the classic intracellular
TTField effects (Rominiyi et al., 2021). Cells
Encouraged by the optimal biocompatibility of ZnO NPs
(≤ 5 μg/mL), GNPs (≤ 50 μg/mL) and SiO2 (≤ 50 μg/mL),
we then investigated the role of these inorganic NPs in
enhancing EF effects in patient derived GBM cells. Dielec-
tric properties of tissues, as well as intracellular machin-
ery, play an important role in determining the efficacy of
TTFields as they are known to inhibit the proliferation of
cancer cells by inducing DEP of proteins involved in the
cell division process (Hershkovich et al., 2019; Wenger
et al., 2015). Therefore, we hypothesised that by intro-
ducing NPs of different electrical conductivity (dielectric
properties), the impedimetric properties of the cells can
be modified, allowing further insights into the EF medi-
ated cellular response. TTFields were delivered in GIN
28 and GCE 28 cells using the Inovitro laboratory system
(Novocure, Haifa, Israel, schematic shown in Fig. 3A) that
replicates the effect of the clinically used technique (the
OptuneTM device). Before the application of TTFields,
cells were incubated for 4 hours with either GNPs/ZnO/
SiO2 NPs to allow uptake. We observed that the applica-
tion of TTFields alone (300 kHz and1V/cm) for 48 hours
decreased the metabolic activity of both GIN 28 (Fig. 3B)
and GCE 28 cells (Fig. 3C) by ~ 25% vs. untreated con-
trol (p < 0.0003). In vitro Toxicity of NPs Error bars represent the standard deviation of the mean n=3; N= 3 concentration of 50 μg/ mL, and at 10 μg/ mL for GIN
28 and GCE 28 cells. This decrease in metabolic activ-
ity by ZnO NPs (concentration = > 5 μg/mL) has been
attributed to the generation of reactive oxygen species
at higher concentrations (Liu et al., 2017). Nevertheless,
the FDA has classified ZnO NPs as a “GRAS” (generally
regarded as safe) at lower concentrations (Rasmussen
et al., 2010). Based on the obtained data, a concentra-
tion of 5 μg/ mL of each type of NPs was chosen for the
in vitro TTFields experiment, as at this concertation no
significant change in the metabolic activity of GBM cells
was observed for all three types of the NPs. not identify the mechanism of change in metabolic activ-
ity. In the cases of GNPs and SiO2, there was no effect on
the metabolic activity of the HA-COR (Fig. S1), GIN 28
(Fig. 2A), and GCE 28 (Fig. 2B) cells across the concentra-
tion range tested. From this, we can infer GNPs and SiO2
are not toxic to the healthy HA-COR and patient derived
GBM cells used in this study at concentrations up to 50
μg/ mL and are therefore biocompatible. In contrast, as
the concentration of ZnO NPs increased, a clear effect
on cellular metabolism was observed for all cell types at
higher concentrations. ZnO nanoparticles significantly
reduced the metabolic activity of healthy HA-COR at a Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine Page 6 of 9 Fig. 2 In vitro toxicity of inorganic NPs on patient derived GBM cells. A GIN 28, and B GCE 28 were incubated with increasing concentration of GNPs,
SiO2 and ZnO NPs for 4 hours, before changing the media containing the NPs with fresh media. Metabolic activity was determined 48 hours after
changing the media, the experiment was run in triplicate, and fluorescence at 590 nm is expressed as % of control (no NPs). Results are expressed as
the mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001 obtained using 2-way ANOVA with a Tukey post-test Fig. 2 In vitro toxicity of inorganic NPs on patient derived GBM cells. In vitro Toxicity of NPs A GIN 28, and B GCE 28 were incubated with increasing concentration of GNPs,
SiO2 and ZnO NPs for 4 hours, before changing the media containing the NPs with fresh media. Metabolic activity was determined 48 hours after
changing the media, the experiment was run in triplicate, and fluorescence at 590 nm is expressed as % of control (no NPs). Results are expressed as
the mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001 obtained using 2-way ANOVA with a Tukey post-test TTFields and NPs Mediated Enhanced EF Effects in GBM
Cells TTFields and NPs Mediated Enhanced EF Effects in GBM
Cells from triplicate or quadruplicate repeats and two independent experiments (
ld
d
l
d
)
d
d
h
d
ff Fig. 3 TTFields and inorganic NPs (Gold – GNPs; Zinc oxide – ZnO; Silica oxide – SiO2) mediated enhanced EF effects on patient-derived GBM cells. A Schematic representation of TTFields setup consisting of – a base plate containing 8 ceramic dishes is connected to TTFields generator via a flat
cable. Cells were seeded on a coverslip placed within a ceramic dish. The base plate is placed inside an incubator where the cells were maintained
at 37°C and 5% CO2. B GIN 28 cells and C GCE 28 cells were treated with TTFields at 300 kHz, 1V/cm, and 48 hours at NPs concentration of 5 μg/
mL. D GNPs mediated enhanced TTFields effect on GIN 28 cells at 300 kHz, 1V/cm after 48 and 72 hours at a concentration of 25 μg/mL. Error bar
represents mean ± S.E.M. from triplicate or quadruplicate repeats and two independent experiments Fig. 3 TTFields and inorganic NPs (Gold – GNPs; Zinc oxide – ZnO; Silica oxide – SiO2) mediated enhanced EF effects on patient-derived GBM cells. A Schematic representation of TTFields setup consisting of – a base plate containing 8 ceramic dishes is connected to TTFields generator via a flat
cable. Cells were seeded on a coverslip placed within a ceramic dish. The base plate is placed inside an incubator where the cells were maintained
at 37°C and 5% CO2. B GIN 28 cells and C GCE 28 cells were treated with TTFields at 300 kHz, 1V/cm, and 48 hours at NPs concentration of 5 μg/
mL. D GNPs mediated enhanced TTFields effect on GIN 28 cells at 300 kHz, 1V/cm after 48 and 72 hours at a concentration of 25 μg/mL. Error bar
represents mean ± S.E.M. from triplicate or quadruplicate repeats and two independent experiments could be due to the enhanced bipolar electrophoretic
(GNPs and ZnO) and dielectrophoretic effects (SiO2)
mediated by inorganic NPs. Further in vitro studies are
required to elucidate the detailed molecular mecha-
nism underlying the observed NPs mediated TTFields
enhancement. TTFields and NPs Mediated Enhanced EF Effects in GBM
Cells Interestingly, inorganic NPs (4 hours)
+ TTFields (48 hours) treated showed a ~ 40% decrease
in metabolic activity compared to untreated control (p
<0.0001). Importantly, this corresponds to a ~15% higher
decrease in metabolic activity compared to TTFields alone
(p = 0.002 for GNPs; 0.0001 for ZnO, and 0.04 for SiO2). f
To further validate and establish the observed
enhanced EF effect on GBM cells with GNPs regard-
ing the conductivity of cells, we incubated GIN 28
cells with GNPs at a higher concentration (25 μg/mL)
to further increase the intracellular concentration of
GNPs cell conductivity. In GNP + TTFields treated
group a ~52% and ~75% decrease in the metabolic
activity of GIN 28 cells was observed after 48- and 72-h
treatment, respectively, which was found to be signifi-
cantly higher than both untreated and TTFields treated
groups (Fig. 3D). Overall, the obtained data suggest that
all three NPs utilized in this work enhance EF mediated
anticancer effects in patient derived GBM cells. From
the observed in vitro effects, we hypothesise that this Together with our previous observations and the
obtained data we tentatively suggest that the inorganic
NPs irrespective of their dielectric properties, could acts
as EF transducers (Robinson et al., 2021). The observed (2022) 8:17 Jain et al. Bioelectronic Medicine (2022) 8:1 Page 7 of 9 Jain et al. Bioelectronic Medicine could be due to the enhanced bipolar electrophoretic
(GNPs and ZnO) and dielectrophoretic effects (SiO2)
Conclusions
In summary, conducting GNPs, semiconducting ZnO,
Fig. 3 TTFields and inorganic NPs (Gold – GNPs; Zinc oxide – ZnO; Silica oxide – SiO2) mediated enhanced EF effects on patient-derived GBM cells. A Schematic representation of TTFields setup consisting of – a base plate containing 8 ceramic dishes is connected to TTFields generator via a flat
cable. Cells were seeded on a coverslip placed within a ceramic dish. The base plate is placed inside an incubator where the cells were maintained
at 37°C and 5% CO2. B GIN 28 cells and C GCE 28 cells were treated with TTFields at 300 kHz, 1V/cm, and 48 hours at NPs concentration of 5 μg/
mL. D GNPs mediated enhanced TTFields effect on GIN 28 cells at 300 kHz, 1V/cm after 48 and 72 hours at a concentration of 25 μg/mL. Error bar
represents mean ± S.E.M. Conclusions In summary, conducting GNPs, semiconducting ZnO,
and insulating SiO2 with excellent physicochemical
properties, colloidal stability, and biocompatibility,
were investigated as a transducer for enhancing EF
activity in vitro. We demonstrated that inorganic NPs Jain et al. Bioelectronic Medicine (2022) 8:17 Jain et al. Bioelectronic Medicine (2022) 8:17 Page 8 of 9 Page 8 of 9 Page 8 of 9 Jain et al. Bioelectronic Medicine irrespective of dielectric permittivity, enhance the effi-
ciency and efficacy of EFs in patient derived GBM cells
isolated from intra-tumour regions. The in vitro efficacy
was significantly enhanced by GNPs and ZnO treat-
ment, which is attributed to the ability of these NPs to
polarize and act as bipolar nanoelectrodes/ transduc-
ers which can sense external EFs, thereby enhancing
electrophoretic effects. We additionally suggest that
SiO2 NPs may enhance EF effects by increasing the
forces exerted due to DEP. Furthermore, we have dem-
onstrated that the FDA approved inorganic NPs can be
used as nano transducers for enhancing intracellular EF
effects. This work further paves the pathway for future
studies to systemically deliver these NPs across the BBB
to determine the in vivo efficacy. Abbreviations
l
ld ad MP, Rao AV, Babu KS, Rao GN. Mater Chem Phys. 2019;224:79–84. Ahmad MP, Rao AV, Babu KS, Rao GN. Mater Chem Phys. 2019;224:79 84. Blatt R, Davidi S, Munster M, Shteingauz A, Cahal S, Zeidan A, et al. Front Oncol. 2021;11:2459. EFs: Electric Fields; FTIR: Fourier-transform infrared spectroscopy; GBM: Glio-
blastoma; GNP: Gold nanoparticles; NPs: Nanoparticles; SiO2: Silicon Dioxide
Nanoparticle; TEM: Transmission Electron Microscopy; TTF: Tumour treating
fields; ZnO: Zinc oxide nanoparticles. Branter J, Estevez-Cebrero M, Diksin M, Griffin M, Castellanos-Uribe M, May S,
et al. Int J Mol Sci. 1982;2022:23. Brassard J-D, Sarkar DK, Perron J. ACS Appl Mat Interfaces. 2011;3:3583–8. Brightman M Reese T J Cell Biol 1969;40:648–77 Brightman M, Reese T. J Cell Biol. 1969;40:648–77. Acknowledgments Guo Q, Lei C, Chen W, Zhang J, Huang B. Cell Rep Phys Sci. 2021;2:100299. g
The authors would also like to thank Michael Fay at the Nanoscale and
Microscale Research Centre, University of Nottingham for assistance with the
TEM analysis. Haiss W, Thanh NT, Aveyard J, Fernig DG. Analytical Chem. 2007;79:4215–2 Hanley C, Layne J, Punnoose A, Reddy K, Coombs I, Coombs A, et al. Nanotech-
nology. 2008;19:295103. Hershkovich HS, Urman N, Yesharim O, Naveh A, Bomzon ZE. Phys Med Biol. 2019;64:185014. Funding Kumthekar P, Ko CH, Paunesku T, Dixit K, Sonabend AM, Bloch O, et al. Science
Transl Med. 2021;13:eabb3945. This work was supported by the Engineering and Physical Sciences Research
Council Grant numbers [EP/R004072/1, EP/S023054/1]. We would like to
acknowledge the support of Novocure Inc. through the loan of the Inovitro
TTFields device for laboratory work. Transl Med. 2021;13:eabb3945. Li M, Anand RK. J Am Chem Soc. 2017;139:8950–9. Libutti SK, Paciotti GF, Byrnes AA, Alexander HR, Gannon WE, Walker M, et al. Clin Cancer Res. 2010;16:6139–49. Linder B, Schiesl A, Voss M, Rödel F, Hehlgans S, Güllülü Ö, et al. Front Oncol. 2021:3083. Availability of data and materials Data supporting results can be found at the University of Nottingham reposi-
tory (https://doi.org/10.17639/nott.7241) and will be publicly available as of
the date of publication. Any additional information required to reanalyse the
data reported in this paper is available from the lead contact upon request. Liu J, Kang Y, Yin S, Song B, Wei L, Chen L, et al. Int J Nanomed. 2017;12:8085. Mittag A, Hoera C, Kämpfe A, Westermann M, Kuckelkorn J, Schneider T, et al. Toxics. 2021;9:96. Murugan C, Venkatesan S, Kannan S. ACS Omega. 2017;2:7959–75. Ostrom QT, Cioffi G, Waite K, Kruchko C, Barnholtz-Sloan JS. Neuro Oncol. 2021;23:iii1-iii105. Competing interests The authors declare no conflict of interest. The funders and Novocure had no
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https://openalex.org/W4386415949
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https://www.nature.com/articles/s41467-023-40670-7.pdf
|
English
| null |
Inherent spatiotemporal uncertainty of renewable power in China
|
Nature communications
| 2,023
|
cc-by
| 14,453
|
Inherent spatiotemporal uncertainty of
renewable power in China Jianxiao Wang
1,2,13, Liudong Chen3,13, Zhenfei Tan
4, Ershun Du5, Nian Liu6,
Jing Ma6, Mingyang Sun7, Canbing Li4, Jie Song8,1,2, Xi Lu
9,10
,
Chin-Woo Tan11
& Guannan He
1,8,12 Jianxiao Wang
1,2,13, Liudong Chen3,13, Zhenfei Tan
4, Ershun Du5, Nian Liu6,
Jing Ma6, Mingyang Sun7, Canbing Li4, Jie Song8,1,2, Xi Lu
9,10
,
Chin-Woo Tan11
& Guannan He
1,8,12 Received: 17 November 2022
Accepted: 7 August 2023
Check for updates Solar and wind resources are vital for the sustainable energy transition. Although renewable potentials have been widely assessed in existing litera-
ture, few studies have examined the statistical characteristics of the inherent
renewable uncertainties arising from natural randomness, which is inevitable
in stochastic-aware research and applications. Here we develop a rule-of-
thumb statistical learning model for wind and solar power prediction and
generate a year-long dataset of hourly prediction errors of 30 provinces in
China. We reveal diversified spatiotemporal distribution patterns of prediction
errors, indicating that over 60% of wind prediction errors and 50% of solar
prediction errors arise from scenarios with high utilization rates. The first-
order difference and peak ratio of generation series are two primary indicators
explaining the uncertainty distribution. Additionally, we analyze the seasonal
distributions of the provincial prediction errors that reveal a consistent law in
China. Finally, policies including incentive improvements and interprovincial
scheduling are suggested. To realize China’s carbon neutrality goal proposed in 20201,
the installed capacity of renewable energy resources should be
significantly increased. As China mentioned in the 2020 Climate
Ambition Summit, the installation of wind and solar energy should
reach no less than 1.2 Terawatt (TW) in 2030, almost 3 times more
than that in 20192, becoming the dominant electricity generation
resource. However, due to the salient intermittency and volatility, wind
and solar energy operation and modeling face the critical challenges of
a high degree of uncertainty, which must be considered in energy
research3–5. Various studies have investigated the generalized spatial and
temporal characteristics of renewable energy resources in regional
areas and compiled standardized test datasets, including statistical
analysis studies of current wind and solar resources6–10 and important
impact factors of renewable energy generation11, current wind and
solar energy resource estimation studies using meteorological data
and prediction methods12–14, and future wind and solar energy resource
assessment studies based on wind speed and solar irradiation data15–19. Article https://doi.org/10.1038/s41467-023-40670-7 Inherent spatiotemporal uncertainty of
renewable power in China However, renewable energy resources rely on weather conditions and
thus are highly unstable, posing great challenges to accurate and 1National Engineering Laboratory for Big Data Analysis and Applications, Peking University, Beijing 100871, China. 2Peking University Ordos Research Institute
of Energy, Ordos 017000, China. 3Department of Earth and Environmental Engineering, Columbia University, New York, NY 10027, USA. 4Key Laboratory of
Control of Power Transmission and Conversion (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China. 5Low-Carbon Energy
Laboratory, Tsinghua University, Beijing 100084, China. 6State Key Laboratory of Alternate Electrical Power System with Renewable Energy Sources, School
of Electrical and Electronic Engineering, North China Electric Power University, Beijing 102206, China. 7College of Control Science and Engineering, Zhejiang
University, Hangzhou 310058, China. 8Department of Industrial Engineering and Management, College of Engineering, Peking University, Beijing 100871,
China. 9School of Environment and State Key Joint Laboratory of Environment Simulation and Pollution Control, Tsinghua University, Beijing 100084, China. 10Institute for Carbon Neutrality, Tsinghua University, Beijing 100084, China. 11Department of Civil and Environmental Engineering, Stanford University, Palo
Alto, CA 94305, USA. 12Institute of Carbon Neutrality, Peking University, Beijing 100871, China. 13These authors contributed equally: Jianxiao Wang, Liudong
Chen. e-mail: xilu@tsinghua.edu.cn; tancw@stanford.edu; gnhe@pku.edu.cn 1National Engineering Laboratory for Big Data Analysis and Applications, Peking University, Beijing 100871, China. 2Peking University Ordos Research Institute
of Energy, Ordos 017000, China. 3Department of Earth and Environmental Engineering, Columbia University, New York, NY 10027, USA. 4Key Laboratory of
Control of Power Transmission and Conversion (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China. 5Low-Carbon Energy
Laboratory, Tsinghua University, Beijing 100084, China. 6State Key Laboratory of Alternate Electrical Power System with Renewable Energy Sources, School
of Electrical and Electronic Engineering, North China Electric Power University, Beijing 102206, China. 7College of Control Science and Engineering, Zhejiang
University, Hangzhou 310058, China. 8Department of Industrial Engineering and Management, College of Engineering, Peking University, Beijing 100871,
China. 9School of Environment and State Key Joint Laboratory of Environment Simulation and Pollution Control, Tsinghua University, Beijing 100084, China. 10Institute for Carbon Neutrality, Tsinghua University, Beijing 100084, China. 11Department of Civil and Environmental Engineering, Stanford University, Palo
Alto, CA 94305, USA. 12Institute of Carbon Neutrality, Peking University, Beijing 100871, China. 13These authors contributed equally: Jianxiao Wang, Liudong
Chen. e-mail: xilu@tsinghua.edu.cn; tancw@stanford.edu; gnhe@pku.edu.cn 1National Engineering Laboratory for Big Data Analysis and Applications, Peking University, Beijing 100871, China. 2Peking University Ordos Research Institute
of Energy, Ordos 017000, China. Inherent spatiotemporal uncertainty of
renewable power in China 3Department of Earth and Environmental Engineering, Columbia University, New York, NY 10027, USA. 4Key Laboratory of
Control of Power Transmission and Conversion (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China. 5Low-Carbon Energy
Laboratory, Tsinghua University, Beijing 100084, China. 6State Key Laboratory of Alternate Electrical Power System with Renewable Energy Sources, School
of Electrical and Electronic Engineering, North China Electric Power University, Beijing 102206, China. 7College of Control Science and Engineering, Zhejiang
University, Hangzhou 310058, China. 8Department of Industrial Engineering and Management, College of Engineering, Peking University, Beijing 100871,
China. 9School of Environment and State Key Joint Laboratory of Environment Simulation and Pollution Control, Tsinghua University, Beijing 100084, China. 10Institute for Carbon Neutrality, Tsinghua University, Beijing 100084, China. 11Department of Civil and Environmental Engineering, Stanford University, Palo
Alto, CA 94305, USA. 12Institute of Carbon Neutrality, Peking University, Beijing 100871, China. 13These authors contributed equally: Jianxiao Wang, Liudong
Chen. e-mail: xilu@tsinghua.edu.cn; tancw@stanford.edu; gnhe@pku.edu.cn Nature Communications| (2023) 14:5379 1 of solar energy according to the prediction error: (i) >8%, (ii) 7–8%, (iii)
6–7%, and (iv) <6%. The details of each group are provided in the
Supplementary Information (SI). reliable prediction. Some studies have examined the uncertainty of
solar and wind power equipped with energy storage to assess their
potential to meet future electricity demand20. Prediction methods
such as linear regression models and eXtreme Gradient Boosting have
been utilized to forecast the uncertainty of wind and solar generation
in specific regional areas, considering seasonal or yearly analyses21,22. However, limited research has focused on analyzing the spatio-
temporal uncertainty distributions of renewable energy23,24. There are
research gaps in terms of error analysis benchmarks that consider
long-term, high-granularity, and nationwide scales of wind and solar
output prediction, particularly within the context of China. The results demonstrate that, except for Southwest China, the
wind prediction error in the other regions is relatively large, especially
large in the eastern area, i.e., Shandong (SD), SH, Jiangsu (JS), Anhui
(AH), and Henan (HA), and Northern area including Beijing (BJ), TJ,
Liaoning (LN), Jilin (JL), Shanxi (SX), and Hebei (HE), ranging from 8.0
to 11.3% and 5.3 to 13.6%, respectively. These two areas account for
25.0% and 27.9%, respectively, of the total prediction error in China. Inherent spatiotemporal uncertainty of
renewable power in China Regarding solar energy, the prediction error is concentrated in the
areas of Central China covering Ningxia (NX), Shaanxi (SN), Hubei
(HB), Jiangxi (JX), and Hunan (HN), North China, and East China, ran-
ging from 6.2 to 9.0%, 7.2 to 9.3%, and 6.8 to 10.0%, respectively,
accounting for 17.5%, 25.0%, and 19.1%, respectively, of the total pre-
diction error in China. Error-analysis benchmarks for wind and solar output forecasting
are of great value in academic research and industry. First, a prediction
error database of the wind and solar output should be obtained via
benchmark prediction methods, e.g., neural network-based25, data
mining26, and regression methods27. Second, a wide variety of studies,
e.g., power system planning and operation28–31, energy scheduling32–34,
and market operation and mechanism design studies35,36, must con-
sider the intermittencyand volatility of renewableenergy resources via
robust optimization37,38, stochastic programming39,40, and statistical
analysis methods41,42. Third, the prediction error of renewable power
determines the revenue risk of power generation companies, espe-
cially in markets with deviation punishment. In this regard, prediction
error analysis can provide an important reference for the decision-
making of intermittent renewables. We compare the prediction errors of various methods, including
random forest (RF), recurrent neural network (RNN), fully-connected
neural network (FCNN), and support vector machine (SVM), for pre-
dicting nationwide renewable energy output. The results are presented
in Fig. 1c and Supplementary Table 1. Our observations indicate that
although each method demonstrates varying prediction error dis-
tributions across different provinces, the overall nationwide predic-
tion errors remain similar among all methods, ranging from 6 to 9%. Further details can be found in the SI. Notably, ARIMA and RNN exhibit
similar prediction errors and outperform other methods, benefiting
from their inherent ability to effectively handle time series data. In the
following part of this paper, we focus on the prediction error with the
ARIMA model as a benchmark method. The motivation of this work is to develop a year-long error-ana-
lysis benchmark for hourly wind and solar generation forecasting in 30
provinces of China, which is expected to constitute a valuable resource
and toolkit for market operators or planners. To this end, we use a one-
year standard dataset including hourly wind and solar output data for
30 provinces of China11. Here, we establish a rule-of-thumb prediction
model to conduct hourly predictions of the wind and solar output in a
rolling fashion and to obtain basic prediction datasets. Results y
g p
Two statistical indicators are proposed to explore the factors
impacting prediction errors. Due to the irregular distribution of the
wind output and the daily periodicity of the solar output, we use hourly
and daily output data to analyze the wind and solar prediction errors,
respectively (Methods and Supplementary Fig. 4). We use the coeffi-
cient of determination (CoD) R2, which measures the linear correla-
tion, to quantify the relationship between the prediction error and
various factors. The installed capacity is independent of the prediction
error, with R2 = 0:002 for wind energy (Fig. 2a) and R2 = 0:076 for solar
energy (Fig. 2b). In addition, the power generation reflected by the
bubble size exhibited no correlation with the prediction error
(Fig. 2a, b). Inherent spatiotemporal uncertainty of
renewable power in China The results
reveal the nationwide spatial distribution of the wind and solar energy
uncertainty through the prediction error. The first-order difference
and peak ratio of output data are determined as primary factors of the
prediction error. To further analyze provincial forecasting character-
istics, we provide the provincial probability distribution function (PDF)
of prediction errors and distribution regularities, the influence of
power generation intervals on prediction in each province, and the
temporal features of uncertainty via seasonal analysis. Moreover, we examine the impact of the prediction time scale on
the distribution of nationwide prediction errors for both wind and
solar energy, as illustrated in Fig. 1d. We observe that prediction error
increases with the prediction time scale, with a 2-h prediction resulting
in a 3.40% error for solar and a 2.83% error for wind, a 6-h prediction
resulting in a 6.14% error for solar and a 6% error for wind, and a 24-h
prediction resulting in a 9.25% error for solar and a 10.86% error for
wind. A detailed analysis of each hour’s prediction error reveals that
the error mainly originates from the ending periods, e.g., during 5–6 h
for the 6-h ahead predictions and during 15–24 h for the 24-h ahead
predictions. Nationwide analysis of the uncertainty of wind and solar
generation g
We obtain an error-analysis benchmark for the forecasting of hourly
wind and solar output potential in 30 provinces of China in 2016 using
the autoregressive integrated moving average (ARIMA) model based
on installation and hourly generation data retrieved from our previous
study11. The spatial distributions of the wind and solar uncertainty
across China are analyzed through the prediction error, as shown in
Fig. 1a, b, respectively, excluding Taiwan, Hong Kong, and Macau, as
well as wind energy in Tibet and solar energy in Chongqing (unsuitable
for wind/solar energy construction10 or data limitations). The predic-
tion error is calculated as the predicted value minus the actual value
(please refer to Methods). The wind prediction error ranges from 2.1 to
13.6%, with the largest error in Tianjin (TJ) and the smallest error in
Yunnan (YN). The overall prediction error of solar energy is smaller
than that of wind energy, ranging from 3.9 to 10.0%, and the largest
provincial prediction error is observed in Shanghai (SH), while the
smallest provincial prediction error comes from Xinjiang (XJ). Detailed
error analysis of wind and solar power for each province is shown in
Supplementary Figs. 1–3, respectively. We divide the 30 provinces into
four groups according to the wind prediction error: (i) >9%, (ii) 7–9%,
(iii) 5–7%, and (iv) <5%. Four groups can also be distinguished in terms As shown in Fig. 2c, d, the results indicate that the first-order
difference is a major influencing factor of the prediction error, which
comprises a series of changes from one period to the next. The rela-
tionship between the prediction error and first-order difference is
approximately linear. Regarding wind power, the relationship between
the prediction error and hourly first-order difference yields R2 = 0:988
(Fig. 2c), while the daily first-order difference does not impact the wind
prediction error (please refer to the bubble size in Fig. 2c). Regarding
solar power, the CoD between the prediction error and the daily first-
order difference is R2 = 0:676 (Fig. 2d). The hourly first-order differ-
ence, however, could not reflect the prediction error, as indicated by Nature Communications| (2023) 14:5379 2 https://doi.org/10.1038/s41467-023-40670-7 Article Fig. 1 | Spatial distributions of wind and solar power prediction errors and the
impacts of different methods and time scales. a Wind energy. b Solar energy. Nationwide analysis of the uncertainty of wind and solar
generation The
larger bubbles indicate the provincial wind and solar energy installations, and the
smaller ones indicate the average wind and solar energy generation (8760 h) by
province. The provinces are divided into four groups according to the provincial
prediction error (average value of 8760 h) and marked with four gradient colors. The thick red line marks the boundaries of the four areas of China, I. North China, II. East China, III. Central China, and IV. Southwest China. Individual provinces are
indicated with lighter white lines. MW Megawatt, BJ Beijing, TJ Tianjin, HE Hebei, SX
Shanxi, IM Inner Mongolia, LN Liaoning, JL Jilin, HL Heilongjiang, SH Shanghai, JS
Jiangsu, ZJ Zhejiang, AH Anhui, FJ Fujian, JX Jiangxi, SD Shandong, HA Henan, HB
Hubei, HN Hunan, GD Guangdong, GX Guangxi, HI Hainan, CQ Chongqing, XZ
Tibet, SC Sichuan, GZ Guizhou; YN Yunnan, SN Shaanxi, GS Gansu, QH Qinghai, NX
Ningxia XJ Xinjiang. c Prediction error distribution across 30 provinces obtained by
different methods. The smoothed curve in the left and right parts represents the
prediction error density function across 30 provinces of solar and wind energy,
respectively. The short black line in the middle of each shape is the median value of
the data distribution, which visualizes the central tendency of the data distribution
of each method. The algorithm used to fit the density function is Kernel Density
Estimation. RF random forest, RNN recurrent neural network, FCNN fully-
connected neural network, SVM support vector machine, ARIMA autoregressive
integrated moving average. d Nationwideprediction error distribution of eachhour
based on 2, 6 and 24-h ahead prediction. Each box includes 1917, 638, and
159 samples for solar energy and 4297, 1432, and 358 samples for wind energy. The
lower/upper end of each box indicates the minimal/maximal value, and the lower
and upper percentiles indicate 25% and 75%, respectively. The short blue line
indicates the median, and the blue points show the outliers. There are blank areas
for the 2-h and 6-h predictions since these two prediction tasks only contain 2 and 6
time periods, respectively. Fig. 1 | Spatial distributions of wind and solar power prediction errors and the Fig. 1 | Spatial distributions of wind and solar power prediction errors and the
impacts of different methods and time scales. a Wind energy. b Solar energy. Nationwide analysis of the uncertainty of wind and solar
generation Each box includes 1917, 638, and
159 samples for solar energy and 4297, 1432, and 358 samples for wind energy. The
lower/upper end of each box indicates the minimal/maximal value, and the lower
and upper percentiles indicate 25% and 75%, respectively. The short blue line
indicates the median, and the blue points show the outliers. There are blank areas
for the 2-h and 6-h predictions since these two prediction tasks only contain 2 and 6
time periods, respectively. prediction error, e.g., TJ: 13.6% and SD: 8.9% occur in both higher and
lower power intervals, and the frequency fluctuates at10%. However, in
provinces with a small prediction error (SX: 5.4% and GS: 4.2%), peaks
are concentrated in lower power intervals from 1 to 4, at 76.76% and
83.48%. In contrast, solar energy peaks are mainly located in higher
power intervals, with the peaks in intervals above 4 accounting for
62.59%, 59.38%, 64.90%, and 89.61% in BJ, JS, HB, and IM, respectively. the bubble size in Fig. 2d. The reason is that wind power prediction is
conducted hour-by-hour, and the daily wind power generation is
irregular and cannot reflect the hourly wind generation pattern. Regarding solar power, power generation varies periodically daily, and
the characteristics of the hourly first-order difference could be masked
by this daily periodicity. Another significant factor influencing the prediction error is the
peak ratio, which reflects the frequency of the tendency changes in the
power output series, with CoD R2 = 0:967 for the hourly wind output
(Fig. 3a) and R2 = 0:558 for the daily solar output (Fig. 3c). Similar to the
first-order difference, wind and solar energy differ in their hourly and
daily features. To further explore the impact of different power gen-
eration levels on the prediction error, we evenly divide the installed
generation capacity into 10 intervals. We also select a representative
province in each wind and solar energy category for detailed analysis. The representative wind energy provinces are TJ, SD, SX, and Gansu
(GS); the representative solar energy provinces are BJ, JS, HB, and Inner
Mongolia (IM). We express the peak distribution in each power gen-
eration interval as a frequency (Fig. 3b for wind energy and Fig. 3d for
solar energy). Regarding wind energy, peaks in provinces with a large Nationwide analysis of the uncertainty of wind and solar
generation The
larger bubbles indicate the provincial wind and solar energy installations, and the
smaller ones indicate the average wind and solar energy generation (8760 h) by
province. The provinces are divided into four groups according to the provincial
prediction error (average value of 8760 h) and marked with four gradient colors. The thick red line marks the boundaries of the four areas of China, I. North China, II. East China, III. Central China, and IV. Southwest China. Individual provinces are
indicated with lighter white lines. MW Megawatt, BJ Beijing, TJ Tianjin, HE Hebei, SX
Shanxi, IM Inner Mongolia, LN Liaoning, JL Jilin, HL Heilongjiang, SH Shanghai, JS
Jiangsu, ZJ Zhejiang, AH Anhui, FJ Fujian, JX Jiangxi, SD Shandong, HA Henan, HB
Hubei, HN Hunan, GD Guangdong, GX Guangxi, HI Hainan, CQ Chongqing, XZ
Tibet, SC Sichuan, GZ Guizhou; YN Yunnan, SN Shaanxi, GS Gansu, QH Qinghai, NX
Ningxia XJ Xinjiang. c Prediction error distribution across 30 provinces obtained by
different methods. The smoothed curve in the left and right parts represents the prediction error density function across 30 provinces of solar and wind energy, prediction error density function across 30 provinces of solar and wind energy,
respectively. The short black line in the middle of each shape is the median value of
the data distribution, which visualizes the central tendency of the data distribution
of each method. The algorithm used to fit the density function is Kernel Density
Estimation. RF random forest, RNN recurrent neural network, FCNN fully-
connected neural network, SVM support vector machine, ARIMA autoregressive
integrated moving average. d Nationwideprediction error distribution of eachhour
based on 2, 6 and 24-h ahead prediction. Each box includes 1917, 638, and
159 samples for solar energy and 4297, 1432, and 358 samples for wind energy. The
lower/upper end of each box indicates the minimal/maximal value, and the lower
and upper percentiles indicate 25% and 75%, respectively. The short blue line
indicates the median, and the blue points show the outliers. There are blank areas
for the 2-h and 6-h predictions since these two prediction tasks only contain 2 and 6
time periods, respectively. Estimation. RF random forest, RNN recurrent neural network, FCNN fully
connected neural network, SVM support vector machine, ARIMA autoregressive
integrated moving average. d Nationwideprediction error distribution of eachhour
based on 2, 6 and 24-h ahead prediction. Nature Communications| (2023) 14:5379 Temporal analysis of provincial prediction errors We examine the PDF and prediction error in each province within the
above 10 power generation intervals to analyze further the spatial
characteristics of the prediction error (Fig. 4 and Supplementary
Table 2). The results reveal that the more concentrated the PDF is
within a certain interval, the smaller the prediction error within this
interval. In terms of wind generation, the average prediction error
within interval 1 in TJ is small (only 10.6%), and the PDFs within this
intervalare concentrated from intervals 1–4; in contrast, the prediction
error within interval 8 reaches 21.5%, and the PDF within this interval is
distributed across almost all intervals. The prediction error within each
interval also reflects the variance and fluctuation magnitude within the Nature Communications| (2023) 14:5379 3 Article https://doi.org/10.1038/s41467-023-40670-7 0.04
0.06
0.08
0.1
0.12
0.14
0.16
Solar Daily First-order Difference (MW)
3
4
5
6
7
8
9
10
11
Predict Error (%)
6
Slope: 43.503, Intercept: 2.086, CoD: 0.676
Fit
Solar Hourly First-order Difference
0
2000
4000
6000
8000
10000
Solar Installed Capacity (MW)
3
4
5
6
7
8
9
10
11
Predict Error (%)
Slope: -1.744e-04, Intercept: 7.136, CoD: 0.076
Fit
Solar Power Generation
0
0.01
0.02
0.03
0.04
0.05
Wind Hourly First-order Difference (MW)
0
2
4
6
8
10
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14
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Slope: 274.445, Intercept: -0.163, CoD: 0.988
Fit
Wind Daily First-order Difference
0
0.5
1
1.5
2
2.5
3
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104
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14
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Slope: -2.390e-05, Intercept: 6.279, CoD: 0.002
Fit
Wind Power Generation
Fig. 2 | Impacts of installed capacity, power generation and first-order differ-
ence of time series. a wind installed capacity, (b) solar installed capacity, (c) wind
hourly first-order difference, and (d), solar daily first-order difference. Here we use
daily and hourly data to analyze solar and wind energy, respectively, which are
presented in the x-axis. Each bubble indicates less influential factors, including wind
or solar generation, wind daily first-order difference, and solar hourly first-order
difference, respectively. The radius of each bubble is the value of each factor. The
number of bubbles is 30, representing the 30 provinces of China, excluding Tibet
(wind), Chongqing (solar), Hong Kong, Macao, and Taiwan. The black linear
regression line fits the center of the bubbles, complemented by the slope, inter-
cept, and coefficient of determination (CoD). Temporal analysis of provincial prediction errors The color of each bubble indicates
the different categories: red—category with the largest prediction error; yellow—
category with the second-largest prediction error; blue—category with the third-
largest prediction error; green—category with the smallest prediction error. MW
Megawatt. 0
2000
4000
6000
8000
10000
Solar Installed Capacity (MW)
3
4
5
6
7
8
9
10
11
Predict Error (%)
Slope: -1.744e-04, Intercept: 7.136, CoD: 0.076
Fit
Solar Power Generation 0
0.01
0.02
0.03
0.04
0.05
Wind Hourly First-order Difference (MW)
0
2
4
6
8
10
12
14
Predict Error (%)
Slope: 274.445, Intercept: -0.163, CoD: 0.988
Fit
Wind Daily First-order Difference
0
0.5
1
1.5
2
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3
Wind Installed Capacity (MW)
104
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14
Predict Error (%)
Slope: -2.390e-05, Intercept: 6.279, CoD: 0.002
Fit
Wind Power Generation 0.04
0.06
0.08
0.1
0.12
0.14
0.16
Solar Daily First-order Difference (MW)
3
4
5
6
7
8
9
10
11
Predict Error (%)
6
Slope: 43.503, Intercept: 2.086, CoD: 0.676
Fit
Solar Hourly First-order Difference
Solar Installed Capacity (MW)
5 Fig. 2 | Impacts of installed capacity, power generation and first-order differ-
ence of time series. a wind installed capacity, (b) solar installed capacity, (c) wind
hourly first-order difference, and (d), solar daily first-order difference. Here we use
daily and hourly data to analyze solar and wind energy, respectively, which are
presented in the x-axis. Each bubble indicates less influential factors, including wind
or solar generation, wind daily first-order difference, and solar hourly first-order
difference, respectively. The radius of each bubble is the value of each factor. The
number of bubbles is 30, representing the 30 provinces of China, excluding Tibet Fig. 2 | Impacts of installed capacity, power generation and first-order differ- (wind), Chongqing (solar), Hong Kong, Macao, and Taiwan. The black linear
regression line fits the center of the bubbles, complemented by the slope, inter-
cept, and coefficient of determination (CoD). The color of each bubble indicates
the different categories: red—category with the largest prediction error; yellow—
category with the second-largest prediction error; blue—category with the third-
largest prediction error; green—category with the smallest prediction error. MW
Megawatt. Fig. 2 | Impacts of installed capacity, power generation and first-order differ-
ence of time series. a wind installed capacity, (b) solar installed capacity, (c) wind
hourly first-order difference, and (d), solar daily first-order difference. Temporal analysis of provincial prediction errors Here we use
daily and hourly data to analyze solar and wind energy, respectively, which are
presented in the x-axis. Each bubble indicates less influential factors, including wind
or solar generation, wind daily first-order difference, and solar hourly first-order
difference, respectively. The radius of each bubble is the value of each factor. The
number of bubbles is 30, representing the 30 provinces of China, excluding Tibet interval. As shown in Fig. 4a, the average prediction error within
interval 8 in TJ is larger than that within interval 1, and the fluctuation
range within these two intervals is 0–72.1% with a variance of 404:2,
and 0–32.9% with a variance of 134:5, respectively. illustrated in Fig. 5b, d. The wind uncertainties in TJ and SD in spring
and summer account for 59.9% and 57.4%, respectively, of the total
prediction error; the solar uncertainties in BJ, HB, and IM in spring and
winter account for 60.4%, 58.0%, and 63.9%, respectively, of the total
prediction error. This occurs because solar irradiation in summer and
autumn is sufficient with fewer rainy days, resulting in more stable
solar power generation and relatively accurate prediction results. As illustrated in Fig. 4 and Supplementary Table 2, we also dis-
cover that most of the provinces with large prediction errors reach
wind and solar prediction errors in high power intervals. The propor-
tions of intervals above 5 in TJ for wind energy, SD for wind energy, SX
for wind energy, BJ for solar energy, JS for solar energy, and HB for
solar energy are 64.9%, 64.0%, 60.3%, 61.2%, 56.9%, and 53.4%,
respectively. This phenomenon is more obvious for wind energy
because solar power never occurs at full generation, and there is
almost no solar power generation within intervals 9–10. Instead, the
prediction errors in provinces with a small prediction error are dis-
tributed almost equally among all intervals, e.g., the wind prediction
error within each interval in GS ranges from 8.3 to 22.8%. This occurs
because high power generation generally exhibits peak or inflection
points, which fluctuate wildly and are difficult to predict. The pro-
portion of peaks within each interval is provided in Supplementary
Table 3. Thus, the uncertainty of power generation can be intuitively
assessed based on power generation. Nature Communications| (2023) 14:5379 https://doi.org/10.1038/s41467-023-40670-7 https://doi.org/10.1038/s41467-023-40670-7 Article Gansu
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Shanxi
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Shandong
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Tianjin
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Inner Mongolia
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Hubei
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Jiangsu
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Beijing
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
u
e
). In (a) and (c), the number of bubbles is 30, representing the 30 provinces of China
excluding Tibet (wind), Chongqing (solar), Hong Kong, Macao, and Taiwan. The
black linear regression line fits the center of the bubbles, complemented by the
slope, intercept, and coefficient of determination (CoD). https://doi.org/10.1038/s41467-023-40670-7 The color of each bubbl
indicates the different categories: red—category with the largest prediction error
yellow—category with the second-largest prediction error; blue—category with th
third-largest prediction error; green—category with the smallest prediction error Gansu
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Shanxi
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Shandong
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Tianjin
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Inner Mongolia
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Hubei
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Jiangsu
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Beijing
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
Number of Wind Hourly Peaks
2
4
6
8
10
12
14
Predict Error (%)
Slope: 165.406, Intercept: 2.251, CoD: 0.967
Fit
Number of Wind Daily Peaks
0.1
0.15
0.2
0.25
0.3
0.35
0.4
Number of Solar Daily Peaks
3
4
5
6
7
8
9
10
11
Predict Error (%)
Slope: 15.782, Intercept: 1.903, CoD: 0.558
Fit
Nmuber of Solar Hourly Peaks
Fig. 3 | Peaks distribution and the impact on the wind and solar power pre-
diction errors. a Influence of the wind hourly peaks. The radius of each bubble
indicates the ratio of the wind daily peaks. b Wind hourly peak distribution in 10
power generation intervals for Tianjin (TJ), Shandong (SD), Shanxi (SX), and Gansu
(GS). c Influence of the solar daily peaks. The radius of each bubble represents the
ratio of the solar hourly peaks. d Solar daily peak distribution in 10 power gen-
eration intervals for Beijing (BJ), Jiangsu (JS), Hubei (HB), and Inner Mongolia (IM). https://doi.org/10.1038/s41467-023-40670-7 The color of each bubble
indicates the different categories: red—category with the largest prediction error;
yellow—category with the second-largest prediction error; blue—category with the
third-largest prediction error; green—category with the smallest prediction error. Number of Wind Hourly Peaks
0.1
0.15
0.2
0.25
0.3
0.35
0.4
Number of Solar Daily Peaks
3
4
5
6
7
8
9
10
11
Predict Error (%)
Slope: 15.782, Intercept: 1.903, CoD: 0.558
Fit
Nmuber of Solar Hourly Peaks Wind Power Generation Range (%)
Wind Power Generation Range (%)
Inner Mongolia
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Hubei
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Jiangsu
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Beijing
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak Jiangsu
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Beijing
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak Predict Error (%) Inner Mongolia
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Hubei
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Solar Power Generation Range (%)
Solar Power Generation Range (%) 10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%) Fig. 3 | Peaks distribution and the impact on the wind and solar power pre-
diction errors. a Influence of the wind hourly peaks. The radius of each bubble
indicates the ratio of the wind daily peaks. b Wind hourly peak distribution in 10
power generation intervals for Tianjin (TJ), Shandong (SD), Shanxi (SX), and Gansu
(GS). c Influence of the solar daily peaks. The radius of each bubble represents the
ratio of the solar hourly peaks. d Solar daily peak distribution in 10 power gen-
eration intervals for Beijing (BJ), Jiangsu (JS), Hubei (HB), and Inner Mongolia (IM). https://doi.org/10.1038/s41467-023-40670-7 In this paper, we focus on the
inherent uncertainty of renewable generation, and the forecasting
errors are obtained merely by time-series analysis. In practice, the
prediction errors of renewable generation may be impacted by more
complicated factors such as weather forecasting quality and opera-
tional curtailment strategies. In some application scenarios, the fore-
casting tools may result in asymmetric errors conservatively. For
instance, a system operator tends to forecast renewable generation
conservatively for the sake of system reliability. These practical factors
may lead to deviations in the distribution of the forecasting error, and p
g
The statistical analysis indicates that the first-order difference and
peak ratio of renewable generation are two primary influencing factors
of prediction errors, both reflecting fluctuations in power generation. The wind prediction error is affected by the hourly power generation
because the prediction model is employed based on the irregular
hourly wind output. In contrast, the solar prediction error is affected
by daily fluctuations since solar generation exhibits daily periodicity. Our results reveal the provincial distribution of the uncertainty of
wind and solar generation, indicating different priorities for renewable
energy development in different areas. Some of the top 10 provinces
with the largest wind prediction error are TJ, SH, JS, and AH, with values
of 13.6%, 11.3%, 9.6%, and 8.4%, respectively. In contrast, the solar
prediction error in these provinces is 9.0%, 10.0%, 7.1%, and 6.8%,
respectively, which indicates that JS and AH should prioritize the
development of solar energy due to the small prediction errors and
fluctuations. SH and TJ are commercial provinces with small areas and The statistical analysis indicates that the first-order difference and
peak ratio of renewable generation are two primary influencing factors
of prediction errors, both reflecting fluctuations in power generation. The wind prediction error is affected by the hourly power generation
because the prediction model is employed based on the irregular
hourly wind output. In contrast, the solar prediction error is affected
by daily fluctuations since solar generation exhibits daily periodicity. Our results reveal the provincial distribution of the uncertainty of
wind and solar generation, indicating different priorities for renewable
energy development in different areas. Some of the top 10 provinces
with the largest wind prediction error are TJ, SH, JS, and AH, with values
of 13.6%, 11.3%, 9.6%, and 8.4%, respectively. https://doi.org/10.1038/s41467-023-40670-7 In (a) and (c), the number of bubbles is 30, representing the 30 provinces of China,
excluding Tibet (wind), Chongqing (solar), Hong Kong, Macao, and Taiwan. The
black linear regression line fits the center of the bubbles, complemented by the
slope, intercept, and coefficient of determination (CoD). The color of each bubble
indicates the different categories: red—category with the largest prediction error;
yellow—category with the second-largest prediction error; blue—category with the
third-largest prediction error; green—category with the smallest prediction error. can be incorporated into the analysis by replacing the benchmark
forecasting model with a more realistic one, which deserves an in-
depth investigation in the future. prediction errors are large and renewable generation is unstable,
renewable projects will take more risks, and the investment should be
reduced. In addition, policy-makers and system planners need infor-
mation contained in the benchmark when determining development
strategies for cleaner energy systems. An emergent and valuable issue
entails the implementation of energy storage devices to mitigate the
power balance stress in power systems with an increasing share of
renewable resources48,49, and the optimal sizing and setting processes
of energy storage devices rely heavily on the spatial and temporal
uncertainties of renewable generation. In this paper, we focus on the
inherent uncertainty of renewable generation, and the forecasting
errors are obtained merely by time-series analysis. In practice, the
prediction errors of renewable generation may be impacted by more
complicated factors such as weather forecasting quality and opera-
tional curtailment strategies. In some application scenarios, the fore-
casting tools may result in asymmetric errors conservatively. For
instance, a system operator tends to forecast renewable generation
conservatively for the sake of system reliability. These practical factors
may lead to deviations in the distribution of the forecasting error, and prediction errors are large and renewable generation is unstable,
renewable projects will take more risks, and the investment should be
reduced. In addition, policy-makers and system planners need infor-
mation contained in the benchmark when determining development
strategies for cleaner energy systems. An emergent and valuable issue
entails the implementation of energy storage devices to mitigate the
power balance stress in power systems with an increasing share of
renewable resources48,49, and the optimal sizing and setting processes
of energy storage devices rely heavily on the spatial and temporal
uncertainties of renewable generation. Discussions We provide an error-analysis benchmark for hourly wind and solar
generation in 30 provinces of China with significance for research,
industry, and policy decision-making. The proposed benchmark
revealsstatistical characteristicsof wind and solar uncertainty, which is
indispensable for academic research. First, it can help to build the PDF
of wind and solar generation, providing scenario basis for stochastic
economic dispatch43. Energy scheduling may also use renewable gen-
eration
and
consider
their
prediction
errors
as a
probability
distribution44. Second, the benchmark is applicable for robust opti-
mization, because the best and worst-case operating conditions can be
obtained through prediction results. It can also replace the assumed
prediction errors to generate reasonable probability distribution and
be used as expected forms in optimization formulations45,46. Third, risk
assessment can also benefit from the benchmark, as the security
region of power systems can be depicted based on the prediction
results and errors47. Without our work, most of these research use
assumed renewable generation and prediction error. In industry, the
benchmark plays a critical role as a guiding reference for intuitive
analysis of resource distributions and fluctuations, which could help to
evaluate investment revenue and the risk of renewable projects. If We also analyze the seasonal characteristics of the generation
uncertainty of solar and wind power on a provincial level. Here, we
compare the provincial prediction error in spring, summer, autumn,
and winter. Nationally, we determine that spring and summer are
dominant seasons for wind uncertainty, accounting for 55.48% of the
total prediction error (Fig. 5a), and spring and winter are dominant
seasons for solar uncertainty, accounting for 57.6% of the total pre-
diction error (Fig. 5c). The provincial characteristics are also similar, as Nature Communications| (2023) 14:5379 4 https://doi.org/10.1038/s41467-023-40670-7 In (a) and (c), the number of bubbles is 30, representing the 30 provinces of China
excluding Tibet (wind), Chongqing (solar), Hong Kong, Macao, and Taiwan. The
black linear regression line fits the center of the bubbles, complemented by the
slope, intercept, and coefficient of determination (CoD). The color of each bubble
indicates the different categories: red—category with the largest prediction error;
yellow—category with the second-largest prediction error; blue—category with the
third-largest prediction error; green—category with the smallest prediction error. https://doi.org/10.1038/s41467-023-40670-7 Gansu
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Shanxi
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Shandong
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Tianjin
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
Number of Wind Hourly Peaks
2
4
6
8
10
12
14
Predict Error (%)
Slope: 165.406, Intercept: 2.251, CoD: 0.967
Fit
Number of Wind Daily Peaks 0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
Number of Wind Hourly Peaks
2
4
6
8
10
12
14
Predict Error (%)
Slope: 165.406, Intercept: 2.251, CoD: 0.967
Fit
Number of Wind Daily Peaks Shandong
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak Tianjin
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak Gansu
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
g
(
) Wind Power Generation Range (%)
Wind Power Generation Range (%)
Inner Mongolia
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Hubei
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Jiangsu
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
Beijing
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
0.1
0.2
0.3
0.4
Frequency
Peak
In (a) and (c), the number of bubbles is 30, representing the 30 provinces of China,
excluding Tibet (wind), Chongqing (solar), Hong Kong, Macao, and Taiwan. The
black linear regression line fits the center of the bubbles, complemented by the
slope, intercept, and coefficient of determination (CoD). Nature Communications| (2023) 14:5379 https://doi.org/10.1038/s41467-023-40670-7 In contrast, the solar
prediction error in these provinces is 9.0%, 10.0%, 7.1%, and 6.8%,
respectively, which indicates that JS and AH should prioritize the
development of solar energy due to the small prediction errors and
fluctuations. https://doi.org/10.1038/s41467-023-40670-7 SH and TJ are commercial provinces with small areas and Nature Communications| (2023) 14:5379 5 https://doi.org/10.1038/s41467-023-40670-7 Article 10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
20
40
60
80
Predict Error (%)
Gansu
Average Prediction Error
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
20
40
60
80
Predict Error (%)
Shanxi
Average Prediction Error
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
20
40
60
80
Predict Error (%)
Shandong
Average Prediction Error
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
20
40
60
80
Predict Error (%)
Inner Mongolia
Average Prediction Error
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
20
40
60
80
Predict Error (%)
Hubei
Average Prediction Error
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
20
40
60
80
Predict Error (%)
Jiangsu
Average Prediction Error
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
20
40
60
80
Predict Error (%)
Beijing
Average Prediction Error
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
20
40
60
80
Predict Error (%)
Tianjin
Average Prediction Error
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
10
20
30
40
50
Frequency
Tianjin
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
10
20
30
40
50
Frequency
Shandong
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
10
20
30
40
50
Frequency
Shanxi
10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
10
20
30
40
50
Frequency
Gansu
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
10
20
30
40
50
Frequency
Beijing
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
10
20
30
40
50
Frequency
Jiangsu
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
10
20
30
40
50
Frequency
Hubei
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
10
20
30
40
50
Frequency
Inner Mongolia
g. https://doi.org/10.1038/s41467-023-40670-7 4 | Provincial probability distribution function (PDF) and prediction errors to the original power generation data in each generation range: pale turquoise: to the original power generation data in each generation range: pale turquoise:
0–10%; cornflower blue: 10–20%; dark salmon: 20–30%; burlywood: 30–40%; pur-
ple: 40–50%; pale green: 50–60%; light sky blue: 60–70%; yellow: 70–80%; deep sky
blue: 80–90%. Different colors mean the frequency of a certain predicted power
generation is composed of data from different power generation ranges. Each box
shows the distribution of the prediction errors. The lower/upper end of each box
indicates the minimal/maximal value, and the lower and upper percentiles indicate
25% and 75%, respectively. The short red line indicates the median and the bubble
line indicates the average prediction error of each box. Fig. 4 | Provincial probability distribution function (PDF) and prediction errors
in each interval. a–d The upper figures show the PDFs of wind prediction in Tianjin
(TJ), Shandong (SD), Shanxi (SX), and Gansu (GS), and the lower figures show the
wind prediction error in each interval. e–h The upper figure shows the PDFs of solar
prediction in Beijing (BJ), Jiangsu (JS), Hubei (HB), and Inner Mongolia (IM), and the
lower figure shows the solar prediction error in each interval. PDFs and box plots
are missing in some intervals because the power generation does not reach that
range of the installed capacity, such as TJ wind generation only covers 0–90%
capacity. The PDFs plot indicates the distribution of predicted generation data. The
x-axis indicates the predicted power generation range, and the color corresponds to the original power generation data in each generation range: pale turquoise:
0–10%; cornflower blue: 10–20%; dark salmon: 20–30%; burlywood: 30–40%; pur-
ple: 40–50%; pale green: 50–60%; light sky blue: 60–70%; yellow: 70–80%; deep sky
blue: 80–90%. Different colors mean the frequency of a certain predicted power
generation is composed of data from different power generation ranges. Each box
shows the distribution of the prediction errors. The lower/upper end of each box
indicates the minimal/maximal value, and the lower and upper percentiles indicate
25% and 75%, respectively. The short red line indicates the median and the bubble
line indicates the average prediction error of each box. exhibits a large prediction error, ranging from 9.3 to 11.4%, with an
average value of 10.4%, larger than the total prediction error of
3.9–10.0%, with an average value of 6.7%. https://doi.org/10.1038/s41467-023-40670-7 4 | Provincial probability distribution function (PDF) and prediction errors
each interval. a–d The upper figures show the PDFs of wind prediction in Tianjin
J), Shandong (SD), Shanxi (SX), and Gansu (GS), and the lower figures show the
nd prediction error in each interval. e–h The upper figure shows the PDFs of solar
ediction in Beijing (BJ), Jiangsu (JS), Hubei (HB), and Inner Mongolia (IM), and the
wer figure shows the solar prediction error in each interval. PDFs and box plots
e missing in some intervals because the power generation does not reach that
nge of the installed capacity, such as TJ wind generation only covers 0–90%
pacity. The PDFs plot indicates the distribution of predicted generation data. The
axis indicates the predicted power generation range, and the color corresponds
to the original power generation data in each generation range: pale turquoise:
0–10%; cornflower blue: 10–20%; dark salmon: 20–30%; burlywood: 30–40%; pur-
ple: 40–50%; pale green: 50–60%; light sky blue: 60–70%; yellow: 70–80%; deep sky
blue: 80–90%. Different colors mean the frequency of a certain predicted power
generation is composed of data from different power generation ranges. Each box
shows the distribution of the prediction errors. The lower/upper end of each box
indicates the minimal/maximal value, and the lower and upper percentiles indicate
25% and 75%, respectively. The short red line indicates the median and the bubble
line indicates the average prediction error of each box. https://doi.org/10.1038/s41467-023-40670-7 10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
20
40
60
80
Predict Error (%)
Shanxi
Average Prediction Error
Wind Power Generation Range (%) 10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
20
40
60
80
Predict Error (%)
Gansu
Average Prediction Error 10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
20
40
60
80
Predict Error (%)
Shandong
Average Prediction Error 10
20
30
40
50
60
70
80
90
100
Wind Power Generation Range (%)
0
20
40
60
80
Predict Error (%)
Tianjin
Average Prediction Error g
(
)
00
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
10
20
30
40
50
Frequency
Jiangsu 00
10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
10
20
30
40
50
Frequency
Inner Mongolia 00
10
20
30
40
50
60
70
80
90
100
S l
P
G
ti
R
(%)
0
10
20
30
40
50
Frequency
Hubei 100
)
10
20
30
40
50
60
70
80
90
100
S l
P
G
ti
R
(%)
0
10
20
30
40
50
Frequency
Hubei
10
20
30
40
50
60
70
80
90
100
Solar Po er Generation Range (%)
0
10
20
30
40
50
Frequency
Inner Mongolia Inner Mongolia 1
Solar Power Generation Range (%) Solar Power Generation Range (%) 10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
20
40
60
80
Predict Error (%)
Jiangsu
Average Prediction Error
Solar Power Generation Range (%) 10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
20
40
60
80
Predict Error (%)
Beijing
Average Prediction Error
Solar Power Generation Range (%) 10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
20
40
60
80
Predict Error (%)
Hubei
Average Prediction Error
Solar Power Generation Range (%) 10
20
30
40
50
60
70
80
90
100
Solar Power Generation Range (%)
0
20
40
60
80
Predict Error (%)
Inner Mongolia
Average Prediction Error
g
(
) Inner Mongolia Inner Mongolia 1
2
3
4
5
6
7
8
9
10
Solar Power Generation Range (%) 1
Solar Power Generation Range (%) 1
Solar Power Generation Range (%) 1
Solar Power Generation Range (%) 1
Solar Power Generation Range (%) Fig. Nature Communications| (2023) 14:5379 https://doi.org/10.1038/s41467-023-40670-7 As the Chinese government
has issued the Electric Heating Policy to provide heat in North China in
winter, the load demands in the power sector have increased
significantly50. The flexibility-adjustable resources and volatility on the
power source side exhibit inverse distributions, which have become a
central problem in the consumption of renewable energy in these
regions. In contrast, Southeast China achieves the smallest prediction
error in regard to both wind and solar energy in winter, with average
values of 2.8% and 5.1%, respectively. Additionally, existing research
has suggested abundant offshore wind power resources in the area,
with wind capacity factors higher than 50%, almost ranking at the top
in China10,11. Due to the obvious seasonal distribution of offshore wind
power, which dominates in spring and winter51, wind power represents are not suitable for wind and solar energy development. YN, Fujian, GS,
Zhejiang (ZJ), and Guizhou (GZ) should develop wind energy due to
their smallest prediction errors of 2.1%. 2.6%, 4.2%, 4.9%, and 3.8%,
respectively. ZJ, SX, GZ, and SH are some of the top 10 provinces with
larger solar prediction errors, namely, 7.1%, 7.2%, 7.4%, and 10.0%,
respectively, while the wind prediction errors in ZJ, SX, and GZ reach
4.9%, 5.3%, and 3.8%, respectively, and the potential wind capacity
factor for Sichuan and GZ is approximately 15–25%10. Therefore, wind
energy development in these provinces is a recommended pathway to
reduce the adverse impact of renewable generation on power system
operation. are not suitable for wind and solar energy development. YN, Fujian, GS,
Zhejiang (ZJ), and Guizhou (GZ) should develop wind energy due to
their smallest prediction errors of 2.1%. 2.6%, 4.2%, 4.9%, and 3.8%,
respectively. ZJ, SX, GZ, and SH are some of the top 10 provinces with
larger solar prediction errors, namely, 7.1%, 7.2%, 7.4%, and 10.0%,
respectively, while the wind prediction errors in ZJ, SX, and GZ reach
4.9%, 5.3%, and 3.8%, respectively, and the potential wind capacity
factor for Sichuan and GZ is approximately 15–25%10. Therefore, wind
energy development in these provinces is a recommended pathway to
reduce the adverse impact of renewable generation on power system
operation. The temporal analysis demonstrates that renewable generation in
spring exerts the greatest impact on the power system, requiring the
proactive deployment of flexible resources. Combined with the spatial
distribution, the solar prediction error in North China in winter Nature Communications| (2023) 14:5379 6 Article https://doi.org/10.1038/s41467-023-40670-7 Fig. https://doi.org/10.1038/s41467-023-40670-7 Different colors in season arcs differentiate each province’s influence. BJ Beijing, TJ
Tianjin, HE Hebei, SX Shanxi, IM Inner Mongolia, LN Liaoning, JL Jilin, HL Fig. 5 | Temporal analysis of wind and solar prediction errors. a Wind, (c) solar
prediction error in the 30 provinces in spring, summer, autumn, and winter. Each
chord and arc represent the prediction error (%) between a province and the sea-
son, where the thickness is proportional to the level of prediction error. Regarding
province arcs, each segment corresponds to the prediction error in each season;
regarding season arcs, each segment corresponds to the prediction error in each
province. The number next to the arc indicates the cumulative prediction error. Different colors in season arcs differentiate each province’s influence. BJ Beijing, TJ
Tianjin, HE Hebei, SX Shanxi, IM Inner Mongolia, LN Liaoning, JL Jilin, HL a suitable alternative resource to offset the winter load peak in North
and Northeast China. 28.1% and 25.0%, respectively, of the total prediction error in China,
especially during winter, with a proportion of 27.4% and 27.7%. How-
ever, during spring and summer, much energy consumption can be
satisfied by renewable energy, resulting in an unbalance in different
seasons and requiring additional energy sources. As such, the gov-
ernment should improve the power system infrastructure, system-
atically evaluate potential transmission projects, and plan additional
power lines according to the resource and load distribution. Based on the prediction error analysis, we summarize two policy
suggestions for China. First, the government should provide adequate
policy support and incentives to encourage wind energy development
in the Southwestern and Central areas of China and solar energy
development in the areas of Southwest and Northwest China. These
areas experience limited fluctuations in wind and solar generation,
around 2.1–6.4% and 4.3–7.4%, reducing the adverse impact on the
power system. However, the current installed capacities in these
regions are insufficient, even lower than East area with less land. Sec-
ond, the government should plan interprovincial energy transmission
in the space dimension to reduce the winter load peak in North China
and reduce the adverse impact of renewable energy. As concluded, the
wind and solar fluctuations in North China are notable, accounting for https://doi.org/10.1038/s41467-023-40670-7 5 | Temporal analysis of wind and solar prediction errors. a Wind, (c) solar
prediction error in the 30 provinces in spring, summer, autumn, and winter. Each
chord and arc represent the prediction error (%) between a province and the sea-
son, where the thickness is proportional to the level of prediction error. Regarding
province arcs, each segment corresponds to the prediction error in each season;
regarding season arcs, each segment corresponds to the prediction error in each
province. The number next to the arc indicates the cumulative prediction error. Different colors in season arcs differentiate each province’s influence. BJ Beijing, TJ
Tianjin, HE Hebei, SX Shanxi, IM Inner Mongolia, LN Liaoning, JL Jilin, HL
Heilongjiang, SHShanghai, JS Jiangsu, ZJ Zhejiang, AH Anhui,FJ Fujian, JX Jiangxi, SD
Shandong, HA Henan, HB Hubei, HN Hunan, GD Guangdong, GX Guangxi, HI Hai
nan, CQ Chongqing, XZ Tibet, SC Sichuan, GZ Guizhou, YN Yunnan, SN Shaanxi, GS
Gansu, QH Qinghai, NX Ningxia XJ Xinjiang. b Hourly prediction error of wind
power in TJ, SD, SX, and GS. d Hourly prediction error of solar in BJ, JS, HB, and IM
Curves indicate hourly prediction errors (left axis), and bars indicate average pre
diction errors (right axis) in the four seasons: Green—spring; red—summer; yellow—
autumn; and blue—winter. Heilongjiang, SHShanghai, JS Jiangsu, ZJ Zhejiang, AH Anhui,FJ Fujian, JX Jiangxi, SD
Shandong, HA Henan, HB Hubei, HN Hunan, GD Guangdong, GX Guangxi, HI Hai-
nan, CQ Chongqing, XZ Tibet, SC Sichuan, GZ Guizhou, YN Yunnan, SN Shaanxi, GS
Gansu, QH Qinghai, NX Ningxia XJ Xinjiang. b Hourly prediction error of wind
power in TJ, SD, SX, and GS. d Hourly prediction error of solar in BJ, JS, HB, and IM. Curves indicate hourly prediction errors (left axis), and bars indicate average pre-
diction errors (right axis) in the four seasons: Green—spring; red—summer; yellow—
autumn; and blue—winter. Fig. 5 | Temporal analysis of wind and solar prediction errors. a Wind, (c) solar
prediction error in the 30 provinces in spring, summer, autumn, and winter. Each
chord and arc represent the prediction error (%) between a province and the sea-
son, where the thickness is proportional to the level of prediction error. Regarding
province arcs, each segment corresponds to the prediction error in each season;
regarding season arcs, each segment corresponds to the prediction error in each
province. The number next to the arc indicates the cumulative prediction error. Comparative prediction models In this paper, we compare four prediction methods including RF,
FCNN, RNN, and SVM. These four methods are all sample-based pre-
diction approaches. We begin by constructing the samples using 168-h
wind and solar generation data as input features and extracting sub-
sequences of 2, 6, and 24 h as output for 2-h, 6-h, and 24-h step pre-
dictions, respectively. The RF method employs a tree-based prediction
model that builds multiple decision trees during training. The struc-
ture of the decision trees is determined by parameters such as tree
depth, the number of trees, and the maximum number of features
considered when splitting nodes. The FCNN method utilizes a network
structure consisting of interconnected perceptron. Each time slot’s
generation data serves as an input feature for the FCNN, and the pre-
dicted generation is the output. The network structure is designed
based on factors such as regularization, batch size during training,
learning rate, and the number of neurons in each layer. The RNN is a
neural network structure specifically designed for time series data,
incorporating hidden variables to carry information from previous
time slots. Similar to the FCNN, the RNN’s network structure is deter-
mined by parameters including the number of neurons, batch size, and
learning rate. The SVM is an initial machine learning method employed
to separate the dataset. The SVM solves an optimization problem to
find an optimal hyperplane. Key considerations for SVM include reg-
ularization parameters, the margin of tolerance around predicted
regression values, and the influence attributed to each sample. Further
details on the network parameters and the tuning process can be
found in the Supplementary Note and Supplementary Table 5. Xt = ð1 BÞdY t,ADFtestðXtÞ = 1,
ð3Þ ð3Þ where X t is the stationary series of the original real data, B is the lag
operator, and ADFtest = 1 passes the stationarity test. Except for the
differential order d, the ARIMA model should also determine the
autoregressive order p and moving average order q, and the ARMA
model for Xt can be expressed as Eq. Wind and solar output data Hourly wind and solar output data for 2016 pertaining to 30 provinces
of China are retrieved from previous work11, except for Tibet wind,
Chongqing solar, Taiwan, Hong Kong, and Macao. The dataset con-
tains 8760 h of wind and solar output data, and wind and solar installed Nature Communications| (2023) 14:5379 7 Article https://doi.org/10.1038/s41467-023-40670-7 generally smaller than three because the greater the difference order,
the more information would be lost52. It should be noted that para-
meter d is completely determined by the properties of the original
sequence, while the selection of p and q should consider the overall
prediction effect. In general, p and q should remain within 1/5 of the
length of the input data. Due to the large amount of wind and solar
power generation data in each province in one year, usually 8760 h, we
separate multiple prediction windows for each province and used the
moving window method to predict wind and solar power generation. At present, the methods for p and q determination usually include the
Akaike information criterion (AIC) and Bayesian information criterion
(BIC), but the optimal parameter configuration can only be provided
for a single prediction window. To unify the prediction models with the
different prediction windows in the same provinces and minimize the
prediction error, we randomly select 5 weeks of data throughout the
year as a sample and traverse p and q for each province to obtain the
best parameters with the minimum prediction error. The detailed
parameters for each province are listed in Supplementary Table 4. generally smaller than three because the greater the difference order,
the more information would be lost52. It should be noted that para-
meter d is completely determined by the properties of the original
sequence, while the selection of p and q should consider the overall
prediction effect. In general, p and q should remain within 1/5 of the
length of the input data. Due to the large amount of wind and solar
power generation data in each province in one year, usually 8760 h, we
separate multiple prediction windows for each province and used the
moving window method to predict wind and solar power generation. capacity data for these 30 provinces are included. Wind and solar output data We denote the
hourly wind output as W i,t + 1,0 and the hourly solar output as Si,t + 1,0,
where i and t are province and time slot indices, respectively, for
i 2 ½1,N,t 2 ½1,T, N = 30, and T = 8760. As previously mentioned, daily
wind and solar output data are also required for the analysis, which can
be calculated as Eqs. (1)-(2): W Day,i,c,0 = maxðW i,t,0,W i,t + 1,0, W i,t + 23,0Þ,t = 24 ðc 1Þ
ð1Þ W Day,i,c,0 = maxðW i,t,0,W i,t + 1,0, W i,t + 23,0Þ,t = 24 ðc 1Þ
ð1Þ At present, the methods for p and q determination usually include the
Akaike information criterion (AIC) and Bayesian information criterion
(BIC), but the optimal parameter configuration can only be provided
for a single prediction window. To unify the prediction models with the
different prediction windows in the same provinces and minimize the
prediction error, we randomly select 5 weeks of data throughout the
year as a sample and traverse p and q for each province to obtain the
best parameters with the minimum prediction error. The detailed
parameters for each province are listed in Supplementary Table 4. SDay,i,c,0 = maxðSi,t,0,Si,t + 1,0, Si,t + 23,0Þ,t = 24 ðc 1Þ
ð2Þ where SDay,i,c,0 and WDay,i,c,0 are the daily solar and wind output,
respectively, of province i in time slot t, and c is a day index,
for c 2 1,C
½
and C = 365. Comparative prediction models (4): 1
Xp
i = 1φiBi
Xt = μ0 + ð1
Xq
i = 1μiBiÞαt,
ð4Þ ð4Þ where φi and μi are the autoregressive parameter and moving average
parameter, respectively, αt is white noise with a mean of 0, μ0 is a
deterministic trend quantity greater than 0, and Bi is the ith power of B. Via the use of the prediction model, we can obtain the predicted series
Xpredict,t, which is a differential series of the predicted wind and solar
power generation. Thus, the predicted power generation can be
obtained through Eq. (5): Y predict,t = ð1 BÞdX predict,t,
ð5Þ ð5Þ where Y predict,t denotes the predicted results of the ARIMA-based
prediction model, and in this paper, this variable indicates the wind
and solar output. Benchmark prediction model Time series prediction is based on historical data, among which the
autoregressive (AR), moving average (MA), and autoregressive moving
average (ARMA) techniques are typical methods to study stationary
time series and are suitable for a large number of problems. However,
the fluctuations in wind and solar energy indicate that their power
generation involves a nonstationary time series with a time-varying
mean value and variance, which is difficult to study with these meth-
ods. Thus, to predict nonstationary sequences, the ARIMA prediction
model is introduced by Box-Jerkins. Considering a certain number of
differences in the ARIMA prediction model, wind and solar power
generation series can be converted into a stationary series, convenient
for prediction analysis. In the literature, the ARIMA model is widely
used in short-term renewable forecasting and is validated to yield
satisfactory results. Other parameters, such as the autoregressive parameter φi and
moving average parameter μi, can vary with the input data. These two
parameters are determined by the autocorrelation coefficient and
autocovariance, respectively, which can be obtained with the
Yule–Walker estimation, least squares estimation or maximum like-
lihood estimation method53. In this paper, we build the ARIMA-based
prediction model, and all the parameters except p, d, and q could be
automatically generated. In this paper, we set 6 h as the prediction time scale and 168 h as
the input data dimension to predict wind and solar power generation. The reason is that 6 h-ahead forecast of renewable generation is widely
used for power system scheduling and electricity trading in practice. The 6 h-ahead forecast also results in moderate errors that can serve as
a benchmark for the uncertainty analysis. In prediction model construction, it is necessary to first determine
whether the series is stationary. If the series is not stationary, it should
be differentiated until the series meets the stationarity requirements. Suppose the real wind and solar power generation series are Y t, the
differential order can be denoted by d, and the differential process can
be expressed as Eq. (3): Nature Communications| (2023) 14:5379 Code availability ð8Þ The code used in this study is available from the authors upon rea-
sonable request. where FDay,i,c is the daily first-order difference in province i on day c. We also calculate the average value over 365 days to evaluate the solar
energy fluctuations in a given province. First-order difference The first-order difference can be used to assess the variation in discrete
time-series data. With the use of the first-order difference, we can
obtain the increment in the original data, which can reflect gradient
information. In this paper, prediction is conducted hour-by-hour, and
the prediction accuracy is primarily determined by the hourly change
in the generation data. Thus, in terms of wind energy, we use the first-
order difference of hourly wind generation data to measure the hourly
change, which can be calculated as Eq. (7): PDay,i,c = 1, SDay,i,c,0 SDay,i,c1,0 < 0,SDay,i,c1,0 SDay,i,c2,0 ≥0,SDay,i,c2,0
SDay,i,c3,0 ≥0,SDay,i,c1,0 SDay,i,c3,0 ≥0:1 CS,i,
ð12Þ ð12Þ PN,Day,i =
X
c2CPDay,i,c,
ð13Þ
PR,Day,i = PN,Day,i=C,
ð14Þ ð13Þ ð14Þ FH,i,t = W i,t + 1,0 W i,t,0
CW,i
,
ð7Þ ð7Þ where PDay,i,c is the daily peak in province i on day c, PN,Day,i is the
number of daily peaks in province i, and PR,Day,i is the ratio of daily
peaks in province i. The average value over 365 days is also calculated
to express the solar energy fluctuations in each province. where FH,i,t is the hourly first-order difference in province i in time
slot t and W i,t + 1,0 and W i,t,0 are the real wind energy generation in
time slots t + 1 and t, respectively. When evaluating the hourly first-
order difference in a province, we calculate the average value
over 8760 h. Data availability The source data underlying Figs. 1–5 and Supplementary Figs. 1-4,
including the data of provincial wind and solar power generation of the
30 provinces in China, are provided as a Source Data file. Other data
used in this study are available from the authors upon reasonable
request. Source data are provided with this paper. Regarding solar energy, power generation exhibits daily periodi-
city, so we use daily solar energy generation data to measure the
fluctuation, which can be expressed as Eq. (8): FDay,i,c = SDay,i,c + 1,0 SDay,i,c,0
CS,i
,
ð8Þ References 1. China Xinhua News. Xi’s statement at the General Debate of the 75th
Session of the United Nations General Assembly (2020). [http://
www.qstheory.cn/yaowen/2020-09/22/c_1126527766.htm] (2022). 1. China Xinhua News. Xi’s statement at the General Debate of the 75th
Session of the United Nations General Assembly (2020). [http://
www.qstheory.cn/yaowen/2020-09/22/c_1126527766.htm] (2022). Article predicted wind and solar energy generation, and the prediction error
can then be calculated as Eq. (6): predicted wind and solar energy generation, and the prediction error
can then be calculated as Eq. (6): PN,H,i =
X
t2TPH,i,t,
ð10Þ ð10Þ εW,i,t = W i,t,* W i,t,0
CW,i
100%, εS,i,t = Si,t,* Si,t,0
CS,i
100%,
ð6Þ ð6Þ PR,H,i = PN,H,i=T
ð11Þ PR,H,i = PN,H,i=T
ð11Þ ð11Þ where PH,i,t denotes the hourly peaks in province i in time slot t, PN,H,i is
the number of hourly peaks in province i, and PR,H,i is the ratio of
hourly peaks in province i. We also calculate the average value over
8760 h to evaluate the wind energy fluctuations in each province. where εW,i,t and εS,i,t are the wind and solar prediction error in province
i in time slot t, W i,t,* and Si,t,* are the predicted wind and solar output,
respectively, of province i in time slot t, and CW,i and CS,i are the wind
and solar installed capacities, respectively, in province i. When deter-
mining the prediction error in a given province, we calculate the
average value over 8760 h. Regarding solar energy, we use daily power generation data to
obtain daily peaks. Similar to the hourly peak calculation, four con-
secutive days are chosen to determine peaks, and similar conditions
should be satisfied, which can be expressed as Eqs. (12)–(14): Analysis and calculation of the peak ratio 2. Climate Ambition Summit. Leaders statements of president Xi
Jinping (2020). [http://www.gov.cn/xinwen/2020-12/13/content_
5569138.htm] (2022). 2. Climate Ambition Summit. Leaders statements of president Xi
Jinping (2020). [http://www.gov.cn/xinwen/2020-12/13/content_
5569138.htm] (2022). In this paper, we use the peak ratio to evaluate the prediction error. It
should be noted that all the prediction methods learn the variation
tendency of a given data series to predict future data. The easier a
tendency is to learn, the more accurate the prediction. Thus, we aim to
obtain a feature that could indicate the change in tendency to better
measure the prediction error. The peaks of series data indicate
inflection points, with previous data exhibiting an upward tendency
and subsequent data exhibiting a downward tendency, which is a key
feature reflecting the tendency change. 3. PrakashKumar, K. & Saravanan, B. Recent techniques to model
uncertainties in power generation from renewable energy sources
and loads in microgrids–A review. Renew. Sustain. Energy Rev. 71,
348–358 (2017). 3. PrakashKumar, K. & Saravanan, B. Recent techniques to model
uncertainties in power generation from renewable energy sources
and loads in microgrids–A review. Renew. Sustain. Energy Rev. 71,
348–358 (2017). 4. Mahdi, S., Helena, L. & Nilay, S. Integrated renewable electricity
generation considering uncertainties: the UK roadmap to 50%
power generation from wind and solar energies. Renew. Sustain. Energy Rev. 72, 385–398 (2017). 4. Mahdi, S., Helena, L. & Nilay, S. Integrated renewable electricity
generation considering uncertainties: the UK roadmap to 50%
power generation from wind and solar energies. Renew. Sustain. Energy Rev. 72, 385–398 (2017). In regard to wind energy, we use four consecutive time slots to
determine hourly peaks and traverse the time series to find all peaks,
i.e., t = t + 1. The power generation in these four time slots should
satisfy the following conditions to reach a peak: the first three hours
should continuously increase, the first three hours should increase by
more than 10% of the installed capacity, and the fourth hour should
decrease, which can be expressed as Eqs. (9)–(11): 5. Salvador, P., Juan, M. & Trine, B. Impact of forecast errors on
expansion planning of power systems with a renewables target.Eur. J. Oper. Res. 248, 1113–1122 (2016). 5. Salvador, P., Juan, M. & Trine, B. Impact of forecast errors on
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model: differential order d, autoregressive order p, and moving aver-
age order q. Parameter d is determined based on the minimum number
of differences required to obtain a stationary time series. The d value is In this paper, the prediction error of wind and solar energy could be
calculated as the unit megawatt (MW) prediction error. When using the
ARIMA-based benchmark prediction model, we could obtain the Nature Communications| (2023) 14:5379 8 https://doi.org/10.1038/s41467-023-40670-7 Article Nature Communications| (2023) 14:5379 Analysis and calculation of the peak ratio Preliminary wind resource
assessment in South Sudan using reanalysis data and statistical
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W i,t3,0 ≥0,W i,t1,0 W i,t3,0 ≥0:1 CW,i,
ð9 PH,i,t = 1, W i,t,0 W i,t1, 0< 0,W i,t1,0 W i,t2,0 ≥0, W i,t2,0
W i,t3,0 ≥0,W i,t1,0 W i,t3,0 ≥0:1 CW,i,
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no. 2022YFB2405600 for supporting J.W. and G.H. and the National
Natural Science Foundation of China under grant No. 72241429,
No. 72271008, No. 72243007, and No. 52277092 for supporting J.S.,
G.H., X.L., and J.W. We also acknowledge the support of State Grid
Corporation of China, State Grid Jiangsu Electric Power Co., LTD. and
State Grid Wuxi Power Supply Company. Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
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use, you will need to obtain permission directly from the copyright
holder. To view a copy of this licence, visit http://creativecommons.org/
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E.D., N.L., J.M., M.S., C.L., J.S., X.L., C.W.T. and G.H. contributed to the
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Supplementary information The online version contains
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https://doi.org/10.1038/s41467-023-40670-7. Peer review information Nature Communications thanks Mingquan Li,
and the other, anonymous, reviewer(s) for their contribution to the peer
review of this work. A peer review file is available. Peer review information Nature Communications thanks Mingquan Li,
and the other, anonymous, reviewer(s) for their contribution to the peer
review of this work. A peer review file is available. 52. Amini, M. H., Kargarian, A. & Karabasoglu, O. ARIMA-based decou-
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378–390 (2016). and the other, anonymous, reviewer(s) for their contribution to the peer
review of this work. A peer review file is available. Additional information 49. Hunt, J. D. et al. Global resource potential of seasonal pumped
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6054 (2020). Correspondence and requests for materials should be addressed to Xi
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The authors declare no competing interests. p
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Polar front associated variation in prokaryotic community structure in Arctic shelf seafloor
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Frontiers in microbiology
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INTRODUCTION The microbial communities in the upper sediment layers in
marine environments show a steeper decay in similarity with
distance than assemblies of the pelagic water masses, which
may be attributed to more pronounced environmental gradients
within the sediments and more restricted dispersal of sediment
microorganisms. Additionally, the more heterogeneous environ-
ments in coastal areas have been found to generate steeper gradi-
ents than such found in the open ocean both in the seawater and
sediments (Zinger et al., 2014). The environmental conditions on
the continental shelf seafloors may in several respects be char-
acterized as intermediate between those of the deep ocean and
the shallow coastal areas. Due to the combination of less water
depth and frequently much higher primary production than in
the open oceans, the shelf sediments will receive higher influxes
of sedimentary material (Suess, 1980) that sustain stronger het-
erotrophic activity. The bottom-dwelling fauna, including bio-
turbating animals (Bertics and Ziebis, 2009) and demersal fishes,
contributes to resuspension of sediment particles into the water
column, as anthropogenic influences (e.g., from bottom trawl-
ing) may do as well. Moreover, because ocean currents, including
tidal currents, have often been found to be of great importance at
the shelf seafloors, microorganisms are likely dispersed quite effi-
ciently, thereby making mass effects a potentially import factor Microbial community similarities tend to show a distance decay
relationship, implying that the phylogenetic composition of
communities becomes increasingly dissimilar with increasing
geographical distance. It is now generally accepted that both
contemporary environmental parameters and historical contin-
gencies, maintained by dispersal limitation, may contribute to
this beta-diversity. Hence, the classical Baas Becking statement
“Everything is everywhere, but the environment selects” is ques-
tioned as a universal model for explaining the observed variation
in microbial community composition (Hedlund and Staley, 2003;
Martiny et al., 2006; Hanson et al., 2012). In the conceptual
framework of metacommunity ecology (Leibold et al., 2004;
Logue and Lindström, 2008) this emphasis on local environmen-
tal factors vs. spatial (regional) effects largely coincides with the
distinction between species sorting and mass effects as the two
models best explaining microbial community assembly dynam-
ics (Lindström and Langenheder, 2012). Edited by: Edited by:
Jürg Brendan Logue, Lund
University, Sweden
Reviewed by:
Mark Alexander Lever, ETH Zürich,
Switzerland
Marie-Ève Garneau, University of
Zurich, Switzerland
*Correspondence:
Tan T. Nguyen, Faculty of
Biosciences, Fisheries and
Economics, Norwegian College of
Fishery Science, UiT The Arctic
University of Norway, Breivika,
N-9037, Tromsø, Norway
e-mail: tan.t.nguyen@uit.no *Correspondence:
Tan T. Nguyen, Faculty of
Biosciences, Fisheries and
Economics, Norwegian College of
Fishery Science, UiT The Arctic
University of Norway, Breivika,
N-9037, Tromsø, Norway
e-mail: tan.t.nguyen@uit.no Tan T. Nguyen 1* and Bjarne Landfald 2 Tan T. Nguyen 1* and Bjarne Landfald 2
1 Centre for Research-based Innovation on Marine Bioactives and Drug Discovery (MabCent-SFI), UiT The Arctic University of Norway, Tromsø, Norway
2 Faculty of Biosciences, Fisheries and Economics, Norwegian College of Fishery Science, UiT The Arctic University of Norway, Tromsø, Norway Spatial variations in composition of marine microbial communities and its causes have
largely been disclosed in studies comprising rather large environmental and spatial
differences. In the present study, we explored if a moderate but temporally permanent
climatic division within a contiguous arctic shelf seafloor was traceable in the diversity
patterns of its bacterial and archaeal communities. Soft bottom sediment samples were
collected at 10 geographical locations, spanning spatial distances of up to 640 km,
transecting the oceanic polar front in the Barents Sea. The northern sampling sites were
generally colder, less saline, shallower, and showed higher concentrations of freshly
sedimented phytopigments compared to the southern study locations. Sampling sites
depicted low variation in relative abundances of taxa at class level, with persistent
numerical dominance by lineages of Gamma- and Deltaproteobacteria (57–66% of
bacterial sequence reads). The Archaea, which constituted 0.7–1.8% of 16S rRNA
gene copy numbers in the sediment, were overwhelmingly (85.8%) affiliated with the
Thaumarchaeota. Beta-diversity analyses showed the environmental variations throughout
the sampling range to have a stronger impact on the structuring of both the bacterial and
archaeal communities than spatial effects. While bacterial communities were significantly
influenced by the combined effect of several weakly selective environmental differences,
including temperature, archaeal communities appeared to be more uniquely structured by
the level of freshly sedimented phytopigments. Keywords: archaea, bacteria, Barents Sea, beta-diversity, sediment, 16S rRNA gene ORIGINAL RESEARCH ARTICLE
bli h d 23 J
2015 Edited by:
Jürg Brendan Logue, Lund
University, Sweden
Reviewed by:
Mark Alexander Lever, ETH Zürich,
Switzerland
Marie-Ève Garneau, University of
Zurich, Switzerland
*Correspondence:
Tan T. Nguyen, Faculty of
Biosciences, Fisheries and
Economics, Norwegian College of
Fishery Science, UiT The Arctic
University of Norway, Breivika,
N-9037, Tromsø, Norway
e-mail: tan.t.nguyen@uit.no INTRODUCTION The disentanglement of
these different effects is, however, not trivial in many systems due
to spatial autocorrelation or co-variations among environmental
variables (Horner-Devine et al., 2004; Böer et al., 2009; Zinger
et al., 2011; Bienhold et al., 2012; Jacob et al., 2013; Wang et al.,
2013). January 2015 | Volume 6 | Article 17 | 1 www.frontiersin.org Prokaryotic diversity in marine sediment Nguyen and Landfald FIGURE 1 | The western Barents Sea with geographical positions of
sampling stations. Red color (southern part); blue color (northern part),
green color (transition temperature zone). in the establishment of microbial community assemblies in this
habitat type. The Barents Sea (1.4 mill km2) is part of the circumpolar
Arctic Continental Shelf. It extends northwards from the north-
ern coasts of Norway and Russia to the Arctic Ocean, and is
delimited by the Novaya Zemlya and the Norwegian Sea along the
east-west axis. With an average depth of 230 m, it is the deepest of
the Arctic shelf seas. It is also characterized by less coastal erosion
and river water inflow than other Arctic shelf seas (Vetrov and
Romankevich, 2004). The most distinctive oceanographic feature
of the Barents Sea is, however, the influx of temperate and salty
Atlantic water from the southwest. These water masses meet and
mix with sub-zero, less saline Arctic Ocean water from the north,
resulting in a coarse division of the Barents Sea into a northern
and a southern region separated by a transition zone named the
polar front (Ingvaldsen and Loeng, 2009). The temperature dif-
ferences are most pronounced in the surface waters, resulting in
winter sea ice covering the northern regions, while the southern
parts of the Barents Sea are ice-free throughout the entire year. Near the seafloor, the temperature difference is modest, i.e., about
2◦C, and it has even shown a diminishing trend in recent years
(Lind and Ingvaldsen, 2012). The overall primary production is
highest in the southern parts (Sakshaug et al., 2009) but the depo-
sition of organic material shows a more patchy pattern, caused by
additional factors like water depth, bottom topography and local
currents (Vetrov and Romankevich, 2004). FIGURE 1 | The western Barents Sea with geographical positions of
sampling stations. Red color (southern part); blue color (northern part),
green color (transition temperature zone). BACTERIAL AND ARCHAEAL ABUNDANCES Quantification of 16S ribosomal RNA genes was used for the
estimation of prokaryotic cell densities. Quantitative real-time
PCR (qPCR) was performed on an ABI 7500Fast real-time PCR
system (Applied Biosystems, NYSE, Waltham, MA, USA) using
primers 27F/338R for Bacteria and A571F/915R for Archaea (see
Supplementary Table S1). The environmental DNA samples were
run in duplicate with three dilutions of the primary extract (10−1
to 10−3). Standard curves for threshold cycle (Ct) vs. logarithm
of the start concentration of 16S rRNA gene copies, from 106
to 101, were established with Escherichia coli K10 for Bacteria
and Methanoplanus petrolearius DSM11571 for Archaea. This cor-
responded to E. coli genomic DNA being serially diluted from
0.76 to 0.76 × 10−5 ng and M. petrolearius diluted from 1.56
to 1.56 ×10−5 ng. Genomic standards were included in each
qPCR run to ensure linearity and expected slope values of the
Ct/log[gDNA] curves. SAMPLING Sediment samples were taken from 10 locations in the western
Barents Sea separated by up to 640 km. Sampling was carried
out over the course of 3 days from 20th to 23rd May 2009. The
sampling was done along a curved transect that followed the grad-
ually more shallow Bear Island – Hopen channel from close to the
continental slope to east of the Svalbard archipelago (Figure 1). Seawater temperature and salinity, as measured within 10 m of the
seafloor by a CTD instrument, were used as proxies for seafloor
values. The upper 4 cm sediment cores of van Veen grab sam-
ples were pressed into sterile plastic tubes. The content of each
core was homogenized by mixing and stored frozen at -80◦C until
processing in the laboratory. INTRODUCTION On this background, the primary aim of present study was to
explore if significant community variations could be detected in a
sampling area, which encompassed the moderate environmental
variations of the Barents Sea polar front. And if such variations
were detectable, should they be attributed to environmental or
spatial effects, or both. Beta-diversity analyses were based on
16S rRNA gene sequence data obtained by 454 pyrosequencing. Additionally, the study provided a comprehensive picture of the
prokaryotic alpha-diversities in the upper centimeters of this kind
of arctic shelf seafloor. (LECO Corporation, St. Joseph, MI, USA). Sediment chlorophyll
a (Chl a) and phaeophytin were determined by a Turner 7000
fluorometer (Turner Designs Inc., Synnyvale, CA, USA) from
readings at 665 nm in ethanol extracts before and after treatment
with 1 M acetic acid (Pápista et al., 2002). DNA EXTRACTION Total DNA was extracted from duplicate 0.5 g samples of each
site using the PowerSoil™DNA Isolation kit (Mo Bio Labs, Inc.,
Carlsbad, CA, USA) according to the manufacturer’s instructions. The concentration and quality of extracted DNA were determined
by a NanoDrop ND-1000 spectrophotometer (Thermo Scientific,
Wilmington, DE, USA). STATISTICAL ANALYSES A geographical distance matrix was calculated from the latitude
and longitude coordinates obtained by the Global Positioning
System by use of the package fossil (Vavrek, 2011) in the R statisti-
cal software (R Development Core Team, 2008). The community
beta-diversities were determined by the Bray-Curtis, Sørensen
and phylogenetic distance based unweighted UniFrac indices, as
implemented in the QIIME and R software packages (Lozupone
et al., 2006; R Development Core Team, 2008). The community
distance matrices were based on jackknifing (100 permutations)
with 75% of the sequence number in the sample with the lowest
number of sequences. To visualize the grouping patterns of the samples based
on community distances, non-metric multidimensional scaling
(NMDS) based on the Hellinger transformed Bray-Curtis dis-
tance metric was used (Legendre and Gallagher, 2001). Vector
fitting was employed to identify directions and strengths of the
effects of environmental factors and geographical distance in
relation to the community-based ordination of samples, in accor-
dance with the procedure of Monier et al. (2014). This included
the use of the envfit function of the vegan package in R (Oksanen
et al., 2012). The combinations of environmental variables that best
explained community variation among the sampling stations
were obtained as the ones generating maximum rank correlations
between the environmental and community distance matrices
(Clarke and Ainsworth, 1993) by employing the bioenv proce-
dure in the vegan R package. Generalized linear models (GLM)
were subsequently constructed in R from the standardized envi-
ronmental variables to quantify their relative importance and test
the significance of the individual environmental factors by using
the glm function. To partition the possible community struc-
turing effects of geography and environmental factors, partial
Mantel tests were used (Legendre and Legendre, 1998; Martiny
et al., 2011). To test if southern and northern communities were
significantly different, a multivariate generalized linear models
approach (Warton et al., 2012) was employed as implemented
in the R package mvabund (Wang et al., 2012). The model that
was fitted is log-linear and assumes a negative binomial distribu-
tion of data. To determine which taxa contributed the most to the
differences between the two regions, the univariate ANOVA func-
tion with adjusted p-values for multiple testing in mvabund was
used. Community distance decays were calculated by regressing
the community distance matrices on the geographical distance
matrices. SEDIMENT CHARACTERISTICS Grain size distribution was determined by dry sieving. The sed-
iment samples were separated into two grain size classes, i.e.,
clay/silt (<63 μm) and sand/gravel (>63 μm). Total organic car-
bon (TOC) content was analyzed by a LECO CS-200 Analyzer January 2015 | Volume 6 | Article 17 | 2 Frontiers in Microbiology | Aquatic Microbiology Prokaryotic diversity in marine sediment Nguyen and Landfald STATISTICAL ANALYSES The significance of these decays was determined by sim-
ple Mantel tests based on Spearman rank correlation coefficients
(ρ) with 104 Monte Carlo permutations. The same procedure was
used for testing the relationships between geographical and envi-
ronmental distances. Tests for correlations between bacterial and
archaeal abundances and environmental variables were also based
on Spearman rank correlation coefficients. AMPLIFICATION AND MULTIPLEX PYROSEQUENCING OF 16S rRNA
GENES 2010). The singletons constituted 62.8 and 38.2% of the primary
bacterial and archaeal datasets, respectively. OTU richness was
calculated by the non-parametric Chao1 estimator (Chao, 1984)
after normalization of the sequence numbers in each sample to
4000 for the Bacteria and 9000 for the Archaea. Tagged PCR primers for each sampling station were constructed
by adding unique oligonucleotides to the universal forward
primers 27F for Bacteria and 571F for Archaea (Supplementary
Table S1). The 25 μL PCR reaction mixtures contained 1X
PCR buffer (Invitrogen, Waltham, MA, USA), 0.2 mM dNTPs
(Invitrogen), 0.5 μM of each primer (Eurofins MWG, Ebersberg,
Germany), 1.25 U of Taq polymerase (Invitrogen), and 10 ng of
genomic DNA template. The thermocycler (Applied Biosystems)
conditions were: initial denaturation step at 94◦C for 5 min; 30
cycles at 94◦C for 30 s, 55◦C for 30 s, and 72◦C for 1 min; a final
extension at 72◦C for 5 min. To minimize potential random PCR
biases, each sample was amplified in sextuplicate (triplicates of
each DNA isolation). Correctly sized amplification products were
extracted from the gel by use of the QIAquick Gel Extraction kit
(Qiagen, Hilden, Germany), and replicate samples were pooled
and purified one more time with Agencourt AMPure XP beads
(Beckman Coulter, Brea, CA, USA). Equal amounts of ampli-
cons from each PCR run were pooled and subjected to multiplex
pyrosequencing using a 454/Roche GS-FLX Titanium instru-
ment (454 Life Sciences, Branford, CT, UAS) installed at the
Norwegian High Throughput Sequencing Centre (NSC, Oslo,
Norway; http://www.sequencing.uio.no). The bacterial (BM) and
archaeal (AM) amplicons were sequenced separately, as was a
second bacterial preparation from sampling station 6 (D6). The
latter was subjected to a deeper sequencing effort than used in the
multiplex analysis. The raw sequence data have been submitted
to the EMBL database under the accession numbers ERP003605
(BM dataset), ERP003606 (AM dataset), and ERP003607 (D6
dataset). www.frontiersin.org PROKARYOTIC DIVERSITY The sequence datasets comprised 65 904, 139 590, and 164
880 qualified reads (excluding reads representing singletons)
from the sequencing of the bacterial (BM) and archaeal (AM)
multiplex amplicons of the transect and a deeper bacterial
sequencing of station 6 (D6). A high bacterial diversity was con-
firmed in this Barents Sea sediment sample as the numbers of
unique OTUs obtained both from the multiplex BM and the
single station D6 material exceeded 5500 at ≥97% sequence
identity (Table 2). The 21-fold deeper D6 sequencing of sta-
tion 6, as compared with the BM data, led to a more than Table 2 | Pyrosequencing statistics, number of operational taxonomic
units at 97% similarity level and richness estimates. St
Bacteria
Archaea
Qualified
OTUs
Chao1*
Qualified
OTUs
Chao1*
reads
reads
1
6148
1799
2842
12,023
253
369
2
6148
1856
2728
13,613
266
343
3
4033
1583
3593
18,502
695
883
4
11,417
2872
3542
18,510
585
594
5
5387
1604
2963
9006
209
353
6
7761
2116
3339
11,821
805
1044
7
7049
2096
3392
18,271
972
1184
8
5157
1578
2918
10,704
492
659
9
8238
2263
3064
14,410
578
696
10
4566
1578
3369
12,730
650
876
D6
164,880
9072
14,016
Abbreviations: St, Station; OTUs, operational taxonomic units. D6; Deeper
sequence at station 6. *Computed on quality read subsampled at an even depth of 4000 sequences
for bacteria and 9000 sequences for archaea. Table 2 | Pyrosequencing statistics, number of operational taxonomic
units at 97% similarity level and richness estimates. St Bacterial 16S rRNA gene copy numbers varied in the range
of 3.1 × 109 to 1.7 × 1010 per g dry sediment, and the Archaea
constituted 0.7 to 1.8% of total 16S rRNA copy numbers in
the corresponding samples (Table 1). If employing the empiri-
cal average rRNA operon numbers of 3.9 for Bacteria and 1.8
for Archaea (Lee et al., 2009), the quantitative PCR figures cor-
responded to 7.9 × 108 to 4.4 × 109 bacterial cells per g and
3.2 × 107 to 1.5 × 108 archaeal cells per g, respectively. A posi-
tive correlation was observed between the bacterial and archaeal
copy number log abundance values (Spearman ρ = 0.75; p =
0.01). ENVIRONMENTAL VARIATION AND PROKARYOTIC ABUNDANCES ONMENTAL VARIATION AND PROKARYOTIC ABUNDANC Sediment samples from 10 stations separated by up to 640 km
were collected during a time period of 3 days, implying that
the impact of temporal changes due to the length of the sam-
pling period was minimized. The temperature recordings through
the sampling area confirmed a consistent drop of roughly 1.6◦C
at the seafloor, when moving from the southern stations (1–4)
to the northern ones (6–10), while station 5 was in a transi-
tional temperature zone (Table 1). The temperature variation
showed significant spatial autocorrelation (Spearman ρ = 0.87;
p = 0.001), as did the additional environmental factors water
depth (Spearman ρ = 0.87; p = 0.001), salinity (Spearman ρ =
0.39; p = 0.03), and Chl a/phaeophytin ratio (Spearman ρ =
0.41; p = 0.02), the latter being used as indicator of freshly sed-
imented phytopigment material. On the other hand, the grain
size distribution and organic content of the sediment showed a
more random variation between the sampling stations. Principal
component ordination, based on the environmental data, sep-
arated the sampling stations in accordance with the south-
north dichotomy along PC1 (Figure S1 in the Supplementary
Information). Noticeably, the peak phytopigment concentration
at station 6 was reflected in the fraction of the putative chloro-
plast 16S rRNA gene sequence reads to the total sequence reads,
which also showed a distinct maximum at station 6 (Table 1). SEQUENCE ANALYSES Quality checks, OTU clusterings and phylogenetic annotations
of the sequences were all done by the Quantitative Insights Into
Microbial Ecology (QIIME v.1.8.0) pipeline (Caporaso et al.,
2010b). In brief, low quality sequences were removed, includ-
ing sequences shorter than 150 bp or with a quality score below
25. Furthermore, sequences containing ambiguous nucleotides or
homopolymers longer than six nucleotides were removed (Huse
et al., 2007) using Denoiser software (v.0.91) (Reeder and Knight,
2010). Putative chimeras were identified by ChimeraSlayer and
discarded (Haas et al., 2011). The overall numbers of pyro-
tags were reduced by 26.0% for Bacteria and 12.9% for Archaea
by removing low-quality, chimeric and chloroplast-affiliated
reads. The qualified sequences were clustered into Operational
Taxonomic Units (OTUs) based on 97% sequence similarity
by the UCLUST algorithm (Edgar, 2010), and representative
sequences from each OTU were aligned to the GreenGenes (ver-
sion May 2013) public database (http://greengenes.lbl.gov) using
the PyNAST tool, as integrated in the QIIME package (DeSantis
et al., 2006; Caporaso et al., 2010a). Taxon assignments were
obtained with 80% bootstrap cutoffs for both Bacteria and
Archaea. Singletons, i.e., OTUs with only one sequence, were removed
as putative sequencing errors or PCR amplification artifacts to
prevent artificial diversity inflation (Huse et al., 2010; Kunin et al., January 2015 | Volume 6 | Article 17 | 3 www.frontiersin.org www.frontiersin.org Prokaryotic diversity in marine sediment Nguyen and Landfald while no correlations were found between the abundance and
environmental data for the Archaea. Abbreviations: St, station; Temp, temperature; TOC, total organic carbon in % of dry weight; Chla, chlorophyll a; Phae, phaeophytin; Chl-16S, chloroplast 16S rRNA
genes as % of total sequence reads in each sample. PROKARYOTIC DIVERSITY For both Bacteria and Archaea, the
communities of stations 1–4 tended to be associated with the
slightly warmer, deeper and more saline conditions in the south-
ern part of the sampling range, while the communities from
stations 6–10 were associated with the observed higher lev-
els of the phytopigment indicators in that region (Figure 3). Statistical comparisons between the southern (1–4) and north-
ern (6–10) communities showed significant differences by the
multivariate generalized linear models approach for Bacteria and
Archaea (ANOVA, p = 0.009 for both groups). The five bacte-
rial taxa that generated most explained difference between the
two regions were the proteobacterial orders Nitrosomonadales
(p = 0.006), Rhodospirillales (p = 0.009), Marinicellales (p =
0.009), Desulfuromonadales (p = 0.010) and the uncultured pro-
teobacterial group Sva0853 (p = 0.010). For the Archaea, just the
variation in the Parvarchaeota (p = 0.001) and Thaumarchaeota
(p = 0.021) tag abundances gave significant contributions to
explained regional difference (Supplementary Figures S2, S3). Distance decays of community similarity were confirmed both by
the Bray-Curtis index (Figures 4A,B) and the phylogeny-based
unweighted UniFrac dissimilarity index (ρ = 0.34; p = 0.02 for
Bacteria; ρ = 0.54; p = 0.01 for Archaea). The zero-distance bac-
terial Bray-Curtis index value of 0.65 (Figure 4A) represents the
similarity between the BM and the D6 sequence pools of station 6. They constituted separate, independently analyzed DNA extracts
from the same, well-mixed sediment sample material. Hence,
the deviation of this value from unity reflects the stochastic
beta-diversity associated with non-exhaustive sequencing within four-fold increase in the OTU richness estimate for this station
by the Chao1 estimator. This suggested that deeper sequenc-
ing of all stations would result in corresponding increases in
richness estimates as observed for station 6. Proteobacteria were
shown to be dominant in the Barents Sea seafloor by compris-
ing an average of 73.8% of bacterial sequence tags (Figure 2A). Gammaproteobacteria and Deltaproteobacteria accounted for
41.1 and 23.2% of the reads, respectively. The taxonomic assign-
ment pointed to the Piscirickettsiaceae as the most prominent
sub-group of the Gammaproteobacteria (43.6% of sequence
reads), while a substantial fraction of the deltaproteobacterial
reads (31.9%) were affiliated with the orders Desulfobacterales
and Desulfuromonadales. Despite the more than two-fold deeper sequencing of the
AM than the BM dataset, archaeal OTU numbers were, on
average, 29% of the bacterial figures for the same stations. PROKARYOTIC DIVERSITY Furthermore, the bacterial gene abundance showed signif-
icant relationships with temperature (Spearman ρ = 0.60; p =
0.04), and phytopigment ratio (Spearman ρ = 0.66; p = 0.04), Table 1 | Geographical locations and environmental characteristics of samples. St
Latitude
Longitude
Depth
Temp
TOC
Clay/silt
Salinity
Chla
Chla:Phae
Chl-16S
16S gene copies/g
(N)
(E)
(m)
(◦C)
(%)
(%)
(%)
(μg/gdw−1)
ratio
(%)
Bacteria (×109)
Archaea (×107)
1
73◦13’52”
16◦20’55”
474
2.7
0.73
67.2
35.01
0.96
0.33
0.1
5.4 ± 0.3
9.7 ± 3.4
2
73◦17’74”
19◦15’59”
460
2.8
2.24
38.7
35.05
0.55
0.42
0.1
3.1 ± 0.5
6.9 ± 2.4
3
73◦23’99”
22◦03’13”
450
2.6
2.44
77.0
35.06
1.04
0.32
0.0
4.5 ± 0.2
5.6 ± 0.9
4
73◦47’55”
24◦35’38”
442
2.5
1.61
86.0
35.05
1.13
0.32
0.2
10.7 ± 3.2
19.0 ± 6.6
5
74◦55’01”
28◦54’52”
364
1.7
1.96
86.9
35.05
3.24
0.54
1.0
10.8 ± 1.0
7.9 ± 0.7
6
75◦38’81”
29◦44’48”
330
1.1
2.21
83.5
35.04
8.10
0.96
8.3
13.7 ± 0.8
12.8 ± 5.3
7
76◦24’12”
30◦37’13”
290
1.2
1.83
86.8
34.98
3.91
0.77
2.5
17.0 ± 0.5
26.7 ± 2.5
8
77◦08’92”
31◦16’67”
189
1.1
1.21
63.5
34.99
2.00
0.53
2.0
10.2 ± 0.9
8.2 ± 3.5
9
77◦20’48”
30◦58’81”
194
1.2
1.21
63.5
34.97
1.92
0.64
0.3
11.5 ± 1.6
18.5 ± 1.4
10
77◦43’10”
30◦56’30”
230
0.9
1.26
66.1
34.97
3.67
0.97
1.8
8.8 ± 0.4
9.4 ± 0.0
Abbreviations: St, station; Temp, temperature; TOC, total organic carbon in % of dry weight; Chla, chlorophyll a; Phae, phaeophytin; Chl-16S, chloroplast 16S rRNA
genes as % of total sequence reads in each sample. Table 1 | Geographical locations and environmental characteristics of samples. al locations and environmental characteristics of sample Abbreviations: St, station; Temp, temperature; TOC, total organic carbon in % of dry weight; Chla, chlorophyll a; Phae, phaeophytin; Chl-16S, chloroplast 16S rRNA
genes as % of total sequence reads in each sample. January 2015 | Volume 6 | Article 17 | 4 Frontiers in Microbiology | Aquatic Microbiology Prokaryotic diversity in marine sediment Nguyen and Landfald different sampling stations for both the Bacteria and Archaea
weighed against strong community structuring forces within this
range of arctic seafloor. However, NMDS ordination based on
the complete sequence information (singletons not included)
indicated some level of clustering of the prokaryotic commu-
nities in accordance with the separation by environmental fac-
tors and spatial distance. PROKARYOTIC DIVERSITY The archaeal communities were highly dominated by a few
phylotypes, as the three most prevalent OTUs constituted 60
to 89% of total sequence reads in the different samples. The
other striking feature of the archaeal communities was the over-
whelming quantitative dominance by the class Thaumarchaeota,
which averaged 85.8% of archaeal sequence reads in the samples
(Figure 2B). A substantial fraction (33.9%) of the thaumarchaeo-
tal reads was affiliated with the marine, ammonia-oxidizing genus
Nitrosopumilus. Besides the Thaumarchaeota, phylotypes repre-
senting the Miscellaneous Crenarchaeotal Group (Inagaki et al.,
2003), the Marine benthic group B (Knittel et al., 2005) and the
candidate phylum Parvarchaeota (Rinke et al., 2013; Hedlund
et al., 2014) constituted significant groups, while less than 1% of
the archaeal sequence reads showed euryarchaeotal affiliation. COMMUNITY STRUCTURE VARIATION FIGURE 4 | Relationships between community similarity (1-Bray
Curtis index) and spatial distance (A,B) and between community
similarity and environmental distance (C,D) for Bacteria (A,C) and
Archaea (B,D). The beta-diversity of the two bacterial station 6
datasets (filled circle in A) is not included in the regression line or
correlation analyses. The significance of the correlations were assessed
by Mantel tests based on Spearman’s rank correlation with 104 Monte
Carlo permutations. FIGURE 3 | Non-metric multidimensional scaling based on Bray Curtis
community distances for Bacteria (A) and Archaea (B). Numbers
represent sampling stations and arrows show vector fitting of the
environmental variables. Abbreviations: Temp, Temperature; Depth, water depth; TOC, total organic carbon; Chl a, chlorophyll a; ratio, chlorophyll
a/phaeophytin ratio; Geo, spatial distance between sampling stations. Red
color (southern part), blue color (northern part), green color (transition
temperature zone). FIGURE 4 | Relationships between community similarity (1-Bray
Curtis index) and spatial distance (A,B) and between community
similarity and environmental distance (C,D) for Bacteria (A,C) and
Archaea (B,D). The beta-diversity of the two bacterial station 6
datasets (filled circle in A) is not included in the regression line or
correlation analyses. The significance of the correlations were assessed
by Mantel tests based on Spearman’s rank correlation with 104 Monte
Carlo permutations. a single community. The |β|coefficients, i.e., the absolute values
of the linear regression coefficients based on the Sørensen simi-
larity index in a double logarithmic plot (Zinger et al., 2014) were
Mantel tests showed that the independence of possible influ-
ential factors on the community structuring was obscured both
by significant collinearities between several of the individual
l f
d
h
l
d FIGURE 4 | Relationships between community similarity (1-Bray
Curtis index) and spatial distance (A,B) and between community
similarity and environmental distance (C,D) for Bacteria (A,C) and
Archaea (B,D). The beta-diversity of the two bacterial station 6 datasets (filled circle in A) is not included in the regression line or
correlation analyses. The significance of the correlations were assessed
by Mantel tests based on Spearman’s rank correlation with 104 Monte
Carlo permutations. Mantel tests showed that the independence of possible influ-
ential factors on the community structuring was obscured both
by significant collinearities between several of the individual
environmental factors, i.e., temperature, depth, salinity and a single community. COMMUNITY STRUCTURE VARIATION The overall stable distribution of phylotypes (Figure 2) and con-
gruent ranking of abundant OTUs (data not shown) at the FIGURE 2 | Distribution of major phylogenetic groups of Bacteria (A)
and Archaea (B) at each sampling station. The analyses of both the
multiplex (BM) and the deeper D6 pyrotag datasets are presented for
Bacteria at station 6. Abbreviations: MCG, Miscellaneous Crenarchaeotal
Group. “Other Crenarchaeota” include Marine benthic group A, Marine
benthic group B and Marine Hydrothermal Vent group. Red color
(southern part), blue color (northern part), green color (transition
temperature zone). www.frontiersin.org
January 2015 | Volume 6 | Article 17 | 5 Group. “Other Crenarchaeota” include Marine benthic group A, Marine
benthic group B and Marine Hydrothermal Vent group. Red color
(southern part), blue color (northern part), green color (transition
temperature zone). Group. “Other Crenarchaeota” include Marine benthic group A, Marine
benthic group B and Marine Hydrothermal Vent group. Red color
(southern part), blue color (northern part), green color (transition
temperature zone). FIGURE 2 | Distribution of major phylogenetic groups of Bacteria (A)
and Archaea (B) at each sampling station. The analyses of both the
multiplex (BM) and the deeper D6 pyrotag datasets are presented for
Bacteria at station 6. Abbreviations: MCG, Miscellaneous Crenarchaeotal January 2015 | Volume 6 | Article 17 | 5 www.frontiersin.org Prokaryotic diversity in marine sediment Nguyen and Landfald depth; TOC, total organic carbon; Chl a, chlorophyll a; ratio, chlorophyll
a/phaeophytin ratio; Geo, spatial distance between sampling stations. Red
color (southern part), blue color (northern part), green color (transition
temperature zone). FIGURE 3 | Non-metric multidimensional scaling based on Bray Curtis
community distances for Bacteria (A) and Archaea (B). Numbers
represent sampling stations and arrows show vector fitting of the
environmental variables. Abbreviations: Temp, Temperature; Depth, water
depth; TOC, total organic carbon; Chl a, chlorophyll a; ratio, chlorophyll
a/phaeophytin ratio; Geo, spatial distance between sampling stations. Red
color (southern part), blue color (northern part), green color (transition
temperature zone). FIGURE 3 | Non-metric multidimensional scaling based on Bray Curtis
community distances for Bacteria (A) and Archaea (B). Numbers
represent sampling stations and arrows show vector fitting of the
environmental variables. Abbreviations: Temp, Temperature; Depth, water
depth; TOC, total organic carbon; Chl a, chlorophyll a; ratio, chlorophyll
a/phaeophytin ratio; Geo, spatial distance between sampling stations. Red
color (southern part), blue color (northern part), green color (transition
temperature zone). COMMUNITY STRUCTURE VARIATION The |β|coefficients, i.e., the absolute values
of the linear regression coefficients based on the Sørensen simi-
larity index in a double logarithmic plot (Zinger et al., 2014) were
0.056 ± 0.013 for the Bacteria and 0.153 ± 0.041 for the Archaea. January 2015 | Volume 6 | Article 17 | 6 Frontiers in Microbiology | Aquatic Microbiology Prokaryotic diversity in marine sediment Nguyen and Landfald hypervariable basepairs than in the complete gene (Youssef et al.,
2009). phytopigment ratio (p = 0.001 for all combinations) and by
spatial autocorrelations of the same environmental factors (p =
0.001). Hence, relationships were optimized between combina-
tions of environmental parameters and community variation by
the bioenv procedure, and significant relationships were found
between these combinations of environmental factors and com-
munity distances (Figures 4C,D). For the Bacteria, the four vari-
ables temperature, phytopigment ratio, %silt/clay and TOC were
maintained in the model, while depth, phytopigment ratio and
Chl a gave positive contributions for the Archaea. The bacterial taxa composition of the Barents Sea samples
was similar to recent reports for marine seafloor upper sedi-
ments, i.e., distinctly higher fractions of Deltaproteobacteria, but
lower abundances of Alphaproteobacteria than commonly found
in the pelagic bacterial communities. This main feature has been
observed from deep ocean seabeds with low influx of water-
column derived sedimenting material to more shallow coastal
areas, where benthic–pelagic coupling likely is strong. It there-
fore seems to reflect a universal environmental adaptation of the
marine sediment bacterial communities (Li et al., 2009; Teske
et al., 2011; Zinger et al., 2011; Bienhold et al., 2012). Hence, the
uppermost centimeters of the sediment appeared to be dominated
by autochtonous bacterial assemblies throughout the sampling
area. g
p
Partial Mantel tests were employed to assess the independent
effects of space and environment on the community structuring. Significant relationships between community and environmen-
tal variation were confirmed when controlling for spatial distance
(Table 3). For the reciprocal tests, i.e., spatial effects when con-
trolling for environmental distance, the null hypothesis could
not be rejected, but the similar magnitudes of the correlation
coefficients for the two bacterial tests indicated comparable con-
tributions to explained variation by environment and space. To
quantitate the relative contributions to explained community
variation by the different environmental factors, general linear
models were established. DISCUSSION Table 4 | General linear model analyses of the effect of individual
environmental variables on bacterial and archaeal communities. Coefficient
p-Value
BACTERIA
Temperature
0.020
0.009
TOC
0.016
0.016
%(Silt/Clay)
0.014
0.019
Phytopigment
0.007
0.432
ARCHAEA
Phytopigment
0.033
0.024
Chl-a
0.014
0.201
Depth
0.017
0.199
Statistically significant relationships are indicated by bold letters; TOC, total
organic carbon in % of dry weight; Chl a, chlorophyll a; Phytopigment, chlorophyll
a/phaeophytin ratio. R2 adj = 0.42 for bacteria, R2 adj = 0.31 for archaea. Table 4 | General linear model analyses of the effect of individual
environmental variables on bacterial and archaeal communities. COMMUNITY STRUCTURE VARIATION These models retained the indepen-
dently varying factors temperature, %silt + clay and TOC as
statistically significant contributors for the Bacteria, while the
phytopigment ratio alone showed significance for the Archaea. The contribution to overall community variation explained by
environmental variables was 42.1% for the bacterial communities
and 31.0% for the archaeal communities (Table 4). The less than 2% of total 16S rRNA genes affiliated with
Archaea seems characteristic of the uppermost layer of marine
sediments. Comparably low presence of Archaea have been found
in other Arctic and Antarctic sediments (Sahm and Berninger,
1998; Ravenschlag et al., 2001; Bowman and McCuaig, 2003). The archaeal communities showed a noticeable skewness in
phylotype distribution, as the three top-ranking OTUs consti-
tuted more than two-thirds of total archaeal sequence reads
and there was an absolute dominance by representatives of the
recently established group Thaumarchaeota (Brochier-Armanet
et al., 2008). The Thaumarchaeota comprise the phylotypes PROKARYOTIC COMMUNITIES IN THE BARENTS SEA SEDIMENT The Thaumarchaeota are associ-
ated with an autotrophic ammonia-oxidizing energy metabolism
with the capacity to utilize very low substrate concentrations
(Könneke et al., 2005; Herfort et al., 2007; Pester et al., 2011). As established ammonia-oxidizing bacterial phylogenetic groups,
like Nitrosomonadales, were very poorly represented among the
Bacteria, the Thaumarchaeota appeared to be the predominant
ammonia oxidizers in this cold shelf sediment. With some reser-
vations regarding seasonal variations or primer bias in the 16S
rRNA gene amplification, the virtual absence of relevant groups
of Euryarchaeota in our material excluded methanogenesis or
anaerobic methane oxidation as significant processes in the top
centimeters of this seafloor. in accordance with the south-north spatial and environmental
separation of the sampling range. However, estimates of the
importance of the factors that gave rise to this structuring
was complicated both by the extensive collinearity between sev-
eral environmental factors, i.e., temperature, phytopigment ratio,
water depth and salinity, and the just as strong spatial autocor-
relation of the same factors. These phenomena weakened the
possibility to disentangle the contributions by the various fac-
tors to the overall beta-diversity and made general linear models
labile, with coefficient estimates that were sensitive to minor
changes in the data or the optimization criteria (Legendre, 1993;
Dormann et al., 2013). The spatial separations of sampling sites in the present study
varied from 23 to 640 km, thereby falling into the intermediate
range (10–1200 km) in which Schauer et al. (2010) have found
dispersal limitation and contemporary environmental selective
forces to show comparable contributions to biogeographic pat-
terning in deep-sea sediments. We found the impact of spatial
effects to be subordinate to the one of environmental factors,
although this ranking was less evident for the Bacteria than the
Archaea. Hence, the data did not exclude our initial assumption
that dispersal is substantial in this kind of shelf sediment, but
dispersal was evidently not strong enough to blur the commu-
nity structuring effects of the moderate environmental differences
along the sampling area. The partitioning of the various environ-
mental factors that contributed to explained community varia-
tion was based on the criterion of Clarke and Ainsworth (1993)
of optimized fit between community and environmentally based
distance matrices, in combination with a general linear model. SOURCES OF COMMUNITY VARIATION The dissimilarity between spatially separated microbial commu-
nities is established in the balance between neutral factors, the
rate of dispersal of the organisms and the strength of local
selective forces (Sloan et al., 2006; Lindström and Langenheder,
2012; Wang et al., 2013). The Barents Sea comprises a con-
tiguous shelf seafloor, where minor differences in the prokary-
otic assemblies were expected due to moderate environmental
variations in combination with an anticipated substantial dis-
persal effected by re-suspension of fine-grained sediment par-
ticles. The stability in higher taxa composition throughout the
sampling range consolidated this presumption. Additionally,
allochtonous influx of microorganisms via particulate pelagic
material may have promoted the high community similarity, as
the bacterioplankton is more weakly biogeographically structured
than the benthic microbiotas across similar distances (Zinger
et al., 2014). However, our phylogenetic data gave no basis to
conclude that bacterial groups that are associable with sedi-
menting planktonic material constituted a significant fraction
of the seafloor microbiota. The frequently cultivable, copi-
otrophic lineages of Gammaproteobacteria, principally mem-
bers of the Alteromonadales, Oceanospirallales, Vibrionales and
Pseudomonadales, are pointed out as characteristic of particle-
bound planktonic Bacteria (Zhang et al., 2007; Lauro et al.,
2011; Teske et al., 2011; Crespo et al., 2013; D’Ambrosio et al.,
2014). These groups constituted minor proportions of the
Gammaproteobacteria in our sediment material, while represen-
tatives of the dominating Piscirickettsiaceae family have not, to
our knowledge, been associated with pelagic particulate material. The temperature difference between the southern and north-
ern sampling stations constitutes a stable oceanographic feature
of the western Barents Sea (Ingvaldsen and Loeng, 2009) but the
observed difference appears to be at best marginal with regards
to leaving a detectable footprint in the prokaryotic assemblies. Previous documentations of temperature effects have largely been
associated with markedly wider ranges (Fuhrman et al., 2008;
Gilbert et al., 2009; Wietz et al., 2010; Agogué et al., 2011), while
Hamdan et al. (2013) found no contribution to beta-diversity by
a ≤2.4◦C temperature variation in arctic marine sediment. At the time of our sampling effort in late May, winter sea ice
had retracted from around sampling stations 5–6 to about sta-
tion 8 and was partly disintegrated even further north. PROKARYOTIC COMMUNITIES IN THE BARENTS SEA SEDIMENT Our data confirmed the initial finding of Torsvik et al. (1996)
that upper marine sediments harbor one of nature’s most diverse
microbiotas. High 16S rRNA gene diversity estimates for sedi-
ments have previously been obtained from rarefaction analyses
of clone libraries (Ravenschlag et al., 2001; Pedrós-Alió, 2006)
and, more recently, by massive parallel sequencing efforts (Zinger
et al., 2011; Bowen et al., 2012; Hamdan et al., 2013). The four-
time increase in the bacterial richness estimate for station 6 when
comparing the one based on the roughly 7700 reads of the BM
with the 21-fold deeper D6 dataset confirmed the strong depen-
dency on sequencing depth that has previously been documented
for the Chao1 estimator (Lemos et al., 2011). As compared with
near full-length amplicons of the bacterial 16S rRNA gene, our
sequence reads of the V1–V2 region may have produced up to
30% overestimations of OTU richness due to a higher fraction of Table 3 | Partial Mantel tests of Spearman’s rank correlations between prokaryotic community distance and either geographical or
environmental distance. Correlation between Prokaryotic community and
Controlling for
Bacteria
Archaea
ρ-Value
p-Value
ρ-Value
p-Value
Geographic distance
Environmental distance
0.28
0.06
0.1
0.27
Environmental distance
Geographic distance
0.3
0.04
0.43
0.005
Statistically significant relationships are indicated by bold letters. www.frontiersin.org
January 2015 | Volume 6 | Article 17 | 7 Table 3 | Partial Mantel tests of Spearman’s rank correlations between prokaryotic community distance and either geographical or
environmental distance. Correlation between Prokaryotic community and
Controlling for
Bacteria
Archaea
ρ-Value
p-Value
ρ-Value
p-Value
Geographic distance
Environmental distance
0.28
0.06
0.1
0.27
Environmental distance
Geographic distance
0.3
0.04
0.43
0.005
Statistically significant relationships are indicated by bold letters. www.frontiersin.org
January 2015 | Volume 6 | Article 17 | 7 sts of Spearman’s rank correlations between prokaryotic community distance and either geographical or January 2015 | Volume 6 | Article 17 | 7 www.frontiersin.org Prokaryotic diversity in marine sediment Nguyen and Landfald that were previously classified as Crenarchaeotal Group 1.1a
(Schleper and Nicol, 2010), which have been identified as
major archaeal constituents in marine pelagic waters and sed-
iments, including polar and other cold regions (Bano et al.,
2004; Galand et al., 2009; Dang et al., 2010; Alonso-Sáez et al.,
2011; Durbin and Teske, 2011). In contrast, Hamdan et al. (2013) did not identify Thaumarchaeota in the sediment of
the Alaska Beaufort Sea shelf. PROKARYOTIC COMMUNITIES IN THE BARENTS SEA SEDIMENT This approach picked two different, covarying environmental
variables, i.e., temperature for the Bacteria and phytopigment
ratio for the Archaea, as the most influential community struc-
turing factors, with some additional contribution to explained
variation by the independently varying factors organic content
and grain size distribution for the Bacteria. Published studies in
the field or additional data acquired through the present study did
not give robust grounds to conclude whether this difference had a
true ecological basis or rather was a consequence of model lability
caused by collinearity. Frontiers in Microbiology | Aquatic Microbiology SOURCES OF COMMUNITY VARIATION The spring
bloom, which is particularly intensive in the 20–50 km marginal
zone south of the ice edge (Sakshaug et al., 2009), was well-
under way and sedimentation from this bloom may explain the
distinctly higher phytopigment and chloroplast-associated 16S
rRNA gene levels in the northern part of the sampling range. In A main objective of our study was, however, to eluci-
date if even these small environmental differences across the
more than 600 km sampling area transecting the Barents Sea
polar front, were reflected in non-random community varia-
tions if analyzed by a next-generation sequencing approach. The
NMDS ordination patterns of the assemblies of both Bacteria
and Archaea suggested some degree of community structuring January 2015 | Volume 6 | Article 17 | 8 Frontiers in Microbiology | Aquatic Microbiology Prokaryotic diversity in marine sediment Nguyen and Landfald addition, the greater water depth in the southern part, with less
sedimented material reaching the seafloor, may have contributed
in the same direction. Both the Chl a/phaeophytin ratio and the
concentration of Chl a have been used as estimators of fresh-
ness of sedimented phytoplanktonic material in e.g., the western
Barents Sea. Positive relationships between the content of sedi-
ment phytopigments and bacterial growth and production has
been demonstrated (Jørgensen and Boetius, 2007; Morata and
Renaud, 2008) and pigment content has been used as a proxy
for available energy to benthic bacteria in arctic marine sediment
(Bienhold et al., 2012). The abundance of Thaumarchaeota in
pelagic marine waters has also been shown to correlate positively
with Chl a (Robidart et al., 2012) but in the present study, the can-
didate phylum Parvarchaeota rather was the group that showed a
marked increase in the northern region. This recently identified
group (Rinke et al., 2013) of very small cells with correspond-
ingly small genomes has as yet only been genetically characterized
through an acid mine drainage single-cell sequencing project
(Hedlund et al., 2014) and these data do not give any hint to its
ecological adaptation in marine sediment. A recent global sampling study employing an analytical approach
highly similar to the one used by us (Zinger et al., 2014) con-
solidates our estimate of 0.056 for the absolute value of the
double-logarithmic distance decay regression coefficient on shelf
seafloor. Our figure was in-between the values of for deep-sea
and coastal sediments estimated by Zinger et al. (2014). SOURCES OF COMMUNITY VARIATION On the
other hand, the corresponding coefficient estimated by Schauer
et al. (2010) for South Atlantic deep-sea sediments was just 0.003,
i.e., at least an order of magnitude smaller than our shelf sea
figure, and the authors associate this low distance decay with
high dispersal rates and low extinction rates of the vast bac-
terial populations in this kind of environment. The archaeal
coefficient of 0.15 estimated in our study corresponded to the
upper extreme bacterial values recorded by Zinger et al. (2014). Together with the above-mentioned assignment of the explained
archaeal community variation solely to environmental factors, the
archaeal beta-diversity appeared as more sensitive to environmen-
tal variation than the one of the Bacteria in this shelf seafloor
environment. To our knowledge, this kind of comparative beta-
diversity observations between Bacteria and Archaea in the same
environment has not previously been reported. No significant community structuring effects of water depth
or salinity were observed. Previous studies documenting effects of
water depth are founded on substantially wider depth ranges than
the less than 300 m in the present study. In two studies based on
pyrosequence data comprising sampling sites from surface level
to the deep ocean floor, up to 3.0% of the sediment bacterial
community variation was found to be explained by water depth
(Zinger et al., 2011; Bienhold et al., 2012). Although consistent,
the shift in salinity close to the seafloor between the southern
and northern parts of the sampling range was below 0.1%, and
we anticipate it generates a negligible structuring effect on the
prokaryotic communities. In conclusion, our data consolidated previous findings regard-
ing the bacterial alpha-diversities of marine shelf seafloor sed-
iments but reinforced the significance of the Thaumarchaeota
as the principal archaeal group in this type of environment. Furthermore, the study confirmed that biogeographical struc-
tures are detectable in marine sediment prokaryotic communities
by deep 16S rRNA gene sequencing, even where high dispersal
rates combined with weak environmental filtration counteract the
build-up of beta-diversity patterns. This may have implications
for the practicality of employing such approaches to monitor-
ing microbial effects of e.g., the predicted rise in air and water
temperatures in the polar regions, including the Barents Sea, in
the years to come. This climate change is expected to be mani-
fested in the microbial communities (Kirchman et al., 2009). ACKNOWLEDGMENTS
h
k h
f We thank the crew of F/F Helmer Hansen for their technical
and logistic assistance during the sampling cruise. Thanks also to
Hans Christian Eilertsen and Gunilla Eriksen for assistance in the
pigment analyses. We kindly thank the reviewers and editor for
constructive and useful comments. Several studies have confirmed microbial community distance
decay relationships in marine habitat types like pelagic water
(Monier et al., 2014; Zinger et al., 2014), salt marshes (Horner-
Devine et al., 2004; Martiny et al., 2011), and oceanic sediments
(Schauer et al., 2010; Zinger et al., 2014). Sapp et al. (2010)
represent an exception as they were unable to detect significant
spatially induced variation of bacterial and archaeal communities
in North Sea sediment by a denaturing gradient gel electrophore-
sis approach. The actual magnitudes of the distance decays are,
however, difficult to compare due to differences in diversity
indices, organismal target groups, genetic entity compared, etc. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online
at:
http://www.frontiersin.org/journal/10.3389/fmicb.2015. 00017/abstract SOURCES OF COMMUNITY VARIATION On
the other hand, the study also emphasized the importance of suf-
ficient prior knowledge of the environmental variations within
the sampling area to avoid complications caused by extensive
co-variations among the spatial and environmental variables. There is the possibility that unmeasured environmental vari-
ables contributed significantly to community variation through
the sampling range. Possible unaccounted variables include the
levels of inorganic nutrients (Wu et al., 2008; Böer et al., 2009)
and the degree of oxygen penetration into the sediment (Durbin
and Teske, 2012). However, no variations in abiotic composition
have been reported along this well-characterized extent of con-
tiguous soft bottom seafloor, which will likely overshadow the
influences by the variables that were included in the study. In
addition, the moderate variation in the fraction of taxonomic
groups associated with anaerobic sulfur compound metabolisms,
e.g., the orders Desulfobacterales and Desulfuromonadales, was
not indicative of major changes in oxgen profiles within the upper
4 cm of the seafloor (data not shown). Agogué, H., Lamy, D., Neal, P. R., Sogin, M. L., and Herndl, G. J. (2011). Water
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in Microbiology.
Copyright © 2015 Nguyen and Landfald. This is an open-access article dis-
tributed under the terms of the Creative Commons Attribution License (CC BY).
The use, distribution or reproduction in other forums is permitted, provided
the original author(s) or licensor are credited and that the original publica-
tion 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. January 2015 | Volume 6 | Article 17 | 12 Received: 27 August 2014; accepted: 07 January 2015; published online: 23 January
2015.
Citation: Nguyen TT and Landfald B (2015) Polar front associated variation in
prokaryotic community structure in Arctic shelf seafloor. Front. Microbiol. 6:17. doi:
10.3389/fmicb.2015.00017 Received: 27 August 2014; accepted: 07 January 2015; published online: 23 January
2015. Conflict of Interest Statement: The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest. Frontiers in Microbiology | Aquatic Microbiology Citation: Nguyen TT and Landfald B (2015) Polar front associated variation in
prokaryotic community structure in Arctic shelf seafloor. Front. Microbiol. 6:17. doi:
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10.1111/mec.12640 January 2015 | Volume 6 | Article 17 | 11 www.frontiersin.org Prokaryotic diversity in marine sediment Nguyen and Landfald Conflict of Interest Statement: The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest. Received: 27 August 2014; accepted: 07 January 2015; published online: 23 January
2015. January 2015 | Volume 6 | Article 17 | 12 Frontiers in Microbiology | Aquatic Microbiology Frontiers in Microbiology | Aquatic Microbiology
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