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https://jneuroengrehab.biomedcentral.com/track/pdf/10.1186/s12984-018-0477-5
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User activity recognition system to improve the performance of environmental control interfaces: a pilot study with patients
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Journal of neuroengineering and rehabilitation
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Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation
https://doi.org/10.1186/s12984-018-0477-5
RESEARCH
(2019) 16:10
Open Access
User activity recognition system to
improve the performance of environmental
control interfaces: a pilot study with patients
Arturo Bertomeu-Motos1* , Santiago Ezquerro1 , Juan A. Barios1 , Luis D. Lledó1 , Sergio Domingo2 ,
Marius Nann4 , Suzanne Martin3 , Surjo R. Soekadar5 and Nicolas Garcia-Aracil1
Abstract
Background: Assistive technologies aim to increase quality of life, reduce dependence on care giver and on the
long term care system. Several studies have demonstrated the effectiveness in the use of assistive technology for
environment control and communication systems. The progress of brain-computer interfaces (BCI) research together
with exoskeleton enable a person with motor impairment to interact with new elements in the environment. This
paper aims to evaluate the environment control interface (ECI) developed under the AIDE project conditions, a
multimodal interface able to analyze and extract relevant information from the environments as well as from the
identification of residual abilities, behaviors, and intentions of the user.
Methods: This study evaluated the ECI in a simulated scenario using a two screen layout: one with the ECI and the
other with a simulated home environment, developed for this purpose. The sensorimotor rhythms and the horizontal
oculoversion, acquired through BCI2000, a multipurpose standard BCI platform, were used to online control the ECI
after the user training and system calibration. Eight subjects with different neurological diseases and spinal cord injury
participated in this study. The subjects performed simulated activities of daily living (ADLs), i.e. actions in the simulated
environment as drink, switch on a lamp or raise the bed head, during ten minutes in two different modes, AIDE mode,
using a prediction model, to recognize the user intention facilitating the scan, and Manual mode, without a prediction
model.
Results: The results show that the mean task time spent in the AIDE mode was less than in the Manual, i.e the users
were able to perform more tasks in the AIDE mode during the same time. The results showed a statistically significant
differences with p < 0.001. Regarding the steps, i.e the number of abstraction levels crossed in the ECI to perform an
ADL, the users performed one step in the 90% of the tasks using the AIDE mode and three steps, at least, were
necessary in the Manual mode. The user’s intention prediction was performed through conditional random
fields (CRF), with a global accuracy about 87%.
Conclusions: The environment analysis and the identification of the user’s behaviors can be used to predict the user
intention opening a new paradigm in the design of the ECIs. Although the developed ECI was tested only in a
simulated home environment, it can be easily adapted to a real environment increasing the user independence at
home.
Keywords: Environment control interface, Brain-computer interface, User intention prediction, Multimodal system,
Brain injury, Spinal-cord injury
*Correspondence: abertomeu@umh.es
Miguel Hernández University of Elche, Av. Universidad w/n, Ed. Innova, 03202
Elche, Spain
Full list of author information is available at the end of the article
1
© 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.
Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation
Background
It is estimated that one in six people in the world are
diagnosed with a neurological disorder and this number
is expected to rise considerable due to extensions of life
expectancy [1]. A neurological condition is a damage to
the brain, spinal column or nerves due to illness or injury
such as spinal cord injury, acquired brain damage, stroke,
motor neurons disease and locked in syndrome. Neurological disorders are considered the primary cause of
disability in modern society [1, 2]. The debilitating consequences of neurological disorders include communication
difficulties, impaired memory, inappropriate behavior, physical disability, restricted independence, social
isolation and poor quality of life.
Assistive technologies aim to increase quality of life
[3–6], reduce dependence on care giver [7] and reduce
dependence on the long term care system [8]. Several
studies have demonstrated the effectiveness in the use
of environment control interfaces (ECI) for environment
control or communication through voice commands [9],
scan interfaces based on grid structure, eye tracking
[10–12] or brain-computer interface (BCI) based on P300
[13], among others. These software platforms actively aid
during the Activities of Daily Living (ADL) improving the
independence both at home and outside. However, these
platforms are based in a manual scan over the different
abstraction levels of the ECIs and the scan speed only
depends on the users familiarization with the system and
the configuration of the grids over the different menus.
Thus, introducing the user environment and behavior into
this loop will help the navigation agility in the ECIs.
On the other hand, The progress of BCI research
together with exoskeleton enables a person with motor
impairment to interact with new elements of the environment [14, 15]. Thus, this progress will deliver new scenarios to BCI systems out of laboratories and move BCI into
the domestic environment. The AIDE project1 aims to
create new shared-control paradigm for assistive devices
that integrates information from identification of residual abilities, behaviors, emotional state and intentions of
the user on one hand and analysis of the environment and
context factors on the other hand. In this context, a hybrid
BCI model was chosen to control the ECI. It was developed as a fusion between non-invasive electroencephalography (EEG) and electrooculography (EOG) system [16].
The EEG records the sensorimotor rhythms (SMR) called
event-related desynchronization (ERD) and event-related
synchronization (ERS) during a motor imagery (MI) task
[17] whilst the EOG records the horizontal oculoVersion (HOV).
This paper aims to evaluate the ECI developed under the
AIDE project conditions, a multimodal system developed
to assist people with acquired brain damage or neurodegenerative diseases that need a wheelchair and has low
(2019) 16:10
Page 2 of 9
or any upper limbs mobility in their ADLs, in a simulated
environment able to detect the user intention through the
environment analysis and the identification of the user’s
behaviors, based on a conditional random fields (CRF)
model [18]. Thus, the handling of the interface was studied in two different ways, with and without the prediction
of the user’s intention. Users with neurological and muscular diseases and spinal cord injury (SCI) tested the
system on a virtual home due to the early phase of the
project.
Methods
This study evaluated the ECI in a simulated scenario
under the AIDE project conditions, a multimodal interface able to analyze and extract relevant information from
the environments as well as from the identification of
residual abilities, behaviors, and intentions of the user. It
consisted in a two screen layout: ECI and simulated room,
with an EEG and EOG data acquisition system (see Fig. 1).
Environment control interface
The environment control system used in this experimentation was based in two main software components:
GRID3 from Smartbox2 , a commercial augmentative and
alternative communication (AAC) solution, and SHX, a
specific developed software, presented in Fig 2. The ECI
had three different abstraction levels: 1) related with the
room (room menu), 2) related to the activities that can
be performed in a specific room (activity menu), and 3)
related to the actions regarding a specific activity (ADL
menu). The jump between two consecutive abstraction
levels will be named as step.
Levels one and two were specific grid sets, created in
GRID3, to be used in the context of the experimentation.
They include grids for the different rooms, communication, control a wheelchair and control an exoskeleton arm.
In all grids, a color code has been used: red for the alarm;
green for communication, computer control and digital
leisure; white for wheelchair and arm control (not used
in this experimentation); light blue for the rooms; dark
blue for environmental control activity. The dark blue cells
are referred to environmental control activities and linked
Grid3 with the SHX application. The user could scan
across the different cells, select one, and then confirm or
cancel the selection using the chosen signals (EOG, EEG,
eye tracking, etc.).
SHX is a custom build solution for environmental control management, level three. It allowed the user to easily
configure and select the actions programmed for a specific
activity. Different scenes for each of the possible activities
in every room were created. Here too, the user could scan
across the different cells, select one, and then confirm or
cancel the selection using the chosen signals (EOG, EEG,
eye tracking, etc.).
(2019) 16:10
Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation
a
Page 3 of 9
b
Fig. 1 Experimentation setup. The experimental setup was composed by two screens layout: ECI & Virtual home simulator, and an eye tracking
placed in the bottom screen. The users performed tasks in the simulated environment (a) and they also interacted with real elements (b)
Data acquisition
The acquisition of the brain activity was performed
with eight solid-gel electrodes placed according to
international 10-20 system placed at F3, C3, Cz, P3,
T7 and Mastoid, a reference electrode was placed
on C4 and the ground on FpZ. Furthermore, two
a
electrodes were placed on the outer canthus of the
eyes to the EOG signal recording. The EEG/EOG
signal was acquired via Bluetooth through the Neuroelectrics amplifier (Enobio, Neuroelectrics, Barcelona,
Spain). Skin/electrode resistance was kept below
12 kOhm.
b
c
Fig. 2 Environment Control Interface. The ECI had three different abstraction levels: 1) related with the room, 2) related to the activities that can be
performed in a specific room and 3) related to the actions regarding a specific activity. An example menu of each level is shown in (a), (b) and (c)
respectively
Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation
A real-time SMR-based BCI was implemented using
BCI2000, a freely distributed software for multipurpose
standard BCI platform [19]. EEG and EOG were recorded
at a sampling rate of 500 Hz, bandpass filtered at 0.470 Hz and pre-processed using a small Laplacian filter.
Based on the maximum values for basal ERD, the ongoing
EEG signal associated with the specified SMR rhythm frequency range (11-14 Hz) calculated from C3 electrode, a
subject’s individual motor imagery discrimination threshold were set. The EOG discrimination thresholds were
calculated regarding the average amplitudes of horizontal saccades. These individual parameters were obtained
from the training session and used for later online BCI
control [20].
(2019) 16:10
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Table 1 Correlation between the ADLs and ADL menu name, in
the third abstraction level
ADLs
ADL menu
ADLs
ADL menu
Open/close
fridge
Drink or eat
Switch on/off air
conditioner
Air conditioner
Open/close
microwave
Drink or eat
Brushing teeth
Teeth
Eating task
Eat
Washing face
Face
Drinking task
Drink
Raise/lower the
bed head
Bed
Switch on/off
Music
Entertainment
Raise/lower the
bed feet
Bed
Switch on/off PC
Entertainment
Open/close the
blinds
Blinds
Switch on/off TV
TV
Switch on/off the Light
light
Prediction model
The proposed ECI combines the environmental information and context factors together with user’s behaviors
in order to detect the user intention. Thus, the input
information of the prediction model was a sequence
of data, the user is moving and looking at the environment, that had to be labeled. In this context, different models were tested (time-delay neural networks,
decision trees, hidden markov model (HMM)...) and
the CRF model was chosen, showing the best results.
The CRF model is a probabilistic model for segmentation and labeling sequence data. This discriminate
model takes into account not only the current state
but also the previous states to perform its prediction.
A conditional model specifies the probabilities of possible label sequences given an observation sequence
in contras of the generative models that make very
strict independence assumptions on the observation, for
instance conditional independence given the labels as
HMM [21].
In our case, the inputs of the system were: localization, objects in the environment, object that the user
is looking at, temperature of the room, brightness of
the room and day time; the output was the ADL menu,
i.e. the most probable action that the user wanted to
perform that were directly linked with a specific ADL
(see Table 1.
Participants
Eight persons with different neurological pathology and
spinal cord injury participated in this study (37 ± 15
years old), their demographic and clinical characteristics are listed in (Table 2). The subjects were evaluated before the experiment with the barthel index [22].
All participants gave informed consent using their standard communication channel prior to participation in the
study. The protocol was approved by the Office Research
Ethics Northern Ireland - approval granted for project
(15/NE/0384).
Experimental protocol
Subjects were sitting in his/her own wheelchair in front of
a table with two screens, as shown in Fig. 1. The screens
were used to show the ECI and the virtual home simulator. Subjects used the AIDE multimodal interface, hybrid
EEG EOG system, to online control the ECI and preform
specific ADLs. Two modes were tested:
A) MANUAL mode: the user had to navigate through
the three abstraction levels in order to accomplish
the task showed in the virtual house. The objects
related with the corresponding task were surrounded
by a green color in the virtual house environment
and the task appeared in the right top corner.
B) AIDE mode: in this mode the prediction model was
used. The user had to look at the objects related to
the specific ADL, showed like in the other mode, and,
after the user’s intention prediction, the ECI directly
jumped to the corresponding ADL menu. Then the
user had to navigate like MANUAL mode. In case of
Table 2 Demographic and clinical characteristics of participants
ID Patient
Sex
Age
Diagnosis
Barthel score
1
Male
32
C4 SCI
4/20
2
Male
22
Duchenne
Muscular
Dystrophy
6/20
3
Male
55
Brain stem
strokes
16.5/20
4
Male
30
C4/C5 SCI
2/20
5
Female
20
C6/C7 SCI
10/20
6
Male
58
Ischemic Stroke
19/20
7
Male
55
Multiple Systems
Atrophy
5/20
8
Male
30
C6/C7 SCI
9/10
Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation
wrong prediction, the user had to manually go back,
the second abstraction level, and complete the
corresponding ADL. The observed objects were
online detected from the eye tracking Tobbi3 PCEye
go, placed on the virtual environment screen, and the
rest of the inputs were online simulated.
Each subject performed two experimental sessions in
two consecutive days. The first session was for training
and calibration purpose as well as for the familiarization with the systems to be controlled. This session lasted
around 60-80 min. In the last part of this session the
user learned how to use the hybrid EEG/EOG interface
in order to control the ECI. An example of MI and EOG
movements in the training session are shown in Fig. 3c
and d, respectively.
The second session lasted a maximum of 60 min. The
setup and familiarization phase took approximately 15
min (subjects have already tested the system in the first
session). They had 10 min to perform a predefined ADLs
list in both AIDE and Manual modes (all ADLs can be
observed in Table 1). Each ADL can be a single action, it
has a visual effect on the house simulator, e.g. swith on a
lamp, or an exoskeleton action, the simulator play a short
video showing the corresponding action, e.g. drink from
a glass. The order of the modes was randomly selected
and, before each mode, a baseline of 3 min was acquired.
During the break (5 min) and at the end of this session
the subjects answered the NASA task load index (tlx)
questionnaire [23].
The scan in the ECI was performed through lookingright eye movements (Fig. 3b) implying a forward displacement of the grid marker (Fig. 3a-e). Once the subject
stopped at one grid a customized time, chosen in the first
session, the ECI ‘switch off ’ the rest of the grids (Fig. 3f ).
Then, the next level or the action in the ECI was achieved
by on-line ERD detection, like the subjects learned during
the first session (Fig. 3a). On the other hand, if the user
did not want to click on this specific grid, in the phase
Fig. 3f, a looking-left eye movement returns the ECI to
the phase Fig. 3a. When the user performs an action, a
visual feedback is presented in the virtual home regarding
to the action performed (Fig. 3g) and he/she waited for the
next task.
After both modes, they were instructed to interact with
real elements through the ECI and watch a video using
objects of a multi-sensory room, as can be observed in
Fig. 1b.
Results
The users performed simulated ADLs during 10 min
in a virtual home using an ECI in both Manual and
AIDE modes. The number of the performed tasks with
respect to the mean time spent per user is presented
(2019) 16:10
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in Fig. 4a. Furthermore, it has been trained a Support
Vector Machine (SVM) model with Gaussian kernel
to estimate the boundary between both modes (yellow line in Fig. 4a). It should be noted that statistically
significant differences between both modes in terms
of number of tasks and mean tasks time is shown
(p − value < 0.001 using Wilcoxon test). The steps distribution that the users performed in both modes are
shown in Fig. 4c. ADLs manually omitted tasks were
excluded from the study due to the subject was blocked
during the ECI scan caused by frustration or fatigue over a
specific task.
On the other hand, the AIDE mode uses a CRF
model, previously trained with simulated data using the
same virtual home, to predict the user intention. Thus,
the confusion matrix of the prediction model regarding
the ADL menus is presented in Fig. 4b. In addition, the
results obtained through the NASA tlx questionnaire are
presented in Fig. 5.
Discussion
The AIDE project aims to develop a multimodal system in
order to help people with neurological diseases wearing a
wheelchair. The presented environment allowed the user
to navigate through a virtual house and perform several
ADLs using a developed ECI. Two modes were estudied,
the AIDE mode, that used a CRF model to predict the user
intention and ease the ECI scan, and the Manual mode,
that needed a complete scan through the ECI to perform
a specific ADL. The ECI was online controlled using the
AIDE multimodal system based on a combination of EEG
and EOG wireless acquisition system [17].
The results presented in Fig. 4a show the mean time
per task spent in the AIDE mode is less than in the Manual mode being able to perform more tasks in the same
time, both modes show statistically significant differences
(p < 0.001). Furthermore, both modes can be easily classified by training a SVM model with Gaussian kernel, the
boundary is presented with a yellow line in Fig. 4a.
On the other hand, Fig. 4c shows the difference between
both groups in terms of steps, i.e. the ECI abstraction levels that the user had to cross in order to perform a specific
ADL. It must be noticed that in the AIDE mode, the users
performed one step in the 90% of the tasks and three steps,
at least, were necessary in the Manual mode. Regarding
the AIDE mode, three steps were necessary only when the
CRF model realized a wrong prediction and, therefore,
the user had to return to the activity menu and select the
proper ADL menu. Although a bad prediction is sometimes performed, the multimodal system helps in terms of
location, i.e. the activity that the system predicts is always
related with the room where the user is, facilitating the
navigation. Perform five or seven steps in the Manual
mode implies that a wrong abstraction level was selected,
Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation
(2019) 16:10
Page 6 of 9
a
c
b
d
Fig. 3 Multimodal system processing for one ADL in AIDE mode. The user had to perform different actions in order to execute the corresponding
ADLs, in this example, the user had to switch on the TV, phases a-g show the behavior of both screens during the task. EEG (a) and EOG (b) signals
were acquired to online control the ECI in order to perform ADLs in a virtual house. When the task started (vertical purple line), the scan through the
ECI was performed by EOG activity detection [orange line in (b)], i.e. when HOV activity exceeded the threshold [indicated by the orange dashed
line in (b)] the grid marker moved forward (phases a-e). Once the subject stopped at one grid, a task confirmation was needed [indicated by the
vertical black line] and the ECI ‘switched off’ the rest of the grids indicating this purpose (phase f). The confirmation was performed by the detection
of SMR-ERD [indicated by red line in (a)] and the action was done, so the ADL finished (vertical dotted purple line). This ADL was performed in one
step, i.e. the user only needed to navigate through the last abstraction level to complete the task. Before the experimentation, the user was trained
in motor imagery (c) and EOG movement (d) to the set up the control system with the personalized parameters
Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation
(2019) 16:10
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a
b
c
Fig. 4 Environment control interface performance. The users performed two different trials with the same goal: complete as many tasks as possible
in 10 min by using the Manual mode and the AIDE mode (a). Both modes have been used to train a SVM model with Gaussian kernel and find the
boundary between them [yellow line in (a)]. The interaction with the ECI was measured by steps that a user had to perform in order to complete the
tasks (c). Furthermore, in AIDE mode the user’s intention prediction was performed through CRF model and, therefore, the confusion matrix of the
model is obtained (b)
due to user confusion or lack of practice, and the user had
to go back in the ECI.
The AIDE mode uses a CRF model to perform the
predictions about the user’s intention. The model uses
the information of the virtual home and the object that
the user is looking at, acquired thought an eye tracking
device. The model was previously trained with simulated
data using the same virtual house environment. Thus,
Fig. 4b shows the confusion matrix of the prediction
model, regarding the ADL menu, with a global accuracy
about 87%. The CRF model, as it takes into account not
only the current state but also the previous states to perform its prediction, it could fail in the prediction of task
with common features. Therefore, the ADLs related to the
Drink menu are sometimes predicted as the ADLs related
with the Eat menu, in this case around the 50% of the
trials. In addition, TV and the Teeth menus are rarely
selected as Entertainment and Face menu, respectively, by
the prediction model.
After each mode, the subjects answered the NASA tlx
questionnaire in order to assess the workload between the
modes, showed in Fig. 5. Unexpectedly, it has not statistically significant differences, so we can say that the
users do not notice subjective differences between both
modes in terms of workload. It can be explained because
it was the first time that the users handle the complex multimodal control system (EEG+EOG) with this
ECI. We assume that, observing the results presented
in Fig 4, the workload should decrease, at least, in the
AIDE mode.
Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation
(2019) 16:10
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Fig. 5 NASA task load index. The subjects answered the NASA tlx questionnaire after each mode
Conclusion
The presented ECI allowed the users to perform simulated
ADLs with a multimodal control system. The platform
was tested in two different scenarios: Manual and AIDE
mode. The first one was presented as a simple ECI where
the user had to achieve the corresponding ADL. The
second mode used a CRF model to predict the user’s
intention through the environment analysis and identification of the user’s behaviors. We conclude that, even the
users do not perceive subjective differences between both
modes in terms of workload, the AIDE mode helps the
user to perform mode ADLs, spending less time per task,
showing statistically significant differences with respect
to the Manual mode. This effect is caused by the user’s
intention prediction as the ECI jumps directly to the last
abstraction level of the ECI. The environment analysis and
the identification of the user’s behaviors can be used to
predict the user intention and will allow to speed up the
ECIs scan opening a new paradigm in the design of these
interfaces. Although the developed ECI was tested only in
a simulated home environment, it can be easily adapted
to a real environment increasing the user independence
at home.
Endnotes
1
http://aideproject.umh.es/
2
http://www.thinksmartbox.com
3
http://www.tobii.com
Abbreviations
AAC: Augmentative and alternative communication; ADL: Activity of daily
living; BCI: Brain computer interface; CRF: Conditional random field; ECI:
Environment control interface; EEG: Electroencephalography; EOG:
Electrooculography; HOV: Horizontal oculoversion; MI: Motor Imagery;
SMR-ERD: Sensorimotor rhythm event-related desynchronization; SVM:
Support vector machine; tlx: task load index
Acknowledgments
The authors thank the subjects for their participation. In addition, we
acknowledge the great support from CEDAR Foundation, Belfast, UK and the
Biomedical Research Networking Center (CIBER).
Funding
This work has been supported by the AIDE project through the grant
agreement no. 645322 of the European Commission and by the Ministry of
Economy and Competitiveness through the project DPI2015-70415-C2-2-R.
Availability of data and materials
Data and materials can be made available upon request to the corresponding
author.
Bertomeu-Motos et al. Journal of NeuroEngineering and Rehabilitation
(2019) 16:10
Authors’ contributions
ABM, SM, SRS and NGA designed the study. ABM, SE, JAB, LDL and SD collect
the data. SD developed the environment control interface. LDL designed the
virtual home environment. ABM, SE and JAB interpreted and analyze the data.
ABM, SE created the figures. ABM, SE, JAB, LDL and SD wrote the manuscript.
SM, NGA and SRS deeply revised the manuscript. ABM, SE, JAB, LDL, SE, MN,
SM, SRS and NGA edited the manuscript. All authors read and approved the
final manuscript.
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20. Barios JA, Ezquerro S, Bertomeu-Motos A, Nann M, Badesa FJ,
Fernandez E, Soekada SR, Garcia-Aracil N. Synchronization of slow
cortical rhythms during motor imagery-based brain-machine interface
control. Int J Neural Syst. 2018. in press.
21. Rabiner LR. A tutorial on hidden Markov models and selected applications
in speech recognition. Proc IEEE. 1989;77(2):257–86.
22. Quinn TJ, Langhorne P, Stott DJ. Barthel index for stroke trials:
development, properties, and application. Stroke. 2011;42(4):1146–51.
23. Hart SG, Staveland LE. Development of nasa-tlx (task load index): Results
of empirical and theoretical research. In: Advances in Psychology, vol 52.
North Holland: Elsevier; 1988. p. 139–83. https://doi.org/10.1016/S01664115(08)62386-9.
Ethics approval and consent to participate
This study was conducted according to the declaration of Helsinki and had
ethical approval by the Office Research Ethics Northern Ireland (June 2016) –
approval granted for AIDE project (15/NE/0384). All subjects gave written
informed consent prior to data collection.
Consent for publication
Participants gave written informed consent to data treatment in this research
study and permission to publish anonymous data and results.
Competing interests
Sergio Domingo is a software developer at BJ Adaptaciones, the developer of
SHX software. Sergio Domingo declares no competing interests. The rest of
the authors declare no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1 Miguel Hernández University of Elche, Av. Universidad w/n, Ed. Innova, 03202
Elche, Spain. 2 BJ Adaptaciones, St Mare de Déu del Coll, 70, 08023 Barcelona,
Spain. 3 The Cedar Foundation, 1 Upper Lisburn Road, BT10 0GW Belfast, UK.
4 University Hospital of Tuebingen, Applied Neurotechnology Lab, Calwerstr.
14, D-72076 Tübingen, Germany. 5 Clinical Neurotechnology Laboratory,
Neuroscience Research Center (NWFZ), Charité University Medicine Berlin,
Charitéplatz 1, 10117 Berlin, Germany.
Received: 25 July 2018 Accepted: 18 November 2018
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CRISPR Explorer: A fast and intuitive tool for designing guide RNA for genome editing
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HIGHLIGHT visualization on the CRISPR-Explorer browser. In the Browse mode,
potential gRNA candidates are color-coded based on their specificities
and efficiencies to facilitate selection, and detailed information for an
individual gRNA design can be displayed in a pop-up. In the Batch
mode, the user can input a gene, a gene list, a genome coordinate, or a
list of genome coordinates. The output of the “Batch Design Exporter”
is an interactive table that allows the user to rank and/or filter his/her
gRNA designs. The table can be exported in a text file format. www.jbmethods.org
POL Scient
How to cite this article: Chen K, Jin Y, Lin YC. CRISPR Explorer: A fa
Biol Methods 2016;3(4):e56. DOI: 10.14440/jbm.2016.138
CRISPR-Explorer facilitates the design of high-quality gRNA for
the low- and high-throughput needs of researchers due to its ease of
use, comprehensiveness, and instantaneous feedback. The advantages
of our web application over other existing web applications are: (1)
fast: gRNA design results based on published empirical studies are
generated instantly, thus removing wait times; (2) intuitive: the user
can visualize the high-quality gRNAs with detailed design information
through an interactive genome browser; and (3) flexible: the user can
define his/her own parameters for gRNA selection and various input
formats are recognized. The scoring schemes that are used include the
specificity score and the efficiency score. Targeting multiple regions
can be achieved simultaneously through Batch Design. Guide RNA
design results can be ranked and filtered on the browser before export. The user will select the genome of interest for genome editing, the
desired gRNA length, and the cognate promoter (U6 or T7) that drives
the gRNA expression. After selecting these options, the user will have
the choice to input a gene name (official gene symbol, Ensembl ID,
or Refseq ID), genome coordinates, or a DNA sequence for gRNA
p
a
i
m
l
i
g
f
f
s
t
a
t
m CRISPR-Explorer facilitates the design of high-quality gRNA for
the low- and high-throughput needs of researchers due to its ease of
use, comprehensiveness, and instantaneous feedback. CRISPR Explorer: A fast and intuitive tool for designing
guide RNA for genome editing
Kenian Chen, Yi Jin, Yin C. Lin*
Baylor Institute for Immunology Research, Dallas, TX 75246, USA Baylor Institute for Immunology Research, Dallas, TX 75246, USA *Corresponding author: Yin C. Lin, Email: Yin.Lin@BSWHealth.org
Competing interests: The authors have declared that no competing interests exist. Abbreviations used: gRNA, guide RNA; PAM, protospacer adjacent motif; DSBs, double-stranded breaks; NHEJ, non-homologous end-joining; HDR, homology-directed Abbreviations used: gRNA, guide RNA; PAM, protospacer adjacent motif; DSBs, double-stranded breaks; NHEJ, non-homologous end-joining; HDR, homology-directed Abbreviations used: gRNA, guide RNA; PAM, protospacer adjacent motif; DSBs, double-stranded breaks; NHEJ, non-homologo Received June 8, 2016; Revision received August 31 2016; Accepted August 31 2016; Published October 10, 2016 Journal of Biological Methods | 2016 | Vol. 3(4) | e56
DOI: 10.14440/jbm.2016.138 Journal of Biological Methods | 2016 | Vol. 3(4) | e56
DOI: 10.14440/jbm.2016.138 Resource ABSTRACT The RNA-guided CRISPR-Cas9 (clustered, regularly interspaced, short palindromic repeat-CRISPR-associated 9) system
has become a revolutionary technology for targeted genome engineering. The critical step of this technology requires
the design of a highly specific and efficient guide RNA (gRNA) that will guide the Cas9 nuclease to the complementary
DNA target sequence. CRISPR-Explorer is a new and user-friendly web server for selecting optimal CRISPR sites. It
implements the latest scoring schemes of gRNA specificity and efficiency based on published empirical studies. The
gRNA design results are generated instantly, thus removing wait times. The user can visualize the high-quality gRNAs
with detailed design information through an interactive genome browser. Furthermore, the user can define and specify
the parameters for gRNA selection in the Batch Design mode, which recognizes various input formats. CRISPR Explorer
is freely accessible at: http://crisprexplorer.org. Keywords: CRISPR-Cas9, guide RNA design, open-source web application HIGHLIGHT The advantages
of our web application over other existing web applications are: (1)
fast: gRNA design results based on published empirical studies are
generated instantly, thus removing wait times; (2) intuitive: the user
can visualize the high-quality gRNAs with detailed design information
through an interactive genome browser; and (3) flexible: the user can
define his/her own parameters for gRNA selection and various input
formats are recognized. The scoring schemes that are used include the
specificity score and the efficiency score. Targeting multiple regions
can be achieved simultaneously through Batch Design. Guide RNA
design results can be ranked and filtered on the browser before export. The user will select the genome of interest for genome editing, the
desired gRNA length, and the cognate promoter (U6 or T7) that drives
the gRNA expression. After selecting these options, the user will have
the choice to input a gene name (official gene symbol, Ensembl ID,
or Refseq ID), genome coordinates, or a DNA sequence for gRNA CRISPR-Cas is an RNA-guided nuclease system that allows ef
ficient perturbation of gene functions and is increasingly being used
for genome-wide functional screens [1-3]. The type II CRISPR-Cas
system from Streptococcus pyogenes Cas9 (SpCas9) has been adapted
to target a specific DNA sequence for Cas9 nuclease cleavage using
a programmable guide RNA (gRNA) [4-7]. Targeting is mediated by
the first 20 nucleotides at the 5’-end of the gRNA, which are comple
mentary to the target DNA sequence, followed by a 3 bp protospacer www.jbmethods.org
1
POL Scientific
How to cite this article: Chen K, Jin Y, Lin YC. CRISPR Explorer: A fast and intuitive tool for designing guide RNA for genome editing. J
Biol Methods 2016;3(4):e56. DOI: 10.14440/jbm.2016.138 How to cite this article: Chen K, Jin Y, Lin YC. CRISPR Explorer: A fast and intuitive tool for designing guide RNA for genome editing. J
Biol Methods 2016;3(4):e56. DOI: 10.14440/jbm.2016.138 1 www.jbmethods.org Journal of Biological Methods | 2016 | Vol. 3(4) | e56
DOI: 10.14440/jbm.2016.138 Resource adjacent motif (PAM) sequence. Cas9 nuclease can be directed by a
programmable gRNA to induce DNA double-stranded breaks (DSBs)
at specific locations in the genome. In mammalian cells, genome ed
iting typically occurs through the repair of the Cas9-induced DSB by
the error-prone non-homologous end-joining (NHEJ) mechanism,
which introduces variable-length insertion/deletion (indel) mutations,
or through homology-directed (HDR) repair in the presence of an
exogenous DNA template. HIGHLIGHT The sequence model predicts that the editing efficiency is influenced by
the DNA target sequence as well as the flanking sequences [13]. Indeed,
such a sequence model has been experimentally validated, and gRNA
efficiency can be calculated based on this model [13]. Therefore, to
generate a highly specific and efficient gRNA design, we believe that it
is important to consider both the specificity and efficiency of the gRNA. To date, a number of web-based applications have been developed
to select highly specific genome-editing sites [9,12,14,15]. Typically,
these applications require the target sequence or gRNA as input. The
potential CRISPR sites on the target sequence are identified, and this
is followed by the searching of potential off-target sites throughout the
reference genome. The wait time depends on the computation time for
each target site calculation. The wait time increases as the number of input
sequences increases, which can result in a long wait time. The output
is often presented on a plain web page that provides a basic display of
the positions of the CRISPR sites that match the input sequence. A few
applications use the UCSC Genome Browser to display results that are
not interactive. Most of these applications only consider the specificity of To achieve fast query speed, the results were sorted and indexed
using the Tabix program [19]. We adopted the WashU Epigenome
Browser [20] as the presentation framework for display customization
and linking to the gRNA database. The batch design exporter that we
implemented is based on the Angular UI Grid (http://ui-grid.info/)
JavaScript framework, which provides an interactive output. PROCEDURE Input The CRISPR-Explorer homepage contains 3 panels. See Figure 1. 1. ‘Basic Options’ panel. Choose the genome that you want to edit and which promoter you are planning to use. Currently, only Cas9 PAM (-NGG) is supported because it is the most widely used and has a more mature
scoring scheme based on empirical studies. However, we will include other PAMs (such as Cpf1) in the future
when possible. By default, repeat regions are excluded for off-target searching. However, the user has the
option to include repeat regions during the gRNA design. 2. ‘Browse gRNAs’ panel. Browse gRNAs for your gene of interest by entering the gene name, the genome
coordinate, or a DNA sequence from the genome. The input format for the gene name can be the official
gene symbol, Ensembl gene name, or refGene name. HIGHLIGHT As for the calculation of the truncated (18 bp) gRNA,
the first two nucleotide positions and their experimentally determined
effects were removed from the calculation of the specificity score. The
efficiency score was calculated using the SSC program [13]. g
p
,
p
y [ ]
Numerous studies have shown that CRISPR-Cas9 not only targets
its intended on-target sites, but also certain off-target sites in the ge
nome that share sequence similarity with the on-target sites [9-12]. This off-target effect is attributed to the ability of Cas9 to recognize
a non-canonical PAM sequence and tolerate the nucleotide mismatch
between gRNA and its target sequence. To achieve productive genome
editing, the efficiency of Cas9-mediated modification is also critical. The sequence model predicts that the editing efficiency is influenced by
the DNA target sequence as well as the flanking sequences [13]. Indeed,
such a sequence model has been experimentally validated, and gRNA
efficiency can be calculated based on this model [13]. Therefore, to
generate a highly specific and efficient gRNA design, we believe that it
is important to consider both the specificity and efficiency of the gRNA. To date, a number of web-based applications have been developed
to select highly specific genome-editing sites [9,12,14,15]. Typically,
these applications require the target sequence or gRNA as input. The
potential CRISPR sites on the target sequence are identified, and this
is followed by the searching of potential off-target sites throughout the
reference genome. The wait time depends on the computation time for
each target site calculation. The wait time increases as the number of input
sequences increases, which can result in a long wait time. The output
is often presented on a plain web page that provides a basic display of
the positions of the CRISPR sites that match the input sequence. A few
applications use the UCSC Genome Browser to display results that are
not interactive. Most of these applications only consider the specificity of Numerous studies have shown that CRISPR-Cas9 not only targets
its intended on-target sites, but also certain off-target sites in the ge
nome that share sequence similarity with the on-target sites [9-12]. This off-target effect is attributed to the ability of Cas9 to recognize
a non-canonical PAM sequence and tolerate the nucleotide mismatch
between gRNA and its target sequence. To achieve productive genome
editing, the efficiency of Cas9-mediated modification is also critical. HIGHLIGHT To identify potential off-targets for each
possible gRNA, we used the “all-mapper” of the Genome Multitool
(GEM) mapper, which gives all alignments of a specific short sequence
with a user-defined number of mismatches [17]. The number of off-target
alignments in the reference genome for a specific CRISPR site grows
rapidly as the number of mismatches increases, and therefore, up to
4 mismatches in the 20 bp gRNA were allowed when searching for
off-target sites. Three mismatches were used for the truncated (18 bp)
gRNA since previous studies showed that a truncated gRNA with more
than 3 mismatches usually does not have a detectable off-target effect
[18]. To calculate the specificity score (or aggregate scores of single
hits), we adopted the algorithm that was developed by the Zhang F
group [12] and incorporated their experimentally determined effect on
targeting of each mismatch position (M= [0, 0, 0.014, 0, 0, 0.395, 0.317,
0, 0.389, 0.079, 0.445, 0.508, 0.613, 0.851, 0.732, 0.828, 0.615, 0.804,
0.685, 0.583]). As for the calculation of the truncated (18 bp) gRNA,
the first two nucleotide positions and their experimentally determined
effects were removed from the calculation of the specificity score. The
efficiency score was calculated using the SSC program [13]. gi
[
]
Briefly, the whole reference genomes of human (hg19) and mouse
(mm10) for every possible CRISPR site (e.g. 5’(N~20)-NGG, 5’(N~20)-
NAG sites, etc.) were scanned. To identify potential off-targets for each
possible gRNA, we used the “all-mapper” of the Genome Multitool
(GEM) mapper, which gives all alignments of a specific short sequence
with a user-defined number of mismatches [17]. The number of off-target
alignments in the reference genome for a specific CRISPR site grows
rapidly as the number of mismatches increases, and therefore, up to
4 mismatches in the 20 bp gRNA were allowed when searching for
off-target sites. Three mismatches were used for the truncated (18 bp)
gRNA since previous studies showed that a truncated gRNA with more
than 3 mismatches usually does not have a detectable off-target effect
[18]. To calculate the specificity score (or aggregate scores of single
hits), we adopted the algorithm that was developed by the Zhang F
group [12] and incorporated their experimentally determined effect on
targeting of each mismatch position (M= [0, 0, 0.014, 0, 0, 0.395, 0.317,
0, 0.389, 0.079, 0.445, 0.508, 0.613, 0.851, 0.732, 0.828, 0.615, 0.804,
0.685, 0.583]). HIGHLIGHT If you know the genomic location of the region, you
can directly query the browser with the genome coordinate (either in bed format or the following format: 2. ‘Browse gRNAs’ panel. Browse gRNAs for your gene of interest by entering the gene name, the genome
coordinate, or a DNA sequence from the genome. The input format for the gene name can be the official
gene symbol, Ensembl gene name, or refGene name. If you know the genomic location of the region, you
can directly query the browser with the genome coordinate (either in bed format or the following format: J Biol Methods | 2016 | Vol. 3(4) | e56
POL Scientific HIGHLIGHT An indel mutation at the spliced coding
exon of the target gene frequently results in a coding frameshift and
the initiation of nonsense-mediated decay of the gene transcript, which
causes gene inactivation. In addition to its utility in genome editing, by
removing the Cas9 nuclease activity, catalytically inactive Cas9 (dCas9)
proteins that are fused to a co-activation or co-repression domain can
be guided by gRNA to a specific DNA sequence to activate or repress
gene transcription, respectively [8]. the gRNA, but not the efficiency. One application, CRISPR-ERA, does
consider the specificity and efficiency for gRNA design but uses an ad
hoc scoring scheme [14]. Finally, most of the existing web applications
lack an intuitive and convenient way for browsing and batch design. the gRNA, but not the efficiency. One application, CRISPR-ERA, does
consider the specificity and efficiency for gRNA design but uses an ad
hoc scoring scheme [14]. Finally, most of the existing web applications
lack an intuitive and convenient way for browsing and batch design. Here, our goal was to develop a guide RNA design web server that
eliminates the above issues and offers the most up-to-date and proven
scoring schemes for gRNA specificity and efficiency. CRISPR-Explorer
is fast, intuitive, and flexible. It is particularly useful for genome-wide
functional screening and high-throughput screening using the CRIS
PR-Cas9 technology. The CRISPR-Cas system is a rapidly moving
technology. We will continue to update the scoring schemes as new
knowledge and data become available, and we expect to incorporate the
scoring schemes for new Cas nucleases that are identified in the future [16]. Here, our goal was to develop a guide RNA design web server that
eliminates the above issues and offers the most up-to-date and proven
scoring schemes for gRNA specificity and efficiency. CRISPR-Explorer
is fast, intuitive, and flexible. It is particularly useful for genome-wide
functional screening and high-throughput screening using the CRIS
PR-Cas9 technology. The CRISPR-Cas system is a rapidly moving
technology. We will continue to update the scoring schemes as new
knowledge and data become available, and we expect to incorporate the
scoring schemes for new Cas nucleases that are identified in the future [16]. Briefly, the whole reference genomes of human (hg19) and mouse
(mm10) for every possible CRISPR site (e.g. 5’(N~20)-NGG, 5’(N~20)-
NAG sites, etc.) were scanned. L Scienti 2 J Biol Methods | 2016 | Vol. 3(4) | e56 2 POL Scientific Resource Journal of Biological Methods | 2016 | Vol. 3(4) | e56
DOI: 10.14440/jbm.2016.138 The ‘SSC score > 0’ option selects gRNAs that have efficiency scores that are greater than zero
[13]. The ‘Mismatch > 1’ option selects for gRNAs that have more than 1 mismatch with the most similar
sequence in the whole genome. Output The two main display options in CRISPR-Explorer are the interactive browser (Fig. 2 and 3) and a table (Fig. 4) 4. After the ‘Browse’ button inside the ‘Browse gRNAs’ panel is clicked, the browser launches (Fig. 2). The
gRNAs are color-coded based on different specificity and efficiency score cutoffs. The legend on the bot
tom left-hand side defines the color codes. Red gRNAs have the highest specificity and efficiency scores,
and green gRNAs have the lowest specificity and efficiency scores. By rolling the cursor over the gRNA
bar, brief information about that gRNA is displayed. To get detailed information about a gRNA, clicking on
the gRNA leads to a pop-up window that shows the gRNA’s genome coordinate, strand-specificity, length,
closest mismatch, specificity and efficiency scores, sequence, and detailed off-target information (#OT)
(Fig. 2). A click on the ‘Details’ link in this pop-up will lead to a page with detailed information about the
off-target sequences of the selected gRNA. The table that is found on this page is interactive and contains
the following information: off-target sequences with the mismatches in upper case letters (‘Off-target se
quence’), genome coordinate with strand specificity that is indicated by “+” or “-” (‘Location’), number of
mismatches (‘Mismatch’), off-target score (‘Score’), and the name of the gene in which the exons are hit by
the off-target mismatch (‘Hit Exon’). 5. To change the gRNA display on the browser, first right-click on the browser track and then click on ‘Config
ure’. One can choose different filters (‘Closes Mismatch’, ‘Specificity’, or ‘Efficiency’) to limit the gRNAs
that are displayed on the browser (Fig. 3). For example, in Figure 3, the selection of ‘Specificity’ with the
scoring range between 50 and 100 restricts the display of gRNAs to those whose scores fall within this range. 5. To change the gRNA display on the browser, first right-click on the browser track and then click on ‘Config
ure’. One can choose different filters (‘Closes Mismatch’, ‘Specificity’, or ‘Efficiency’) to limit the gRNAs
that are displayed on the browser (Fig. 3). Journal of Biological Methods | 2016 | Vol. 3(4) | e56
DOI: 10.14440/jbm.2016.138 Figure 1. A screen shot of the
CRISPR-Explorer homepage
that shows the three input
panels: “Basic Options”,
“Browse gRNAs”, and “Batch
Design Exporter”. Figure 1. A screen shot of the
CRISPR-Explorer homepage
that shows the three input
panels: “Basic Options”,
“Browse gRNAs”, and “Batch
Design Exporter”. chrX:NNNNN-NNNNN). When the target sequence (with a limit of 20000 bases) is supplied as input, the
Blat program (an alignment tool) from the UCSC Genome Browser is used to map the sequence to the genome
location. The speed of this part depends on the UCSC Blat server. A guide RNA ID that is generated from
Batch Design Exporter can be used to access the detailed information about the gRNA. chrX:NNNNN-NNNNN). When the target sequence (with a limit of 20000 bases) is supplied as input, the
Blat program (an alignment tool) from the UCSC Genome Browser is used to map the sequence to the genome
location. The speed of this part depends on the UCSC Blat server. A guide RNA ID that is generated from
Batch Design Exporter can be used to access the detailed information about the gRNA. 3. 3. ‘Batch Design Exporter’ panel. Multiple genome coordinates or gene entries can be entered simultaneously. Currently, a maximum number of 100 entries is allowed per run to prevent server overload. You can also
reduce the output and get a faster response by applying pre-defined filters. The ‘Exonic guide only’ option
selects gRNAs that fall within the exonic regions. RefGene provides the gene structure to define the exonic
regions. The ‘MIT score ≥ 50’ option selects gRNAs that have specificity scores greater than or equal to
50 [12]. The ‘SSC score > 0’ option selects gRNAs that have efficiency scores that are greater than zero
[13]. The ‘Mismatch > 1’ option selects for gRNAs that have more than 1 mismatch with the most similar
sequence in the whole genome. 3. ‘Batch Design Exporter’ panel. Multiple genome coordinates or gene entries can be entered simultaneously. Currently, a maximum number of 100 entries is allowed per run to prevent server overload. You can also
reduce the output and get a faster response by applying pre-defined filters. The ‘Exonic guide only’ option
selects gRNAs that fall within the exonic regions. RefGene provides the gene structure to define the exonic
regions. The ‘MIT score ≥ 50’ option selects gRNAs that have specificity scores greater than or equal to
50 [12]. Journal of Biological Methods | 2016 | Vol. 3(4) | e56
DOI: 10.14440/jbm.2016.138 For example, in Figure 3, the selection of ‘Specificity’ with the
scoring range between 50 and 100 restricts the display of gRNAs to those whose scores fall within this range. 6. After the ‘Submit’ button inside the ‘Batch Design Exporter’ panel is clicked, an interactive table launches
(Fig. 4). When a gene name is used as input, the output will contain information about the exon (ranked in 6. After the ‘Submit’ button inside the ‘Batch Design Exporter’ panel is clicked, an interactive table launches
(Fig. 4). When a gene name is used as input, the output will contain information about the exon (ranked in Resource Resource Figure 2. An example of the browser track dis
play of “Browse gRNA”. The human IL1A gene
was used as an input. By clicking on the gRNA,
the pop-up box reveals detailed information about
it. The “Details” link directs the user to off-target
sequence information. Figure 2. An example of the browser track dis
play of “Browse gRNA”. The human IL1A gene
was used as an input. By clicking on the gRNA,
the pop-up box reveals detailed information about
it. The “Details” link directs the user to off-target
sequence information. Figure 2. An example of the browser track dis
play of “Browse gRNA”. The human IL1A gene
was used as an input. By clicking on the gRNA,
the pop-up box reveals detailed information about
it. The “Details” link directs the user to off-target
sequence information. Figure 3. An example of the browser track dis
play of “Browse gRNA”. The human IL1A gene
was used as an input. Selecting “Configure” after
right clicking on the track allows the user to choose
the gRNAs that are displayed based on closest
mismatch, specificity and efficiency scores. Figure 3. An example of the browser track dis
play of “Browse gRNA”. The human IL1A gene
was used as an input. Selecting “Configure” after
right clicking on the track allows the user to choose
the gRNAs that are displayed based on closest
mismatch, specificity and efficiency scores. Figure 4. The interactive table of the IL1A gene. The filters of “Exonic guide only”, “MIT score ≥ 50”, “SSC score > 0”, and “Mismatch > 1” are applied
before the generation of this table. Figure 4. The interactive table of the IL1A gene. The filters of “Exonic guide only”, “MIT score ≥ 50”, “SSC score > 0”, and “Mismatch > 1” are applied
before the generation of this table. Figure 4. The interactive table of the IL1A gene. The filters of “Exonic guide only”, “MIT score ≥ 50”, “SSC score > 0”, and “Mismatch > 1” are applied
before the generation of this table. composite gene model) in which the target site falls (‘HitExon’ column). If a transcript name is use as input
(e.g. Ensembl transcript name), the ‘HitExon’ will rank by that transcript. Genome coordinates is the ideal
input format for gRNA design in unannotated genomic regions. When genome coordinates are supplied as
input, the ‘HitExon’ information is not reported. J Biol Methods | 2016 | Vol. 3(4) | e56 3 3 POL Scientific Resource
Journal of Biological Methods | 2016 | Vol. 3(4) | e56
DOI: 10.14440/jbm.2016.138
Figure 2. An example of the browser track dis
play of “Browse gRNA”. The human IL1A gene
was used as an input. By clicking on the gRNA,
the pop-up box reveals detailed information about
it. The “Details” link directs the user to off-target
sequence information. Figure 3. An example of the browser track dis
play of “Browse gRNA”. The human IL1A gene
was used as an input. Selecting “Configure” after
right clicking on the track allows the user to choose
the gRNAs that are displayed based on closest
mismatch, specificity and efficiency scores. Figure 4. The interactive table of the IL1A gene. The filters of “Exonic guide only”, “MIT score ≥ 50”, “SSC score > 0”, and “Mismatch > 1” are applied
before the generation of this table. Journal of Biological Methods | 2016 | Vol. 3(4) | e56
DOI: 10.14440/jbm.2016.138 J Biol Methods | 2016 | Vol. 3(4) | e56 Resource composite gene model) in which the target site falls (‘HitExon’ column). If a transcript name is use as input
(e.g. Ensembl transcript name), the ‘HitExon’ will rank by that transcript. Genome coordinates is the ideal
input format for gRNA design in unannotated genomic regions. When genome coordinates are supplied as
input, the ‘HitExon’ information is not reported. 7. 7. The interactive table generated by the ‘Batch Design Exporter’ contains the following information: gRNA
sequence (‘Guide RNA’), genome coordinates (‘Location’), strand specificity (‘Strand’), target’s name
(‘Target’), exon number where the gRNA falls (‘HitExon’), number of mismatches (‘MM’), specificity score
(‘SpecMIT’), efficiency score (‘EffiSSC’), exonic or not (‘Exon’), number of potential off-targets that is
exonic (‘OtExon’), number of mismatches contained by the potential off-targets that fall in exons (‘OtEx
onDist’), number of potential off-targets in the whole genome (‘OT’), number of mismatches contained by
the potential off-targets that fall in the genome (‘OtDist’), and the link to the detailed information about the
off-target sequences (‘GuideID’) (Fig. 4). The description in the ‘OtExonDist’ column (for example, 0:0:0:0:4 7. The interactive table generated by the ‘Batch Design Exporter’ contains the following information: gRNA
sequence (‘Guide RNA’), genome coordinates (‘Location’), strand specificity (‘Strand’), target’s name
(‘Target’), exon number where the gRNA falls (‘HitExon’), number of mismatches (‘MM’), specificity score
(‘SpecMIT’), efficiency score (‘EffiSSC’), exonic or not (‘Exon’), number of potential off-targets that is
exonic (‘OtExon’), number of mismatches contained by the potential off-targets that fall in exons (‘OtEx
onDist’), number of potential off-targets in the whole genome (‘OT’), number of mismatches contained by
the potential off-targets that fall in the genome (‘OtDist’), and the link to the detailed information about the
off-target sequences (‘GuideID’) (Fig. 4). The description in the ‘OtExonDist’ column (for example, 0:0:0:0:4 J Biol Methods | 2016 | Vol. 3(4) | e56
POL Scientific L Sc ent J Biol Methods | 2016 | Vol. 3(4) | e56 References 1. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, et al. (2013) Genome-
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web application for at least two years and may provide updates that
include the newly identified CRISPR systems that target RNA. 9. Bae S, Park J, Kim J (2014) Cas-OFFinder: a fast and versatile algorithm that
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vice versa. This can be achieved by clicking on the right of the individual header of the first row of the table
where an up or down arrow is. 8. Each column of the interactive table can be ranked alphabetically or numerically from lowest to highest or
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where an up or down arrow is. 9. Different features can be selected for the ‘Guide RNA’, ‘Location’, ‘Strand’, ‘Target’, ‘Exon’, ‘OtExon’,
‘OtExonDist’, ‘OT’, and ‘OtDist’ columns. The filtering functions (equals/less/or greater) are available for
the ‘HitExon’, ‘MM’, ‘SpecMIT’, and ‘EffiSSC’ columns. This can be achieved by rolling the cursor to the
left of the individual column header. Then, click on the ‘Ξ’ icon that appears and the filtering option will
be revealed. 10. After filtering and ranking, the personally customized table can be exported as a ‘.csv’ file by clicking the
“Export to CSV” button at the bottom of the table. The user can keep the default file name or type in the file
name inside the textbox that is above the “Export to CSV” button. 10. After filtering and ranking, the personally customized table can be exported as a ‘.csv’ file by clicking the
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Intimate partner violence against adolescents and young women in sub-Saharan Africa: who is most vulnerable?
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eCommons@AKU
eCommons@AKU eCommons@AKU
eCommons@AKU eCommons@AKU
eCommons@AKU Medical College, East Africa Medical College, East Africa Obstetrics and Gynaecology, East Africa Authors
Authors
Yohannes Dibaba Wado, Martin K. Mutua, Abdu Mohiddin, Macellina Y. Ijadunola, Cheikh Faye, Carolina V.
N. Coll, Aluisio J. D. Barros, and Caroline W. Kabiru Intimate partner violence against adolescents and young women
Intimate partner violence against adolescents and young women
in sub-Saharan Africa: who is most vulnerable?
in sub-Saharan Africa: who is most vulnerable? See next page for additional authors Follow this and additional works at: https://ecommons.aku.edu/eastafrica_fhs_mc_obstet_gynaecol
Part of the Obstetrics and Gynecology Commons Abstract Background: Intimate partner violence (IPV) is a global public health and human rights issue that affects millions
of women and girls. While disaggregated national statistics are crucial to assess inequalities, little evidence exists on
inequalities in exposure to violence against adolescents and young women (AYW). The aim of this study was to deter-
mine inequalities in physical or sexual IPV against AYW and beliefs about gender based violence (GBV) in sub-Saharan
Africa (SSA). Methods: We used data from the most recent Demographic and Health Surveys (DHS) conducted in 27 countries in
SSA. Only data from surveys conducted after 2010 were included. Our analysis focused on married or cohabiting AYW
aged 15–24 years and compared inequalities in physical or sexual IPV by place of residence, education and wealth. We
also examined IPV variations by AYW’s beliefs about GBV and the association of country characteristics such as gender
inequality with IPV prevalence. Results: The proportion of AYW reporting IPV in the year before the survey ranged from 6.5% in Comoros to 43.3%
in Gabon, with a median of 25.2%. Overall, reported IPV levels were higher in countries in the Central Africa region
than other sub-regions. Although the prevalence of IPV varied by place of residence, education and wealth, there was
no clear pattern of inequalities. In many countries with high prevalence of IPV, a higher proportion of AYW from rural
areas, with lower education and from the poorest wealth quintile reported IPV. In almost all countries, a greater pro-
portion of AYW who approved wife beating for any reason reported IPV compared to their counterparts who disap-
proved wife beating. Reporting of IPV was weakly correlated with the Gender Inequality Index and other societal level
variables but was moderately positively correlated with adult alcohol consumption (r = 0.48) and negative attitudes
towards GBV (r = 0.38). Conclusion: IPV is pervasive among AYW, with substantial variation across and within countries reflecting the role of
contextual and structural factors in shaping the vulnerability to IPV. The lack of consistent patterns of inequalities by
the stratifiers within countries shows that IPV against women and girls cuts across socio-economic boundaries sug-
gesting the need for comprehensive and multi-sectoral approaches to preventing and responding to IPV. Keywords: Physical or sexual violence, Attitudes, SSA, Adolescents and young women, Equiplot Authors
Authors Wado et al. Reprod Health 2021, 18(Suppl 1):119
https://doi.org/10.1186/s12978-021-01077-z Open Access Open Access © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://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. Intimate partner violence
against adolescents and young women
in sub‑Saharan Africa: who is most vulnerable? Carolina V. N. Coll4, Aluisio J. D. Barros4 and Caroline W. Kabiru1 Plain English summary Intimate partner violence (IPV) is a global public health
and human rights issue that affects millions of women
and girls. Disaggregated national data are needed to *Correspondence: ywado@aphrc.org
1 African Population and Health Research Center, Nairobi, Kenya
Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://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. Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 2 of 13 assess inequalities in exposure to violence against adoles-
cents and young women (AYW). and violated because they have less bargaining power
within their relationships [10, 11]. Data from the WHO
violence against women surveys show that globally 30%
of adolescent girls (aged 15 to 19 years) have experi-
enced physical and/or sexual violence by an intimate
partner in their lifetime [3]. Moreover, one study that
used Demographic and Health Survey (DHS) data from
30 developing countries estimated that 28% of ado-
lescents (15–19 years) and 29% of young women (20–
24 years) had experienced physical or sexual intimate
partner violence (IPV) [12]. In this study, we examined inequalities in physical or
sexual IPV against AYW in sub-Saharan Africa (SSA)
using data from the most recent national surveys from 27
countries. Our analyses focused on married or cohabit-
ing adolescents and young women aged 15–24 years and
compared inequalities in IPV by place of residence, edu-
cation and wealth. We also examined IPV variations by
AYW’s beliefs and country characteristics such as gender
inequality.h Much research on the determinants of IPV against
women has been informed by an ecological framework
that outlines multiple factors operating at different lev-
els—individual, relationship, community, and societal
levels—that explain why some groups of people are at
greater risk [13–15]. For instance, socioeconomic ine-
qualities and socio-cultural norms such as those around
male dominance over women contribute to the high
prevalence of GBV in SSA [12, 16]. Evidence from a sys-
tematic review also shows that individual characteristics
such as age, age difference with the partner, and educa-
tion level are risk factors of GBV [17]. Moreover, there
are various contextual and country-specific drivers of
violence in SSA. There is a strong link between pov-
erty and violence among young women with those from
poor households and communities being at greater risk
[18]. Low education, exposure to violence in childhood,
unequal power in intimate relationships, and attitudes
and norms accepting violence and gender inequality also
increase the risk of experiencing IPV and sexual violence
[13]. The percentage of AYW reporting IPV in the year
before the survey ranged from 6.5% in Comoros to 43.3%
in Gabon. Overall, IPV levels were higher in countries
in the Central Africa region than other sub-regions. IPV
levels varied by place of residence, education and wealth
although there was no clear pattern of inequalities. A
higher percentage of AYW from rural areas, with lower
education and from the poorest households reported
IPV. In almost all countries, a higher percentage of AYW
who approved wife beating reported IPV compared to
their counterparts who disapproved wife beating. IPV is pervasive with substantial variations between
and within countries reflecting the role of contextual
and structural factors in shaping the vulnerability to IPV. There is a need for comprehensive and multi-sectoral
approaches to preventing and responding to IPV against
AYW. Background Education was coded into two categories (primary
or less, and secondary or more) based on the distribu-
tion of AYW’s education. In the majority of the coun-
tries included in this analysis, only a small proportion
of AYW had no formal education. Age was recoded
into two categories: adolescents (15–19 years) and
young women (age 20–24 years). Age difference with
the partner was categorized into two groups based on
the distribution of the data (a difference of more than
5 years vs. a difference of 5 years or less). Demographic and Health Surveys are an important
source of data to study cross-national and regional ine-
qualities in exposure to IPV because they are nation-
ally-representative and use standardized tools that
follow ethical and safety recommendations for research
on domestic violence against women [24]. Such cross-
national and regional comparisons will enable the identi-
fication of groups of AYW that are most affected. As the
DHS are conducted about every 5 years, they are valuable
in monitoring the progress and effectiveness of inter-
ventions targeting the protection and empowerment of
AYW to prevent violence. Population-based surveys that
highlight differences in IPV by wealth index, residence
and education and other individual- and community-
level determinants of violence against AYW are useful
for informing the design and targeting of interventions. In this study, we drew on DHS data to examine inequali-
ties in IPV against AYW in SSA and identify groups that
experience the highest levels of violence in different
contexts. f
The main outcome variable was intimate partner vio-
lence (physical, sexual and physical or sexual violence)
against AYW in the past year. The current prevalence of
IPV was defined as the percentage of currently married
or cohabiting AYW who reported having experienced
at least one act of IPV in the 12 months before the sur-
vey. In the DHS, violence information is obtained from
ever-married and cohabiting respondents on violence
committed by their current and former spouses/partners
and by others. Physical IPV is measured using a short-
ened, modified version of the Conflict Tactics Scale [26]
which asks the respondent if she has ever been—pushed,
shaken, slapped, punched with a fist or something that
could hurt, kicked, dragged or beaten up, choked, burned
on purpose or had something thrown at her [27]. Background Violence against women and girls is a global public health
and human rights issue that affects millions of women
and girls. According to the World Health Organization
approximately one-third of women globally have expe-
rienced some form of violence (physical or sexual) from
a partner or non-partner in their lifetime [1]. While the
effects of gender based violence (GBV) on the physical,
mental health and social well-being of women and girls
are relatively well-documented [2–4], its health conse-
quences continue to be unabated due to the persistent
high prevalence. For instance, young women in sub-
Saharan Africa (SSA) continue to carry the brunt of high
HIV infection due to sexual violence, poverty and social
norms around marriage, gender inequalities and harm-
ful traditional practices that reinforce unequal power
dynamics with young women particularly disadvantaged
[5–8]. With the Sustainable Development Goals (SDGs), the
international community committed to the achieve-
ment of gender equality and elimination of all forms of
violence against women and girls by 2030. Promoting
gender equality, preventing violence against women and
girls (SDG, goal 5) and ensuring responsive and inclusive
societies (SDG target 16.1) are far-reaching goals in the
SDGs to ensure gender equity [19]. While the relation-
ship between gender and violence is complex, evidence
indicates that gender inequalities increase the risk of
violence by men against women and inhibit the ability
of those affected to seek protection [9, 20]. SDG target
17.18 also calls for disaggregated national statistics by
income, rural–urban residence, gender and other vari-
ables to assess inequalities. Yet, little evidence exists on
inequalities in exposure to violence against AYW in SSA. In this regard, the agenda to “leave no one behind” and
Countdown to 2030 are well-timed to provide inequality
data for the purpose of designing effective interventions
to improved gender equity and address violence against
AYW in SSA countries [21] since existing evidence of Gender inequalities increase the risk of violence
against women and girls and inhibit the ability of those
affected to seek protection [9, 10]. Adolescent girls and
young women (AYW), particularly those married to
older men, and/or married as children or adolescents,
may be disproportionately at risk of being exploited Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 3 of 13 interventions are skewed towards high income countries
[20, 22, 23]. and the fifth quintile represents the wealthiest 20%
[25]. Methods
D t We used data from the most recent DHS with the vio-
lence against women module. We limited our analyses
to data from 27 countries in SSA whose most recent
survey was conducted after 2010. Eight countries with
surveys were excluded because the violence module was
not applied (Guinea, Lesotho, Madagascar, Maurita-
nia and Guinea) or only had data from national surveys
carried out before 2010 (Ghana, Liberia and Sao Tome). We also included similar number of countries with at
least one round of surveys since 2010 for the analysis of
AYW’s attitudes towards wife beating. The study popula-
tion included married or cohabiting adolescent girls aged
15–19 years and young women aged 20–24 years. We
used the United Nations Population Division grouping
of countries in SSA into four sub-regions: Central Africa,
Eastern Africa, Southern Africa and Western Africa. Table 1 presents the countries included in the study by
region and year of survey.hi The DHS collects various proxy indicators of women’s
empowerment including attitudes towards wife beating,
also named beliefs towards gender based violence. As
attitudes towards violence is one of the key predictors of
exposure to GBV [15, 28], we examine variation in atti-
tudes towards wife beating by the stratifiers. In the DHS,
all respondents are asked a series of questions to assess
their attitudes to wife-beating. The questions ask whether
a husband is justified in hitting or beating his wife in any
of the following scenarios; if she goes out without tell-
ing him; if she neglects the children; if she argues with
him; if she refuses to have sex with him and if she burns
the food. A single composite variable ‘disagreement with
wife-beating’ was constructed by grouping women into
two categories: women who endorse at least one reason
for wife-beating and women who reject all reasons of
wife-beating. Unlike the IPV, which is administered to a
sub-sample of households, the attitudes towards violence
data is collected from all women. The equity stratifiers used in this analysis included
household wealth quintiles, rural–urban residence,
woman’s education, age and age difference with the
partner. Accordingly, inequality in this analysis refers
to differences in the outcome indicator (IPV against
AYW) between two or more sub-groups. In the DHS,
the wealth index is coded into five quantiles, however,
in our analyses, we compared the two extreme catego-
ries (the first quintile vs. the fifth quintile). Background Sexual
violence was assessed using the following items: physi-
cally forced to have unwanted sex, or forced into other
unwanted sexual acts. An affirmative answer to one or
more of the items listed in the Conflict Tactics Scale
constitutes evidence of physical and sexual violence (see
DHS reports, www.dhsprogram.org). We also looked at
the prevalence of a combination of the two types of vio-
lence (physical or sexual).h Methods
D t We analyzed survey data for each country at the
national level, to compare the prevalence of IPV (physi-
cal or sexual violence) among AYW by the equity strati-
fiers—wealth quintiles, education, place of residence,
and age. We also examined how attitudes towards wife
beating, one of the major predictors of IPV, vary by the
stratifiers. Data presented are weighted using the domes-
tic violence weight, to adjust for within country sample
selection and nonresponse. We use equiplots and charts
to visualize inequalities in IPV against AYW by the strati-
fiers. Equiplots are used to present intervention cover-
age by groups, making it possible to visualize both the
level of coverage in each group and the distance between
groups, which represents absolute inequality [34]. We
also use other relative measures of inequality such as
ratios to demonstrate how one sub-group differs from the In addition, we performed an ecological analysis
and used multiple data sets to examine the correlation
between country IPV prevalence with key societal char-
acteristic: Gross National Income per capita extracted
from the World Bank [29]; Gender Inequality Index (GII)
an index that measures gender inequalities in reproduc-
tive health, empowerment and labor force participation,
from the UNDP [30]; Fragile States Index—a measure of
state fragility and instability—extracted from the 2019
Fragile States Index annual report [31]; urbanization
levels from PRB’s world population data sheet [32]; edu-
cational attainment and AYW’s beliefs about GBV both
extracted from the DHS; and adult male alcohol con-
sumption per capita extracted from the WHO data base
[33]. Methods
D t The first
quintile represents the poorest 20% in the population Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 4 of 13 Table 1 List of countries by year of DHS surveys for the intimate partner violence analysis
Region
Country
IPV data
Most
recent
survey
Eastern and Southern Africa
Eastern Africa
Rwanda
2014
2014
Malawi
2015
2015
Kenya
2014
2014
Ethiopia
2016
2016
Zambia
2013
2013
Burundi
2016
2016
Tanzania
2015
2015
Uganda
2016
2016
Mozambique
2015
2015
Comoros
2012
2012
Southern Africa
Zimbabwe
2016
2016
Namibia
2013
2013
South Africa
2016
2016
West and Central Africa
West Africa
Mali
2018
2018
Senegal
2017
2017
Burkina Faso
2010
2010
Sierra Leone
2013
2013
Nigeria
2018
2018
Togo
2013
2013
Cote d’ivoire
2011
2011
Benin
2017
2017
Gambia
2013
2013
Central Africa
Congo Brazzaville
2011
Gabon
2012
2012
Cameroon
2011
2011
Angola
2015
2015
Congo DRC
2013
2013
Chad
2014
2014 Table 1 List of countries by year of DHS surveys for the intimate partner violence analysis Table 1 List of countries by year of DHS surveys for the intimate partner violence analysis West and Central Africa Central Africa prevalence of IPV at country level by the stratifiers. The
median and the interquartile range were used to sum-
marize IPV prevalence at regional and sub-regional
levels. We analyzed survey data for each country at the
national level, to compare the prevalence of IPV (physi-
cal or sexual violence) among AYW by the equity strati-
fiers—wealth quintiles, education, place of residence,
and age. We also examined how attitudes towards wife
beating, one of the major predictors of IPV, vary by the
stratifiers. Data presented are weighted using the domes-
tic violence weight, to adjust for within country sample
selection and nonresponse. We use equiplots and charts
to visualize inequalities in IPV against AYW by the strati-
fiers. Equiplots are used to present intervention cover-
age by groups, making it possible to visualize both the
level of coverage in each group and the distance between
groups, which represents absolute inequality [34]. We
also use other relative measures of inequality such as
ratios to demonstrate how one sub-group differs from the prevalence of IPV at country level by the stratifiers. The
median and the interquartile range were used to sum-
marize IPV prevalence at regional and sub-regional
levels. West and Central Africa Violence against adolescents and young women Across the 27 countries, the reported prevalence of phys-
ical or sexual IPV ranged from 6.5% in Comoros (2012)
to 43.3% in Gabon (2012). It varied widely between the
countries. The median prevalence of combined physical
or sexual IPV against AYW was 25.2%. The prevalence
was highest in Central Africa (39.8%), followed by South-
ern Africa (28.4%) respectively (Fig. 1; Additional file 1:
Table S1). In 5 of the 27 countries (Gabon, Burundi, DRC
Congo, Cameroon, and Sierra Leone) more than 35%
of AYW reported experiencing either physical or sex-
ual IPV in the 12 months before the survey. Within the
sub-regions, large inequalities between countries were
observed in Eastern Africa. In East Africa, the prevalence
of physical or sexual IPV varied from 36.4% in Burundi to
only 6.5% in Comoros (Table 2). A close examination of the prevalence of IPV by the
different stratifiers showed no clear overall pattern and
little within-country variations in AYW’s experience of
physical or sexual IPV in the 12 months preceding the
survey (see Additional file 1: Table S1). For instance,
there is no clear pattern in rural–urban inequalities in
the prevalence of physical or sexual IPV. In the major-
ity of countries (15 of the 27) the prevalence was higher
in urban areas although the differences were statistically
significant in only three of the 17 countries; Mozam-
bique, Angola, and Cote d’Ivoire (Fig. 2). The prevalence of physical IPV varied from 5.2% in
Comoros (2012) to 38.8% in Gabon (2012). The preva-
lence of physical IPV was higher in the Central Africa
region where AYW in three of the five countries (Congo
DRC, Cameroon and Gabon) reported a prevalence of
over 30%. The reported prevalence of sexual IPV ranged
from 1.5% in Burkina Faso (2010) to 23.9% in DRC Congo In 11 of the 27 countries, a higher proportion of AYW
from rural areas experienced physical or sexual IPV in
the past year with differences being statistically signifi-
cant in three countries (Burundi, Ethiopia and Uganda). The pattern of inequality by education was similarly
mixed. In 14 out of 27 countries, the prevalence of IPV
was higher among AYW with primary education, and the
differences were significant in four countries (Uganda,
Burundi, Tanzania and Malawi). Data analysis Data were analyzed using STATA software version 14
(StataCorp, 2015). We used proportions to estimate Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 5 of 13 (2013). The prevalence of sexual IPV was highest in Cen-
tral Africa (16.1%), followed by Southern Africa (10.4%),
Eastern Africa (10.1%) and West Africa (7.7%) respec-
tively (Fig. 1). Outside Central Africa (DRC Congo,
Gabon and Cameroon) more than one sixth of AYW in
countries such as Burundi, Uganda and Malawi reported
sexual violence (Table 2). (2013). The prevalence of sexual IPV was highest in Cen-
tral Africa (16.1%), followed by Southern Africa (10.4%),
Eastern Africa (10.1%) and West Africa (7.7%) respec-
tively (Fig. 1). Outside Central Africa (DRC Congo,
Gabon and Cameroon) more than one sixth of AYW in
countries such as Burundi, Uganda and Malawi reported
sexual violence (Table 2). other. Moreover, proportions of key outcome indicators
are presented with their confidence intervals in tables. Accordingly, significant differences between sub-groups
are determined based on non-overlapping confidence
intervals. To assess the association of IPV with selected
societal determinants (at the country level) we used
scatter plots, Pearson correlation coefficient and linear
regression analyses to test associations with IPV preva-
lence at the 5% level. For some of the indicators, we did
log transformations to reduce the influence of outliers. There is a high correlation between the prevalence of
sexual IPV and physical IPV in SSA (Pearson’s correla-
tion, coefficient, r = 0.66), and several countries with
high physical IPV (DRC, Gabon, Burundi, Uganda) also
reported high levels of sexual IPV. In a few countries, for
instance in Burundi and Malawi, AYW reported nearly
equal or higher level of sexual IPV than physical IPV. On the other hand, there are a few countries with very
low sexual IPV overall; AYW in Burkina Faso, Comoros,
Mozambique and Gambia reported sexual IPV preva-
lence of about 2% or less (Table 2). Violence against adolescents and young women Physical or sexual IPV
prevalence was higher among AYW with secondary and
above education in 11 countries but the differences were
not statistically significant except in Mozambique (Addi-
tional file 1: Table S1). 0
5
10
15
20
25
30
35
40
45
50
IPV (%)
Median
Central
Eastern
Southern
West
Fig. 1 Median prevalence of physical or sexual intimate partner
violence against AYW by region, SSA i
The patterns by wealth quintile shows that in about
three-fifth of countries, a higher proportion of AYW
from the poorest wealth quintile reported physical or
sexual IPV although the differences were significant for
only four countries (Uganda, Tanzania, Rwanda, and
Gabon). In nine countries, the reported prevalence of
physical or sexual IPV was higher among AYW from
the richest wealth quintile. However, the difference was
statistically significant in four countries; Mozambique,
Senegal, Sierra Leone, and Cameroon (Additional file 1:
Table S1). Fig. 1 Median prevalence of physical or sexual intimate partner
violence against AYW by region, SSA Wado et al. Violence against adolescents and young women However, in many coun-
tries, IPV prevalence was higher among AYW whose age
difference with the partner was less than 5 years and the
differences were statistically Significant in three coun-
tries; Cameroon, Burundi, Angola (Additional file 1:
Table S1). i
The prevalence of physical or sexual IPV varied mark-
edly by attitudes towards wife beating. As shown in
Fig. 3, a higher proportion of AYW who approved wife-
beating for any reason reported physical or sexual IPV
than their counterparts who disapproved wife beating
for any reason. The differences were significant at the
5% level in 15 of the 27 countries and remarkable dis-
parities were seen in countries such as Namibia, Mali, Violence against adolescents and young women Reprod Health 2021, 18(Suppl 1):119 Page 6 of 13 Table 2 Percentage of adolescents and young women 15–24 years who experienced intimate partner violence, national
DHS surveys, 2010–2018 (in parenthesis 95% confidence interval)
Country
Physical and or sexual IPV
Physical IPV
Sexual IPV
N
Gabon
43.4 [36.7,50.3]
38.8 [32.3,45.6]
16.1 [11.6,21.9]
573
Congo DRC
40.9 [36.8,45.1]
32.6 [29.1,36.4]
23.9 [20.0,28.3]
1136
Cameroon
39.8 [36.1,43.5]
33.4 [29.7,37.4]
16.3 [13.6,19.5]
943
Sierra Leone
36.7 [32.2,41.4]
32.9 [28.7,37.4]
8.6 [6.3,11.7]
681
Burundi
36.4 [33.4,39.6]
23.6 [20.9,26.5]
23.2 [20.6,26.1]
1010
Uganda
32.5 [29.6,35.6]
23.9 [21.5,26.4]
18.3 [16.1,20.7]
1675
Tanzania
32.4 [29.2,35.7]
28.9 [25.9,32.0]
11.6 [9.7,13.8]
1491
Zambia
31.8 [29.0,34.8]
26.2 [23.5,29.0]
14.9 [13.0,17.0]
1694
Angola
30.6 [27.2,34.2]
28.5 [25.3,31.9]
9.8 [8.1,11.8]
2086
Namibia
29.1 [20.9,38.9]
26.7 [19.2,36.0]
10.1 [5.5,17.9]
125
Zimbabwe
28.4 [25.2,31.9]
21.5 [18.7,24.6]
12.9 [10.7,15.5]
1081
Malawi
26.1 [23.3,29.1]
15.8 [13.6,18.4]
16.9 [14.5,19.6]
1265
Côte d’Ivoire
25.3 [22.2,28.8]
24.4 [21.3,27.7]
5.9 [4.2,8.1]
960
Kenya
25.2 [21.1,29.8]
21.1 [17.2,25.6]
9.1 [6.5,12.6]
676
Mali
24.7 [20.6,29.2]
20.3 [17.0,24.1]
12.9 [10.3,16.1]
847
Rwanda
23.9 [18.9,29.8]
19.1 [14.7,24.4]
8.4 [5.5,12.8]
219
Ethiopia
21.7 [17.8,26.1]
18.7 [14.9,23.1]
8.4 [5.8,12.0]
814
Chad
18.2 [14.9,22.2]
14.2 [11.5,17.3]
8.7 [6.4,11.9]
948
Nigeria
17.0 [14.8,19.4]
12.8 [10.9,14.9]
8.4 [6.7,10.4]
1530
South Africa
15.8 [9.8,24.5]
12.2 [7.2,19.8]
8.0 [3.6,16.8]
185
Togo
15.1 [12.5,18.0]
11.9 [9.6,14.5]
7.1 [5.3,9.4]
785
Benin
14.2 [11.5,17.3]
9.1 [7.0,11.7]
7.7 [5.7,10.3]
832
Senegal
14.1 [10.3,19.0]
10.4 [6.9,15.2]
8.2 [5.4,12.2]
504
Mozambique
12.4 [9.6,16.0]
11.8 [9.0,15.3]
2.1 [1.2,3.5]
661
Burkina Faso
8.4 [7.1,9.9]
7.8 [6.6,9.1]
1.5 [1.0,2.2]
2475
Gambia
7.1 [5.2,9.6]
5.3 [3.7,7.4]
2.2 [1.2,4.0]
869
Comoros
6.5 [4.4,9.6]
5.2 [3.5,7.7]
2.0 [1.0,4.1]
479 We also examined inequalities in physical or sexual IPV
by AYW’s attitudes towards wife beating, age difference
between the partner and AYW’s age. Overall, the preva-
lence of IPV was higher among young women than ado-
lescents, although the differences were significant in only
four countries (Nigeria, Tanzania, Angola and Burkina
Faso). In the four countries (Zimbabwe, Malawi, Benin
and Senegal) where a greater proportion of adolescents
than young women reported IPV, the differences were
not statistically significant. Angola, Burundi and Uganda. In Namibia for instance,
the proportion of AYW who reported physical or sexual
IPV varied from 13.3% among those who disapproved
wife-beating to 50.2% among those who accepted wife-
beating. The prevalence of physical or sexual violence
did not vary significantly by the age difference between
the respondent and her partner. Societal characteristics and reporting IPV While there were observable variations between coun-
tries, the within countries variation by the stratifiers
was relatively inconsistent. Key questions emerging
from this observation are: what explains the large Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 7 of 13 Fig. 2 Percentages of AYW reporting physical or sexual intimate partner violence by stratifiers, SSA the 27 countries in the analysis. Moreover, wide within-
country inequalities in attitudes towards wife-beating
existed by place of residence, wealth quintile and educa-
tion. In almost all the 27 countries, a higher proportion of
AYW residing in urban areas rejected wife-beating com-
pared to their rural counterparts and the differences were
statistically significant for 18 countries. Wider rural–
urban differences of over 20 or more percentage points
were observed in countries such as Ethiopia, Nigeria,
Gambia and Namibia (Fig. 5, Additional file 3: Table S3). variation between countries? We examined the corre-
lation between reporting IPV at the country level and
selected societal characteristics—Gender Inequality
Index (GII), educational attainment (proportion of AYW
with secondary and above education), GNP per capita,
urbanization levels, adult male alcohol consumption per
capita and the prevalence of negative attitudes towards
wife beating. All bivariate analyses showed no or weak
associations between the societal characteristics and
IPV among AYW. Moderate or weak correlations were
observed with adult male alcohol consumption per capita
(Pearson correlation coefficient, r = 0.48) and the preva-
lence of negative attitudes towards wife beating (Pearson
correlation coefficient, r = 0.38). There was no or very
weak association with the GII, GNP per capita, Fragile
States Index, and level of education of young people in
the society (see Fig. 4 and Additional file 2: Table S2). i
The disparity by wealth quintile was also remarkable. In 17 of the 27 countries, the proportion of AYW who
rejected wife-beating was significantly higher among the
richest quintile compared to the poorest. Large dispari-
ties by wealth were observed in Ghana, Nigeria, Angola,
Ethiopia, Namibia, Zambia and Senegal. Inequalities by
education were also notable with a higher proportion of
AYW with secondary education rejecting wife-beating. The differences between AYW with secondary education
and those with primary or lower education were statis-
tically significant for 17 countries (see Additional file 3:
Table S3). Attitude towards wife beatingh The median proportion of AYW who rejected all rea-
sons of wife-beating ranged from 17.9% in Mali (2018) to
92.5% (2016) in South Africa with a median of 47.7% in Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 8 of 13 Fig. 3 Percentages of AYW reporting physical or sexual IPV by empowerment measures, SSA
r=0.3831
r=0.4809
Fig. 4 Scatterplot matrix of the correlation between the societal determinant and IPV Fig. 3 Percentages of AYW reporting physical or sexual IPV by empowerment measures, SSA Fig. 3 Percentages of AYW reporting physical or sexual IPV by empowerment measures, SSA r=0.3831
r=0.4809
Fig. 4 Scatterplot matrix of the correlation between the societal determinant and IPV Fig. 4 Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 9 of 13 Fig. 5 Percentage of adolescents and young adult women who reject wife-beating by stratifiers observable regional and between-country variation,
the reporting of IPV by AYW was not strongly corre-
lated with societal characteristics such as the Gender
Inequality Index, GNP per capita, Fragile States Index,
or aggregate levels of AYW educational attainment at
the national level. It was moderately correlated with
male adult alcohol consumption per capita and approval
of wife beating at the national level. The poor correla-
tion with Gender Inequality Index is unexpected. Some
studies have reported a moderate correlation between
the index and IPV among women aged 15–49 in low
and middle income countries [36, 37]. Our study shows
that conventional indicators of socio-economic develop-
ment may not explain large inter-country differences in
reported IPV against AYW in the region (Fig. 5). Discussion As documented by several studies [15,
16] social norms play a significant role with a large pro-
portion of AYW from the poorest households viewing
spousal violence as a normal and justified occurrence
in marriage. Traditional beliefs that men have a right to
control women and that increase vulnerability to violence
are more common among the poorest, less educated and
rural adolescents in SSA [9, 20, 37, 43]. Consistent with
the findings of our ecological analysis, the association
between partner alcohol use and increased risk of IPV
victimization at the individual level has been reported by
various studies [44, 45]. These findings have implications
for the targeting of violence prevention interventions
aimed at promoting more equitable gender norms.i respectively. In particular, in many countries with a very
high prevalence of IPV (Gabon, DRC Congo, Uganda,
Burundi and others) a higher proportion of AYW from
the poorest wealth quintile experienced IPV compared
to their counterparts. Previous studies have highlighted
associations between low education, poverty and violence
among young women and noted that young women from
poor households may have low decision-making abili-
ties, resources and empowerment, which increases their
vulnerability to violence [12, 38]. Thus, interventions
that broaden women’s access to economic resources
and opportunities may help empower women and help
reduce the risk of IPV. The finding that in countries like Angola, Cote d’ivoire,
Mali, Mozambique and Burkina Faso, more educated,
urban and wealthier AYW were more likely to report IPV
is noteworthy. These AYW may challenge the traditional
status quo and may be considered more “empowered”. Their male partners may therefore resort to using vio-
lence to maintain a dominant position in contexts where
male dominance is normative [8, 15]. “Empowered” AYW
may also be more willing to disclose IPV [39]. However,
these inconsistent patterns also suggest that violence
against women and girls is pervasive across all socio-eco-
nomic backgrounds [3, 20, 23].fl While the regional differences in reporting may reflect
important cultural, political, or religious differences
[40, 41] differential reporting by women of different
socio-economic groups within a country is also possi-
ble depending on cultural and social norms that under-
lie the acceptance of violence in each settings [37, 40]. The culture of silence that affects the reporting and what
constitutes violence varies across cultures and can make
comparability difficult. Discussion Findings from these analyses indicate that IPV against
AYW is a pervasive problem in SSA with large between
and within-country variations. Across the 27 countries
included in our analyses, more than one in four AYW
reported physical or sexual IPV in the 12 months before
the surveys. National prevalence of physical or sexual IPV
varied from 6.5% in Comoros to 43.3% in Gabon. Over-
all, the prevalence of physical or sexual IPV was higher
in Central Africa region compared to other sub-regions. In countries such as Gabon, Cameroon, Sierra Leone and
Congo DRC over one-third of AYW reported experienc-
ing physical or sexual IPV in the past year. Many of these
countries are in conflict or post conflict situations that
might have exacerbated pre-existing patterns of violence
against women and girls as conflicts can result in higher
levels of violence against women and girls, including arbi-
trary killings, torture, sexual violence and forced mar-
riage [35].f While the between countries variation in the preva-
lence of IPV is large, the analysis demonstrated that the
within country inequalities by the stratifiers are not con-
sistent. However, in close to half of the countries, AYW
residing in rural areas, with lower education and those
from the poorest wealth quintile experienced more IPV
than their counter-parts from urban areas, with higher
education and those from the richest wealth quintiles, Previous studies show that differences in contextual
and structural factors may explain some of the differ-
ences observed in IPV prevalence between countries
[12, 20, 23]. Our analysis, however, showed that despite Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 10 of 13 Page 10 of 13 specific and vary among and within countries [14, 42]. The disaggregated analysis showed greater inequality in
IPV by AYW’s attitudes towards wife beating, with lower
IPV levels among those who reject wife beating for any
reason compared to those who accept wife beating. In
most countries, trend analysis of DHS data shows that
attitudes towards wife beating are changing. However,
we found that approval of wife beating is more common
among rural dwellers, those from poor households, and
those with lower levels of education. Similar findings
were recently reported in a publication describing the
most vulnerable groups in low and middle-income coun-
tries that high IPV prevalence among those who accept
wife beating [37]. Discussion Evidences show that levels of IPV
may be under reported due to fear of retaliation by part-
ners, shame and stigma, lack of awareness of available
services or access to such services among other reasons
[2, 40]. Interestingly, as approval of wife beating is asso-
ciated with IPV prevalence, it is possible that AYW who
accept wife beating are more willing to disclose experi-
ence of IPV than their counterparts who disapprove
wife beating. However, while the magnitude of under-
reporting is unknown, the DHS violence against women
module uses validated and standardized questions that
are implemented following WHO recommendations of
studies on violence against women and girls to improve
data quality, protect the safety of respondents and enable
comparability across countries. Overall, the findings demonstrate that IPV is pervasive
among AYW, with substantial variation between coun-
tries and regions reflecting the role of contextual and
structural factors in shaping vulnerability to IPV. The
lack of consistent pattern of inequalities by the stratifiers
within countries shows that IPV against women and girls
cuts across socio-economic boundaries suggesting the
need for comprehensive and multi-sectoral approaches
to preventing and responding to IPV in line with the
ecological framework [46, 47]. Moreover, the observed
variation by attitudes towards wife-beating shows that
promoting gender equitable norms from early childhood
through multi-sectoral strategies (including school-based
interventions that address gender norms and attitudes
from younger ages, and community interventions that
can empower women such as microfinance schemes; and
media interventions to increase public awareness) can
help in reducing violence against women and girls [9, 20,
48]. However, the effectiveness of IPV mitigation actions
and care services in SSA remain to be evaluated. Globally, efforts to prevent and respond to cases of
violence against women and girls have increased in the
last few decades. Many countries in SSA have adopted
laws and policies addressing different forms of violence,
including rape, child sexual abuse, and domestic and/or
intimate partner violence. However, the implementation The ecological model, which is most widely used for
understanding the causes of violence, proposes that vio-
lence is a result of factors operating at various levels. While there is limited research on community and soci-
etal influences, many of the factors identified are context Wado et al. Availability of data and materials The dataset used for the current study is available for free from https://dhspr
ogram.com/data/available-datasets.cfm and can be provided up on request
by the corresponding author. Consent for publication
Not required. Consent for publication
Not required. Discussion Reprod Health 2021, 18(Suppl 1):119 Page 11 of 13 Page 11 of 13 of these laws is hampered by weak institutional capaci-
ties and limited reporting by victims of violence [30, 48]. Nonetheless, there is an increasing availability of data
and lessons from programmatic responses that can be
used to scale up prevention and response mechanisms to
attain the SDG goal of eliminating all forms of violence
against women and girls by 2030. Funding The Countdown to 2030 for Women’s, Children’s and Adolescents’ Health is
the recipient of an investment of the Bill & Melinda Gates Foundation. This
study also benefitted from funding of Canada Partnership for Women’s and
Children’s Health. The contents of this research article only reflect the authors’
opinions but do not show interest/s of either of the organizations involved in
the funding. Acknowledgements The authors gratefully acknowledge Ties Boerma for the review and com-
ments he provided on the various drafts of the manuscript. g
g
y
This study is not without limitations. It is important
to note that rates of IPV may be under-reported due to
cultural and social norms that underlie the acceptance
of violence. Although the DHS violence against women
module is implemented following WHO recommenda-
tions of research, the fact that the module is implemented
within a wide range of health modules means that women
are likely to under report due to social desirability bias
[49]. Evidence from countries with two or more sur-
veys with the module (e.g., Nigeria, Malawi, Kenya and
Rwanda) also shows that prevalence of IPV against AYW
between consecutive surveys were not consistent indica-
tive of reporting issues. Moreover, as these modules are
implemented in a sub-sample of households and individ-
uals, the sample of married adolescents is relatively small
which may have resulted in wide confidence intervals for
some parameters. But, we have excluded surveys with
less than 30 observations from the analysis. Abbreviations AYW: Adolescents and young women; DHS: Demographic and Health Surveys;
GBV: Gender based violence; IPV: Intimate partner violence; SDGs: Sustainable
Development Goals; SSA: Sub-Saharan Africa; GII: Gender Inequality Index;
GNP: Gross National Product. Competing interests
h
h
d
l
h p
g
The authors declare that they have no competing interests. p
g
The authors declare that they have no competing interests. About this supplement This articles has been published as part of Reproductive Health Volume 18
Supplement 1 2021: ASRH for all in SSA: are inequalities reducing?. The full
contents of the supplement are available at https://reproductive-health-journ
al.biomedcentral.com/articles/supplements/volume-18-supplement-1. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12978-021-01077-z. The online version contains supplem
org/10.1186/s12978-021-01077-z. Authors’ contributions YDW, MK, AM, MC and CF conceptualized the study. MK and YDW conducted
data analysis and interpretation. AM and MYI performed literature review. YDW wrote the initial draft of the manuscript. CK, CC, AB and DM participated
in interpretation of findings and revised the manuscript. All authors read and
approved the final manuscript. Author details
1 1 African Population and Health Research Center, Nairobi, Kenya. 2 Aga Khan
University, Nairobi, Kenya. 3 Department of Epidemiology and Medical Sta-
tistics, University of Ibadan, Ibadan, Nigeria. 4 International Centre for Equity
in Health, Universidade Federal de Pelotas, Pelotas, RS, Brazil. Received: 10 January 2021 Accepted: 11 January 2021
Published: 17 June 2021 Ethics approval and consent to participate Overall, the findings demonstrate that IPV is pervasive
among AYW, with substantial variation between coun-
tries and regions reflecting the role of contextual and
structural factors in shaping vulnerability to IPV. The
lack of consistent pattern of inequalities by the strati-
fiers within countries shows that IPV against AYW cuts
across socio-economic boundaries suggesting the need
for comprehensive and multi-sectoral approaches to
preventing and responding to IPV. The between country
variation however is poorly measured by conventional
development indicators. On the other hand, the observed
variation by attitudes towards wife-beating show that
promoting gender equitable norms from early childhood
helps in reducing violence against women and girls. Ethical approval for the DHS is obtained from national ethics review boards
through the national institutions involved in the survey and from ICF Interna-
tional. The description of consent process is available from https://www.dhspr
ogram.com/What-We-Do/Protecting-the-Privacy-of-DHS-Survey-Respondent
s.cfm. 2004/11/20). 29. WorldBank. GNI per capita, Atlas method (current US$), World Bank national
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norms are associated with increased male-perpetrated rape and sexual risks
for HIV infection in Botswana and Swaziland. PLoS ONE. 2012;7:e28739. 30. UNDP. Violence against women, a cause and consequence of inequality,
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2019. https://fundforpeace.org/2019/04/10/fragile-states-index-2019/. Accessed 7 Feb 2020. 9. WHO. Promoting gender equality to prevent violence against women. WHO; 2009. http://www.who.int/violenceprevention/publications/en/index
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lished maps and institutional affiliations. Wado et al. Reprod Health 2021, 18(Suppl 1):119 Wado et al. Reprod Health 2021, 18(Suppl 1):119 Page 13 of 13 Page 13 of 13 2019/07/03). The political, research, programmatic, and social responses to
adolescent sexual and reproductive health and rights in the 25 years since 24. WHO. Putting women’s safety first: ethical and safety recommendations
for research on domestic violence against women. Geneva: World Health
Organization; 1999. Report No, 1999 Contract No.: WHO/EIP/GPE/99.2,
Accessed 5 Feb 2020. 24. WHO. Putting women’s safety first: ethical and safety recommendations
for research on domestic violence against women. Geneva: World Health
Organization; 1999. Report No, 1999 Contract No.: WHO/EIP/GPE/99.2,
Accessed 5 Feb 2020. Wado et al. Reprod Health 2021, 18(Suppl 1):119 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub-
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Supplementary Figure 4 from Nutlin-3a Activates p53 to Both Down-regulate Inhibitor of Growth 2 and Up-regulate <i>mir-34a, mir-34b,</i> and <i>mir-34c</i> Expression, and Induce Senescence
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interactions of model cationic drug with newly synthesized starch derivatives
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Keywords potato starch, adsorption, interaction, methylene blue 1Department of Physical Chemistry and Biophysics, Wrocław Medical University, Borowska 211A, 50-556 Wrocław,
Poland 2Department of Food Storage and Technology, Faculty of Biotechnology and Food Science, Wroclaw University of
Environmental and Life Sciences, Chełmońskiego 37, 51-630 Wrocław, Poland *Corresponding Author: E-mail: witold.musial@umw.edu.pl; Tel.: + 48-717-840-228
Received: June 26 2023; Revised: September 8 2023; Published: September 20 2023 *Corresponding Author: E-mail: witold.musial@umw.edu.pl; Tel.: + 48-717-840-228 *Corresponding Author: E-mail: witold.musial@umw.edu.pl; Tel.: + 48-717-840-228
Received: June 26, 2023; Revised: September 8, 2023; Published: September 20, 2023 Received: June 26, 2023; Revised: September 8, 2023; Published: September 20, 2023 Abstract Background and purpose: The aim of the work was to compare the interactions of three newly synthesized
non-toxic starch derivatives, with varied anionic and non-ionic functional groups with methylene blue (MB) as
a model cationic drug, and selection of starch derivative with highest affinity to the MB. Experimental
approach: The native potato starch (SN), modified via acetylation (SM1), esterification and crosslinking (SM2)
and crosslinking (SM3), was evaluated in MB adsorption studies and assessed by FTIR, PXRD, and DSC. Key
results: The adsorption of MB on SM2 and SM3 matched the BET isotherm model, which confirmed
physisorption on the low-porous surface. In the case of SM1, adsorption took place via electrostatic attraction
between the heterogeneous adsorbent surface and the adsorbate, as demonstrated by the Freundlich plot. The FTIR confirmed vibrations assigned to N=C stretching bonds at 1600 cm-1 in the case of MB adsorbed on
the SN and SM2. The most intense PXRD peaks belonged to SN and the least to SM2. In the DSC study, the
thermal stability via ΔT was assessed, with SM2 of lowest ΔT value (179.8 °C). Conclusion: SM2 presented the
best adsorption capacity, followed by SM3 and the weakest SM1. The interactions were confirmed in the
adsorption studies and may reflect applications of the modified starches as drug carriers. In the FTIR study, a
probable interaction between the OH- groups of SM2 and N+ of MB was revealed. The most amorphous struc-
ture was shown for SM2, which was correlated with the lowest thermal stability provided by the DSC study. ©2023 by the authors. This article is an open-access article distributed under the terms and conditions of the Creative Commons
Attribution license (http://creativecommons.org/licenses/by/4.0/). doi: https://doi.org/10.5599/admet.1950 Original scientific paper ADMET & DMPK 11(3) (2023) 387-407; https://doi.org/10.5599/admet.1950 ADMET & DMPK 11(3) (2023) 387-407; https://doi.org/10.5599/admet.1950 Introduction There are numerous newly developed carriers for therapeutic substances [1-3]. They are often completely
new synthetic polymers [4,5]. Among polymers of plant origin, there are natural examples of potential
carriers [6-10]. Wheat, maize and potato starch are available in large quantities from natural sources [11-13]. So far, starch has been modified by Utomo, Odeniyi, Singh et al. [14-16], among others. Previous publications
have presented new methods for obtaining a number of starch derivatives [17-19] and proposed the
potential use of some of them, including acetylated starch [20,21] and starch citrate [22]. The figure below
shows the structural models of the modified potato starches created by the team from the University of
Environmental and Life Science (Wrocław, Poland) (Figures 1a,b,c). doi: https://doi.org/10.5599/admet.1950 387 ADMET & DMPK 11(3) (2023) 387-407 J. Kobryń et al. a
b
c
d
Figure 1. Structural models of the adsorbates as: a - starch acetate (SM1), b - starch citrate (SM2),
c - starch diphosphate (SM3) and the d - adsorbent methylene blue tautomers (MB). b a d c c Figure 1. Structural models of the adsorbates as: a - starch acetate (SM1), b - starch citrate (SM2),
c - starch diphosphate (SM3) and the d - adsorbent methylene blue tautomers (MB). Some drugs have a 1,4-thiazine ring, which is included in the structure of methylene blue (MB). The study
that used the similarity between MB and other molecules was performed on acridine and phenothiazine
derivatives for anti-prion therapy [23]. Selected drugs include the 1,4-thiazine ring: phenothiazine, which has
antiparasitic, antiseptic and antioxidant effects [24], promethazine, which has antihistamine and anti-allergic
effects [25], chlorpromazine and [26] thioridazine - as psychotropic drugs. The ionic interaction between the
promethazine and Eudragit® was ascribed to the electrostatic interaction between the drug and ammonium
groups in the polymer backbone [27]. The interactions of chlorpromazine with cyclodextrin were realized via
ionic bonds between the ionized amine group of chlorpromazine and anionic group of cyclodextrin sulfate
[28]. Another study, which exploited cyclic voltammetry, interpreted the interactions of chlorpromazine and
thioridazine with bovine serum albumin nanoparticles as a result of hydrophobic interaction [29]. Therefore,
research has been undertaken on MB as a model drug representing the structural features of some active
pharmaceutical ingredients (API). These studies may determine whether similar drugs could adsorb on
modified starch. Preparation of citrate starch (SM2) Native potato starch (SN) was esterified with citric acid. 10 g of citric acid per 100 g of starch dry matter
was dissolved in 90 mL of water and thoroughly mixed. The resulting starch paste was left for 12 h at room
temperature and then dried in an air dryer (Memmert, Germany) at 50 °C for 12 h. The starches were calcined
for 3 h at 100 °C. The sample was rinsed three times with ethyl alcohol with a concentration of 95 mL of
ethanol per 100 mL of solution, and each time, the solution was poured over the sediment. The washed
precipitate was dried in an air dryer at 30 °C for 12 h. The obtained citrate starch was hardened at a
temperature 2 degrees below the gelatinization temperature (determined by DSC), dried in an air dryer at
30 °C, ground in a laboratory mill and passed through a sieve with a mesh size of 400 μm [17]. Preparation of diphosphate starch (SM3) Native potato starch (SN) (94.6 wt.%), sodium trimetaphosphate (1.2 wt.%), sodium carbonate (2.1 wt.%)
and sodium chloride (2.1 wt.%) were introduced into the reaction vessel, 500 mL of distilled water at 45 °C
was added. The mixture was adjusted to pH 10.5 with a 3 % NaOH solution. It was kept at 45 °C for 30 minutes
with constant stirring, then neutralized with 8 % HCl to pH 6.5-6.8. The cross-linked starch was washed several
times with distilled water on a funnel under vacuum and dried for 48 h at 25 °C. After obtaining starch
diphosphate, it was hardened for 24 h at 2 degrees below the gelatinization temperature, dried in an air dryer
at 30 °C, milled and sieved through a sieve with a mesh size of 200 μm. Preparation of the modified starches The thermal and chemical modifications of native potato starch were conducted and resulted in
acetylation (SM1), esterification and crosslinking (SM2), and crosslinking (SM3). Preparation of acetylated starch (SM1) The fractions of starch particles with an average volume moment diameter D [3,4] equal to 39.1 and
61.7 µm were separated from the native potato starch (SN) by measuring the volume diameter (Malvern
laser particle size analyzer). Larger starch particles were acetylated with acetic anhydride in the amount of
13 cm3 /100 g of starch. After drying the acetylated starch, an initial gelatinization temperature of 49.17 °C
was determined by differential scanning calorimeter Flash DSC (Mettler Toledo, Poland). The acetylated
starch has been stirred for 24 h at 48 °C. It was then washed three times with five-liter portions of distilled
water and separated from the slurry using the Contifuge Stratos (Heraeus, Germany) flow centrifuge. The
obtained starch was dried for 24 h in an air dryer at 30 °C. Materials The following materials were employed in the study: methylene blue (MB, Reko, Dzierżoniów, Poland) - a
model active pharmaceutical ingredient (API), native potato starch (SN) (PPZ, Niechlów, Poland), modified
potato starches (starch acetate - SM1, starch citrate - SM2 and starch diphosphate - SM3) prepared according
to below-described methods. Introduction To date, studies have analyzed the adsorption of MB and other dyes on activated carbon,
minerals of natural origin such as perlite, clay and its derivatives, bentonite [30,31], and agricultural solid
wastes [32]. In summary, the important group that led to the intermolecular activity may be the ammonia
group, which specifically interacts with anionic functionals. Because of the wide potential application of
modified potato starch [33-36], we decided to compare the adsorption capacity of selected starch
derivatives, characterized by a distinct negative charge of carboxyl and phosphate groups, or a characteristic
ester group, towards methylene blue as a cationic substance. The aim of the work was the evaluation of the prospective pharmaceutical applicability of three newly
synthesized non-toxic starch derivatives, assessed via analysis of interactions of the starches, modified by
varied acidic and non-ionic functional groups, with methylene blue, as a model cationic drug. The main
question was the selection of the starch derivative with the highest affinity to the MB molecule. The possible
interactions were evaluated in the terms of spectroscopic structural studies, as well as thermal assessments. The interactions, which may lead to drug release prolongation, were practically confirmed in the adsorption
studies, which may reflect use of the above-mentioned modified starches as drug carriers for topical
application, with retardant and prolonging effect against drug release process. 388 ADMET & DMPK 11(3) (2023) 387-407 Model cationic drug and newly synthesized starch derivatives Model cationic drug and newly synthesized starch derivatives Powder X-ray diffraction (PXRD) analysis Powder X-ray diffraction (PXRD) analysis The analysis of starch was supplemented by X-ray diffraction measurements (PXRD). The powder PXRD
data were recorded on a Bruker D2 PHASER diffractometer (Bruker AXS, Karlsruhe, Germany) with a Lynxeye
detector using Cu Kα radiation (0.15418 nm). All samples were measured at 295 K with 3.0 mm slit and
1.0 mm shutter. Diffractograms were obtained between 7.5° and 40° (2) (step size of 0.02° (2) and 0.25 s
per step). The X-ray generator operated at 30 kV and 10 mA. The PXRD patterns were processed using the
software Diffrac.Eva V 3.2. (Bruker AXS). The percentage of crystallinity and amorphousness of the tested
starch samples in relation to SN was determined. Spectroscopic evaluation Fourier transform infrared spectroscopy (FTIR) Fourier-transform infrared spectroscopy (FTIR) and attenuated total reflectance (ATR) appetizer (Nicolet
380 FTIR, Thermo Scientific, Waltham, MA, USA) with OMNIC ™ software were used to determine possible
interactions between MB particles and starch. The formulations of MB adsorbed on every 50 mg of starch
(SN, SM1, SM2 and SM3) were dried at 40 °C and compared with a physical mixture of MB and starch in a
weight ratio of 1:10 and with the standards of the pure substances. The spectra of powders were recorded
at wavelengths of 400 to 4000 cm-1 at 32 scans per sample and a resolution of 4 cm−1. Determination of the potential number of functional groups of modified starches Based on the recipe for preparing individual modified starches, the mass values of substrates attached to
native starch (SN), such as acetic anhydride, trimethaphosphate and citric acid, were converted to moles. The
converted values per gram of SN are given in Table 1. doi: https://doi.org/10.5599/admet.1950 389 ADMET & DMPK 11(3) (2023) 387-407 J. Kobryń et al. Table 1. Number of moles of individual functional groups of modified starches per 1 g of SN. Modified starch
Concentration, mmol g-1
Carbonyl groups
Carboxyl groups
Phosphate groups
SM1
2.74
-
-
SM2
-
1.56
-
SM3
-
-
0.12
Spectroscopic evaluation Table 1. Number of moles of individual functional groups of modified starches per 1 g of SN. Differential scanning calorimetry (DSC) Differential scanning calorimetry (DSC 214 Polyma, Netzsch, Selb, Germany) was performed to investigate
the samples of dried adsorbed MB on 50 mg starches (SN, SM1, SM2, SM3), their physical mixtures of
1:10 w/w and pure ingredients. The grated samples of 3 to 5 mg were explored in aluminium pans with lids
under a nitrogen atmosphere, with a flow rate of 50 mL min-1. The thermograms were recorded at a constant
heating rate of 5 °C min-1 in the temperature range from 0 to 350 °C. Determination of the effect of MB adsorption on the pH of starch Determination of the effect of MB adsorption on the pH of starch The effect of MB adsorption on starch pH was investigated. The suspensions of pure starch in distilled
water and suspensions of starch with adsorbed MB were prepared according to adsorption conditions (50
mg starch per 40 mL of water). pH measurements were taken after 1 and 72 h at 22±2 °C. Each measurement
was taken three times. An ANOVA test with α = 0.05 was performed for these measurements. Evaluation of the adsorption kinetics of MB on the modified starches 1/
F
s
=
n
tq
k C
1
a
n
=
F
ln
b
k
=
(
)
m
L
s
L
s
=
1
q k C
q
k C
+
m
1
a
q
=
m
L
1
b
q k
=
( )
( )
(
)
( )
( )
(
)
m
s
BET s
s
s
s
BET L
BET L
BET s
=
1-
1-
+
q C k
q
C k
C k
C k
( )
( )
( )
( )
BET L
BET L
BET s
m
BET s
2-
=
k
k
k
a
q k
( )
( )
( )
BET s
BET L
m
BET s
-2
=
k
k
b
k
q
( )
m
BET s
1
=
c
k
q
2
2
1
=
+
t
t
t
q
k q
q
The values of q and qt were calculated by the equations (1) and (2): 1/
F
s
=
n
tq
k C
1
a
n
=
F
ln
b
k
=
(
)
m
L
s
L
s
=
1
q k C
q
k C
+
m
1
a
q
=
m
L
1
b
q k
=
( )
( )
(
)
( )
( )
(
)
m
s
BET s
s
s
s
BET L
BET L
BET s
=
1-
1-
+
q C k
q
C k
C k
C k
( )
( )
( )
( )
BET L
BET L
BET s
m
BET s
2-
=
k
k
k
a
q k
( )
( )
( )
BET s
BET L
m
BET s
-2
=
k
k
b
k
q
( )
m
BET s
1
=
c
k
q
2
2
1
=
+
t
t
t
q
k q
q
The values of q and q were calculated by the equations (1) and (2): The values of q and qt were calculated by the equations (1) and (2): (
)
0
s
C -C
=
V
q
m
(1)
(
)
0
C -C
=
t
t
V
q
m
(2) (2) m
where C0 is initial concentration of the solution (mg L-1), Ct is concentration of the solution in time t (mg L-1),
V is volume of solution (L) and m is adsorbent mass (g). doi: https://doi.org/10.5599/admet.1950 Evaluation of the adsorption kinetics of MB on the modified starches Evaluation of the adsorption kinetics of MB on the modified starches The absorbance study was performed using four 50 mL conical flasks (A, B, C, D) as a series. 40 mL 6 mg L-1
MB was added for each flask. The SN, SM1, SM2 and SM3 starch probes of 2, 5, 10, 25, 50, 125, 250, 500, 750
and 1000 mg were placed in the flasks during the series, respectively. The flasks were put on the orbital
shaker (at 100 rpm) at 22±0.5 °C. Four measurements of absorbance were proceeded based on the
pharmacopoeial method by sampling the MB solution volumes of 3 mL in every 5, 10, 15, and 20 minutes ,
and than in equal periods up to 4 h. The solution was returned to the flasks. Analysis of taken samples was
done by the spectrophotometer UV/VIS Jasco V-530 (Tokyo, Japan) at 663 nm, according to the available
bibliography [37-39], and compared to the absorption spectrum of MB. A standard curve based on three
series of measurements with five concentration points from 0.5 to 5.0 mg L-1 was prepared. The results were
examined according to pseudo-zero-order kinetics and first-order kinetics. ANOVA statistical test was
performed for independent groups at the significance level α = 0.05. Freundlich, Langmuir and BET isotherm
models were taken for analysis of adsorption processes (Table 2). BET isotherm equation was adapted to 390 ADMET & DMPK 11(3) (2023) 387-407 Model cationic drug and newly synthesized starch derivatives form BET isotherm for liquid phase adsorption [40], where kBET(s) applies to the equilibrium constant of
adsorption of the first layer and kBET(L) refers to the equilibrium constant of adsorption of upper layers. Table 2. Isotherm and kinetics models applied for evaluation of obtained data, Cs - concentration of substance in the
solution in the equilibrium state (e.s.), qt - adsorbed quantity of adsorbate in time, q -adsorbed quantity of adsorbate in
the e.s., qm - maximum monolayer capacity, kF, kL and kBET - adsorption equilibrium constants, k1, k2 - equilibrium rate
constants, 1/n - constant, t - time, a - slope, b - intercept, r2 - regression coefficient. Evaluation of the adsorption kinetics of MB on the modified starches where C0 is initial concentration of the solution (mg L-1), Ct is concentration of the solution in time t (mg L-1),
V is volume of solution (L) and m is adsorbent mass (g). Equation (3) was used for the adsoprption (%) calculation: Equation (3) was used for the adsoprption (%) calculation: 0
0
Adsorption
100
t
m
m
m
−
=
0
0
Adsorption
100
t
m
m
m
−
=
0
0
Adsorption
100
t
m
m
m
−
=
(3) (3) where m0 - the initial mass of the adsorbate, mt - is the adsorbate mass after time t. Determination of the effect of MB adsorption on the pH of starch Results The results of the starch synthesis procedures yielded products with structures that conformed to the
patterns (Figure 1 in the Introduction), and the appearance was as shown in Figures 2A and 2B. The molar ratio of functional groups added to the modified starches was converted per 1 g of native starch. Spectroscopic evaluations The molar ratio of functional groups added to the modified starches was converted per 1 g of native starch. Spectroscopic evaluations The molar ratio of functional groups added to the modified starches was converted per 1 g of native starch. Spectroscopic evaluations FTIR spectroscopy FTIR spectroscopy The FTIR spectrum of pure MB showed a broad peak from 3219 to 3364 cm-1, indicating an O-H bending
bond. The sharp peaks at 3050 cm-1 and in the range of 817 to 884 cm-1 were responsible for the presence of
C-H or =C-H bonds in the aromatic ring. Similarly, a sharp peak around 2700 cm-1 may have occurred due to the
C-H stretching bonds [41]. doi: https://doi.org/10.5599/admet.1950 391 ADMET & DMPK 11(3) (2023) 387-407 J. Kobryń et al. Figure 2. Images of actual appearance (above) and images magnified 10 times with a stereoscopic microscope
(SMZ-171-TLED, Motic, Hongkong, China) (below) of A - SM1, B - SM2, C - SM3; petri dish diameter = 10 cm. Figure 2. Images of actual appearance (above) and images magnified 10 times with a stereoscopic microscope
(SMZ-171-TLED, Motic, Hongkong, China) (below) of A - SM1, B - SM2, C - SM3; petri dish diameter = 10 cm. Characteristic peaks interacting at 1596 cm-1 might have been assigned to N=C stretching bonds. The peaks
in the range 1420-1490 cm-1 most likely belonged to the stretching C=C-C bonds in the aromatic ring. Vibration
from 1340 to 1360 cm-1 revealed the presence of C-N stretching bonds in aromatic tertiary amine [41] and peaks
from 1059 to 1064 cm-1 and 661 cm-1 are related to C-S-C stretching bonds in heterocycle [42]. The pure starches
FTIR spectra indicated a broad peak of O-H stretching bond at about 3300 cm-1, C-O stretching vibration at the
range 1185-1347 cm-1, especially the strong peak at 1240 cm-1 for SM1 (Figure 3 b), C-O stretching bond at
930 cm-1 [43] and O-H bending bond at 988 cm-1 [44] (Figure 3). 92
a
b
c
d
Figure 3. The FTIR spectra of methylene blue (MB), the pure starches (a) SN, (b) SM1, (c) SM2 and (d) SM3,
the physical mixture and the experimental formulation of the starches with adsorbed MB, respectively. b
d b a c d c d Figure 3. The FTIR spectra of methylene blue (MB), the pure starches (a) SN, (b) SM1, (c) SM2 and (d) SM3,
the physical mixture and the experimental formulation of the starches with adsorbed MB, respectively. FTIR spectroscopy 392 ADMET & DMPK 11(3) (2023) 387-407 Model cationic drug and newly synthesized starch derivatives The plot of the spectrum of adsorbed MB on the starch (red line) mostly followed the plot of the spectrum
of the respective starch in its pure state (green line). However, there were locations where individual peaks
were in excess. The peaks assigned to N=C stretching bonds at about 1600 cm-1 appeared in the case of MB
adsorbed on the SN and SM2, however, to a very slight extent (Figure 3 a,c). The peak indicating the presence
of the C=O bond of the acetyl group at 1730 cm-1 [45] was spotted on the SM1 spectra (Figure 3b). PXRD Diffractograms of modified starches were compared to those of SN (Figure 4). The peaks with the highest
intensity were in the 2 range from 17.2 to 17.4°. The most intense belonged to SN and the least to SM2. The proportion of crystallinity and amorphousness for the tested samples was determined using the
amorphous subtraction method [46,47] and presented in Table 3. Table 3. Composition of crystallinity and amorphousness for SM1, SM2 and SM3 samples compared to SN (n = 3). Starch types
SN
SM1
SM2
SM3
Crystallinity, %
14.3 ± 0.80
9.8 ± 0.14
3.2 ± 0.26
5.7 ± 0.75
Amorphousness, %
85.7 ± 0.80
90.2 ± 0.14
96.8 ± 0.26
94.3 ± 0.75
2 / °
Figure 4. The diffractograms of acetylated starch (SM1), starch citrate (SM2) and starch diphosphate (SM3)
compared to native starch (SN). Table 3. Composition of crystallinity and amorphousness for SM1, SM2 and SM3 samples compared to SN (n = 3). Starch types
SN
SM1
SM2
SM3
Crystallinity, %
14.3 ± 0.80
9.8 ± 0.14
3.2 ± 0.26
5.7 ± 0.75
Amorphousness, %
85.7 ± 0.80
90.2 ± 0.14
96.8 ± 0.26
94.3 ± 0.75 Table 3. Composition of crystallinity and amorphousness for SM1, SM2 and SM3 samples compared to SN (n = 3). Starch types
SN
SM1
SM2
SM3
Crystallinity, %
14.3 ± 0.80
9.8 ± 0.14
3.2 ± 0.26
5.7 ± 0.75
Amorphousness, %
85.7 ± 0.80
90.2 ± 0.14
96.8 ± 0.26
94.3 ± 0.75
2 / °
Figure 4. The diffractograms of acetylated starch (SM1), starch citrate (SM2) and starch diphosphate (SM3)
compared to native starch (SN). Figure 4. The diffractograms of acetylated starch (SM1), starch citrate (SM2) and starch diphosphate (SM3)
compared to native starch (SN). Thermal properties DSC analysis results shown in Figure 5 compared pure MB and evaluated starches with adsorbed MB and
physical mixtures of these components. The thermograms showed the results of heat flow measurements
from 0 to 350 °C taken in two cycles, with a cooling interval in between. The exothermic direction has been
marked with an appropriate arrow (exo). The results of DSC testing of native and modified starches under two heating cycles are shown in Figure 6. The glass transition midpoint temperatures Tg, the difference in the heat capacity between the transition
from glass to the liquid state ΔCp and ΔT - difference between onset crystallization temperature and Tg were
analyzed using Proteus 7.0 software (Netzsch, Selb, Germany). The results are shown in the Supplementary
material, Table S1. doi: https://doi.org/10.5599/admet.1950 393 ADMET & DMPK 11(3) (2023) 387-407 J. Kobryń et al. bryń et al. ADMET & DMPK 11(3) (2023) 387-407
a
b
c
d
e
f
g
h
Figure 5. The DSC thermograms of methylene blue (MB), the pure starches (SN, SM1, SM2 and SM3), physical
mixtures, and the experimental formulations of the starches with adsorbed MB. The subfigures (a,b,c,d)
represent thermograms of the first heating cycle and (e,f,g,h) thermograms of the second heating cycle. a b a c
e d d c f
h f e e h g h g Figure 5. The DSC thermograms of methylene blue (MB), the pure starches (SN, SM1, SM2 and SM3), physical
mixtures, and the experimental formulations of the starches with adsorbed MB. The subfigures (a,b,c,d)
represent thermograms of the first heating cycle and (e,f,g,h) thermograms of the second heating cycle. 394 Model cationic drug and newly synthesized starch derivatives ADMET & DMPK 11(3) (2023) 387-407 b a
b
Figure 6. The DSC thermograms of the starches (SN, SM1, SM2 and SM3). The subfigure (a) represents
thermograms of the first heating cycle and (b) the second heating cycle. a a Figure 6. The DSC thermograms of the starches (SN, SM1, SM2 and SM3). The subfigure (a) represents
thermograms of the first heating cycle and (b) the second heating cycle. doi: https://doi.org/10.5599/admet.1950 Adsorption tests The graphs showing the course of MB adsorption on starch showed the lowest saturation for SN and the
highest for SM2 for 2 mg starch samples. It was 7.60 mg g-1 for SN and 22.86 mg g-1 for SM2 (Figure 7). The graphs showing the course of MB adsorption on starch showed the lowest saturation for SN and the
highest for SM2 for 2 mg starch samples. It was 7.60 mg g-1 for SN and 22.86 mg g-1 for SM2 (Figure 7). The graphs showing the course of MB adsorption on starch showed the lowest saturation for SN and the
highest for SM2 for 2 mg starch samples. It was 7.60 mg g-1 for SN and 22.86 mg g-1 for SM2 (Figure 7). b
d a
b
c
d
Figure 7. The plot of MB adsorption on the starch samples with time: (a) SN, (b) SM1, (c) SM2, (d) SM3;
● - 2 mg, ○ -5 mg, ■ - 10 mg, □ - 25 mg, ▲ - 50 mg, Δ - 125 mg, ♦ - 250 mg, ◊ - 500 mg, - - 750 mg, + - 1000 mg,
n = 4, at 22±0.5 °C and 6.0 mg L-1 MB solution with pH 4.26. b a a c d d c Figure 7. The plot of MB adsorption on the starch samples with time: (a) SN, (b) SM1, (c) SM2, (d) SM3;
● - 2 mg, ○ -5 mg, ■ - 10 mg, □ - 25 mg, ▲ - 50 mg, Δ - 125 mg, ♦ - 250 mg, ◊ - 500 mg, - - 750 mg, + - 1000 mg,
n = 4, at 22±0.5 °C and 6.0 mg L-1 MB solution with pH 4.26. The amount of adsorbed MB increased with the enhancement of the mass of starch samples. The
parameter values, adjusted to the tested kinetics models, based on the correlation factor, are given in the
Supplementary material, Table S2. Table 4 exhibits the parameters adapted to the chosen isotherm models. The samples of the starches
were divided into two categories dependent on the mass; “A” was between 2 and 125 mg and “B” was
between 250 and 1000 mg. Freundlich isotherm described in a satisfactory way adsorption equilibriums of doi: https://doi.org/10.5599/admet.1950 395 ADMET & DMPK 11(3) (2023) 387-407 J. Kobryń et al. MB adsorbed on the SN A, SM1, SM2 and SM3 B. Determination of the effect of MB adsorption on the pH of starch The pH of starch suspensions with adsorbed MB compared to that of pure starch suspensions was higher
for SM2 and lower for SN and SM1. In addition, there was a proportional increase in pH values after 72 h
(Figure 8). a
b
Figure 8. Comparison of pH values of starch suspensions with adsorbed MB to pure starch suspensions after
(a) 1 h and (b) 72 h at 22±2 °C (n = 3). NosignificantdifferenceswereobservedbetweenthepHvaluesof SN-MBandSM1-MBafter1h(Figure8a) a Figure 8. Comparison of pH values of starch suspensions with adsorbed MB to pure starch suspensions after
(a) 1 h and (b) 72 h at 22±2 °C (n = 3). No significant differences were observed between the pH values of SN-MB and SM1-MB after 1 h (Figure 8a). Adsorption tests The Freundlich exponent 1/n between 0.5723 and 0.9221
for SN “A” and SM1 “B” indicated favorable adsorption for which 1 ≤ 1/n. The adsorption on SM2 “B” had the
largest value of kF. The adsorption on SM1 “A” had the lowest kF-value. In the case of the Langmuir isotherm,
only SM2 “B” and SM3 “B” gave a satisfactory value of r2. However, in this case, the values of qm and kL were
negative. The largest r2 values for the BET isotherm belonged to SM2 “B” and SM3. Table 4. Parameters of selected isotherm models for adsorption of 6 mg L-1 MB solution on SN, SM1, SM2 and SM3
starches. A - applies to 2 to 125 mg, B - applies to 250 to 1000 mg of the adsorbents (n = 4). Isotherm
model
Parameters
SN
SM1
SM2
SM3
A
B
A
B
A
B
A
B
Freundlich
1/n
0.5723
1.0254
4.8442
0.9221
2.4503
3.5491
2.4769
1.5960
kF / L g-1
2.1632
0.9702
0.0010
0.3727
0.3964
7.7983
0.1407
0.5681
r2
0.9071
0.8096
0.8467
0.8834
0.8954
0.9162
0.9083
0.9981
Langmuir
kL / L g-1
0.3020
-0.0712
-0.1593
0.0488
-0.1671
-1.2145
-0.1317
-0.2738
qm / mg g-1
8.9767
-12.594
-0.3920
7.9177
-4.7619
-0.4387
-3.4807
-1.5094
r2
0.6793
0.0771
0.5560
0.0836
0.7378
0.9220
0.8701
0.9684
BET
kBET(s) / L mg-1
1.4106
4.7971
0.5151
0.7223
0.0216
5.1912
1.8188
0.7352
kBET(L) / L mg-1
0.0821
0.6768
0.0741
0.1666
0.1399
1.2943
0.1539
0.2591
qm / mg g-1
3.3822
0.0318
0.0290
0.3175
27.676
0.2730
1.4069
0.4535
r2
0.7331
0.3582
0.6907
0.2395
0.7706
0.9334
0.9434
0.9813 meters of selected isotherm models for adsorption of 6 mg L-1 MB solution on SN, SM1, SM2 and SM3
applies to 2 to 125 mg, B - applies to 250 to 1000 mg of the adsorbents (n = 4). Discussion Spectroscopic evaluations Spectroscopic evaluations DSC The DSC thermograms of pure MB in the first heating cycle displayed an endothermic peak at about 80 °C
responsible for the dehydration (Figure 5 a-d). The thermogram of the second heating cycle no longer showed
this peak, while an endothermic peak in the 100 °C range appeared (Figure 5 e-h). MB forms several hydrates. In addition, it reacts with an aluminum container for DSC [51]. This endothermic peak may indicate a
transition from one form of MB hydrate to another. In the thermogram of the first heating cycle, it was most
likely obscured by the broad peak of dehydration. These speculations are confirmed by the shifted
exothermic peaks occurring at 171 and 233 °C in the first heating cycle (Figure 5 a-d) and 201 °C in the second
cycle (Figure 5 e-h). They testify to the decomposition of MB [52]. Omer et al. [53] indicated the occurrence
of oxidation of MB at 260 °C. A difference was observed between the spectrum of adsorbed MB and physical
mixed MB. An exothermic peak appeared, indicating the presence of MB in the physical mixture with SN
(177.6 °C), SM1 (175.9 °C), SM2 (174.0 °C) and SM3 (177.2 °C) (Figure 5 a,b,c,d respectively). No MB peak was
observed in MB adsorbed at SN and SM1 (Figure 5 a,b), which may indicate the absence or complete
decomposition of MB. For MB adsorbed on SM2 and SM3, the course of these peaks was flattened at 174.4
and 176.7 °C, respectively, which may indicate the interaction of MB with these starches (Figure 5 c,d). Glass transition temperature Tg value readings showed the lowest Tg for SM2 (47.2 °C) and the highest for
SM3 (55.9 °C) (Table S1). Hydrogen bonds are among the factors affecting Tg [54]. Taylor et al. [55] observed
significantly lower Tg of sucrose compared to other disaccharides. This was due to the less extensive hydrogen
bonding network in the amorphous state. Similarly, Zhou et al. [56] demonstrated the dependence of Tg on
hydrogen bonds occurring between polymer and copolymer particles (maleimide-isobutene alternating
copolymers). A network of hydrogen bonds of carboxyl groups resulted in a higher Tg value than hydrogen bonds
between hydroxyl groups. The lowest Tg occurred in the absence of hydrogen bonds between polymer
molecules. PXRD According to Table 3, the SN was the most crystalline, and SM2 the least. The crystallinity of SM increased
according to the following pattern, SM2<SM3<SM1<SN, which suggests that the implementation of ionic
groups resulted in the highest amorphicity, whereas the non-ionic acetyl groups in the lowest extent
influenced the increase of amorphicity. This remark agrees with the results of Bhargav et al. [49], who
assessed the combination of PVA with NaI. As the salt content of the polymer increased, the DSC diffraction
peaks became less intense. They suggested high susceptibility of the amorphous structure of the polymer to
ionic diffusivity of ionic particles. Similarly, Hodge et al. [50] found a correlation between peak intensity and
the degree of crystallinity. They observed that the addition of water resulted in a decrease in XRD peak
intensity and an increase in PVA amorphousness. Model cationic drug and newly synthesized starch derivatives Model cationic drug and newly synthesized starch derivatives FTIR spectroscopy FTIR spectroscopy Due to the appearance of peaks at 1599 cm-1 assigned to the C=N bond on the FTIR spectrum of samples
of adsorbed MB on SN and SM2, we postulate the existence of an interaction between the N+ ion derived
from MB and the OH- group belonging to starch, which probably leads to enhanced adsorption of the dye on
the polymer. This observation coincides with the mechanism of MB adsorption on black olive stone carried
out by Al-Ghouti et al. [48]. 396 ADMET & DMPK 11(3) (2023) 387-407 DSC In our case, the holders of the hydroxyl groups were SN and SM1 (Tg 52.2 and 51.15 °C respectively)
(Table S1) and the carboxyl groups were present in the SM2 molecule. In addition, phosphate groups appeared
in the SM3 molecule. High Tg values for SM3 may suggest the self-association of these starch molecules, whose
Tg decreased to 53.0 °C after bond formation with MB [55]. In the case of SN, Tg values increased after MB
adsorption (54.7 °C), what may indicate low self-association of SN particles in the pure state. In the present work, the values of ΔT were assessed to determine the thermal stability of the tested
samples. The higher the value of ΔT, the better the thermal stability. The value for SN was the highest
(221.07 °C), demonstrating the highest thermal stability of this starch. At the same time, its highest
percentage of crystallinity was confirmed (Table 3). On the contrary, SM2 showed the lowest value of
ΔT (179.8 °C) - the lowest thermal stability. Keshk et al. [43] demonstrated the dependence of thermal doi: https://doi.org/10.5599/admet.1950 397 ADMET & DMPK 11(3) (2023) 387-407 J. Kobryń et al. stability on ΔT-values, which correlated with the degree of crystallinity of corn starch-cellulose mixtures,
depending on the mixture ratio. They found an inversely proportional relationship between ΔT values and
ΔCp values. Our ΔCp studies confirmed such a relationship outside of the SM2 samples having the lowest ΔCp
value (0.465 J g-1·K-1) according to the lowest ΔT (Table S1). This phenomenon may be supported by the
observation of Liu et al. [57]. They studied various starches whose structure was more or less amorphous. According to them, amorphousness gives the compounds a liquid-like structure. More amorphous
compounds have a less ordered structure than those with a crystalline structure. They showed that the
process of lowering the temperature below Tg leads to the reorganization of the structure. In our study, SM1,
SM2 and SM3 starches were hardened at 2 degrees below Tg. Moreover, the research is confirmed by PXRD
studies, where SM2 had the highest degree of amorphousness (Table 3). The thermograms of the second heating cycle can show the structure of the compound when structural
reorganization occurs after the cooling cycle. In the case of SM2 there was an endothermic peak at 215 °C,
from which an increase in heat flow is observed. DSC The thermograms of adsorbed MB on starches are
characterized by different temperatures of the start of the exothermic process. The increase in heat flow
started fastest for SM2_MB (adsorbed) at 157 °C and slowest for SM3_MB (adsorbed) at 163 °C (Figures 5g
and 5h, respectively). This endothermic event might be attributed to reduced hydrogen bonding as well as
the interference of molecular organization due to the interaction between SM2, SM3 and MB. For SN_MB
(adsorbed) and SM1_MB (adsorbed), no increase in heat flow was observed (Figures 5e and 5f). Adsorption tests 398 Model cationic drug and newly synthesized starch derivatives ADMET & DMPK 11(3) (2023) 387-407 Figure 9. Isotherms of MB adsorbed on the starches: SN, SM1, SM2 and SM3 at 22±0.5 °C and 6.0 mg L-1 MB
solution with pH 4.26 (n = 4). Figure 9. Isotherms of MB adsorbed on the starches: SN, SM1, SM2 and SM3 at 22±0.5 °C and 6.0 mg L-1 MB
solution with pH 4.26 (n = 4). Table 5. Maximum adsorption capacities (qm) of different starches and other polysaccharides toward MB. Type of adsorbent
qm / mg g-1
Ref. Hydrogel poly(sodium methacrylate) with wheat starch and eggshell
1.95
[66]
Crosslinked porous corn starch
9.46
[67]
Starch cryogel of a rice flour and a tapioca starch
34.84
[68]
Honeycomb biomass adsorbent based on oxidized corn starch-gelatin
1551.5
[69]
Hydrogel poly(acrylamide) with cassava starch
1917
[70]
Hydrogel poly(acrylic acid) with potato starch crosslinked with N,N’-methylene-bisacrylamide
2967.66
[71]
Xanthate modified magnetic chitosan
197.8
[72]
Chitosan/activated charcoal
500
[73]
Cellulose-grafted poly 4-hydroxybenzoic acid magnetic nanohybrid
7.5
[74]
Hydrogel of hydroxypropyl cellulose (HPC) composited with graphene oxide
6.59
[75]
Native potato starch
7.60
This work
Acetylated potato starch
8.57
This work
Diphosphate potato starch
16.10
This work
Citrate potato starch
22.86
This work The Freundlich model describes multilayer adsorption on a surface, with an inhomogeneous distribution of
binding sites and, therefore, an inhomogeneous energy distribution. The constant kF expresses the maximum
adsorption on the surface of the adsorbent. The BET isotherm describes the phenomenon of physisorption on
a non-microporous surface. The constant kBET is related to the adsorption energy. Bhattacharyya et al. [36]
found graphene oxide-potato starch almost perfectly fitted to the Freundlich isotherm model. Dipa et al. [37]
adapted MB adsorption on kaolinite to the Langmuir model. Bestani et al. [62] studied MB adsorption on
carbon-activated leaves, which they fitted to the Langmuir and Freundlich models. El Qada et al. [31] matched
MB adsorption on activated carbon to Redlich-Peterson and Langmuir isotherm models. The Redlich-Peterson
model is a compilation of the elements in the equations of the Langmuir and the Freundlich isotherms. Langmuir
isotherm mostly applies to chemisorption on a monolayer. Activated carbon is highly porous and is a well-
known adsorbent used in industry as a filter [77]. The dependence of the isotherm model on the structure of
the adsorbent can also be supported by the study of Ma et al. Adsorption tests MB adsorption on starch predominantly matched a pseudo-second-order model. A study by Lin et al. [58]
of MB adsorption on activated carbon indicated a better pseudo-second-order fit to both linear and nonlinear
models. Similarly, Ma et al. [45] matched the adsorption of heavy metal ions on modified potato starch to
the pseudo-second-order model. In our study, the highest k2 values obtained for SN, SM3, SM2 and SM1 were
1.9623, 1.5367, 0.9240, 0.1848 g mg-1·min-1, respectively, and occurred at a weight of 1000 mg for SN and
SM1 and 2.0 mg for SM2 and SM3 (Table S2). This suggests that MB adsorbed fastest on SN, but this was
dependent on the surface area of the adsorbent: the larger the surface area, the faster the saturation. In the
case of SM2 and SM3, MB adsorbed fastest already on the smallest starch particle, suggesting a high affinity
of the adsorbate to the adsorbent. The slowest adsorption was observed in SM1. The q values in Table S2 differ from qm obtained according to Langmuir and BET isotherms. Moreover,
some of the Langmuir adsorption constants (qm and KL) showed negative values (Table 4). Langmuir isotherm
is known to get different parameters according to the calculation method [59-61], therefore, we used the
course of the isotherm of MB on the starches to establish qm values (Figure 9). Methylene blue is a substance that has been thoroughly studied by scientists because of its harmfulness
to the environment. As a dye used in industry, it leaks into water waste in large quantities. Their purification
is a major challenge for researchers. Many study substances that are adsorbents for MB and can serve as
filters [32,61-65]. Some of these include starches and other polysaccharides. Table 5 shows the qm values of
example adsorbents for MB. The sigmoidal course of the SM2 and SM3 starch isotherms falls into type IV according to the IUPAC
classification. SN and SM1 isotherms are more aligned with type VI [76]. Type IV represents single and
multilayer adsorption plus capillary condensation. Type VI illustrates that the adsorption isotherm can have
one or more steps. In our study, the adsorptions on SN and SM1 starches best fitted to the Freundlich
isotherm model. On the other hand, for SM2 and SM3 starches, the adsorption courses more closely matched
the BET isotherm (Table 4). J. Kobryń et al. J. Kobryń et al. ADMET & DMPK 11(3) (2023) 387-407 Determination of the effect of MB adsorption on the pH of starch Determination of the effect of MB adsorption on the pH of starch Determination of the effect of MB adsorption on the pH of starch The measured pH and pKa values of the acids from the literature enabled us to estimate the number of
moles of functional groups derived from the molecules and finally present at the modified starches. The
calculated values are gathered in Table 6. Table 6. Concentrations of functional groups located on 50 mg of modified starches recalculated on the basis of
parameter values of the Henderson-Hasselbalch equation [78] (n = 3). SM1
SM2
SM3
pH after 1h
6.44 ± 0.05
5.19 ± 0.09
5.98 ± 0.07
pKa1*
4.76
3.13
2.16
pKa2*
-
4.76
7.21
pKa3*
-
6.40
12.32
Concentration of functional
groups, mmol mg-1
6.07 ± 1.64 ×10-7
7.07 ± 2.82 × 10-6
1.90 ± 0.62 × 10-8
*according to database in LibreTexts™ chemistry [79] Table 6. Concentrations of functional groups located on 50 mg of modified starches recalculated on the
parameter values of the Henderson-Hasselbalch equation [78] (n = 3). The initial assumptions of the ratio of the number of millimoles of functional groups on the modified
starches used to react with the native starch during its modification was 23:13:1 for SM1:SM2:SM3,
respectively (Table 1). The millimole ratio of the same groups, calculated from the pH values of the modified
starch suspensions after 1 h, was 1:13:0.03. This shows that the activity of acyl and phosphate groups
decreased 23 and 33 times compared to carboxyl groups, respectively. MB solution with pH of 4.26 resulted
in the acidification of SN and SM1 suspensions. It did not change the pH for SM3. The increase in the pH value
of the suspension of adsorbed MB on SM2 starch may indicate the most effective binding of MB to the
carboxyl groups of SM2 (Figure 9). Adsorption tests [45], who used potato starch modified with high
temperature to increase its porosity. Adsorption of heavy metal ions on such modified starch showed good
adherence to the Langmuir model. In the case of our experiment, only SM2 was subjected to high-temperature
modification at 100 °C. This could most likely increase its adsorption properties. doi: https://doi.org/10.5599/admet.1950 399 Effect of type of functional groups of modified starches on interactions with MB The MB adsorbed on the 1000 mg of the starch decreased according to the following pattern:
SN > SM2 > SM3 > SM1 (Figure 7). The hydroxyl, carboxyl and phosphate groups of starch favored MB binding
to the polymer to the greatest extent, while the acetyl groups had the greatest effect on the reduction of
adsorption capacity. The rationale for the decreased sorption capacity could be the blocking of starch
functional groups in the structure of starch polymer or the specific activity of functional groups towards MB. Dipa et al. [37] observed the effect of NaOH-derived hydroxyl groups on the increase of MB adsorption on
kaolinite. However, these were groups added to kaolinite. In our case, only the carboxyl, phosphate and acyl
groups originated from acids and were added during the starch modification process. Physisorption may be
taken into account in the case of SN and SM3. It differs for SM2 and SM1, where acid groups can affect MB
adsorption. Similar conclusions were issued by Huang et al. [80] after studying the adsorption of Pb2+ ions on
activated carbon with additional functional groups. Pb2+ ions were adsorbed according to three mechanisms:
(1) in mesopores, (2) via bonds between -OH and C=O and Pb2+, (3) H+ from the acidic carboxyl and phosphate
groups is replaced by the Pb2+ ion. An additional element worth highlighting is the effect of the method of preparation of modified starches on
their structure. Two of our SM2 and SM3 starches were prepared using the crosslinking method, which provides
increased starch stabilization by forming bridges between hydroxyl groups and anhydroglucoses
[17] found crosslinking reactions caused a decrease in solubility in water and a decrease in phase
transition. Shen et al. [82] used corn starch and citric acid, malic acid, succinic acid and 1,2,3,4-butanetetra-
carboxylic acid (BTCA) as cross-linkers. They observed that the acid with more carboxyl groups (citric acid and
BTCA) showed higher crosslinking degrees and enhanced mechanical properties than acids with two
carboxylic groups. Moreover, as they are used in food modification, poly-carboxylic acids are in the category 400 ADMET & DMPK 11(3) (2023) 387-407 Model cationic drug and newly synthesized starch derivatives of safe chemicals [83]. In the case of SM1 and SM3, the chemically modified starches with a low degree of
substitution (DS) are permitted for use in the food industry and are denoted with the symbol “E” followed by
an appropriate number. Conclusions Citrate starch (SM2) presented the best adsorption capacity, followed by phosphate starch (SM3), and
acetylated starch (SM1) showed the weakest adsorption capacity, according to the adsorption results. MB
adsorption on SM2 and SM3 followed the BET isotherm model, which shows the physical adsorption of the
cationic dye on the low-porous surface. In the case of SM1, MB adsorption probably took place via
electrostatic attraction between the heterogeneous adsorbent surface and the adsorbate molecules, as
indicated by the Freundlich adsorption model. Spectral methods confirmed the adsorption capacity of SM2. In the FTIR study, a probable interaction
between the OH- groups of SM2 starch and N+ of MB was revealed on the basis of characteristic vibration of
the C=N bond in the adsorbent derived from MB at 1600 cm-1. The DSC results showed the lowest thermal stability of SM2 correlated with its amorphous structure. Moreover, according to DSC, the possibility of interaction between SM2 or SM3 and MB was demonstrated
via the existence of an exothermic peak belonging to MB in samples of these two adsorbents. The appearance
of endothermic peaks in the case of SM2 and SM3 samples with adsorbed MB in the second heating cycle
confirmed the interaction between the adsorbents and the adsorbates. The effect of the manufacturing
method of the modified starches on their durability was noted. SM2 and SM3 starches were prepared using
the crosslinking method, which provided increased starch stabilization. The above presented properties and, in addition, the "safe chemistry" and slightly acidic pH of the tested
starches make them useful, especially citrate starch, as carriers for biologically active substances containing
cationic groups, in topical skin applications. The present research confirms our previous studies on lidocaine
hydrochloride. The modified starches may be the potential carriers in the form of hydrogels, similar to the
hydrocolloid dressings. Author contributions: Conceptualization, J.Kobryń and W.Musiał; methodology, J.Kobryń, W.Musiał and
T.Zięba; formal analysis, J.Kobryń, M.Rzepczyńska and W.Musiał; investigation, J.Kobryń, W.Musiał,
M.Rzepczyńska and T.Zięba; resources, W.Musiał; data curation, J.Kobryń and W.Musiał; writing—original
draft preparation, J.Kobryń and W.Musiał; writing—review and editing, J.Kobryń, W.Musiał and T.Zięba;
visualization, J.Kobryń, W.Musiał and T.Zięba; supervision, W.Musiał; project administration, W.Musiał;
funding acquisition, W.Musiał. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Wroclaw Medical University, grant No SUBZ.D060.23.025. Effect of type of functional groups of modified starches on interactions with MB Among others, acetylated starch (E1420) and di-starch phosphate (E1412) belong to
this group [84]. Conclusions Acknowledgement: The authors gratefully acknowledge the Laboratory of Elemental Analysis and Structural
Research at the Faculty of Pharmacy at Wroclaw Medical University for allowing access to DSC, FTIR and PXRD
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©2023 by the authors; licensee IAPC, Zagreb, Croatia. This article is an open-access article distributed under the terms and
conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/) ©2023 by the authors; licensee IAPC, Zagreb, Croatia. This article is an open-access article distributed under the terms and
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Individual differences in the influence of task-irrelevant Pavlovian cues on human behavior
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Keywords: Pavlovian-to-instrumental transfer, cue-controlled behavior, Sign-Tracker, Goal-Tracker, reinforcement
learning Gregory B. Bissonette,
University of Maryland, USA Reviewed by:
Jeremy J. Clark,
University of Washington, USA
Donna J. Calu,
National Institute on Drug Abuse,
USA *Correspondence:
Sara Garofalo,
Department of Psychiatry, University
of Cambridge, Forvie Site, Robinson
Way, Cambridge CB2 0SR, UK
garofalosara56@gmail.com Individual differences in the influence
of task-irrelevant Pavlovian cues on
human behavior Sara Garofalo 1,2,3* and Giuseppe di Pellegrino 1 1 Center for Studies and Research in Cognitive Neuroscience, Department of Psychology, University of Bologna, Cesena,
Italy, 2 Department of Psychiatry, University of Cambridge, Cambridge, UK, 3 Behavioural and Clinical Neuroscience Institute,
Department of Psychology, University of Cambridge, Cambridge, UK Pavlovian-to-instrumental transfer (PIT) refers to the process of a Pavlovian reward-
paired cue acquiring incentive motivational proprieties that drive choices. It represents a
crucial phenomenon for understanding cue-controlled behavior, and it has both adaptive
and maladaptive implications (i.e., drug-taking). In animals, individual differences in
the degree to which such cues bias performance have been identified in two
types of individuals that exhibit distinct Conditioned Responses (CR) during Pavlovian
conditioning: Sign-Trackers (ST) and Goal-Trackers (GT). Using an appetitive PIT
procedure with a monetary reward, the present study investigated, for the first time,
the extent to which such individual differences might affect the influence of reward-
paired cues in humans. In a first task, participants learned an instrumental response
leading to reward; then, in a second task, a visual Pavlovian cue was associated with
the same reward; finally, in a third task, PIT was tested by measuring the preference
for the reward-paired instrumental response when the task-irrelevant reward-paired cue
was presented, in the absence of the reward itself. In ST individuals, but not in GT
individuals, reward-related cues biased behavior, resulting in an increased likelihood to
perform the instrumental response independently paired with the same reward when
presented with the task-irrelevant reward-paired cue, even if the reward itself was no
longer available (i.e., stronger PIT effect). This finding has important implications for
developing individualized treatment for maladaptive behaviors, such as addiction. Introduction Goal-directed behavior can be variably influenced by external and internal factors which impact
the values and priorities assigned to rewards and goals (Doya, 2008). One of the most simple and
effective mechanisms for influencing choice is reinforcement learning. Reinforcement learning
allows animals to connect spatially and/or temporally related events in order to predict future
events. Given the complexity of the animal’s environment, learning that an arbitrary cue (e.g., a
sound) is predictive of a certain goal (e.g., obtain a reward, such as food), allows the animal to learn
a flexible response that facilitates achievement of the goal itself. In most cases such cue-controlled
behavior is adaptive; for example it helps one obtain food when hungry (Perks and Clifton, 1997;
Holmes et al., 2010). However, an inflexible association can lead to perseverance in the same choice Received: 07 April 2015
Accepted: 08 June 2015
Published: 24 June 2015 Edited by:
Gregory B. Bissonette,
University of Maryland, USA ORIGINAL RESEARCH
published: 24 June 2015
doi: 10.3389/fnbeh.2015.00163 Citation: PIT has been mainly studied
in non-human animals (Rescorla and Solomon, 1967; Lovibond,
1981; Colwill and Rescorla, 1988; Balleine, 1994; Rescorla, 1994a,
1997, 2000; Delamater, 1995, 1996; Holland et al., 2002; Corbit
and Balleine, 2003; Holland and Gallagher, 2003; Holland, 2004;
Delamater and Holland, 2008; for review, see Dickinson and
Balleine, 1994, 2002; Holmes et al., 2010), but some recent studies
have also reported this effect in humans (Paredes-Olay et al.,
2002; Hogarth et al., 2007, 2010, 2013a,b; Bray et al., 2008; Allman
et al., 2010; Nadler et al., 2011; Prévost et al., 2012; Lovibond and
Colagiuri, 2013). An important, but still neglected, aspect in the human
literature about PIT concerns individual differences In the engage the location of reward delivery (the Goal), even if it is not
yet available. The first CR has been categorized as Sign-Tracking
behavior, while the second CR has been categorized as Goal-
Tracking behavior. ST and GT can be conceived of as different
learning styles, expressed through a specific CR during Pavlovian
learning. ST behavior is thought to arise from the attribution
of incentive salience to Pavlovian reward-paired cues, which
consequently become a powerful source of motivation for future
behavior (Flagel et al., 2011). In ST, incentive stimuli become
attractive, eliciting approach towards them and promoting
potentially maladaptive cue-controlled behaviors; ST individuals,
indeed, are generally more vulnerable to addiction and relapse
(Tomie et al., 1998; Flagel et al., 2008; Robinson and Flagel, 2009). The ST and GT profiles do not seem to be limited to the CR
expressed, but are also associated with differences in traits such
as impulsivity; ST individuals are characterized by higher levels
of impulsive behavior compared to GT individuals (Tomie et al.,
2000; Flagel et al., 2009). A
deeper
investigation
into
individual
differences
in
attributing incentive salience to reward-paired stimuli would
thus be important for understanding and reducing the propensity
to develop maladaptive behaviors. The aim of the present study was to investigate individual
differences in human PIT. Specifically, the present study
explored, for the first time in humans, whether individual
differences in the propensity to approach and engage a Sign
(cue-predicting reward) or a Goal (reward) are predictive of
cue-controlled behavior. To this end, a typical PIT experimental
design was used, comprising three tasks. Citation: Garofalo S and di Pellegrino G (2015)
Individual differences in the influence
of task-irrelevant Pavlovian cues on
human behavior. Front. Behav. Neurosci. 9:163. doi: 10.3389/fnbeh.2015.00163 Garofalo S and di Pellegrino G (2015)
Individual differences in the influence
of task-irrelevant Pavlovian cues on
human behavior. Front. Behav. Neurosci. 9:163. doi: 10.3389/fnbeh.2015.00163 June 2015 | Volume 9 | Article 163 Frontiers in Behavioral Neuroscience | www.frontiersin.org 1 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino even if the goal itself is no longer available, or has negative long-
term consequences (Holmes et al., 2010). For example, a cue
associated with drugs can induce relapse even when the drug
is not voluntary sought, and a sign associated with food can
induce craving in the absence of hunger, leading to compulsive
over-eating (Volkow et al., 2008). These biases on voluntary
choice are also implemented in marketing strategies, such as
advertisements, to influence consumer behavior (Smeets and
Barnes-Holmes, 2003; Bray et al., 2008; de Wit and Dickinson,
2009). Cue-controlled behaviors have been interpreted as the
endpoint of an initial intentional seeking behavior (of a reward),
which leads to habitual, and ultimately compulsive, conduct
characterized by a loss of control over behavior (Everitt and
Robbins, 2005). This interesting framework proposes that the
transition from intentional volition to habit and compulsion can
be explained by interactions between Pavlovian and instrumental
learning processes: a reward acts as an instrumental reinforcer
by enhancing actions that are able to produce it, while Pavlovian
learning confers incentive salience to cues (Conditioned Stimuli
or CS) closely associated with the reward (Everitt and Robbins,
2005). Such cues can elicit craving and motivation towards the
associated reward, thus biasing choice. Well-known evidence
of this effect can be found in the so-called Pavlovian-to-
Instrumental Transfer (PIT) effect (Estes, 1943, 1948). PIT
captures the ability of a Pavlovian cue (i.e., a CS associated
with a reward) to increase the likelihood of an instrumental
response independently paired with the same (specific-PIT), or
a similar (general-PIT), reward (Rescorla and Solomon, 1967;
de Wit and Dickinson, 2009; Holmes et al., 2010). This effect
emerges without any formal association between Pavlovian and
instrumental contingencies, and even when the reward itself is no
longer available (Talmi et al., 2008). Citation: In the first phase,
participants performed an Instrumental Conditioning task, in
which they were presented with two possible choices, one
paired with an actual monetary win (Rewarded Choice) and
the other paired with a neutral outcome (Unrewarded Choice). In a subsequent session, participants performed a Pavlovian
Conditioning task, during which they learned to associate a
specific visual cue with an actual monetary win (CS+), and
another visual cue with a neutral outcome (CS−). During
this phase, eye-movements were recorded and subsequently
analyzed in order to identify the expressed CR and characterize
participants as ST or GT. Mirroring previous studies conducted
in animals (Boakes, 1977; Flagel et al., 2007, 2008, 2011;
Saunders and Robinson, 2013), in which the CR is identified
based on the amount of approaching behavior expressed
during CS presentation, in the present study ST and GT
participants were distinguished based on a learned oculomotor
CR. Specifically, it was measured the tendency to direct
contiguous eye-gazes toward the location where the visual
CS (Sign) or the reward (Goal) would be presented. Finally,
PIT was tested in an extinction phase (without any rewards),
during which participants had to choose between the same
two options given during instrumental conditioning, while
presented with the task-irrelevant CS. In this final phase, PIT
would be observed if presentation of the CS+, compared to the
CS−, enhanced instrumental responses to the choice rewarded
during instrumental conditioning (Congruent Choice), relative
to the previously unrewarded choice (Incongruent Choice). If
consistent with animal literature, this effect should be stronger An important, but still neglected, aspect in the human
literature about PIT concerns individual differences. In the
animal literature, the extent to which a Pavlovian cue becomes
attractive and exerts a biasing effect varies between individuals. In particular, Sign-Trackers (ST) and Goal-Trackers (GT) have
been shown to have different learning styles, consisting of a
tendency to attribute more or less incentive salience to Pavlovian
reward-associated cues. In a typical Pavlovian conditioning
paradigm, a CS (e.g., lever presentation) is paired with a reward
(e.g., food pellet), which is delivered in a different spatial position. In such a situation, two different Conditioned Responses (CR;
i.e., learned responses to a previously neutral stimulus) might be
expressed. Frontiers in Behavioral Neuroscience | www.frontiersin.org Citation: Some animals approach and engage the CS (the Sign)
itself and, only after its termination, reach the location of reward
delivery; other animals, upon CS presentation, immediately June 2015 | Volume 9 | Article 163 Frontiers in Behavioral Neuroscience | www.frontiersin.org 2 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino in ST individuals than in GT individuals, possibly indicating a
stronger biasing effect of Pavlovian cues over behavior in the first
group relative to the second. FIGURE 1 | Graphical illustration of the three tasks: Instrumental
Conditioning Task (Panel A), Pavlovian Conditioning Task (Panel B);
Pavlovian-to-Instrumental Transfer (PIT) Task (Panel C). Stimuli and Procedure The whole experiment consisted of three tasks. The same visual
background was used in all three tasks. Four black squares
(4 cm2) were displayed on a 17-inch color monitor with a
black background. The squares were highlighted by a white
frame and positioned as follows: top center, bottom center,
right center, left center. Two black-and-white fractal images
(balanced for luminance, complexity and color saturation) were
used as Pavlovian cues (CS) and presented within the top
center square. An image of a 10 euro cent coin was used
as the reward, and a light-yellow circle (equally sized) was
used as the neutral outcome (no-reward). Both these visual
cues appeared within the bottom center square (Figure 1). A computer running Presentation software (Neurobehavioral
Systems, Albany, CA, USA) controlled stimulus presentation. On
arrival, participants were comfortably seated in a silent room and
their position was centered relative to the screen, at a viewing
distance of 60 cm from the eye-tracker and 75 cm from the
screen. The eye-tracker was positioned under the screen, and
was centered relative to both the screen and the participant. Eye-
movements and behavioral responses were collected throughout
the experiment and stored for offline analysis. Participants
were asked to remain as still as possible to avoid confounding
effects on eye-movements. The whole experiment was conducted
in a dark room to facilitate eye-movement recording. The
experimental session began with calibration of the eye-tracker
device, during which the participant fixated nine specific points
on the computer screen. The experimental session followed the
standard paradigm for testing PIT. It was composed of three tasks
administered in succession: an Instrumental Conditioning task,
in which participants learned a response-contingent reward; a
Pavlovian Conditioning task, in which participants learned a cue-
contingent reward; and a PIT task, during which the influence
of irrelevant Pavlovian cues on instrumental responding was
tested. In each task, participants were required to pay attention
to the screen and follow the instructions reported at the
beginning of the task. A few example trials were always
performed and, if necessary, further clarifications were given
before beginning each task. At the end of the experimental FIGURE 1 | Graphical illustration of the three tasks: Instrumental
Conditioning Task (Panel A), Pavlovian Conditioning Task (Panel B);
Pavlovian-to-Instrumental Transfer (PIT) Task (Panel C). Stimuli and Procedure FIGURE 1 | Graphical illustration of the three tasks: Instrumental
Conditioning Task (Panel A), Pavlovian Conditioning Task (Panel B);
Pavlovian-to-Instrumental Transfer (PIT) Task (Panel C). session, participants completed the Barratt Impulsiveness Scale
(BIS-11; Patton et al., 1995). Previous studies on animals reported
an association between Sign-Tracking behavior and reduced
impulse control (Flagel et al., 2011). Thus, this measure allowed
further investigation into the differences between ST and GT
individuals. Participants Forty-five volunteers (27 female; 2 left-handed; mean age = 24.87,
sd = 2.5; mean education = 17.53, sd = 1.5) with no history of
neurological diseases were recruited from the student population
at the University of Bologna. All participants gave written
informed consent to take part in the experiment and received
payment corresponding to the amount earned during the tasks. The study was conducted in accordance with institutional
guidelines and the 1964 Declaration of Helsinki. It was approved
by the Ethics Committee for Psychological Research at the
University of Bologna. Frontiers in Behavioral Neuroscience | www.frontiersin.org Eye Tracking
E
t Eye movements were recorded in a dimly lit room using a
Pan/Tilt optic eye-tracker (Eye-Track ASL-6000) which registers
real-time gaze at 50 Hz. Data acquired during the Pavlovian
Conditioning task were analyzed offline using EyeNal Analysis
Software (ASL). Dwell time during the 5 s of CS presentation was
then measured for two specific areas of interest (AOI): ‘‘Sign’’,
corresponding to the 4 cm square at the top center, plus a 1 cm
margin; ‘‘Goal’’, corresponding to the 4 cm square at the bottom
center, plus a 1 cm margin. Dwell time was defined as the amount
of time during which a series of contiguous fixations remained
within the same AOI. The Pavlovian speeded reaction time response described
above (‘‘press the button upon patch presentation’’) has been
successfully used in previous studies Talmi et al. (2008) and
was introduced to obtain a behavioral measure of Pavlovian
conditioning. The main reason for using a speeded response
was to mirror PIT studies on animals, in which Pavlovian
conditioning is measured by a behavior performed to gain the
reward (e.g., latency of the first nose-poke or frequency of nose-
pokes; Dickinson et al., 2000; Holland, 2004; Corbit and Balleine,
2005). The rationale here is to observe a faster reaction times
when a reward was predicted (CS+ condition) than when a
neutral outcome was predicted (CS−condition). To avoid a
possible instrumental influence on the task, participants were
explicitly told that, in this task, the reward was not contingent
on their response. It was demonstrated that, if no answer was
given, the patch would disappear anyway after 1.5 s, revealing
the outcome. Importantly, this speeded reaction time response Sign-Tracker and Goal-Tracker Categorization
Participants were categorized as ST or GT based on the
oculomotor CR expressed during the Pavlovian Conditioning
task. Previous studies used approaching and engaging behaviors
during Pavlovian Conditioning to identify ST and GT. In these
studies, the numbers of contacts with the Sign (i.e., lever)
and the Goal (i.e., food tray) were compared to obtain an
index of behavior, and divide the subjects into ST (i.e., high
probability to engage the lever) and GT (i.e., high probability
to engage the food-tray) individuals (Flagel et al., 2007, 2008,
2011; Robinson and Flagel, 2009; Saunders and Robinson, 2013;
Robinson et al., 2014). Pavlovian-to-Instrumental Transfer (PIT) Task Participants received exactly the same instructions as in the
Instrumental Conditioning phase requiring them to choose
between the right and left white squares. The task was identical
to the Instrumental Conditioning task, except in two aspects:
first, the task-irrelevant Pavlovian CS were presented sequentially
within the top square, changing every 30 s, the task was
completely performed in extinction, so all choices always lead
to no-reward. (Figure 1C). Extinction is a standard procedure
for assessing PIT, both in human and animal research, since it
allows one to test the influence of Pavlovian cues on instrumental
responding without the confounding effects of the reward
(Rescorla, 1994a,b; Corbit et al., 2001; Bray et al., 2008; Talmi
et al., 2008). Indeed, the rationale here is to test the ability of
a task-irrelevant Pavlovian cue to drive choices (presumably,
towards the response previously associated with a reward) even if
the reward is not available anymore. The PIT task lasted about 6
min, during which subjects were free to perform as many choices
as they wished, with no time pressure. Instrumental Conditioning Task Participants were instructed to choose between two squares to
gain a reward. One square was paired with an actual monetary Frontiers in Behavioral Neuroscience | www.frontiersin.org June 2015 | Volume 9 | Article 163 3 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino win (Rewarded Choice), while the other was paired with a neutral
outcome (Unrewarded Choice). The right and left squares were
presented in white and indicated as possible choices to be
selected by a mouse click. The mouse pointer was centrally
positioned before each choice, in order to not encourage a
specific choice. Only one square was associated with a reward
following a partial reinforcement schedule, so that between
one reward and the next a variable interval between 4 and
12 s was always associated with no-reward. After each choice,
a corresponding neutral image (light-yellow circle) or reward
image (10 euro cents coin) appeared for 1 s in the bottom square
(Figure 1A). Participants were aware that they would receive an
actual payment corresponding to the amount of coins collected
during the task. The association between square and outcome
was counterbalanced across subjects. The rationale of this task
was to make participants learn an association between a specific
response (left or right square) and the reward; thus, participants
would get a higher frequency of Rewarded Choices if they learned
the correct association. The task lasted about 6 min, during which
subjects were free to perform as many choices as they wished,
with no time pressure. allowed us to obtain a measure of the learning rate that is
independent from ST/GT behavior. To identify ST and GT CR, eye-movements were recorded
in order to evaluate contiguous eye-gazes directed toward
the ‘‘Sign’’ (top center square) and the ‘‘Goal’’ (bottom
center square). Mirroring animal studies, these two CR were
subsequently used to distinguish participants as ST or GT,
depending on the tendency to direct eye-gaze toward the Sign
or the Goal during the 5 s of CS presentation (Flagel et al., 2011). Pavlovian Conditioning Task In each trial, one of two possible visual cues (fractal images)
appeared for 5 s within the top square, followed by a white
patch within the bottom square. Upon presentation of the patch,
participants were instructed to press the left-Ctrl button on
the keyboard as quickly as possible to remove the patch and
discover the outcome hidden below. To perform this button
press, participants did not need to remove their gaze from the
screen. The outcome was then presented for 1 s. One fractal was
associated with a reward (10 euro cent coin) on 80% of trials
(CS+), while the other fractal was associated with no-reward
(light-yellow circle) on all trials (CS−; Figure 1B). The task
consisted of 40 trials (20 per condition) with a variable inter-trial-
interval between 0.5 and 4 s. Participants were aware that they
would receive an actual payment corresponding to the amount
of coins collected during the task. The association between visual
cue and outcome was counterbalanced across subjects. The whole
task lasted around 6 min. Frontiers in Behavioral Neuroscience | www.frontiersin.org Eye Tracking
E
t This method was adapted in the present
experiment by calculating contiguous eye-gazes (Dwell Time)
toward the cue (Sign) and the reward (Goal) AOI, during CS Frontiers in Behavioral Neuroscience | www.frontiersin.org Frontiers in Behavioral Neuroscience | www.frontiersin.org June 2015 | Volume 9 | Article 163 4 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino presentation (see above). ST behavior has been defined as a
CR to approach and engage ‘‘the cue or sign that indicates
impending reward delivery’’; while GT behavior has been defined
as a tendency to ‘‘engage the location of unconditioned cue
delivery, even though it is not available until conditioned cue
termination’’ (Flagel et al., 2011). Thus, a learned oculomotor
CR towards the location of the Sign or the Goal is a practical
method for distinguishing between ST and GT individuals. On
this basis, an eye-gaze index was created based on the Dwell
Time spent on the Sign and Goal locations. An individual
dwell is defined as the time period during which a fixation
or series of temporally contiguous fixations remain within an
AOI. That is, an individual dwell is defined as the sum of
the durations across all fixations within the current AOI, from
entry to exit. To compute fixations, EyeNal ASL was used,
which defines a fixation if the observer’ s gaze position remains
within a diameter of 0, 5◦of visual angle for at least 120 ms
(six consecutive samples, at 50 Hz sampling rate; Eye-Analysis
software Manual, v. 1.41, Applied Science Laboratories, 2007). The Dwell Time spent on the Sign and Goal locations was
calculated for each trial and then averaged for each participant. The eye-gaze index was calculated as the difference between the
Dwell Time on Sign minus the Dwell Time on Goal over the
total Dwell Time (Sign −Goal/Sign + Goal), so that a higher
value corresponded to a higher Dwell Time toward the Sign
(Sign-Tracking behavior) and a lower value corresponded to a
higher Dwell Time toward the Goal (Goal-Tracking behavior). Since the interest here was to disentangle two reward-specific
CR, only CS+ trials in the second half of the task were
considered, when contingency learning was more established. Based on this index, the top and bottom 50% of the total
sample were categorized as ST (eye-gaze index between 0.38
and 1.00) and GT (eye-gaze index between −1.00 and 0.27),
respectively. FIGURE 2 | Oculomotor response. Eye Tracking
E
t (Panels A and B) show the eye-gaze
index in the two groups (ST = Sign-Trackers; GT = Goal-Trackers) and the two
task hemiblocks. (Panel A) represents CS+ trails and (Panel B) represents
CS−trials. (Panels C and D) show the eye-gaze index in the two conditions
(CS+ = reward-associated cue; CS−= neutral cue) in ST and GT, respectively. (Panel E) shows visual exploratory behavior in the two groups
(ST = Sign-Trackers; GT = Goal-Trackers) throughout the task. Bars indicate
standard error of the mean. *p < 0.05; **p < 0.01. FIGURE 2 | Oculomotor response. (Panels A and B) show the eye-gaze FIGURE 2 | Oculomotor response. (Panels A and B) show the eye-gaze
index in the two groups (ST = Sign-Trackers; GT = Goal-Trackers) and the two FIGURE 2 | Oculomotor response. (Panels A and B) show the eye-gaze
index in the two groups (ST = Sign-Trackers; GT = Goal-Trackers) and the two
task hemiblocks. (Panel A) represents CS+ trails and (Panel B) represents
CS−trials. (Panels C and D) show the eye-gaze index in the two conditions
(CS+ = reward-associated cue; CS−= neutral cue) in ST and GT, respectively. (Panel E) shows visual exploratory behavior in the two groups
(ST = Sign-Trackers; GT = Goal-Trackers) throughout the task. Bars indicate
standard error of the mean. *p < 0.05; **p < 0.01. Frontiers in Behavioral Neuroscience | www.frontiersin.org ST and GT CR In both groups a significant difference Frontiers in Behavioral Neuroscience | www.frontiersin.org Frontiers in Behavioral Neuroscience | www.frontiersin.org June 2015 | Volume 9 | Article 163 5 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino between the two conditions was found. The ST group showed
a significantly higher eye-gaze index in the CS+ condition than
in the CS−condition (t(21) = 1.69; one-tailed p = 0.03; Cohen’s
d = 0.19), indicating a greater tendency to direct contiguous
eye-gazes towards the Sign during CS+ trials than during CS−
trials (Figure 2C). The GT group showed a significantly lower
eye-gaze index in the CS+ condition than in the CS−condition
(t(21) = 2.21; one-tailed p = 0.01; Cohen’s d = 0.24), indicating a
greater tendency to direct contiguous eye-gazes towards the Goal
during CS+ trials than during CS−trials (Figure 2D). FIGURE 3 | Learning rates in the two groups (ST = Sign-Trackers;
GT = Goal-Trackers) during Instrumental Conditioning (Panel A) and
Pavlovian Conditioning (Panel B). Bars indicate standard error of the mean. ***p < 0.001. g
g
g
Given the specific spatial locations of the Sign and the Goal
in the present paradigm, visual exploratory behavior was also
considered by analyzing the total dwell time spent on the top
and the bottom portions of the screen, in order to exclude the
presence of a spatial bias that could account for ST and GT
behavior. A mixed-effects model was used, with Group (ST/GT)
and AOI (Top/Bottom) as independent variables and Total Dwell
Time as dependent variable. Subjects were modeled as a random
effect. Assumptions of normal distribution, independence of
residuals and sphericity were verified. Results showed a marginal
main effect of AOI (F(1,42) = 4.01; two-tailed p = 0.05; part. η2 = 0.09), with more Dwell Time spent on the Top of the screen
(mean = 0.76; sd = 0.91) than on the Bottom (mean = 0.41;
sd = 0.64) in both groups (Figure 2E). Neither group differences,
nor interaction effects emerged (ps > 0.87). These results
strengthen the evidence that the behavioral differences observed
between ST and GT cannot be ascribed to a mere spatial bias
towards the upper or the lower part of the screen. The general
difference in time spent looking at the Top and the Bottom
of the screen is compatible with the fact that dwell time was
calculated during the 5 s of CS presentation. ST and GT CR To ensure that the oculomotor responses used to categorize
ST and GT individuals were learned CRs, eye-gaze indices
were separately analyzed for CS+ and CS−trials in the
first and second halves of the Pavlovian Conditioning task. Two separate mixed-effects models with Group (ST/GT) and
Hemiblock (1/2) as independent variables were performed for
CS+ and CS−conditions. The eye-gaze index described above
was the dependent variable. Subjects were modeled as a random
effect. Assumptions of normal distribution, independence of
residuals and sphericity were verified. Results from CS+ trials
showed a significant interaction effect (F(1,42) = 14.75; two-
tailed p = 0.0004; part. η2 = 0.26). Bonferroni-corrected post hoc
tests revealed a significant difference (p = 0.003) between ST
(mean = 0.35; sd = 0.77) and GT (mean = −0.06; sd = 0.79) in the
second Hemiblock (Figure 2A). No other post hoc comparisons
were significant (ps > 0.15). Results from CS−trials did not
show any significant effects (ps > 0.05; Figure 2B). Overall,
these results indicate two important points: first, a bias toward
either the Sign or the Goal is a learned CR, since it is not present at the beginning of the task but emerges later in time,
when contingencies have been learned (Figure 2A); moreover,
this looking bias is specific to the reward-paired cue (CS+),
as no differences were observed for the unpaired cue (CS−;
Figure 2B). In Figure 2A it is evident how, at the beginning
of the Pavlovian task, during CS+ presentation, no tendency
seems evident, while, towards the end ST show higher Dwell
Time towards the Sign (eye-gaze index increases) while GT
show higher Dwell Time towards the Goal (eye-gaze index
decreases). Figure 2B, on the other hand, shows that the same
pattern is not observable during the presentation of the neutral
stimulus (CS−). To further test that this behavior is a reward-specific CR, the
eye gaze index was also directly compared between CS+ and
CS−trials from the second hemiblock (when contingencies had
been learned) within each group. Two separate paired t-tests
were performed for the ST and GT groups, using Condition
(CS+/CS−) as the independent variable and the eye-gaze index
as the dependent variable. ST and GT CR These results thus
indicate that both groups spent more time visually exploring
the region of the screen where a stimulus was being presented
(Top), rather than where there was no stimulus (Bottom). No
difference in this spatial bias was found between the two groups
(Figure 2E). FIGURE 3 | Learning rates in the two groups (ST = Sign-Trackers;
GT = Goal-Trackers) during Instrumental Conditioning (Panel A) and
Pavlovian Conditioning (Panel B). Bars indicate standard error of the mean. ***p < 0.001. (mean = 22.09; sd = 9.10; Figure 3A). Neither group differences,
nor interaction effects emerged (ps > 0.55). These results
indicate that the ST and GT groups learned to discriminate
between the rewarding and non-rewarding choices equally
well. Pavlovian Conditioning To ensure that Pavlovian learning occurred in both ST and GT
groups, reaction times to patch presentation were analyzed. If
participants correctly learned to discriminate between the two
Pavlovian cues, faster reaction times should be observed for CS+
trials relative to CS−trials. A mixed-effects model was used,
with Condition (CS+/CS−) and Group (ST/GT) as independent
variables, and reaction times as the dependent variable. Subjects
were modeled as a random effect. Assumptions of normal
distribution, independence of residuals and sphericity were
verified. Results showed a significant main effect of Condition
(F(1,842) = 110.24; two-tailed p = 0.0001; part. η2 = 0.72),
with faster reaction times for CS+ trials (mean = 306.33;
sd = 44.41) relative to CS−trials (mean = 351.21; sd = 50.05;
Figure 3B). Neither group differences, nor interaction effects
emerged (ps > 0.29). These results indicate that participants
generally reacted more quickly to the patch on trials with
the reward-paired cue (CS+) than on trials with the unpaired
cue (CS−). This reward-specific response facilitation indicates
successful Pavlovian conditioning in both ST and GT. Taken together, the last two analyses demonstrated that group
differences in the tendency to direct contiguous eye-gazes to the
location of the Sign or the Goal cannot be ascribed to a mere
spatial bias, but rather reflect a learned reward-related CR. Instrumental Conditioning To ensure that instrumental conditioning was successful in both
the ST and the GT groups, so that all participants learned
which response leads to a reward, the number of choices
(mouse clicks) made on the two white squares were compared. Choosing the square associated with reward was considered
a Rewarded Choice, and choosing the square associated with
no-reward was considered an Unrewarded Choice. A mixed-
effects model was used, with Choice (Rewarded/Unrewarded)
and Group (ST/GT) as independent variables and the number
of choices as the dependent variable. Subjects were modeled
as a random effect. Assumptions of normal distribution,
independence of residuals and sphericity were verified. Results
showed a main effect of Choice (F(1,42) = 20.88; two-tailed p <
0.0001; part η2 = 0.33), with Rewarded Choices (mean = 32.80;
sd = 9.38) occurring more frequently than Unrewarded Choices Pavlovian-to Instrumental Transfer Pavlovian-to Instrumental Transfer
To test for PIT, the numbers of Congruent choices (associated
with the reward during Instrumental Conditioning) and
Incongruent
choices
(associated
with
no-reward
during
Instrumental Conditioning) during CS+ and CS−presentation
were compared. A response index was calculated as the
probability of selecting the Congruent choice minus the
probability of selecting the Incongruent choice (number of
congruent—incongruent
choices/total
number
of
choices). Higher values correspond to a higher probability of making
the Congruent choice, while lower values correspond to
a higher probability of making the Incongruent choice. A
mixed-effects model was used, with Condition (CS+/CS−) and June 2015 | Volume 9 | Article 163 Frontiers in Behavioral Neuroscience | www.frontiersin.org 6 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino FIGURE 4 | Pavlovian-to-Instrumental Transfer (PIT). (Panel A) shows
the response index (Congruent-Incongruent/Total) in the two groups
(ST = Sign-Trackers; GT = Goal-Trackers) during CS−and CS+ trials. (Panels
B and C) show the response index over time by dividing the task into three
blocks of two trials. (Panels D and E) show the number of responses. Bars
indicate standard error of the mean. *p < 0.05; ***p < 0.001. Subjects were modeled as a random effect. Assumptions of
normal distribution, independence of residuals and sphericity
were verified. Results showed a significant main effect of
Condition (F(1,42) = 6.39; two-tailed p = 0.02; part. η2 = 0.13), a
significant Condition × Group interaction (F(1,42) = 7.69; two-
tailed p = 0.008; part. η2 = 0.15), and a significant Block ×
Group interaction (F(1.27,53.32) = 50.61; two-tailed p < 0.001;
part. η2 = 0.5; Figures 4B,C). Bonferroni-corrected post hoc tests
on the Condition × Group interaction revealed a significant
difference (p = 0.003) between CS+ and CS−in ST group but
not the GT group, and a significant difference (p = 0.02) between
ST and GT groups in CS+ trials (Figures 4B,C). Bonferroni-
corrected post hoc tests on the Block × Group interaction
revealed a significant difference (p < 0.0001) between ST and GT
groups in the third block, but not in the first and second blocks
(Figures 4B,C). Figures 4D,E show the number of responses. In line with the results of the first analysis, these results
showed that, unlike GT, ST group was more likely to choose the
congruent option when they saw the task-irrelevant CS+ than
when they saw the CS−, throughout the entire PIT task. Pavlovian-to Instrumental Transfer The only
effect of time revealed by this analysis was in the last block, where
a group difference in responses emerged. Since this difference
was unrelated to the displayed stimulus (CS+/CS−), it does not
constitute a difference in PIT. This result instead indicates that
the ST and GT groups differed in the proportion of congruent
choice made towards the end of the task. Frontiers in Behavioral Neuroscience | www.frontiersin.org Impulsiveness To
further
investigate
differences
between
ST
and
GT
individuals, self-reported impulsiveness, as rated by the BIS-11
questionnaire (Patton et al., 1995), was compared between the
two groups. A two-sample t-test was performed using Group
(ST/GT) as the independent variable and BIS-11 scores as the
dependent variable. Results revealed a significant difference
between the two groups (t(28.75) = 2.06; two-sided p = 0.04,
with the ST group (mean = 61.0; sd = 9.91) showing higher
impulsiveness than the GT group (mean = 54.09; sd = 8.86;
Figure 5). This finding is consistent with previous studies
showing significantly higher levels of impulsiveness as compared
to GT (Tomie et al., 2000; Flagel et al., 2009). FIGURE 4 | Pavlovian-to-Instrumental Transfer (PIT). (Panel A) shows
the response index (Congruent-Incongruent/Total) in the two groups
(ST = Sign-Trackers; GT = Goal-Trackers) during CS−and CS+ trials. (Panels
B and C) show the response index over time by dividing the task into three
blocks of two trials. (Panels D and E) show the number of responses. Bars
indicate standard error of the mean. *p < 0.05; ***p < 0.001. FIGURE 4 | Pavlovian-to-Instrumental Transfer (PIT). (Panel A) shows Group (ST/GT) as independent variables and the response
index, described above, as the dependent variable. Subjects
were modeled as a random effect. Assumptions of normal
distribution, independence of residuals and sphericity were
verified. Results showed a significant Condition × Group
interaction (F(1,42) = 8.22; two-tailed p = 0.006; part. η2 = 0.16). Bonferroni-corrected post hoc comparisons revealed a significant
difference (p = 0.001) between CS+ (mean = 0.18; sd = 0.12) and
CS−(mean = 0.04; sd = 0.13) only in ST group, and a significant
difference (p = 0.04) between ST (mean = 0.18; sd = 0.12) and
GT (mean = 0.08; sd = 0.12) during CS+ (Figure 4A). No other
comparisons were significant (ps > 0.13). These results indicate
that the ST group was more likely to choose the congruent
option when they saw the task-irrelevant CS+ than when they
saw the CS−. thus revealing a PIT effect. Critically, this bias was
stronger in ST than in GT individuals. Group (ST/GT) as independent variables and the response
index, described above, as the dependent variable. Subjects
were modeled as a random effect. Assumptions of normal
distribution, independence of residuals and sphericity were
verified. Results showed a significant Condition × Group
interaction (F(1,42) = 8.22; two-tailed p = 0.006; part. Discussion Importantly, the present results
demonstrate that this oculomotor CR was: (i) acquired over
time (i.e., learned), since a specific CR towards the Sign or the
Goal only emerged towards the end of the task, when stimulus-
reward associations had been acquired selectively during the
presentation of reward-paired cues (CS+; Figures 2A,B); and
(ii) reward specific, since the CR was only evident when
participants saw the reward-related cue (CS+) and not when
they saw the neutral cue (CS−; Figures 2C,D). Coherently with
what expected, the task-irrelevant CS had a much stronger
influence on the ST group than on the GT group during the
PIT task. Since the ‘‘Sign’’ and the ‘‘Goal’’ had specific spatial locations
(the top and bottom portions of the screen, respectively), it is
important to rule out the possibility that spatial biases in gaze
direction might account for the difference in the PIT effect
between groups. A bias towards looking at the top of the screen
might cause result in a stronger influence of the Sign on the ST
group just because they spent more time looking at it. Analysis
of visual exploratory behavior during Pavlovian Conditioning,
however, revealed that the ST and GT groups did not differ in
the total amount of time spent looking at the top and bottom
of the screen (Figure 2E). Critically, behavioral differences only
emerged during CS+ trials towards the end of the task, once the
association between the cue and the reward had been learned. Consequently, it is concluded that there was no a priori bias in
gaze direction; rather, such a bias emerged during the Pavlovian
Conditioning task as a learned reward-specific CR. Moreover, a recent study (Trick et al., 2011) directly
investigated the relation between fixation times during Pavlovian
learning and the PIT effect. The authors found that fixation
times during Pavlovian learning increased with uncertainty
(that is, more attention was paid to stimuli with uncertain
outcome probabilities, e.g., 50%, than to stimuli with more
certain outcome probabilities, e.g., 90%). In contrast, the PIT
effect increased with the probability of reward (that is, it
was stronger for stimuli associated with a high probability of
reward, e.g., 90%, than for stimuli associated with uncertain
outcomes, e.g., 50%, or a low probability of reward, e.g.,
10%). Thus, Trick et al. Discussion contingencies. In line with the animal literature (Robinson and
Flagel, 2009), the Pavlovian cue (CS+) was clearly predictive of
reward, since it elicited faster reaction times during Pavlovian
conditioning than the neutral stimulus (CS−) did in both groups,
along with a CR corresponding to the behavioral profile of each
group (ST/GT). Motivated behavior is characterized by a wide span of inter-
individual differences in both human and non-human animals. In the present study, the PIT paradigm was used to examine
individual differences in the excitatory influence that signals
associated with reward can exert on human choices. PIT is
a well-known procedure for testing the ability of a Pavlovian
reward-paired cue to acquire incentive motivational properties
and influence instrumental performance (Estes, 1943, 1948;
Rescorla and Solomon, 1967; de Wit and Dickinson, 2009;
Holmes et al., 2010). Here, participants performed a standard
PIT paradigm composed of three tasks: an Instrumental
Conditioning task, during which response-outcome associations
were learned; a Pavlovian Conditioning task, during which
stimulus-outcome associations were leaned; and a PIT task,
in which the ability of a Pavlovian cue to drive instrumental
responses was tested. Individual differences were characterized
by two distinct oculomotor CR exhibited during Pavlovian
Conditioning, corresponding to two different learning styles
previously identified and described in animal literature: Sign-
Tracking (ST) and Goal-Tracking (GT; Estes, 1943, 1948; Boakes,
1977; Flagel et al., 2011). In the present study, ST behavior
consisted of a tendency to direct contiguous eye-gazes towards
the cue (CS) that indicated impending reward delivery (Sign);
in contrast, GT behavior was characterized by a tendency to
direct contiguous eye-gazes towards the location of reward (US)
delivery (Goal), even if not available until CS termination. An eye-gaze index was based on the emergence of these two
behavioral patterns during presentation of the reward-paired
stimulus (CS+) in the second half of the task (when contingencies
had been learned), and a median split was used to categorize
participants as ST or GT. Impulsiveness η2 = 0.16). Bonferroni-corrected post hoc comparisons revealed a significant
difference (p = 0.001) between CS+ (mean = 0.18; sd = 0.12) and
CS−(mean = 0.04; sd = 0.13) only in ST group, and a significant
difference (p = 0.04) between ST (mean = 0.18; sd = 0.12) and
GT (mean = 0.08; sd = 0.12) during CS+ (Figure 4A). No other
comparisons were significant (ps > 0.13). These results indicate
that the ST group was more likely to choose the congruent
option when they saw the task-irrelevant CS+ than when they
saw the CS−. thus revealing a PIT effect. Critically, this bias was
stronger in ST than in GT individuals. FIGURE 5 | Impulsiveness levels in the two groups (ST = Sign-Trackers;
GT = Goal-Trackers) as measured by the Barratt Impulsiveness Scale
(BIS-11). Bars indicate standard error of the mean. *p < 0.05. While the first analysis on PIT focused on the overall effect,
a second analysis divided the task into three equal blocks of
2 min (four trials) to check for differences in task performance
over time. A mixed-effects model was used, with Condition
(CS+/CS−), Group (ST/GT) and Block (1/2/3) as independent
variables, and the response index as the dependent variable. FIGURE 5 | Impulsiveness levels in the two groups (ST = Sign-Trackers;
GT = Goal-Trackers) as measured by the Barratt Impulsiveness Scale
(BIS-11). Bars indicate standard error of the mean. *p < 0.05. June 2015 | Volume 9 | Article 163 Frontiers in Behavioral Neuroscience | www.frontiersin.org 7 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino Frontiers in Behavioral Neuroscience | www.frontiersin.org Discussion suggesting that the group difference in the PIT effect most likely
reflects greater incentive salience to reward cues in ST than in
GT individuals. A group difference in the overall amount of
congruent responses (during both CS+ and CS−presentation,
thus not reflecting PIT) emerged towards the end of the task
(Figures 4B,C). for differences in dopamine responsiveness, it has been argued
that higher reward-related dopamine release before conditioning
might increase attribution of incentive salience to reward-related
cues (Wyvell and Berridge, 2000, 2001). Additionally, Flagel et al. (2011) directly demonstrated that dopaminergic transmission is
not involved in all forms of learning, but it is necessary for
the acquisition of a sign-tracking CR, playing a crucial role
in the assignment of incentive salience to reward-related cues. The same study also showed that dopaminergic prediction-error
signals, coded by activity in the nucleus accumbens, are present in
ST individuals, but not in GT individuals. In the present study, a
similar mechanism might occur: high levels of dopamine release
might boost attribution of incentive salience to reward-related
cues, increasing their ability to motivate and drive behavior. Previous studies have found an association between ST
behavior and other traits, such as higher levels of behavioral
impulsivity and a greater propensity to develop addiction (Tomie
et al., 1998; Flagel et al., 2008; Robinson and Flagel, 2009). In
line with these studies, the present study found reduced self-
reported impulse control in the ST group than in the GT group
(Figure 5). These findings seem to corroborate the idea that ST
and GT behaviors are just one expression of a broader profile of
individual differences, which might be clinically relevant. Many
studies have reported that ST individuals are more impulsive
and prone to develop potentially maladaptive behaviors, such
as addiction (Tomie et al., 1998; Robinson and Flagel, 2009;
Flagel et al., 2011). For example, the propensity to sign-track is
associated with a stronger effect of psychomotor sensitization,
a higher susceptibility to a form of cocaine-induced plasticity
that may contribute to the development of addiction (Flagel
et al., 2008). Furthermore, ST behavior in relation to a specific
Pavlovian cue (i.e., a cue predicting monetary reward) is also
predictive of the propensity to attribute incentive salience to
other reward-paired cues, such as food-related or drug-related
cues (e.g., cocaine and alcohol; Uslaner et al., 2006; Cunningham
and Patel, 2007; Flagel et al., 2008; Clark et al., 2013). Discussion The extent
to which such individual differences might play a role in the
development of addiction and in the propensity to relapse is
not yet clear, but their implications for developing individually
targeted treatment programs are promising. g
y
Future studies might further investigate individual differences
in the influence of Pavlovian cues on behavior by taking
additional measures into account, such as phasic dopamine
levels, psychophysiological indices (e.g., galvanic skin response
and heart rate) and as neuroimaging measurements. These
methods would allow better comparisons between human and
non-human animal research on individual differences in ST/GT
behavior and learning styles. A general limitation in the standard
PIT paradigm is that the ‘‘Sign’’ and the ‘‘Goal’’ are presented
in distinct spatial locations. Thus, unrelated spatial biases in
gaze direction might obscure the effect of interest. Although
the analysis conducted in this study already confirmed that
the present findings cannot be accounted for by any a priori
difference in spatial bias between groups, another way to control
for this possibility would be to replicate the experiment with the
spatial positions of the ‘‘Sign’’ and the ‘‘Goal’’ inverted in the
three tasks. In conclusion, the individual differences demonstrated here
offer a promising direction for further investigating the degree
to which incentive salience is attributed to environmental
stimuli associated with rewards, as well as the link between
this process and maladaptive behaviors, ranging from over-
eating to pathological gambling and addiction (Saunders and
Robinson, 2013). Further, the present findings have important
implications for the treatment of impulse-control disorders. Overall, these individual differences in PIT offer new insights
into the mechanisms underlying the transition from intentional
to habitual/compulsive behavior. It should be noted that some recent studies highlighted a more
complex scenario relating ST and GT behaviors to addiction. While ST individuals are more susceptible to the influence of
discrete cues, GT individuals are more influenced by contextual
cues, which can motivate drug-seeking behavior (Robinson
et al., 2014). Consequently, these learning styles seem to reflect
differences in the kinds of triggers to which the individual is
susceptible (e.g., discrete/contextual), rather than a propensity to
addiction per se. This finding emphasizes that there are diverse
pathways to addiction, and has remarkable implications for the
development of personalized treatments in the future. Author Contributions But what exactly is the mechanism underlying the attribution
of incentive salience to discrete stimuli, such as Pavlovian cues? A large amount of evidence points to the role of dopaminergic
transmission within circuits known to be involved in addiction. The core of the nucleus accumbens, for example, was reported
to be involved in ST behavior, and mediates the reinstatement
of drug-seeking and drug-taking behavior (Flagel et al., 2007,
2008, 2011; Clark et al., 2013). Furthermore, various studies have
supported the involvement of the mesolimbic dopamine system
in the emergence of ST behavior. ST individuals are characterized
by stronger dopaminergic gene expression and increased levels
of dopamine in the nucleus accumbens (correlated with the vigor
with which the CR is performed; Flagel et al., 2007, 2008). Even
if differences in basic dopaminergic levels cannot fully account All authors conceived of and designed the experiment; S. G. programmed the task, ran the experiment, analyzed the data,
wrote the main manuscript text and prepared the figures; all
authors read, corrected and approved the final manuscript. Discussion (2011) concluded that the behavioral
influence exerted by CS (i.e., the PIT effect) is dissociated
from attention to Pavlovian stimuli in humans, (see Kaye and
Pearce, 1984, for similar findings in animals). Instead, PIT
is linked to the predictive value acquired by stimuli during
learning. ST behavior has been explained as a consequence of
attributing incentive salience to reward-paired cues (Pavlovian
CS), arising from the interaction between previous experience
(reinforcement learning processes) and individual propensities
(Berridge, 2001; Berridge and Robinson, 2003; Flagel et al., 2011). This incentive salience motivates reward-related action (Tomie
et al., 2000; Flagel et al., 2008; Robinson and Flagel, 2009). In the
present study, ST and GT groups differed in the extent to which
Pavlovian reward-paired cues biased their behavior: relative to
the GT group, the ST group showed an increased likelihood of
performing the instrumental response independently paired with
the same reward when presented with the task-irrelevant reward-
paired cue, even if the reward itself was no longer available
(i.e., a stronger PIT effect; Figure 4A). Therefore, reward-paired
cues exerted a stronger source of influence on the behavior of
ST individuals, as predicted. Importantly, time course analysis
revealed that this effect occurred early and remained stable
throughout the entire PIT test session (Figures 4B,C), thereby Group differences in the PIT effect are not attributable to
differences in the strength of Instrumental or Pavlovian learning
between the groups, which could have potentially induced a bias
towards the rewarded choice in the Instrumental Conditioning
task, or a stronger influence of the reward-paired cue in
the second Pavlovian Conditioning task. Analyses of both the
number of rewarded choices during Instrumental Conditioning,
and reaction times during Pavlovian Conditioning, exclude
such a possibility by revealing that both the ST and GT
groups learned the response-outcome and stimulus-outcome
contingencies equally well (Figure 3). Consequently, differences
in the PIT effect cannot be explained by group differences in
the ability to learn either the instrumental or the Pavlovian June 2015 | Volume 9 | Article 163 8 Individual differences in human cue-controlled behavior Garofalo and di Pellegrino suggesting that the group difference in the PIT effect most likely
reflects greater incentive salience to reward cues in ST than in
GT individuals. A group difference in the overall amount of
congruent responses (during both CS+ and CS−presentation,
thus not reflecting PIT) emerged towards the end of the task
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conducted in the absence of any commercial or financial relationships that could
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instrumental transfer. J. Neurosci. 28, 360–368. doi: 10.1523/JNEUROSCI. 4028-07.2008 Copyright © 2015 Garofalo and di Pellegrino. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC
BY). The use, distribution and reproduction in other forums is permitted, provided
the original author(s) or licensor are credited and that the original publication
in this journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these
terms. Copyright © 2015 Garofalo and di Pellegrino. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC
BY). The use, distribution and reproduction in other forums is permitted, provided
the original author(s) or licensor are credited and that the original publication
in this journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these
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differences in pavlovian autoshaping of lever pressing in rats predict June 2015 | Volume 9 | Article 163 Frontiers in Behavioral Neuroscience | www.frontiersin.org 11
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Novel Geometric Series for Application of Computational Science
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MSC Classification codes: 40A05 (65B10) Keywords: computation, theorem, geometric series 1. Introduction Geometric series played a vital role in differential and integral calculus at the earlier stage of
development and still continues as an important part of the study in science, engineering,
management and its applications. In this article, a new geometric series [1-6] is constructed for
application [7] of computational science and engineering. Novel Geometric Series for Application of Computational Science Chinnaraji Annamalai
School of Management, Indian Institute of Technology, Kharagpur, India
Email: anna@iitkgp.ac.in
https://orcid.org/0000-0002-0992-2584 Chinnaraji Annamalai
School of Management, Indian Institute of Technology, Kharagpur, India
Email: anna@iitkgp.ac.in
https://orcid.org/0000-0002-0992-2584 Abstract: Computational science is a rapidly growing multi-and inter-disciplinary area where
science, engineering, information technology, management and its collaboration use advance
computing capabilities to understand and solve the most complex real life problems. In this
article, a new geometric series is constituted for application of computational science and
engineering. 3. Conclusion Nowadays, the growing complexity of mathematical and computational modelling demands the
simplicity of mathematical and combinatorial equations for solving today’s scientific problems
and challenges. This article provides a novel geometric series and its theorem. This idea can
enable the scientific researchers for further involvement in research and development. For example, For example, For example, For example, ∑
∑
∑ ∑ 2. Novel Geometric Series In today’s world, the growing complexity of mathematical modelling demands the simplicity of
mathematical equations for solving today’s scientific problems. For this purpose, the following
theorem [1-6] is introduced. ∑ Proof. Let us prove this theorem by the equality given below: If we continue this expansion again and again, we can obtain the following series. p
g
g
g
∑ p
g
g
g
∑ Now, let us prove the theorem shown below: ,
p https://doi.org/10.33774/coe-2023-l5mxr ORCID: https://orcid.org/0000-0002-0992-2584 Content not peer-reviewed by Cambridge University Press. License: CC BY 4.0 https://doi.org/10.33774/coe-2023-l5mxr ORCID: https://orcid.org/0000-0002-0992-2584 Content not peer-reviewed by Cambridge University Press. License: CC BY 4.0 ∑ From the Equation (3), we conclude that From the Equation (3), we conclude that From the Equation (3), we conclude that ∑ Hence, theorem is proved. Hence, theorem is proved. Hence, theorem is proved. ∑ Hence, theorem is proved. ∑
F
l ∑ ∑ https://doi.org/10.33774/coe-2023-l5mxr ORCID: https://orcid.org/0000-0002-0992-2584 Content not peer-reviewed by Cambridge University Press. License: CC BY 4.0 References [1]
Annamalai, C. (2018) Annamalai’s Computing Model for Algorithmic Geometric Series
and Its Mathematical Structures. Journal of Mathematics and Computer Science, 3(1),1-6
https://doi.org/10.11648/j.mcs.20180301.11. Page | 2 https://doi.org/10.33774/coe-2023-l5mxr ORCID: https://orcid.org/0000-0002-0992-2584 Content not peer-reviewed by Cambridge University Press. License: CC BY 4.0 [2]
Annamalai, C. (2018) Algorithmic Computation of Annamalai’s Geometric Series and
Summability. Journal of Mathematics and Computer Science, 3(5),100-101. https://doi.org/10.11648/j.mcs.20180305.11. [3]
Annamalai, C. (2017) Analysis and Modelling of Annamalai Computing Geometric
Series and Summability. Mathematical Journal of Interdisciplinary Sciences, 6(1), 11-15. https://doi.org/10.15415/mjis.2017.61002. [4]
Annamalai, C. (2018) Novel Computation of Algorithmic Geometric Series and
Summability. Journal of Algorithms and Computation, 50(1), 151-153. https://www.doi.org/10.22059/JAC.2018.68866. [5]
Annamalai, C. (2017) Computational modelling for the formation of geometric series
using Annamalai computing method. Jñānābha, 47(2), 327-330. https://zbmath.org/?q=an%3A1391.65005. [6]
Annamalai, C. (2018) Computing for Development of A New Summability on Multiple
Geometric Series. International Journal of Mathematics, Game Theory and Algebra,
27(4), 511-513. [7]
Annamalai C (2010) “Application of Exponential Decay and Geometric Series in
Effective Medicine”, Advances in Bioscience and Biotechnology, Vol. 1(1), pp 51-54. https://doi.org/10.4236/abb.2010.11008. Page | 3
https://doi.org/10.33774/coe-2023-l5mxr ORCID: https://orcid.org/0000-0002-0992-2584 Content not peer-reviewed by Cambridge University Press. License: CC BY 4.0 Page | 3
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Phytohormone up-regulates the biochemical constituent, exopolysaccharide and nitrogen metabolism in paddy-field cyanobacteria exposed to chromium stress
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cc-by
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Phytohormone up-regulates the biochemical
constituent, exopolysaccharide and nitrogen
metabolism in paddy-¦eld cyanobacteria exposed to
chromium stress Sanjesh Tiwari ( tiwarisanjesh@gmail.com )
Ranjan Plant Physiology and Biochemistry Laboratory
Anuradha Patel
University of Allahabad
Sheo Mohan Prasad
University of Allahabad Anuradha Patel
University of Allahabad
Sheo Mohan Prasad
University of Allahabad Research article Version of Record: A version of this preprint was published on July 13th, 2020. See the published version
at https://doi.org/10.1186/s12866-020-01799-3. The authors have withdrawn the journal submission associated with this preprint
and requested that the preprint also be withdrawn.
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Effects of COVID19 Pandemic on Pediatric Kidney Transplant in the United States
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Effects of COVID19 Pandemic on P
Transplant in the United States
Olga Charnaya
(
ocharna1@jhmi.edu
)
Johns Hopkins University
https://orcid.org/0000-0003-1104-2882
Teresa Po-Yu Chiang
Johns Hopkins University
https://orcid.org/0000-0003-0601-7420
Richard Wang
Johns Hopkins University
Jennifer Motter
Johns Hopkins University
https://orcid.org/0000-0003-1750-0119
Brian Boyarsky
Johns Hopkins University
https://orcid.org/0000-0001-6902-9854
Elizabeth King
Johns Hopkins University
William Werbel
Johns Hopkins University
https://orcid.org/0000-0003-2943-5895
Christine M. Durand
Johns Hopkins University
https://orcid.org/0000-0003-2605-9257
Robin Avery
Johns Hopkins University
https://orcid.org/0000-0001-7692-3619
Dorry Segev
Johns Hopkins University
https://orcid.org/0000-0002-1924-4801
Allan Massie
Johns Hopkins University
https://orcid.org/0000-0002-5288-5125
Jacqueline Garonzik-Wang
Johns Hopkins University
https://orcid.org/0000-0002-2789-7503 Effects of COVID19 Pandemic on Pediatric Kidney
Transplant in the United States Olga Charnaya
(
ocharna1@jhmi.edu
) Olga Charnaya
(
ocharna1@jhmi.edu
)
Johns Hopkins University
https://orcid.org/0000-0003-1104-2882 Elizabeth King
Johns Hopkins University Introduction As the death toll from COVID-19 in the United States surpasses 180,000 fatalities, the pandemic continues to place
considerable burden on the health care system. Many transplant centers have been challenged to determine how to
proceed with organ transplantation during a time of limited data and resources. These decisions have had profound
implications for many patients awaiting transplantation, including the pediatric population, where kidney
transplantation (KT) remains the optimal treatment for end-stage renal disease (ESRD) [1]. Adult KT has been significantly impacted by COVID-19, with reduced new wait-list addition and transplantation at many
transplant centers across the nation [2,3]. A national survey of adult transplant centers showed that 72% had
suspended live donor kidney transplants (LDKT) and 84% had placed significant restrictions on deceased donor kidney
transplants (DDKT) by April 2020 [4]. The stringency of these restrictions varied by center, with more restrictive
measures in areas with a higher incidence of COVID-19. Several reasons have been cited for these changes. First, there
is concern that organ transplant recipients may be at a greater risk for acquiring and experiencing worse clinical
outcomes to COVID-19 infection [5-7]. Second, immunosuppressed KT recipients may have prolonged viral shedding
and transmissibility, potentially posing a greater risk to public health [8,9]. Third, there is concern that hospitals will not
have the resources to safely perform KT and provide the necessary post-operative care [10]. These changes are not
without consequences; postponing KT can lead to a higher waitlist mortality, and diverting resources away from
transplant recipients may worsen post-operative outcomes [11,12]. It is unclear if the pandemic has resulted in similar challenges and restrictions for the pediatric population. For the most
part, children are asymptomatic or have milder infection and fewer complications from COVID-19 compared to adult
patients [13,14]. From the limited data available, it appears that disease severity even among immunocompromised
children remains less severe than that of the general adult population and remains similar to healthy children [15-17]. Furthermore, while pediatric patients in general have less dialysis-associated morbidity and mortality compared to
adults, delaying transplantation and prolonging time on dialysis is associated with increased morbidity and repetitive
risk of COVID exposure during in-center dialysis sessions [18,19]. The response of the pediatric transplant community is
likely different than adult practices and therefore needs to be studied separately. Research Article Version of Record: A version of this preprint was published at Pediatric Nephrology on September 8th, 2020. See the
published version at https://doi.org/10.1007/s00467-020-04764-4. ersion of Record: A version of this preprint was published at Pediatric Nephrology on S
ublished version at https://doi.org/10.1007/s00467-020-04764-4. Page 1/14 Abstract In March 2020, COVID-19 infections began to rise exponentially in the United States, placing substantial burden on the
healthcare system. As a result, there was a rapid change in transplant practices and policies, with cessation of most
procedures. Our goal was to understand changes to pediatric kidney transplantation (KT) at the national level during
the COVID-19 epidemic. Using SRTR data, we examined changes in pediatric waitlist registration, waitlist removal or
inactivation, and deceased donor and living donor (DDKT/LDKT) events during the start of the disease transmission in
the United States compared to the same time the previous year. We saw an initial decrease in DDKT and LDKT by 47%
and 82% compared to expected events and then a continual increase, with numbers reaching expected pre-pandemic
levels by May 2020. In the early phase of the pandemic, waitlist inactivation and removals due to death or deteriorating
condition rose above expected values by 152% and 189%, respectively. There was a statistically significant decrease in
new waitlist additions (IRR 0.49 0.65 0.85) and LDKT (IRR 0.17 0.38 0.84) in states with high vs low COVID
activity. Transplant recipients during the pandemic were more likely to have received a DDKT, but had similar cPRA,
waitlist time and cause of ESRD as before the pandemic. The COVID-19 pandemic initially reduced access to kidney
transplantation among pediatric patients in the United States, but has not had a sustained effect. activity. Transplant recipients during the pandemic were more likely to have received a DDKT, but had similar cPRA,
waitlist time and cause of ESRD as before the pandemic. The COVID-19 pandemic initially reduced access to kidney
transplantation among pediatric patients in the United States, but has not had a sustained effect. Data Source This study used data from the Scientific Registry of Transplant Recipients (SRTR). The SRTR data system includes data
on all donor, wait-listed candidates, and transplant recipients in the US, submitted by the members of the Organ
Procurement and Transplantation Network (OPTN). The Health Resources and Services Administration (HRSA), U.S. Department of Health and Human Services provides oversight to the activities of the OPTN and SRTR contractors. This
dataset has previously been described elsewhere [20]. National cumulative incidence of COVID-19 National COVID-19 incidence data from January – June 2020 were extracted from USA FACTS
(https://usafacts.org/issues/coronavirus/) [21]. COVID-19 incidence is reported for all patients and not restricted to
children, as many states are not providing data of infections by age groups. Based upon cumulative incidence of
COVID-19 positive cases per-million state population (PMP) between March 15 and June 30, states were stratified to be
having low (<8000 PMP) or high (>8000 PMP) COVID-19 burden. This cutoff was selected based on an visible
difference in distribution of cumulative incidence of COVID- 19 burden in the United States. Introduction To investigate center-specific changes in pediatric transplant practices during the COVID-19 pandemic, we used
national registry data to quantify changes to KT waitlist registration, waitlist deaths or removal, rates of DDKT and Page 2/14 LDKT, between February and June 2020. We examined rates of these waitlist events among the overall patient
population, and evaluated for differences in patient characteristics during the COVID-19 pandemic. This information
will allow us to better understand of the pandemic’s impact on the pediatric kidney transplant population. LDKT, between February and June 2020. We examined rates of these waitlist events among the overall patient
population, and evaluated for differences in patient characteristics during the COVID-19 pandemic. This information
will allow us to better understand of the pandemic’s impact on the pediatric kidney transplant population. Study Population For waitlist analysis, we included DDKT waitlist registrants aged 0-17 at time of listing (new listing; changed to inactive
status; removed due to DDKT, LDKT, death or deteriorating condition, and other causes) were included. For analysis of
transplant volume (DDKT, LDKT, DCD, national or regional import) recipients aged 0-17 at time of transplant were
included. Weekly counts of waitlist and transplant changes For each week (Sunday-Saturday) starting February 2, 2020 until June 27, 2020, we plotted cumulative counts of new
waitlist additions, newly inactive patients, waitlist removal due to death or deteriorating condition, and waitlist removal
due to transplant or other causes, using a Lowess smoothing function. We made similar plots for weekly counts of
DDKT, LDKT, DCD donor, and regional and national imports. Weekly instead of daily counts were used due to the low
number of daily events in pediatric patients to enable statistical analysis. On each plot we also included the average
counts during the same period in 2017-2019 as a visual reference of national pre-pandemic pediatric KT volume. Characteristics of pediatric transplant patients during
COVID-19 Patient characteristics in three time periods (“Early” Jan 1 – Mar 15 2020, “Middle” Mar 16 – Apr 30 2020, and “Late”
May 1 – Jun 30 2020) were examined (Table 1). Patients who received a kidney transplant during the first COVID-19
peak in the United States, Middle period, had similar waitlist time, cPRA and blood type compared to Early and Late
periods (p > 0.1). Higher proportion of Black patients received a transplant in the Middle (30.6%) compared to Early
(13.1%) and Late (20.7%), (p=0.28). Living donor transplants made up a smaller proportion of total transplants during
the Middle period 13.9%, compared to 29.5% and 36.4% in Early and Late, respectively (p=0.035). Median cold ischemia
time (CIT) was longer in the Middle period 10.2 hours (IQR 6.5-17.4), compared to the Early 9.0 hours (IQR 4.0-13.2) and
Late 7.6 hours (IQR 2.4-10.7) periods, (p=0.02). Weekly count of waitlist changes National weekly pediatric KT waitlist additions ranged from 7 - 41 cases per week between February 2 and June 30,
2020. There was a trend of decreasing new pediatric DDKT registrations, following the national rise of COVID-19 cases
mid-March. Since April, none of the weekly pediatric KTs exceeded 21, the 2017- 2019 average counts for the same
period (Figure 1A). The numbers of registrants who changed to inactive status also increased in March, with 77.2% of
registrants who changed to inactive status in the third and fourth week of March indicating COVID-19 as reason of
inactivation (Figure 1C). COVID-19 was added as a refusal code or cause for change in status in UNET on March 25,
2020; however, this classification does not differentiate new COVID-19 infection in the patient vs precaution secondary
to the pandemic. Percentage of inactive waitlist registrants rose from 72% to 77% between March 1 and April 15, and remained elevated
above previous baseline thereafter (Figure 1D). We observed an increasing trend in waitlist removal due to death or
deteriorating condition since March, followed by a trend that returned to previous benchmarks by late-April (Figure 1B). Statistical analysis We presented characteristics of pediatric kidney transplant recipients separately in three time periods: January 1 -
March 15, 2020 (“Early”); March 16 - April 30, 2020 (“Middle”); and May 1 – June 30, 2020 (“Late”). Continuous
variables were presented as median and interquartile range, and categorical variables were presented as counts and
proportion. Comparison between groups were tested using Kuskal-Wallis or Mann Whitney-U, as appropriate, for
continuous variables and Fisher's exact test for categorical variables. We used 2015 as reference year to calculate KDPI
[22]. We obtained pediatric kidney waitlist changes or transplant volume by center, month and year from January 1,
2016 to February 28, 2020, and constructed a mixed-effects Poisson regression with a center-level random intercept to
obtain expected daily counts by center (monthly counts divided by 31), using methods previously described [23]. The
expected counts of each time period were the sum of expected center-level counts during the corresponding length of
time (March 1 to April 30: 47 days; May 1 – June 30: 61 days). We then compared the observed and expected counts of
each time period using Chi-square testing. We used an α of 0.05 to define statistical significance. All analyses were
performed using Stata 16.0/MP for Linux (College Station, Texas). Weekly count of transplant events Page 4/14 The national weekly pediatric DDKT volume ranged from 0-16 cases per week between February 1 and June 30, 2020. On average, the weekly DDKT volume in 2017-2019 was 9.6 cases. Between mid-March and the end of June 2020,
DDKT volume remained lower than 9.6 except for 4 weeks out of the 15 during observation. There was a trend of
decreasing DDKT and LDKT volume seen since March, followed by increase from mid-April to end of June (Figure 2A,
2B). For LDKT, the weekly volumes were never above the 2017-2019 average between mid-March and May 31, but
consistently surpassed this volume in June 2020 (Figure 2B). Comparing the observed and predicted waitlist changes The overall observed national volume of waitlist registration was lower (-13.3%, p=0.021) and change to inactive
waitlist status was higher (57.2%, p<0.001) compared to the expected volume during March 15 – June 30, 2020 (Table
2). When stratified into the earlier (March 15 – April 30, 2020) and the latter (May 1– June 30, 2020) periods, 6
candidates were removed from the waitlist during the earlier period due to death or deteriorated condition, which was
189% more than the expected 2.1 cases (p=0.005). Similarly, 83 candidates had changed to inactive status during the
earlier period, which was 152% more than the expected 32.9 cases (p<0.001). In both cases, the significance was not
achieved in the latter period (11.3%, p=0.3 and -11.1%, p=0.5, respectively). Contrarily, the observed counts of new
waitlist registration was 23.8% lower compared to the expected during the latter period (p=0.002), though not
significantly different in the earlier period (0.4%, p=1.0). Comparing the observed and predicted transplant events There were 157 pediatric KTs performed during March 15-June 30, 2020 (108 DDKT, 49 LDKT), which was 22.8% fewer
than the expected 203.3 cases (p=0.001). The 108 DDKT performed during the same period was 29.2% fewer than the
expected 103.1 cases (p=0.03), whereas the 49 LDKTs was not significantly different from the expected 64.2 cases
(p=0.058). When stratified to the earlier and the latter COVID-19 eras, the observed DDKT, LDKT, and combined total
transplant were all significantly less than expected in the earlier era (total: 36 vs. 88.5, -59.3%, p<0.001; LDKT: 5 vs. 27.9,
-82.1%, p<0.001; DDKT: 31 vs. 59.1%, -47.6%, p<0.001) but not during the latter period. Regional and National Imports Overall numbers of regional and national imports were extremely low (0-4 per week), with average <1 import per week in
2017-2019 (Figure 2C). During the early period of COVID-19 disease activity in the United States, imports were more
common than in previous years. As the pandemic progressed, there was a decline in imports, however the average
number of imports continue to remain higher than previous years. Discussion In this national registry study of pediatric KT trends during the COVID-19 pandemic, we found an increase in patients
being changed to inactive status by 152%, increase in mortality on the waitlist by 189%, decrease in DDKT by 48% and
LDKT by 82% compared to expected in the early COVID-19 time period without a significant impact on new waitlist
additions. The COVID-19 pandemic has substantially limited access to KT and increased waitlist mortality in pediatric
patients. Many transplant programs significantly altered their routine protocols and stopped performing kidney transplants
thereby restricting access to KT during the COVID-19 pandemic [4]. A recent registry of adult SOT recipients infected
with COIVD-19 did not show any significant difference in mortality or morbidity compared to non-SOT patients [24]. While extensive data in pediatric solid organ transplant (SOT) recipients is not available, several case reports show that
pediatric SOT patients infected with COVID-19 showed only mild disease, even while on immunosuppressive therapy
[25,26]. As children have different etiologies of ESRD, different comorbidities and seem to be affected differently by
COVID-19, uniform policies affecting access to transplantation for both children and adults are not appropriate nor in
the best interest of pediatric patients [16]. The increased mortality while on the waitlist that we showed in pediatric
patients is a striking metric that supports the need for an individualized approach for pediatric KT patients. As pediatric
KT events are relatively rare compared to adult KT, this affords an opportunity for patient- level versus center-level
decisions about risk and benefit of KT in a pandemic setting. The reduction of transplant events in children seen during the Middle (March 15 – April 30, 2020) COVID- 19 era was
likely secondary to a combination of factors. As hospitals shifted resources to treat COVID-19 patients there was a
decrease in available ICU beds for post-operative management and restrictions on operating room availability. Pediatric
KT patients require intensive post-operative care, which may strain healthcare systems already overburdened by COVID-
19 patients [27]. In addition, there was a notable decline in deceased donor organs recovered during March and April
[28]. Shortages of COVID-19 testing or delayed results for deceased donors may have impacted center willingness to
accept an organ from what would otherwise be an acceptable donor. As testing capacity increased across the country,
this limitation was ameliorated. Regional differences in transplant practice and waitlist
death by COVID-19 burden Centers situated in states with high COVID-19 burden (NY, NJ, RI, MA, DC, CT,LA, DE, IL, MD, AZ, NE, IA, NS) between
March 15 and June 30 had significantly fewer new waitlist registrations (incidence rate ratio (IRR): 0.49 0.65 0.85) and
LDKT (IRR: 0.17 0.38 0.84) compared to centers in states with low burden (IRR: 0.82 0.94 1.08) (Table 3). There were no
differences in the proportion of expected DDKT and waitlist death between centers in states with high and low COVID-
19 burden. Page 5/14 Page 5/14 Discussion Finally, many centers stopped or significantly reduced elective and non-emergent
surgical procedures, which likely had a significant impact on LDKT. For pediatric patients scheduled to receive an LDKT,
it should be argued that transplant is not truly an elective procedure whether pre-emptive or not. Dialysis initiation
would require at least one surgical procedure to establish dialysis access, and initiation or continuation of hemodialysis
would result in a much higher COVID-19 exposure risk [29]. While peritoneal dialysis patients do not require frequent in-center visits and thereby can have minimal healthcare
exposure, especially with the advancement of telehealth capabilities across the country, the benefit of transplant over
dialysis has been well established in the pediatric ESRD population [30,31]. As healthcare centers lifted restrictions on
elective cases and practice patterns changed, there was a large increase in LDKT in June 2020 suggesting cases had
been postponed due to the pandemic. Our hypothesis that children who received a transplant during the peak of the pandemic would differ in some
characteristics compared to pre-pandemic patients was not supported. There was not a statistically significant
difference in donor KDPI, recipient cPRA, or etiology of ESRD. While cold ischemia time in the Middle time period was
longer, this is unlikely to be a clinically significant difference. This is the first study to describe characteristics of donors
and recipients receiving a KT during the pandemic. As this is a registry study, we are limited in the information that is available for analysis and are not able to delve into
granular details of waitlist removal or patient death. While transplant centers have mandatory reporting requirements to Page 6/14 Page 6/14 mission may be delayed due to center practices and pandemic effect on workflow. UNOS/OPTN, data transmission may be delayed due to center practices and pandemic effect on workflow. Nevertheless, we are able to make generalized conclusions about the effects of COVID-19 on access to KT in pediatric
patients. We were unable to differentiate regional variability in transplant rates and waitlist changes due to the overall
low number of events in pediatric patients. In summary, we found that the COVID-19 pandemic has had a significant impact on pediatric KT waitlist mortality,
waitlist registration, DDKT and LDKT. DCD: donation after circulatory death IRR: incidence rate ratio ICU: intensive care unit OR: operating room Discussion Further studies to assess outcomes of pediatric patients who received a KT
during this time are necessary to inform changes in policies and practices to optimize pediatric transplant outcomes
and ensure access to this life-saving treatment. DISCLAIMER The analyses described here are the responsibility of the authors alone and do not necessarily reflect the views or
policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or
organizations imply endorsement by the U.S. Government. The data reported here have been supplied by the Hennepin
Healthcare Research Institute (HHRI) as the contractor for the Scientific Registry of Transplant Recipients (SRTR). The
interpretation and reporting of these data are the responsibility of the author(s) and in no way should be seen as an
official policy of or interpretation by the SRTR or the U.S. Government. Abbreviations COVID-19: coronavirus disease 2020 KT: kidney transplant LDKT: live donor kidney transplantation DDKT: deceased donor transplantation ESR y
p
p
g
SRTR: Scientific Registry of Transplant Recipients OPTN: Organ Procurement and Transplantation Network PMP: per
million population DCD: donation after circulatory death IRR: incidence rate ratio DCD: donation after circulatory death IRR: incidence rate ratio Declarations Funding: This work was supported by grant number KL2TR003099 (Charnaya) from Johns Hopkins Institute for Clinical
and Translational Research (ICTR) as well as F32DK124941 (Boyarsky), K23DK115908-01 (Garonzik-Wang), and
K01DK101677 (Massie) from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and
K24AI144954 (Segev) from National Institute of Allergy and Infectious Diseases (NIAID). Conflicts of interest/Competing interests: The authors of this manuscript have no conflicts of interest to disclose. onflicts of interest/Competing interests: The authors of this manuscript have no conf Ethics approval: Approved by the institutional review board at Johns Hopkins University School of Medicine. Consent to participate: Not applicable Consent for publication: Not applicable Availability of data and material: Data is available for review upon request to the corresponding author
Code availability: Not applicable Availability of data and material: Data is available for review upon request to the corresponding author Page 7/14 Authors' contributions: Charnaya, Chiang, Motter, Wang and Massie participated in study design, analysis and
manuscript preparation. Boyarsky participated in study design and manuscript preparation. Wang, Motter, King, Werbel,
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6736(20)31040-0 10. Loupy A, Aubert O, Reese PP, Bastien O, Bayer F, Jacquelinet C (2020) Organ procurement and transplantation
during the COVID-19 pandemic. Lancet (London, England) 395 (10237):e95-e96. doi:10.1016/s0140-
6736(20)31040-0 11. Massie AB, Boyarsky BJ, Werbel WA, Bae S, Chow EK, Avery RK, Durand CM, Desai N, Brennan D, Garonzik-Wang
JM, Segev DL (2020) Identifying scenarios of benefit or harm from kidney transplantation during the COVID-19
pandemic: a stochastic simulation and machine learning study. American journal of transplantation : official
journal of the American Society of Transplantation and the American Society of Transplant Surgeons. doi:10.1111/ajt.16117 11. Massie AB, Boyarsky BJ, Werbel WA, Bae S, Chow EK, Avery RK, Durand CM, Desai N, Brennan D, Garonzik-Wang
JM, Segev DL (2020) Identifying scenarios of benefit or harm from kidney transplantation during the COVID-19
pandemic: a stochastic simulation and machine learning study. American journal of transplantation : official
journal of the American Society of Transplantation and the American Society of Transplant Surgeons. doi:10.1111/ajt.16117 Page 8/14
12. de Vries APJ, Alwayn IPJ, Hoek RAS, van den Berg AP, Ultee FCW, Vogelaar SM, Haase-Kromwijk B, Heemskerk MBA,
Hemke AC, Nijboer WN, Schaefer BS, Kuiper MA, de Jonge J, van der Kaaij NP, Reinders MEJ (2020) Immediate Page 8/14
12. de Vries APJ, Alwayn IPJ, Hoek RAS, van den Berg AP, Ultee FCW, Vogelaar SM, Haase-Kromwijk B, Heemskerk MBA,
Hemke AC, Nijboer WN, Schaefer BS, Kuiper MA, de Jonge J, van der Kaaij NP, Reinders MEJ (2020) Immediate Page 8/14
12. Page 8/14
12. de Vries APJ, Alwayn IPJ, Hoek RAS, van den Berg AP, Ultee FCW, Vogelaar SM, Haase-Kromwijk B, Heemskerk MBA,
Hemke AC, Nijboer WN, Schaefer BS, Kuiper MA, de Jonge J, van der Kaaij NP, Reinders MEJ (2020) Immediate References Massie AB, Kucirka LM, Segev DL (2014) Big data in organ transplantation: registries and administrative claims. Am J Transplant 14 (8):1723-1730. doi:10.1111/ajt.12777 20. Massie AB, Kucirka LM, Segev DL (2014) Big data in organ transplantation: registries and administrative claims. Am J Transplant 14 (8):1723-1730. doi:10.1111/ajt.12777 20. Massie AB, Kucirka LM, Segev DL (2014) Big data in organ transplantation: registries and administrative claims. Am J Transplant 14 (8):1723-1730. doi:10.1111/ajt.12777 21. USAFACTS (2020) Coronavirus Stats & Data. https://usafacts.org/issues/coronavirus/. 2020 21. USAFACTS (2020) Coronavirus Stats & Data. https://usafacts.org/issues/coronavirus/. 2020 21. USAFACTS (2020) Coronavirus Stats & Data. https://usafacts.org/issues/coron 22. Massie AB, Leanza J, Fahmy LM, Chow EKH, Desai NM, Luo X, King EA, Bowring MG, Segev DL (2016) A Risk Index
for Living Donor Kidney Transplantation. American Journal of Transplantation 16 (7):2077-2084. doi:10.1111/ajt.13709 22. Massie AB, Leanza J, Fahmy LM, Chow EKH, Desai NM, Luo X, King EA, Bowring MG, Segev DL (2016) A Risk Index
for Living Donor Kidney Transplantation. American Journal of Transplantation 16 (7):2077-2084. doi:10.1111/ajt.13709 23. Boyarsky BJ, Werbel WA, Durand CM, Avery RK, Jackson KR, Kernodle AB, Snyder J, Hirose R, Massie IM, Garonzik-
Wang JM, Segev DL, Massie AB (2020) Early national and center-level changes to kidney transplantation in the
United States during the COVID-19 epidemic. American journal of transplantation : official journal of the American
Society of Transplantation and the American Society of Transplant Surgeons. doi:10.1111/ajt.16167 23. Boyarsky BJ, Werbel WA, Durand CM, Avery RK, Jackson KR, Kernodle AB, Snyder J, Hirose R, Massie IM, Garonzik-
Wang JM, Segev DL, Massie AB (2020) Early national and center-level changes to kidney transplantation in the
United States during the COVID-19 epidemic. American journal of transplantation : official journal of the American
Society of Transplantation and the American Society of Transplant Surgeons. doi:10.1111/ajt.16167 24. Molnar MZ, Bhalla A, Azhar A, Tsujita M, Talwar M, Balaraman V, Sodhi A, Kadaria D, Eason JD, Hayek SS, Coca SG,
Shaefi S, Neyra JA, Gupta S, Leaf DE, Kovesdy CP, Investigators TS-C Outcomes of Critically Ill Solid Organ
Transplant Patients with COVID-19 in the United States. American Journal of Transplantation n/a (n/a). doi:10.1111/ajt.16280 24. Molnar MZ, Bhalla A, Azhar A, Tsujita M, Talwar M, Balaraman V, Sodhi A, Kadaria D, Eason JD, Hayek SS, Coca SG,
Shaefi S, Neyra JA, Gupta S, Leaf DE, Kovesdy CP, Investigators TS-C Outcomes of Critically Ill Solid Organ
Transplant Patients with COVID-19 in the United States. References de Vries APJ, Alwayn IPJ, Hoek RAS, van den Berg AP, Ultee FCW, Vogelaar SM, Haase-Kromwijk B, Heemskerk MBA,
Hemke AC, Nijboer WN, Schaefer BS, Kuiper MA, de Jonge J, van der Kaaij NP, Reinders MEJ (2020) Immediate Page 8/14 impact of COVID-19 on transplant activity in the Netherlands. Transplant immunology 61:101304. doi:10.1016/j.trim.2020.101304 impact of COVID-19 on transplant activity in the Netherlands. Transplant immunology 61:101304. doi:10.1016/j.trim.2020.101304 13. Zimmermann P, Curtis N (2020) Coronavirus Infections in Children Including COVID-19: An Overview of the
Epidemiology, Clinical Features, Diagnosis, Treatment and Prevention Options in Children. The Pediatric infectious
disease journal 39 (5):355-368. doi:10.1097/inf.0000000000002660 14. Dong Y, Mo X, Hu Y, Qi X, Jiang F, Jiang Z, Tong S (2020) Epidemiology of COVID-19 Among Children in China. Pediatrics 145 (6). doi:10.1542/peds.2020-0702 15. Marlais M, Wlodkowski T, Vivarelli M, Pape L, Tönshoff B, Schaefer F, Tullus K (2020) The severity of COVID-19 in
children on immunosuppressive medication. The Lancet Child & adolescent health 4 (7):e17-e18. doi:10.1016/s2352-4642(20)30145-0 16. Zachariah P, Johnson CL, Halabi KC, Ahn D, Sen AI, Fischer A, Banker SL, Giordano M, Manice CS, Diamond R,
Sewell TB, Schweickert AJ, Babineau JR, Carter RC, Fenster DB, Orange JS, McCann TA, Kernie SG, Saiman L
(2020) Epidemiology, Clinical Features, and Disease Severity in Patients With Coronavirus Disease 2019 (COVID-
19) in a Children's Hospital in New York City, New York. JAMA pediatrics:e202430. doi:10.1001/jamapediatrics.2020.2430 17. Melgosa M, Madrid A, Alvárez O, Lumbreras J, Nieto F, Parada E, Perez-Beltrán V (2020) SARS- CoV-2 infection in
Spanish children with chronic kidney pathologies. Pediatr Nephrol:1-4. doi:10.1007/s00467-020-04597-1 18. Weaver DJ, Jr., Somers MJG, Martz K, Mitsnefes MM (2017) Clinical outcomes and survival in pediatric patients
initiating chronic dialysis: a report of the NAPRTCS registry. Pediatr Nephrol 32 (12):2319-2330. doi:10.1007/s00467-017-3759-4 19. Prezelin-Reydit M, Combe C, Harambat J, Jacquelinet C, Merville P, Couzi L, Leffondré K (2019) Prolonged dialysis
duration is associated with graft failure and mortality after kidney transplantation: results from the French
transplant database. Nephrology, dialysis, transplantation : official publication of the European Dialysis and
Transplant Association - European Renal Association 34 (3):538-545. doi:10.1093/ndt/gfy039 19. Prezelin-Reydit M, Combe C, Harambat J, Jacquelinet C, Merville P, Couzi L, Leffondré K (2019) Prolonged dialysis
duration is associated with graft failure and mortality after kidney transplantation: results from the French
transplant database. Nephrology, dialysis, transplantation : official publication of the European Dialysis and
Transplant Association - European Renal Association 34 (3):538-545. doi:10.1093/ndt/gfy039 20. Transplant Surgeons. doi:10.1111/ajt.16003 27. Pathak EB, Salemi JL, Sobers N, Menard J, Hambleton IR (2020) COVID-19 in Children in the United States:
Intensive Care Admissions, Estimated Total Infected, and Projected Numbers of Severe Pediatric Cases in 2020. Journal of public health management and practice : JPHMP 26 (4):325-333. doi:10.1097/phh.0000000000001190 27. Pathak EB, Salemi JL, Sobers N, Menard J, Hambleton IR (2020) COVID-19 in Children in the United States:
Intensive Care Admissions, Estimated Total Infected, and Projected Numbers of Severe Pediatric Cases in 2020. Journal of public health management and practice : JPHMP 26 (4):325-333. doi:10.1097/phh.0000000000001190 28. UNOS (2020) Number of deceased donors recovered in the US by week. https://public.tableau.com/shared/8MW3ZBGK9?:toolbar=n&:display_count=n&:origin=viz_share_link&: embed=y. Accessed 6/23/2020 2020 29. Ikizler TA, Kliger AS (2020) Minimizing the risk of COVID-19 among patients on dialysis. Nature reviews Nephrology
16 (6):311-313. doi:10.1038/s41581-020-0280-y 30. Lew SQ, Sikka N (2019) Operationalizing Telehealth for Home Dialysis Patients in the United States. American
journal of kidney diseases : the official journal of the National Kidney Foundation 74 (1):95-100. doi:10.1053/j.ajkd.2019.01.023 31. Verghese PS (2017) Pediatric kidney transplantation: a historical review. Pediatric research 81 (1- 2):259-264. doi:10.1038/pr.2016.207 References American Journal of Transplantation n/a (n/a). doi:10.1111/ajt.16280 25. D’Antiga L (2020) Coronaviruses and Immunosuppressed Patients: The Facts During the Third Epidemic. Liver
Transplantation 26 (6):832-834. doi:10.1002/lt.25756 25. D’Antiga L (2020) Coronaviruses and Immunosuppressed Patients: The Facts During the Third Epidemic. Liver
Transplantation 26 (6):832-834. doi:10.1002/lt.25756 26. Bush R, Johns F, Acharya R, Upadhyay K (2020) Mild COVID-19 in a pediatric renal transplant recipient. American
journal of transplantation : official journal of the American Society of Transplantation and the American Society of 26. Bush R, Johns F, Acharya R, Upadhyay K (2020) Mild COVID-19 in a pediatric renal transplant recipient. American
journal of transplantation : official journal of the American Society of Transplantation and the American Society of Page 9/14 Page 9/14 Transplant Surgeons. doi:10.1111/ajt.16003 Transplant Surgeons. doi:10.1111/ajt.16003 Tables Table 1. Patient and donor characteristics broken down by three time periods of COVID-19
activity in 2020. Page 10/14 Early
Jan 1 – Mar 15
Middle
Mar 15 – Apr 30
Late
May 1 – Jun 30
p-
value
N
122
36
121
Recipient Factors
Wait time (IQR)
202.5 (81, 618)
201.5 (42.5, 547)
227 (92, 495)
0.87
Female Sex
59 (48.4%)
14 (38.9%)
56 (46.3%)
0.61
Race
0.28
White
95 (77.9%)
21 (58.3%)
86 (71.1%)
Black
16 (13.1%)
11 (30.6%)
25 (20.7%)
Asian
7 (5.7%)
3 (8.3%)
8 (6.6%)
Others
4 (3.3%)
1 (2.8%)
2 (1.7%)
ABO Blood Type
0.94
Type O
70 (57.4%)
21 (58.3%)
71 (58.7%)
Type A
30 (24.6%)
10 (27.8%)
34 (28.1%)
Type B
16 (13.1%)
4 (11.1%)
13 (10.7%)
Type AB
6 (4.9%)
1 (2.8%)
3 (2.5%)
Primary diagnosis
0.065
Glomerulonephritis
21 (17.2%)
2 (5.6%)
17 (14.0%)
FSGS
15 (12.3%)
5 (13.9%)
15 (12.4%)
Hypoplasia
32 (26.2%)
7 (19.4%)
14 (11.6%)
Obstructive
19 (15.6%)
8 (22.2%)
19 (15.7%)
Polycystic
9 (7.4%)
2 (5.6%)
18 (14.9%)
Others
26 (21.3%)
12 (33.3%)
38 (31.4%)
cPRA, mean (SD)
12% (24)
9% (26)
7% (20)
0.29
cPRA ≠ 0, median (IQR)
44% (5, 64) (n=36)
61% (5, 99) (n=7)
11% (0, 52)
(n=31)
0.087
cPRA ≥ 0.2
23 (18.9%)
4 (11.1%)
15 (12.4%)
0.33
Donor Factors
Age (Yr), median (IQR)
28 (20, 34)
25.50 (21.50, 32)
28 (22, 35)
0.68
Female Gender
52 (42.6%)
16 (44.4%)
51 (42.1%)
0.97
Living donor
36 (29.5%)
5 (13.9%)
44 (36.4%)
0.035
Race
0.55
White
103 (84.4%)
29 (80.6%)
102 (84.3%)
Black
13 (10.7%)
3 (8.3%)
15 (12.4%)
Asian
3 (2.5%)
3 (8.3%)
3 (2.5%)
Other race
3 (2.5%)
1 (2.8%)
1 (0.8%)
KDPI
n=86
n=31
n=77
median (IQR)
13.73 (7.65, 26.72)
14.57 (5.93, 28.99)
17.56 (9.71, 28.34)
0.46
mean (SD)
17.30 (13.46)
17.38 (12.63)
19.21 (12.18)
0.61
Cold Ischemic Time (Hours), median
(IQR)
9.0 (4.0, 13.2)
(n=121)
10.2 (6.5, 17.5)
(n=34)
7.6 (2.4, 10.7)
(n=57)
0.021 Table 2. Tables Observed compared to expected events in early and later COVID-19 eras of (A) waitlist changes,
(B) transplant events
Waitlist
changes
(pediatric
age
at
listing)
March 15- April 30, 2020
May 1 – June 30, 2020
Total
Observed expected %
change
p
value
observed expected %
change
p
value
observed expected %
change
p
value
New listing
132
131.5
0.4
0.967
130
170.7
-23.8
0.002
262
302.2
-13.3
0.021
Causes
of
removal
Death 6
2.1
189.0
0.006
3
2.7
11.3
0.852
9
4.8
88.7
0.053
DDKT 42
64.5
-34.9
0.005
93
83.7
11.1
0.309
135
148.2
-8.9
0.279
LDKT 5
27.8
-82.0
< 0.01
44
36.1
21.9
0.188
49
63.9
-23.3
0.062
Changed to
inactive status
83
32.9
152.0
< 0.01
38
42.7
-11.1
0.468
119
75.7
57.2
< 0.01 rved compared to expected events in early and later COVID-19 eras of (A) waitlist changes,
t events Table 2. Observed compared to expected events in early and later COVID-19 eras of (A) waitlist changes,
(B) transplant events ompared to expected events in early and later COVID-19 eras of (A) waitlist changes, Page 11/14 Page 11/14 Transplant
(pediatric age
at transplant)
March 15- April 30, 2020
May 1 – June 30, 2020
Total
Observed expected %
change
p
value
observed expected %
change
p
value
observed expected %
change
p
value
Total transplant
36
88.5
-59.3
<
0.01
121
114.8
5.4
0.566
157
203.3
-22.8
0.001
LDKT
5
27.9
-82.1
<
0.01
44
36.3
21.3
0.199
49
64.2
-23.7
0.058
DDKT
31
59.1
-47.6
<
0.01
77
76.8
0.3
1.000
108
135.9
-20.5
0.017
DCD
1
3.2
-68.4
0.224 5
4.1
21.6
0.663
6
7.3
-17.6
0.632
Regional or
national import
7
3.7
90.7
0.082
4
4.8
-16.1
0.729
11
8.4
30.4
0.377 Table 3. Observed center-level events as a proportion of expected events, March 15 -
June 30, 2020. Bold denotes IRRs that are statistically significantly different from the IRR
in states with low COVID-19 disease burden (<8000 cases PMP). COVID-19 rates
New listings
DDKT
LDKT
Waitlist death
Overall
0.77 0.87 0.98
0.66 0.79 0.96
0.58 0.76 1.01
0.98 1.89 3.63
Low
0.82 0.94 1.08
0.66 0.81 1.00
0.66 0.89 1.20
0.84 1.87 4.16
High*
0.49 0.65 0.85
0.48 0.73 1.12
0.17 0.38 0.84
0.62 1.92 5.96
*States with high COVID-19 burden: NY, NJ, RI, MA, DC, CT, LA, DE, IL, MD, AZ, NE, IA, NS Table 3. Tables Observed center-level events as a proportion of expected events, March 15 -
June 30, 2020. Bold denotes IRRs that are statistically significantly different from the IRR
in states with low COVID-19 disease burden (<8000 cases PMP). Table 3. Observed center-level events as a proportion of expected events, March 15 -
June 30, 2020. Bold denotes IRRs that are statistically significantly different from the IRR
in states with low COVID-19 disease burden (<8000 cases PMP). COVID-19 rates
New listings
DDKT
LDKT
Waitlist death
Overall
0.77 0.87 0.98
0.66 0.79 0.96
0.58 0.76 1.01
0.98 1.89 3.63
Low
0.82 0.94 1.08
0.66 0.81 1.00
0.66 0.89 1.20
0.84 1.87 4.16
High*
0.49 0.65 0.85
0.48 0.73 1.12
0.17 0.38 0.84
0.62 1.92 5.96 *States with high COVID-19 burden: NY, NJ, RI, MA, DC, CT, LA, DE, IL, MD, AZ, NE, IA, NS *States with high COVID-19 burden: NY, NJ, RI, MA, DC, CT, LA, DE, IL, MD, AZ, NE, IA, NS States with high COVID-19 burden: NY, NJ, RI, MA, DC, CT, LA, DE, IL, MD, AZ, NE, IA, NS *States with high COVID-19 burden: NY, NJ, RI, MA, DC, CT, LA, DE, IL, MD, AZ, NE, IA, NS
Figures *States with high COVID-19 burden: NY, NJ, RI, MA, DC, CT, LA, DE, IL, MD, AZ, NE, IA, NS
Figures Figures Pediatric patient deceased donor KT waitlist status change; (A) new waitlist additions, (B) removal due to death or
deteriorating condition, (C) changed to inactive status, (D) percentage inactive. A. Figures Page 12/14 Figure 1
Pediatric patient deceased donor KT waitlist status change; (A) new waitlist additions, (B) removal due to death or
deteriorating condition, (C) changed to inactive status, (D) percentage inactive. A. Figure 1
Pediatric patient deceased donor KT wait
deteriorating condition, (C) changed to in Figure 1 Pediatric patient deceased donor KT waitlist status change; (A) new waitlist additions, (B) removal due to death or
deteriorating condition, (C) changed to inactive status, (D) percentage inactive. A. Page 13/14 Figure 2
Pediatric transplant events cumulatively by week
imports A. Figure 2
Pediatric transplant events cumulatively by week starting Feb 2, 2020; (A) DDKT, (B) LDKT,
(C) Regional and National
imports A. Figure 2 Pediatric transplant events cumulatively by week starting Feb 2, 2020; (A) DDKT, (B) LDKT,
(C) Regional and National
imports A. Pediatric transplant events cumulatively by week starting Feb 2, 2020; (A) DDKT, (B) LDKT,
(C) Regional and National
imports A. Pediatric transplant events cumulatively by week starting Feb 2, 2020; (A) DDKT, (B) LDKT,
(C) Regional and National
imports A. Pediatric transplant events cumulatively by week starting Feb 2, 2020; (A) DDKT, (B) LDKT,
(C) Regional and National
imports A Page 14/14
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https://europepmc.org/articles/pmc6748250?pdf=render
|
English
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The Mersin Greenhouse Workers Study. Surveillance of Work-related Skin, Respiratory, and Musculoskeletal Diseases
|
Annals of global health
| 2,018
|
cc-by
| 5,493
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* Ministry of Health, Mersin Public Health Directorate, Mersin, TR
† Ministry of Health, Mardin Public Health Directorate, Mardin, TR
‡ Ministry of Health, Manisa Public Health Directorate, Manisa, TR
§ Ministry of Health, Turkish Public Health Institution, Ankara, TR
‖ Hacettepe University, Faculty of Medicine, Department of
Internal Medicine, Division of Occupational Medicine, Ankara, TR The Mersin Greenhouse Workers Study. Surveillance
of Work-related Skin, Respiratory, and Musculoskeletal
Diseases Farmers and farm work
ers experience high rates of low back, shoulder, and upper
extremity disorders [6]. Pesticide-related illnesses refer to
a broad group of health outcomes, including dermatitis,
eye injuries, respiratory diseases, and cancers [7]. The use to hazardous chemicals, particularly pesticides [3]. Agri
culture and forestry, as an occupation, consistently ranks
as the third or fourth most hazardous occupations in the
European Union [4]. Agricultural workers suffer from sev
eral conditions and diseases [5]. Farmers and farm work
ers experience high rates of low back, shoulder, and upper
extremity disorders [6]. Pesticide-related illnesses refer to
a broad group of health outcomes, including dermatitis,
eye injuries, respiratory diseases, and cancers [7]. The use Nuraydın A, et al. The Mersin Greenhouse Workers Study. Surveillance of Work-
related Skin, Respiratory, and Musculoskeletal Diseases. Annals of Global Health. 2018; 84(3), pp. 504–511. DOI: https://doi.org/10.29024/aogh.2315 Nuraydın A, et al. The Mersin Greenhouse Workers Study. Surveillance of Work-
related Skin, Respiratory, and Musculoskeletal Diseases. Annals of Global Health. 2018; 84(3), pp. 504–511. DOI: https://doi.org/10.29024/aogh.2315 ORIGINAL RESEARCH ¶ Department of Health Sciences of the University of Milan, Milan, IT
** Medical Centre of Vrije Universiteit Amsterdam, Amsterdam, NL
†† Hacettepe University, Faculty of Medicine, Department of
Public Health, Ankara, TR
Corresponding author: Ali Naci Yıldız, MD, PhD
(alinaciyildiz@gmail.com) The Mersin Greenhouse Workers Study. Surveillance
of Work-related Skin, Respiratory, and Musculoskeletal
Diseases Aydın Nuraydın*, Özgür Bilek†, Ali Koray Kenziman*, Mehmet Ali Korkusuz‡, Ali İhsan
Atagün*, Nezaket Özpolat Çakar*, Naci Özer*, Serdar Deniz*, Mustafa Kemal Başaralı§,
Ahmet Özlu§, Abdulsamet Sandal‖, Gert van der Laan¶,** and Ali Naci Yıldız†† Background: Skin, respiratory, and musculoskeletal diseases in greenhouse workers are frequently observed
due to exposure to plant products and pesticides in enclosed conditions and ergonomic risks. Current
studies on occupational health risks of greenhouse workers in Turkey are insufficient. p
g
yfi
Objectives: The aim of the present study was to assess work-related skin, respiratory, and musculoskel
etal diseases in greenhouse workers in the Erdemli province, Mersin, an area with a workforce predomi
nantly active as greenhouse workers. y
g
Methods: The study population consists of adult greenhouse workers, who visited their family physician
between June 12–14, 2017 and were diagnosed with dermatological, respiratory, and musculoskeletal
diseases. Immediately after this consultation, occupational physicians conducted face-to-face interviews,
asking questions about sociodemographic features and occupational factors including current and previous
work, current exposures and relation of current symptoms with work. Results: In total, 423 workers with 555 diagnoses were included in the study. Percentages of diagnoses
were 30.1%, 21.6% and 48.3%, for skin, respiratory and musculoskeletal diseases respectively. Nearly half
of participants had taken an absence from work due to those diseases. Mean age of onset for greenhouse
working was 15.5 years. Almost all participants (96.2%) reported contact with chemicals, and usage of
respiratory protection was low (17.3%). Pesticides were regarded as a risk factor by nearly two-thirds of
workers with skin or respiratory system disorders. Participants’ answers to questions regarding the rela
tionship between their diseases and their work was positive for more than half of patients and patients
with skin and respiratory diseases. Nearly half of the patients with musculoskeletal diseases attributed
their complaints to physical overload at work. p
p y
Conclusions: We found evidence of work-relatedness in almost half of the 555 greenhouse workers with
dermatological, respiratory and musculoskeletal diseases. These findings are helpful in creating an aware
ness program. There is a need for a more detailed assessment of the cases and the related working condi
tions to start a tailored prevention program. to hazardous chemicals, particularly pesticides [3]. Agri
culture and forestry, as an occupation, consistently ranks
as the third or fourth most hazardous occupations in the
European Union [4]. Agricultural workers suffer from sev
eral conditions and diseases [5]. Introduction The share of the population working in agriculture greatly
varies among countries: while the vast majority of the pop
ulation in low-income countries work in agriculture, this
rate drops to less than 5% in high-income countries [1]. In Turkey, agriculture employs nearly one-fifth (18.9%) of
the registered workforce [2]. The work is generally heavy,
working hours can be very long, workers are exposed to
extreme climatic conditions, and many are also exposed Nuraydın et al: The Mersin Greenhouse Workers Study 505 of pesticides causes at least 7 million cases of acute and
long-term nonfatal illness [8]. occupational physicians for data collection via face-to-face
interview in FHCs. Data collection forms had 23 questions
divided into in two parts, including sociodemographic fea
tures and features related to work including current and
previous work, current exposures, and relation of current
diagnosis with work. Diagnoses obtained from the fam
ily physician visit were listed according to 10th edition
of the International Statistical Classification of Diseases
and Related Health Problems (ICD-10) codes [13], but no
additional examinations were performed due to limited
resources, nor was any treatment recommended by the
researchers. The preliminary study was performed on ten
patients from a different FHC. Greenhouse cultivation, a form of agricultural
production, is commonly preferred among farmers
because it allows farmers to grow vegetables, fruit, flow
ers, rare and exotic plants outside the normal seasons in
areas covered by glass or plastic, regardless of weather con
ditions. The work has many physical, chemical, biological
and ergonomic risks, and those risks result in a variety of
occupational accidents and work-related or occupational
diseases [4, 9]. Although climate conditions can be con
trolled in greenhouse cultivation, the level of occupational
exposures to pesticides and plant products and related
health risks in greenhouse workers are higher than those
of outdoor farm workers due to the enclosed working
conditions. The main hazards of working in greenhouses
include: injury from materials, problems related to work
ing position, chemicals including pesticides, endotoxins,
dusts, and work at height [4, 10]. Dermatitis, musculoskele
tal complaints, respiratory system diseases, and neurological
diseases are the most common diseases among greenhouse
workers [11]. However, the number of studies on health and
safety issues of greenhouse workers is limited. Researchers obtained informed consent from each of the
participants, whose names were not recorded. Results In three FHCs, 487 greenhouse workers over 18 years of
age were diagnosed with skin, respiratory or musculo
skeletal disease in 23 work days; however, 64 workers did
not agree to participate in the study. Some demographic
characteristics of 423 participants were summarized in
Table 1. More than two-thirds (63.3%) of the participants
were between 30–49 years of age and 30 participants
(7.1%) were 60 years of age or older. A total of 74.9% of
participants started to work in greenhouses at 18 years of
age or younger, while 37.5% began such work at 15 years
of age or younger. Of all participants, 57.4% who worked in the current
greenhouses had previously worked in another workplace
(Table 1); 169 people worked at one other workplace,
11 people worked at two and two participants worked
at three different workplaces. Approximately one-fifth of
these workplaces (22.8%) were greenhouses. Other sectors
(n = 152) were outdoor agriculture and livestock (75.0%),
industry (10.5%), service sector (10.5%) and construc
tion sector (3.9%). The mean working duration at previ
ous workplace was 12.1 ± 7.4 years (min = 1, max = 41,
median = 10). There were 60 participants (14.2%) working
in additional jobs (40.0% service sector, 31.6% outdoor
agriculture and livestock, 13.3% greenhouse manufactur
ing, 8.3% industry, 6.6% construction). Introduction After the inter
views, all participants were informed about occupational
diseases and advised to refer to the related department of
the nearest authorized hospital (Mersin University Hospital)
to prepare reference files for final diagnosis of occupational
diseases by SSI. Permissions were obtained from Ministry
of Health Turkish Public Health Institution, Mersin Public
Health Directorate and Erdemli District Governorship, and
the results of the research were presented to those units. Ethical approval was obtained from Hacettepe University
Ethics Committee for Non-Interventional Clinical Studies
(30 May 2017, Report No. 2017/14-846). In Turkey, there are approximately 5 million agricultural
workers, but there was only one recognized case of occu
pational disease in the agricultural work sector between
2012 and 2015, according to official statistics of the Social
Security Institution (SSI) [2, 12]. There are no official sta
tistics of occupational diseases in greenhouse workers. Greenhouse agriculture is common in the southern Turkey
where Mersin is located. The Erdemli province of Mersin
is one of the leading regions of Turkey’s greenhouse pro
duction. The main economic activity in the district is agri
culture, for which greenhouse cultivation is an important
form of production. Enterprises are mostly small-scale
family enterprises. The main products are tomato, cucum
ber, pepper, and banana. Despite the extent of this eco
nomic activity in this region, there is no data about the
frequency occupational diseases of greenhouse workers. This gap in the literature was recently noticed by the occu
pational physicians in the region who are also part of the
current research team. Thus, in this study, we aimed to
assess the greenhouse workers’ perception and evaluation
of occupational risk factors for dermatological, respira
tory, and musculoskeletal diseases. Statistical analyses were performed using the SPSS
software version 24.0. Chi-square test was performed for
categorical comparisons and logistic regression analyses
were used for multiple comparisons. A 5% type-1 error
level was used to infer statistical significance. Materials and Methods The research was conducted between June 12–July 14,
2017 (during 23 work days) in three family health centers
(FHCs), which offer health service to a region with numer
ous greenhouses in the Erdemli province. Nearly 21,000
people are served by nine family physicians working in
those three FHCs. Greenhouse workers over 18 years of
age who were diagnosed with dermatological, respira
tory, and musculoskeletal diseases by family physicians
within the study period and who agreed to participate to
the study were included. After the family physician visit,
patients meeting specified criteria were asked whether
they wanted to participate in the ongoing research. Patients who accepted were directed to one of the six In terms of duties listed in Table 1, three of four partici
pants (75.5%) stated that they performed all of the jobs 506 Nuraydın et al: The Mersin Greenhouse Workers Study Table 1: Demographic and work characteristics of participants (n = 423). Table 1: Demographic and work characteristics of participants (n = 423). Characteristic
Statistics
Age (years), mean ± SD (min-median-max)
41.2 ± 11.2 (18–41–75)
Sex, n (%)
Male
247 (58.4)
Female
176 (41.6)
Business type, n (%)
Family business
355 (83.9)
Business owner
28 (6.6)
Salaried seasonal worker
26 (6.1)
Paid permanent worker
13 (3.1)
Age started to work (years), mean ± SD (min-median-max)
15.5 ± 3.6 (5–15–35)
Total working duration in greenhoouses (months),
mean ± SD (min-median-max)
215.5 ± 116.1 (12–216–732)
Working duration in current greenhouse (months),
mean ± SD (min-median-max)
187.5 ± 111.5 (1-180–480)
Prior working history,
n (%)
Absent
243 (57.4)
Present*
180 (42.6)
Duties performed in
greenhouses, n (%)
Dibbling
413 (97.6)
Weeding
379 (89.6)
Spraying
364 (86.1)
Spinning
411 (97.2)
Fertilization
353 (83.5)
Harvesting
415 (98.1)
Sorting
403 (95.3)
Roofing
323 (76.4)
Occupational health
practice†, n (%)
Contact with chemical substances
405 (96.2)
Using work clothes
293 (69.6)
Using gloves
266 (62.9)
Using work clothes in daily life/at home
192 (45.6)
Using a mask
73 (17.3)
Consuming food and beverage in the
greenhouse
282 (67)
min, minimum; max, maximum; SD, standard deviation. *Duration of prior working history (years), mean ± SD (min-median-max) was 12.1 ± 7.4 (1–10–41). †Two cases were missing (n = 421). With 113 people having more than one diagnosis (94
people with two, and 19 people with three systemic dis
eases), a total of 555 diseases were diagnosed in 423 indi
viduals (Table 2). Materials and Methods *10th revision of the International Statistical Classification of Diseases and Related Health Proble Of the cases referred by family physicians to the Mersin
University Hospital, three were related to musculoskeletal
diseases, one for respiratory system diseases, and one for
skin diseases. The frequency of any workday loss due to illness in past
year was 19.7% (n = 33) for skin diseases, 55.8% (n = 67)
for respiratory diseases, 57.5% (n = 153) for musculoskel
etal diseases, and 45.7% (n = 253) overall. The frequency
of workday loss from respiratory and musculoskeletal
disorders was significantly higher than that from skin dis
ease (p < 0.001). Materials and Methods Durations of the complaints in months
[n (mean ± standard deviation-median)] were 167 (80.05
± 66.19–60) for skin diseases, 120 (112.32 ± 85.0–16) for
respiratory diseases, and 266 (92.39 ± 68.90–72) for mus
culoskeletal diseases. listed. In the analysis of duties according to sex, statisti
cally significant differences (p < 0.0001) were observed
for weeding (63.1%–36.9%), spraying (66.5%–33.5%),
fertilization (68.8%–31.2%), roofing (72.1%–27.9%) and
workers who did all the jobs listed (72.3%–27.7%) (order
of percentages follows as men to women). Some features of participants related to occupational
health practices are given in Table 1. Logistic regression
analysis did not reveal any statistically significant rela
tionship between presence of the dermatological or res
piratory disease and contact with chemical substances,
features related to personal protectors (e.g. usage of spe
cial clothes, gloves, and masks at work or changing clothes
after work), and consuming food or beverages in green
houses (p > 0.05). More than half of the patients with skin or respiratory
diseases answered open-ended questions about their own
beliefs regarding possible occupational factors causing
the disease as pesticides (61.1% and 66.7% respectively). People with musculoskeletal disease most frequently indi
cated heavy lifting (46.6%) and bending (45.1%) as occu
pational risk factors (Table 3). 507 Nuraydın et al: The Mersin Greenhouse Workers Study Table 2: Distribution of the diseases according to ICD-10* codes. Disease
n
%
Skin (n = 167)
L23.9 Allergic contact dermatitis, unspecified cause
125
74.9
L30. 9 Dermatitis, unspecified
11
6.6
L50 Urticaria
9
5.4
L29.9 Pruritus, unspecified
8
4.8
L20.9 Atopic dermatitis, unspecified
6
3.6
R23.8 Other skin changes
5
3.0
R21 Rash and other nonspecific skin eruption
3
1.8
Respiratory
System (n = 120)
R06.0 Dyspnea
68
56.7
J45 Asthma
32
26.7
J30.4 Allergic rhinitis, unspecified
8
6.7
R05 Cough
7
5.8
J40 Bronchitis, not specified as acute or chronic
2
1.7
R07.0 Pain in throat
2
1.7
J44 Other chronic obstructive pulmonary disease
1
0.8
Musculoskeletal
System (n=268)
M54.5 Low back pain
195
72.8
M25.5 Pain in joint
25
9.3
M51.9 Unspecified thoracic, thoracolumbar and
lumbosacral intervertebral disc disorder
22
8.2
M79.1 Myalgia
20
7.5
M79.6 Pain in limb, hand, foot, fingers and toes
4
1.5
M50 Cervical disc disorders
2
0.7
*10th revision of the International Statistical Classification of Diseases and Related Health Problems. Table 2: Distribution of the diseases according to ICD-10* codes. Discussion In the present study, we evaluated 423 greenhouse work
ers with total number of 555 diagnoses of skin, respira
tory, and musculoskeletal diseases. Approximately half
of the diagnoses (48.3%) were musculoskeletal diseases,
30.1% were skin diseases, and 21.6% were respiratory
diseases. Mean age of onset for working in greenhouses
was 15.5 years. Percentages of participants with a previ
ous history of working in a different workplace and with
a current additional job were 42.6% and 14.2% respec
tively. Nearly half of participants had workday loss due to
those diseases. Almost all participants in the survey were
exposed to hazardous chemicals, especially pesticides via
routes of inhalation and skin contact as well as ingestion
with food and beverages consumed in the workplace. Pes
ticides were claimed as an occupational risk factor trig
gering the disease by nearly two-thirds of patients with
skin or respiratory system disorders. More than half of
all patients and patients with skin diseases or patients
with musculoskeletal diseases answered positively to all
five questions about work-relatedness of their disease,
although the number of the cases referred to authorized
university hospital by family physicians was quite few. Analysis of answers of participants to questions asking
the relationship between their complaints or diseases and
their work revealed that more than half of the all patients
and patients with skin diseases or patients with muscu
loskeletal diseases answered positive to all five questions
(50.1%, 50.9%, and 57.1% respectively). Only five people
(1.2%) answered negatively to all questions (Table 4). In
the logistic regression analysis, a positive response to the
occupational relationship for all five problems involving
the work performed by the patient was not associated
with sex, age, previous job, previous occupation, other
job, current disease duration, the number of days in which
the worker did not work in the last year and the working
position (p > 0.05). Compared with the lesser duration of
greenhouse work history, those who worked more than
20 years in greenhouses and compared to those diagnosed
with a respiratory system disease, those diagnosed with a
musculoskeletal system disease were 3.05 times higher
(95% CI: 1.55–6.00, p = 0.001) and 2.43 times (95% CI:
1.30–4.50, p = 0.005) answered as yes to all questions
respectively. Nuraydın et al: The Mersin Greenhouse Workers Study 508 Table 3: Distribution of factors participants’ answers about their own beliefs regarding possible occupational factors
causing the disease. Nuraydın et al: The Mersin Greenhouse Workers Study 509 Nuraydın et al: The Mersin Greenhouse Workers Study A person with an undefined occupational or work-
related disease is expected to seek care from a physician
in the primary health care services [14]. The nature of
work-related or occupational diseases is often not recog
nized when a patient is consulting a general physician. Lack of awareness and lack of taking a proper occupa
tional history generally are the causes of underreporting. Several studies address this problem. In general, one of
the main problems for underdiagnosis of occupational
diseases is the lack of proper occupational history and
other related information [15]. Although some basic ques
tions on whether symptoms arise at work or whether
other colleagues have similar symptoms could be impor
tant clues, occupational history is not usually obtained
at the expected level, due to intensive work conditions. Several studies address this problem. A study performed
in the UK established that 25% of family physicians did
not ask about occupation during their examination [16]. In Turkey, a study showed that 43.9% of the physicians
who provided outpatient care didn’t ask patients’ occupa
tions [17]. Other problems for underdiagnosis of occupa
tional diseases may include unawareness of notification
procedures, need for additional time and effort to com
plete notification procedures, and unavailability of sys
tems promoting notification [18]. Although notification
of occupational diseases is defined by law and reporting
is mandatory in Turkey, notification system has some bar
riers. The occupational or family physicians should direct
the patients with occupational disease to the authorized
hospitals for the preparation of the files. The file, which is
the basis for the recognition of the occupational disease,
is sent to SSI which makes the final decision on suspected
occupational disease. an early age and spend time with their parents in an indoor
greenhouse environment where they are faced with many
unfavorable conditions for every age group. Family mem
bers can spend the day and even sleep in the greenhouse,
especially during the winter months. Moreover, small
farmers live where they work, so workplace exposures all
too easily migrate into the home.Future studies may be
planned to evaluate women’s and children’s health issues
related to greenhouse working. The rates of working in another workplace or having
additional work and the frequency of workplace change
were high in our study. Nuraydın et al: The Mersin Greenhouse Workers Study This phenomenon, which is wide
spread in the agricultural sector, poses a problem for the
implementation and sustainability of occupational health
and safety practices and monitoring efforts. Although the
average daily working time was within the legal limits on
the dates of the research, the workload and the duration
of daily working can change according to the seasonal
and production stage and can exceed the legal limits. Three-quarters of the participants indicated that they did
all kind of work in the greenhouse, although it can be
inferred that most of the participants performed all kinds
of duties and were exposed to all kinds of effects in the
same indoor environment. Lack of statistical significance
in the distribution of diseases according to the work may
be related to the aforementioned issues. Participants reported higher exposure frequencies
than protective device use. Lack of any statistically sig
nificant difference between groups using and not using
personal protective equipment in regards to the presence
of skin or respiratory disease may directly be related to
the characteristics of the equipment used. Two-thirds of
the respondents said they used work clothes, but not all
were work-specific clothes. Similarly, although two-thirds
of participants reported the usage of gloves, the gloves
were mostly inappropriate for the task and of poor quality. Less than one-fifth of participants stated that they used
masks. It is common practice for farmers in developing
countries to apply hazardous pesticides while working
barefoot. The level of skin exposure varies with the fre
quency of pesticide application, the pesticide-active ingre
dient concentration and whether personal protectors or
other protector equipment is being used correctly [3]. In
order to prevent the spread of pesticide-related health
conditions, precautions such as providing workers with
less toxic substances (such as biological control meth
ods [23, 24]) adequate protective equipment, training on
prevention of pesticide exposures, and implementation
of administrative restrictions of working in fields where
exposure may occur, ould be taken [3]. In the present study, greenhouse workers with skin,
respiratory, and musculoskeletal diseases were evaluated
through questions about their work. Although some of
the diseases required additional diagnostic procedures
(e.g. patch tests for allergic contact dermatitis, serial Peak
Expiratory Flow measurements or bronchial challenge
tests for asthma), others like low back pain did not, and
the algorithm for the diagnosis of occupational diseases
began with compatible occupational history [19, 20]. Discussion g
Disease
n
%
Skin (n = 167)
Pesticides
102
61.1
Dust
58
34.7
Hot working environment
27
16.2
Contact with the produced plant
18
10.8
Humidity
9
5.4
Bending work
2
1.2
Chemical fertilizer
2
1.2
Working upwards
2
1.2
Heavy lifting
1
0.6
Respiratory
System (n = 120)
Pesticides
80
66.7
Dust
54
45
Humidity
19
15.8
Hot working environment
18
15
Chemical fertilizer
3
2.5
Heavy lifting
1
0.8
Musculoskeletal
System (n = 268)
Heavy lifting
125
46.6
Bending work
121
45.1
Hot working environment
15
5.6
Working upwards
8
3
Dust
2
0.7
Humidity
2
0.7
Pesticides
2
0.7
Table 4: Distribution of positive answers for questions asking work-relatedness and for all questions about the relation
ship of diseases to work. Table 4: Distribution of positive answers for questions asking work-relatedness and for all questions about the relation
ship of diseases to work. tion of positive answers for questions asking work-relatedness and for all questions about the relation
s to work Table 4: Distribution of positive answers for questions asking work-relatedness and for all questions about the relation
ship of diseases to work. Skin
(n* = 167)
Respiratory
System (n* = 120)
Musculoskeletal
System (n* = 268)
Total
(n† = 423)
Question
n
%
n
%
n
%
n
%
Did disease start after started to work
in the greenhouse? 141
84.4
97
80.8
230
85.8
353
83.5
Does the disease exacerbated as you
work in the greenhouse? 155
92.8
117
97.5
258
96.3
402
95
Are there any others working in the
same workplace have similar disease? 102
61.1
56
46.7
186
69.4
262
61.9
Do your complaints decline when
you do not work in the greenhouse? 152
91
112
93.3
249
92.9
387
91.5
Is there at least one factor in your
workplace causative this disease? 148
88.6
110
91.7
248
92.5
380
89.8
Total number of positive answers
n*
%
n*
%
n*
%
n†
%
5
85
50.9
46
38.3
153
57.1
212
50.1
4
54
32.3
53
44.2
84
31.3
144
34,1
3
14
8.4
12
10
17
6.3
36
8.5
2
4
2.4
6
5
7
2.6
14
3.3
1
7
4.2
2
1.7
5
1.9
12
2.8
–
3
1.8
1
0.8
2
0.7
5
1.2
*Number of diagnoses. †Number of workers. Nuraydın et al: The Mersin Greenhouse Workers Study By analyzing occupational histories of participants, we
obtained clues to diagnose occupational or work-related
diseases. Our results also showed that those diseases cause
workday loss; thus future studies are needed to evaluate
and enhance working conditions of greenhouse workers
and to implement specific diagnostic guidelines. Within the scope of the research, we also obtained data
on working life, working environment and working con
ditions that could affect the health level of greenhouse
employees. Although all participants were adults, the
mean age of onset for working in greenhouses was 15.5
years. Some participants reported much younger starting
ages for greenhouse working, which was part of family
business, despite strict prohibitions of current legislation
[21]. Agricultural work is one of the most common forms
and the most dangerous form of child labor [22]. Most of
the participants were business owners or unpaid family
workers. In the family business, children begin to work at Our study’s strengths include the original study design,
the large study population size, its impact in raising
awareness among stakeholders, and encouragement of
further studies. After the research was completed, basic
occupational health and safety trainings were held, bro
chures and banners about occupational diseases were
delivered. Thanks to the efforts of the Mersin Public
Health Directorate, the frequency of obtaining occupa
tion and work records of family physicians in the region
has increased. A workshop demonstrating good practice Nuraydın et al: The Mersin Greenhouse Workers Study 510 examples was shared with all stakeholders was also held. Additionally, results of current study will be reinforced by
several future intervention studies. 5. Hansen E and Donohoe M. Health issues of migrant
and seasonal farmworkers. Journal of Health Care
for the Poor and Underserved. 2003; 14(2): 153–164. DOI: https://doi.org/10.1353/hpu.2010.0790 A limitation of the study is that the period of collection
of the data, corresponded with both summer months with
38°C in the temperature and the Ramadan month of fast
ing, so the time period reduced the number of referrals
to FHCs compared to other months, despite the absence
of any factors causing a selection bias. Additionally, there
were no records of occupational health and safety and a
lack of information on risk assessments and hazard identi
fication studies and precautionary measures of entry and
periodic examinations, as well as family physicians’ work
load, duration of loss of capacity due to illness, which may
have limited number of referred patients. Competing Interest 13. WHO. International Statistical Classification of Dis
eases and Related Health Problems 10th Revision
Version; 2016. http://apps.who.int/classifications/
icd10/browse/2016/en. Accessed September 12, 2017. The authors have no competing interests to declare. Author Contribution All authors had access to the data and a role in writing the
manuscript. 14. Lax MB, Grant WD, Manetti FA and Klein R. Recognizing occupational disease--taking an effec
tive occupational history. Am Fam Physician. 1998;
58(4): 935–944. Nuraydın et al: The Mersin Greenhouse Workers Study Other limita
tions include usage of diagnoses made by family physicians
without any further standardized verification, inclusion of
participants who were currently at a health care facility far
from workplace, lack of standardized evaluation of work
places, possible increased awareness of participants about
the relationship between work and their diseases due to
family physicians’ awareness about the study. 6. Davis KG and Kotowski SE. Understanding the
ergonomic risk for musculoskeletal disorders in the
United States agricultural sector. American Journal
of Industrial Medicine. 2007; 50(7): 501–511. DOI:
https://doi.org/10.1002/ajim.20479 7. Kim K-H, Kabir E and Jahan SA. Exposure to pes
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Press Release ILO/WHO Number of Work-related
Accidents and Illnesses Continues to Increase ILO
and WHO Join in Call for Prevention Strategies,
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To conclude, factors such as younger starting ages of
working due to family businesses, frequent job changes
due to insecurity, and increased physical risk factors such
as heavy lifting, bending, or extreme temperature or
chemical risk factors like pesticides and chemical fertiliz
ers, demonstrate that greenhouse work is related to a vari
ety of diseases. Results of our study emphasize the need
to objectively evaluate both health conditions and risk fac
tors in greenhouse workplaces and to develop solutions. Our findings are important for awareness raising and ini
tiation of a program for improvement of health and safety
in greenhouse workers in the Mersin area, which may
be implemented later in the entire greenhouse sector in
Turkey. 10. Nordgren TM and Bailey KL. Pulmonary health
effects of agriculture. Current Opinion in Pulmonary
Medicine. 2016; 22(2): 144–149. DOI: https://doi. org/10.1097/MCP.0000000000000247 11. Jurewicz J, Kouimintzis D, Burdorf A, Hanke W,
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https://doi.org/10.1007/s10389-007-0129-x 12. Republic of Turkey Social Security Institution. SGK İstatistik Yıllığı 2016. http://www.sgk.gov.tr/
wps/portal/sgk/tr/kurumsal/istatistik/sgk_istatis
tik_yilliklari. Accessed September 10, 2017. Competing Interest How to cite this article: Nuraydın A, Bilek Ö, Kenziman AK, Ali Korkusuz M, Atagün Aİ, Çakar NÖ, Özer N, Deniz S, Başaralı
MK, Özlü A, Sandal A, van der Laan G and Yıldız AN. The Mersin Greenhouse Workers Study. Surveillance of Work-related Skin,
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Yetiştiriciliğinde Biyolojik Mücadele. 2012; 13–25. Ankara: T.C. Gıda Tarım ve Hayvancılık Bakanlığı. 24. Tuncer C, Mennan S, Akça İ, Saruhan İ and
Akyazı F. Seralarda Zararlılar ile Biyolojik Mücadele. The Journal of Agricultural Facullty of Ondokuz Mayıs
University. 2004; 19(2): 83–89. 21. Republic of Turkey Ministry of Labor and Social
Security. Regulations on the Fundamentals and
Principles of the Employment of Children and Young How to cite this article: Nuraydın A, Bilek Ö, Kenziman AK, Ali Korkusuz M, Atagün Aİ, Çakar NÖ, Özer N, Deniz S, Başaralı
MK, Özlü A, Sandal A, van der Laan G and Yıldız AN. The Mersin Greenhouse Workers Study. References Surveillance of Work-related Skin,
Respiratory, and Musculoskeletal Diseases. Annals of Global Health. 2018; 84(3), pp. 504–511. DOI: https://doi.org/10.29024/
aogh.2315 Published: 31 August 2018 Published: 31 August 2018 Published: 31 August 2018 Copyright: © 2018 The Author(s). This is an open-access article distributed under the terms of the Creative Commons
Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium,
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The use of heat from the CO<sub>2</sub>compression system for production of system heat
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© 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/). The use of heat from the CO2 compression
system for production of system heat
Robert Zarzycki1,*
1Energy Engineering Department, Faculty of Infrastructure and Environment, Czestochowa
University of Technology, Brzeznicka Street 60A, 42-200 Czestochowa, Poland Robert Zarzycki1,*
1Energy Engineering Department, Faculty of Infrastructure and Environment, Czestochowa
University of Technology, Brzeznicka Street 60A, 42-200 Czestochowa, Poland Robert Zarzycki1,*
1Energy Engineering Department, Faculty of Infrastructure and Environment, Czestochowa
University of Technology, Brzeznicka Street 60A, 42-200 Czestochowa, Poland Abstract. The study presents the concept and computations of the CO2
compression for the purposes of transport and underground storage of the
gas. Cooling between individual stages is needed to reduce the power needed
for CO2 compression. Heat obtained from the cooling process can be used
to provide heat to the municipal heat power systems. A design of a heat
accumulator for storage of excess heat was proposed in order to improve
heat supply safety. The solution proposed in the study allows for heating
condensing power units using waste heat from the CO2 cooling process. * Corresponding author: zarzycki@is.pcz.pl E3S Web of Conferences 49, 00135 (2018)
SOLINA 2018 E3S Web of Conferences 49, 00135 (2018)
SOLINA 2018 https://doi.org/10.1051/e3sconf/20184900135 2 Conventional energy generation As mentioned in the Introduction section, processes of combustion of fossil fuels for the
purposes of electricity and heat generation contribute to emissions of various pollutants,
including CO2. All the newly built power plants have to be prepared for construction of
installations of CO2 capture and separation. With the already existing energy installations,
one should take into consideration the increase in unit costs of electricity and heat
generation connected with buying permits for CO2 emissions to the atmosphere or the
necessity to install CO2 capture and separation systems. y
p
p
y
The conventional energy sector can limit CO2 emissions through [8]: • CO2 capture from flue gas. Nowadays, it is possible to use absorption or adsorption
technologies; • CO2 capture from flue gas. Nowadays, it is possible to use absorption or adsorption
technologies; • Oxygen combustion. In this case, flue gas contains 90–95% CO2; • CO2 capture before combustion. Regardless of the method of limitation of CO2 emissions, it is necessary to compress the
gas in order to transport it and store underground. In order to demonstrate the scale of the
problem, one can use the following example of newly built power units. Depending on the
fuel burnt, a power unit with a capacity of 900 MWe emits around 175 kg CO2/s (hard coal),
225 kg CO2/s (brown coal). The major task of power units in industrial power plants is to generate electricity. Depending on the location (power plant near a big city) and local infrastructure (presence
of heat power systems), power units may, besides their main function of electricity
generation, be a source of heat needed for municipal heat power system. In the case of big power objects (with condensing turbines), the opportunities for
generating additional heat is connected with collecting the steam from the turbine outlet and
transfer of the steam to heat exchanger where the water for the network is heated while
steam collected from the turbine outlet is condensed. This solution is popular in steam
power units and allows for generation of the required amount of heat. This system allows
for improving the efficiency of electricity and heat generation. A drawback of this solution
is a decline in electricity generation with the increase in heat generation, related to the
increased stream of steam transferred to the heat exchanger. 1 Introduction In many countries, including Poland, combustion of fossil fuels have been used to generate
electricity and heat. Fossil fuel combustion processes contribute to degradation of the
natural environment and climate changes through emissions of combustion products to the
atmosphere, including: CO2, SO2, NOx, dusts, and heavy metals, including Hg. Modern
technologies of flue gas treatment allow for a substantial limitation of emissions of SO2,
NOx and dusts. Technologies of Hg emissions limitation and other heavy metals have been
developed [1–3]. The problem of CO2 emissions to the atmosphere has not been solved yet. The regulations known as climate package require limitations of CO2 emissions by 20%,
the increase in the use of renewable energy sources to 20% and the increase in energy
efficiency to 20% compared to prognoses for 2020. In the development of renewable
energy sources, it is mainly the energy sector that uses wind force which helps meet the
requirement of 20% energy production from renewable sources. The percentage of
electricity generated from renewable sources in Germany increased from 6.3% in 2000 to
ca. 41% in 2018, which is double the value of the assumed level of 20% [4]. One should
expect further dynamic increase in renewable energy in other countries of the European
Union. The studies have been conducted in recent years to improve efficiency of energy
generation in power plants and combined heat and power plants [5–7]. Ultra-supercritical
power unit technologies are being developed in order to exceed the level of efficiency of 50%. The increase in the use of fossil fuel energy can be achieved by the combined generation of
electricity and heat and construction of gas and steam systems. Limitation of CO2 emissions
to the atmosphere can be achieved by its capture from flue gas, followed by storage [8]. The process of CO2 capture from flue gas can be performed using various technologies: E3S Web of Conferences 49, 00135 (2018)
SOLINA 2018 https://doi.org/10.1051/e3sconf/20184900135 adsorption, absorption, membrane, and cryogenic. Carbon dioxide isolated from flue gas
should be then transported to places where it can be permanently stored. The study presents a proposal for the use of heat from the CO2 combustion process for
the purposes of supplying heat to the steam power unit. Furthermore, the opportunities for
storing the heat using a heat accumulator were also discussed. E3S Web of Conferences 49, 00135 (2018)
SOLINA 2018 E3S Web of Conferences 49, 00135 (2018)
SOLINA 2018 https://doi.org/10.1051/e3sconf/20184900135 Another method to provide heat to the steam power unit is to install an absorption heat
pump which is fuelled by steam from the turbine outlet and allows for using a low-
temperature heat contained in the water that cools the condenser [12]. Compared to
a conventional steam-fuelled heat exchanger, this solution allows electrical power of the
steam power unit to be reduced at similar heat generation. Providing heat to a steam power
unit using an absorption heat pump helps prepare water with maximal temperature of up to
95°C but it requires installation of a peak heat exchanger for ensuring the required
parameters of the network water and heat supply safety. The presented methods to provide heat to condensing steam power units allow first and
foremost to improve the efficiency of the combined electricity and heat generation. However, they require additional investment expenditure for building installations of heat
exchangers or installations of absorption heat pumps. In order to limit costs of these
installations (reduction of installed capacity) and their negative effect on operation of
a steam power unit (reduction in electrical power), it is necessary to store heat generated. Installation of the system of heat storage helps make electricity and heat generation process
independent in the steam power unit. Therefore, it is possible to ensure optimal electricity
and heat generation at higher thermodynamic and economic efficiency. Heat supplies to heat networks and next to receivers require ensuring adequate
parameters (temperature, mass flux) of the heat carrier (water). Parameters of heat network
operation are contained in the regulation tables which are prepared individually for each
heat network and heat consumer. An example of a regulation table is presented in Table 1. From the standpoint of work and installed capacity of the heat source, in addition to the
required temperature of network water, important factors also include durations of
individual values of external temperatures. Fig. 1 presents an example diagram of external
temperatures and the number of hours these temperatures are observed [13]. Table 1. Regulation table [10]. No
Air temp. [oC]
Feed water temp. 2 Conventional energy generation Another method to provide
heat for power unit is to use waste heat contained in flue gas or water that cools the turbine
condenser. The efficiency of power units depends on several factors, such as power, combustion
technology (pulverized coal-fired boiler, fluidized-bed boiler) and parameters of generated
steam or water. However, the biggest effect on energy boiler efficiency is from the fuel and
its water content. The most advanced brown coal-fired power boilers reach the efficiency of
ca. 88%, whereas those hard coal-fired have an efficiency of even 95% [9]. Despite such
high efficiency, the amount of heat contained in flue gas, especially in boilers fired by
brown coal or biomass with high water content is sufficient for heat production [10] or
using it for other purposes, e.g. preparation of flue gas for CO2 separation using adsorption
methods [11]. In this case, the amount of heat generated is proportional to boiler power and,
therefore, the capacity of steam power unit. Using this solution, there is a necessity to
install a peak heat exchanger that allows for reaching required parameters (temperature,
power) of the water in the network. 2 E3S Web of Conferences 49, 00135 (2018)
SOLINA 2018 [oC]
Return water temp [oC]
1
-16
125
66
2
-15
123
65
3
-14
121
64
4
-13
118
63
5
-12
115
62
6
-11
113
61
7
-10
111
60
8
-9
108
59
9
-8
105
58
10
-7
102
57
11
-6
99
56
12
-5
97
55
13
-4
95
54
14
-3
93
53
15
-2
91
52
16
-1
88
51
17
0
86
50
18
1
84
49
19
2
81
48
20
3
79
47
21
4
76
46
22
5
72
45
23
6
69
44
24
7
66
43
25
8
63
42
26
9
59
41
27
10
56
40
28
11
53
39
29
-12
115
62 Table 1. Regulation table [10]. 3 3 E3S Web of Conferences 49, 00135 (2018) E3S Web of Conferences 49, 00135 (2018)
SOLINA 2018 https://doi.org/10.1051/e3sconf/20184900135 SOLINA 2018 a)
0
1000
2000
3000
4000
5000
6000
7000
8000
35
30
25
20
15
10
5
0
-5
-10
-15
-20
Air temperature [
oC]
Time [h]
b)
-16
-14
-12
-10
-8
-6
-4
-2
0
2
4
6
8
10
12
0
50
100
150
200
250
300
350
400
450
Hours of air temperature [h]
Air temperature [
oC]
Fig. 1. The structured graph of outer air temperature for location of a dedicated heat recipient a),
hours of presence of external air temperature during the central heating season b) [13]. b) Fig. 1. The structured graph of outer air temperature for location of a dedicated heat recipient a),
hours of presence of external air temperature during the central heating season b) [13]. 3 Storing heat For economic and constructional
reasons, heat accumulators are unpressurised tanks, and, consequently, maximal
temperature of the water stored cannot exceed 100°C. The upper part of the heat accumulator contains hot water with the temperature of the
heat network feed, whereas the lower part of the tank is filled with water with the return
temperature. There is also a mixture of both types of water between each other, termed
thermocline. The difference between temperature of hot and cold water in unpressurised
tanks ranges from 30–40°C. The phenomenon of thermal stratification of water is
demanded in heat accumulators. In properly designed containers, this phenomenon occurs
independently through gravitational forces (differences in hot water and cold water
densities). For functional reasons, the heat accumulator should be smooth. The optimal
height-to-diameter ratio of heat accumulator ranges from 3 to 4 [15]. The Polish energy
system uses heat accumulators with capacities from 12,000 to over 30,000 m3. The heat
accumulator installed in Elektrociepłownia Siekierki has capacity of 30,400 m3, with tank
diameter of 30 m and height of 47 m. Heat capacity of the accumulator is 1,600 MWh
whereas its heat power is 300 MWt. 3 Storing heat Due to substantial capacities of heat power systems, storage of heat requires the equipment
and installations which allow for storing huge amounts of heat. There is a number of heat
storing methods [14]. However, for technical reasons, the best solution is to store heat in
hot water in heat accumulators. Heat accumulators are mostly built near combined heat and
power units, which allows for separation of electricity and heat generation. A heat accumulator is a tank which accumulates heat contained in hot water. The accumulator ensures smooth operation of the unit between night and day, allowing for
switching the generation to more efficient units and increasing production in co-generation. The heat is collected when the demand for heat is lower and returned when its consumption
increases (in the mornings and evenings). Heat accumulator capacities have to be chosen
based on the analyses of heat demand and opportunities for operation with the generating
unit and heat network and economic analysis connected with investment costs. A heat accumulator is usually an unpressurised water tank included in the heat power
system. During "charging" the accumulator (Fig. 2a), hot water is supplied to the upper part
of the tank, whereas cold water is received from its bottom part. Therefore, the intermediate
layer between the hot and cold water is moved to the lower part of the tank, while the
amount of heat collected in the accumulator is increased (level of water in the tank is
virtually unchanged). The accumulator is discharged (Fig. 2b) by reception of the hot water Fig. 2. Processes of (a) charging and (b) discharging of a heat accumulator. hot water
cold water
thermocline
hot water
cold water
thermocline Fig. 2. Processes of (a) charging and (b) discharging of a heat accumulator. 4 4 E3S Web of Conferences 49, 00135 (2018)
SOLINA 2018 https://doi.org/10.1051/e3sconf/20184900135 from the upper part of the tank and supply of the cooled water to its lower part. A direct
layer is moved towards the upper part of the container. For economic and constructional
reasons, heat accumulators are unpressurised tanks, and, consequently, maximal
temperature of the water stored cannot exceed 100°C. from the upper part of the tank and supply of the cooled water to its lower part. A direct
layer is moved towards the upper part of the container. n – number of compression stages. The most popular compression systems are those with 3 to 6 compression degrees with
cooling between stages, whereas the use of a higher number of compression stages does not
produce substantial thermodynamic effects [16] and significantly improves the costs of
installation and reduces its efficiency. In the case of multi-stage systems, adequate division
of pressures between individual compression stages is critical to minimize compression. It is necessary to conduct multi-dimensional optimization, with optimization criterion being
minimization of power necessary for CO2 stream compression. The CO2 obtained from flue gas during separation has to be compressed to reach the
parameters that allow for its transport in the liquid phase. With the necessity of supplying
the compressed gas to more remote destinations (200 to 300 km), it is critical to obtain high
initial pressure which allows for overcoming CO2 flow resistance in the piping and ensures
minimal pressure in the pipeline which is not lower than 8 MPa. Therefore, for the purposes
of computations presented in the study, the initial CO2 pressure of 15 MPa was adopted,
with temperature of 20°C. Computation of the multi-stage compression with cooling between stages was
performed for the 900 MWe power unit fired with hard coal (emits CO2 around 175 kg/s)
using the dedicated computation software where necessary physical parameters of CO2
were implemented based on the studies [17, 18]. The process of multi-stage CO2 compression is performed near the steam power unit
that generates power necessary for driving the compressors (6 to 8% of the power unit
capacity) [8]. Depending on the parameters, the heat obtained during cooling in inter-stage
coolers can be used in the system for regeneration of water that fuels the steam water, in
ORC cycle, and system heat generation or for the purposes of cold generation. The process of multi-stage compression with cooling between stages was performed for
the systems containing from 4 to 7 compression stages in the 900 MWe. power unit. A diagram of an example structure of a 7-stage compression system with cooling between
steps is presented in Fig. 3. The system is composed of seven compressors denoted with
symbols of C1 to C7, with heat exchangers between each other, marked HE1 to HE7. The HE0 heat exchanger is installed before the first compression stage. The temperature of
the compressed CO2 after each heat exchanger and before the next compression stage is 60°C. 4 Compression of CO2 The CO2 separated from flue gas, with pressure and temperature similar to conditions of the
environment has to be compressed to the pressure of 12–15 MPa and cooled to the
temperature of 10–25°C that ensures its transport in the liquid phase. The process of CO2 compression can be performed by means of positive displacement
compressors and flow compressors. However, due to substantial fluxes of the compressed
CO2, the flow compressors are more often used. The thermodynamic fundamentals of the
multi-step CO2 compression were presented in a study [16]. The work [J/(kg CO2)] of the
polytropic compression which is actually performed is given by (1): (1)
−
⋅
−
⋅
⋅
=
−
1
1
1
0
0
m
m
t
p
p
m
m
T
R
L (1) R – individual gas constant;
T0 – initial suction temperature;
p0 – initial pressure (suction);
p – final pressure (pumping);
m – polytropic exponent, has value from 1.4 to 1. R – individual gas constant; R – individual gas constant; T0 – initial suction temperature; p0 – initial pressure (suction); p – final pressure (pumping); m – polytropic exponent, has value from 1.4 to 1. m – polytropic exponent, has value from 1.4 to 1. A multi-stage compression process with cooling between stages is used to limit the
demand for power for compression of CO2 stream. This solution substantially limits the
power needed to drive compressors and allows for the reduction of their size and helps use
the heat stream obtained in inter-stage coolers in e.g. the system of boiler water
regeneration for heating purposes or supplying heat to a cooler or absorption coolers for
cold generation. The increase in the number of compression stages with cooling between
stages allows for a reduction in the demand for power while reducing gas temperature
following the compression process, which directly impacts maximal temperature of the
cooling medium at the outlet from the inter-stage cooler. 4 Compression of CO2 g
g
The demand for work [J/(kg CO2)] at multi-stage adiabatic compression with n degrees
is described by the relationship (2): 5 E3S Web of Conferences 49, 00135 (2018)
SOLINA 2018 https://doi.org/10.1051/e3sconf/20184900135 (2)
−
⋅
⋅
−
⋅
⋅
=
−
1
1
1
0
0
κ
κ
κ
κ
n
t
p
p
n
T
R
L (2) κ – adiabatic exponent; n – number of compression stages. n – number of compression stages. n – number of compression stages. Adoption of this value results from the assumption of the network water temperature of
55°C for the ambient temperature of -5°C (Table 1). It was assumed that polytropic efficiency for the first compressor is 85% and it is
gradually reduced to 70% in the last compression stage. This assumption was adopted for
all the analysed multi-stage compression systems. 6 https://doi.org/10.1051/e3sconf/20184900135 E3S Web of Conferences 49, 00135 (2018) E3S Web of Conferences 49, 00135 (2018)
SOLINA 2018 SOLINA 2018 Fig. 3. The system structure for seven-stage compression process with cooling between stages. C1
C2
C3
C4
C5
C6
C7
HE0
HE7
HE1
HE2
HE3
HE4
HE5
HE6 Fig. 3. The system structure for seven-stage compression process with cooling between stages. For this complex system, a comprehensive optimization analysis of pressures of inter-
stage compression pi was performed, with its criterion being a minimal demand for
compression power P. The result of the optimization process (minimum power necessary to
carry out the compression of CO2 process, mass flow equal to 175 kg/s) are optimal inter-
stage pressure values pi opt. Details of the optimization procedure can be found in [16]. opt
i
i
p
p
f
P
)}
(
min{
=
(3) (3) The results of computations for four computational cases for complete optimization,
from seven-stage compression to four-stage compression are presented in Figs. 4 to 8. The results of computations for four computational cases for complete optimization,
from seven-stage compression to four-stage compression are presented in Figs. 4 to 8. Fig. 4 presents the values of inter-stage pressures for four analysed cases. Figure 5
presents values of CO2 temperature following the compression stages for the four analysed
cases. It can be observed that for the system composed of four compression degrees,
maximal gas temperature exceeds the level of 190°C, and reduces following individual
stages, eventually reaching 150°C following the fourth compression stage. Analysis of the
results for greater number of compression stages (5, 6, 7) reveals a reduction in CO2
temperatures after compression. In the case of seven compression degrees, CO2 temperature
following the last stage is ca. 95°C. Fig. 6 presents the values of heat flux which should be
received in coolers between stages from the compressed CO2 in order to reduce its
temperature to 60°C. Figure 7 presents the values of compression power at individual
stages depending on the number of compression stages. n – number of compression stages. It can be observed that the increase
in the number of compression stages reduces the power of individual stages. Fig. 8
illustrates the demand for power for CO2 compression and total heat flux which should be
received in the process of CO2 cooling. It is noticeable that the increase in the number of
compression stages leads to the reduction in the demand for power and heat flux from the
process of CO2 cooling. Assuming the temperature in the heat accumulator at the level of 97°C, it is possible to
use heat from systems composed of 4, 5 or 6 compression stages due to the temperature of
CO2 after the compression process following individual stages (Fig. 5). From the standpoint
of the demand for power for driving compressors, the best solution is the system composed
of 6 compression stages. The demand for power for compression is ca. 65.6 MW, whereas
heat flux from CO2 cooling is ca. 98.8 MW. The heat obtained from the compression
process can be directly used in the heat network or be stored in the heat accumulator. Assuming the operation of the power unit at maximal power output for 16 hours and
minimal power output (40%) for 8 hours over the day, it is necessary to collect 6,829 GJ
(1897 MWh) from the compressed CO2 for a system with 6 compression stages. In order to
store this amount of heat in the heat accumulator, the required capacity of 41,000 m3 of
water is needed assuming the difference between water temperatures (of hot and cold
water) of 40°C. Due to the variable demand for heat in the heating season and opportunity 7 E3S Web of Conferences 49, 00135 (2018) E3S Web of Conferences 49, 00135 (2018)
SOLINA 2018 https://doi.org/10.1051/e3sconf/20184900135 of storing heat from the CO2 cooling process, it is advisable to increase the accumulator
capacity to ca. 50,000 m3. 0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Pressure [MPa]
Compression stage number [-]
number of compression stages
4
5
6
7
Fig. 4. Distributions of pressure between stages for the analysed four structures of the compression system. Pressure [MPa] Compression stage number [-] . 4. Distributions of pressure between stages for the analysed four structures of the compression system. The research was financed by grants No.BS/PB-404-301/11. n – number of compression stages. 0
1
2
3
4
5
6
7
90
100
110
120
130
140
150
160
170
180
190
CO2 temperature after compression [
oC]
Compression stage number [-]
number of compression stages
4
5
6
7
Fig. 5. Distributions of temperatures between stages for the analysed four structures of the
compression system. Fig. 5. Distributions of temperatures between stages for the analysed four structures of the
compression system. 0
1
2
3
4
5
6
7
12
14
16
18
20
22
24
26
28
30
32
34
36
Heat [MW]
Compression stage number [-]
number of compression stages
4
5
6
7
Fig. 6. Values of CO2 cooling at individual stages depending on the number of compression stages. Fig. 6. Values of CO2 cooling at individual stages depending on the number of compression stages 8 8 E3S Web of Conferences 49, 00135 (2018)
SOLINA 2018 https://doi.org/10.1051/e3sconf/20184900135 0
1
2
3
4
5
6
7
2
4
6
8
10
12
14
16
18
20
22
Power [MW]
Compression stage number [-]
number of compression stages
4
5
6
7
Fig. 7. Values of compression power at individual stages depending on the number of compression stages. Fig. 7. Values of compression power at individual stages depending on the number of compression stages. 4
5
6
7
65
70
75
80
85
90
95
100
105
Heat, power [MW]
Number of compression stages [-]
Heat
Power
Fig. 8. Demand for power for compression, total heat flux from CO2 cooling between steps. Fig. 8. Demand for power for compression, total heat flux from CO2 cooling between steps. 5 Conclusion The computations of the CO2 compression process for the purposes of transport and
underground storage presented in this study demonstrated the opportunities for the use of
heat from the CO2 cooling system for the municipal heat network. The choice of optimal
compression system should take into consideration the parameters of heat network
operation, temperatures and opportunities for storing heat and minimal power needed for
the CO2 compression system. The system composed of 6 compression stages seems to be
an optimal solution for the analysed case. It allows for reaching the heat power at the level
of 98.8 MWt. Assuming variability of the load to the steam power unit, it is possible to
reach daily heat generation from the compression system of 6829 GJ. In order to store this
amount of heat it is necessary to use a heat accumulator with a water capacity of at least
41,000 m3. If power unit had to be performed, the process of CO2 capture and compression for the
purposes of transport and storage, this solution would allow for the use of waste heat from
the compression process for heating the condensing steam power units. 9 9 https://doi.org/10.1051/e3sconf/20184900135 E3S Web of Conferences 49, 00135 (2018)
SOLINA 2018 References 1. R. Zarzycki, M. Wichliński, Energ. Policy Journal, 19(4), 75–86 (2016) 2. R. Zarzycki, M. Wichliński, Energ. Policy Journal, 17(4), 303–316 (2014) 3. R. Zarzycki, M. Wichliński, (Cyclone furnance as a way for mercury removal from
lignite, ICCHMT 2018, Cracow, Poland, 21-24 May 2018) 4. https://www.energy-charts.de/energy_pie.htm?year=2018 (access 04. 2018) 5. W. Nowak, T. Czakiert, (Spalanie tlenowe dla kotłów pyłowych i fluidalnych zintegrowanych
z wychwytywaniem CO2. Wydawnictwo Politechniki Częstochowskiej, 2012) 6. W. Nowak, W. Rybak, T. Czakiert, (Spalanie tlenowe dla kotłów pyłowych i fluidalnych
zintegrowanych z wychwytywaniem CO2. Kinetyka i mechanizm spalania tlenowego oraz
wychwytu CO2. Wydawnictwo Politechniki 2013) 7. W. Nowak, M. Chorowski, T. Czakiert, (Spalanie tlenowe dla kotłów pyłowych i fluidalnych
zintegrowanych z wychwytywaniem CO2. Produkcja tlenu na potrzeby spalania tlenowego. Wydawnictwo Politechniki Częstochowskiej, 2014) 8. T. Chmielniak, H. Łukowicz, (Modelowanie i optymalizacja węglowych bloków
energetycznych z wychwytem CO2. Wydawnictwo Politechniki Śląskiej, 2015) 9. www.rafako.com.pl (access 04.2018) 10. R. Zarzycki, M. Panowski, (The use of waste heat in the process of combined
generation of heat and electricity, ICCHMT 2018, Cracow, Poland, 21-24 May 2018) R. Zarzycki, M. Panowski, Energy Res. Technol. 140 (3), 032008-1–032008-7, (2018) 11. R. Zarzycki, M. Panowski, Energy Res. Technol. 140 (3), 032008-1–032008-7, (2018)
12 R
Zarzycki
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(Waste heat utilisation for cogeneration of energy 11. R. Zarzycki, M. Panowski, Energy Res. Technol. 140 (3), 032008-1–032008-7, (2018)
12. R. Zarzycki, M. Panowski, (Waste heat utilisation for cogeneration of energy,
MECHANIK 3 (2017) 12. R. Zarzycki, M. Panowski, (Waste heat utilisation for cogeneration of energy,
MECHANIK, 3, (2017) 13. R. Zarzycki, M. Panowski, P. Komur, Energ. Policy Journal, 17, 375–389 (2014) 14. I. Sarbu, C. Sebarchievici, (A Comprehensive Review of Thermal Energy Storage,
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13-15 września 2004) 16. M. Panowski, R. Zarzycki, Energ. Policy Journal, 16(4), 243–256 (2013) 17. R. Span, W. Wagner, J. Phys. Chem. Ref. Data, 25(6), (1996) 18. V. Vesovic, W. Wakeham, G. Olchowy, J. Sengers, J. Watson, J. Millat, J. Phys. Chem. Ref. Data, 19(3), (1990) 10
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English
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Enhancing the corrosion resistance of reinforcing steel under aggressive operational conditions using behentrimonium chloride
|
Scientific reports
| 2,019
|
cc-by
| 9,333
|
Enhancing the corrosion resistance
of reinforcing steel under
aggressive operational conditions
using behentrimonium chloride A. Bahgat Radwan1, Mostafa H. Sliem1, Noor S. Yusuf1, Nasser A. Alnuaimi1,2 &
Aboubakr M. Abdullah1* A. Bahgat Radwan1, Mostafa H. Sliem1, Noor S. Yusuf1, Nasser A. Alnuaimi1,2 &
Aboubakr M Abdullah1* Aggressive operational conditions e.g. saline media and acidic gases, e.g., CO2 can increase the
corrosion rate of reinforcing steel. Accordingly, the necessity to protect the steel under the above
conditions without affecting the mechanical properties of the concrete is growing. Herein, the inhibition
efficiency of a new corrosion inhibitor, behentrimonium chloride (BTC, C25H54ClN), is explored in a
simulated-concrete pore solution (SCP) with 3.5 wt.% NaCl at different pH using electrochemical
impedance spectroscopy (EIS) and polarization methods. Using only a 50 μmol L−1 of BTC, we are able
to measure an inhibition efficiency of 91, 79, and 71% in SCP solution with 3.5% NaCl at pH of 12.5,
10 and 7, respectively without showing any effect on the mechanical properties on the cured mortars. Temkin isotherm is used to describe the physisorption of BTC inhibitor on the steel surface. Also, the
adsorption and influence of the inhibitor on the metal surface are characterized using the scanning
electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. In conclusion,
this new inhibitor shows high corrosion inhibition efficiencies under different aggressive conditions
and can be used in concrete to reduce the corrosion rate of reinforcing steel without decreasing the
mechanical properties of the concrete. Corrosion mitigation has attained a vast interest due to the high economic impact of replacing the damaged parts
with new ones especially in reinforced concrete structures1. It is well-known that once corrosion starts in reinforc-
ing steel, the rust (corrosion product) occupies two to three times more volume than the un-corroded steel. This
higher volume induces pressures around the reinforcing bar and causes cracking of the surrounding concrete. In general, a concrete solution has an alkaline nature (pH ~ 13) owing to the existence of sodium oxide
(Na2O), and potassium oxide (K2O) in addition to calcium hydroxide as a result of the hydration reaction of cal-
cium silicate hydrate in cement (CSH) with water from the surrounding environment2–7. Accordingly, an oxide
layer is existing on the reinforcing steel surface within concrete8–11. However, penetrations of aggressive anions
like chloride (Cl−) and sulfate (SO4
2−) ions can lead to localized damage of the passive film which increases the
corrosion rate of steel1,8,10. www.nature.com/scientificreports www.nature.com/scientificreports 1Center for Advanced Materials, Qatar University, Doha, P.O. Box 2713, Qatar. 2Department of Civil and Architectural
Engineering, Qatar University, Doha, P.O. Box 2713, Qatar. *email: bakr@qu.edu.qa Experimentalh p
The reinforcing steel samples were abraded by silicon carbide grit papers using a grinding machine (Jean Wirts
TG 200, Germany), sonicated with acetone, rinsed by deionized water and after that desiccate in air. The mild
steel rebar contains (wt.%) C = 0.128, Si = 0.25, Mn = 0.7, Cu = 0.15, P = 0.04, S = 0.03, and rest was Fe. Saturated
calcium hydroxide (Ca(OH)2), were used as an electrolyte to mimic SCP in 3.5 wt.% NaCl. NaCl was purchased
from Sigma Aldrich and Ca(OH)2 from Riedel-de Haën. The pH of the SCP solution under investigation was 12.5
for the saturated Ca(OH)2, 10 or 7. The pH was reduced by addition of NaHCO3 powder to the SCP28. The electro-
chemical measurements were done at ambient temperatures using a GAMRY 3000 potentiostat/galvanostat/ZRA
(Warminster, PA, USA). EIS measurements were investigated in a frequency range of 100 kHz to 0.01 Hz with an
AC amplitude of 5 mV. In all electrochemical measurements, a saturated calomel electrode (SCE) and a graphite
rod were employed as a reference and counter electrodes, respectively. The mild steel samples with surface areas of
0.765 cm2 were subjected to the SCP. All the mild steel coupons were sited under open circuit (OCP), conditions
for 30 min before initiating any electrochemical test to attain the steady-state conditions. Polarization curves
were attained from −0.25 to + 0.25 V against the open circuit potential (OCP), with a scan rate of 0.167 mV s−1. Various concentrations (0, 2.5, 5, 10, and 20 ppm) of BTC (molecular weight = 404.164 g mol−1), which are equiv-
alent to 12, 24, 37 and 50 μmol L−1, respectively, were synthesized in the simulated saline SCP solutions. The BTC
inhibitor was attained from Shanghai Dejun Chemical Technology Co., Ltd, Shanghai China, and its chemical
formula is displayed in Fig. 1. Each electrochemical measurement was repeated three times to confirm the repro-
ducibility, and the average values were reported. Mechanical characterization. The effect of BTC inhibitor on the compressive and flexural strength of the
mortars prepared according to the ASTM C109/C109M and ASTM C348, respectively, was evaluated after differ-
ent exposure times in the existence and absence of the BTC corrosion inhibitor29,30. The compressive experiments
were performed using a 300 KN Tecnotest 3 compression testing machine (Tecnotest, Modena, Italy). The flexural
strength experiments were utilized by a Lloyd LR 50 K universal testing machine (Ametec Inc. USA). Enhancing the corrosion resistance
of reinforcing steel under
aggressive operational conditions
using behentrimonium chloride Anitha et al.23, used the extract of rosa damascene leaves as nature-friendly inhibitor
in SCP which achieved an IE% of 82. Wang et al.24, found that using 0.0008 mol L−1 of calcium lignosulfonate
(CLS) showed a high IE% of 93.7 after immersion of carbon steel in SCP for 7200 h in comparsion to sodium
oleate (SO), that exhibited an IE% of 40–60. Cao et al.25 explored the inhibition behaiour of phytic acid in car-
bonated concrete pore solution containing 0.6 mol L−1 NaCl on 20SiMn steel, which displayed an IE% of 84.0 after
immersion for 72 h. Asaad et al.26, prepared non- poisonous corrosion inhibitor of silver nano-particles doped
palm oil leaf extracts for renforcing steel in salin water. It was found that the addition of silver nanoparticles in
the green inhibitor lead to increase the IE% to 94.7 after immersion for 365 d, owing to the presence of excess
calcium silicate hydrate and the enhancement of the pore construction and therefore decrease the conductivity
of the pore solution. p
In this work, the effectiveness of a new inhibitor (behentrimonium chloride, C25H54ClN) for the cor-
rosion of reinforcing steel in highly saline SPCs at ambient temperature and different pH values is explored. Behentrimonium chloride (BTC) is commonly used in hundreds of personal care products as conditioning and
anti-static agents. Interestingly, Cameron et al.27, found that BTC is biologically safe for humans when used in
a concentration range up to 5%. However, the European Union recently restricted its use for more than ≥ 1%. Consequently, we, for the first time, report the use of BTC as a corrosion inhibitor for reinforcing steel in saline
SPC solutions of different pH values at significantly low concentrations of 2.5, 5, 10, and 20 ppm using electro-
chemical and surface analysis techniques. Enhancing the corrosion resistance
of reinforcing steel under
aggressive operational conditions
using behentrimonium chloride On
the other hand, the chemi-physisorped polymethacrylic acid co-acrylamide corrosion inhibitor displayed an IE%
of 92.35 in SCP containing 1.8 wt.% chlorides21. Shanmugapriya et al.22, achieved an IE% of 98 in SCP using an
aqueous extract of turmeric. Anitha et al.23, used the extract of rosa damascene leaves as nature-friendly inhibitor
in SCP which achieved an IE% of 82. Wang et al.24, found that using 0.0008 mol L−1 of calcium lignosulfonate
(CLS) showed a high IE% of 93.7 after immersion of carbon steel in SCP for 7200 h in comparsion to sodium
oleate (SO), that exhibited an IE% of 40–60. Cao et al.25 explored the inhibition behaiour of phytic acid in car-
bonated concrete pore solution containing 0.6 mol L−1 NaCl on 20SiMn steel, which displayed an IE% of 84.0 after
immersion for 72 h. Asaad et al.26, prepared non- poisonous corrosion inhibitor of silver nano-particles doped
palm oil leaf extracts for renforcing steel in salin water. It was found that the addition of silver nanoparticles in
the green inhibitor lead to increase the IE% to 94.7 after immersion for 365 d, owing to the presence of excess
calcium silicate hydrate and the enhancement of the pore construction and therefore decrease the conductivity
of the pore solution. et al.10,15, highlighted the influence of benzotriazole (C6H5N3) and its derivatives, 5-nitrobenzotriazole (C6H4N4O2)
and 5-chlorobenzotriazole (C6H4ClN3), on reinforcing steel inhibition in an SCP solution containing 1 M NaCl. It was found that the IE% of the explored inhibitors dwindled accordingly: 5-chlorobenzotriazole ˃ benzotriazole
˃ 5-nitrobenzotriazole. The maximum attained IE% was 69% in the presence of 5×10−4 M 5-chlorobenzotriazole. Interestingly, 0.0025% of deoxyribonucleic acid (DNA), showed IE% of 94% in SCP solution containing 3.5 wt. % NaCl with an increase of 3.61% in the compressive strength (Fc), after 28 days18. Zhang et al.19, achieved an
inhibition efficiency of 83.15% using maize gluten meal extract as an ecologically friendly inhibitor for reinforcing
steel in SPC containing 3.5 wt.%NaCl. The synthesized inhibitor of 4-(1-(4-methoxyphenyl) cyclohexyl)phenyl
9-oxodecanoate (MPOD) by Unnisa et al.20, exhibited and IE% of 71.81 in SCP solution including 0.5 M NaCl. On
the other hand, the chemi-physisorped polymethacrylic acid co-acrylamide corrosion inhibitor displayed an IE%
of 92.35 in SCP containing 1.8 wt.% chlorides21. Shanmugapriya et al.22, achieved an IE% of 98 in SCP using an
aqueous extract of turmeric. Enhancing the corrosion resistance
of reinforcing steel under
aggressive operational conditions
using behentrimonium chloride A corrosion current density (icorr), of around 0.2 μA cm−2, indicates active corrosion12,
0.1 μA cm−2, is safe for typical design life requirements of reinforced concrete structures13, while icorr, less than
0.01 μA cm−2, is low enough to avoid corrosion-induced cracking indefinitely14. Consequently, the inhibitors to
be used in simulated concrete pore solution should satisfy two conditions; (i) a high inhibition efficiency in the
existence of destructive ions, e.g. Cl− ions, at different pH values (from 7 to 12.5) and (ii) no influence on the
mechanical attributes of the concrete8,15,16. Abd El Haleem et al.9, used different inorganic inhibitors in saturated
calcium hydroxide. The outcomes pointed out that the inhibition efficiency (IE%), of the inhibitors, improved
in the following order MoO4
−2 > WO4
−2 > HPO4
−2 > CrO4
−2. However, the disadvantages of using inorganic
inhibitors in concrete environments are their toxicity to living beings, cost, and inefficiency for localized corro-
sion8,17. Ormellese et al.1, has studied the long-term inhibition effectiveness of over 80 organic compounds from
three main categories: amines and alkanoamines, amino alcohols, and carboxylates in SCP solution containing
0.01 M NaOH at pHΣ12.6 in the absence of chloride ions. The results showed an increase in the effectiveness of
the inhibitors in the following order: carboxylates > amino acids > amines and alkanolamines. Abd El Haleem Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports/ Figure 1. The chemical structure of behentrimonium chloride surfactant used as a corrosion inhibitor. Figure 1. The chemical structure of behentrimonium chloride surfactant used as a corrosion inhibitor. et al.10,15, highlighted the influence of benzotriazole (C6H5N3) and its derivatives, 5-nitrobenzotriazole (C6H4N4O2)
and 5-chlorobenzotriazole (C6H4ClN3), on reinforcing steel inhibition in an SCP solution containing 1 M NaCl. It was found that the IE% of the explored inhibitors dwindled accordingly: 5-chlorobenzotriazole ˃ benzotriazole
˃ 5-nitrobenzotriazole. The maximum attained IE% was 69% in the presence of 5×10−4 M 5-chlorobenzotriazole. Interestingly, 0.0025% of deoxyribonucleic acid (DNA), showed IE% of 94% in SCP solution containing 3.5 wt. % NaCl with an increase of 3.61% in the compressive strength (Fc), after 28 days18. Zhang et al.19, achieved an
inhibition efficiency of 83.15% using maize gluten meal extract as an ecologically friendly inhibitor for reinforcing
steel in SPC containing 3.5 wt.%NaCl. The synthesized inhibitor of 4-(1-(4-methoxyphenyl) cyclohexyl)phenyl
9-oxodecanoate (MPOD) by Unnisa et al.20, exhibited and IE% of 71.81 in SCP solution including 0.5 M NaCl. Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y Results and Discussion EIS. Figures 2 and 3 display the Bode and Nyquist plots, respectively, for the reinforcing steel in SCP solutions
containing 3.5 wt.%NaCl and BTC inhibitor concentrations of 12, 24, 37, 50 μmol L−1 at pH values of 12.5, 10 and
7 within a frequency range from 0.01 Hz to 100 k Hz at an Ac amplitude of 5 mV. Figure 4 exhibits the equivalent
circuit (EC) utilized to fit the measured EIS data to obtain the different parameters that explain the metal/solution
interface. The parameters are listed in Table 1 in which Rs and Rct, are credited to the electrolyte resistance and
the charge transfer resistance, respectively. However, the constant phase element is expressed by (CPE), which is
used for a non-ideal double layer. The imperfectness behavior of the double layer is accredited to the following
parameters (i) a non-uniform surface coverage, (ii) surface roughness, and (iii) nonuniform current distribution
or corrosion rate.h The admittance and impedance of the CPE is given by34,35: The admittance and impedance of the CPE is given by34,35: ω
=
Z
Y
1/
(j )
CPE
p
n ω
=
Z
Y
1/
(j )
(1)
CPE
p
n (1) where ZCPE is the CPE impedance (Ω cm−2); Yp is the numerical value of the admittance 1/│Z│, at ω = 1 (rad s−1)
and j2 = −1. ω is the angular frequency and n is the deviation element which varies from 0 and 1. When n = 1 or
0, ZCPE is corresponding to an ideal capacitor or resistor, respectively.hli The influence of the thickness and dielectric constant of the double layer is defined by the Helmholtz regime
iven by the following formula: εε
δ
=
C
A
(2)
dl
o εε
δ
=
C
A
dl
o (2) where Ɛo and Ɛ are the dielectric constant of air and electrolyte (mainly water), respectively and A is the surface
rea of the working electrode.hfi g
The inhibition efficiency (IE%), is calculated using Eq. 3, g
The inhibition efficiency (IE%), is calculated using Eq. Experimentalh The results
are obtained by averaging three repeated tests. The mortar with 50 µmol L−1 of BTC was prepared by the mechan-
ical mixing, in a stainless steel mixer, one part mass of cement and one and a half part mass of standard sand, with
a water/cement ratio of 0.48531. Then, the mold was filled with the mixture under vibration to release air bubbles,
and thereafter stored in a moist atmosphere for 2 days. After that, the demolding of the prepared specimens was
conducted, and the samples were kept under tap water over the test period32. The cured mortar samples were
removed from the water and located in a drying oven at 60 °C for 24 h before the strength test in order to shun the
impact of the hydration of the concrete and to increase the strength of the measured samples33. Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports/ Figure 2. (a) Bode spectra for reinforcing steel in 3.5 wt.%NaCl at ambient temperature using variable
concentrations of BTC inhibitor (12, 24, 37, 50 μmol L−1), at variable pH values of (a) 12.5, (b) 10 and (c) 7. Figure 2. (a) Bode spectra for reinforcing steel in 3.5 wt.%NaCl at ambient temperature using variable
concentrations of BTC inhibitor (12, 24, 37, 50 μmol L−1), at variable pH values of (a) 12.5, (b) 10 and (c) 7. Results and Discussion 3, =
−
×
IE
R
R
R
%
100
(3)
ct1
ct2
ct1 (3) Table 1 exhibits that the higher the corrosion inhibitor concertation is, the higher the Rct and lower Cdl values
are which indicates that the ability of Cl− ions to attack the reinforcing steel surface declines due to the presence
of a protective adsorbed layer of BTC inhibitor. It is worth to mention that lowering the pH lead to alleviat-
ing the IE% from 91% at pH=12.5 to 79% and 72% at pH 10 and 7, respectively. Three reasons can justify the
chloride-induced loss of passivity of the reinforcing steel. First, an induced de-passivation owing to adsorption of
Cl− ion on the passive film at potential values higher than a critical value. Second, the penetration of Cl− ions into
the oxide layer leading to the formation of chloride-contaminated oxides. Finally, a mechanical film breakdown Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports
cientificreports/
Figure 3. (a) Nyquist plots for reinforcing steel in SCP solution with 3.5 wt.%NaCl at ambient temperature
using variable concentrations of BTC (12, 24, 37, 50 μmol L−1), at variable pH values of (a) 12.5, (b) 10 and (c) 7. www.nature.com/scientificreports/ Figure 3. (a) Nyquist plots for reinforcing steel in SCP solution with 3.5 wt.%NaCl at ambient temperature
using variable concentrations of BTC (12, 24, 37, 50 μmol L−1), at variable pH values of (a) 12.5, (b) 10 and (c) 7. Figure 4. Equivalent circuit utilized to fit the EIS data for reinforcing steel in SCP containing 3.5 wt. % NaCl at
variable pH. igure 4. Equivalent circuit utilized to fit the EIS data for reinforcing steel in SCP containing 3.5 wt. % NaCl at
ariable pH. due to Cl− ions adsorption which can attenuate the surface tension, thus leading to a localized disturbance in the
mechanical stability of the passive layer36.h due to Cl− ions adsorption which can attenuate the surface tension, thus leading to a localized disturbance in the
mechanical stability of the passive layer36.h y
p
y
The polarization curves for the reinforced steel in SCP solutions containing 3.5 wt.%NaCl and BTC inhib
concentrations of 12, 24, 37, 50 μmol L−1 at pH values of 12.5, 10 and 7 are shown in Fig. Results and Discussion 5.h y
p
y
The polarization curves for the reinforced steel in SCP solutions containing 3.5 wt.%NaCl and BTC inhibitor
oncentrations of 12, 24, 37, 50 μmol L−1 at pH values of 12.5, 10 and 7 are shown in Fig. 5.h The electrochemical corrosion factors such as as the corrosion free potential (Ecorr), pitting potential (Epit), cor-
rosion current density (icorr), the polarization resistance, (Rp), cathodic and anodic Tafel slopes (bc and ba, respec-
tively), the corrosion inhibition efficiency (IE%) and the surface coverage area (θ), are calculated from Fig. 5, and
recorded in Table 2. Additionally, the passive potential window is calculated using the following formula: Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports/ Inhibitor
concertation
(µmol L−1)
Rct,
Ω cm2
Y × 10−6
sn ohm−1
cm−2
n
Cdl, × 10−5 F
Inhibition
efficiency
(IE%)
Surface
coverage
(ϴ)
Goodness
of fit (χ2)
pH = 7
Blank
377 ± 5.3
96 ± 22
0.88 ± 0.02
6.1
—
—
2.1 × 10−5
12
461 ± 42
78 ± 10
0.84 ± 0.11
4.1
18
0.18
1.5 × 10−5
24
637 ± 52
70 ± 12
0.81 ± 0.08
3.4
41
0.41
3.4 × 10−6
37
924 ± 28
66 ± 6
0.78 ± 0.12
3.1
59
0.59
5.2 × 10−5
50
1362 ± 100
59 ± 8
0.75 ± 0.21
2.5
72
0.72
1.8 × 10−5
pH = 10
Blank
643 ± 18
89 ± 11
0.85 ± 0.02
5.3
—
—
6.5 × 10−6
12
863 ± 24
74 ± 15
0.81 ± 0.09
3.8
25
0.25
4.2 × 10−5
24
1349 ± 31
61 ± 17
0.80 ± 0.03
3.2
52
0.52
1.1 × 10−4
37
1953 ± 52
50 ± 13
0.79 ± 0.05
2.7
67
0.67
2.4 × 10−6
50
3105 ± 120
44 ± 28
0.72 ± 0.12
2.1
79
0.79
1.1 × 10−5
pH = 12.5
Blank
960 ± 66
80 ± 32
0.82 ± 0.06
4.5
—
—
8.1 × 10−5
12
2678 ± 133
64 ± 9
0.75 ± 0.13
3.5
64
0.64
7.7 × 10−6
24
5009 ± 149
49 ± 15
0.72 ± 0.09
2.8
80
0.80
3.6 × 10−5
37
7680 ± 352
39 ± 10
0.66 ± 0.17
2.1
87
0.87
8.1 × 10−6
50
10526 ± 420
28 ± 15
0.56 ± 0.27
1.1
91
0.91
1.9 × 10−5
Table 1. Results and Discussion The electrochemical factor derived from polarization plots of the reinforcing steel in SCP solutions
including 3.5 wt.%NaCl and BTC inhibitor concentrations of 12, 24, 37, 50 μmol L−1 at pH values of 12.5, 10 and 7 Table 2. The electrochemical factor derived from polarization plots of the reinforcing steel in SCP solutions
including 3.5 wt.%NaCl and BTC inhibitor concentrations of 12, 24, 37, 50 μmol L−1 at pH values of 12.5, 10 and 7. Figure 6. Temkin adsorption plots for reinforcing steel in SCP solution conatining 3.5 wt.% NaCl in the
existence of 12, 24, 37, 50 μmol L−1 of BTC corrosion inhibitor at variable pH values of 12.5, 10, and 7. Figure 6. Temkin adsorption plots for reinforcing steel in SCP solution conatining 3.5 wt.% NaCl in the
existence of 12, 24, 37, 50 μmol L−1 of BTC corrosion inhibitor at variable pH values of 12.5, 10, and 7. =
−
×
IE
i
i
i
%
100
(5)
1
2
1 (5) where i1 and i2 are the corrosion current densities of reinforcing steel in the absence and existence of the BTC
corrosion inhibitor, respectively.h where i1 and i2 are the corrosion current densities of reinforcing steel in the absence and existence of the BTC
corrosion inhibitor, respectively. Th
f
(θ) i
l
l t d tili i
E
611 y
The surface coverage area (θ) is calculated utilizing Eq. 6 θ = IE%
100
(6) (6) and the polarization resistance (Rp), was detected using the Stern–Geary equation37. and the polarization resistance (Rp), was detected using the Stern–Geary equation37. =
. +
R
b b
i
b
b
2 303
(
)
(7)
p
c
a
corr
c
a (7) The polarization curves show the breakdown of the passive film before and after the addition of BTC inhibitor
at pH 12.5, see Table 2. However, increasing the inhibitor concentration shifts the pitting potentials (Epit), towards
the more noble values indicating that the passive layer formed more stabilized by the presence of BTC inhibitor. Moreover, the passive potential window at pH 12.5 is 0.18, which increased to 0.25, 0.27, 0.32 and 0.38 by the
addition of 12, 24, 37, 50 μmol L−1 of the BTC inhibitor, respectively. Results and Discussion Electrochemical elemen ts attained from the EIS spectra of the reinforcing steel in SCP solution with
3.5 wt.% NaCl in the existence of 12, 24, 37, 50 μmol L−1 of BTC corrosion inhibitor at variable pH values. Table 1. Electrochemical elemen ts attained from the EIS spectra of the reinforcing steel in SCP solution with
3.5 wt.% NaCl in the existence of 12, 24, 37, 50 μmol L−1 of BTC corrosion inhibitor at variable pH values. Table 1. Electrochemical elemen ts attained from the EIS spectra of the reinforcing steel in SCP solution with
3.5 wt.% NaCl in the existence of 12, 24, 37, 50 μmol L−1 of BTC corrosion inhibitor at variable pH values. Figure 5. Polarization curves for reinforced steel in SCP solutions containing 3.5 wt.%NaCl and BTC inhibitor
concentrations of 12, 24, 37, 50 μmol L−1 at pH values of (a) 12.5, (b) 10 and (c) 7. Figure 5. Polarization curves for reinforced steel in SCP solutions containing 3.5 wt.%NaCl and BTC inhibitor
concentrations of 12, 24, 37, 50 μmol L−1 at pH values of (a) 12.5, (b) 10 and (c) 7. Results and Discussion −
E
E
(4)
pit
corr (4) The corrosion inhibition efficiency (IE%), is calculated using the following formula37, The corrosion inhibition efficiency (IE%), is calculated using the following formula37, Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports/ pH
values
Inhibitor concertation
(µmol L−1)
icorr (µA
cm−2)
ipit (µA
cm−2)
Epit (mV)
SCE
Ecorr (mV)
SCE
Epit-Ecor
(mV) SCE
Bc (V
decade−1)
Ba (V
decade−1)
Rp Ω
cm2
IE%
surface
coverage (ϴ)
12.5
Blank
13
2.0 × 10−4
−0.41
−0.601
0.187
0.11
0.076
1502
—
—
12
5.2
6.7 × 10−5
−0.34
−0.598
0.254
0.081
0.061
2888
56
0.56
24
3.2
5.0 × 10−5
−0.33
−0.601
0.271
0.077
0.059
4590
76
0.76
37
2.5
4.2 × 10−5
−0.28
−0.602
0.322
0.072
0.056
5471
81
0.81
50
1.6
3.9 × 10−5
−0.21
−0.594
0.384
0.068
0.048
7636
88
0.88
10
Blank
20
—
—
−0.71
—
0.091
0.091
988
—
—
12
15
—
—
−0.639
—
0.087
0.081
1214
22
0.22
24
10
—
—
−0.665
—
0.075
0.079
1671
52
0.52
37
8
1.23 × 10−5
—
−0.635
0.058
0.061
0.066
1721
63
0.63
50
5
9.7 × 10−5
—
−0.585
0.376
0.058
0.058
2518
78
0.78
7
Blank
33
—
—
—
—
0.098
0.120
709
—
—
12
28
—
—
—
—
0.087
0.110
755
15
0.15
24
21
—
—
—
—
0.080
0.109
958
36
0.36
37
14
—
—
—
—
0.071
0.111
1344
57
0.57
50
9.5
—
—
—
—
0.116
0.091
2332
71
0.71
Table 2. The electrochemical factor derived from polarization plots of the reinforcing steel in SCP solutions
including 3.5 wt.%NaCl and BTC inhibitor concentrations of 12, 24, 37, 50 μmol L−1 at pH values of 12.5, 10 and 7. Table 2. The electrochemical factor derived from polarization plots of the reinforcing steel in SCP solutions
l di
3 5
t %N Cl
d BTC i hibit
t ti
f 12 24 37 50
l L−1 t H
l
f 12 5 10
d Table 2. The electrochemical factor derived from polarization plots of the reinforcing steel in SCP solutions
including 3.5 wt.%NaCl and BTC inhibitor concentrations of 12, 24, 37, 50 μmol L−1 at pH values of 12.5, 10 and 7 Table 2. Results and Discussion The attack of chloride species to the reinforc-
ing steel surface in SCP can lead to loss of its passive layer if the concentration of the chloride species is adequately
high. For reinforcing steel in concrete, the degree to which Cl¯ ions can damage the passive layer is related to
the alkalinity of the environment. In chloride-free alkaline conditions, the passive layer on the mild steel breaks Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports/ pH
values
Slope
α
Intercept
Kads × 104,
(L mole−1)
∆G°ads,
(kJ mol−1)
12.5
−0.33
0.17
−3
0.88
−32
10
−0.36
0.14
−3.5
1.6
−33
7
−0.39
0.13
−3.9
2.2
−34
Table 3. The calculated thermodynamic parameters derived from Temkin plot. pH
values
Slope
α
Intercept
Kads × 104,
(L mole−1)
∆G°ads,
(kJ mol−1)
12.5
−0.33
0.17
−3
0.88
−32
10
−0.36
0.14
−3.5
1.6
−33
7
−0.39
0.13
−3.9
2.2
−34
Table 3. The calculated thermodynamic parameters derived from Temkin plot. Table 3. The calculated thermodynamic parameters derived from Temkin plot. Table 3. The calculated thermodynamic parameters derived from Temkin plot. Table 3. The calculated thermodynamic parameters derived from Temkin plot. Figure 7. SEM images for the reinforcing steel (a,c,e) before and (b,d,f) after immersion in SCP solution
containing 3.5 wt.% NaCl in existence of 50 μmol L−1 of BTC corrosion inhibitor at variable pH values of (a,b)
12.5, (c,d) 10, and (e,f) 7 for 24 h. Figure 7. SEM images for the reinforcing steel (a,c,e) before and (b,d,f) after immersion in SCP solution
containing 3.5 wt.% NaCl in existence of 50 μmol L−1 of BTC corrosion inhibitor at variable pH values of (a,b)
12.5, (c,d) 10, and (e,f) 7 for 24 h. Figure 7. SEM images for the reinforcing steel (a,c,e) before and (b,d,f) after immersion in SCP solution
containing 3.5 wt.% NaCl in existence of 50 μmol L−1 of BTC corrosion inhibitor at variable pH values of (a,b)
12.5, (c,d) 10, and (e,f) 7 for 24 h. Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports/ www.nature.com/scientificreports
ure.com/scientificreports/
Figure 8. AFM images for the reinforcing steel surface (a,c,e) before and (b,d,f) after immersing the reinforcing
steel for 24 h in SCP solution containing 3.5 wt.% NaCl in existence of 50 μmol L−1 of BTC corrosion inhibitor at
variable pH values of (a,b) 12.5, (c,d) 10, and (e,f) 7. Figure 8. Results and Discussion In order to understand and estimate the adsorption route of BTC on steel surface,
different adsorption isotherms are checked using the measured data from the poanlarization plots e.g. Langmuir,
Frumkin and Temkin isotherms using the following equations: mkin and Temkin isotherms using the following equations:
Langmuir Langmuir θ
=
+
C
K
C
1
(8)
inh
ads
inh (8) Frumkin α
×
θ
−θ
=
. +
θ
log C
logK
{
1
}
2 303
2
(9) (9) Temkin α
−
θ = K
C
exp(
2
)
(10)
ads (10) where θ is the surface coverage of the reinforcement steel, C is the concentration of the BTC inhibitor species, α is
the adsorbate interaction factor and Kads is the adsorption– desorption equilibrium constant. The fitting outcomes
showed that BTC inhibitor obeys Temkin isotherm, see Fig. 6.t y
g
After reorganizing Eq. (10), the following expression is attained: After reorganizing Eq. (10), the following expression is attained: θ
α
α
=
−
+
−
lnc
lnK
1
2
1
2
(11)
ads (11) It can be deduced from Eq. (11) that both of the slope and intercept are calculated from 1/−2α and (1/−2α)
lnKads, respectively. It can be deduced from Eq. (11) that both of the slope and intercept are calculated from 1/−2α and (1/−2α)
lnK
respectively Knowing the Kads values at various pH values, the standard Gibbs free energy change of adsorption (∆G°ads)
are calculated using Eq. 12. =
. −∆
K
e
1
55 5
(12)
G
RT
ads
ads
o (12) Table 3 summarizes the values of the α, Kads and ∆G°ads. Values of ∆G°ads ≥ −20 kJ mol−1, showing a phy-
sisorption adsorption, while ∆G°ads ≤ −40 kJ mol−1 depicts chemisorption adsorption reactions. Consequently,
the intermediate values of ∆G°ads shown in Table 3 (−32, −33 and −34 kJ mol−1), usually elucidate that
chemi-physisorption of BTC inhibitor occurs on the reinforcing steel at different pH values of 12.5, 10 and 7,
respectively. However, since there is no free electron pair existing in the molecular structure of the BTC inhib-
itor that can form coordinated covalent bond with the vacant d-orbitals in Fe (chemisorption), therefore it is
more favorable that the adsorption mechanism is a strong physisorption ratherthan a chemi-physisorption one. Results and Discussion AFM images for the reinforcing steel surface (a,c,e) before and (b,d,f) after immersing the reinforcing
steel for 24 h in SCP solution containing 3.5 wt.% NaCl in existence of 50 μmol L−1 of BTC corrosion inhibitor at
variable pH values of (a,b) 12.5, (c,d) 10, and (e,f) 7. pH
Ra in absence of BTC (nm)
Ra in presence of BTC (nm)
12.5
27
15
10
47
23
7
70
36
Table 4. The surface roughness values (Ra), of the reinforcing steel before and after addition of BTC inhibitor in
saline water for 24 h at variable pH values. Table 4. The surface roughness values (Ra), of the reinforcing steel before and after addition of BTC inhibito
saline water for 24 h at variable pH values. down at a potential of +560 mV SCE38. The highest attained IE% was 88% at 50 μmol L−1 of the BTC inhibitor at
pH = 12.5. It can be seen that the values of ba and bc diminished upon the addition of the inhibitor indicating that
BTC is a mixed type inhibitor. it is noteworthy to mention that the tabulated values of the corrosion density (icorr),
shifts towards decreases with increasing the concertation of the BTC inhibitor. However, these values are not in
accordance with reported articles in references12,39. In fact, the diffusion of chloride species (Cl−), in cementitious
materials immersed in saline water is a difficult process, which includes numerous chemical and physical inter-
actions. Cl− ions can bound chemically or physically through the cement paste, thus reducing the segment of free Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y www.nature.com/scientificreports/ Figure 9. XPS survey scan composition of the mild steel after immersion for 24 h in SCP solution containing
3.5 wt.%NaCl in the existence of 50 μmol L−1 of BTC corrosion inhibitor at pH 12.5. Figure 9. XPS survey scan composition of the mild steel after immersion for 24 h in SCP solution containing
3.5 wt.%NaCl in the existence of 50 μmol L−1 of BTC corrosion inhibitor at pH 12.5. Cl− species that can diffuse easily in the concrete pore solutions. Furthermore, the internal electric field generated
from the anions and cations will accelerate the ions that possess low diffusion coefficients and decelerate the ions
that have high diffusion coefficients in order to keep the electro-neutrality status40. Adsorption isotherm. Results and Discussion Physical adsorption takes place rapidly because of weak bondings such as Van der Waal’s or electrostatic attrac-
tive forces between inhibitor species and metal surface, and is directly influenced by the electronegativity of the
inhibitor compounds. The residence time for a physically adsorbed inhibitor is short, and its interaction with Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y 9 www.nature.com/scientificreports/ Figure 10. High resolution XPS spectrum of (a) Cl 2p, (b) C 1 s, (c) Ca 2p, (d) N 1 s (e) O 1 s and (f) Fe 2p after
immersing the reinforcing steel for 24 h in SCP solution containing 3.5 wt.% NaCl in existence of 50 μmol L−1 of
BTC corrosion inhibitor at pH Σ12.5. Figure 10. High resolution XPS spectrum of (a) Cl 2p, (b) C 1 s, (c) Ca 2p, (d) N 1 s (e) O 1 s and (f) Fe 2p after
immersing the reinforcing steel for 24 h in SCP solution containing 3.5 wt.% NaCl in existence of 50 μmol L−1 of
BTC corrosion inhibitor at pH Σ12.5. the steel surface is directly associated with the corrosion free potential of the metal corrosion with respect to the
potential of zero charge.t Figure 7 exhibits the SEM of the reinforcing steel coupons after immersion in SCP including 3.5 wt.% NaCl
at different pH values of 12.5, 10, 7 in the existence and absence of 50 μmol L−1 of the BTC inhibitor for 24 h. It is
clear that in case of the absence of the corrosion inhibitor, deep pits were formed and their number is suppressed
as the pH of the medium increases. Nonetheless, in the existence of the corrosion inhibitor, the number and pits
size are considerably reduced at the same pH values. Moreover, the pH values before and after immersion are
measured. It is found that the pH values in the absence of the corrosion inhibitors are lowered from 12.5, 10 and
7 to 10, 8.7 and 5.7, respectively. However, in the existence of the BTC inhibitor the pH values slightly dropped
from 12.5, 10 and 7 to 11.5, 9.3 and 6.3, respectively. p
y
Surface topography and surface roughness of the reinforcing steel are explored after immersion in 3.5
wt.%NaCl of variable pH values for 24 h in the existence and absence of 50 μmol L−1 of the BTC inhibitor using
AFM, as depicted in Fig. 8. Results and Discussion It is noted that the surface roughness (Ra), escalates as the pH alleviates in the absence Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y 10 www.nature.com/scientificreports/ Exposure
time (days)
Flexural strength
Maximum Bending
Stress at Break, (MPa)
Compression strength
(N mm−2)
Blank
BTC
Blank
BTC
7
5.9
5.6
42.2
39.1
14
6.3
6.3
44. 2
44.2
21
6.5
6.2
47.31
47.1
28
6.6
6.4
47.4
47.3
Table 5. Flexural and compressive strength results of the as-prepared cured mortars after different curing
times. Exposure
time (days)
Flexural strength
Maximum Bending
Stress at Break, (MPa)
Compression strength
(N mm−2)
Blank
BTC
Blank
BTC
7
5.9
5.6
42.2
39.1
14
6.3
6.3
44. 2
44.2
21
6.5
6.2
47.31
47.1
28
6.6
6.4
47.4
47.3
Table 5. Flexural and compressive strength results of the as-prepared cured mortars after different curing
times. Table 5. Flexural and compressive strength results of the as-prepared cured mortars after different curing
times. Table 5. Flexural and compressive strength results of the as-prepared cured mortars after different curing
times. of the corrosion inhibitor, see Table 4. However, Ra is decreased significnatly in the existence of the BTC inhibitor
in the deleterious medium signifying the construction of an adsorbed protective layer of BTC inhibitor on the
metal surfaces, which retards the attack of the Cl− species
Th
d
(
)
d h h h
l
(
)
b
d ft of the corrosion inhibitor, see Table 4. However, Ra is decreased significnatly in the existence of the BTC inhibitor
in the deleterious medium signifying the construction of an adsorbed protective layer of BTC inhibitor on the
metal surfaces, which retards the attack of the Cl− speciesht The wide scan spectrum (Fig. 9) and the high resolution XPS spectra (Fig. 10) are obtained after immersing
the reinforcing steel for 24 h in SCP including 3.5 wt.% NaCl in the existence of 50 μmol L−1 of BTC corrosion
inhibitor at pH 12.5.h p
The Cl 2p spectrum is deconvoluted into two components at BE of 199.1 and 200.7 eV which are attributed to
Cl− and FeClx, respectively41, see Fig. 10a. It is noteworthy that the peak intensity of Cl− species is low; indicating
that the adsorbed BTC inhibitor lowers the adsorption affinity of the Cl− ions to the metal surface which subse-
quently lessens the corrosion rate. Results and Discussion The adsorption of the BTC inhibitor on the reinforcing steel surface is further
confirmed by the analysis of C spectra, which showed the presence of CN+-R3 and (C-C & C-H) at BE of 284.5
and 287.7 eV, respectively42,43, see Fig. 10b. However, Ca 2p spectrum shown in Fig. 10c is decomposed into two
bands at 347.8 eV and 351.5 eV that are credited to Ca 2P3/2 and 2P1/2 respectively, of CaO/Ca(OH)2
8. Figure 10d
shows the appearance of N 1 s peaks at 399.4 and 402.5 eV, characteristics of N-CH2 and N+ quaternary nitrogen,
respectively43-46. This approves the adsorption of the BTC inhibitor on the reinforcing steel surface. There is no
peak for C–N–Fe bonding was observed at 397.7–398.6 eV, suggesting that BTC inhibitor was adsorbed on the
reinforcing steel through physisorption47. The XPS spectrum of O 1 s in Fig. 10e, exhibits three peaks at 530.3,
531.7 and 534.2 eV, that are credited to O2− of iron oxides, OH− of hydrous iron oxides (FeOOH), and H2O,
respectively43,46. On the other hand, Fe 2p spectra in Fig. 10f is deconvoluted into six peaks. In fact, the interpre-
tation of Fe 2p spectra is a complex owing to the existence of iron (Fe), in variable oxidation states of Fe°, Fe2+,
Fe3+, and satellites of Fe3+ species. The (Fe 2p3/2), XPS spectra at high resolution involves four bands at 707.1 eV
that is related to the metallic iron, 710.9 eV for Fe3+ of Fe2O3/ FeOOH and 713.9 eV, which could be attributed
to a mixture of (Fe2+ & Fe3+), in different forms of iron (II) oxide (FeO), iron (II) hydroxide Fe(OH)2, iron (III)
hydroxide Fe(OH)3, FeOOH, iron (III) oxide (Fe2O3), and magnetite (Fe3O4)48. The shake up phenomenon found
at 716.6 and 719.8 eV is ascribed to Fe2+ and Fe3+, respectively. The spectra of the Fe 2p1/2 peaks at BE of 722.7 and
725.3 eV can be ascribed to Fe2O3 and FeO(OH), respectively49. Mechanical properties. Although corrosion inhibitors can protect steel against corrosion, however it can
badly affect its mechanical features50. Therefore, the mechanical characteristics of cured mortars are investigated
in the presence and absence of a 20 ppm (50 μmol L−1), of the BTC inhibitor. This is achieved through measuring
the flexural and compressive strength of cement, see Table 5. Results and Discussion It can be observed that there is almost no change in
the mechanical properties of the concrete after the addition of the inhibitor, which is attributed to the low con-
centration of the BTC. Conclusions A new BTC corrosion inhibitor for reinforcing steel is investigated in 3.5 wt.% NaCl at different pH values. Taflel
plots indicated that BTC is a mixed type inhibitor. The BTC inhibitor showed a corrosion inhibition efficiency
(IE%) of 88, 78 and 71% in 3.5 wt.% NaCl using 50 μmol L−1 at pH values of 12.5, 10 and 7, respectively, which is
effective if carbonation of the concrete happens and the pH of the concrete is lowered. Based on the adsorption
isotherm calculations, BTC inhibitor showed the best fitting with Temkin isotherm. XPS results illustrate that
BTC inhibitor is physisorbed on the reinforcing steel surface, which is matching with the ∆G°ads calculations. The
surface roughness of the metal surface is significantly decreased upon the addition of BTC inhibitor as shown
in the AFM results confirming a high corrosion inhibition efficiency of BTC. Additionally, no change in the
mechanical features of concrete is observed upon the addition of BTC inhibitor, which allows using it in concrete
without any reservation. Data availability The raw/processed data required to reproduce these findings could not be shared at this time due to time
limitations. However, it will be available on request. Received: 31 July 2019; Accepted: 30 October 2019;
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47. Finšgar, M., Fassbender, S., Hirth, S. & Milošev, I. References ASTM C348-18, Standard Test Method for Flexural Strength of Hydraulic-Cement Mortars, ASTM International, Wes
Conshohocken, PA, doi:www.astm.org (2018). 30. ASTM C109/C109M-16a, Standard Test Method for Compressive Strength of Hydraulic Cement Mortars (Using 2-in or [50-mm]
Cube Specimens), ASTM International, West Conshohocken, PA, doi:www.astm.org (2018). 1. Poursaee, A. & Hansson, C. M. Reinforcing steel passivation in mortar and pore solution. Cement and Concrete Research 37
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2. Ghantous, R. M., Poyet, S., L’Hostis, V., Tran, N.-C. & François, R. Effect of crack openings on carbonation-induced corrosion
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Research 53, 44–50, https://doi.org/10.1016/j.cemconres.2013.06.003 (2013). 34. Macdonald, J. R. Impedance spectroscopy and its use in analyzing the steady-state AC response of solid and liquid electrolytes. Journal of Electroanalytical Chemistry and Interfacial Electrochemistry 223, 25–50, https://doi.org/10.1016/0022-0728(87)85249-X
(1987). 35. J. R. & MacDonald. Impedance Spectroscopy Emphasizing Solid Materials and Systems. John Wiley & Sons, New York (1987).h 36. Frankel, G. S. Pitting Corrosion of Metals: A Review of the Critical Factors. Journal of The Electrochemical Society 145, 2186–2198,
https://doi.org/10.1149/1.1838615 (1998). 37. Ahamad, I., Prasad, R. & Quraishi, M. A. Adsorption and inhibitive properties of some new Mannich bases of Isatin derivatives on
corrosion of mild steel in acidic media. Corrosion Science 52, 1472–1481, https://doi.org/10.1016/j.corsci.2010.01.015 (2010). 38. Martin, F. & Olek, J. The Nature of Passivity of Reinforcing Steel. ASCE Materials Congress, Washington, D.C (1996). 37. Ahamad, I., Prasad, R. & Quraishi, M. A. Adsorption and inhibitive properties of some new Mannich bases of Isatin derivatives on
corrosion of mild steel in acidic media. Corrosion Science 52, 1472–1481, https://doi.org/10.1016/j.corsci.2010.01.015 (2010).h p
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38. Martin, F. & Olek, J. The Nature of Passivity of Reinforcing Steel. ASCE Materials Congress, Washington, D.C (1996). Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y Acknowledgementsh g
The publication of this article was funded by the Qatar National Library. Author contributions Ahmed Bahgat Radwan, Mostafa Sliem and Noor Yusuf were responsible for the experimental work including
preparation and characterizataion. Dr. Nasser Alnuaimi supervised the concrete preparation and mechanical
testing section. Dr. Aboubakr Abdullah was resposnsible for the corrosion section and the overall manuscript
gathering, reviewing and submission. Additional information Correspondence and requests for materials should be addressed to A.M.A. Correspondence and requests for materials should be addressed to A.M.A. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
i
tit ti
l ffili ti Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional 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
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-
ative Commons license, and indicate if changes were made. The images or other third party material in this
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mitted 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/. © The Author(s) 2019 Scientific Reports | (2019) 9:18115 | https://doi.org/10.1038/s41598-019-54669-y 13
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THE USE OF CONSONANT ELISION BY SRI MULYANI DURING HER INTERVIEW SESSION WITH THE BANKER
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Eltin Journal : Journal of English Language Teaching in Indonesia
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Firdhani, Indrayani & Mahdi: The Use of Consonant… Firdhani, Indrayani & Mahdi: The Use of Consonant… ABSTRACT This research is entitled “The Use of Consonant Elision by Sri Mulyani During Her
Interview Session with The Banker”. The objects of this research are the consonant elisions
that are produced by Sri Mulyani when she was interviewed by The Banker. The objectives
of this research are to describe and analyze the frequency of consonant elision used by Sri
Mulyani and to find out the phonemes that are often being elision-ed by Sri Mulyani in her
interview with The Banker. The analysis of this research was done by using descriptive
qualitative analysis method. This study uses Carr’s (2008), Giegerich’s (1992), and
Roach’s (2001) theories about phonological rules and elision. This study shows that during
her interview, Sri Mulyani uses consonant elision for 26 times and she tends to do the
omission to the phoneme /t/ (19 times), /d/ (5 times), /r/ (1 times), and /s/ (1 times). Keywords: Consonant Elision, Elision, Phonology, Phonological Rules, Sri
Mulyani Keywords: Consonant Elision, Elision, Phonology, Phonological Rules, Sri
Mulyani THE USE OF CONSONANT ELISION BY SRI MULYANI
DURING HER INTERVIEW SESSION WITH THE BANKER Anggi Rizky Firdhani1, Lia Maulia Indrayani2 , Sutiono Mahdi3
anggi12001@mail.unpad.ac.id, lia.maulia@unpad.ac.id, sutiono.mahdi@unpad.ac.id Keywords: Consonant Elision, Elision, Phonology, Phonological Rules, Sri
Mulyani B. LITERATURE REVIEW
1. Elision In English pronunciation, sometimes a speaker simplifies the pronunciation of some words. According to Carr (2008), this process of simplification is counted as a part of the
phonological rules. In his book, Carr mentions that simplification consists of various types,
those are reduction, lenition, assimilation, and elision. Sometimes, the simplification
occurs in order to ease the native speakers in expressing their feelings. It is common for
them to speak English in high speed along with their emotions, and as the result, they make
a ‘shortcut’ to ease their pronunciation. Sometimes, the native speakers of English make a
complete disappearance of one or more sounds in a word or phrase as an attempt to make
a word or phrase easier to pronounce, and that phenomenon is called elision (Crystal,
2008). According to Giegerich (1992) in Febriyanti (2015), one of the most common
elisions in English is the omission of phoneme /t/ and /d/. Although the use of elision is often used by native speakers, in fact, native speakers are not
the only person who use elision in uttering some words in English. Many non-native
speakers often use elision when they are speaking in English, including Indonesians. One
example of the native speakers of Indonesian who use elision while speaking in English is
the Minister of Finance of the Republic of Indonesia, Sri Mulyani. Sri Mulyani is one of
the prominent figures in Indonesia, so it is not surprising that Sri Mulyani is often required
to speak English. Sri Mulyani's speaking style in English caught the attention of the researcher, especially
when the researcher watched her being interviewed about the tax amnesty in Indonesia by
The Banker, a British financial media, for approximately nine minutes. In the interview,
the researcher saw that Sri Mulyani had her own pattern when she tried to speak English
and she used a lot of elisions, particularly consonant elision when she was pronouncing
certain words. The researcher saw that the use of elision by Sri Mulyani while being
interviewed by The Banker is interesting to be examined in this research. Based on the explanation above, the researcher is interested to find out how often Sri
Mulyani used elision while being interviewed and to find out the phonemes that are often
being elision-ed by Sri Mulyani in her interview with The Banker. A. INTRODUCTION In English pronunciation, there are various ways of pronunciation for each individual. This
phenomenon occurs because of numerous factors, such as areas of origin, cultural
influences, and social environment. English pronunciation involves the production of each
sound and pronunciation of words, phrases, and sentences with correct spelling, emphasis
and/or intonation. In addition, there is a way to read the word correctly called 'phonetic
transcription', which is defined as a kind of alphabetic writing in which each letter
represents a sound (Fromkin, Rodman, & Hyams, 2003). The purpose of phonetic
transcriptions is to provide clear and unambiguous information to language learners, such
as which sounds should be used on a word or phrase, and in what order to use the sound. The branch of linguistics which studies the sound system of languages is called phonology. The pronunciation of speech sound in English is governed by certain rules, and the rule is
called the phonological rules. This statement was approved by Fromkin, Rodman, and
Hyams (2003) because, in their book, they state that "phonological rules relate to phonemic
representations and phonetic representations of the speaker’s knowledge of the language."
With the existence of these phonological rules, we cannot pronounce any speech sounds
carelessly, especially in pronouncing speech sounds in English. There are several
phonological rules that we need to obey in pronouncing English speech sound, such as the
use of pressure, intonation, and so forth. The aim of studying the rules of phonology is to
give a phonetic information or guideline on how to pronounce a word in a correct way, so 53 ELTIN Journal, Volume 6/II, October 2018 the meaning that conveyed from the spoken word can be conveyed correctly. Although the
specific rules of phonology differ from language to language, the kind of rules, what they
do, and the natural classes they refer to are the same cross-linguistically (Fromkin,
Rodman, & Hyams, 2003). the meaning that conveyed from the spoken word can be conveyed correctly. Although the
specific rules of phonology differ from language to language, the kind of rules, what they
do, and the natural classes they refer to are the same cross-linguistically (Fromkin,
Rodman, & Hyams, 2003). 2. Types of Simplification As the researcher mentioned in the explanation above that in English pronunciation,
sometimes, a speaker simplifies the pronunciation of some words. The simplification
occurs in order to ease the native speakers in expressing their feelings. It is common for
them to speak English in high speed along with their emotions, and as the result, they make
a ‘shortcut’ to get the ease of their pronunciation. According to Carr (2008), this process
of simplification is counted as a part of the phonological rules. In his book, Carr mentions
that simplification consists of various types, those are reduction, lenition, assimilation, and
elision. Elision is going to be the focus of this research. 54 Firdhani, Indrayani & Mahdi: The Use of Consonant… Firdhani, Indrayani & Mahdi: The Use of Consonant… Crystal (2008) defines that elision is a complete disappearance of one or more sounds in a
word or phrase as an attempt to make a word or phrase easier to pronounce. The process of
elision occurs when speakers leave or delete a phoneme out of pronunciation as an attempt
to fasten and get rid of difficulties in speech. Elision happens with both consonants and
vowels. Carr (2008) states that where the cluster of consonants arise through the
combination of words into phrases, they are frequently reduced in this way. For example,
the phrase crisp bowl is often pronounced as [khrɪsboʊl], with the final stop in crisp elided. Carr also states that vowel elision may result in the loss of an entire syllable as in university
when it is pronounced as [juː.nɪˈvɜː.sti]. According to Giegerich (1992) cited in Febriyanti (2015), elision often happens before
sonorant consonants which give the consonants an opportunity to be syllabic so that they
will occupy the peak of the syllable. The process is called vowel elision, for examples are
button /bʌtən/ or /bʌtn/ and little /lɪtəl/ or /lɪtl/. Giegerich also explains that the most
common elision in English words is the omission of sounds /t/ and /d/ at the word boundary:
they are positioned at end of a word before going to the next word. For example, the elision
of phoneme /t/ in the word postman is missing, so its pronunciation from [poʊstmən]
changed to [poʊsmən] because of the speed of the speaker’s speech. In the case of phoneme
/t/, the preceding consonant must be voiceless; in the case of /d/, the preceding consonant
must be voiced. 2. Types of Simplification In both cases, elision happens with any following consonant, except /h/
and sometimes /j/, e.g. Christmas becomes [’krɪsməs], listen becomes [lɪsən], left foot
becomes [lefʊ:t], but it doesn’t happen in left hand [left hænd], left you [lefʧʊ], and old
man [oʊld mæn]. Roach (2001) agreed with that explanation: Roach states the first type of
elision that can be found in connected speech involves a loss of weak vowels after /p/, /t/,
/k/. Roach pointed out that the vowel in the first syllable may be omitted. That is, the
aspiration of the initial plosive may take up the whole of the middle portion of the syllable
as in “potato”, “tomato”, “canary” “perhaps” and “today” C. RESEARCH METHODOLOGY The method used in this research is descriptive qualitative analysis method. The descriptive
qualitative analysis method is a research method that produces descriptive data in the form
of written words or oral things that can be observed (Bogdan and Taylor, 1975). Qualitative
descriptive analysis research relates to ideas, perceptions, opinions, and all of them cannot
be measured by numbers. This qualitative descriptive study aims to obtain information
about existing circumstances (Mardalis, 1999) and is designed to collect information about
current ongoing realities (Cevilla, 1993). In doing this study, the researcher transcribes the audio via video obtained from Youtube. The video that is chosen by the researcher is an interview video between Sri Mulyani and
The Banker entitled View from IMF: Interview with Sri Mulyani Indrawati, Finance
Minister, Indonesia. To perform the audio transcription, the researcher listened to the
speech sounds produced by Sri Mulyani carefully, then the researcher marked the
pronunciation of any elision-ed words that are produced by Sri Mulyani. After that, the
pronunciation of some words spoken by Sri Mulyani compared with the General American
English (GA) pronunciation. 55 ELTIN Journal, Volume 6/II, October 2018 D. FINDINGS AND DISCUSSION The process of elision involves the omission of a phoneme in its pronunciation. The
researcher found several cases which belong to elision in Sri Mulyani’s utterance during
her interview, and most of them the elision of phoneme /t/ and /d/. In Sri Mulyani's
interview with The Banker, which lasted approximately for 9 minutes, the researcher found
out that Sri Mulyani used elision 26 times. More detailed information can be seen in the
table below. Table 1. The frequency of elision uttered by Sri Mulyani
during her interview with The Banker
Frequency of
the Elision of
Phoneme /t/
Frequency
of the
Elision of
Phoneme
/d/
Frequency
of the
Elision of
Phoneme /r/
Frequency
of the
Elision of
Phoneme
/s/
19
5
1
1
TOTAL
26 Table 1. The frequency of elision uttered by Sri Mulyani From the table above, we can see that the phoneme that is often omitted by Sri Mulyani is
phoneme /t/, followed by phoneme /d/, /r/, and /s/. Here is the list of pronunciation
realizations by Sri Mulyani that are being elision-ed, compared with General American
English Pronunciation (GA). Table 2. Li
f
i
i
li
i
b S i M l
i h
b i
li i
d Table 2. List of pronunciation realizations by Sri Mulyani that are being elision-ed
No
Word
GA English
Pronunciation
Realization by
Sri Mulyani
1. Months
/mʌnθs/
[mʌns]
2. Month
/ mʌnθ/
[mʌn]
3. Went
/went/
[wen]
4. Export
/ek.spɔːrt/
[ekspɔr]
5. Import
/ɪm.pɔːrt/
[ɪmpor]
6. Left
/left/
[lef]
7. Lengthen
/leŋ.θən/
[leŋhən]
8. Project
/prɑː.dʒekt/
[prɑː.dʒek]
9. Affect
/əˈfekt/
[əˈfek]
10. Past
/pæst/
[pæs]
11. Percent
/pəˈsent/
[pəˈsen]
12. Wealth
/welθ/
[welh]
13. Robust
/roʊˈbʌst/
[roʊˈbʌs]
14. Government
/ɡʌv.ɚn.mənt/
[ɡʌvən.mən]
15. Department
/dɪˈpɑːrt.mənt/
[dɪpɑːrmən]
16. Important
/ɪmˈpɔːr.tənt/
[ɪmpɔrtən]
17. Conduct
/kənˈdʌkt/
[kənˈdʌk]
18. Want
/wɑːnt/
[won] Table 2. List of pronunciation realizations by Sri Mulyani that are being elision-ed 56 Firdhani, Indrayani & Mahdi: The Use of Consonant… Firdhani, Indrayani & Mahdi: The Use of Consonant… 19. Trust
/trʌst/
[trʌs]
20. Behind
/bɪˈhaɪnd/
[bɪˈhaɪn]
21. Land
/lænd/
[læn]
22. Spending
/spen.dɪŋ/
[spenɪŋ]
23. Abandoned
/əˈbæn.dənd/
[əˈbæn.dən]
24. Second
/sek.ənd/
[sek.ən]
25. Course
/kɔːrs/
[kɔs]
26. Maintenance
/meɪn.tən.əns/
[meɪn.tən.ən] From the table above, the researcher sees the phonological pattern that is used by Sri
Mulyani when she is talking. E. CONCLUSION Based on the explanations above, the researcher can conclude that in Sri Mulyani's interview
with The Banker, which lasted approximately for 9 minutes, Sri Mulyani used elision for 26
times and most of them are the elision of phoneme /t/ (19 times) and /d/ (5 times). Mostly,
the omission of the phoneme /t/ and /d/ was done by Sri Mulyani when she had to utter the
sonorant consonantal sounds such as the liquid sounds (l, r) and nasal sounds (n, m, ŋ) that
appear before the phoneme /t/ and /d/. That phenomena show that plosive alveolar sounds
such as /t/ and /d/ is quite difficult to pronounce when those sounds are placed after the
liquid sounds or after the nasal sound. Therefore, the elision occurs as the way of Sri
Mulyani in maintaining the speed of her speech in order to make the pronunciation easier. D. FINDINGS AND DISCUSSION According to the table, we can see that mostly, the omission
of the phoneme /t/ and /d/ was done by Sri Mulyani when she had to utter the sonorant
consonantal sounds such as the liquid sounds (l, r) and nasal sounds (n, m, ŋ) that appear
before the phoneme /t/ and /d/. The phenomena indicate that plosive alveolar sounds such
as /t/ and /d/ is quite difficult to pronounce when those sounds are placed after the liquid
sounds or after the nasal sound. Therefore, the elision occurs as the way of Sri Mulyani in
maintaining the speed of her speech in order to make the pronunciation easier. Roach, P. (2001). English Phonetics and Phonology: A Practical Course.
Cambridge: Cambridge University Press. ELTIN Journal, Volume 6/II, October 2018 F. REFERENCES Bogdan, R., & Taylor, S. (1975). Introduction to Qualitative Research Methods. New York: John Willey and Sons. Carr, P. (2008). English Phonetics and Phonology: An Introduction. West Sussex:
Blackwell Publishing. Cevilla, C. G. (1993). Pengantar Metode Penelitian. Jakarta: Universitas
Indonesia. Crystal, D. (2008). A Dictionary of Linguistics and Phonetics. Oxford: Blackwell
Publishing. Febriyanti, D. N. (2015). Assimilation, Reduction and Elision Reflected in the
Selected Song Lyrics of Avenged Sevenfold. Journal of Language and Literature,
14-19. Fromkin, V., Rodman, R., & Hyams, N. (2003). An Introduction to Language. Boston: Thomson/Heinle. Giegerich, H. (1992). English Phonology: An Introduction. Cambridge:
Cambridge University Press. Mardalis. (1999). Metode Penelitian Suatu Pendekatan Proposal. Jakarta: Bumi
Aksara. Bogdan, R., & Taylor, S. (1975). Introduction to Qualitative Research Methods. New York: John Willey and Sons. Carr, P. (2008). English Phonetics and Phonology: An Introduction. West Sussex:
Blackwell Publishing. Cevilla, C. G. (1993). Pengantar Metode Penelitian. Jakarta: Universitas
Indonesia. Crystal, D. (2008). A Dictionary of Linguistics and Phonetics. Oxford: Blackwell
Publishing. Febriyanti, D. N. (2015). Assimilation, Reduction and Elision Reflected in the
Selected Song Lyrics of Avenged Sevenfold. Journal of Language and Literature,
14-19. Fromkin, V., Rodman, R., & Hyams, N. (2003). An Introduction to Language. Boston: Thomson/Heinle. Giegerich, H. (1992). English Phonology: An Introduction. Cambridge:
Cambridge University Press. g
y
Mardalis. (1999). Metode Penelitian Suatu Pendekatan Proposal. Jakarta: Bumi
Aksara. 57 57 ELTIN Journal, Volume 6/II, October 2018
Roach, P. (2001). English Phonetics and Phonology: A Practical Course.
Cambridge: Cambridge University Press. ELTIN Journal, Volume 6/II, October 2018 Roach, P. (2001). English Phonetics and Phonology: A Practical Course. Cambridge: Cambridge University Press. 58
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Have Host Defense Peptides Been Acting in Innate Immunity Since the Trilobites of the Cambrian Period 540 Million Years Ago?
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Juntendo iji zasshi
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Current Topics: Current
Status and Future Perspective
on the Application of Host
Defense Peptides to Medicine
Juntendo Medical Journal
2016. 62(2), 96-97 Current Topics: Current
Status and Future Perspective
on the Application of Host
Defense Peptides to Medicine Juntendo Medical Journal
2016. 62(2), 96-97 ISAO NAGAOKA*
*Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine, Tokyo, Japan ISAO NAGAOKA*
*Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine, Tokyo, Japan Antimicrobial peptides (AMPs), molecules evolu-
tionarily conserved among various species, are
usually positively charged, having both hydropho-
bic and hydrophilic domains. They are also called
host defense peptides (HDPs), because they partici-
pate in the innate immune response by exerting
antimicrobial activities against Gram-negative and
-positive bacteria, viruses and fungi. Moreover,
some HDPs (such as human cathelicidin peptide
LL-37 and guinea pig cathelicidin CAP11) directly
bind
with
Gram-negative
bacterial
endotoxin
(lipopolysaccharides, LPS) and neutralize its biolog-
ical activity
1)-5). Based on their potent and broad
spectrum of activities, they are expected to become
novel therapeutic agents. In fact, HDP derivatives
are being developed as antibiotics, anti-inflammato-
ries and antifungals. In addition to their direct
antimicrobial properties, HDPs have the ability to
enhance immunity and link innate and adaptive
immunity by functioning as immunomodulators. dins are characterized by a highly conserved N-
terminal cathelin-domain, and a variable C-terminal
antimicrobial peptide domain (12-80 amino acid
residues). Cathelicidin peptides have been isolated
from many different mammalian species. In this issueʼs Current Topics, findings about the
“Current status and future perspective on the appli-
cation of host defense peptides to medicine”are
reviewed. First, Dr. Hu demonstrated LL-37 to be a
promising candidate for sepsis, based on its poten-
tial abilities, including the inhibition of pyroptosis (a
recently identified caspase-1 dependent macro-
phage cell death pathway), and modulation of
inflammatory cytokine production and antimicro-
bial activity. Second, Dr. Suzuki suggests the
possibility that during sepsis (endotoxemia) LL-37
may not only suppress LPS-induced endothelial cell
apoptosis through the inhibition of LPS-binding to
the cells but also induce the clearance (uptake) of
LPS by endothelial cells via complex formation with
LPS. Third,
Dr. Ishibashi
characterizes
insect
(beetle)
defensin-derived
AMPs,
and
demon-
strates their therapeutic applications to bacterial
infections and their use as novel antimicrobial
materials such as AMP-immobilized fibers. Fur-
thermore, Dr. Niyonsaba reviews the roles of HDPs
in human skin diseases, based on their antimicrobial There are two major families of HDPs, defensins
and cathelicidins. Defensins are small cysteine-rich
cationic peptides consisting of 18-45 amino acids, and
include six to eight conserved cysteine residues with
intramolecular disulfide bonds. Defensins are found
in both vertebrates and invertebrates (including
arthropods), and also in plants. In contrast, cathelici- Copyright © 2016 The Juntendo Medical Society. Isao Nagaoka
Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine
2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
TEL: +81-3-5802-1032
FAX: +81-3-3813-3157
E-mail: nagaokai@juntendo.ac.jp
〔Received
Apr. 27, 2016〕 ISAO NAGAOKA*
*Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine, Tokyo, Japan This is an open access article distributed under the terms of Creative Commons Attribution Li-
cense (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original source is properly credited. doi: 10.14789/jmj. 62.96 96 Juntendo Medical Journal 62(2), 2016 Dermis
A
B
C
Figure-1
Horseshoe crab and trilobite
A. A skeletal specimen of a horseshoe crab (Tachyples tridentatus) known as the Japanese, Chinese, or tri-spine horseshoe crab; B. A
decorative panel depicting a horseshoe crab (Limulus polyphemus) known as the Atlantic horseshoe crab; C. A trilobite fossil (Elrathia
kingi) that lived during the middle Cambrian of Utah, USA (530 million years ago). Dermis
B C C B A Figure-1
Horseshoe crab and trilobite Figure-1
Horseshoe crab and trilobite
A. A skeletal specimen of a horseshoe crab (Tachyples tridentatus) known as the Japanese, Chinese, or tri-spine horseshoe crab; B. A
decorative panel depicting a horseshoe crab (Limulus polyphemus) known as the Atlantic horseshoe crab; C. A trilobite fossil (Elrathia
kingi) that lived during the middle Cambrian of Utah, USA (530 million years ago). Figure-1
Horseshoe crab and trilobite
A. A skeletal specimen of a horseshoe crab (Tachyples tridentatus) known as the Japanese, Chinese, or tri-spine horseshoe crab; B. A
decorative panel depicting a horseshoe crab (Limulus polyphemus) known as the Atlantic horseshoe crab; C. A trilobite fossil (Elrathia
kingi) that lived during the middle Cambrian of Utah, USA (530 million years ago). Figure 1
Horseshoe crab and trilobite
A. A skeletal specimen of a horseshoe crab (Tachyples tridentatus) known as the Japanese, Chinese, or tri-spin
decorative panel depicting a horseshoe crab (Limulus polyphemus) known as the Atlantic horseshoe crab; C. A
kingi) that lived during the middle Cambrian of Utah, USA (530 million years ago). and immunomodulatory actions in the skin. By
understanding the roles of HDPs in the pathophysi-
ology of skin diseases, novel therapeutic targets can
be identified, and potential treatments can be
developed for these skin conditions. Lastly, Dr. Tamura reviews the Limulus amoebocyte lysate
(LAL) test, which is the most sensitive and reliable
assay for the detection of trace amounts of bacterial
endotoxin (LPS); thus, the LAL test can be utilized
for the diagnosis of sepsis, or endotoxemia. ISAO NAGAOKA*
*Department of Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine, Tokyo, Japan How-
ever, it is important to note that the LAL test can be
affected by the presence of HDPs, since HDPs with
potent LPS-neutralizing activities (such as LL-37
and CAP11) can mask the endotoxin active site
when they bind. relatives of trilobites (ancient arthropods), which
appeared in the Cambrian period, 540 million years
ago (https://en.wikipedia.org/wiki/Trilobite, http:
//www.uh.edu/engines/epi2496.htm)(Figure-1C). Thus, it is reasonable to speculate that AMPs/
HDPs were present in trilobites and have been
acting in innate immunity since the Cambrian
period. Now, we are attempting to apply these
ancient host defense peptides as novel therapeutic
agents to medicine. References 1) Nagaoka I, Hirota S, Niyonsaba F, et al: Cathelicidin
family of antibacterial peptides CAP18 and CAP11
inhibit the expression of TNF-alpha by blocking the
binding of LPS to CD14 + cells. J Immunol, 2001; 167:
3329-3338. Interestingly, LAL is prepared from amoebocytes
(hemocytes) of horseshoe crabs (Figure-1A and
B). Horseshoe crab hemocytes contain two types of
granules; large
granules
contain
the
essential
components of the LAL test, including serine
protease zymogens (factor C, factor G and factor
B), proclotting enzyme and the clotting factor
coagulogen, whereas small (dense) granules con-
tain AMPs
6). Hoemocytes are highly sensitive to
LPS, and LPS stimulation induces the degranulation
and secretion of granular components and results in
clot formation. These responses are important for
host defense of horseshoe crabs in engulfing and
killing invading microbes as well as preventing
hemolymph leakage. Horseshoe crabs are called
living fossils, since they have been living on earth
since the late Ordovician period, 450 million years
ago (https://en.wikipedia.org/wiki/Horseshoe_crab). Notably, horseshoe crabs are the closest living 2) Nagaoka I, Hirota S, Niyonsaba F, et al: Augmentation of
the lipopolysaccharide-neutralizing activities of human
cathelicidin CAP18/LL-37-derived antimicrobial pepti-
des by replacement with hydrophobic and cationic amino
acid residues. Clin Diagn Lab Immunol, 2002; 9: 972-982. 3) Nagaoka I, Kuwahara-Arai K, Tamura H, Hiramatsu K,
Hirata M: Augmentation of the bactericidal activities of
human cathelicidin CAP18/LL-37-derived antimicrobial
peptides by amino acid substitutions. Inflamm Res, 2005;
54: 66-73. 4) Okuda D, Yomogida S, Tamura H, Nagaoka I: Determi-
nation of the antibacterial and lipopolysaccharide-neu-
tralizing regions of guinea pig neutrophil cathelicidin
peptide CAP11. Antimicrob Agents Chemother, 2006;
50: 2602-2607. 5) Okuda D, Yomogida S, Kuwahara-Arai K, Hiramatsu K,
Tamura H, Nagaoka I: Augmentation of the antimicro-
bial activities of guinea pig cathelicidin CAP11-derived
peptides by amino acid substitutions. Int J Mol Med,
2009; 23: 501-508. 6) Kawabata S: Immunocomponent molecules and their
response network in horseshoe crabs. Adv Exp Med
Biol, 2010; 708: 122-136. 97
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https://www.scielo.br/j/pab/a/CDVPmtmxCCQnr8LhRPgqkVC/?lang=pt&format=pdf
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unk
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Efeito do ácido indolbutírico no enraizamento de estacas de ramos semilenhosos de pessegueiro
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Pesquisa Agropecuária Brasileira
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https://www.epj-conferences.org/10.1051/epjconf/202226901023/pdf
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English
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An experimental analysis of the fluid flow on performance and frost formation in exhaust air energy recovery heat exchanger
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EPJ web of conferences
| 2,022
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cc-by
| 3,690
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1 Introduction efficiency values are usually evaluated without taking
into account a dynamic change of fluid flow velocity or
flow rate as well as outdoor and indoor air conditions
which significantly affecting the reduction of heat
recovery efficiency. In 2010 the Energy Performance of Buildings Directive
(EPBD) [1] had been introduced by the European
Commission. This document states that from 2019 all
public buildings in UE will have to meet the
requirements for nearly zero-energy buildings. Additionally during the winter air temperature in cold
and moderate climate zones may drop below minus 5℃
which results in danger for HE icing. This phenomenon
is caused by water dropping out from the exhaust air and
occurs at the contact of the heat exchanger surface with
cold air drawn from the outside [15,16]. Under the
temperatures below zero Celsius, the condensate
deposited in HE change its phase and cause a significant
decrease in the heat recovery and finally increase the
flow resistance. During low outdoor air temperatures,
there is a risk of frost large formation on the heat
exchanger surface which can lead to high-pressure drop,
flow velocity decrease or even serious unit damage. To
prevent heat exchanger from this situation and to keep
constant fluid flow velocity, additional energy supply
has to be implemented. Such a system is usually energy
consuming and significantly reduce system performance. In the literature, various solutions have been proposed to
melt ice deposited on HE surface. One of the most
popular solutions includes the implementation of an
electrical heater inside of HE in the supply air duct. Protection systems warm up the heat exchanger surface
and melt the ice layer. The electrical heater significant
increase the energy consumption and decrease central
unit efficiency. Often a fresh air bypass is also applied. Different manufacturers also apply own solution, in
which to warm-up the heat exchanger they turn-off the
fresh air fans. However, the fresh air flow prevention Heating, as well as air conditioning systems, are
responsible for substantial energy consumption. In many
cases, this consumption exceeds 60% of the total energy
in operated buildings. Due to new regulations, such
systems have to be designed to meet the requirements for
the maximum use of non-renewable energy for heating,
ventilation, cooling and hot water purpose. On the other
hand, heating/cooling and ventilation units are often
increasingly applicable in the domestic application as
well as in industry [2-5]. An experimental analysis of the fluid flow on performance and
frost formation in exhaust air energy recovery heat exchanger
Marek Jaszczur1,*, Marek Borowski2, Michał Karch2, Sławosz Kleszcz1,3
1AGH University of Science and Technology, Faculty of Energy and Fuels, Kraków, Poland
2AGH University of Science and Technology, Faculty of Mining and Geoengineering, Kraków, Poland
3Frapol Sp. z o.o., Kraków, Poland Marek Jaszczur1,*, Marek Borowski2, Michał Karch2, Sławosz Kleszcz1,3 1AGH University of Science and Technology, Faculty of Energy and Fuels, Kraków, Poland
2AGH University of Science and Technology, Faculty of Mining and Geoengineering, Kraków, Poland
3Frapol Sp. z o.o., Kraków, Poland 1AGH University of Science and Technology, Faculty of Energy and Fuels, Kraków, Poland
2AGH University of Science and Technology, Faculty of Mining and Geoengineering, Kraków, Poland
3Frapol Sp. z o.o., Kraków, Poland Abstract. In the present paper, results for fluid flow, temperature and air humidity experimental
measurements for innovative energy recovery counterflow air-to-air heat exchanger have been shown. For
the low outdoor air temperatures case, frost formation is also observed and analysed. Base on the results of
analysis the temperature, humidity and total efficiency of the exchanger are presented. The analysis showed
that fluid flow rate, humidity and system efficiency significantly drop down under unfavourable conditions
in the winter period when the outdoor temperature decrease below -5oC. In the present system, the heat
exchanger equipped with a set of opposing air dampers can use intake air to absorb the moisture from the
exhaust air. This solution has the advantage to prevent the exchanger frosting as well as melt ice layer if any
growth on heat exchanger walls. * Corresponding author: jaszczur@agh.edu.pl © 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/). EPJ Web of Conferences 26
EFM 2019
, 01023 (2022)
9 EPJ Web of Conferences 26
EFM 2019
, 01023 (2022)
9 https://doi.org/10.1051/epjconf/202226901023 DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
ses/by/4 0/) 1 Introduction Public buildings as also quite
often with modern ventilation systems [6,7]. With increasing share of ventilation, the efficient
energy recovery appears to be an essential issue and the
unique solutions for energy saving [7-9]. But such
ventilation type is necessary to keep certain indoor air
quality [10-13]. With the implementation of energy
recovery exchanger, it is a possibly significant decrease
building energy consumption. The typically used for this
purpose heat exchanger (HE) are characterised by high
(often above 90%) recuperation efficiency [14]. Catalogue sheets of heat exchangers typically provide
the temperature efficiency under optimum fluid flow
conditions. Such high-efficiency also results from the
fact that it is usually determined under laboratory
conditions and for very low fluid flow velocity through
the heat exchanger. However, these relatively high- EPJ Web of Conferences 26 , 01023 (2022)
9 EPJ Web of Conferences 26
EFM 2019
, 01023 (2022)
9 https://doi.org/10.1051/epjconf/202226901023 https://doi.org/10.1051/epjconf/202226901023 The experimental measurements were done under real
conditions reproduced in the laboratory to generate low
air temperature and the possibility of occurrence of the
phenomenon of heat exchanger frosting (Table 1). The experimental measurements were done under real
conditions reproduced in the laboratory to generate low
air temperature and the possibility of occurrence of the
phenomenon of heat exchanger frosting (Table 1). fully disturbs the air flow in the entire building not
ensuring acceptable comfort [17]. In many situations, the
ice layer can also be melted by different the ratio of air
supplied to the exhausted application. But such a
solution reduces the amount of fresh air delivery to the
building as well as create substantial negative pressure in
the ventilated space. p
g
g (
)
The test section consisted of the central unit with a
control system, which activates the anti-icing mode
when the fresh air temperature 𝑡21 was below zero
Celsius. The air handling unit was also equipped with a
fresh air bypass, which was closed and deactivated for
the current analysis. The time of dampers switching was
set to 300s. The central unit was placed in an insulated
chamber, inside which the air temperature was about
20+/-1oC. The air was supplied to the air handling unit
by two ducts equipped with glycol heaters/coolers. Figure 1 shows the measurement section of the heat
exchanger. 1 Introduction The temperature of the exhaust air from the
room was maintained on a constant level of 23oC, while
the temperature of the fresh air was equal to -18oC. JUMO EE650-T2L200 airspeed converters were used to
record the volume flow rates (𝑉11 ,𝑉12, 𝑉21, 𝑉22). JUMO
EE210-HT3xPBFxB
hygroscopic
humidity
and
temperature converters were used to measure the air
temperature and humidity (𝑡11, 𝑡12, 𝑡21, 𝑡22 and 𝜙12, 𝜙12,
𝜙21, 𝜙22). The data acquisition from sensors was carried
out using a Mitsubishi Electric FX5U module and a
MAPS HMI 750 panel. The measured values were
recorded with 1-second intervals during the time equal to
2000s. The data recording was activated at the time of
receiving constant temperature of the fresh air. p
In most of the central units, parallel plate counterflow
air-to-air energy recovery heat exchanger are used. This
type of heat exchanger was analysed in [18] in order to
provide a guideline for optimal ventilation unit design. In their study two-dimensional model has was used in
order to find analytical expressions. In other work [19]
authors examined a new type of geometry for HE used in
ventilation systems in cold and moderate climate zones. The methodology for system performance was presented
base on the theoretical model and verified with
experimental data. Fluid flow in parallel-plate turbulent
open channel [20] has been studied successfully using
Direct Numerical Simulation methods which provide
very accurate results. In [21] the influence of channel
geometrical configuration on the counter flow heat
exchanger efficiency has been studied. In this work, flow, temperature and humidity for
innovative energy recovery counterflow air-to-air heat
exchanger equipped with a set of opposing air dampers
have been studied for the selected set of temperatures. Base on the results of analysis the temperature, humidity
and total efficiency of the exchanger is evaluated. For
the low outdoor air temperatures case, frost formation is
also analysed. The present study was aimed to
implement this solution to large industrial units. Fig. 1. Diagram of the test section. Fig. 1. Diagram of the test section. Moisture content in the air: The absolute humidity is defined as a ratio of water
vapour mass in the air to the dry air mass. Using
Dalton’s law and the gas equation of state, it is possible
to obtain a formula to evaluate the moisture content x in
the air based on temperature and relative humidity as
follows: Additionally, the central unit is equipped with four
counter-rotating aluminium dampers controlled by the
actuators. Switching position allows changing the
direction of air in different part of the exchanger. The
fourth tightness class (according to EN-1751) prevent
fresh and used air mixing. The measurement was done to
determine the efficiency of the air handling unit with a
new generation of the high-performance periodic
counter-flow air-to-air heat exchanger with dampers. 𝑥= 0.622 ∙
𝜑∙𝑝𝑔𝑠
𝑝𝑏−𝜑∙𝑝𝑔𝑠 (1) (1) where 𝑝𝑔𝑠= 6.1121 ∙𝑒
17,502𝑇
𝑇+240,97 (2)
𝑝𝑏= 1013.25 (2) 2 Methodology The
periodic-flow
heat
exchanger
unit
under
consideration consists of a standard counter-flow
exchanger equipped with a set of opposing air dampers
with a short opening/closing time, used in order to
cyclically modify air flow direction. With this solution,
the heat exchanger can use intake air to absorb the
moisture from the exhaust air. It has also advantage to
prevent the exchanger frosting as well as melt ice layer if
any appears. The analysed HE used four identical
counterflow exchanger arranged in the right order. Fig. 1. Diagram of the test section. where V, t, ϕ are volume flow rate, air temperature and
humidity respectively. 3 Results and discussion It is worth notice that despite critical air conditions due
to dumpers implementation and flow direction variation,
no frost layer was observed on the heat exchanger
surfaces (see Fig. 7). Despite of not full water droplets
evaporation (condensate), heat exchanger surface was at
good condition. The average amount of condensed water
from extract air was about 5.9 g/kg, and the evaporated
water in supply air increase was about 4.72 g/kg. The
deposited water content was observed as the iced surface
of drip tray shown in Figure 8. It is worth notice that despite critical air conditions due
to dumpers implementation and flow direction variation,
no frost layer was observed on the heat exchanger
surfaces (see Fig. 7). Despite of not full water droplets
evaporation (condensate), heat exchanger surface was at
good condition. The average amount of condensed water
from extract air was about 5.9 g/kg, and the evaporated
water in supply air increase was about 4.72 g/kg. The
deposited water content was observed as the iced surface
of drip tray shown in Figure 8. Figures 2 to 5 shows the experimental results of the air
flow rate of fresh air V21, and exhaust air V11,
temperatures t21, t11 and relative humidity ϕ21, ϕ11 of the
air on each side involved in the energy exchange
process. The volume flow rate of both flows was set-up
to be 2000 m3/h. On the basis of laboratory and
experimental test measurements, the interval of rapid air
flow damper position changes was set-up at the level of
300 s. Different the dumpers position results in different
the direction of air flow through the air-to-air heat
exchanger,
enabling
the
condensate
accumulated
evaporation from the walls of the exchanger in both
operating modes. The deposited at the wall surface water
droplets are evaporated and absorbed by the stream of
outside air. The results in Figure 2 shows that by
switching the air damper position result in the air flow
direction change through the heat exchanger. At the same
time the resistance of the channel flows has changed
which impact on the fluid flow velocity on both sides of
the heat exchanger. The relative humidity is presented in Figure 4. During
the measurements, it changes dynamically between
40%–60% on supply air and 80–90% on waste air. Temperature efficiency: Table 1. Flow Conditions during the study. Parameter
V21
t21
ϕ 21
V11
t11
ϕ 11
Unit
m3/h
oC
%
m3/h
oC
%
Value
2000
-18
70
2000
23
46 The temperature 𝜂𝑡 efficiency is calculated according to
the following equations: The temperature 𝜂𝑡 efficiency is calculated according to
the following equations: η𝑡=
𝑡22−𝑡21
𝑡11−𝑡21 (3) (3) where V, t, ϕ are volume flow rate, air temperature and
humidity respectively. where 𝑡22, 𝑡21, are the temperature of supply and
outdoor air, while 𝑡11 is exhaust air temperature. where 𝑡22, 𝑡21, are the temperature of supply and
outdoor air, while 𝑡11 is exhaust air temperature. 2 EPJ Web of Conferences 26 , 01023 (2022)
9 EPJ Web of Conferences 26
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9 https://doi.org/10.1051/epjconf/202226901023 https://doi.org/10.1051/epjconf/202226901023 3 Results and discussion During the study, the supplied air had the average
humidity level higher than 50%, which effectively
prevented the
drying
of
the
ventilated rooms,
contributing to maintaining the feeling of thermal
comfort for the users. Fig. 4. Fresh 𝜑21, supply 𝜑22, exhaust 𝜑11 and waste 𝜑12 air
relative humidity changes. 0
20
40
60
80
100
3000
3500
4000
4500
5000
Relative humidity, %
Time, s
φ21
φ22
φ11
φ12 Fig. 2. Fresh V21 and exhaust V11 air volume flow rates. 0
1000
2000
3000
3000
3500
4000
4500
5000
Airflow, m3/h
Time, s
V21
V11 Fig. 4. Fresh 𝜑21, supply 𝜑22, exhaust 𝜑11 and waste 𝜑12 air
relative humidity changes. Fig. 2. Fresh V21 and exhaust V11 air volume flow rates. On the basis of the measurements of temperature and
relative humidity values of the air flowing through the
heat exchanger, the moisture content changes in each of
the four air flow streams were evaluated. All these
values, which contribute to the heat and moisture
exchange processes, are presented in Figure 5. The temperature of the supply air t22 presented in Figure
3 was about 14oC, with the temperature values for the
intake t21 and extract air t11 stable all equal to -18oC (+/-
0.5oC), and 23oC (+/-0.5oC), respectively. The above
conditions result in the estimated temperature efficiency
of the exchanger with dumpers in the range of 76-78%
(see Fig. 6). Fig. 5. Fresh 𝑥21, supply 𝑥22, exhaust 𝑥11 and waste 𝑥12 air
humidity changes. 0
2
4
6
8
10
3000
3500
4000
4500
5000
Humidity, g/kg
Time, s
x21
x11
x12
x22 0
2
4
6
8
10
3000
3500
4000
4500
5000
Humidity, g/kg
Time, s
x21
x11
x12
x22 of the exchanger with dumpers in the range of 76 78%
(see Fig. 6). Fig. 3. Fresh t21, supply t22, exhaust t11 and waste t12 air
temperature changes. Fig. 5. Fresh 𝑥21, supply 𝑥22, exhaust 𝑥11 and waste 𝑥12 air
humidity changes. -30
-20
-10
0
10
20
30
3000
4000
5000
Temperature, oC
Time, s
T21
T22
T11
T12
0
2
4
6
8
10
3000
3500
4000
4500
5000
Humidity, g/kg
Time, s
x21
x11
x12
x22 Fig. 3. Fresh t21, supply t22, exhaust t11 and waste t12 air
temperature changes. 3 Results and discussion -30
-20
-10
0
10
20
30
3000
4000
5000
Temperature, oC
Time, s
T21
T22
T11
T12 -30
-20
-10
0
10
20
30
3000
4000
5000
Temperature, oC
Time, s
T21
T22
T11
T12 Fig. 5. Fresh 𝑥21, supply 𝑥22, exhaust 𝑥11 and waste 𝑥12 air
humidity changes. Fig. 3. Fresh t21, supply t22, exhaust t11 and waste t12 air
temperature changes. 3 EPJ Web of Conferences 26 , 01023 (2022)
9 EPJ Web of Conferences 26
EFM 2019
, 01023 (2022)
9 https://doi.org/10.1051/epjconf/202226901023 Fig. 9. Air process heating on Mollier chart (x=const). Pressure
Temperature
Density
Humidity
Ethalpy Fig. 6. The temperature efficiency over time. 0%
20%
40%
60%
80%
100%
3000
3500
4000
4500
5000
Efficiency, %
Time, s Fig. 6. The temperature efficiency over time. Based on the experimental measurement the average
humidity efficiency was evaluated to be equal to approx. 64%. According to Table 2 when humidity is not
exchanged (as the case in traditional standard ventilation
units), the process of heat exchange between fresh and
exhaust air can be shown on Mollier chart - Figure 9. On
these terms, the relative humidity would be only 10%. Table 2. Average value of temperature, relative humidity and
humidity. 𝑡21
−17.8℃
𝜑21
70.7%
𝑥21
0.66 g/kg
𝑡22
13.8℃
𝜑22
55.4%
𝑥22
5.38 g/kg
𝑡11
23.3℃
𝜑21
45.6%,
𝑥11
8.04 g/kg
𝑡12
−5.6℃,
𝜑22
85.7%
𝑥12
2.13 g/kg Table 2. Average value of temperature, relative humidity and
humidity. Fig. 9. Air process heating on Mollier chart (x=const). 4 Conclusions Fig. 7. The heat exchanger surface at the end of measurements. In
this
research,
the
results
of
experimental
measurements of an air handling unit equipped with an
innovative periodic parallel-plate counterflow heat
exchanger with dumpers are presented. Based on the
analysis it is seen that traditional “anti-freezing” system
which utilises different flow rates or even prevents fresh
air flow to melt ice layer on heat exchanger surface
should not be used at low-temperature conditions and
can’t be applied without an additional usually energy
consuming protection method. Additionally, such a
solution doesn’t fulfil its primary role - proper building
ventilation. The deficit of fresh air will result in
significant growth of CO2 concentration in the room as
well as in the air temperature drop. The implementation
of the heat exchanger with a system of air dampers
which change air flow direction may solve wholly issue
of heat exchanger icing. With dumpers, it is possible to
achieve very high-efficiency values - not available by
any other commercial unit. The proposed solution,
remove anti-freeze systems (usually electrical heaters)
completely. For the stable fresh air temperature equal to
-18oC the evaluated temperature efficiency of the
exchanger with dumpers was in the range of 76-78%. In addition to that, based on the experimental
measurement the average humidity efficiency is equal to
approx. 64%. The mean amount of condensed water
from extract air was 5.9 g/kg, while the evaporated water
in supply air increase was 4.72 g/kg. During the analysis, Fig. 7. The heat exchanger surface at the end of measurements. Fig. 8. Iced drip tray at the end of measurements. Fig. 8. Iced drip tray at the end of measurements. 4 EPJ Web of Conferences 26 , 01023 (2022)
9 EPJ Web of Conferences 26
EFM 2019
, 01023 (2022)
9 https://doi.org/10.1051/epjconf/202226901023 19. J. Kragh, J. Rose, T. R. Nielsen, S. Svendsen,
Ener. Build. 39, 1151 (2007)
20. M. Jaszczur, Arch. of Mech. 63,77 (2011)
21. M. I. Hasan, A. A. Rageb, M. Yaghoubi, H. Homayoni, Int. J. Therm. Sci. 48, 1607 (2009) the supplied air the average humidity was higher than
50%, which entirely prevents the drying of the ventilated
space. It is important to notice that low moisture content
in air flow stream may lead to health problems, malaise
and worsens the thermal comfort. At autumn and winter
seasons there is even recommended to humidify the air
in dry rooms. 4 Conclusions However using additional devices people
are exposed to unnecessary investment, operational and
maintenance costs. The proposed periodic-flow heat
exchanger with flow direction switching using dampers
may be an excellent solution for all types of buildings
(public, private, schools or hospitals), allowing to keep
the necessary measure at a comfort level. It has been found that it is important to precisely
determine the time of changing the direction of the fluid
flow (the positions of the air dampers) with correlation
to the outside and indoor conditions thus enabling a
controlled recovery of moisture from the extract air. The works on the periodic-flow heat exchanger are supported
in the Smart Growth Operational Programme 2014-2020 by the
Polish National Centre for Research and Development and
presentation by the Polish Ministry of Science Grant AGH No. 11.11.210.312. 19. J. Kragh, J. Rose, T. R. Nielsen, S. Svendsen,
Ener. Build. 39, 1151 (2007)
20. M. Jaszczur, Arch. of Mech. 63,77 (2011)
21. M. I. Hasan, A. A. Rageb, M. Yaghoubi, H.
Homayoni, Int. J. Therm. Sci. 48, 1607 (2009) References 1. Directive 2012/27/EU of the European Parliament
and of the Council of 25 Oct. (2012) 2. M. Mijakowski, J. Sowa, P. Narowski, Civil
Engineering 4, 107 (2010) 3. G. P. Vasilyev, A. Tabunshchikov, M. M. Brodach,
V. A. Leskov, N. V. Mitrofanova, N. A. Timofeev,
V. F. Gornov, G. V. Esaulov, Ener. Build. 112, 96
(2016) 4. V.Yu. Borodulin, M.I. Nizovtsev, App. Therm. Eng. 130, 1246 (2018) 5. S. Koestera, M. Falkenberga, M. Logemanna, M
Wessling, J. Memb. Sci. 525, 68 (2017) 5. S. Koestera, M. Falkenberga, M. Logem Wessling, J. Memb. Sci. 525, 68 (2017) 6. B. R Hughes, H. N. Chaudhry, J. K. Calautit, Appl. Energy 113,127 (2014) Energy 113,127 (2014) 7. C. Simonson, Energy Build. 37, 23 (2005) 8. X. P. Liu, J.L. Niu, Appl. Energy 129, 364 (2014) 9. El Fouih, Y. Stabat, P. Rivie`re, P. Hoang, P. Archambault, Ener. Build. 54, 29 (2012) 10. M. Orme, Energy Build. 33, 199 (2001) 11. J. K. Calautit, B. R. Hughes, H. N. Chaudhry, S. A. Ghani, Appl. Energy 112, 576 (2013) 12. M Jaszczur, M Branny, M Karch, M Borowski, J. Phys. Conf. Ser. 745, 032049 (2016) 13. M. Borowski,
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The Role of Methyl Jasmonate During the Biotic Stress in Matricaria Chamomilla L.
|
Research Square (Research Square)
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cc-by
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The Role of Methyl Jasmonate During the Biotic
Stress in Matricaria Chamomilla L. Zuzana Dučaiová
(
zuzana.kovalikova@uhk.cz
) Pavol Jozef Stafarik University in Kosice Faculty of Science: Univerzita Pavla Jozefa Safarika v
Kosiciach Prirodovedecka fakulta Highlights MeJA pre-treatment ameliorate negative effect of thrips thrips feeding enhanced PAL activity and coumarins content diploid cultivar reacted more significantly compared to the tetraploid one Research Article Keywords: Matricaria chamomilla, Echinothrips americanus, methyl jasmonate, coumarin-related
compounds
Posted Date: September 13th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-3333626/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Posted Date: September 13th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3333626/v1 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/20 Abstract The objective of the present study was to evaluate the effect of methyl jasmonate (MeJA) in plant
resistance to polyphagous thrips. Eight-weeks old diploid and tetraploid chamomile plants (Matricaria
chamomilla) in stage of leaf-rosettes were pre-treated with 0.22 mM MeJA for 72 h and then co-cultivated
with thrips for next 96 h. In both cultivars, there was an increase in the content of hydrogen peroxide and
superoxide radical, with a maximum in plants after thrips infestation. The pre-treatment with MJ led to a
reduction, but the measured values were still higher than those in the control plants. The accumulation of
total phenols and flavonoids corresponded with the activity of phenylalanine ammonia-lyase (PAL). The
highest PAL activity and the highest levels of total phenols were observed in plants infected with thrips. MeJA pre-treatment slightly decreased their values. Within the cultivars we noticed differences in the
accumulation of monitored coumarins: (Z)- and (E)-GMCA, herniarin and umbelliferone. In diploids MeJA
significantly ameliorated their accumulation caused by thrips. On the other hand, in tetraploids, changes
were minimal. The amount of umbelliferone sharply increased in both cultivars which points to its role as
a stress metabolite. INTRODUCTION Various fungal, bacterial and parasitic diseases degrade the quality of important medical herbs and
crops. During the long co-evolution plants have developed various defensive strategies. In addition to
always expressed constitutive defence represented by physical (e.g. thorns, trichomes) or chemical (e.g. repellent or toxic chemicals) barriers, plants respond with a more specific induced defence. It is triggered
specifically after an herbivore attack and thus allows plants to effectively manage their energy reserves
via synthesis defence compounds only when needed. At the same time, volatile substances can act as
attractants for herbivore predators (Mouden and Leiss 2021; War et al. 2012). In general, feeding stimuli by chewing herbivores or certain phloem feeder are transduced via a signalling
pathway mediated through the synthesis of jasmonate (JA) compounds (Lazebnik et al. 2014). Artificial
manipulation of jasmonates induces the expression of JA-responsive genes, resulting in the formation of
chemical and physical barriers in a pattern similar to herbivory. For example, triggering of synthesis of
primary metabolites such as amino acids or sugars (Chen et al. 2020; Wei et al. 2021), secondary
metabolites (Chen et al. 2020), enhancing antioxidant activity (Tianzi et al. 2018) or trichome density
(Mouden et al. 2020). This state of preparedness for the attack of herbivores can subsequently manifest
itself as a higher percentage of plant survival after the insect attack (Tierranegra-García et al. 2011),
reducing oviposition or increasing larval and adult’s mortality (Kawazu et al. 2013; Senthil-Nathan 2019). Page 2/20 Page 2/20 In addition, some author suggests that application of volatile methyl jasmonate (MeJA) in inducing
jasmonate response could be more effective than that of JA itself due to its high volatility and easy entry
through stomata (Jang et al. 2014; Jiang and Yan 2018). In addition, some author suggests that application of volatile methyl jasmonate (MeJA) in inducing
jasmonate response could be more effective than that of JA itself due to its high volatility and easy entry
through stomata (Jang et al. 2014; Jiang and Yan 2018). Echinothrips americanus is a polyphagous thrip species that in a very short time occupy large areas of
plants. Their original territory was east part of North America and their first European catch was recorded
in 1989. Since then, this species infested rapidly at least in 19 European countries. It can be easily
recognized due to the white stripes pass across the chest and at the base of the wings. INTRODUCTION It has piercing-
sucking mouthparts and feed on plant juices sucking from a wide range of host plants (Varga et al. 2010) German chamomile (Matricaria chamomilla L.) belongs to a major group of cultivated medicinal plants. It
owes its therapeutic activity to different groups of effective substances; such as sesquiterpenes,
flavonoids, coumarins (herniarin and its glucosidic precursors (Z)- and (E)-2-β-d-glucopyranosyloxy-4-
methoxycinnamic acid (GMCA); umbelliferone) and polyacetylenes (Franke and Schilcher 2012). Several
papers, dealing with the changes of mentioned metabolites as a result of plant reaction to biotic stimuli,
approved their involvement in plant defence processes (Repcak and Suvak 2013; Repcak et al. 2001). The
results of previously published works (Repcak et Suvak 2013; Ducaiova et al. 2016) pointed out that the
impact of phytophagous insects or/and at the same time MeJA increased the level of chamomile
secondary metabolites. Since the used MeJA concentration (0.4 mM) significantly affected the
accumulation of the total phenols and coumarins (Ducaiova et al. 2016), here we selected lower
concentration, only 0.2 mM, for stimulation the defence processes. Based on these studies, we examined
whether the exogenously added low concentration of MeJA before attack of E. americanus could
ameliorate its negative impact. The induced stress intensity was evaluated at biochemical level by
measuring of reactive oxygen species and total proteins content, as well as phenolic metabolites involved
in defence responses, i.e. total phenolic and flavonoid content and coumarin content. Polyploidisation, or genome duplication, is common in higher plants and is considered to be an
adaptation mechanism which played significant role in plant speciation. Induces polyploidisation can
influence overall plant physiological and biochemical processes and affect the biosynthetic pathways of
primary and secondary metabolites (Madani et al. 2021). In several species the production of primary and
secondary metabolites was higher in polyploids than in their diploid counterparts, even qualitative
differences were displayed (Moetamedipoor et al. 2022; Park et al. 2021). Although overall enlargement
of the plant organs was observed in chamomile in tetraploid cultivars, a clear correlation between the
increase in the level of active compounds was not recorded (Repčák and Krausová 2009). However, under
the impact of environmental stimuli or hormone treatment, the responses of diploid plants were more
stimulated (Ducaiova et al. 2020; Petruľová et al. 2014). Therefore, the second aim of the present study
was to evaluate defence responses of two chamomile cultivars differing in ploidy level (diploid vs. tetraploid). MATERIAL AND METHODS MATERIAL AND METHODS Page 3/20 Plant cultivation and experimental design Seeds of chamomile (Matricaria chamomilla L.), diploid cv. 'Novbona' and tetraploid cv. 'Lutea', were
germinated in sterile sands. Three-week-old plants were transferred into plastic pots filled with garden
substrate. Eight-week-old plant in stage of leaf rosettes were used for the experiment. The cultivation was
held in a growing chamber (Fitotron®, Weiss Technik) under controlled 12-h photoperiod lighting
conditions and temperature 25/20°C (day/night). The soil water-holding capacity was maintained to 60%
with daily moistening. Thrips (Echinothrips americanus Morgan) were collected on the leaves of the
Alocasia macrorrhiza in the greenhouse of the Botanical Garden using the exhauster. Overall, four
different variants were tested: C – control plants sprayed with distil water C – control plants sprayed with distil water MeJA – plant treated with 0.22 mM aq. solution of MeJA T – plant treated with 100 pieces of thrips for 96 hours T – plant treated with 100 pieces of thrips for 96 hours MJT – plant pre-treated with MeJA for 72 hours and subsequently treated with 100 pieces of thrips for
the next 96 hours. All plants were placed under the glass bells to prevent the spread of volatile MeJA and insect. A total of
three glass bells with 8 plants were used for each variant. Whole leaf rosettes were used for all analyses. Damage caused by thrips feeding (hereinafter referred to as “silver damage”) was evaluated visually and
expressed as total damaged leaf area in mm2. Quantification of stress-related parameters Potassium phosphate buffer (50 mM; pH 7.0) homogenates were used for hydrogen peroxide and
superoxide radical determination. The accumulation of hydrogen peroxide was estimated according to
titanium chloride method (410 nm) and superoxide radical was estimated using hydroxylamine for the
nitrite formation (530 nm). The amount of malondialdehyde (MDA) monitored by thiobarbituric acid
reactive substance assay (TBARS) was used as an expression of the level of membrane lipid
peroxidation (Dučaiová et al. 2016). Quantification of total phenols, flavonoids a Total soluble phenols (TP) and flavonoids (TF) were extracted from fresh leaf rosettes with 80%
methanol. Their content was determined spectrophotometrically at 750 nm using the Folin–Ciocalteu
method (Ducaiova et al. 2016) with gallic acid as standard (Sigma-Aldrich) for TP, and at 420 nm using
aluminium chloride method (Ordoñez et al. 2006) with quercetin (Sigma-Aldrich) as standard for TF. The activity of phenylalanine ammonia-lyase (PAL) was measured as a production of t-cinnamic acid (t-
CA) from phenylalanine in sodium borate buffer (Ducaiova et al. 2016). The HPLC conditions: column
Kromasil SGX C18 7 µm (150 × 4.6 mm); flow rate 0.8 ml min-1; mobile phase: 55% acetonitrile. Detection Page 4/20 was performed at 275 nm using t-CA standard compound (Sigma-Aldrich). The activity of PAL was
expressed as nmol t-CA min-1 mg-1 proteins. Total soluble proteins were quantified in phosphate buffer
extracts according to Bradford (1976) with bovine serum albumin (Bio Rad) as standard. Quantification of secondary metabolites Quantification of secondary metabolites The 80% methanol was used for extraction of secondary metabolites. The separation of coumarin-related
compounds was performed using HPLC-DAD (Agilent Technologies 1260 Infinity device) on a reversed-
phase column LiChroCart 4–4, C-18 (5 µm). The mixture of mobile phases A (water with 1% trifluoroacetic
acid) and B (55% acetonitrile) was used in gradient programme with flow rate of 0.7 ml min-1: 0–15 min
40% B; 15–25 min 100% B; 25–35 min 100% B; 35–40 min 0% B. A wavelength of 320 nm was used for
quantitative analysis. For the separation of dicycloethers the mobile phase was changed to the mixture of
A (acetonitrile:water:trifluoroacetic acid; 19:80:1) and B (90% acetonitrile) in gradient programme: 0–23
min 70% B; 23–25 min 0% B (Ducaiova et al. 2020). Commercially available standards were used to construct calibration curves: herniarin (≥ 98%;
Extrasynthese), umbelliferone (≥ 99%; Fluka), skimmin (Apin Chemicals), daphnin (≥ 95%;
Extrasynthese), chlorogenic acid (≥ 98%; Fluka) and 1,5-dicaffeoylquinic acid (≥ 95%; Fluka). For
commercially unavailable standards (Z) - and (E) -GMCA, 5-feruloylquinnic acid and dicycloethers
isolated and purified substances with confirmed identity based on NMR analyses were used. Statistical analysis The comparison of differences in experiment was based on one-way analysis of variance (ANOVA) and
Tukey’s test at the significance level p < 0.05 (GraphPad Prism 9 Software, LLC, San Diego, CA, USA). Six
individual plants mixed from three glass bells were used for analyses of each parameter. The whole
experiment was repeated twice. Effect of thrips on plant oxidative status During the experiment, any significant increment or drop of biomass compared to the control plants was
record (0.11 g ± 0.003 for both cultivars). MeJA treated plants exhibited mild leaves curling with
prolonged isolation inside a glass bell. Exposure to thrips led to the visible changes in plants. On a closer
study, numerous silver or brownish diffuse spots were observed, which in older leaves changed into the
small necrotic lesions. This negative effect was in MJ pre-treated plants significantly reduced (Fig. 1). In both cultivars, there was an increase in the content of hydrogen peroxide, with a maximum in plants
after thrips infestation, more than 2-fold for diploid and 1.5-fold for tetraploid plants (Fig. 2). The pre-
treatment with MeJA led to a reduction, but the measured values were still higher than those in the control
plants. In the case of the superoxide radical (Fig. 2), a similar accumulation trend was observed. The
highest values were found in T variants, 2-fold for diploid and 1.2-fold for tetraploid plants. In the diploid Page 5/20 Page 5/20 cultivar, the addition of MeJA significantly reduced the level of superoxide radical, while in tetraploid
plants the reduction was negligible. Changes in reactive oxygen species (ROS) accumulation led to a
membrane damage only in the plants affected by thrips, where an approximately 1.3-fold increase in
MDA content in both cultivars was observed (Fig. 2). The addition of MeJA reduced the content of MDA,
but significantly only in diploid cultivar. Overall, the MDA content in tetraploid plants was approximately
twice as high as in diploid ones. The maximum values of total soluble proteins were recorded in T variants and MeJA pre-treatment
reduced their values, but significantly only in case of diploid plants. Overall diploid plants showed higher
values and subsequent increment was much more pronounced here, almost 2-fold. Effect of thrips on phenol metabolism The content of TP (Fig. 3) sharply increased and the maximum values were recorded in T variants, 1.6-
fold increase for diploid and 2-fold increase for tetraploid plants. A similar accumulation trend was
observed for TF (Fig. 3). Overall, the addition of MeJA before the thrips ameliorate their negative impact
only in case of TF, where there was a significant decrease, but the values were still higher compared to
control values. The accumulation of TP and TF corresponded to the PAL enzyme activity (Fig. 3). The
highest enzymatic activity was observed in plants infested with thrips, 1.8-fold for diploids and 1.3-fold
for tetraploids. Pre-treatment with MeJA slightly decreased their values. Surprisingly, a significant drop in
the activity after application of MeJA alone was monitored in both cultivars. Overall, the values in tetraploid plants were lower compared to diploid ones. A more pronounced
difference occurred in the case of flavonoids and PAL activity, where the values in MJT and T variants did
not rise as rapidly as in the case of the diploid cultivar. The effect of thips and MeJA pre-treatment was variously reflected in the accumulation of coumarins and
their glucosidic precursors (Fig. 4) in both cultivars. In diploid plants, thrips infestation resulted in a
significant increment compared to control almost in all monitored coumarins, with exception for (Z)-
GMCA where the increase was only modest but still significant. Pre-treatment with MeJA reduce elevated
level, but significantly only in case of umbelliferone and its glucosidic precursor skimmin, 3.7-times and
3.2-times, respectively. MeJA alone increased the values of coumarins, but their levels still remained lower
in comparison to MJT and T variants. The accumulation trend in tetraploid plants was similar to diploids, with exception for skimmin and (Z)-
GMCA. Here, a significant increment over control values was observed, but with moderate differences
between tested variants. Like in diploids, sharp, almost 2-fold increment was in case of stress coumarin
umbelliferone. Changes of three simple phenylpropanoids, namely chlorogenic acid, 1,5-dicaffeoylquinic acid, and 5-
feruloylquinic acid, we monitored. The thrips invasion significantly raised their levels (Fig. 5). Pre- Page 6/20 treatment with MeJA reduced their levels, more pronounced in diploid plants in the case of 1,5-
dicaffeoylquinic acid and 5-feruloylquinic acid, more then 1.5-times. Effect of thrips on dicycloethers As shown in Fig. 6, more enhanced accumulation of both (Z)- and (E)- isoforms of dicycloethers after
thrips infestation was in the case of diploid plants, 3.5-fold and 1.8-fold, respectively. The protective
effect of MeJA showed a significant decrease only in the case of (E)-isoform. In tetraploid plants, the
increment was moderate, around 1.4-times for both isoforms. Here, in (Z)-isoform, the MeJA pre-
treatment reduced its level to the control values. DISCUSSION (2018) pointed out that MeJa-treated leaves of Populus x
euramericana led to increased ROS generation, which was subsequently reflected in enhanced synthesis
of antioxidant enzymes in Clostera anchoreta larvae fed on these leaves. et al. 2014). The study of Tianzi et al. (2018) pointed out that MeJa-treated leaves of Populus x
euramericana led to increased ROS generation, which was subsequently reflected in enhanced synthesis
of antioxidant enzymes in Clostera anchoreta larvae fed on these leaves. Overall, the characteristics of ROS accumulation can vary depending on the plant type and the invading
insect. For example, in Arabidopsis, accumulation maximum of hydrogen peroxide after the oviposition of
P. brassicae was after 72 hours (Little et al. 2006), while the attack of the phloem feeding aphid
Brevicoryne brassicae showed changes after only 48 hours (Kunierczyk et al. 2008). In the case of
Bemisia tabaci, no oxidative burst occurred even after 21 days of feeding (Kempema et al. 2006). In the
aforementioned works, in parallel with the increasing concentration of ROS, an increased activity of not
only antioxidant enzymes, specifically superoxide dismutase, catalase, peroxidase or polyphenoloxidase,
but also other effective antioxidants, such as ascorbate and glutathione, was recorded. At the same time
a number of corresponding genes associated with oxidative stress were reported to be upregulated
(Kempema et al. 2006; Kunierczyk et al. 2008). Malondialdehyde (MDA) represents a decomposition product of peroxidation of polyunsaturated fatty
acid, and its concentration is usually correlated with the degree of membrane lipid degradation. Moreover,
it has been suggested that elevated level of MDA may assist in the synthesis of more complex defence
compounds and activate antioxidative enzymes. In addition, the emissions of leaf volatiles partially
involved in indirect plant defence, are induced by lipid peroxidation after herbivore damage. Our results
are in line with earlier reports, where MDA level was induced in cucumber plants infested with Bemisia
tabaci (Zhang et al. 2008) or in rice plants attacked by Cnaphalocrocic medinalis (Chen et al. 2022). The
rate of MDA formation can also depend on the insect type, e.g. scraping-sucking feeding activity is more
stressful due to its concertation on large surface of plant tissues than the phloem-sucking insects which
affect only vascular bundles (Kuster et al. 2022). In addition to secondary metabolites, which are the main components of chemical defence, proteins also
play an important role. DISCUSSION The ability of plants to recognize and adequately respond to herbivores attack is essential for their
survival. Important hormones in such induced resistance are JA or salicylic acid, which trigger many
defence mechanisms that reduce herbivore survival, reproductive capacity, or preference for a plant. It has
been well documented that exogenous applications of JAs can protect plants against herbivores by
inhibiting or disturbing their normal development and reproduction processes, e.g. reduction in egg
numbers (Shahabinejad et al. 2014), larval and pupal average weight (Jiang and Yan 2018) or fecundity
(Tianzi et al. 2018). Even with increasing MeJA concentration, the total weight of larvae and adults and
their survival time decreased (Senthil-Nathan 2019). These results suggest that pre-treatment of plants
with JAs could provide a greater opportunity for plant survival via the activation of JA-responsive genes
(Selig et al. 2016). E. americanus represent phloem feeding insect whose infestation of piercing-sucking mouthparts is
manifest as silver dots, which later turn into necrotic lesions. In our work, numerous silver or brownish
diffuse spots were observed which were slightly reduced in MeJA pre-treated plants. Similarly, a foliar
application of JA caused a marked reduction in silver damage caused by western flower thrips which had
dose-dependent trend (Mouden et al. 2020) or locally-dependent trend (Chen et al. 2020). Insect feeding activity provokes a local wound, and consequently triggers various metabolic responses
driven by ROS formation. Apoplastic ROS burst acts as a first barrier against the pathogens and
herbivores attack. Their elevated levels are not only toxic to the invasive organisms, but also to the plants
themselves. Oxidative damage to significant biomolecules can lead to programmed cell death, which is
part of a hypersensitive reaction (War et al. 2012). Among ROS, highly stabile and freely diffusible
hydrogen peroxide may also act as secondary messenger by triggering the expression of defence genes
and synthesis of various secondary metabolites (Fürstenberg-Hägg et al. 2013). Moreover, hydrogen
peroxide has been found to have direct toxicity against insects (Maffei et al. 2007). In our experiment, we
noted significantly higher ROS level after thrips invasion, which was ameliorated via MeJA pre-treatment. Similar results were also recorded in white cabbage infected with larvae of Pieris brassicae or Phyllotreta
nemorum (Kovalikova et al. 2019) or in sorghum plants attacked by the butterfly Chlio partellus (Hussain Page 7/20 Page 7/20 et al. 2014). The study of Tianzi et al. DISCUSSION Proteins are the main nutrient for insect growth and changes in their content can
affect the overall physiological state of herbivores by reducing their growth rate, adult size or survival
rate. In this study, a sharp increase in protein content was noted. Hussain et al. (2014) reported similar
results, where their content increased even more after the addition of JA. Increased levels of proteins may
reflect the plants' current need for antioxidant enzymes or other protein-based defensive compounds. For
example, MeJA spraying on Larix olgensis seedlings significantly increased the bio-activities of
superoxide dismutase, polyphenol oxidase and two proteinase inhibitors, which resulted in strongly
reduced larval/pupal weights and survivals, as well as the fecundity of Lamantria dispar (Jiang and Yan
2018). Among the secondary metabolites, plant phenols constitute one of the most common and widespread
group of defensive compounds, which play a major role in plant resistance against herbivores, including
insects. In plants, polyphenols are synthesized through the phenylpropanoid pathway in which L-
phenylalanine is converted to cinnamic acid via PAL enzyme. Further, different enzymes at different levels Page 8/20 Page 8/20 modulate the structure of the compounds formed at different levels of the pathway (Singh et al. 2021). PAL together with polyphenol oxidase have been considered as key enzymes in phenol metabolism, and
their activity has been correlated to plant protection against insect feeding. In our study, the accumulation
of phenols and flavonoids corresponded with the enzymatic activity of PAL with the maximum in plants
attacked by thrips. Pre-treatment with MeJA slightly reduced PAL activity together with phenol levels. Similar results were also found in sorghum plants (Hussain et al. 2014), but on the contrary, an increased
level of phenols and flavonoids was recorded in lettuce under the influence of MeJA and pill bugs
(Tierranegra-García et al. 2011). Moreover, the level of activity may also depend on the type of plant
cultivar. Aphid induced stronger enzyme elevation during the early period after infestation in resistant
cultivars of chrysanthemum than to susceptible (He et al. 2011). Changes were also noted at the level of
gene expression. For example, in pepper plants, the relative expression of PAL gene increased with
prolonged invasion of western flower thrips (Zheng et al. 2019). Surprisingly, Selig et al. DISCUSSION This negative effect is reduced by their sequestration into an inactive form in vacuoles,
mostly by conjugation with glucose. With an increased need for active aglycones, the bond is broken by a
specific glucosyltransferase (Singh et al. 2021). Occurrence of cellular membrane disruption, monitored
here as an elevated level of MDA, should lead to a decrease in glycosidic form, GMCA and skimmin, and
an increase in aglycones, herniarin and umbelliferone. Surprisingly, continuous increment in all
mentioned coumarins was monitored here. Similar results were found in chamomile plants treated with
salicylic acid (Ducaiova et al. 2013) or MeJA (Ducaiova et al. 2016). The involvement of coumarins in defence processes suggests both their elevated concentrations in
stressful conditions and also their antioxidant potential linked with the presence of conjugated system,
ester bonds and with the number of bound hydroxyl groups. According to Lin et al. (2008), coumarins
with higher amount of hydroxyl groups express better scavenging properties than unhydroxylated
coumarins. From hydroxylated coumarins, we observed strong increment in the content of umbelliferone
(7-hydroxycoumarin), but surprisingly, daphnetin (7,8-dihydroxycoumarin), was found only in trace
amounts, but its glucosidic precursor daphnin (daphnetin-7-O-glucoside) significantly rose in MJT and T
variants. The work of Petruľová-Poracká et al. (2013), points out that there are significantly higher
concentrations of daphnetin in the anthodia than in the leaves. Umbelliferone plays an important role, both as a stress coumarin and as a precursor for the synthesis of
other coumarins (herniarin, esculetin or skimmin) (Chu et al. 2017). In this way, low amounts of
umbelliferone under normal physiological conditions and elevated levels under stressful conditions were
expected. Both in our experiments and in abovementioned works, a significant increase in umbelliferone
was noted. Polyacetylenes of spiroketal enol ether type are known secondary metabolites synthesized by Asteraceae
plants. Together with other substances such as monoterpenes or sesquiterpenes, they are part of the
volatile substances that create the typical aroma. In addition, they play an important role in plant defence
thanks to their toxic properties, such as growth and reproductive inhibitors, repellents, and oviposition-
deterrent (Minto and Blacklock 2008). Several studies showed that essential oils or substances alone
showed strong larvicidal potential against mosquito (Ali et al. 2018; Govindarajan and Benelli 2016). DISCUSSION (2016) pointed
out that the expression level can also be influenced by the type of insect itself, when phloem-feeder thrips
upregulated and cell-content feeder aphid downregulated soybean PAL2 expression. Among the phenolic acids, chlorogenic acid is the most abundant among the caffeoylquinic acid isomers
in nature. It is considered as a defence metabolite in plants that provides protection against different
herbivores via its oxidation to chlorogenoquinone which binds to free amino acids and proteins and
reduced their digestibility. Higher accumulation of chlorogenic acid has been described in several plant
species regardless of the feeding method, i.e. stem boring (Lee et al. 2017), piercing–sucking (Leiss et al. 2009) and chewing (Kundu et al. 2018). In these studies, anti-herbivore effect of chlorogenic acid was
also confirmed by dietary tests, when a growth retardation, survival rate or larval weight reduction was
detected. In our work, we noted an increase not only in chlorogenic acid, but also in 1,5-dicaffeoylquinic
acid and 5-ferruloylquinic acid, both due to MeJA and thrips. Overall, the levels were higher in the case of
the diploid cultivar, which, according to the conclusions of the study by Leiss et al. (2009), indicated that
cultivars with higher values of the mentioned metabolites showed higher resistance. In addition to
chlorogenic acid, other phenolic acids can also play an important role in protection. For example,
exogenous application of cinnamic and vanillic acid negatively affected the oviposition and feeding
preference of the larvae of Achaea janata and Spodoptera litura on castor plants (Usha Rani and
Pratyusha 2014). Among other phenolic metabolites we focused on coumarins, which content was significantly influenced
by thrips infestation. Almost in all cases, pre-treatment with MeJA reduced their levels. A significant
elevation not only due to the effect of thrips, but also due to the effect of MeJA itself was monitored in
the work Repčák and Suvák (2013), so we can conclude that concentration (0.4 mM) used here could
already have a stressful rather than acclimatizing effect. Increased levels of coumarins in chamomile
plants were also observed under the influence of various abiotic, e.g. heavy metals (Kováčik et al. 2012),
nitrogen nutrition (Sajko et al. 2018) or UV radiation (Petruľová et al. 2014), and biotic stimuli, e.g. Erypside cichoracearum infection (Repčák et al. 2001). Page 9/20 Page 9/20 Many phenolic metabolites, including coumarins, are toxic not only to herbivores, but also to the plants
themselves. DISCUSSION The
literature data about the changes of dicycloethers in chamomile plants during the stress conditions are
expressed in two way, as their sum or as free form, so it is difficult to compare to our results. Similar to
our results, an increasing trend was observed after biotic stress (Repčák et al. 2001). However, MeJA
application alone resulted in different accumulation trends, an increase in the (Z)- isoforms and a
decrease in the (E)-isoforms (Dučaiová et al., 2016). In the case of other abiotic stressors, a
predominantly declining trend was observed (Repčák et al. 2001, Sajko et al. 2018). An increased ploidy level not only leads to a higher accumulation of pharmaceutically important
metabolites, but can also help better adaptation to environmental stimuli. Yang et al 2021 showed that
plants with higher ploidy level produced more phenolic metabolites with allelopathic effects, which Page 10/20 Page 10/20 allowed seedlings to survive better and colonize new habitats faster. The effect of ploidy level on the rate
of attack by herbivores is not yet completely clear. Authors report both a lower (Nuismer et Thompson
2001) and a higher attack rate (Arvanitisa et al. 2010) in polyploid plants. Furthermore, it is known that
polyploidy can cause several changes in physiology, morphology, and gene expression that can impact
resistance to herbivores. For example, in Brassicales, genome duplication led to an increased rate of
glucosinolate synthesis, which play a key role in defence against invading butterflies (Edger et al. 2015). Qualitative changes of terpenes in Solidago gigantea, namely an increase in sesquiterpenes in tetraploids
and, conversely, an increase in monoterpenes and diterpenes in hexaploids, led to changes in the survival
and growth of invading herbivores (Hull-Sanders et al. 2009a, b). Due to the fact that in the case of our experiment diploid and tetraploid plants were separated, it is not
possible to clearly determine the preference of invading thrips. Macroscopically observable
manifestations of feeding in the form of silver and brown spots did not show significant differences in
number between cultivars. Activation of defence mechanisms was manifested by increased
accumulation of total proteins (possibly increased synthesis of antioxidant enzymes) and increased
accumulation of coumarins and phenolic acids with potential antioxidant properties (chlorogenic acid, 5-
dicaffeoylquinic acid, and 5-feruloylquinic acid). A higher content of phenolic metabolites in diploid
Centaurea phrygia plants compared to tetraploid plants was also noted by Münzbergová et al. DISCUSSION (2015),
which led to increased resistance to insects. However, the total content of hydrogen peroxide and the rate
of its increase was significantly higher in the case of diploid plants, which indicates a higher level of
stress. Overall, we can say that diploid plants responded to stress to a higher extent, but the use of MeJA
as a protective agent alleviated the subsequent insect-induced stress more significantly than tetraploid
plants. ACKNOWLEDGEMENTS The authors are grateful to Excellence project PřF UHK 2216/2022-2023 for the financial support. We the authors declare that this manuscript is original, has not been published before, and is not currently
being considered for publication elsewhere. We confirm that the manuscript has been read and approved by all named authors and that there are no
other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order
of authors listed in the manuscript has been approved by all of us. The authors have no competing interests to declare that are relevant to the The authors have no competing interests to declare that are relevant to the content of this article. competing interests to declare that are relevant to the content of this article. editing: Zuzana Kovalikova. All authors revised the manuscript and approved its final version. editing: Zuzana Kovalikova. All authors revised the manuscript and approved its final version. AUTHOR CONTRIBUTION Page 11/20
Conceptualization: Zuzana Kovalikova, Veronika Petrulova; methodology: Zuzana Kovalikova, Veronika
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Figure 1
Feeding damage by thrips on chamomile leaf with or without MeJA pre-treatment. Data are means ± SDs
(n = 12). Values within column, followed by the same regular or italic letter(s), are not significantly
different according to Tukey’s test (p< 0.05) References Wei X, Vrieling K, Kim HK, Mulder PPJ, Klinkhamer PGL (2021) Application of methyl jasmonate and
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59. Zhang SZ, Hua BZ, Zhang F (2008) Induction of the activities of antioxidative enzymes and the
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pathways, western flower thrips, and thrips-transmitted tomato zonate spot virus infection in
Capsicuum annuum. Arthropod-Plant Interact 13:289–297 Aleyrodidae) infestation. Arthropod-Plant Interact 2:209–213. Aleyrodidae) infestation. Arthropod-Plant Interact 2:209–213. Aleyrodidae) infestation. Arthropod-Plant Interact 2:209–213. 60. Zheng X, Chen Y, Zhao L et al. (2019) Tripartite interactions between jasmonic/salicylic acid
pathways, western flower thrips, and thrips-transmitted tomato zonate spot virus infection in
Capsicuum annuum. Arthropod-Plant Interact 13:289–297 60. Zheng X, Chen Y, Zhao L et al. (2019) Tripartite interactions between jasmonic/salicylic acid
pathways, western flower thrips, and thrips-transmitted tomato zonate spot virus infection in
Capsicuum annuum. Arthropod-Plant Interact 13:289–297 Figure 1 Feeding damage by thrips on chamomile leaf with or without MeJA pre-treatment. Data are means ± SDs
(n = 12). Values within column, followed by the same regular or italic letter(s), are not significantly
different according to Tukey’s test (p< 0.05) Page 16/20 Figure 2 Figure 2 Effect of MeJA pre-treatment and thrips infestation on the content of hydrogen peroxide (µmol g-1 DW),
superoxide radical (µg g-1 DW), malondialdehyde (µmol g-1 DW), and total soluble proteins (mg g-1 DW) in
chamomile leaf rosettes. Data are means ± SDs (n = 12). Values within column, followed by the same
regular or italic letter(s), are not significantly different according to Tukey’s test (p < 0.05) Effect of MeJA pre-treatment and thrips infestation on the content of hydrogen peroxide (µmol g-1 DW),
superoxide radical (µg g-1 DW), malondialdehyde (µmol g-1 DW), and total soluble proteins (mg g-1 DW) in
chamomile leaf rosettes. Data are means ± SDs (n = 12). Values within column, followed by the same
regular or italic letter(s), are not significantly different according to Tukey’s test (p < 0.05) Page 17/20 Page 17/20 Figure 3
Effect of MeJA pre-treatment and thrips infestation on the content of total soluble phenols (mg g-1 DW),
flavonoids (mg g-1 DW) and PAL activity (nmol t-CA min-1 mg-1 prot.) in chamomile leaf rosettes. Data are
means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not
significantly different according to Tukey’s test (p < 0.05) Figure 3 Effect of MeJA pre-treatment and thrips infestation on the content of total soluble phenols (mg g-1 DW),
flavonoids (mg g-1 DW) and PAL activity (nmol t-CA min-1 mg-1 prot.) in chamomile leaf rosettes. Data are
means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not
significantly different according to Tukey’s test (p < 0.05) Effect of MeJA pre-treatment and thrips infestation on the content of total soluble phenols (mg g-1 DW),
flavonoids (mg g-1 DW) and PAL activity (nmol t-CA min-1 mg-1 prot.) in chamomile leaf rosettes. Data are
means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not
significantly different according to Tukey’s test (p < 0.05) Page 18/20 Figure 4
Effect of MeJA pre-treatment and thrips infestation on the content of coumarin-related compounds (m
1 DW). Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic
letter(s), are not significantly different according to Tukey’s test (p < 0.05) Figure 4 Effect of MeJA pre-treatment and thrips infestation on the content of coumarin-related compounds (mg g-
1 DW). Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic
letter(s), are not significantly different according to Tukey’s test (p < 0.05) Page 19/20 Page 19/20 Page 19/20 pre-treatment and thrips infestation on the content of simple phenylpropanoids (mg g-1
means ± SDs (n = 12). Values within column, followed by the same regular or italic
significantly different according to Tukey’s test (p < 0.05)
pre-treatment and thrips infestation on the content of dicycloethers (mg g-1 DW). Data are
= 12). Values within column, followed by the same regular or italic letter(s), are not
ferent according to Tukey’s test (p < 0 05) Figure 5
Effect of MeJA pre-treatment and thrips infestation on the content of simple phenylpropanoids (mg g-1
DW). Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic
letter(s), are not significantly different according to Tukey’s test (p < 0.05) Figure 5 Figure 5 Effect of MeJA pre-treatment and thrips infestation on the content of simple phenylpropanoids (mg g-1
DW). Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic
letter(s), are not significantly different according to Tukey’s test (p < 0.05) Effect of MeJA pre-treatment and thrips infestation on the content of simple phenylpropanoids (mg g-1
DW). Data are means ± SDs (n = 12). Values within column, followed by the same regular or italic
letter(s), are not significantly different according to Tukey’s test (p < 0.05) Figure 6
Effect of MeJA pre-treatment and thrips infestation on the content of dicycloethers (mg g-1 DW). Data are
means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not
significantly different according to Tukey’s test (p < 0.05) Figure 6 Effect of MeJA pre-treatment and thrips infestation on the content of dicycloethers (mg g-1 DW). Data are
means ± SDs (n = 12). Values within column, followed by the same regular or italic letter(s), are not
significantly different according to Tukey’s test (p < 0.05) Page 20/20
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Malaria Epidemics and Surveillance Systems in Canada
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J. Dick MacLean,* Anne-Marie Demers,* Momar Ndao,* Evelyne Kokoskin,* Brian J. Ward,*
and Theresa W. Gyorkos† J. Dick MacLean,* Anne-Marie Demers,* Momar Ndao,* Evelyne Kokoskin,* Brian J. Ward,*
and Theresa W. Gyorkos† tem should be strengthened and used more proactively to
help identify appropriate preventive measures. In the past decade, fluctuations in numbers of import-
ed malaria cases have been seen in Canada. In 1997 to
1998, malaria case numbers more than doubled before
returning to normal. This increase was not seen in any
other industrialized country. The Canadian federal malaria
surveillance system collects insufficient data to interpret
these fluctuations. Using local (sentinel), provincial, feder-
al, and international malaria surveillance data, we evaluate
and interpret these fluctuations. Several epidemics are
described. With an ever-increasing immigrant and refugee
population of tropical origin, improved surveillance will be
necessary to guide public health prevention policy and
practice. The Canadian experience is likely to be general-
izable to other industrialized countries where malaria is a
reportable disease within a passive surveillance system. All 10 provincial and 3 territorial health authorities in
Canada are required by law to report diagnoses of malaria
and other selected diseases to federal authorities at Health
Canada (2). Summary reports of these diseases are pub-
lished by both levels of reporting in provincial and territo-
rial news bulletins and by Health Canada in the Canada
Communicable Diseases Report. In several instances over the past decade, malaria inci-
dence in Canada as a whole, or in individual provinces,
reached epidemic levels (7). Why some were not immedi-
ately identified and why no comprehensive analysis has
been published as part of government surveillance systems
are questions that will be addressed. Failing to recognize
these epidemics has limited the ability of public health
officials to assess and intervene appropriately to control
the illness and death associated with imported malaria in
Canada. M
alaria has been a reportable communicable disease in
Canada since 1929, when a surveillance system for
communicable diseases was first developed. Although no
longer endemic in Canada, malaria has remained an impor-
tant imported disease, principally in immigrants and travel-
ers (1–3). Rarely, it has been transmitted in blood products
(4). Published reports document delays in clinical and lab-
oratory diagnoses of malaria and lack of understanding of
malaria prophylaxis and fever management in travelers (3). J. Dick MacLean,* Anne-Marie Demers,* Momar Ndao,* Evelyne Kokoskin,* Brian J. Ward,*
and Theresa W. Gyorkos† The Canadian infectious disease surveillance system has
reported an average of 538 malaria cases per year since
1990, and Statistics Canada reported an average of one
death per year (5,6, Carole Scott [Division of Disease
Surveillance, Health Canada], pers. comm.). The present
federal surveillance system reports the age and sex of a
patient and does not document malaria death rate, malaria
species, nor the likely country of acquisition. While malar-
ia-related deaths may be few, that any exist is a matter of
concern. The continued incidence of malaria cases and
deaths in Canada suggests that the malaria surveillance sys- M This study evaluated and summarized data collected
over the past 22 years by local, provincial, and federal
malaria surveillance systems, from Canadian federal
immigration and refugee data resources and from interna-
tional tourist resources, to identify and explore the causes
of malaria epidemics. In addition, geographic patterns and
Plasmodium spp. profiles of malaria are examined. This
analysis led us to conclude that changes are needed in both
the surveillance reporting instruments and how these sur-
veillance results are analyzed and used. *Montreal General Hospital, McGill University Centre for Tropical
Diseases, Montreal, Québec, Canada; and †McGill University,
Montreal, Québec, Canada TDC Database Overall, 553 clinical cases of malaria were seen at TDC
from 1981 to 2002, with some fluctuation over time but an
overall gradual increase (Figure 1). In these 553 cases, 562
microscopy diagnoses were made; Plasmodium falci-
parum 295 (52%), P. vivax 218 (39%), P. ovale 26 (5%), P. malariae 16 (3%), and unknown species 7 (1%). Nine
(2%) of the clinical cases were mixed infections, involving
P. falciparum with either P. malariae or P. vivax. Seven
patients were seen two or three times with relapses of P. vivax (recurrence >2 months later). The relative frequency
of species changed over time, with a gradual increase in
the proportion of P. falciparum cases from 20% to 30% in
the early 1980s to 60% to 70% in the 1990s and to 70% to
80% in the present decade (Figure 2). Over this 22-year
period, only one fatality occurred (3). Sixty-one countries were identified as the most likely
sources of the malaria exposure. Sub-Saharan Africa was
the region where most patients contracted malaria, 353
case-patients (65%), followed by south Asia (23%),
Southeast Asia (6%), Central America (5%), and South
America (1%). However, India, with 110 cases (20%), was
the single most frequent source country. Tourists (29%),
immigrants or refugees (29%), and foreign workers (24%)
represented the categories most frequently reported. A shift
over time occurred in the importance of sub-Saharan Africa
as a source of malaria cases. In the 1980s, 50% of malaria
infections were acquired in Africa; in the 90s, 70%; and,
since 2000, 85%. Patients of all categories were more like-
ly to contract P. falciparum in Africa (74.3%) while it has
been an uncommon species in south Asia (5.8%). The
increase in P. falciparum cases over time correlated with Provincial reportable disease databases have included,
in the past 10 years, patient characteristics such as age,
sex, and malaria species, but not the likely country of
acquisition. Because 90% of all malaria cases in Canada
were reported by the Provinces of British Columbia
(Monica Naus [British Columbia Centre for Disease
Control], pers. comm.), Ontario (Lorraine Schiedel
[Ontario Ministry of Health and Long-Term Care], pers. comm.), and Québec (Colette Colin [Ministère de la santé
et des services sociaux, Québec], pers. comm.), the present
analysis focuses on their data, primarily for 1990–2002
(11). Methods The databases used for the present analysis include 22
years of records from a local malaria reference center in
Montreal, Canada (the McGill University Centre for
Tropical Diseases [TDC]) and up to 13 years of quality
assurance and notifiable disease surveillance databases of
the provincial and federal governments of Canada, France,
India, Switzerland, the United States, and the United
Kingdom. TDC is a clinical and laboratory facility that *Montreal General Hospital, McGill University Centre for Tropical
Diseases, Montreal, Québec, Canada; and †McGill University,
Montreal, Québec, Canada 1195 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 7, July 2004 PERSPECTIVES terns of Canadians to the tropics by the World Tourism
Organization, Madrid (27). provides care to 800 to 1,100 new patients per year
(approximately 60% primary cases and 40% consult
cases), drawn mainly from the Montreal region. The TDC
database from 1981 to 2002 has allowed previous detailed
reviews of changing patterns of malaria in its patient pop-
ulations (8–10). Malaria-relevant data captured include
category of traveler (tourist, immigrant, refugee, expatri-
ate, missionary, and volunteer), countries visited, and
malaria species. A diagnosis of malaria is made if para-
sites are noted on a blood smear (thin, thick, or buffy
coat) or if, in the last 5 years, the patient had a positive
result on a malaria antigen-capture test (e.g., Macromed
[Nova Century Scientific, Inc., Burlington, Ontario,
Canada], ICT Malaria P.f. [ICT Diagnostics, Brookvale,
New South Wales, Australia], or OptiMAL [Flow Inc.,
Portland, OR]). While active surveillance studies during
this period included polymerase chain reaction (PCR) as
a screening tool, PCR-positive cases were not included in
any of the passive surveillance statistics unless they were
also independently confirmed by either malaria antigen
capture or smear. TDC Database Quality assurance data for the province of Québec
(1994–2002) were provided by TDC and the Laboratoire
de Santé Publique du Québec. The federal government’s
notifiable disease database from 1990 to 2002 is a compi-
lation of selected information from individual provincial
databases and includes patient age and sex for each report
but no malaria species or country of acquisition (Carole
Scott [Division of Disease Surveillance, Health Canada],
pers. comm.). Figure 1. McGill University Centre for Tropical Diseases malaria
cases by year and origin (N = 553). International malaria surveillance data (1990–2002)
were acquired from the World Health Organization (WHO)
Regional Office for South East Asia (Rakish Mani Rastogi,
pers. comm.), the WHO Regional Office for Europe (12),
and the United States (13–24). Malaria rates for all coun-
tries were based on population data of the U.S. Census
Bureau (25). Trends in Canadian immigration and refugee data for
the years 1990–2002 were provided by Citizen and
Immigration, Canada (Karen Tremblett [Medical Services
Branch, Citizen and Immigration Canada], pers. comm.),
data on language by Statistics Canada (26), and travel pat- Figure 1. McGill University Centre for Tropical Diseases malaria
cases by year and origin (N = 553). 1196 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 7, July 2004 Malaria Epidemics and Surveillance Systems in Canada Figure 2. Relative rates of Plasmodium species (N = 553). malaria incidence was observed in males and females. British Columbia had the highest rate per 100,000 (3.6 ±
2.8) over this period, followed by Ontario (2.2 ± 0.98), and
Québec (1.3 ± 0.67) (Figure 3). However, the highest
cumulative numbers for the 12-year period were reported
from Ontario (N = 3,222), followed by British Columbia
(N = 1,763), and Québec (N = 1,246). The Canadian data
suggest that an epidemic occurred from 1995 to 1997,
reflecting higher than average numbers of malaria cases in
these years from British Columbia, Ontario, and to a less-
er extent, from Québec (Figure 3). This epidemic was
almost entirely due to increased P. vivax being reported in
these provinces (Figure 4). From 1990 to 1999, two events
occurred in Québec that did not occur in other Canadian
provinces. In 1994, a quality assurance program for the
province was initiated by TDC, in collaboration with the
Laboratoire de Santé Publique du Québec. TDC Database Provincial malaria rates for Québec, Ontario, and British
Columbia (6,11, Colette Colin [Ministère de la santé et des servic-
es sociaux, Québec], pers. comm.; Lorraine Schiedel [Ontario
Ministry of Health and Long Term Care], pers. comm.; Monica
Naus [British Columbia Centre for Disease Control], pers. comm.;
Carole Scott [Division of Disease Surveillance, Health Canada],
pers. comm.). TDC Database This three-
pronged program provided: 1) a free, rapid turnaround
confirmation service for positive or equivocal malaria
diagnoses from any laboratory in Québec, 2) a biannual
malaria-training course for clinical laboratory technolo-
gists, and 3) a voluntary proficiency testing program for
Québec hospital laboratories, in which once or twice a year
they are sent unknown positive and negative smears for
identification and receive extensive feedback. From the
inception of the quality assurance program, a parallel
increase was seen in numbers of specimens being sent to
the reference laboratory and to the Québec surveillance
program (Figure 5). This fourfold increase represented an
epidemic attributable to improved diagnosis and reporting. The second event in Québec was another epidemic, in this
case of falciparum malaria, observed in 2000 to 2001 and
associated with a large influx of refugees from Tanzanian
refugee camps (Figure 4) (29). the increase in the total number of malaria cases contracted
in Africa; P. falciparum represented ≈30% of all cases in
the early 1980s and increased to 70% in the late 1990s. From 1981 to 2002, 96% of malaria infections acquired in
south Asia were non-falciparum malarias, while only 29%
of infections from Africa were non-falciparum. None of the
553 cases of malaria originated from China, Malaysia,
Saudi Arabia, Peru, or Venezuela, which are frequent trav-
el destinations of Québecers. Other common travel destina-
tions contributed little to the 20-year malaria case total
(e.g., Philippines [1 case], South Africa [1], Costa Rica [2],
Mexico [2], and Dominican Republic [3]). Malaria cases
from Africa from 1992 to 2000 came predominantly (69%)
from the French-speaking African countries, notably high-
er than the proportion of U.S. travelers (18%) who acquired
malaria in these countries (13–24). Two “epidemics” were observed at TDC during this
period (Figures 1 and 2). The first was in 1986 to 1990 and
resulted from increasing numbers of P. falciparum infec-
tions from Africa, thought to be due to increasing chloro-
quine-resistant P. falciparum in chloroquine-prophylaxed
travelers (28), and the second was in 1999 through 2000,
resulting from increased numbers of P. falciparum infec-
tions associated with the arrival in Québec of large num-
bers of refugees from Tanzanian refugee camps (29). Both
epidemics were recognized and reported in the literature
soon after their appearance. Figure 3. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 7, July 2004 Federal and Provincial Databases A review of the Federal Health Canada databases for
the incidence of malaria in Canada, from 1990 through
2002, documents a range from 364 to 1,029 cases per year,
with an average of 538 cases per year during the period (or
an average of ≈1.8 cases per 100,000 population per year)
(6) (Carole Scott [Division of Disease Surveillance, Health
Canada], pers. comm.). Figure 3. Provincial malaria rates for Québec, Ontario, and British
Columbia (6,11, Colette Colin [Ministère de la santé et des servic-
es sociaux, Québec], pers. comm.; Lorraine Schiedel [Ontario
Ministry of Health and Long Term Care], pers. comm.; Monica
Naus [British Columbia Centre for Disease Control], pers. comm.;
Carole Scott [Division of Disease Surveillance, Health Canada],
pers. comm.). Figure 3. Provincial malaria rates for Québec, Ontario, and British
Columbia (6,11, Colette Colin [Ministère de la santé et des servic-
es sociaux, Québec], pers. comm.; Lorraine Schiedel [Ontario
Ministry of Health and Long Term Care], pers. comm.; Monica
Naus [British Columbia Centre for Disease Control], pers. comm.;
Carole Scott [Division of Disease Surveillance, Health Canada],
pers. comm.). Figure 3. Provincial malaria rates for Québec, Ontario, and British
Columbia (6,11, Colette Colin [Ministère de la santé et des servic-
es sociaux, Québec], pers. comm.; Lorraine Schiedel [Ontario
Ministry of Health and Long Term Care], pers. comm.; Monica
Naus [British Columbia Centre for Disease Control], pers. comm.;
Carole Scott [Division of Disease Surveillance, Health Canada],
pers. comm.). Figure 3. Provincial malaria rates for Québec, Ontario, and British
Columbia (6,11, Colette Colin [Ministère de la santé et des servic-
es sociaux, Québec], pers. comm.; Lorraine Schiedel [Ontario
Ministry of Health and Long Term Care], pers. comm.; Monica
Naus [British Columbia Centre for Disease Control], pers. comm.;
Carole Scott [Division of Disease Surveillance, Health Canada],
pers. comm.). While all ages were affected, patients were mostly
adults from 20 to 59 years of age. A similar pattern of 1197 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 7, July 2004 PERSPECTIVES Figure 4. Plasmodium species provincial trends (6,11, Colette
Colin [Ministère de la santé et des services sociaux, Québec],
pers. comm.; Monica Naus [British Columbia Centre for Disease
Control], pers. comm.; Lorraine Schiedel [Ontario Ministry of
Health and Long Term Care], pers. comm.). known to have important immigration and travel links with
North America (Figure 7). Federal and Provincial Databases During the 1990s, France had a
60% increase in malaria in the latter part of the decade
(31), reportedly caused by African travel, and Denmark
experienced an increase of 68%; Germany, Italy, Spain,
Sweden, the Netherlands, and Belgium, however, had sta-
ble rates during this time (12,31). None of these countries
had the increase in rates of malaria seen in Canada and, to
a lesser degree, in the United States from 1995 to 1997. Discussion Malaria importations into Canada can occur by either
immigration or travel, and changing malaria attack rates in
the countries of exposure are likely to influence the inci-
dence of imported disease. Changes in Canadian immigra-
tion and refugee patterns from 1990 to 2002 are notable for
a threefold increase in annual immigrant numbers from the
Indian subcontinent and relatively stable numbers from
sub-Saharan Africa. Neither combined nor separate
provincial immigration and refugee patterns explain the
important swings in annual Canadian malaria rates. Figure 4. Plasmodium species provincial trends (6,11, Colette
Colin [Ministère de la santé et des services sociaux, Québec],
pers. comm.; Monica Naus [British Columbia Centre for Disease
Control], pers. comm.; Lorraine Schiedel [Ontario Ministry of
Health and Long Term Care], pers. comm.). While the geographic origins of immigrants and
refugees do not immediately explain the epidemic changes
in P. vivax malaria seen in the mid-1990s, their nonrandom
aggregation in certain provinces allows additional insights. African immigrants and refugees have settled all across
Canada in every province in numbers that paralleled the
province’s population. Immigrants and refugees from the
Indian subcontinent did not: 84% settled in Ontario and
British Columbia, the provinces with the most pronounced
P. vivax epidemics. Canadian travelers to malaria-endemic
areas have gradually but steadily increased during the past
15 years, most notably with a threefold increase to
Southeast Asia and Central and South America, a twofold
increase to the important malarial region of south Asia, and
a smaller increase to Africa. Travel patterns did not offer
an explanation for either the P. vivax epidemics in British While the geographic origins of immigrants and
refugees do not immediately explain the epidemic changes
in P. vivax malaria seen in the mid-1990s, their nonrandom
aggregation in certain provinces allows additional insights. Figure 4. Plasmodium species provincial trends (6,11, Colette
Colin [Ministère de la santé et des services sociaux, Québec],
pers. comm.; Monica Naus [British Columbia Centre for Disease
Control], pers. comm.; Lorraine Schiedel [Ontario Ministry of
Health and Long Term Care], pers. comm.). International Malaria Surveillance However, the major fluctuations in federal and provincial
malaria rates from 1990 to 2002, and, in particular, during
the epidemic years, were not found to be directly linked to
provincial immigration numbers or to the travel destina-
tions of Canadians in general. Unfortunately, no mecha-
nism records the destinations of travelers from specific
provinces. Ontario and British Columbia are home to 86%
of the Punjabi-speaking Canadian population. If provincial
travel destination data were available, it would likely show
that these provinces were the source of most Canadian
travelers to the Indian Punjab (27). Figure 6. Malaria cases/100,000 relative to 1990 (6,12–25,30,
Carole Scott [Division of Disease Surveillance, Health Canada],
pers. comm.). Columbia and Ontario in the late 1990s or the P. falci-
parum epidemic in Québec from 2000 to 2001. The World
Tourism Organization data do not break down Canadian
travel by traveler’s province of origin; however, compar-
ing U.S. malaria surveillance data with TDC surveillance
data, both of which track the likely country of origin of a
malaria case, Québec travelers acquire most African
malaria in French-speaking African countries (69%), a
minor source of malaria for Americans (18%). English-
speaking Ontario and British Columbia likely have more
“American” travel patterns than francophone Québecers. However, no fluctuations were seen in rates of travel to
either East or West Africa or to the Indian subcontinent, the
major source of Canada-acquired P. vivax malaria, which
would explain the impressive change in Canadian malaria
reporting from 1995 through 1997. Working back from individual case data in each
province seems to be the most accurate way to identify
countries where large numbers of imported malaria may
originate. Country of likely origin of the malaria should be
indicated on all requisitions for malaria laboratory diagno-
sis, and this information and the malaria species should be
reported to provincial and then federal surveillance bodies. The fact that the 1995–1997 epidemic was primarily due to
P. vivax, the predominant malaria species in India, and that
it occurred at the same time as the P. vivax epidemic in the
Indian Punjabi states of Punjab and Haryana, is strong evi-
dence to conclude that the Canadian epidemic was
an extension of the Punjab epidemic. This association is The two surveillance sources of India and the United
States were also reviewed for malaria incidence trends. American malaria surveillance includes the likely country
of origin of a malaria case. International Malaria Surveillance International Malaria Surveillance National surveillance systems for malaria are far from
universal, and compliance with national surveillance
instruments, when measured, is low. The stability of the
degree of underreporting over time has been not been eval-
uated. Despite these limitations, trends in malaria inci-
dence over time in different countries can provide useful
information. From 1995 to 1997, when parts of Canada
were having malaria epidemics, similar but smaller
changes in malaria rates were observed in the United
States and United Kingdom (Figure 6). An examination of
the geographic origin of malaria cases reported in the
United States in the mid-1990s showed a more than
twofold increase in malaria cases imported from India in
1995 through 1997, with an abrupt drop in these cases in
1998 (12–23). During this same period, a similar epidem-
ic of P. vivax malaria occurred in certain states in India Figure 5. Malaria surveillance of Québec province, McGill Centre
for Tropical Diseases (TDC), and Québec quality assurance (QA)
program (Colette Colin [Ministère de la santé et des services soci-
aux, Québec], pers. comm.). Figure 5. Malaria surveillance of Québec province, McGill Centre
for Tropical Diseases (TDC), and Québec quality assurance (QA)
program (Colette Colin [Ministère de la santé et des services soci-
aux, Québec], pers. comm.). 1198 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 7, July 2004 Malaria Epidemics and Surveillance Systems in Canada Figure 6. Malaria cases/100,000 relative to 1990 (6,12–25,30,
Carole Scott [Division of Disease Surveillance, Health Canada],
pers. comm.). epidemics in Canada in the last decade. One was a P. vivax
epidemic, the epicenter of which was almost certainly in
the Punjab, India. The second was a P. falciparum epidem-
ic in Québec related to an increased influx of Central
African refugees from Tanzanian refugee camps. At the
time, neither of these epidemics was brought to the atten-
tion of health practitioners in travel clinics through publi-
cation or other standard channels. Consequently, possible
explanations and potential interventions were not dis-
cussed. Trends in immigration do not explain the malaria inci-
dence changes seen in Canada. These trends differ for each
province both in terms of country of origin and numbers. International Malaria Surveillance 1995;53:518–21. 10. Gyorkos TW, Svenson JE, MacLean JD, Mohamed N, Remondin
MH, Franco ED. Compliance with antimalarial chemoprophylaxis
and the subsequent development of malaria: a matched case-control
study. Am J Trop Med Hyg. 1995;53:511–7. 11. Annual summary of reportable diseases [monograph on the Internet]. British Columbia Centre for Disease Control. 2000 [cited 2003 Aug
20]. Available from: http://www.bccdc.org/downloads/pdf/cdc/
reports/rpt-annual2000.pdf 12. Computerized information system on infectious diseases (CISID)
[database on the Internet]. Copenhagen, WHO Regional Office for
Europe. 2003
[cited
2003
Aug
3]. Available
from:
http://data.euro.who.int/cisid/ For surveillance data to be useful and cost-effective, it
must be both available in a timely fashion and interpretable. Local surveillance systems have obvious benefits when
increased water- and foodborne infections or vaccine-pre-
ventable diseases lead to quick public health action. Malaria surveillance differs in two major ways from these
classical scenarios. Malaria is an imported disease, and no
immediate intervention (e.g., vaccine, chemical disinfec-
tant, and handwashing) will affect an epidemic. As with
sexually transmitted infections, the control of a malaria epi-
demic in Canadian travelers requires public education. In
the United States, both malaria speciation and country of
likely acquisition of the malaria case are part of surveil-
lance. Such information, if part of the Canadian system,
would allow rapid appreciation of the etiology of epidemics
such as those reviewed here, which would potentially lead
to appropriate public health response. 13. Centers for Disease Control and Prevention. Notice to readers: final
2002 reports of notifiable diseases. Morb Mortal Wkly Rep Surveill
Summ. 2003;52:741–50. 14. Filler S, Causer LM, Newman RD, Barber AM, Roberts JM,
MacArthur J, et al. Malaria surveillance—United States, 2001. Morb
Mortal Wkly Rep Surveill Summ. 2003;52:1–14. 15. Causer LM, Newman RD, Barber AM, Roberts JM, Stennies G,
Bloland PB, et al. Malaria surveillance—United States, 2000. Morb
Mortal Wkly Rep Surveill Summ. 2002;51:9–21. 16. Newman R, Barber AM, Roberts JM, Holtz T, Steketee RW, Parise
ME. Malaria surveillance—United States, 1999. Morb Mortal Wkly
Rep Surveill Summ. 2002;51:15–28. 17. Holtz TH, Kachur SP, MacArthur JR, Roberts JM, Barber AM,
Steketee RW. Malaria surveillance—United States, 1998. Morb
Mortal Wkly Rep Surveill Summ. 2001;50;1–18. 18. MacArthur JR, Levin AR, Mungai M, Roberts J, Barber AM, Bloland
PB. Malaria surveillance—United States, 1997. Morb Mortal Wkly
Rep Surveill Summ. 2001;50:25–44. 19. Mungai M, Roberts J, Barber AM, Williams HA, Ruebush TK, Chang
M-H. Malaria surveillance—United States, 1996. Morb Mortal Wkly
Rep Surveill Summ. 2001;50:1–22. Dr. International Malaria Surveillance An obvious increase in P. vivax
cases from India was seen in the United States, from 150
cases to 371 and down to 123, during 1995 to 1997. This
increase paralleled the epidemic peak seen in Canada, pri-
marily in Ontario and British Columbia. In India, an epi-
demic of P. vivax malaria occurred during this same period
(1995–1997) in the Punjabi states of Punjab and Haryana
(Figure 7). With negligible changes in travel destination or
immigration numbers to explain the 1995–1997 epidemic
in Canada, the explanation is probably an increased P. vivax attack rate in Canadians traveling to the Punjab,
where a P. vivax epidemic occurred and ended at the same
time as the Canadian epidemic. Figure 7. Plasmodium vivax incidence relative to 1998 (6,11,
Colette Colin [Ministère de la santé et des services sociaux,
Québec], pers comm.; Monica Naus [British Columbia Centre for
Disease Control], pers. comm.). Figure 7. Plasmodium vivax incidence relative to 1998 (6,11,
Colette Colin [Ministère de la santé et des services sociaux,
Québec], pers comm.; Monica Naus [British Columbia Centre for
Disease Control], pers. comm.). Canadian notifiable diseases surveillance data generat-
ed by local, provincial, and federal sources provided evi-
dence for the occurrence of two as-yet unreported malaria Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 7, July 2004 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 7, July 2004
1199 1199 PERSPECTIVES supported by the abrupt halt of both Canadian and Punjabi
epidemics in the same year. 7. Last J, editor. Dictionary of epidemiology. 4th ed. Oxford:
International Epidemiological Association; 2001. 8. Svenson JE, MacLean JD, Gyorkos TW, Keystone J. Imported malar-
ia. Clinical presentation and examination of symptomatic travelers. Arch Intern Med. 1995;155:861–8. The surveillance process for notifiable diseases in
Canada and in other countries where malaria is now an
imported disease should be reviewed. Specific conditions,
such as the frequency of analysis of surveillance data, need
to be discussed and agreed on by collectors of these data at
each level of government. Without a firm plan in place for
analysis and dissemination of results, the validity, not to
mention the utility of the entire surveillance system, is
placed in jeopardy. One approach could be the American
emerging infections programs, a link between public
health, academic, and clinical communities (32). 9. Svenson JE, Gyorkos TW, MacLean JD. Diagnosis of malaria in the
febrile traveler. Am J Trop Med Hyg. International Malaria Surveillance MacLean is professor of medicine and director of the
McGill University Centre for Tropical Diseases. His research
interests are parasitic disease outbreak investigation (trichinosis,
Metorchis infections, and malaria) and the development of diag-
nostic tests for the clinical parasitology laboratory. 20. Williams HA, Roberts J, Kachur SP, Barber AM, Barat LM, Bloland
PB, et al. Malaria surveillance—United States, 1995. Morb Mortal
Wkly Rep Surveill Summ. 1999;48:1–21. 21. Kachur SP, Reller ME, Barber AM, Barat LM, Koumans EHA, Parise
ME. Malaria surveillance—United States, 1994. Morb Mortal Wkly
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dick.maclean@mcgill.ca Address for correspondence: J. Dick MacLean, McGill University Centre
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Characterization of the chloroplast genome of Gleditsia species and comparative analysis
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Scientific reports
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Characterization of the chloroplast
genome of Gleditsia species
and comparative analysis
Feng Xiao 1, Yang Zhao 1*, Xiurong Wang 1 & Xueyan Jian 2
OPEN Feng Xiao 1, Yang Zhao 1*, Xiurong Wang 1 & Xueyan Jian 2 The genus Gleditsia has significant medicinal and economic value, but information about the
chloroplast genomic characteristics of Gleditsia species has been limited. Using the Illumina
sequencing, we assembled and annotated the whole chloroplast genomes of seven Gleditsia species
(Gleditsia sinensis, Gleditsia japonica var. delavayi (G. delavayi), G. fera, G. japonica, G. microphylla,
Fructus Gleditsiae Abnormalis (Zhū Yá Zào), G. microphylla mutant). The assembled genomes revealed
that Gleditsia species have a typical circular tetrad structure, with genome sizes ranging from 162,746
to 170,907 bp. Comparative genomic analysis showed that most (65.8–75.8%) of the abundant
simple sequence repeats in Gleditsia and Gymnocladus species were located in the large single copy
region. The Gleditsia chloroplast genome prefer T/A-ending codons and avoid C/G-ending codons,
positive selection was acting on the rpoA, rpl20, atpB, ndhA and ycf4 genes, most of the chloroplast
genes of Gleditsia species underwent purifying selection. Expansion and contraction of the inverted
repeat (IR)/single copy (SC) region showed similar patterns within the Gleditsia genus. Polymorphism
analysis revealed that coding regions were more conserved than non-coding regions, and the IR
region was more conserved than the SC region. Mutational hotspots were mostly found in intergenic
regions such as “rps16-trnQ”, “trnT-trnL”, “ndhG-ndhI”, and "rpl32-trnL” in Gleditsia. Phylogenetic
analysis showed that G. fera is most closely related to G. sinensis,G. japonica and G. delavayi are
relatively closely related. Zhū Yá Zào can be considered a bud mutation of the G. sinensis. The albino
phenotype of G. microphylla mutant is not caused by variations in the chloroplast genome, and that
the occurrence of the albino phenotype may be due to mutations in chloroplast-related genes involved
in splicing or localization functions. This study will help us enhance our exploration of the genetic
evolution and geographical origins of the Gleditsia genus. The plants in the genus Gleditsia, mainly distributed in central and Southeast Asia and North and South America,
they have been used as local and traditional medicines in many regions, particularly in China1. The genus rec-
ognized 13 species2. There are 6 species and 2 varieties of Gleditsia plants in China, including Gleditsia sinensis,
G. australis, G. fera, G. japonica, G. microphylla, Gleditsia japonica var. delavayi (G. delavayi), Gleditsia japonica
var. velutina (G. velutina), and 1 species (G. triacanthos) which is introduced3,4. G. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | (2024) 14:4262 Plant materials, genomic DNA isolation and genome sequencing Plant materials, genomic DNA isolation and genome sequencing , g
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q
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We collected various species seed of Gleditsia in China, including G. sinensis (Luodian City, Guizhou Province)
(E 106.7379, N 25.2454), G. fera (Ceheng City, Guizhou Province) (E 105.9642, N 24.9327), G. japonica (Guiyang
City, Guizhou Province) (E 106.9382, N 26.6969), G. microphylla (Zhijin City, Guizhou Province) (E 106.0246,
N 26.5328), G. japonica var. delavayi (G. delavayi) (Xinyi City, Guizhou Province) (E 104.9381, N 25.1755). In
addition, there were no seeds of Zhū Yá Zào, we directly collected the leaves of the plant Zhū Yá Zào (Tongren
City, Guizhou Province) (E 108.0349; N 27.9852) (Fig. S1). Gymnocladus chinensis (Gen.: Gymnocladus) and
Gymnocladus dioicus (Gen.: Gymnocladus) were collected as out group. After bringing various seeds back to the
laboratory, then placed in the plant greenhouse incubator for cultivation. In the process of raising seedlings in
the greenhouse, a albino mutant plant of G. microphylla was obtained (labeled G. microphylla mutant), which was
characterized by albino whole plant, obvious dwarfing, and natural death after 1–1.5 months of growth. Taking
single plant leaves of above all species for DNA extraction. Total DNA of each sample was isolated according to
the instructions of the DNA extraction kit (EasyPure® Plant Genomic DNA Kit, Beijing Quanshijin Biological
Co., Ltd.). Nandrop 2000 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used to determine the
concentration and purity of the DNA. The DNA integrity was assessed by agarose gel electrophoresis. According
to the Illumina standard protocol, total DNA was used to generate libraries after DNA extraction which were
sequenced using the Illumina NovaSeq 6000 platform, and the sequencing read length was 150 bp. q
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p
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All methods were performed on Gleditsia plants that were cultivated for the purposes of the experiments,
including the collection of plant material for all analysis, and all relevant institutional, national, and international
guidelines and legislation were complied with. Comparative analysish The genome sequences were initially aligned using MAFFT (v7.310)22. The complete chloroplast genomes were
compared by the mVISTA program (http://genome.lbl.gov/vista/mvista/submit.shtml). DnaSP (DNA Sequences
Polymorphism)23 was used to calculate the nucleotide diversity (Pi) of coding, non-coding regions and whole
complete chloroplast genomes (all chloroplast genome sequences were adjusted to start with LSC, the step size
was set to 200 bp with a window length of 600 bp). Analysis of codon usage bias and selective pressures in the evolution y
g
p
Extracted the full-length coding sequences, with an ATG start codon, a stop codon (TGA/TAG/TAA). The
nucleotide compositions at the third position (A3s, U3s, C3s and G3s), GC content at third codon positions
(GC3s), codon adaptation index (CAI), Codon Bias Index (CBI), effective number of codon (ENC) were deter-
mined with CodonW20. Relative synonymous codon usage (RSCU) was analyzed with bioPython. KaKs calculator
program21 with the NG model to calculate the rates of nonsynonymous (Ka), synonymous (Ks), and their ratio
(Ka/Ks). When Ks = 0, the value cannot be computed and was represented by *. When Ka = 0 and Ks = 0, the
value was represented by NaN. G. sinensis was used as a reference. www.nature.com/scientificreports/ remain relatively independent of species. The use of DNA barcodes of the chloroplast genome will help identify
varieties and resources12. In recent years, with the development of high-throughput sequencing technology, an
increasing number of plant chloroplast genome DNA sequences have been obtained. However, in the Gleditsia
genus, only the chloroplast genomes of G. sinensis and G. japonica have been reported so far13,14, this limits our
understanding of the genetic evolution of Gleditsia. The development of genomic resources for Gleditsia can also
assist in molecular breeding studies of this genus. The collected seven Gleditsia species (Gleditsia sinensis, G. delavayi, G. fera, G. japonica, G. microphylla, Zhū Yá Zào, G. microphylla mutant) and two Gymnocladus species
(Gymnocladus chinensis, Gymnocladus dioicus) were sequencing, assembled and analyzed, complete chloroplast
genomes were obtained. In this study, the seven species chloroplast genome of Gleditsia genus were sequenced,
assembled and analyzed to study any common features or differences between species, which helps in the genetic
breeding and molecular evolution of Gleditsia. Chloroplast genome assembly, annotation and sequence analysishh p
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The raw data quality control was performed by fastp (v0.12.4)15. The clean paired-end reads were assembled with
GetOrganelle (v1.7.6.1)16. The complete chloroplast genome was annotated in CPGAVAS217 (http://www.herba
lgenomics.org/cpgavas2/). The the transfer RNA (tRNA) genes were verified with tRNAscan-SE18. REPuter19
(https://bibiserv.cebitec.uni-bielefeld.de/reputer/) was used to find the sizes and locations of forward, reverse, pal-
indromic, and complementary repeats. Simple sequence repeat (SSRs) were determined using a Perl script MISA
(MIcroSAtellite identification tools), including mono-, di-, tri-, tetra-, penta-, and hexa-nucleotides, minimum
numbers (thresholds) were 10, 5, 4, 3, 3, and 3, respectively. The chloroplast genome sequences were deposited
in GenBank (Accession Numbers: OP722579–OP722582). Characterization of the chloroplast
genome of Gleditsia species
and comparative analysis
Feng Xiao 1, Yang Zhao 1*, Xiurong Wang 1 & Xueyan Jian 2
OPEN sinensis (Fam.: Leguminosae;
Gen.: Gleditsia), deciduous tree or shrub-like, contains both diploids and having 2n = 28 chromosomes5. G. sin-
ensis is widely distributed in China and is resistant to drought, cold, pollution, and has strong stress resistance,
it integrates medicinal, edible, chemical, material, and ornamental purposes6. Fructus Gleditsiae Abnormalis
(Zhū Yá Zào), it is the dried and sterile fruit of G. sinensis, there are significant differences in the morphology,
structure and composition of G. sinensis and Zhū Yá Zào7, Li et al.8 suggested that it should be a variant of G. sinensis. However, the way in which Zhū Yá Zào came into being is still unknown. y
g
Identifying the genetic evolutionary relationship between Gleditsia is the key to distinguish varieties. Chlo-
roplast gene sequences (ndhF and rpl16) are selected to test biogeographic hypotheses, there is a fundamental
division of the genus Gleditsia into three clades9. Based on the ITS sequence, Schnabel10 conducts a systematic
evolutionary study on the 11 species of Gleditsia and the results shows that the Gleditsia and Gymnocladus
appear to have originated in eastern Asia during the Eocene. Xing11 selected three fragments of chloroplast
DNA, MatK, PsbA-trnH, and TrnL-F to establish a phylogenetic tree of Gleditsia, the results shows that the
Gymnocladus has a longer evolutionary time than the Gleditsia. The complete chloroplast genome contains a
large amount of genetic information and is highly conserved. The self-replication and evolution of its genome 1Institute for Forest Resources and Environment of Guizhou, Key Laboratory of Forest Cultivation in Plateau
Mountain of Guizhou Province, College of Forestry, Guizhou University, Guiyang 550025, Guizhou, China. 2College
of Continuing Education, Yanbian University, Yanji 133002, Jilin, China. *email: zhy737@126.com | https://doi.org/10.1038/s41598-024-54608-6 Scientific Reports | (2024) 14:4262 www.nature.com/scientificreports/ Codon bias analysis and selective pressures in the evolutionh y
p
The GC and GC3s content in the codons of the 9 chloroplast genomes studied were both less than 0.5, indicat-
ing a preference for A/T bases and A/T-ending codons in Gleditsia and Gymnocladus chloroplast genomes. We
used the CDS of the chloroplast genome to estimate the codon usage frequency of the seven taxa of Gleditsia. All
20 amino acids were encoded by codons in the Gleditsia chloroplast genome and the synonymous codon usage
(RSCU value) values were similar. Of the 29 codons with an RSCU value > 1, only one ended with G (TTG). The
codons with an RSCU value < 1, except for ATA and CTA ending in A, ended in C or G. Codon pairs ending with
C and G in the Gleditsia chloroplast genome had low bias and were non-preferred codons. We analyzed the ka/ks ratio of the 76 unique protein-coding genes in the 9 chloroplast genome (Fig. 3), using
G. sinensis as the reference, five genes (rpoA, rpl20, atpB, ndhA, ycf4) were identified under positive selection
(Ka/Ks > 1). Ka/Ks ratio of most gene was less than 1. Codon bias analysis and selective pressures in the evolutionh )
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)
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S
G. Assembly and annotation of the Gleditsia chloroplast genomeh Assembly and annotation of the Gleditsia chloroplast genomeh The seven chloroplast genome of Gleditsia genus and two species from Gymnocladus genus both showed the
typical quadripartite structure of angiosperms, consisting of a large single copy (LSC) region (91,203–91,436 bp)
and a small single copy (SSC) region (18,845–19,561 bp), which separated by two inverted repeat (IRA and
IRB) regions (26,122–26,619 bp); The chloroplast genome sizes of Gleditsia species ranged from 162,746 bp (G. sinensis) to 170,796 (G. delavayi) (Table S1). The GC content of genomes ranged from 33.9 to 35.64%. G. sinensis
encoded 84 protein-coding genes, 8 rRNA, 37 tRNA, of which three genes (ycf1, ycf3, clpP) have two introns. G. fera, G. delavayi, Zhū Yá Zào, and G. microphylla encoded 85 protein-coding genes, 8 tRNA genes, and 37 tRNA
genes, respectively. G. microphylla mutant encoded 84 protein-coding genes, 8 tRNA genes and 37 tRNA genes. Analysis of the chloroplast genome structure of Gleditsia y
p
g
Using GCview to visualize the sequence alignment between multiple chloroplast genomes, it was found that
the sequence between different species of Gleditsia were similar. Through SSR identification, in Gleditsia plas-
tomes, the total number of SSRs ranges from 85 to 133 SSRs, while in the Gymnocladus it varied from 109 to
125. Moreover, most (65.8–75.8%) of the SSRs in Gleditsia and Gymnocladus species were located in the LSC. In
Gleditsia, the IR regions include between 2.2 and 5.5% SSRs loci, while the SSC region included between 17.9
and 23.3% (Fig. 1a). In the Gymnocladus sequenced here, 69.9–75.3% of the SSRs were situated in the LSC. A
total of 89 SSR sites were detected in G. sinensis, including 85 mononucleotide and 4 dinucleotide repeat units. The most abundant repeats were mononucleotide repeats in the Gleditsia genus (Fig. 1b). There were 50 repeats
in G. sinensis (Fig. 1c), which included complementary, forward, palindromic, reverse repeats.h The chloroplast genome sequence in Gleditsia was analyzed using the chloroplast genome of G. sinensis as
a reference with mVISTA. It was found that the chloroplast genome sequences within the genus Gleditsia were
highly similar and conserved, with the coding region being more conserved than the non-coding region, and
the IR region being more conserved than the SC region. The IR/SC junctions of the chloroplast genome within
Gleditsia showed similar features (Fig. 2). The lengths of the IR regions in the Gleditsia chloroplast genome
ranged from 26,122 to 26,619 bp. The rps3 gene was present in the LSC region in G. sinensis, Zhū Yá Zào, G. fera,
G. japonica, G. delavayi, G. microphylla, and G. microphylla mut., and all IRs contained a gene rps19, ranging
from 61 to 221 bp from the JLB (junction between LSC and IRb) junction. In the sequenced Gleditsia species,
the ndhF gene was completely present in the SSC and away from the junction, and the trnH gene was entirely
located in the LSC region. These data suggest that the expansion and contraction of the IR/SC region exhibit
similar patterns within Gleditsia. Phylogenetic analysis y
g
y
All chloroplast genome sequences were aligned by MAFFT software. The shared CDS genes were extracted by
python and aligned using MAFFT. Phylogenetic analysis and tree models was conducted by using the IQTREE
software (v2.0.3)24. The phylogenetic tree was visualized using ggtree R package (v3.2.1)25. Scientific Reports | (2024) 14:4262 | https://doi.org/10.1038/s41598-024-54608-6 www.nature.com/scientificreports/ Codon bias analysis and selective pressures in the evolutionh sinensis
Zhuyazao
G. microphylla mut. G. microphylla
G.japonica
Gy. dioicus
G. fera
G. chinensis
G. delavayi
type
IRa
SSC
IRb
LSC
shape
IRa
IRb
LSC
SSC
(a)
10
94
5
14
101
10
5
79
1
5
104
4
97
2
27
101
5
27
101
5
5
84
5
86
0
50
100
G. sinensis
zhuyazao
G. delavayi
G. fera
G. japonica
G. microphylla
G. microphylla mut. Gy. dioicus
Gy. chinensi
Number of SSRs
type
di
mono
tri
(b)
39
6
5
2
29
8
11
44
6
2
17
17
14
35
12
3
29
6
15
29
6
15
2
26
15
7
1
31
14
4
0
10
20
30
40
50
G. sinensis
zhuyazao
G. delavayi
G. fera
G. japonica
G. microphylla
G. microphylla mut. Gy. dioicus
Gy. chinensi
Type of repeat elements
type
C
D
F
P
R
(c)
Figure 1. Analysis of SSR sites and repetitive sequences in 9 chloroplast genomes. (a): Distribution of SSRs in
the Gleditsia and two plastomes from Gymnocladus; (b): Number of different SSRs loci types; (c): Number of
different repeats types. Note In a, different shapes represented the position of SSR, and the proportion of text
displayed; In (c), F: forward repeats, P: palindromic repeats, R: reverse repeats, C: complementary repeats. Codon bias analysis and selective pressures in the evolutionh )
%
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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C
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6
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C
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R
I
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R
I
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R
I
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6
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6
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C
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2
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2
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R
I
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2
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R
I
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R
I
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%
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R
I
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2
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S
IRa:(5.5%)
)
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7
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7
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L
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L
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L
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L
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L
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L
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L
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L
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7
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C
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L
)
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L
)
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2
(:
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S
S
)
%
8.2
2
(:
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S
S
)
%
8.2
2
(:
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S
S
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%
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(:b
R
I
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%
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(:b
R
I
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%
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7
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S
L
)
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2
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S
S
)
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2
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S
S
)
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2
(:
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S
S
)
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2
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S
S
)
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2
(:
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S
S
)
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2
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S
S
)
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2
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S
S
)
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2
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S
S
)
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2
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C
S
S
)
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2
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S
S
)
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2
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S
S
)
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2
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S
S
)
%
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2
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C
S
S
)
%
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2
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C
S
S
)
%
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(:a
R
I
)
%
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(:a
R
I
)
%
8.2
2
(:
C
S
S
)
%
8.5
7
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C
S
L
)
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8.5
7
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C
S
L
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7
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C
S
L
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7
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C
S
L
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7
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C
S
L
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7
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C
S
L
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7
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C
S
L
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7
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C
S
L
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%
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7
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C
S
L
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7
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C
S
L
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%
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7
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C
S
L
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%
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C
S
L
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7
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C
S
L
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7
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C
S
L
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%
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7
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C
S
L
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%
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7
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C
S
L
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%
8.5
7
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C
S
L
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8.5
7
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C
S
L
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%
8.5
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C
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L
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%
8.5
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C
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L
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%
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C
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L
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C
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%
8.5
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C
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%
8.5
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C
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8.5
7
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C
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8.5
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C
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8.5
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C
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8.5
7
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C
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8.5
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C
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8.5
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C
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8.5
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C
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L
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C
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C
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L
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C
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L
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C
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C
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C
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C
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C
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C
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8.5
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C
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L
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8.5
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C
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L
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8.5
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C
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8.5
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C
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C
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8.5
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C
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8.5
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8.5
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8.5
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8.5
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8.5
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8.5
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8.5
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8.5
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8.5
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L
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8.5
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L
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8.5
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L
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8.5
7
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S
L
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1
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S
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1
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S
S
)
%
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R
I
)
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R
I
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R
I
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8.5
7
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S
L
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8.5
7
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L
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1
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1
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1
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1
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1
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1
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S
S
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1
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C
S
S
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%
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(:a
R
I
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%
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R
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R
I
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1
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S
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8.5
7
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S
L
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7
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L
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7
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1
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S
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R
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R
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R
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8.5
7
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C
S
L
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7
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C
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L
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1
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S
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1
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C
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S
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1
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C
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1
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1
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1
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S
L
)
%
9.9
6
(:
C
S
L
)
%
9.9
6
(:
C
S
L
)
%
9.9
6
(:
C
S
L
)
%
9.9
6
(:
C
S
L
)
%
9.9
6
(:
C
S
L
)
%
9.9
6
(:
C
S
L
)
%
9.9
6
(:
C
S
L
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
2.3
(:b
R
I
)
%
2.3
(:b
R
I
)
%
2.3
(:b
R
I
)
%
9.9
6
(:
C
S
L
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
)
%
2.3
(:a
R
I
)
%
2.3
(:a
R
I
)
%
7.3
2
(:
C
S
S
)
%
7.3
2
(:
C
S
S
IRa:(3.2%)
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
9.0
2
(:
C
S
S
)
%
9.0
2
(:
C
S
S
)
%
2.2
(:b
R
I
)
%
2.2
(:b
R
I
)
%
7.4
7
(:
C
S
L
)
%
7.4
7
(:
C
S
L
)
%
9.0
2
(:
C
S
S
)
%
9.0
2
(:
C
S
S
)
%
9.0
2
(:
C
S
S
)
%
9.0
2
(:
C
S
S
)
%
9.0
2
(:
C
S
S
)
%
9.0
2
(:
C
S
S
)
%
9.0
2
(:
C
S
S
)
%
9.0
2
(:
C
S
S
)
%
9.0
2
(:
C
S
S
)
%
9.0
2
(:
C
S
S
)
%
9.0
2
(:
C
S
S
)
%
9.0
2
(:
C
S
S
)
%
9.0
2
(:
C
S
S
)
%
9.0
2
(:
C
S
S
)
%
9.0
2
(:
C
S
S
)
%
9.0
2
(:
C
S
S
)
%
2.2
(:a
R
I
)
%
2.2
(:a
R
I
)
%
9.0
2
(:
C
S
S
G. Codon bias analysis and selective pressures in the evolutionh sinensis
Zhuyazao
G. microphylla mut. G. microphylla
G.japonica
Gy. dioicus
G. fera
G. chinensis
G. delavayi
type
IRa
SSC
IRb
LSC
shape
IRa
IRb
LSC
SSC
(a)
10
94
5
14
101
10
5
79
1
5
104
4
97
2
27
101
5
27
101
5
5
84
5
86
0
50
100
inensis
huyazao
delavayi
G. fera
aponica
ophylla
lla mut. dioicus
hinensi
Number of SSRs
type
di
mono
tri
(b)
39
6
5
2
29
8
11
44
6
2
17
17
14
35
12
3
29
6
15
29
6
15
2
26
15
7
1
31
14
4
0
10
20
30
40
50
inensis
huyazao
delavayi
G. fera
aponica
ophylla
lla mut. dioicus
hinensi
Type of repeat elements
typ
(c) Figure 1. Analysis of SSR sites and repetitive sequences in 9 chloroplast genomes. (a): Distribution of SSRs in
the Gleditsia and two plastomes from Gymnocladus; (b): Number of different SSRs loci types; (c): Number of
different repeats types. Note In a, different shapes represented the position of SSR, and the proportion of text
displayed; In (c), F: forward repeats, P: palindromic repeats, R: reverse repeats, C: complementary repeats. Codon bias analysis and selective pressures in the evolutionh https://doi.org/10.1038/s41598-024-54608-6 Scientific Reports | (2024) 14:4262 | www.nature.com/scientificreports/ Gleditsia delavayi
rps3
657 bp
574 bp
rps19
279 bp
66 bp
ycf1
432 bp
39 bp
ndhF
2241 bp
93 bp
ycf1
5028 bp
432 bp
trnN
72 bp
782 bp
rps19
279 bp
66 bp
trnH
75 bp
14 bp
170796 bp
LSC
IRb
SSC
IRa
LSC
98889 bp
26173 bp
19561 bp
26173 bp
JLB
JSB
JSA
JLA
Gymnocladus chinensis
rps19
182 bp
97 bp
rpl2
1490 bp
184 bp
ycf1
807 bp
54 bp
ndhF
2238 bp
754 bp
ycf1
4677 bp
807 bp
trnN
72 bp
1162 bp
rpl2
1490 bp
184 bp
trnH
75 bp
7 bp
165299 bp
LSC
IRb
SSC
IRa
LSC
92340 bp
26255 bp
20449 bp
26255 bp
JLB
JSB
JSA
JLA
Gleditsia fera
rps3
657 bp
560 bp
rps19
279 bp
69 bp
ycf1
432 bp
33 bp
ndhF
2241 bp
70 bp
ycf1
5028 bp
432 bp
trnN
72 bp
782 bp
rps19
279 bp
69 bp
trnH
75 bp
49 bp
165027 bp
LSC
IRb
SSC
IRa
LSC
93345 bp
26154 bp
19374 bp
26154 bp
JLB
JSB
JSA
JLA
Gleditsia triacanthos
rps19
182 bp
97 bp
rpl2
1490 bp
159 bp
ycf1
800 bp
37 bp
ndhF
2232 bp
161 bp
ycf1
4684 bp
800 bp
trnN
72 bp
1128 bp
rpl2
1490 bp
159 bp
trnH
75 bp
7 bp
157472 bp
LSC
IRb
SSC
IRa
LSC
87239 bp
25882 bp
18469 bp
25882 bp
JLB
JSB
JSA
JLA
Gleditsia japonica
rps3
657 bp
564 bp
rps19
279 bp
86 bp
ycf1
432 bp
54 bp
ndhF
2241 bp
70 bp
ycf1
5028 bp
432 bp
trnN
72 bp
782 bp
rps19
279 bp
86 bp
trnH
75 bp
13 bp
166112 bp
LSC
IRb
SSC
IRa
LSC
94420 bp
26122 bp
19448 bp
26122 bp
JLB
JSB
JSA
JLA
Gleditsia microphylla
rps3
657 bp
541 bp
rps19
279 bp
221 bp
ycf1
756 bp
6 bp
ndhF
2241 bp
7 bp
ycf1
4704 bp
756 bp
trnN
72 bp
1106 bp
rps19
279 bp
221 bp
trnH
75 bp
134 bp
170713 bp
LSC
IRb
SSC
IRa
LSC
98595 bp
26619 bp
18880 bp
26619 bp
JLB
JSB
JSA
JLA
Gleditsia microphylla mutant
rps3
657 bp
541 bp
rps19
279 bp
221 bp
ycf1
756 bp
6 bp
ndhF
2241 bp
7 bp
ycf1
4704 bp
756 bp
trnN
72 bp
1106 bp
rps19
279 bp
221 bp
trnH
75 bp
134 bp
170713 bp
LSC
IRb
SSC
IRa
LSC
98595 bp
26619 bp
18880 bp
26619 bp
JLB
JSB
JSA
JLA
Gleditsia sinensis
rps3
657 bp
559 bp
rps19
279 bp
77 bp
trnN
72 bp
782 bp
ndhF
2241 bp
51 bp
ycf1
4830 bp
219 bp
trnN
72 bp
782 bp
rps19
279 bp
77 bp
trnH
75 bp
10 bp
162746 bp
LSC
IRb
SSC
IRa
LSC
91203 bp
26136 bp
19271 bp
26136 bp
JLB
JSB
JSA
JLA
Zhu Ya Zao
rps3
657 bp
580 bp
rps19
279 bp
61 bp
ycf1
432 bp
33 bp
ndhF
2241 bp
51 bp
ycf1
5028 bp
432 bp
trnN
72 bp
782 bp
rps19
279 bp
61 bp
trnH
75 bp
46 bp
162995 bp
LSC
IRb
SSC
IRa
LSC
91436 bp
26144 bp
19271 bp
26144 bp
JLB
JSB
JSA
JLA
Figure 2. Nucleic acid polymorphism analysis Nucleic acid polymorphism analysis
We conducted an analysis of Pi values to measure the divergence level in protein-coding genes (Fig. 4a), inter-
genic regions (Fig. 4b) and whole chloroplast genome sequences (Fig. 4c) of the 7 Gleditsia species. Taking the
common protein coding sequence of Gleditsia as the research object, ycf1 and petL were found as mutational
hotspots. Through gene sequence alignment and polymorphism analysis using the chloroplast genome of Gledit-
sia as a reference, we found that mutational hotspots occurred in the intergenic regions such as "rps16-trnQ",
"trnT-trnL", "ndhG-ndhI", "rpl32-trnL", etc. The IR region was conserved relative to the SC region. Codon bias analysis and selective pressures in the evolutionh Comparison of the border regions of LSC, IR and SSC. Note JLA: junction between LSC and IRa; JLB:
junction between LSC and IRb; JSA: junction between SSC and IRa; JSB: junction between SSC and IRb. The
numbers above the gene features indicated the distance from the end of the gene to the boundary sites; these
features were not scaled. Gymnocladus chinensis
165299 bp Figure 2. Comparison of the border regions of LSC, IR and SSC. Note JLA: junction between LSC and IRa; JLB:
junction between LSC and IRb; JSA: junction between SSC and IRa; JSB: junction between SSC and IRb. The
numbers above the gene features indicated the distance from the end of the gene to the boundary sites; these
features were not scaled. Phylogenetic analysis https://doi.org/10.1038/s41598-024-54608-6 Scientific Reports | (2024) 14:4262 | www.nature.com/scientificreports/ 0
2
4
6
(a)
GCT
GCC
GCA
GCG
CGT
CGC
CGA
CGG
AGA
AGG
AAT
AAC
GAT
GAC
TGT
TGC
CAA
CAG
GAA
GAG
GGT
GGC
GGA
GGG
CAT
CAC
ATT
ATC
ATA
TTA
TTG
CTT
CTC
CTA
CTG
AAA
AAG
ATG
TTT
TTC
CCT
CCC
CCA
CCG
TCT
TCC
TCA
TCG
AGT
AGC
ACT
ACC
ACA
ACG
TGG
TAT
TAC
GTT
GTC
GTA
GTG
Ala
Arg
Asn
Asp
Cys
Gln
Glu
Gly
HIS
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
GCA
GCC
GCG
GCT
AGA
AGG
CGA
CGC
CGG
CGT
AAC
AAT
GAC
GAT
TGC
TGT
CAA
CAG
GAA
GAG
GGA
GGC
GGG
GGT
CAC
CAT
ATA
ATC
ATT
CTA
CTC
CTG
CTT
TTA
TTG
AAA
AAG
ATG
TTC
TTT
CCA
CCC
CCG
CCT
AGC
AGT
TCA
TCC
TCG
TCT
ACA
ACC
ACG
ACT
TGG
TAC
TAT
GTA
GTC
GTG
GTT
0.5
1.0
1.5
Ala Arg Asn Asp Cys Gln Glu Gly HIS Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val
RSCU
(b)
0.0
0.5
1.0
1.5
2.0
accD
atpA
atpB
atpE
atpF
atpH
atpI
ccsA
cemA
clpP
matK
ndhA
ndhB
ndhC
ndhD
ndhE
ndhF
ndhG
ndhH
ndhI
ndhJ
ndhK
petA
petB
petD
petG
petL
petN
psaA
psaB
psaC
psaI
psaJ
psbA
psbB
psbC
psbD
psbE
ka/ks
0.0
0.5
1.0
1.5
2.0
psbF
psbH
psbI
psbJ
psbK
psbM
psbN
psbT
psbZ
rbcL
rpl14
rpl16
rpl2
rpl20
rpl23
rpl32
rpl33
rpl36
rpoA
rpoB
rpoC1
rpoC2
rps11
rps12
rps14
rps15
rps16
rps18
rps19
rps2
rps3
rps4
rps7
rps8
ycf1
ycf2
ycf3
ycf4
ka/ks
group
G. sinensis - Fructus Gleditsiae Abnormalis
G. sinensis - G. delavayi
G. sinensis - G. fera
G. sinensis - G. japonica
G. sinensis - G. microphylla
G. sinensis - G. microphylla tubianti
G. sinensis - Gy. chinensis
G. sinensis - Gy. dioicus
(c)
Figure 3. Codon bias analysis and selective pressures in the evolution. (a): Codon content of 20 amino
acids and stop codons in all protein-coding genes of Gleditsia chloroplast genome; (b): Distribution of codon
preference in Gleditsia; (c): Ka/Ks values of protein-coding genes of the seven comparative combinations. Note
In the a, the top panel shows the RSCU for the corresponding amino acids, the colored block which are shown
in the below represent different codons; In (c), Ka: nonsynonymous; Ks: synonymous. Phylogenetic analysis 0
2
4
6
(a)
GCT
GCC
GCA
GCG
CGT
CGC
CGA
CGG
AGA
AGG
AAT
AAC
GAT
GAC
TGT
TGC
CAA
CAG
GAA
GAG
GGT
GGC
GGA
GGG
CAT
CAC
ATT
ATC
ATA
TTA
TTG
CTT
CTC
CTA
CTG
AAA
AAG
ATG
TTT
TTC
CCT
CCC
CCA
CCG
TCT
TCC
TCA
TCG
AGT
AGC
ACT
ACC
ACA
ACG
TGG
TAT
TAC
GTT
GTC
GTA
GTG
Ala
Arg
Asn
Asp
Cys
Gln
Glu
Gly
HIS
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
GCA
GCC
GCG
GCT
AGA
AGG
CGA
CGC
CGG
CGT
AAC
AAT
GAC
GAT
TGC
TGT
CAA
CAG
GAA
GAG
GGA
GGC
GGG
GGT
CAC
CAT
ATA
ATC
ATT
CTA
CTC
CTG
CTT
TTA
TTG
AAA
AAG
ATG
TTC
TTT
CCA
CCC
CCG
CCT
AGC
AGT
TCA
TCC
TCG
TCT
ACA
ACC
ACG
ACT
TGG
TAC
TAT
GTA
GTC
GTG
GTT
0.5
1.0
1.5
Ala Arg Asn Asp Cys Gln Glu Gly HIS Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val
RSCU
(b)
0.0
0.5
1.0
1.5
2.0
accD
atpA
atpB
atpE
atpF
atpH
atpI
ccsA
cemA
clpP
matK
ndhA
ndhB
ndhC
ndhD
ndhE
ndhF
ndhG
ndhH
ndhI
ndhJ
ndhK
petA
petB
petD
petG
petL
petN
psaA
psaB
psaC
psaI
psaJ
psbA
psbB
psbC
psbD
psbE
ka/ks
0.0
0.5
1.0
1.5
2.0
psbF
psbH
psbI
psbJ
psbK
psbM
psbN
psbT
psbZ
rbcL
rpl14
rpl16
rpl2
rpl20
rpl23
rpl32
rpl33
rpl36
rpoA
rpoB
rpoC1
rpoC2
rps11
rps12
rps14
rps15
rps16
rps18
rps19
rps2
rps3
rps4
rps7
rps8
ycf1
ycf2
ycf3
ycf4
ka/ks
group
G. sinensis - Fructus Gleditsiae Abnormalis
G. sinensis - G. delavayi
G. sinensis - G. fera
G. sinensis - G. japonica
G. sinensis - G. microphylla
G. sinensis - G. microphylla tubianti
G. sinensis - Gy. chinensis
G. sinensis - Gy. dioicus
(c)
Figure 3. Codon bias analysis and selective pressures in the evolution. (a): Codon content of 20 amino
acids and stop codons in all protein-coding genes of Gleditsia chloroplast genome; (b): Distribution of codon
preference in Gleditsia; (c): Ka/Ks values of protein-coding genes of the seven comparative combinations. Note
In the a, the top panel shows the RSCU for the corresponding amino acids, the colored block which are shown
in the below represent different codons; In (c), Ka: nonsynonymous; Ks: synonymous. 0
2
4
6
(a) RSCU G. sinensis - G. fera
G. sinensis - G. Phylogenetic analysis japonica Figure 3. Codon bias analysis and selective pressures in the evolution. (a): Codon content of 20 amino
acids and stop codons in all protein-coding genes of Gleditsia chloroplast genome; (b): Distribution of codon
preference in Gleditsia; (c): Ka/Ks values of protein-coding genes of the seven comparative combinations. Note
In the a, the top panel shows the RSCU for the corresponding amino acids, the colored block which are shown
in the below represent different codons; In (c), Ka: nonsynonymous; Ks: synonymous. Phylogenetic analysis Fourteen chloroplast genome sequences were used for constructing the systematic evolutionary tree, nine of
which were provided by this study and five were provided by other studies, the accession numbers can be found
at the end of each branch. Based on the full-length chloroplast genome and shared CDS sequences, the optimal
model TVM + F + I was calculated by IQTREE according to BIC. Phylogenetic analysis was conducted using the
maximum likelihood (ML) based on the full-length chloroplast genome and shared CDS sequences, and the
resulting trees (Fig. 5) showed the two datasets produced similar phylogenetic trees with high support and only
differed for some nodes’ supporting values, the topologies of the ML based on the full-length chloroplast genome
and ML based on the shared CDS sequence were essentially consistent. G. sinensis and Zhū Yá Zào clustered into
a subclade. G.fera was most closely related to G. sinensis. Two G. microphylla (OP722576.1, NC_047369.1) and
G. microphylla mutant formed a single branch. Discussionh australis seed implantation site is obviously swollen, few fruitless necks40. G. velutina is endemic to Hunan
Province, China, and is a rare and endangered plant under national key protection41. Stress-related genes had
been positively selected during the evolution through comparative transcriptome analysis of Gleditsia genus42. In this study, positive selection was acting on five genes (rpoA, rpl20, atpB, ndhA, ycf4), which were identified
under positive selection (Ka/Ks > 1), Ka/Ks ratio of most gene were less than 1, pairwise Ka/Ks analysis showed
that most of the chloroplast genes of Gleditsia species underwent purifying selection, the purifying selection
on most chloroplast genes within Gleditsia would be evolutionary result of the preservation of the adaptive
characteristics of Gleditsia species.h the four Carya species, indicating that purifying selection were acting on these genes in C. illinoinensis36. Most
of the CDS genes in Chrysosplenium had a Ka/Ks ratio range from 0.1 to 0.3, implying strong purification37. The
average Ka/Ks ratio was 0.17, indicating that the genes in the Eruca sativa were subject to strong purifying selec-
tion pressures38. Purifying selection constantly sweeps away deleterious mutations in population, the purifying
selection on most chloroplast genes within Chrysosplenium would be evolutionary result of the preservation
of the adaptive characteristics of Chrysosplenium species37. G. microphylla is used currently for food, health
care products, and cosmetics, as well as for the treatment of various cancers and heart, vascular, and infec-
tious diseases39. G. japonica pod flat, irregularly twisted; G. delavayi distributed only in Yunnan and Guizhou
Province, China; G. fera distributed gentle slopes, mountain valleys, forests, beside villages, near roads, sunny
places, occasionally cultivated, among the species studied, G. fera can be divided into fast-growing genotype5;
G. australis seed implantation site is obviously swollen, few fruitless necks40. G. velutina is endemic to Hunan
Province, China, and is a rare and endangered plant under national key protection41. Stress-related genes had
been positively selected during the evolution through comparative transcriptome analysis of Gleditsia genus42. Discussionh The chloroplast genome generally ranges in size from 120 to 160 kb and exhibits a highly conserved structure26. The sequencing, assembly, and analysis of chloroplast genomes can identify common features or differences
between species, which can be used as DNA barcodes. Seven Gleditsia species and two Gymnocladus species
both have a circular tetrad structure, consisting of one LSC and SSC region, separated by two IR inverted repeat
regions, the size of the Gleditsia chloroplast genome ranged from 162,746 to 170,907 bp. Most of the SSRs were
located in the intergenic areas27. Based on SSR identification and examination of their location on the chloroplast
genome, it was found that Mononucleotide repeats were the most abundant SSR type in the Gleditsia genus. The
majority of SSRs (65.8–75.8%) in Gleditsia and Gymnocladus species were located in the LSC region, which is
consistent with previous reports on chloroplast SSRs in other plants28,29.fi Codon usage of highly expressed genes was selected in evolution to maintain the efficiency of global protein
translation30. The RSCU values of the CDSs of Gleditsia in the present study were similar, the RSCU values of
tryptophan and methionine amino acids were 1, they were the only amino acids with no codon bias. There were
29 codons with an RSCU value > 1, only one of which ended with G (TTG); The codons with an RSCU value < 1,
except for ATA and CTA ending in A, ended in C or G, the codon pairs ending with C and G in the Gleditsia
chloroplast genome had low bias, and they were nonpreferred codons. The codons with an RSCU value > 1 were
prefer A/T-ending codons in Gleditsia genus (Fig. 3b). Six Euphorbiaceae plant species31 and seven Miscanthus
species32 were biased towards A/T bases and A/T-ending codons. Quercus chloroplast genomes prefer A/T-ending
codons and avoid C/G-ending codons33. Additionally, the Ka/Ks revealed selection pressure on protein-coding
genes34, Ka/Ks ratios > 1, close to 1, or < 1 indicate that the gene has undergone positive selection, neutral selec-
tion, or purifying selection, respectively35. The Ka/Ks ratios for the majority (74 of 79) genes were below 1 for https://doi.org/10.1038/s41598-024-54608-6 Scientific Reports | (2024) 14:4262 | www.nature.com/scientificreports/ the four Carya species, indicating that purifying selection were acting on these genes in C. illinoinensis36. Most
of the CDS genes in Chrysosplenium had a Ka/Ks ratio range from 0.1 to 0.3, implying strong purification37. Discussionh The
average Ka/Ks ratio was 0.17, indicating that the genes in the Eruca sativa were subject to strong purifying selec-
ycf1
petL
psbM
psbT
rps15
ndhF
psbK
ccsA
ycf4
ndhH
0.000
0.003
0.006
0.009
Nucleotide diversity
(a)
accD_-_--psaI_+_
rpl36_-_--rps11_+_
psbK_+_--rps16__exon1_+_
rpl2__exon2_-_--rps19_+_
psbZ_-_--rps14_-_
ndhG_+_--ndhI_-_ rpl14_-_--rps8_+_
rpl33_-_--rps18_+_
atpF__exon1_+_--atpH_-_
ndhD_+_--psaC_-_
0.00
0.02
0.04
0.06
0.08
Nucleotide diversity
(b)
rps16-trnQ
trnT-trnL
psbZ-trnG
rps4-ndhj
0.00
0.05
0.10
0
6500
13000
19500
26000
32500
39000
45500
52000
58500
65000
71500
78000
84500
91000
97500
104000
110500
117000
123500
130000
136500
143000
149500
156000
162500
Nucleotide diversity (Pi)
LSC
IRb
SSC
IRa
(c)
Figure 4. Nucleotide diversity of chloroplast genomes in Gleditsia. (a): Pi in CDS; (b): Pi in intergenic regions;
(c): chloroplast genome Pi values. Note G. sinensis was used as a reference genome for comparison, window
length: 300 bp, step length: 200 bp; X axis: position of the midpoint of each window; Y axis: Pi of each window. ycf1
petL
psbM
psbT
rps15
ndhF
psbK
ccsA
ycf4
ndhH
0.000
0.003
0.006
0.009
Nucleotide diversity
(a)
accD_-_--psaI_+_
rpl36_-_--rps11_+_
psbK_+_--rps16__exon1_+_
rpl2__exon2_-_--rps19_+_
psbZ_-_--rps14_-_
ndhG_+_--ndhI_-_ rpl14_-_--rps8_+_
rpl33_-_--rps18_+_
atpF__exon1_+_--atpH_-_
ndhD_+_--psaC_-_
0.00
0.02
0.04
0.06
0.08
Nucleotide diversity
(b) ycf1
petL
psbM
psbT
rps15
ndhF
psbK
ccsA
ycf4
ndhH
0.000
0.003
0.006
0.009
Nucleotide diversity
(a) accD_-_--psaI_+_
rpl36_-_--rps11_+_
psbK_+_--rps16__exon1_+_
rpl2__exon2_-_--rps19_+_
psbZ_-_--rps14_-_
ndhG_+_--ndhI_-_ rpl14_-_--rps8_+_
rpl33_-_--rps18_+_
atpF__exon1_+_--atpH_-_
ndhD_+_--psaC_-_
0.00
0.02
0.04
0.06
0.08
Nucleotide diversity
(b) rps16-trnQ
trnT-trnL
psbZ-trnG
rps4-ndhj
0.00
0.05
0.10
0
6500
13000
19500
26000
32500
39000
45500
52000
58500
65000
71500
78000
84500
91000
97500
104000
110500
117000
123500
130000
136500
143000
149500
156000
162500
Nucleotide diversity (Pi)
LSC
IRb
SSC
IRa
(c) (c) Nucleotide diversity (Pi) Figure 4. Nucleotide diversity of chloroplast genomes in Gleditsia. (a): Pi in CDS; (b): Pi in intergenic regions;
(c): chloroplast genome Pi values. Note G. sinensis was used as a reference genome for comparison, window
length: 300 bp, step length: 200 bp; X axis: position of the midpoint of each window; Y axis: Pi of each window. the four Carya species, indicating that purifying selection were acting on these genes in C. illinoinensis36. Most
of the CDS genes in Chrysosplenium had a Ka/Ks ratio range from 0.1 to 0.3, implying strong purification37. The
average Ka/Ks ratio was 0.17, indicating that the genes in the Eruca sativa were subject to strong purifying selec-
tion pressures38. Discussionh Purifying selection constantly sweeps away deleterious mutations in population, the purifying
selection on most chloroplast genes within Chrysosplenium would be evolutionary result of the preservation
of the adaptive characteristics of Chrysosplenium species37. G. microphylla is used currently for food, health
care products, and cosmetics, as well as for the treatment of various cancers and heart, vascular, and infec-
tious diseases39. G. japonica pod flat, irregularly twisted; G. delavayi distributed only in Yunnan and Guizhou
Province, China; G. fera distributed gentle slopes, mountain valleys, forests, beside villages, near roads, sunny
places, occasionally cultivated, among the species studied, G. fera can be divided into fast-growing genotype5;
G. australis seed implantation site is obviously swollen, few fruitless necks40. G. velutina is endemic to Hunan
Province, China, and is a rare and endangered plant under national key protection41. Stress-related genes had
been positively selected during the evolution through comparative transcriptome analysis of Gleditsia genus42. In this study, positive selection was acting on five genes (rpoA, rpl20, atpB, ndhA, ycf4), which were identified
under positive selection (Ka/Ks > 1), Ka/Ks ratio of most gene were less than 1, pairwise Ka/Ks analysis showed
that most of the chloroplast genes of Gleditsia species underwent purifying selection, the purifying selection
on most chloroplast genes within Gleditsia would be evolutionary result of the preservation of the adaptive
characteristics of Gleditsia species. d
h d
b
43 Th h hl
bl the four Carya species, indicating that purifying selection were acting on these genes in C. illinoinensis36. Most
of the CDS genes in Chrysosplenium had a Ka/Ks ratio range from 0.1 to 0.3, implying strong purification37. The
average Ka/Ks ratio was 0.17, indicating that the genes in the Eruca sativa were subject to strong purifying selec-
tion pressures38. Purifying selection constantly sweeps away deleterious mutations in population, the purifying
selection on most chloroplast genes within Chrysosplenium would be evolutionary result of the preservation
of the adaptive characteristics of Chrysosplenium species37. G. microphylla is used currently for food, health
care products, and cosmetics, as well as for the treatment of various cancers and heart, vascular, and infec-
tious diseases39. G. japonica pod flat, irregularly twisted; G. delavayi distributed only in Yunnan and Guizhou
Province, China; G. fera distributed gentle slopes, mountain valleys, forests, beside villages, near roads, sunny
places, occasionally cultivated, among the species studied, G. fera can be divided into fast-growing genotype5;
G. Discussionh DNA barcodes derived from chloroplast genomes will be useful for identifying varieties and
resources12. DNA barcodes with the largest nucleotide diversity are considered to be the focus of phylogenetic
analysis and plant identification47. Chloroplast gene sequences (ndhF and rpl16) are selected to test biogeographic
hypotheses, there is a fundamental division of the genus Gleditsia into three clades9. According to sliding win-
dow analysis, rps16-trnQ, rpl32-trnL, ndhD-psaC and ycf1 showed the greatest variations in Ilex48. The several
non-coding sites (psbI–atpA, atpH–atpI, rpoB–petN, psbM–psbD, ndhf–rpl32, and ndhG–ndhI) and three genes
(ycf1, ycf2, and accD) showed significant variation49. Positive selection is observed in 14 protein coding genes
(accD, ccsA, ndhA, ndhB, psbJ, rbcL, rpl20, rpoC1, rpoC2, rps12, rps18, ycf1, ycf2 and ycf4) in nine species of
subfamily Zingiberoideae50. Ka/Ks values of three genes petL, rpl20, and ycf4 were higher than one in the pair-
wise comparation of Galegeae officinalis and other three Galegeae species51. Mutational hotspots of shared genes
and intergenic spacers of the chloroplast genomes of the Gleditsia species were identified. Taking the common
protein coding sequence of Gleditsia as the research object, ycf1 and petL were found as mutational hotspots. ycf1
encodes unknown function proteins. The petL gene encodes the 3.5 kDa subunit of cytochrome b6/f complex52. In other studies, two regions of the plastid gene ycf1, ycf1a and ycf1b, were the most variable loci and of 420 tree
species, 357 species could be distinguished using ycf1b53. The polymorphism of chloroplast genome is useful for
evolutionary analysis of Gleditsia. Mutational hotspots in Gleditsia were found in the intergenic regions such as
"rps16-trnQ", "trnT-trnL", "ndhG-ndhI", and "rpl32-trnL". trnK-rps16 (exon2-intron), trnT-trnL and ycf1 are also
reported in Allium54. These hypervariable regions as potential DNA barcode regions for Gleditsia. ph
yp
g
p
g
A genetic distance analysis based on the ISSR genetic diversity revealed that G. japonica and G. delavayi had
a closer genetic relationship55. By using the complete chloroplast genomes and shared CDS genes, phylogenetic
analysis was performed. The results showed that the two datasets produced similar phylogenetic trees, the rela-
tionships of genus were consistent with high support and only differed for some nodes’ supporting values. Based
on morphology and phylogenetic analysis, G. japonica and G. delavayi appear most closely related. Zhū Yá Zào
is derived from the plant G. Discussionh In this study, positive selection was acting on five genes (rpoA, rpl20, atpB, ndhA, ycf4), which were identified
under positive selection (Ka/Ks > 1), Ka/Ks ratio of most gene were less than 1, pairwise Ka/Ks analysis showed
that most of the chloroplast genes of Gleditsia species underwent purifying selection, the purifying selection
on most chloroplast genes within Gleditsia would be evolutionary result of the preservation of the adaptive
characteristics of Gleditsia species.h p
IR region can indicate the distance between species to a certain extent43. The highly variable regions can
provide useful plastid markers for studies regarding the identification, phylogeny, and population genetics44. Using mVISTA to analyze chloroplast genome sequences within the genus Gleditsia, coding regions were more
conserved than non-coding regions, and IR regions were more conserved than SC regions. Analysis of IR ampli-
fication data indicates that expansion and contraction of IR/SC regions show similar patterns within the genus,
which is also proved from the polymorphism analysis, in which the IR regions were conserved relative to the https://doi.org/10.1038/s41598-024-54608-6 Scientific Reports | (2024) 14:4262 | www.nature.com/scientificreports/ Gleditsia delavayi(OP722574.1)
Gleditsia fera(OP722575.1)
Gleditsia velutina(OP764692.1)
Gleditsia japonica(OP722577.1)
Gleditsia sinensis(OP722579.1)
Gleditsia sinensis zhuyazao(OP722580.1)
Gleditsia sinensis(MT483984.1)
Gleditsia sinensis(NC_047282.1)
Gleditsia sinensis(MZ636527.1)
Gleditsia microphylla(OP722576.1)
Gleditsia microphylla var(OP722581.1)
Gleditsia microphylla(NC_047369.1)
Gymnocladus chinensis(OP722582.1)
Gymnocladus dioicus(OP722578.1)
(a)
Gleditsia delavayi(OP722574.1)
Gleditsia fera(OP722575.1)
Gleditsia velutina(OP764692.1)
Gleditsia japonica(OP722577.1)
Gleditsia sinensis zhuyazao(OP722580.1)
Gleditsia sinensis(MT483984.1)
Gleditsia sinensis(NC_047282.1)
Gleditsia sinensis(OP722579.1)
Gleditsia sinensis(MZ636527.1)
Gleditsia microphylla var(OP722581.1)
Gleditsia microphylla(OP722576.1)
Gleditsia microphylla(NC_047369.1)
Gymnocladus chinensis(OP722582.1)
Gymnocladus dioicus(OP722578.1)
(b)
Figure 5. Gleditsia phylogenetic tree analysis using the maximum likelihood (ML). (a): Phylogenetic analysis
based on chloroplast genome sequence; (b): Phylogenetic analysis based on shared CDS sequence. Gleditsia delavayi(OP722574.1)
Gleditsia fera(OP722575.1)
Gleditsia velutina(OP764692.1)
Gleditsia japonica(OP722577.1)
Gleditsia sinensis(OP722579.1)
Gleditsia sinensis zhuyazao(OP722580.1)
Gleditsia sinensis(MT483984.1)
Gleditsia sinensis(NC_047282.1)
Gleditsia sinensis(MZ636527.1)
Gleditsia microphylla(OP722576.1)
Gleditsia microphylla var(OP722581.1)
Gleditsia microphylla(NC_047369.1)
Gymnocladus chinensis(OP722582.1)
Gymnocladus dioicus(OP722578.1)
(a) Gleditsia delavayi(OP722574.1)
Gleditsia fera(OP722575.1)
Gleditsia velutina(OP764692.1)
Gleditsia japonica(OP722577.1)
Gleditsia sinensis zhuyazao(OP722580.1)
Gleditsia sinensis(MT483984.1)
Gleditsia sinensis(NC_047282.1)
Gleditsia sinensis(OP722579.1)
Gleditsia sinensis(MZ636527.1)
Gleditsia microphylla var(OP722581.1)
Gleditsia microphylla(OP722576.1)
Gleditsia microphylla(NC_047369.1)
Gymnocladus chinensis(OP722582.1)
Gymnocladus dioicus(OP722578.1)
(b) (b) (a) Figure 5. Gleditsia phylogenetic tree analysis using the maximum likelihood (ML). (a): Phylogenetic analysi
based on chloroplast genome sequence; (b): Phylogenetic analysis based on shared CDS sequence. SSC and LSC regions, similar to studies in other plants45. Mutation hotspots can be used as suitable loci for
population genetics and phylogenetic studies. Hypervariable regions can be as candidates for DNA barcode
development46. Conclusion
h
d In this study, we sequenced and compared the complete chloroplast genomes of seven genotypes from Gleditsia. Assembly and annotation of the chloroplast genomes found that Gleditsia species chloroplast genomes have a
typical circular tetrad structure, the size of the chloroplast genomes ranged from 162,746 to 170,907 bp. Through
comparative genomic analysis, most (65.8–75.8%) of the SSRs in Gleditsia and Gymnocladus species are located
in the LSC. The codon pairs ending with C and G in the Gleditsia chloroplast genome have low bias which are
nonpreferred codons, the genus Gleditsia prefer T/A-ending codons and avoid C/G-ending codons. The selec-
tion pressure estimation (Ka/Ks ratios) of genes in the Gleditsia species showed that rpoA, rpl20, atpB, ndhA and
ycf4 were subjected to positive selection, most of the chloroplast genes of Gleditsia species underwent purifying
selection. The genus Gleditsia face relatively weak selection pressure. Mutational hotspots mostly occurred in
"rps16-trnQ", "trnT-trnL", "ndhG-ndhI", "rpl32-trnL" and other intergenic regions in Gleditsia. Phylogenetic
analysis shows that G. fera was most closely related to G. sinensis, G. japonica and G. delavayi were relatively
close, Zhū Yá Zào can be considered a bud mutation of the G. sinensis. Data availability
Th d
d y
The datasets generated and analyzed in this study are available in the GenBank of NCBI, and the complete chlo-
roplast genome sequence were available under the accession Number OP722579-OP722582. Received: 15 July 2023; Accepted: 14 February 2024 Received: 15 July 2023; Accepted: 14 February 2024 Discussionh sinensis, produced by old or injured plants, there was no significant difference in
the contents of saponin compounds between Fructus Gleditsiae abnormalis and Fructus Gleditsiae sinensis by
LC-ELSD7,56. Li et al.8 suggested that Zhū Yá Zào should be a variant of G. sinensis. The evolutionary relationship
between G. sinensis and Zhū Yá Zào was the closest, G. sinensis and Zhū Yá Zào clustered into a subclade (Fig. 5). Zhū Yá Zào can be considered a bud mutation of the G. sinensis.t Albino phenotypes often occur in nature. In the process of raising seedlings in the greenhouse, a albino
mutant plant of G. microphylla was obtained (labeled G. microphylla mutant), which was characterized by albino
whole plant, obvious dwarfing, and natural death after 1–1.5 months of growth. OsSLC1 is responsible for the
seedling-lethal chlorosis phenotype in the rice seedling-lethal chlorosis 1 mutant, loss-of-function of OsSLC1
affected the intron splicing of multiple group II introns, and especially precluded the intron splicing of rps1657. https://doi.org/10.1038/s41598-024-54608-6 Scientific Reports | (2024) 14:4262 | www.nature.com/scientificreports/ The albinism of Camellia sinensis cv. Baiye1 was due to chloroplast dysplasiaand the blocking synthesis of Pchlide
a from Mg-proto IX58. Deficiency in grana stacking in chloroplasts and inhibition of gene expression related to
chloroplast localization may also lead to the production of albino seedlings59. By assembling and comparing the
chloroplast genomes of the G. microphylla mutant and G. microphylla, we found that their sequences were com-
pletely identical. This suggests that the albino phenotype is not caused by variations in the chloroplast genome,
and that the occurrence of the albino phenotype may be due to mutations in chloroplast-related genes involved
in splicing or localization functions. This requires further experimental validation in the future. References 1. Zhang, J.-P. et al. Gleditsia species: An ethnomedical, phytochemical and pharmacological review. J. Ethnopharmacol. 178, 155–171
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1016/j.xplc.2023.100611 (2023). https://doi.org/10.1038/s41598-024-54608-6 Scientific Reports | (2024) 14:4262 | Author contributions Conceptualization, X.F. and Z.Y.; methodology, X.F.; software, X.F., Z.Y.; validation, X.F., Z.Y.; formal analy-
sis, W.X.; investigation, X.F.; resources, X.F., J.X.Y.; data curation, Z.Y.; writing-original draft preparation, X.F.;
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9. Li, N.-N. et al. Dissection of chemical composition and associated gene expression in the pigment-deficient tea cultivar ‘Xiaoxueya
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The authors declare no competing interests. Scientific Reports | (2024) 14:4262 | https://doi.org/10.1038/s41598-024-54608-6 www.nature.com/scientificreports/ Additional informationh Additional information
Supplementary Information The online version contains supplementary material available at https://doi.org/
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ANALISIS NILAI PERUSAHAAN BANK UMUM SYARIAH DI INDONESIA
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ANALISIS NILAI PERUSAHAAN
BANK UMUM SYARIAH DI INDONESIA ALFIAN HALID SOFIAN Alfianhalid@yahoo.com Keyword : CSR, profitability, liquidity, leverage, growth, firm value ISSN 1412-2936
EISSN 2549-7308
ANALISIS NILAI PERUSAHAAN
BANK UMUM SYARIAH DI INDONESIA ISSN 1412-2936
EISSN 2549-7308 ISSN 1412-2936
EISSN 2549-7308 Abstract Company value may reflect the assets owned by a company in the form of
securities of one of them in the form of shares. The stock price traded on the stock
exchange is an indicator of corporate value. Corporate value is influenced by
many factors. This study aims examine the effect csr, profitability, liquidity,
leverage and growth for the value of the company. This research is a research using quantitative approach. The population in
this study were all companies of Sharia Banks registered in Bank Indonesia, from
the population used the total sampling method and the total population of 11
Sharia Commercial Banks became the sample. The data used is secondary data. Data analysis used is path analysis with SPSS 23 The results show that CSR can contribute to corporate value, profitability
can not contribute to corporate value, liquidity can contribute to firm value,
leverage can contribute to firm value, and growth can not contribute to firm value. 193 Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 ISSN 1412-2936
EISSN 2549-7308
Syariah
7. PT. Bank
Panin
Syariah
29,93
32,90
23,53
20,91
8. PT. Bank
Bukopin
Syariah
31,74
33,47
34,26
32,87
9. PT. Bank
Victoria
Syariah
25,28
26,76
27,85
29,34
10. PT. BCA
Syariah
24,72
29,78
31,75
34,87
11. PT. Maybank
Indonesia
Syariah
27,38
29,83
35,47
34,54 PENDAHULUAN Nilai
perusahaan
dapat
mencerminkan nilai asset atau aktiva
yang dimiliki perusahaan seperti surat-
surat berharga salah satunya berupa
saham. Kemampuan
perusahaan
membayar dividen merupakan salah
satu faktor yang mempengaruhi harga
saham. Bagi
perusahaan
yang
menerbitkan saham di pasar modal,
harga saham yang diperjualbelikan di
bursa efek merupakan indikator nilai
perusahaan. Memaksimumkan
nilai
pasar
perusahaan
sama
dengan
memaksimumkan harga pasar saham. Perusahaan yang memiliki total
aset dengan jumlah besar akan lebih
banyak mendapat perhatian investor,
kreditor maupun para pemakai informasi
keuangan
lainnya,
serta
pihak
manajemen akan lebih leluasa dalam
menggunakan aset yang ada untuk
meningkatkan nilai perusahaan. Kondisi
perekonomian
yang
semakin
tidak
menentu atau unpredictable perusahaan
bersaing
dalam
mencapai
keberhasilannya. Hal ini tentunya dapat
dilihat dengan nilai perusahaan itu
sendiri. Nilai perusahaan dapat menjadi
pedoman atau acuan bagi investor atau
stakeholders lainnya di dalam menilai
keberlangsungan
(going
concern)
masing-masing perusahaan. Pergerakan saham bank umum
syariah
juga
dapat
menunjukkan
eksistensi dari nilai perusahaan itu
sendiri. Adapun nilai perusahaan Bank
Umum Syariah dapat dilihat dalam tabel
1 berikut: Tabel. 1 Nilai Perusahaan Bank Umum Tahun 2012-2015 (persen)
No. Nama
Perusahaan
Tahun
2012
2013
2014
2015
1. PT. Bank
Syariah
Mandiri
46,27
40,80
37,34
49,52
2. PT. Bank
Muamalat
Indonesia
49,93
50,07
57,12
58,59
3. PT. Bank
BNI Syariah
35,20
37,03
40,76
44,22
4. PT. Bank
BRI Syariah
29,35
34,31
38,80
39,67
5. PT. Bank
Mega
Syariah
20,73
22,76
24,89
30,43
6. PT. Bank
Jabar
Banten
26,74
29,77
31,43
33,79 Beberapa
penelitian
menyebutkan ada banyak faktor yang
dapat mempengaruhi nilai perusahaan. Diantaranya
yaitu
CSR,
yang
disebutkan bahwa CSR berpengaruh
signifikan terhadap nilai perusahaan
(Arafat et al, 2012; Kusumadilaga, Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 194 ISSN 1412-2936
EISSN 2549-7308
Kadek
Ayu
dan
Ni
Putu
Ayu
Darmayanti. 2015), dengan variabel
independen
yang
digunakan
yaitu
Profitabilitas, Likuiditas, Pertumbuhan,
dan
Investment
Oppurtunity
Set
Terhadap variabel dependen yaitu Nilai
Perusahaan, sedangkan pada penelitian
ini variabel independen yang digunakan
CSR, Profitabilitas, Likuiditas, Leverage
dan
Growth,
terhadap
variabel
dependen
yaitu
nilai
perusahaan. Perbedaan
lain
dengan
penelitian
sebelumnya adalah objek penelitian. Bank Umum Syariah menjadi objek
dalam penelitian ini, hal ini dikarenakan
Bank
umum
syariah
merupakan
lembaga keuangan yang berasaskan
prinsip-prinsip
syariah,
yang
telah
dijelaskan di dalam Undang-undang
No.10/1998 tentang perbankan Islam
dan disempurnakan dalam Undang-
undang No. 7 tahun 1992, serta diikuti
pula
dengan
Undang-undang
No.23/1999 mengenai Bank Indonesia
yang memberikan peluang kepada Bank
Umum Nasional untuk membuka Unit
Usaha Syariah (UUS). Di samping itu,
Bank
Umum
Syariah
semakin
mengalami perkembangan
dan ikut
serta dalam memajukan perekonomian
dunia,
khususnya
di
Indonesia. Perkembangan Bank Umum syariah ISSN 1412-2936
EISSN 2549-7308 ISSN 1412 2936
EISSN 2549-7308
Kadek
Ayu
dan
Ni
Putu
Ayu
Darmayanti. 2015), dengan variabel
independen
yang
digunakan
yaitu
Profitabilitas, Likuiditas, Pertumbuhan,
dan
Investment
Oppurtunity
Set
Terhadap variabel dependen yaitu Nilai
Perusahaan, sedangkan pada penelitian
ini variabel independen yang digunakan
CSR, Profitabilitas, Likuiditas, Leverage
dan
Growth,
terhadap
variabel
dependen
yaitu
nilai
perusahaan. Perbedaan
lain
dengan
penelitian
sebelumnya adalah objek penelitian. Bank Umum Syariah menjadi objek
dalam penelitian ini, hal ini dikarenakan
Bank
umum
syariah
merupakan
lembaga keuangan yang berasaskan
prinsip-prinsip
syariah,
yang
telah
dijelaskan di dalam Undang-undang
No.10/1998 tentang perbankan Islam
dan disempurnakan dalam Undang-
undang No. 7 tahun 1992, serta diikuti
pula
dengan
Undang-undang
No.23/1999 mengenai Bank Indonesia
yang memberikan peluang kepada Bank
Umum Nasional untuk membuka Unit
Usaha Syariah (UUS). Di samping itu,
Bank
Umum
Syariah
semakin
mengalami perkembangan
dan ikut
serta dalam memajukan perekonomian
dunia,
khususnya
di
Indonesia. ISSN 1412-2936
EISSN 2549-7308 ISSN 1412-2936
EISSN 2549-7308 akan
dengan
sengaja
memberikan
signal ke pasar, agar pasar dapat
membedakan
kualitas
perusahaan
tersebut dengan perusahaan lainnya
(Hartono,
2005)
dalam
Adnantara
(2013). Signal ini dapat berupa promosi
atau informasi lainnya, salah satunya
adalah informasi tentang Corporate
Social Responsibility (CSR), dengan
harapan
dapat
meningkatkan
nilai
perusahaan (Adnantara, 2013). saham bank umum syariah dari tahun
ke tahun. Berdasarkan latar belakang yang
telah diuraikan di atas, maka rumusan
masalah dalam penelitian ini sebagai
berikut: 1. Apakah
Corporate
Social
Responsibility berpengaruh terhadap
nilai perusahaan? 2. Apakah Profitabilitas berpengaruh
terhadap nilai perusahaan? Rustiarini (2010) dalam Umro
dan
Fidiana
(2016)
teori
sinyal
membahas
mengenai
dorongan
perusahaan
untuk
memberikan
informasi
kepada
pihak
eksternal
maupun
pihak
internal. Dorongan
tersebut disebabkan karena terjadinya
asimetri
informasi
antara
pihak
manajemen dan pihak eksternal. Untuk
mengurangi asimetri informasi maka
perusahaan
harus
mengungkapkan
informasi yang dimiliki, baik informasi
keuangan maupun non keuangan. 3. Apakah
leverage
berpengaruh
terhadap nilai perusahaan? 4. Apakah
likuiditas
berpengaruh
terhadap nilai perusahaan? 5. Apakah
Growth
berpengaruh
terhadap nilai perusahaan? Teori Sinyal (Signaling Theory) Teori ini menjelaskan mengapa
perusahaan mempunyai dorongan untuk
memberikan
informasi
laporan
keuangan
pada
pihak
eksternal. Dorongan
perusahaan
untuk
memberikan informasi karena terdapat
asimetri informasi antara perusahaan
dan pihak luar karena perusahaan
mengetahui lebih banyak mengenai
perusahaan dan prospek yang akan
datang daripada pihak luar. Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019
19
perusahaan mempunyai dorongan untuk
memberikan
informasi
laporan
keuangan
pada
pihak
eksternal. Dorongan
perusahaan
untuk
memberikan informasi karena terdapat
asimetri informasi antara perusahaan
dan pihak luar karena perusahaan
mengetahui lebih banyak mengenai
perusahaan dan prospek yang akan
datang daripada pihak luar. Teori Signal menyatakan bahwa
perusahaan yang memiliki kualitas baik
informasi yang dimiliki, baik informasi
keuangan maupun non keuangan. Teori Stakeholder
Secara
umum
dalam
teori
stakeholder
menunjukkan
bahwa
perusahaan tidak hanya bertanggung
jawab pada kesejahteraan perusahaan
saja, melainkan harus memiliki tanggug
jawab
sosial
dengan
mempertimbangkan kepentingan semua
pihak
yang
terkena
dampak
dari
tindakan
atau
kebijakan
strategi Tabel. 1 Nilai Perusahaan Bank Umum Perkembangan Bank Umum syariah
juga ditunjukkan dengan naik turunnya 2010), namun dalam penelitian yang
dilakukan
oleh
(Agustine,
2012;
Maspupah, 2014) menyebutkan bahwa
CSR tidak berpengaruh terhadap nilai
perusahaan. Sebagian
besar
penelitian
menunjukkan
hasil
yang
beragam. Menurut (Umro, Madinatul dan Fidiana,
2016) Corporate Social Responsibility
(CSR) berpengaruh positif terhadap nilai
perusahaan
dan
profitabilitas
tidak
berpengaruh terhadap nilai perusahaan. Adapun menurut Agustine (2010) dalam
Cecilia (2014), profitabilitas sebagai
indikator
kinerja
perusahaan
berpengaruh
positif
terhadap
nilai
perusahaan. Fandini
(2013)
dan
Hermunigsih (2013) juga melakukan
penelitian
tentang
nilai
perusahaan
tetapi dengan variabel independen yang
berbeda, yang menunjukkan bahwa
profitabilitas
berpengaruh
positif
terhadap
nilai
perusahaan. Hasil
penelitian ini sejalan dengan penelitian
yang dilakukan Kurnia (2015), Pangulu
(2014), dan Hemastuti (2014) yang
menunjukkan
bahwa
profitabilitas
berpengaruh
positif
terhadap
nilai
perusahaan. Perbedaan penelitian ini dengan
penelitian
sebelumnya
yaitu
pada
variabel
yang
digunakan
dalam
mengukur nilai perusahaan, seperti
halnya yang dilakukan oleh (Sudiani, Ni 195 Corporate
Social
Responsibility
(CSR) Corporate Social Responsibility
(CSR) dicetuskan sebagai suatu visi
akuntabilitas suatu bisnis. Kuncinya
adalah
terfokus
pada
proteksi
lingkungan dan keselamatan kerja para
pekerja, dan pengembangan komunitas
dan masyarakat secara umum baik saat
ini maupun masa depan (Lilis, 2012). Menurut Djajadiningrat dan Melia, 2004:
35,36 dalam Lilis (2012), Konsep CSR
ini
mengarahkan
bahwa
suatu
perusahaan tidak akan dapat bertahan
lama apabila dia mengisolasikan dan
membatasi
dirinya
dari
masyarakat
sekitarnya. Teori Stakeholder │
Secara
umum
dalam
teori
stakeholder
menunjukkan
bahwa
perusahaan tidak hanya bertanggung
jawab pada kesejahteraan perusahaan
saja, melainkan harus memiliki tanggug
jawab
sosial
dengan
mempertimbangkan kepentingan semua
pihak
yang
terkena
dampak
dari
tindakan
atau
kebijakan
strategi Teori Signal menyatakan bahwa
perusahaan yang memiliki kualitas baik 196 ISSN 1412-2936
EISSN 2549-7308
(Cecilia,
dkk,
2014). Dengan
menerapkan
CSR,
diharapkan
perusahaan
akan
memperoleh
legitimasi sosial dan memaksimalkan
kekuatan keuangannya dalam jangka
panjang
(Kiroyan,
2006). Hal
ini
mengindikasikan
bahwa
perusahaan
yang menerapkan CSR mengharapkan
akan direspon positif oleh para pelaku
pasar. ISSN 1412-2936
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(Cecilia,
dkk,
2014). Dengan
menerapkan
CSR,
diharapkan
perusahaan
akan
memperoleh
legitimasi sosial dan memaksimalkan
kekuatan keuangannya dalam jangka
panjang
(Kiroyan,
2006). Hal
ini
mengindikasikan
bahwa
perusahaan
yang menerapkan CSR mengharapkan
akan direspon positif oleh para pelaku
pasar. perusahaan. Kesuksesan
suatu
perusahaan sangat tergantung pada
kemampuannya
dalam
menyeimbangkan beragam kepentingan
dari para stakeholder atau pemangku
kepentingan (Lako, 2011). ISSN 1412-2936
EISSN 2549-7308 ISSN 1412-2936
EISSN 2549-7308 Dengan rasio profitabilitas yang tinggi
yang dimiliki sebuah perusahaan akan
menarik
minat
investor
untuk
menanamkan modalnya diperusahaan
(Dewi dan Wirajaya, 2013) dalam (I
Gusti Ngurah Agung Dwi Pramana,
2016). Growth Wahyudi
dan
Hartini
(2006)
dalam Ni Kadek Ayu Sudiani dan Ni
Putu
Ayu
Darmayanti
(2016)
menyebutkan
bahwa
investasi
perusahaan dalam aktiva tetap adalah
potensi keuntungan bagi perusahaan di
masa
depan,
sehingga
growth
perusahaan adalah hal positif yang
dapat meningkatkan nilai perusahaan. Semakin
besar
tingkat
growth
perusahaan maka akan semakin tinggi
potensi
profitabilitas
perusahaan
di
masa depan sehingga semakin tinggi
pula nilai perusahaan. LIkuiditas Nilai
likuiditas
yang
tinggi
mencerminkan kemampuan perusahaan
yang tinggi untuk memenuhi kewajiban
jangka pendeknya. Perusahaan yang
memiliki nilai likuiditas yang baik akan
dianggap memiliki kinerja yang baik oleh
investor. Menurut Sartono (2010:114)
dalam Sudiyani dan Darmayanti (2016)
Likuiditas
merupakan
kemampuan
perusahaan untuk memenuhi kewajiban
finansial yang berjangka pendek tepat
pada
waktunya. Ukuran
likuiditas
perusahaan yang diproksikan dengan
current ratio (CR) yang merupakan
perbandingan
antara
aktiva
lancar
(current asset) dengan hutang lancar
(current liabilities). Likuiditas yang tinggi
menunjukkan kekuatan perusahaan dari
segi
kemampuan
untuk
memenuhi
hutang lancar dari harta lancar yang
dimiliki sehingga hal ini meningkatkan
kepercayaan
pihak
luar
terhadap
perusahaan tersebut. Profitabilitas Profitabilitas adalah kemampuan
perusahaan dalam menghasilkan laba
(Suharli, 2007). Perusahaan yang dapat
mengelola
asetnya
dengan
baik,
cenderung
menghasilkan
kinerja
keuangan
yang
baik
pula,
yang
diwujudkan dengan adanya laba yang
tinggi (ROA yang tinggi). Profitabilitas
digunakan
untuk
mengukur
sampai
sejauh
mana
keefektifan
dari
keseluruhan
manajemen
dalam
menciptakan
keuntungan
bagi
perusahaan setelah bunga dan pajak
(Sianturi, 2015)dalam (Mariani, 2016). Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019
19
sekitarnya. Pengungkapan
atas
kegiatan
CSR adalah sinyal yang bagus bagi
investor
dan
stakeholder
bahwa
perusahaan
aktif
dalam
melakukan
kegiatan
CSR,
serta
nilai
pasar
perusahaan berada dalam posisi yang
bagus. Kinerja sosial perusahaan yang
bagus membantu perusahaan untuk
memperoleh reputasi dari pasar modal
dan pasar utang sehingga membuat
nilai
suatu
perusahaan
meningkat
menciptakan
keuntungan
bagi
perusahaan setelah bunga dan pajak
(Sianturi, 2015)dalam (Mariani, 2016). Profitabilitas
yang
tinggi
mencerminkan kemampuan perusahaan
dalam menghasilkan keuntungan yang
tinggi bagi pemegang saham. Semakin
besar
keuntungan
yang
diperoleh
semakin
besar
pula
kemampuan
perusahaan
untuk
membayarkan
devidennya, dan hal ini berdampak
pada
kenaikan
nilai
perusahaan. Pengungkapan
atas
kegiatan
CSR adalah sinyal yang bagus bagi
investor
dan
stakeholder
bahwa
perusahaan
aktif
dalam
melakukan
kegiatan
CSR,
serta
nilai
pasar
perusahaan berada dalam posisi yang
bagus. Kinerja sosial perusahaan yang
bagus membantu perusahaan untuk
memperoleh reputasi dari pasar modal
dan pasar utang sehingga membuat
nilai
suatu
perusahaan
meningkat Profitabilitas
yang
tinggi
mencerminkan kemampuan perusahaan
dalam menghasilkan keuntungan yang
tinggi bagi pemegang saham. Semakin
besar
keuntungan
yang
diperoleh
semakin
besar
pula
kemampuan
perusahaan
untuk
membayarkan
devidennya, dan hal ini berdampak
pada
kenaikan
nilai
perusahaan. 197 ISSN 1412-2936
EISSN 2549-7308
perusahaan. Leverage dihitung dari total
utang
dibagi
dengan
total
aset. Perusahaan dengan tingkat leverage
yang
tinggi
menunjukkan
bahwa
perusahaan lebih banyak bergantung
pada utang dalam membiayai aset
perusahaan. Utang bagi perusahaan
memiliki beban tetap yang berupa
beban bunga. Semakin besar utang
yang dimiliki perusahaan maka beban
bunga yang harus dibayarkan juga
semakin
tinggi. Perusahaan
yang
memiliki utang tinggi akan mendapatkan
insentif pajak berupa potongan atas
bunga pinjaman sehingga perusahaan
yang memiliki beban pajak tinggi dapat
melakukan penghematan pajak dengan
cara
menambah
utang
perusahaan
(Suyanto dan Suparmono, 2012). Pengaruh Likuiditas Terhadap Nilai
Perusahaan Likuiditas
dalam
teori
berhubungan
positif
dengan
nilai
perusahaan. Semakin tinggi likuiditas
maka
nilai
perusahaan
tinggi
dan
semakin rendah likuiditas maka nilai
perusahaan rendah. Kemampuan kas
yang tinggi akan berdampak terhadap
kemampuan kewajiban jangka pendek
perusahaan
dan
berdampak
positif
terhadap nilai perusahaan (Mahendra,
2012). H 1 : Ada pengaruh corporate sosial
responsibility (CSR) terhadap nilai
perusahaan ISSN 1412-2936
EISSN 2549-7308 ISSN 1412-2936
EISSN 2549-7308 ISSN 1412-2936
EISSN 2549-7308 ISSN 1412 2936
EISSN 2549-7308
maka nilai perusahaan rendah. Semakin
baik
perusahaan
membayar
return
terhadap
pemegang
saham
akan
meningkatkan nilai perusahaan. Nilai
perusahaan dapat pula dipengaruhi oleh
besar
kecilnya
profitabilitas
yang
dihasilkan oleh perusahaan. Weston
dan Copeland (1992) dalam Prasetyorini
(2013)
mendefinisikan
profitabilitas
sejauh mana perusahaan menghasilkan
laba
dari
penjualan
dan
investasi
perusahaan. Apabila
profitabilitas
perusahaan
baik
maka
para
stakeholders yang terdiri dari kreditur,
supplier, dan juga investor akan melihat
sejauh
mana
perusahaan
dapat
menghasilkan laba dari penjualan dan
investasi perusahaan. Dengan baiknya
kinerja perusahaan akan meningkatkan
pula nilai perusahaan. EISSN 2549-7308
maka nilai perusahaan rendah. Semakin
baik
perusahaan
membayar
return
terhadap
pemegang
saham
akan
meningkatkan nilai perusahaan. Nilai
perusahaan dapat pula dipengaruhi oleh
besar
kecilnya
profitabilitas
yang
dihasilkan oleh perusahaan. Weston
dan Copeland (1992) dalam Prasetyorini
(2013)
mendefinisikan
profitabilitas
sejauh mana perusahaan menghasilkan
laba
dari
penjualan
dan
investasi
perusahaan. Apabila
profitabilitas
perusahaan
baik
maka
para
stakeholders yang terdiri dari kreditur,
supplier, dan juga investor akan melihat
sejauh
mana
perusahaan
dapat
menghasilkan laba dari penjualan dan
investasi perusahaan. Dengan baiknya
kinerja perusahaan akan meningkatkan
pula nilai perusahaan. Pengembangan Hipotesis
Pengaruh Pengungkapan Corporate
Sosial Responsibility Terhadap Nilai
Perusahaan
Frekuensi
dilakukannya
Corporate Social Responsibility oleh
perusahaan akan mendongkrak citra
perusahaan. Investor lebih berminat
pada perusahaan yang memiliki citra
baik di masyarakat karena semakin baik
citra perusahaan, loyalitas konsumen
semakin tinggi sehingga dalam waktu
lama
penjualan
perusahaan
akan
meningkat dan profitabilitas perusahaan
juga
akan
meningkat
(Retno
dan
Priantinah, 2012) dalam (Mariani,dkk,
2016). Pengungkapan Corporate Social
Responsibility perusahaan diharapkan
dapat memberikan sinyal yang positif
kepada
investor,
sehingga
dapat
meningkatkan nilai perusahaan (Cheng
dan
Christiawan,
2011).Berdasarkan
uraian
di
atas
dapat
dirumuskan
hipotesis sebagai berikut. Pengembangan Hipotesis
Pengaruh Pengungkapan Corporate
Sosial Responsibility Terhadap Nilai
Perusahaan Leverage menggambarkan
yang
dimiliki
oleh Leverage
menggambarkan
struktur
modal
yang
dimiliki
oleh 198 Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 Pengaruh ProfitabilitasTerhadap Nilai
Perusahaan Profitabilitas
dalam
teori
berhubungan
positif
dengan
nilai
perusahaan. Semakin
tinggi
profitabilitas maka nilai perusahaan
tinggi dan semakin rendah profitabilitas Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 199 EISSN 2549-7308
Leverage
dalam
teori
berhubungan
negatif
dengan
nilai
perusahaan. Semakin tinggi leverage
maka nilai perusahaan rendah dan
semakin rendah leverage maka nilai
perusahaan tinggi. Penggunaan hutang
harus hati-hati oleh pihak manajemen,
karena semakin besar hutang akan
menurunkan nilai perusahaan. Leverage
yang parsial dalam hasil penelitian yang
dilakukan
(Mahendra,
dkk,
2012)
menemukan memiliki pengaruh negatif
tidak
signifikan
terhadap
nilai
perusahaan. Berdasarkan uraian di atas
dapat dirumuskan hipotesis sebagai
berikut. Likuiditas
menunjukkan
kemampuan
perusahaan
untuk
memenuhi
kewajiban
jangka
pendeknya. Tingkat likuiditas yang tinggi
akan menunjukkan bahwa perusahaan
berada
dalam
kondisi
yang
baik
sehingga akan menambah permintaan
saham dan tentunya akan menaikkan
harga saham (Mahendra, 2011) dalam
(Mariani, dkk, 2016). Kemampuan kas
yang tinggi akan berdampak pada
kemampuan
memenuhi
kewajiban
jangka
pendek
perusahaan
dan
berdampak positif pada perusahaan
(Mahendra, 2012) dalam (Mariani, dkk,
2016). Semakin
tinggi
likuiditas
perusahaan,
semakin
banyak
dana
yang tersedia bagi perusahaan untuk
membayar dividen, membiayai operasi
dan investasinya, sehingga persepsi
investor pada kinerja perusahaan akan
meningkat (Nurhayati, 2013) dalam
(Mariani, dkk, 2016). H 3 : Ada pengaruh leverage terhadap
nilai perusahaan Pengaruh
Growth
Terhadap
Nilai
Perusahaan Pertumbuhan perusahaan pada
penelitian ini diukur dengan growth
(pertumbuhan
asset). Pertumbuhan
perusahaan adalah selisih total aset
yang dimiliki oleh perusahaan pada
periode
sekarang
dengan
periode
sebelumnya terhadap total aset periode
sebelumnya pada perusahaan Bank
Umum Syariah
di Bank Indonesia
periode 2012-2015. ISSN 1412-2936
EISSN 2549-7308
Populasi dan Sampel Penelitian
Populasi perusahaan dalam aktiva tetap adalah
potensi keuntungan bagi perusahaan di
masa depan, sehingga pertumbuhan
perusahaan adalah hal positif yang
dapat meningkatkan nilai perusahaan. Berdasarkan
uraian
di
atas
dapat
dirumuskan hipotesis sebagai berikut. Populasi menurut Iqbal dalam
Aulia (2008:45) adalah totalitas dari
semua objek atau individu yang memiliki
karakteristik tertentu, jelas, dan lengkap
yang akan diteliti (bahan penelitian). Populasi dalam penelitian ini adalah
semua
peruasahaan
Bank
Umum
Syariah
yang
terdaftar
di
Bank
Indonesia sebanyak 11 perusahaan. H 5 : Ada pengaruh growth terhadap nilai
perusahaan Pengaruh Leverage Terhadap Nilai
Perusahaan Menurut Jensen (1976) dalam
Febrianti (2012), nilai perusahaan dapat
meningkat
dengan
adanya
hutang
apabila
hutang
tersebut
digunakan
bukan
untuk
investasi
yang
tidak
menguntungkan, melainkan digunakan
untuk mengendalikan penggunaan arus
kas
yang
berlebihan
oleh
pihak
manajemen perusahaan. Wahyudi
dan
Hartini
(2006)
dalam Ni Kadek Ayu Sudiani dan Ni
Putu
Ayu
Darmayanti
(2016)
menyebutkan
bahwa
investasi 200 Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 ISSN 1412-2936
EISSN 2549-7308 ISSN 1412-2936
EISSN 2549-7308 TABEL 2. Daftar total sampling
Bank Umum Syariah di Indonesia
No
Nama Bank
1
PT. Bank Syariah Mandiri
2
PT. Bank Syariah Muamalat Indonesia
3
PT. Bank Syariah BNI
4
PT. Bank Syariah BRI
5
PT. Bank Syariah Mega Indonesia
6
PT. Bank Jabar Banten Syariah
7
PT. Bank Panin Syariah
8
PT. Bank Syariah Bukopin
9
PT. Bank Victoria Syariah
10
PT. BCA Syariah
11
PT. Maybank Indonesia Syariah TABEL 2. Daftar total sampling
Bank Umum Syariah di Indonesia TABEL 2. Daftar total sampling untuk mencari adanya hubungan antara
variabel dependen dengan satu atau
lebih variabel independen. Persamaan
yang digunakan untuk menguji hipotesa
adalah: Y = a +b 1X 1+ b 2 X 2 + b 3 X 3 + b 4 X 4 + e Y = a +b 1X 1+ b 2 X 2 + b 3 X 3 + b 4 X 4 + e
Keterangan : Y = a +b 1X 1+ b 2 X 2 + b 3 X 3 + b 4 X 4 + e Keterangan : Keterangan : Keterangan : Y
: Nilai perusahaan
a
: Intersep (atau nilai Y pada saat
X=0)
b
: Koefisien regresi
X 1
: Corporate Social Responsibility
(CSR)
X 2
: Profitabilitas
X 3
: Leverage
X 4
: Likuiditas
e
: Galat / standart error Y
: Nilai perusahaan
a
: Intersep (atau nilai Y pada saat
X=0)
b
: Koefisien regresi
X 1
: Corporate Social Responsibility
(CSR) Y
: Nilai perusahaan
a
: Intersep (atau nilai Y pada saat
X=0) Y
: Nilai perusahaan b
: Koefisien regresi
X 1
: Corporate Social Responsibility
(CSR) Uji Asumsi Klasik Uji
asumsi
klasik
dilakukan
untuk
mengetahui data yang layak untuk
dianalisis. Pengujian
data
dalam
penelitian ini yaitu:Uji Normalitas, Uji
Multikolinearitas, Uji Autokolerasi, dan
Uji Heterokedastisitas. e
: Galat / standart error Untuk kepentingan analisis data
tersebut,
dipergunakan
program
komputer SPSS 23. Pengujian hipotesis
dilakukan dengan uji t, namun sebelum
meregresi data, dilakukan uji asumsi
klasik regresi. Uji Hipotesis Penelitian
ini
menggunakan
regresi berganda. Bentuk persamaan
dalam penelitian ini adalah: Teknik Analisis Data dan Uji Statistik
Statistik Deskriptif Statistik
deskriptif
digunakan
untuk menjelaskan deskripsi data dari
seluruh variabel yang akan dimasukkan
dalam model penelitian yang dilihat dari
nilai minimum, nilai maksimum, nilai
rata-rata dan standar deviasi. Jenis dan Desain Penelitian Sampel
adalah
bagian
dari
jumlah karakteristik yang dimiliki oleh
populasi tersebut (Sugiyono, 2013:116). Teknik
penentuan
sampel
yang
digunakan dalam penelitian ini adalah
total sampling dimana pemilihan elemen
populasi dilakukan sedemikian rupa
sehingga
setiap
elemen
tersebut
mempunyai kesempatan yang sama
untuk terpilih. Sampel akhir adalah 11
perusahaan Bank Umum Syariah yang
terdaftar di Bank Indonesia tahun 2012-
2015. Jenis penelitian yang digunakan
dalam penelitian ini adalah penelitian
kuantitatif. Analisis
data
dalam
penelitian kuantitatif yaitu menggunakan
statistik deskriptif. Variabel dependen penelitian ini
adalah
nilai
perusahaan. Nilai
perusahaan
merupakan
persepsi
investor terhadap perusahaan yang
sering dikaitkan harga saham. Harga
saham
yang
tinggi
membuat
nilai
perusahaan juga tinggi. Harga saham
merupakan harga yang terjadi pada saat
saham diperdagangkan di pasar. Variabel
independen
yang
digunakan dalam penelitian ini yaitu
Corporate Social Reponsibility (CSR),
profitabilitas, likuiditas, leverage dan
growth. 201 Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 Uji multikolinearitas Uji
multikolinearitas
bertujuan
menguji
apakah
model
regresi
ditemukan adanya korelasi antarvariabel
bebas
atau
independen
(Ghozali,
2006:91). Jika hasil menunjukkan nilai
Variance Inflation Factor (VIF) ≥ 10
berarti ada multikolinearitas, sebaliknya
jika nilai VIF < 10 berarti tidak ada
multikolinearitas. PEMBAHASAN Metode
analisis
data
yang
digunakan
dalam
penelitian
ini
menggunakan path analysis dengan
SPSS 23. e : Error Penelitian
ini
menggunakan
analisis regresi linier berganda karena
untuk mengetahui pengaruh variabel
independen
(CSR,
profitabilitas,
leverage,
dan
likuiditas)
terhadap
variabel dependen (nilai perusahaan). Alasan lain peneliti menggunakan
analisis regresi linier berganda karena
variabel independennya lebih dari satu. Gambar 1. Hasil Pengujian Normalitas Pada gambar 1 grafik normal plot
terlihat titik-titik menyebar di sekitar
garis diagonal dan mengikuti arah garis
diagonal. Dengan
demikian
dapat
diketahui bahwa variabel independen
dan dependen memiliki distribusi normal
sehingga dari segi normalitas, model
regresi layak dipakai untuk menganalisis
pengaruh variabel independen terhadap
variabel dependen. ISSN 1412-2936
EISSN 2549-7308 ISSN 1412-2936
EISSN 2549-7308 X2 : Profitabilitas (Return on Total
Leverage/ROA)
X3 : Leverage
X4 : Likuiditas (Current Ratio/CR)
b1,…,b5 : Koefisien regresi Gambar 1. Hasil Pengujian Normalitas X4 : Likuiditas (Current Ratio/CR)
b1,…,b5 : Koefisien regresi Uji Regresi Y = a + b1+ b2X2 + b3X3 + b4D1+e Berdasarkan pada hipotesis yang
diajukan,
metode
analisis
yang
digunakan untuk membuktikan hipotesa
adalah metode statistik regresi linier
berganda. Analisis regresi bertujuan Keterangan:
Y : Nilai Perusahaan
X1 : Corporate Social Responsibility
Disclosure (CSRD) 202 Tabel 4. Hasil Uji Regresi Tabel 4. Hasil Uji Regresi
Coefficientsa
Model
Unstandardized
Coefficients
T
Sig. B
Std. Error
1 (Constant)
30.544
2.448
12.476
.000
CSR
1.831E-9
.000
6.925
.000
Profitabilitas
3.563
18.263
.195
.846
Likuiditas
.039
.018
2.218
.033
Leverage
-2.437
1.038
-2.347
.024
Growth
-2.054
1.728
-1.189
.242
a. Dependent Variable: NP Uji Normalitas Uji
normalitas
dilakukan
untuk
menguji apakah dalam model regresi
variabel
pengganggu
atau
residual
memiliki distribusi normal. Uji normalitas
data menggunakan analisis statistik
non-parametrik uji Kolmogorov-Smirnov. Dalam uji ini variabel yang mempunyai
asymp. Sig < 0,05 maka diartikan
bahwa
variabel-variabel
tersebut
memiliki distribusi tidak normal (Ghozali,
2006:110). Hasil uji normalitas berikut
ini. Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 203 ISSN 1412-2936
EISSN 2549-7308
independen. Model regresi yang baik
adalah yang homokedastisitas atau
tidak
terjadi
heteroskedastisitas. Penelitian ini menggunakan Grafik Plot,
dasar analisis: ISSN 1412-2936
EISSN 2549-7308 Tabel 3. Hasil Uji multikolinearitas Tabel 3. Hasil Uji multikolinearitas
No
Variabel Independen
VIF
1
CSR
1.181
2
Profitabilitas
1.072
3
Likuiditas
1.108
4
Leverage
1.152
5
Growth
1.063 independen. Model regresi yang baik
adalah yang homokedastisitas atau
tidak
terjadi
heteroskedastisitas. Penelitian ini menggunakan Grafik Plot,
dasar analisis: Gambar 2. Hasil Uji Heterokedastisitas Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 Uji Autokolerasi Uji
autokolerasi
bertujuan
menguji apakah dalam model regresi
linear ada korelasi antara kesalahan
pengganggu pada periode t dengan
kesalahan pengganggu pada periode t-1
(sebelumnya). Deteksi
adanya
autokorelasi dapat dilihat dari angka DW
(Durbin-Watson). Angka D-W dibawah -
2 berarti ada autokorelasi positif. Berikut
hasil pengujian autokorelasi. Gambar 2. Hasil Uji Heterokedastisitas Berdasarkan gambar 2 grafik
scatterplots
terlihat
bahwa
titik-titik
menyebar secara acak serta tersebar
baik di atas maupun di bawah angka 0
pada sumbu y dan titik membentuk pola
tertentu. Dengan
demikian
dapat
disimpulkan
bahwa
tidak
terjadi
heterogenitas pada model persamaan
regresi yang digunakan. Tabel 4. Hasil Uji Autokorelasi Tabel 4. Hasil Uji Autokorelasi
Model Summary
b
Model
R
R Square
Adjusted R
Square
Std. Error of
the Estimate
Durbin-
Watson
1
.845
a
.714
.676
5.11482
1.704 SARAN Bagi
perusahaan
khususnya
Bank Umum Syariah yang memiliki
tingkat
profitabilitas
yang
rendah,
sebaiknya lebih baik dalam pengelolaan
asetnya dan kebijakan hutangnya . Hal
ini dikarenakan ROE adalah fungsi dari
profit margin, assets turnover dan equity
multiplier. Adapun
langkah-langkah
yang dilakukan untuk meningkatkan
profitabilitas yaitu mengurangi harga
pokok penjualan atau beban operasi
perusahaan, meningkatkan penjualan
tanpa adanya peningkatan beban atau
biaya
secara
proposional,
dan
meningkatkan
penggunaan
utang
relative terhadap ekuitas sampai titik
yang tidak membahayakan keuangan
perusahaan. Tabel tersebut juga menunjukkan
signifikansi antar variabel, yaitu CSR,
likuiditas
dan
leverage
memiliki
signifikansi sebesar 0.000 0.033 dan
0.024 yang artinya CSR, likuiditas dan
leverage berpengaruh positif terhadap
nilai
perusahaan,
sedangkan
profitabilitas dan growth berpengaruh
negatif
terhadap
nilai
perusahaan
dengan signifikansi sebesar 0.846 dan
0.242. SIMPULAN Hasil
penelitian
menunjukkan
bahwa CSR, likuiditas, dan leverage
memberikan
kontribusi
dalam
meningkatkan nilai perusahaan. Hasil
penelitian
ini
juga
menunjukkan
profitabilitas
dan
growth
tidak
memberikan
kontribusi
dalam
meningkatkan nilai perusahaan. Bagi
perusahaan
khususnya
Bank Umum Syariah yang memiliki
tingkat growth yang rendah, sebaiknya
memilih strategi pertumbuhan
yang
tepat, fokus pada penetrasi pasar,
perkuat
pada
segmen
mikro,
dan
inovasi produk. Uji Heterokedastisitas Uji heteroskedastisitas bertujuan
menguji apakah dalam model regresi
terjadi ketidaksamaan variance dari
residual
satu
pengamatan
ke
pengamatan yang lain. Pengujian ini
juga bertujuan untuk menguji apakah
dalam
model
regresi
terjadi
ketidaksamaan deviasi standar nilai
variabel dependen pada setiap variabel Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 204 ISSN 1412-2936
EISSN 2549-7308
sehingga
ada
perusahaan
yang
mengalami kerugian. Berdasarkan persamaan tersebut
nilai
30.544
merupakan
intersep/konstanta,
nilai
tersebut
merupakan
nilai dari variabel
nilai
perusahaan apabila CSR, profitabilitas,
likuiditas,
leverage
dan
growth
ditetapkan sama dengan nol, maka nilai
perusahaan diproksikan dengan return
saham sebesar 30.544. Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 ISSN 1412-2936
EISSN 2549-7308 Ekonomi, Vol. 18, 108 No. 2,
Agustus 2013 Ekawati, Lilis, et al. 2012. Pengaruh
Profitabilitas, Likuiditas, Growth, dan
Media
Exposure
Terhadap
Pengungkapan
Tanggungjawab
Sosial Perusahaan. g
Ajija, Shochrul R. 2011. Cara Cerdas
Menguasai
E-Views. Jakarta:
Salemba Empat. Ajija, Shochrul R. 2011. Cara Cerdas
Menguasai
E-Views. Jakarta:
Salemba Empat. Kusumayanti & Astika. 2016 Corporate
Social
Responsibility
sebagai
Pemediasi
Pengaruh
Ukuran
Perusahaan,
Profitabilitas,
dan
Leverage Pada Nilai Perusahaan. E-
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Corporate
Social
Responsibility,
Profitabilitas
dan
Ukuran
Perusahaan
Terhadap
Nilai
Perusahaan
pada
Perusahaan
Perkebunan yang Go Public di
Indonesia,
Malaysia,
dan
Singapura.SNA
18. Universitas
Sumatera Utara. Cecilia, dkk. 2014. Analisis Pengaruh
Corporate
Social
Responsibility,
Profitabilitas
dan
Ukuran
Perusahaan
Terhadap
Nilai
Perusahaan
pada
Perusahaan
Perkebunan yang Go Public di
Indonesia,
Malaysia,
dan
Singapura.SNA
18. Universitas
Sumatera Utara. Lako, Andreas 2011. Dekonstruksi CSR
& Reformasi Paradigma Bisnis &
Akuntansi. Jakarta:
Penerbit
Erlangga Cheng, Ming-Chang & Zuwei-Ching
Tzeng. 2011. The Effect of Leverage
on Firm Value and How The Firm
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of
Management
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Alfredo,
et
al. 2012. Pengaruh
Kinerja
Keuangan
Terhadap Nilai Pada Perusahaan
Manufaktur di Bursa Efek Indonesia. Jurnal Manajemen, Strategi Bisnis,
dan Kewirausahaan Vol. 6, 137, No. 2 Agustus 2012 Febrianti, Meiriska. 2012. Faktor-faktor
yang
Mempengaruhi
Nilai
Perusahaan
Pada
Industri
Pertambangan
di
Bursa
Efek
Indonesia. Jurnal
bisnis
dan
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2012 Mardiyati,
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al. 2012. Pengaruh
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Deviden,
Kebijakan
Hutang dan Profitabilitas Terhadap
Nilai Perusahaan Manufaktur yang
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Pancawati. 2012. Determinan Nilai Perusahaan. JAI
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Governance dan CSR Memoderasi
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Likuiditas,
Solvabilitas,
dan
Profitabilitas
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Perusahaan. E-Jurnal Ekonomi dan
Bisnis
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Udayana
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(2016): 2801-2836. ISSN : 2337-
3067 Hardiningsih,
Pancawati. 2012. Determinan Nilai Perusahaan. JAI
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Titin. 2012. Pengaruh
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Hutang, dan Profitabilitas Terhadap
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modal
yang
dimiliki
untuk meningkatkan profit dengan baik Makro, Jurnal Manajemen & Kewirausahaan, Vol. 4 No. 2│November 2019 205 ISSN 1412-2936
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2303-1174 Sugiyono. 2010. Metode
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Kuantitatif,
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EISSN 2549-7308 4 No. 2│November 2019 207
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https://openalex.org/W2181008001
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https://hal.science/hal-03390518/document
|
English
| null |
Modulation of Tonically Active Neurons of the Monkey Striatum by Events Carrying Different Force and Reward Information
|
The journal of neuroscience/The Journal of neuroscience
| 2,015
|
cc-by
| 15,377
|
Systems/Circuits
Modulation of Tonically Active Neurons of the Monkey
Striatum by Events Carrying Different Force and Reward
Information Simon Nougaret and Sabrina Ravel
Institut de Neurosciences de la Timone, UMR7289, CNRS, Aix-Marseille Universite´, 13005 Marseille, France Simon Nougaret and Sabrina Ravel
Institut de Neurosciences de la Timone, UMR7289, CNRS, Aix-Marseille Universite´, 13005 Marseille, France The role of basal ganglia in motivational processes has been under scrutiny in recent decades, with increasing evidence from clinical
studies of cognitive and motivational deficits in patients with basal ganglia lesions. Tonically active neurons (TANs), the presumed
striatal cholinergic interneurons, could be important actors in integrating and relaying motivational information arising from various
modalities. Their multiphasic responses to rewards and to conditioned stimuli associated with reward conferred them a role in limbic
processes.Theyarealsomodulatedbyatask’smotoraspect.Recentstudiessuggesttheyareinfluencedbythecontextinwhichbehavioral
responses are expressed. To investigate the role of TANs in motor-limbic interaction processes, we recorded 169 TANs in the striatum of
two monkeys performing a motivational task, in which they had to develop a variable force to receive different amounts of reward in
response to visual stimuli. Our results reveal new features of TANs response properties. First, TANs usually responded either by a pause
or an elevation of discharge rate to the visual cues and the reward, with few neurons combining both pause and rebound. Second, the
elevations of discharge rate after the cues were most sensitive to the least valuable (high force or small reward) task conditions. Finally,
the responses of TANs to the visual cues were time locked on the onset of the animal’s movement. TANs’ population and responses could
thus play a role in signaling less attractive situations, those with either a high motor demand and/or small reward. ey words: basal ganglia; cholinergic interneurons; effort; electrophysiology; motivation; nonhuman primat Tonically active neurons (TANs) are known for their responses to unpredictable positive or negative events. However, here we
show that TANs respond by a pause or an increase in their activity to all rewarding events in a task in which combined visual cues
indicate to the monkeysthelevelsofforcetoproduceandtheupcomingreward.Unlikethepause,theincreaseinactivityismodulated
by task parameters and is most sensitive to the least attractive task conditions (high force and/or small reward). TANs’ responses
triggered by cue occurrence are also modulated by movement-related information (movement onset). We therefore propose here that
TANscouldplayarole,viatheiractiononstriatalprojectionsneurons,inmaintaininghighcost/lowbenefitratiobehaviors. Clinical studies have reported that basal ganglia disorders not
only trigger motor, but also cognitive and motivational deficits
(Brown et al., 1997; Pessiglione et al., 2006). 15214 • The Journal of Neuroscience, November 11, 2015 • 35(45):15214–15226 15214 • The Journal of Neuroscience, November 11, 2015 • 35(45):15214–15226 Systems/Circuits Systems/Circuits
Modulation of Tonically Active Neurons of the Monkey
Striatum by Events Carrying Different Force and Reward
Information Tonically active
neurons (TANs), presumed to be cholinergic interneurons, are
distributed throughout the striatum, and their responses have
generally been described as homogeneous (Aosaki et al., 1995;
Apicella, 2002; Adler et al., 2012). TANs are known to be involved
in reward-related processes and associative learning. Most stud-
ies have shown that their activity is modulated by the temporal
prediction of a conditioned stimulus or a reward, its probability
of occurrence, and the affective significance of stimuli. Their re-
sponses are also modulated by the general context in which learn-
ing or the expression of the learned behavior take place (Aosaki et
al., 1994; Apicella et al., 1997, 2009, 2011; Sardo et al., 2000;
Shimo and Hikosaka, 2001; Ravel et al., 2003; Morris et al., 2004; Received Jan. 6, 2015; revised Sept. 12, 2015; accepted Oct. 6, 2015.
Authorcontributions:S.R.designedresearch;S.N.andS.R.performedresearch;S.N.andS.R.analyzeddata;S.N.
and S.R. wrote the paper.
This work was supported by Centre National de la Recherche Scientifique and the Fondation de France (Grant
2008 005902). We thank Drs. Paul Apicella, Christelle Baunez, and Janine Simmons for helpful comments and
discussions.
The authors declare no competing financial interests.
Correspondence should be addressed to Sabrina Ravel, Institut de Neurosciences de la Timone, UMR 7289,
Aix-Marseille Université and CNRS, Campus Santé Timone, 27 bd Jean Moulin, 13385 Marseille, France. E-mail:
Sabrina.Ravel@univ-amu.fr.
DOI:10.1523/JNEUROSCI.0039-15.2015
Copyright © 2015 the authors
0270-6474/15/3515214-13$15.00/0 Authorcontributions:S.R.designedresearch;S.N.andS.R.performedresearch;S.N.andS.R.analyzeddata;S.N.
and S.R. wrote the paper. Received Jan. 6, 2015; revised Sept. 12, 2015; accepted Oct. 6, 2015. Introduction In the same way, a red filled circle indicated to the animals
that the reward delivered would be small [0.3 ml of water; “small reward”
(r)]; a filled red square indicated that it would be large [1.2 ml of water;
“large reward” (R)]. The four possible combinations of visual stimuli (fr,
fR, Fr, FR) set the four different conditions of the task. In response to
these stimuli, monkeys had to increase their pressing force on the lever to
reach the required force in a period shorter than 1 s [maximal reaction
time (RT)] and hold this force for 1 s (holding time) to receive the
reward. For each correct trial, monkeys were rewarded with the small or
large reward according to the shape of the red stimuli. Both visual stimuli
were extinguished as soon as the reward was delivered. To monitor force,
monkeys were helped by visual feedback, a vertical rectangle representing
the range of the required force located just below the visual stimuli. In
this rectangle, a white cursor indicated in real time the force developed on
the lever when in the required force range. After receiving the reward, the
monkeys returned to a basal pressing force in preparation for the next
trial, which did not begin until the total duration of the current trial (4.5
s) had elapsed. The responses of TANs classically consist of a pause followed
by a rebound in activity, sometimes preceded by a short initial
increase. TANs could, in turn, modulate the activity of phasically
active neurons (PANs), the striatal projection neurons, by a mod-
ification of their excitability state during the pause displayed by
TANs in relation with stimuli associated with reward or reward
itself, resulting in a time window during which other afferences
could be expressed (Akins et al., 1990). PAN responses show
various modulations in relation to movement, reward, or reward
expectancy, depending on their location in the striatum (Hiko-
saka et al., 1989; Crutcher and Alexander, 1990; Apicella et al.,
1991). Thus, TANs could potentially directly, or indirectly by
acting on presynaptic afferences on PANs, participate in motor,
cognitive, and/or motivational processes, via the PANs. Introduction The role of TANs in motivational processes has been mainly
studied in the context of classical conditioning or instrumental
tasks in which the reward has been manipulated, in its occurrence
or size for instance, but very little attention has been devoted to
their response properties in more complex tasks modulating both
motor and reward aspects. However, in day to day life, animals
and humans must often apply different levels of effort in response
to environmental stimuli to achieve variable quantities of reward. To investigate this situation, we studied the activity of TANs in a
single experimental protocol in which visual cues provide infor-
mation about both required effort and expected reward. If TANs
can encode complex motivational value, we thus expect that they
would respond to visual cues predictive of the reward as a func-
tion of their rewarding value (computed from the effort required
and the expected reward). Moreover, the development of the
required force is necessary to succeed in our task. Consequently,
the activity of TANs, in this context, could also be modulated by
the movement as it has been reported previously. There were three different cases in which a trial was considered as
failed and no reward given. First, trials in which the required force was
not reached within a 1 s period were considered omission errors. Second,
trials in which the required force was not held for at least 1 s (holding
time) were considered holding errors. Last, trials in which the force de-
veloped was greater than the upper limit of the required force (495 and
810 g, respectively, for the low and the high forces) were considered
threshold errors. Both holding and threshold errors were considered as
execution errors. After an error, a correction trial ensued; the same asso-
ciation of visual stimuli was presented again to the monkeys until they
performed the trial correctly. Moreover, trials in which the monkeys
began to increase their pressing force on the lever within 100 ms after the
occurrence of the visual stimuli were considered as anticipations and
were excluded from analysis. Before the electrophysiological recordings began, the monkeys were
extensively trained (4 to 6 months) until a performance threshold of 80%
correct trials was achieved, in which the preparatory period, the maximal
reaction time, and the holding time were all of 1 s. Introduction Basal ganglia, and more specifically one of their input structures,
the striatum, have been considered to be important for perform-
ing and maintaining adapted motivated behaviors. Authorcontributions:S.R.designedresearch;S.N.andS.R.performedresearch;S.N.andS.R.analyzeddata;S.N. and S.R. wrote the paper. This work was supported by Centre National de la Recherche Scientifique and the Fondation de France (Grant
2008 005902). We thank Drs. Paul Apicella, Christelle Baunez, and Janine Simmons for helpful comments and
discussions. The authors declare no competing financial interests. Correspondence should be addressed to Sabrina Ravel, Institut de Neurosciences de la Timone, UMR 7289,
Aix-Marseille Université and CNRS, Campus Santé Timone, 27 bd Jean Moulin, 13385 Marseille, France. E-mail:
Sabrina.Ravel@univ-amu.fr. DOI:10.1523/JNEUROSCI.0039-15.2015 DOI:10.1523/JNEUROSCI.0039-15.2015
Copyright © 2015 the authors
0270-6474/15/3515214-13$15.00/0 J. Neurosci., November 11, 2015 • 35(45):15214–15226 • 15215 Nougaret and Ravel • TANs’ Modulation by Force and Reward Yamada et al., 2004; Joshua et al., 2008; Deffains et al., 2010). An
important feature of the responses of TANs is that the responses
are usually transferred from the reward to the preceding condi-
tioned stimulus during learning (Apicella et al., 1997; Ravel et al.,
2003), and TANs’ modulations are more pronounced in response
to unpredictable, highly rewarding stimuli. It has also been re-
ported that the activity of TANs can be modulated, under some
conditions, by movement in both nonhuman primates and ro-
dents (Lee et al., 2006; Benhamou et al., 2014). between 0 and 20% of the maximal force, defined experimentally at 900 g
based on the capabilities of the animals, during a 1 s preparatory period. After this period, two visual stimuli, a green one and a red one, each being
either a filled circle or filled square, were presented vertically in the center
of the screen. The shape of the green stimulus indicated the amount of
force the animals had to develop on the lever; that of the red stimulus
indicated the amount of the upcoming reward. A green filled circle indi-
cated that the animals had to develop a force between 20 and 55% of the
maximal force [180 to 495 g; “low force” (f)], and a green filled square a
force between 55 and 90% of the maximal force [495 to 810 g; “high
force” (F)]. Introduction In each recording
session, the four different combinations of visual stimuli were presented
pseudorandomly, from trial to trial. The first trial of a session was ran-
domly chosen from a list of trials where each condition was present in the
same proportion. If trials were performed correctly, the same visual stim-
uli were not presented more than times sequentially. Surgery Surgery
Initial anesthesia was administered by an intramuscular injection of ket-
amine (10 mg/kg) and xylazine (0.5 mg/kg), followed by deep anesthesia
induced by isoflurane. A polyether ether ketone recording chamber (19
mm inner diameter) was implanted over the left hemisphere. Recording
chambers in both monkeys were positioned with a 20° angle laterally in
the coronal plane. The targeted stereotaxic coordinates, relative to ear
bars, were as follows: Monkey M, anterior, 18 mm; lateral, 16 mm; Mon-
key Y, anterior, 14 mm; lateral, 16 mm [based on the atlas of Saleem and
Logothetis (2007)]. During the same surgery, two titanium cylinders
were embedded in the orthopedic cement (Palacos with gentamicin) and
fixed to the skull with titanium orthopedic bone screws for subsequent
head restraint during neuronal recordings. Following surgery, monkeys
were given antibiotics (Marbocyl, 2 mg/kg) and analgesics (Tolfedine, 4
mg/kg) on the day of the surgery and for the following 4 d. The recording
chamber was filled with an antibiotic solution (Marbocyl, 2 mg/ml) and
sealed with a removable cap. Materials and Methods
Animal and apparatus pp
Two male rhesus monkeys (Macaca mulatta) weighing 8 and 7 kg at the
beginning of the experiments (Monkeys M and Y, respectively) were
trained to exert and maintain a force on a lever in response to visual
stimuli to receive a liquid reward. All experimental procedures were in
compliance with the National Institutes of Health’s Guide for the Care
and Use of Laboratory Animals, the French laws on animal experimenta-
tion, and the European directive on the protection of animals used for
scientific purposes. Behavioral procedures Behavioral procedures p
The monkeys were seated in a Plexiglas primate chair and faced a panel
supporting a 17 inch screen on which visual stimuli could be presented. The screen was positioned 18 cm from the monkey; a lever equipped with
strain gauges in the lower part of the panel was positioned at waist level. A sliding door at the front of the primate chair could be opened to allow
the animal to position his hand on the lever. The liquid reward (water)
was delivered via a metal spout positioned directly in front of the mon-
key’s mouth. The liquid was delivered through a solenoid valve located
outside the recording room. The trial structure is illustrated in Figure 1A. At the beginning of each
trial, the monkey had to develop a basal pressing force on the lever, 15216 • J. Neurosci., November 11, 2015 • 35(45):15214–15226 Nougaret and Ravel • TANs’ Modulation by Force and Reward Preparatory
period
Cue-threshold
period
Holding
period
Post-reward
period
Beginning
of the trial
Occurence of
the visual stimuli
Force
development
Reward
A
AC + 2
AC + 3
AC +4
AC +5
AC +6
AC -1
AC
AC + 1
Monkey Y
B
Monkey M
Associative neurons
Sensorimotor neurons
AC -2
C
D
AC -1
AC
AC +1
AC +2
AC +3
re1. Taskdesignandreconstructionofrecordinglocations.A,Taskdesign.Apairofvisualstimuliappearedafterthemaintenanceofabasalpressingforcebytheanimalfor1s.Inrespo
esestimuli,themonkeyincreasedhispressingforce,reachedtherequiredforcerange,andheldthisforcefor1stoobtainthereward.Fourpossiblecombinationsofvisualstimuliindicate
nimaltheforcehehadtodevelopandthesizeoftheupcomingreward.Thetopstimulusrepresentedtheforceandthebottomonethereward.Afilledcirclerepresentedasmallamount,
ed square a large one. B–D, Reconstruction of recording locations of TANs. All electrode recording sites were obtained from MR images. Electrode artifacts are visible in MR images of b
keys,andthelocationofeachrecordedneuronwasextrapolatedfromthetipoftheseelectrodes.B,RecordingsitesforMonkeyMfromAC1toAC6(onemoreneuron,recordedinAC
t represented on the figure). Each dot represents the location of one neuron recorded during the task. The white ones represent the locations of associative neurons, and the black ones
ons of sensorimotor neurons. C, Recording sites for Monkey Y from AC 2 to AC 3 (same representation as in B). D, Three-dimensional reconstruction of the anterior part of the stria
sparent gray) of Monkey Y, AC 4 in the background. 6 • J. Behavioral procedures Neurosci., November 11, 2015 • 35(45):15214–15226
Nougaret and Ravel • TANs Modulation by Force and Rew Preparatory
period
Cue-threshold
period
Holding
period
Post-reward
period
Beginning
of the trial
Occurence of
the visual stimuli
Force
development
Reward
A A Reward Post-reward
period Force
development Holding
period Cue-threshold
period Preparatory
period AC + 2
AC + 3
AC +4
AC +5
AC +6
AC -1
AC
AC + 1
Monkey Y
B
Monkey M
Associative neurons
Sensorimotor neurons
AC -2
C
D
AC -1
AC
AC +1
AC +2
AC +3 Monkey M B Associative neurons
Sensorimotor neurons
D Associative neurons
Sensorimotor neurons Monkey Y C D AC +3 AC +1 Figure1. Taskdesignandreconstructionofrecordinglocations.A,Taskdesign.Apairofvisualstimuliappearedafterthemaintenanceofabasalpressingforcebytheanimalfor1s.Inresponse
tothesestimuli,themonkeyincreasedhispressingforce,reachedtherequiredforcerange,andheldthisforcefor1stoobtainthereward.Fourpossiblecombinationsofvisualstimuliindicatedto
theanimaltheforcehehadtodevelopandthesizeoftheupcomingreward.Thetopstimulusrepresentedtheforceandthebottomonethereward.Afilledcirclerepresentedasmallamount,and
a filled square a large one. B–D, Reconstruction of recording locations of TANs. All electrode recording sites were obtained from MR images. Electrode artifacts are visible in MR images of both
monkeys,andthelocationofeachrecordedneuronwasextrapolatedfromthetipoftheseelectrodes.B,RecordingsitesforMonkeyMfromAC1toAC6(onemoreneuron,recordedinAC3
is not represented on the figure). Each dot represents the location of one neuron recorded during the task. The white ones represent the locations of associative neurons, and the black ones the
locations of sensorimotor neurons. C, Recording sites for Monkey Y from AC 2 to AC 3 (same representation as in B). D, Three-dimensional reconstruction of the anterior part of the striatum
(transparent gray) of Monkey Y, AC 4 in the background. Data analyses Data analyses
All data analyses were performed using conventional statistical proce-
dures with the R statistical computing environment (R Development
Core Team, 2011). Behavioral analyses. Behavioral analyses were performed on data col-
lected during the recordings of 169 TANs (81 from Monkey M; average
number of trials, 92; range, 52–131; 88 from Monkey Y, average number
of trials, 95; range, 55–117). Force and reward selectivity. To examine the force and reward sensi-
tivities of TANs, we performed a two-way ANOVA on the spike count
of each of the four determined response periods. We used the amount
of force and the amount of reward as the two factors. We considered
a TAN as modulated by the amount of force during a period if the p
value “force” of the ANOVA was 0.05 in this period. In the same
way, a TAN was considered as modulated by the amount of reward in
a period if the p value “reward” of the ANOVA was 0.05 in this
period. Finally, a TAN was considered as modulated by an interaction
between the amount of force and the amount of reward in a period if
the p value “interaction” of the ANOVA was 0.05 in this period. This analysis was performed on the entire population of TANs, not
only those categorized as responsive, to avoid omission of nonrespon-
sive TANs that could nevertheless be sensitive to the amount of a force
and/or reward. RTs, which were the times between the onset of the visual stimuli and
the time at which the monkey started to increase his pressing force on the
lever, were measured only for correct trials. The time at which monkeys
started to increase their pressing force on the lever to reach the required
force was determined manually by moving a cursor on the force graph
trial by trial using LabVIEW. An automatic detection procedure, using
the force derivate compared to a threshold, failed to give reliable values of
RTs since monkeys were sometimes slightly releasing the lever (and de-
creasing their force) before pressing to reach the required force. Thus,
manual detection was required. Since, on trials in which RTs could be
automatically detected, values were identical to those that we could de-
termined manually, we estimated that it was more reliable to analyze all
trials with the same method, and did it manually. Data analyses RTs were changed into
z-scores for normalization purposes, and a two-way ANOVA was per-
formed with required force and expected reward as the two factors. Error
rates (ERs; i.e., the total number of errors performed in a condition
divided by the total number of trials, both correct and error trials, per-
formed in this condition) were calculated and compared with a Pearson’s
2 test. Each p value was corrected by Bonferroni correction and differ-
ences considered as significant when p 0.0083 (0.05/6, six possible
comparisons). In each condition, the proportion of omission and execu-
tion errors was determined by dividing the number of one type of error
(execution or omission) by the total number of errors in the condition. For each neuron and each period, we estimated a force selectivity
index (FSI) and a reward selectivity index (RSI) to determine the
weight of these modulations based on the firing rate for pairs of
conditions with a common factor (same reward or same force; Peck et
al., 2013). For each selectivity index (SI), we computed SI (1
2)/[(SS1 SS2)/(df1 df2)], where x is the firing rate during a
given period, SSx is the sum of squares, and dfx is the degree of
freedom (number of trials minus 1) for each pair of conditions de-
scribed below (Peck et al., 2013). To calculate the FSI, we compared
the neuronal activity during trials in the high force conditions (Fr and
FR) to the neuronal activity during trials in the low force conditions
(fr and fR). In the same way, to calculate the RSI, we compared, in the
same periods, the neuronal activity during trials in the large reward
conditions (fR and FR) with the neuronal activity during trials in the
small reward conditions (fr and Fr). An index superior to zero indi-
cated, for the force, a stronger modulation in the high force condi-
tions, and for the reward, a stronger modulation in the large reward
conditions. Similarly, an index inferior to zero indicated, for the
force, a stronger modulation in the low force conditions, and for the
reward, a stronger modulation in the small reward conditions. Electrophysiological analyses. Electrophysiological data were analyzed
only for correct trials performed during the recording sessions. Determination of response periods. Data analyses TAN responses to rewarding stimuli
have been described previously as being mainly multiphasic, with a pause
followed by a rebound (Aosaki et al., 1995; Apicella et al., 1997, Ravel et
al., 1999). This biphasic response could rarely be preceded by a short
initial excitation. Thus, we will focus on the classically described pause
and rebound (when it follows the pause) or elevation of discharge rate in
the following analyses. To evaluate statistically the responses to the oc-
currence of the cues and the reward, we set unique test windows for all
recorded TANs, and for each TAN, we counted the number of spikes
within these windows for each neuron. The test windows were deter-
mined on the basis of the population histogram aligned on cues and
reward onsets averaged across all recorded TANs, using the following
procedure [based on the procedure of Shimo and Hikosaka, (2001)]. A
sliding time window with a duration of 100 ms was moved in 10 ms steps
starting at the onset of the cue, for the cue responses, and starting at the
occurrence of the reward, for reward responses. We established four test
windows, two for the decrease of TAN activity after cue and reward
occurrence and two for the increase. The sliding window was moved until
the averaged firing rate within the window was significantly different
from the baseline firing rate (within a 900 ms window before the occur-
rence of the cues) for five consecutive steps (two-tailed t test, p 0.001). For detection of a decrease in the firing rate, these significant differences
had to be combined with a negative t value, and for detection of an Relation to the time of onset of the change in force. Based on visual
inspection of the rasters, the activity of some neurons seemed to be
more aligned on the onset of the change in force (OCF) than on the
occurrence of the cues. To investigate the influence of the OCF on the
neuronal responses and determine whether those responses were trig-
gered by the movement or by sensory information (cues), we per-
formed the same series of analysis as described previously with the
neuronal activity aligned on the OCF of the animals. Different meth-
ods described in the literature have been successfully used to investi-
gate this question in other brain structures (DiCarlo and Maunsell,
2005; Lee et al., 2010). Electrophysiological recordings
hil
h
k
f
i The recording electrode was lowered to isolate neurons while the mon-
key performed the task. We isolated single neurons by continuously
monitoring the waveform of the recorded neuronal impulses on an os-
cilloscope. Tonic striatal neurons were distinguished on the basis of their
spontaneous firing rate and the duration of their spikes (Kimura et al. 1984; Apicella et al. 1997). The activity of the first well-isolated neuron in
a trajectory was recorded for at least ten trials per condition. After re-
cording from a TAN, the electrode was moved forward until another
TAN was encountered. Data from all TANs recorded were included in
analyses. p y
g
g
While the monkeys were performing the task, with head immobilization,
extracellular activity of single neurons was recorded with custom-made
glass-insulated tungsten microelectrodes based on the technique of Mer-
rill and Ainsworth (1972). To record from the basal ganglia structures, a
stainless steel guide tube (diameter, 0.6 mm) was lowered below the
surface of the dura, and the microelectrode was passed inside the
guide and advanced using a manual hydrolic microdrive (M096, Na-
rishige). The microelectrode was connected to a preamplifier located
in close proximity to the microdrive. The neuronal signal was then
amplified 5000 times and filtered at 0.3–1.5 kHz, and spikes were
detected on-line by threshold crossing using a window discriminator
(Neurolog, Digitimer). The presentation of visual stimuli, the force
developed by the animal, the delivery of the reward, and digital pulses
from neuronal activity were controlled/collected by a computer using
custom-designed software written in LabVIEW (National Instru-
ment). The signals from the strain gauge allowing the measure of the
force were digitized at 500 Hz. Localization of recordings To assess the localization of our recordings, we obtained a high-
resolution magnetic resonance imaging (MRI) scan for each monkey
with electrodes positioned (five for Monkey M, six for Monkey Y) in
trajectories on which we recorded TANs. Based on the localization of the
tip of these electrodes, we extrapolated the inferior/superior, anterior/
posterior, and medial/lateral position of each recorded neuron to gener- J. Neurosci., November 11, 2015 • 35(45):15214–15226 • 15217 Nougaret and Ravel • TANs’ Modulation by Force and Reward ate a three-dimensional reconstruction using Brainsight software (Rogue
Research; Fig. 1B). One hundred and sixty-nine neurons were localized
to the striatum and identified as TANs. Electrophysiological recordings
hil
h
k
f
i In the anteroposterior plane from
the anterior commissure (AC), neurons were recorded from AC 3 to
AC 6 and AC 2 to AC 3, for Monkey M and Monkey Y, respectively. For Monkey M, only one neuron was recorded in AC 3 and none in AC
2; these two anteroposterior planes are not illustrated in Figure 1B. Neurons from AC 1 and posterior locations located in the putamen
were considered as located in the sensorimotor part of the striatum. Neurons from AC 1 and posterior locations located in the caudate
nucleus and all neurons anterior to AC 1 were considered as located in
the associative part of the striatum (Parent and Hazrati, 1995). increase in the firing rate, with a positive t value. The onset of the test
windows were taken to be the beginning of the window that was the
earliest among the five consecutive steps. The 100 ms window was further
moved until the averaged firing rate was not significantly different from
the baseline firing rate or the t value was no longer of the same sign for
five consecutive steps. The offset of the test window was taken to be the
beginning of the window that was the earliest among the five consecutive
steps. Presence or absence of cues responses. We then determined whether each
TAN showed a response. For each trial, we calculated the response firing
rate in the four test windows (converted from the spike count within the
window) and the baseline firing rate in the control window (900 ms
period before onset of the cues). If the difference between the response
and the baseline firing rate was statistically significant (Wilcoxon signed
rank test, p 0.05), it was judged that the TAN showed a response during
this window. For the two windows targeting the decrease of TAN activity,
it was verified that significant responses corresponded to decreases of the
firing rate. For the two windows targeting the increases of TAN activity, it
was also verified that significant responses were increases of the firing
rate. Data analyses C, G, Acceptance lev
oftheanimalsinthefourconditionsofthetask(fR,lowforce/largereward;FR,highforce/largereward;fr,lowforce/smallreward;Fr,highforce/smallreward).D,H,Meanoftheforcedevelop
bytheanimalsinthefourconditionsofthetask.Darkgraylines,Largereward;lightgraylines,smallreward;thicklines,highforce;thinlines,lowforce.Asterisksindicateforthereactiontimest
influenceofeachfactorontheanimal’sbehavior(two-wayANOVA.**p0.05;***p0.001)andfortheerrorratesthedifferencesamongconditions(Pearson’s 2test;***p0.001,corrected fR
FR
fr
Fr
Percentage of acceptance
100
95
90
85
80
l
l
l
l
e
e
C -1
0
1
2
3
1000
800
600
400
200
0
4
FR
Fr
fR
fr
Mean force developped on the lever (grams)
D 0
10
20
30
40
50
Percentage of error rates
l
l
***
***
***
***
r
R
Low Force
High Force
B 270
280
290
300
310
Average reaction time (ms)
l
l
r
R
p.reward ***
p.force > 0.05
A fR
FR
fr
Fr
Percentage of acceptance
100
95
90
85
80
l
l
l
l
-1
0
1
2
3
1000
800
600
400
200
0
4
FR
Fr
fR
fr
e
Mean force developped on the lever (grams)
C
D C B D Average reaction time (ms) Percentage of error rates Percentage of acceptance r
R
270
280
290
300
310
Average reaction time (ms)
l
l
r
R
p.reward > 0.05
p.force > 0.05
E 0
10
20
30
40
50
Percentage of error rates
l
l
r
R
**
***
***
r
R
F fR
FR
fr
Fr
Percentage of acceptance
100
95
90
85
80
l
l
l
l
Time
-1
0
1
2
3
1000
800
600
400
200
0
4
Mean force developped on the lever (grams)
G
H
Time E F G Percentage of error rates Percentage of acceptance Figure 2. A–H, Behavioral results for Monkey M (A–D) and Monkey Y (E–H). A, E, Reaction times of the animals in the four conditions of the task. R, Small reward; R, large reward; solid black
line, high force; dashed gray line, low force. The error bars represent the SEM. B, F, Error rates of the animals in the four conditions of the task (same conventions as in A, E). C, G, Acceptance level
oftheanimalsinthefourconditionsofthetask(fR,lowforce/largereward;FR,highforce/largereward;fr,lowforce/smallreward;Fr,highforce/smallreward).D,H,Meanoftheforcedeveloped
bytheanimalsinthefourconditionsofthetask.Darkgraylines,Largereward;lightgraylines,smallreward;thicklines,highforce;thinlines,lowforce.Asterisksindicateforthereactiontimesthe
influenceofeachfactorontheanimal’sbehavior(two-wayANOVA.**p0.05;***p0.001)andfortheerrorratesthedifferencesamongconditions(Pearson’s 2test;***p0.001,corrected). trials only (7480 from Monkey M, 8468 from Monkey Y). RTs
were significantly shorter in the large reward conditions than in
the small reward ones in Monkey M (two-way ANOVA on RT
z-score, preward 7.97.10 15, F 60.59), and there was no sig-
nificant difference among the RTs in the low or high force con-
ditions for this monkey (Fig. 2A). There was no significant
difference among the RTs of Monkey Y (Fig. 2E). Error rates A complementary analysis was performed, in situations in which a
response was detected when the neuronal activity was aligned on cue
occurrence and on the OCF, to specify which of these two events was
most likely to modulate the response. A Wilcoxon rank sum test was
performed to determine whether the detected pauses, aligned on the cues
and on the OCF, were significantly different (in terms of magnitude) and
which one was more pronounced. The same tests were performed for the
elevation of the discharge rate. ERs were computed from the total number of trials performed by
the animals (9115 from Monkey M, 10221 from Monkey Y),
including correct and error trials. The ERs were significantly
higher in the small reward than large reward conditions for the
same force developed (low force, p 3.27.10 14 and 1.44.10 17,
2 57.57 and 72.79 for Monkeys M and Y, respectively; high
force, p 5.43.10 43 and 1.49.10 31, 2 188.94 and 136.57 for
Monkeys M and Y, respectively; Fig. 2B,F). Moreover, for the
same expected reward, the ERs were significantly higher in the
high than low force conditions (small reward, p 8.05.10 53 and
0.00058, 2 233.99 and 11.85 for Monkeys M and Y, respec-
tively; large reward, p 9.28.10 21, 2 87.31 for Monkey M,
nonsignificant for Monkey Y). Data analyses However, the electrophysiological properties
of TANs made those methods unsuitable. Thus, we used an alternative
method in which we successively defined periods of decrease and
increase of activity around the OCF, determined whether each TAN Nougaret and Ravel • TANs’ Modulation by Force and Reward 15218 • J. Neurosci., November 11, 2015 • 35(45):15214–15226 270
280
290
300
310
Average reaction time (ms)
l
l
r
R
0
10
20
30
40
50
Percentage of error rates
l
l
270
280
290
300
310
Average reaction time (ms)
l
l
r
R
0
10
20
30
40
50
Percentage of error rates
l
l
r
R
p.reward ***
p.force > 0.05
p.reward > 0.05
p.force > 0.05
***
***
***
***
**
***
***
fR
FR
fr
Fr
Percentage of acceptance
100
95
90
85
80
r
R
l
l
l
l
fR
FR
fr
Fr
Percentage of acceptance
100
95
90
85
80
l
l
l
l
-1
0
1
2
3
1000
800
600
400
200
0
4
Time
FR
Fr
fR
fr
Low Force
High Force
Mean force developped on the lever (grams)
-1
0
1
2
3
1000
800
600
400
200
0
4
Mean force developped on the lever (grams)
A
B
C
D
E
F
G
H
Time
Figure 2. A–H, Behavioral results for Monkey M (A–D) and Monkey Y (E–H). A, E, Reaction times of the animals in the four conditions of the task. R, Small reward; R, large reward; solid bla
line, high force; dashed gray line, low force. The error bars represent the SEM. B, F, Error rates of the animals in the four conditions of the task (same conventions as in A, E). Data analyses showed a pause or an elevation of discharge rate, and examined the
force and reward sensitivities of each TAN when its activity was
aligned on the time of the OCF. To compare the amplitude of the
response period of the whole population of TANs aligned on the
occurrence of the visual stimuli versus that aligned on the OCF, a
two-tailed t test was performed between the average firing rates of
each significant bin defining the test window aligned on the cues
versus each significant bin defining the test window aligned on the
OCF. Behavioral results: modulation of the behavioral responses by
the force required and the expected reward size Reaction times Average RTs to start increasing the force on the lever after the
occurrence of visual stimuli were computed from the correct J. Neurosci., November 11, 2015 • 35(45):15214–15226 • 15219 Nougaret and Ravel • TANs’ Modulation by Force and Reward 0
4.5
5.0
5.5
6.0
6.5
7.0
200
400
600
800
-30
-20
-10
0
10
20
Spike/s
t-value
Time from cues (ms)
4.5
5.0
5.5
6.0
6.5
7.0
-30
-20
-10
0
10
20
t-value
0
200
400
600
800
Cues
Reward
Spike/s
A
B
f R
F R
f r
F r
Time from reward (ms)
−1000
0
2000
3000
0
10
0
10
0
10
0
10
Spk density
Spk density
Spk density
Spk density
C
1000
−1000
0
2000
3000
0
10
0
10
0
10
0
10
f R
F R
f r
F r
D
1000
0
10
0
10
0
10
0
10
−1000
0
1000
Time from
reward (ms)
Time from
reward (ms)
E
F
f R
F R
f r
F r
−1000
0
1000
0
15
0
15
0
15
0
15
f R
F R
f r
F r
Time from
cues (ms)
Time from
cues (ms)
ure3. Periodsofanalysisaftertheoccurrenceofcuesandreward.A,Populationactivityalignedoncueoccurrenceandcorrespondingtvaluesofthecomparisonbetweenthebaselineactiv
theactivityduringa100msslidingwindowbeginningaftertheoccurrenceofthecues.Thetwograyrectanglesillustratethetwoperiodsofanalysisbasedonthestatisticaldifferencefoundw
baseline. The pause occurred between 60 and 280 ms, and the elevation of the discharge rate between 300 and 530 ms after cue occurrence. B, Population histogram aligned on rewa
rrence (same conventions as in A). The pause occurred between 20 and 280 ms, and the elevation of the discharge rate between 450 and 610 ms after reward occurrence. C–F, Raster plots
examplesofTANs.Eachlinerepresentsatrial,andeachpointrepresentsaspike;thesmoothedlinesontherasterrepresentthespikedensity(50).Trialsarerankedaccordingtothefo
itionsofthetask.C,Neuronshowingapositiveforceeffectduringtheelevationofthedischargerateaftertheoccurrenceofthevisualstimuli.D,Neuronshowinganegativerewardeffectduri
elevation of the discharge rate after the occurrence of the cues E Neuron showing an interaction effect during the pause after reward occurrence Its activity decreased critically in the
garet and Ravel TANs Modulation by Force and Reward
J. Behavioral results: modulation of the behavioral responses by
the force required and the expected reward size Neurosci., November 11, 2015 35(45):15214 15226 1521 0
4.5
5.0
5.5
6.0
6.5
7.0
200
400
600
800
-30
-20
-10
0
10
20
Spike/s
t-value
Time from cues (ms)
Cues
A 4.5
5.0
5.5
6.0
6.5
7.0
-30
-20
-10
0
10
20
t-value
0
200
400
600
800
Reward
Spike/s
B
Time from reward (ms) B Reward A Time from cues (ms)
f R
F R
f r
F r
−1000
0
2000
3000
0
10
0
10
0
10
0
10
Spk density
Spk density
Spk density
Spk density
C
1000
−1000
0
0
10
0
10
0
10
0
10
D
1000
f R
F R
f r
F r
Time from
cues (ms)
Time from
cues (ms) Time from reward (ms)
2000
3000
f R
F R
f r
F r
0
10
0
10
0
10
0
10
−1000
0
1000
Time from
reward (ms)
Time from
reward (ms)
E
F
−1000
0
1000
0
15
0
15
0
15
0
15
f R
F R
f r
F r E C ewar
F m cue
D Time from
reward (ms) Time from
reward (ms) Figure3. Periodsofanalysisaftertheoccurrenceofcuesandreward.A,Populationactivityalignedoncueoccurrenceandcorrespondingtvaluesofthecomparisonbetweenthebaselineactivity
andtheactivityduringa100msslidingwindowbeginningaftertheoccurrenceofthecues.Thetwograyrectanglesillustratethetwoperiodsofanalysisbasedonthestatisticaldifferencefoundwith
the baseline. The pause occurred between 60 and 280 ms, and the elevation of the discharge rate between 300 and 530 ms after cue occurrence. B, Population histogram aligned on reward
occurrence (same conventions as in A). The pause occurred between 20 and 280 ms, and the elevation of the discharge rate between 450 and 610 ms after reward occurrence. C–F, Raster plots of
fourexamplesofTANs.Eachlinerepresentsatrial,andeachpointrepresentsaspike;thesmoothedlinesontherasterrepresentthespikedensity(50).Trialsarerankedaccordingtothefour
conditionsofthetask.C,Neuronshowingapositiveforceeffectduringtheelevationofthedischargerateaftertheoccurrenceofthevisualstimuli.D,Neuronshowinganegativerewardeffectduring
the elevation of the discharge rate after the occurrence of the cues. E, Neuron showing an interaction effect during the pause after reward occurrence. Its activity decreased critically in the FR
condition. F, Neuron showing a negative reward effect during the elevation of the discharge rate after the reward occurrence. Table 1. Responses of TANs in both monkeys, after cue and reward occurrence
Responsive
Cues
Reward
Cues and reward
Pause
105/169 (62.1%)
57/169 (33.7%)
67/169 (39.6%)
19/169 (11.2%)
Elevation
87/169 (51.5%)
58/169 (34.4%)
47/169 (27.8%)
18/169 (10.7%)
Pause and
elevation
36/169 (21.3%)
23/169 (13.6%)
16/169 (9.5%)
3/169 (1.8%)
At least one
136/169 (80.5%)
92/169 (54.4%)
98/169 (58.0%)
54/169 (32.0%)
Therowheadingsindicatethetypeofmodulation(apause,anelevationofthedischargerate,both,oratleastone
of the two). The columns indicate the period of modulation (after the cues and/or the reward, only after the cues,
only after the reward, or after cues and reward). Behavioral results: modulation of the behavioral responses by
the force required and the expected reward size Table 1. Responses of TANs in both monkeys, after cue and reward occurrence reward (fR); high force/large reward (FR); low force/small reward
(fr); high force/small reward (Fr) (Fig. 2C,G). In both monkeys,
the amount of reward seemed to be more important than the
level of effort in the decision of animals to perform the task. In
the fR condition, monkeys performed the action in 99.3%
(Monkey M) and 97.6% (Monkey Y) of trials presented to
them. On the contrary, in the Fr conditions, monkeys per-
formed the action in 82.4% (Monkey M) and 83.7% (Monkey
Y) of trials. For both monkeys, “intermediate” conditions (FR
and fr) were ranked following the same order: they accepted
FR trials more frequently (97% for Monkey M, 95.3% for
Monkey Y) than fr trials (92% for Monkey M, 94.7% for Mon-
key Y). The relatively low level of acceptance in the Fr condi-
tion suggests that an higher effort and smaller amount of
reward are perceived by the animals as being associated with a
negative motivational value. Electrophysiological results
l
f h
d f
g
One-hundred and sixty-nine neurons
were localized to the striatum and identi-
fied as TANs. All recorded neurons were located in
the associative (138 neurons, 81.7%) and
the sensorimotor (31 neurons, 18.3%)
parts of the striatum. We did not investi-
gate the ventral striatum. No obvious dif-
ference
between
each
population
of
neurons during the defined periods of
analysis in the encoding of the force and
the reward was found. Neurons with pos-
itive and negative FSI (indicating a stron-
ger variation in activity in high force
conditions if positive, and in low force
conditions if negative) and RSI (indicat-
ing a stronger variation in activity in large
reward conditions if positive, and in small
reward conditions if negative) were inter-
mixed throughout the regions of the stria-
tum from which we recorded. −1.5
1.5
−1.5
1.5
C
Reward Selectivity Index
−1.5
1.5
−1.5
1.5
Elevation of discharge rate
following cues occurrence
Elevation of discharge rate
following reward occurrence
D C
Reward Selectivity Index
−1.5
1.5
−1.5
1.5
Elevation of discharge rate
following cues occurrence Elevation of discharge rate
following cues occurrence Force Selecti it Inde
−1.5
1.5
−1.5
1.5
Elevation of discharge rate
following reward occurrence
D C D Elevation of discharge rate
following reward occurrence Response of TANs to motivational relevant
stimuli and to reward We recorded from 169 TANs in two mon-
keys. The firing rate of TANs was 6.01
1.1 (mean SD; n 169), ranging from
2.7 to 9.1 spikes s 1. Force Selectivity Index
0
3000
4000
1
-1
Spk density
4
8
7
6
5
4
Fr
fr
FR
fR
E
0
Spk density
8
7
6
5
1
Time from cues (s)
-1
Time from cues (s)
Force effect only (n = 29)
Reward effect only (n = 20)
Force Selectivity Index
F
Figure 4. A–D, Force and reward selectivity indices (FSI and RSI) of TANs during the pause (A, B) and the elevation of the
discharge rate (C, D) after cue (A, C) and reward occurrence (B, D). Scatter plots of force versus reward selectivity indices for each
individual neuron in both periods. Electrophysiological results
l
f h
d FSIs of 0 indicate higher firing rate in the high force conditions, and indices of 0 indicate Force Selectivity Index Force Selectivity Index Force Selectivity Index
0
3000
4000
1
-1
Spk density
4
8
7
6
5
4
Fr
fr
FR
fR
E
0
Spk density
8
7
6
5
1
Time from cues (s)
-1
Time from cues (s)
Force effect only (n = 29)
Reward effect only (n = 20)
Force Selectivity Index
F F E Data from the literature have shown
that TANs respond to unpredictable stim-
uli, associated with reward (Aosaki et al.,
1995; Apicella et al., 1997). In line with
previous reports, we found that most
TANs responded to motivational relevant
visual stimuli (called “cues” in the follow- Spk density higher firing rates in the low force conditions. RSIs of 0 in-
dicate higher firing rates in the large reward conditions, and
indices of 0 indicate higher firing rates in the small reward
conditions. Symbols indicate the significance of the modula-
tion for each neuron when we performed a two-way ANOVA
(squares, force; filled circles, reward; crosses, interaction;
smallfilledcircles,neuronswithoutsignificantmodulation).E,
Average spike density ( 80) of the neurons modulated
only by the amount of force (n 29) during the elevation of
the discharge rate after the occurrence of the cues. The hori-
zontaldashedlinerepresentsthebaselineactivityofthispop-
ulation of neurons. The four shades represent, from the black
to the lightest gray, the high force/small reward, low force/
small reward, high force/large reward, and small force/large
reward conditions, respectively. F, Average spike density
( 80) of the neurons modulated only by the amount of
reward (n 20) during the elevation of the discharge rate
after the occurrence of the cues (same conventions as in E). higher firing rates in the low force conditions. RSIs of 0 in-
dicate higher firing rates in the large reward conditions, and
indices of 0 indicate higher firing rates in the small reward
conditions. Symbols indicate the significance of the modula-
tion for each neuron when we performed a two-way ANOVA
(squares, force; filled circles, reward; crosses, interaction;
smallfilledcircles,neuronswithoutsignificantmodulation).E,
Average spike density ( 80) of the neurons modulated
only by the amount of force (n 29) during the elevation of
the discharge rate after the occurrence of the cues. The hori-
zontaldashedlinerepresentsthebaselineactivityofthispop-
ulation of neurons. Acceptance level Acceptance level p
For each monkey, we ranked the level of motivation in each of the
four conditions by computing an acceptance level from the num-
ber of omission errors. For both monkeys, the four conditions
could be ranked in the same order of preference: low force/large 15220 • J. Neurosci., November 11, 2015 • 35(45):15214–15226 Nougaret and Ravel • TANs’ Modulation by Force and Reward Force Selectivity Index
−1.5
1.5
−1.5
1.5
Force
Reward
Interaction
C
ensity
8
7
6
E
ensity
8
7
6
Force effect only (n = 29)
Reward effect only (n = 20)
N. S. Reward Selectivity Index
−1.5
1.5
−1.5
1.5
−1.5
1.5
−1.5
1.5
Force Selectivity Index
Reward Selectivity Index
−1.5
1.5
−1.5
1.5
Pause following cues
occurrence
Pause following reward
occurrence
Elevation of discharge rate
following cues occurrence
Elevation of discharge rate
following reward occurrence
D
F
A
B Figure 2, D and H, represents the aver-
age force developed by each monkey in
the four conditions and shows that the
monkeys did not modulate their force on
the lever as a function of the expected
reward. Force
Reward
Interaction
N. S. Reward Selectivity Index
−1.5
1.5
−1.5
1.5
−1.5
1.5
−1.5
1.5
Pause following cues
occurrence
Pause following reward
occurrence
A
B Force Selectivity Index
−1.5
1.5
−1.5
1.5
Force
Reward
Interaction
C
0
3000
4000
1
-1
Spk density
4
8
7
6
5
4
Fr
fr
FR
fR
E
0
Spk density
8
7
6
5
1
Time from cues (s)
-1
Time from cues (s)
Force effect only (n = 29)
Reward effect only (n = 20)
N. S. Reward Selectivity Index
−1.5
1.5
−1.5
1.5
−1.5
1.5
−1.5
1.5
Force Selectivity Index
Reward Selectivity Index
−1.5
1.5
−1.5
1.5
Pause following cues
occurrence
Pause following reward
occurrence
Elevation of discharge rate
following cues occurrence
Elevation of discharge rate
following reward occurrence
D
F
A
B
Figure 4. A–D, Force and reward selectivity indices (FSI and RSI) of TANs during the pause (A, B) and the elevation of the
discharge rate (C, D) after cue (A, C) and reward occurrence (B, D). Scatter plots of force versus reward selectivity indices for each
individual neuron in both periods. FSIs of 0 indicate higher firing rate in the high force conditions, and indices of 0 indicate 5
Pause following reward
occurrence B A Electrophysiological results
l
f h
d The four shades represent, from the black
to the lightest gray, the high force/small reward, low force/
small reward, high force/large reward, and small force/large
reward conditions, respectively. F, Average spike density
( 80) of the neurons modulated only by the amount of
reward (n 20) during the elevation of the discharge rate
after the occurrence of the cues (same conventions as in E). Figure 4. A–D, Force and reward selectivity indices (FSI and RSI) of TANs during the pause (A, B) and the elevation of the
discharge rate (C, D) after cue (A, C) and reward occurrence (B, D). Scatter plots of force versus reward selectivity indices for each
individual neuron in both periods. FSIs of 0 indicate higher firing rate in the high force conditions, and indices of 0 indicate J. Neurosci., November 11, 2015 • 35(45):15214–15226 • 15221 Nougaret and Ravel • TANs’ Modulation by Force and Reward responded to the cues, to the reward, or to both. Fifty-seven
neurons responded by a pause only to the cues, 67 neurons re-
sponded only to the reward, and 19 neurons showed a pause to
both the cues and the reward. Similarly, 58 neurons responded by
an elevation of the discharge rate only to the cues, whereas 47 only
showed this elevation after the reward. Eighteen neurons pre-
sented an elevation of the discharge rate to both the cues and the
reward. The number of neurons showing modulations to both
events (N 19 and 18, respectively, for pause and elevation of the
discharge rate) is not significantly different from the number we
could have observed by chance. We calculated the percentile re-
flecting the chance to find this number of neurons that would
respond to both events, replicated 20,000 times. We got an equiv-
alent p value of 0.025, which means that the population of
neurons showing a response to the cues is independent of the one
showing a response to the reward for both pause and elevation of
the discharge rate. ing text) while the monkeys performed the instrumental task. Based on the population histogram aligned on the cues onset
(Fig. 3A), a significant decrease (called “pause” in the following
text) in TAN activity after the occurrence of the cues was found
between 60 and 280 ms. Electrophysiological results
l
f h
d In summary, in our task, TANs did not respond preferentially
to the cues or the reward, and responses to each event were inde-
pendent; i.e., a response to one event did not preclude nor predict
a response to the other, unlike what has been shown in previous
reports using different behavioral tasks, in which neurons tend to
lose their responses to the reward during learning of the associa-
tion of reward to a conditioned stimulus. After the occurrence of
the cues, we observed an equivalent number of neurons showing
a pause or an elevation of the discharge rate, whereas after the
reward occurrence, the predominant response was a pause. Modulation of TAN activity by the amounts of force and reward
Responses of TANs have been shown to be modulated by the
motivational significance of stimuli (Ravel et al., 1999, 2003;
Blazquez et al., 2002; Joshua et al., 2008). In our task, the visual
cues carried different motivational significance depending on the
required force and the expected reward. We examined whether
this motivational significance, translated behaviorally as shown
by the acceptance levels and error rates of the animals and which
should be computed from the information on force and reward,
would also modulate the activity of TANs, qualitatively or quan-
titatively. A two-way ANOVA on the spike count within each of
the four determined response periods was performed. For better
reading, a modulation of the neuronal activity by the amount of
force will be called a “force effect,” and a modulation by the
amount of reward a “reward effect.” All neurons, not only those
categorized as responsive, were taken into account to include
neurons that would respond in only two conditions and show a
modulation by the amount of force or reward but might not be
considered as responsive if all conditions were combined. Results
described in the following paragraphs are displayed in Table 2. After the occurrence of the cues, only 12% (21 of 169) of TANs
showed a modulation during the pause: 4% (7 of 169) showed a
force effect, 3% (6 of 169) a reward effect, and 5% (8 of 169) an
interaction effect. Conversely, during the elevation of the dis-
charge rate after the occurrence of the cue, we observed a strong
modulation of the neuronal activity, with 37% (62 of 169) of
TANs modulated. Electrophysiological results
l
f h
d Surprisingly, we observed a similar proportion
of TANs showing a reward effect (29 of 169; 17%) and a force
effect (34 of 169; 20%). TANs showed an interaction effect (10 of
169; 6%) usually associated with a force or reward main effect (7
of 169; 4%). We noticed that very few neurons showed both force
and reward main effects (4 of 169; 1%), suggesting that these
types of information were encoded by independent groups of
neurons. We calculated the percentile reflecting chance to find
the number of neurons that would encode both force and reward g
p
)
TANs show responses to unpredictable rewards and usually
lose their responses to reward when it is associated with a condi-
tioned stimulus (Apicella et al., 1997; Ravel et al., 2003). Here, the
majority of TANs (98 of 169; 58%) showed a modulation of their
activity in response to the reward, even though it was preceded by
visual cues and predictable. The population of TANs showing a
pause (67 of 169; 40%) was larger than that showing an elevation
of the discharge rate (47 of 169; 28%; 2 4.78, p 0.029). Nine
percent (16 of 169) showed a pause followed by a rebound. There
was no significant difference between the number of responses to
the cues and to the reward ( 2 0.30, p 0.58), for both pauses
( 2 1.03, p 0.31) and elevations of the discharge rate ( 2
1.38, p 0.24). As for the cues, we checked, for modulations after
the reward, that the neurons showing a pause or an elevation in
discharge rate were belonging to comparable populations. We
found no difference in the average firing rate nor in the average
ISI of neurons showing a pause or a combination of a pause
followed by a rebound (N 138; average firing rate, 6 1.2;
average ISI, 159.13 34.38) and of neurons showing only an
elevation of discharge rate (N 31; average firing rate, 6.07
0.77; average ISI, 156.77 19.26; t test, p 0.05). It has been shown that TAN responses transfer from the re-
ward to the preceding conditioned stimulus during learning
(Apicella et al., 1997). However, in our task, we found a large
number of neurons presenting modulations after cues and also
after reward occurrence. Electrophysiological results
l
f h
d A significant increase (called “rebound”
if preceded by a pause or “elevation of discharge rate” in the
following text) occurred between 300 and 530 ms. Following the
same procedure for the reward occurrence (Fig. 3B), the pause in
TAN activity was found between 20 and 280 ms, and the elevation
of the discharge rate occurred between 450 and 610 ms. In re-
sponse to cues and reward, no significant difference in the num-
ber of neurons showing a pause ( 2 1.85, p 0.17 for cues, 2
0, p 1 for reward) or in the number of neurons showing an
elevation of the discharge rate ( 2 3.42, p 0.06 for cues, 2
0.49, p 0.49 for reward) was found between the two monkeys. Consequently, data from Monkey M and Monkey Y were pooled
for further analyses of cues and reward responses. Results described below are summarized in Table 1. The ma-
jority of TANs (92 of 169; 54%) showed a modulation of their
activity in response to the cues: 33% (57 of 169) showed a pause,
34% (58 of 169) an elevation of the discharge rate, and 14% (23 of
169) a pause followed by a rebound. Previous reports on TAN
responses to appetitive stimuli have mainly described decreases
in activity (pauses; Aosaki et al., 1995; Apicella et al., 1997; Ravel
et al., 1999; Morris et al., 2004). Given the large number of eleva-
tions in the discharge rate observed in our data, we checked that
neurons recorded were not belonging to different populations by
comparing the average firing rates and interspike intervals (ISIs). There was no difference in the average firing rate nor in the aver-
age ISI of neurons showing a pause or a combination of a pause
followed by a rebound (N 134; average firing rate, 6.02 1.1;
average ISI, 158.17 31.86) or of neurons showing only an ele-
vation of discharge rate (N 35; average firing rate, 5.99 1.21;
average ISI, 160.72 33.43; t test, p 0.05). Electrophysiological results
l
f h
d “All”correspondstoneuronsshowingaforce,areward,and/oraninteractioneffect.Thecolumnsindicateperiods(cuesandreward)andtypesofmodulation(pauseorelevationindischargerate).Ontheright,Venndiagramsreprensent
the distribution of neurons showing a force (top) or a reward (bottom) effect during the elevations of the discharge rate after the cues, the reward, or both. tions (Fig. 4A,B). During the elevation of the discharge rate fol-
lowing the occurrence of the cues, more force-modulated
neurons exhibited a positive FSI than a negative one (27 of 34,
79% vs 7 of 34, 21%; mean, 0.36; binomial test, p 0.001; Fig. 4C). Additionally, during the same period, more reward-
modulated neurons exhibited a negative RSI than a positive one
(22 of 29, 76% vs 7 of 29, 24%; mean, 0.38; binomial test, p
0.01; Fig. 4C). None of the neurons was part of the two groups of
neurons (positive FSI and negative RSI). We calculated the per-
centile reflecting the chance to find no neuron that would encode
both a positive FSI and negative RSI (replicated 20,000 times). We got an equivalent p value of 0.025, meaning that the group
of neurons showing a positive FSI is different from the one show-
ing a negative RSI. Thus, the encoding of a positive FSI excludes
the encoding of a negative RSI, and vice versa. This result sup-
ports the idea that positive force and negative reward information
were integrated by two populations of neurons during the eleva-
tion of the discharge rate in neuronal activity after the cues (Fig. 4E,F). One population of neurons encoded the force informa-
tion carried by the cues when the force to exert on the lever, the
cost of the action, was high, whereas another one encoded the
reward information carried by the cues when the expected re-
ward, the benefit of the action, was small (Fig. 4C). information (replicated 20,000 times). We got an equivalent p
value of 0.025, meaning that the group of neurons showing a
force effect is independent of the one showing a reward effect. Thus, the encoding of force was neither predictive nor preclusive
to the encoding of reward and vice versa. Figure 3 shows two
examples of neurons illustrating the force effect (Fig. 3C) and the
reward effect (D) during this period. Electrophysiological results
l
f h
d The neuron in Figure 3C has
more pronounced pauses and elevations of the discharge rate in
the high force conditions (FR and Fr) compared to the low force
conditions (fR and fr), whereas the neuron in Figure 3D shows a
stronger elevation of the discharge rate in the small reward con-
ditions (Fr and fr) than in the large reward ones (fR and FR). After the reward occurrence, 24% (40 of 169) of TANs showed
a modulation during the pause: 7% (12 of 169) showed a force
effect, 11% (18 of 169) a reward effect, and 9% (15 of 169) an
interaction effect. Only 10% (4 of 40) of the modulated neurons
showed a combination of two of the three effects (force, reward,
and/or interaction). Figure 3E shows an example of neuron with
an interaction effect during the pause following the reward oc-
currence. This neuron shows a stronger response in the FR con-
dition compared to the fR condition and does not respond in fr
and Fr conditions. During the elevation of the discharge rate after
the reward occurrence, 25% (43 of 169) showed a modulation. A
larger proportion of neurons showed a reward effect (26 of 169;
15%) than a force effect (12 of 169; 7%; 2 5.01; p 0.025) or
an interaction effect (9 of 169; 2 8.16; p 0.004). Only 9% (4
of 43) of the modulated neurons showed a combination of two of
the three effects (force, reward, and/or interaction). Figure 3F
shows an example of neuron with a reward effect during the
elevation of the discharge rate following the reward occurrence,
with a stronger elevation in the small reward conditions (fr/Fr)
compared to the large ones (fR/FR). During the pause following the reward occurrence, 83% (10 of
12) of TANs modulated by the amount of force exhibited a neg-
ative FSI, against only 17% (2 of 12) exhibiting a positive FSI
(mean, 0.45; binomial test, p 0.05). During the same period,
similar proportions of neurons modulated by the amount of re-
ward exhibited a negative RSI (8 of 18; 44%) and a positive one
(10 of 18; 56%). Only two neurons were included in both popu-
lations. Electrophysiological results
l
f h
d We thus examined whether the same
population of neurons responded to the cues, the reward, or both
or whether there were distinct populations. We found that neu-
rons showing significant pause or elevation in their firing rate 15222 • J. Neurosci., November 11, 2015 • 35(45):15214–15226 Nougaret and Ravel • TANs’ Modulation by Force and Reward Table 2. The row headings indicate the type of modulation (all, force effect, positive FSI, negative FSI, reward effect, positive RSI, and negative RSI) or an interaction effect
e
c
r
o
f
a
g
ni
w
o
h
s s
n
o
r
u
e
n
f
o
n
oit
u
birtsi
D
d
r
a
w
e
R
s
e
u
C
or a reward effect during the elevations
of discharge rate
Pause
Elevation
Pause
Elevation
%
7
. 6
3
(
9
6
1
/
2
6
)
%
4
. 2
1
(
9
6
1
/
1
2
ll
A
)
40/169 (23.7%)
43/169 (25.4%)
Force effect
Force effect
7/21 (33.3%)
34/62 (54.8%)
12/40 (30%)
12/43 (27.9%)
FSI+
4/7 (57.1%)
27/34 (79.4%)
2/12 (16.7%)
5/12 (41.7%)
)
%
3
. 8
5
(
2
1
/
7
)
%
3
. 3
8
(
2
1
/
0
1
)
%
6
. 0
2
(
4
3
/
7
)
%
9
. 2
4
(
7
/
3
-I
S
F
Reward effect
6/21 (28.6%)
29/62 (46.8%)
18/40 (45%)
26/43 (60.5%)
Reward effect
RSI+
5/6 (83.3%)
7/29 (24.1%)
10/18 (55.6%)
6/26 (23.1%)
)
%
9
. 5
7
(
9
2
/
2
2
)
%
7
. 6
1
(
6
/
1
-I
S
R
8/18 (44.4%)
20/26 (76.9%)
Interaction effect
8/21 (38.1%)
10/62 (16.1%)
15/40 (37.5%)
9/43 (20.1%)
33
1
11
d
r
a
w
e
R
s
e
u
C
18
11
15
Cues
Reward
“All”correspondstoneuronsshowingaforce,areward,and/oraninteractioneffect.Thecolumnsindicateperiods(cuesandreward)andtypesofmodulation(pauseorelevationindischargerate).Ontheright,Venndiagramsreprensent
the distribution of neurons showing a force (top) or a reward (bottom) effect during the elevations of the discharge rate after the cues, the reward, or both. Table 2. The row headings indicate the type of modulation (all, force effect, positive FSI, negative FSI, reward effect, positive RSI, and negative RSI) or an interaction effect 15 “All”correspondstoneuronsshowingaforce,areward,and/oraninteractioneffect.Thecolumnsindicateperiods(cuesandreward)andtypesofmodulation(pauseorelevationindischargerate).Ontheright,Venndiagramsreprensent
the distribution of neurons showing a force (top) or a reward (bottom) effect during the elevations of the discharge rate after the cues, the reward, or both. Electrophysiological results
l
f h
d During the elevation of the discharge rate following the
reward occurrence, a similar proportion of neurons modulated
by the amount of force exhibited a positive FSI (5 of 12; 42%) and
a negative FSI (7 of 12; 58%), whereas most of the neurons mod-
ulated by the amount of reward exhibited a negative RSI (20 of 26;
77%), and few a positive one (6 of 26; 23%; mean, 0.28; bino-
mial test, p 0.01; Fig. 4D). The encoding of the amount of force
during the elevation of the discharge rate following the cues and
the encoding of the amount of force during the elevation of the Weight and direction of activity modulation by force and reward
after the occurrence of the cues and the reward
An FSI and an RSI were estimated for each neuron in each period
(see Materials and Methods). Results are detailed in the following
section and are summarized in Table 2. During the pause follow-
ing the occurrence of the cues, the small number of significant
neurons did not allow us to test the direction of these modula- Weight and direction of activity modulation by force and reward
after the occurrence of the cues and the reward An FSI and an RSI were estimated for each neuron in each period
(see Materials and Methods). Results are detailed in the following
section and are summarized in Table 2. During the pause follow-
ing the occurrence of the cues, the small number of significant
neurons did not allow us to test the direction of these modula- J. Electrophysiological results
l
f h
d Neurosci., November 11, 2015 • 35(45):15214–15226 • 15223 Nougaret and Ravel • TANs’ Modulation by Force and Reward 2000
3000
−1
0
1
Time from OCF (s)
Aligned on the OCF B
0.7
t-value
20
10
0
-0.5
-0.3
-0.1
0.1
0.3
0.5
0.9
Spike/s
4.5
5
5.5
6
6.5
7
-10
-20
-30
Time from OCF (s) Aligned on the OCF B B A Time from
cues (s)
Time from
cues (s)
Time from OCF (s)
C
Cues
OCF
Pause
9
48
48
10
9
Cues > OCF
OCF > Cues
Cues
Cues > OCF
29
Cues = OCF
Cues
Cues > OCF
Cues = OCF Reward effect (N = 45)
Force effect (N = 50)
N = 16
N = 6
N = 16
N = 12
N = 12
N = 7
N = 16
N = 10
D
Reward effect (N = 45)
N = 12
N = 7
N =
N = cues (s)
cues (s)
C
Cues
OCF
Pause
9
48
48
10
9
Cues > OCF
OCF > Cues
29
Cues = OCF
Cues = OCF D Force effect (N = 50)
N = 16
N = 6
N = 16
N = 12
D C Force effect (N = 50) Cues
Cues > OCF
Cues
Cues > OCF Reward effect (N = 45)
N = 12
N = 7
N = 16
N = 10
Reward effect (N = 45)
N = 12
N = 7
N =
N = Reward effect (N = 45)
N = 12
N = 7
N = 16
N = 10
Reward effect (N = 45)
N = 12
N = 7
N =
N = Figure5. Influenceoftheonsetofthechangeinforceonthepauseandtheelevationofthedischargerateaftercueoccurrence.A,ExampleoftheactivityofaTAN(allconditionsmingled).Left,
Activityisalignedonthecueoccurrence.Center,ActivityisalignedoncueoccurrenceandsortedbythetimeoftheOCFoftheanimal(down,trialwiththeshortestRT;up,trialwiththelongestRT). Right, Activity aligned on the OCF. This neuron exhibited a more pronounced pause and a more pronounced elevation of the discharge rate. B, Population histogram aligned on the OCF (same
conventionsasinFig.3A).Thepauseoccurredbetween190and10ms,andtheelevationofthedischargeratebetween0and250msaroundtheOCF.Theperiodofpauseisnarrowerandmore
pronounced when aligned on the OCF. C, Venn diagrams reprensent the number of neurons showing a pause (top) or an elevation of the discharge rate (bottom) when the activity was aligned on
cue occurrence (left), the OCF (right), and both. Electrophysiological results
l
f h
d Neurosci., November 11, 2015 • 35(45):15214–15226 • 15223 3000
−1
0
2000
1
−1
0
2000
3000
1
−1
0
1
10
0
2
4
6
8
Spk density
Time from
cues (s)
Time from
cues (s)
Time from OCF (s)
B
0.7
t-value
20
10
0
-0.5
-0.3
-0.1
0.1
0.3
0.5
0.9
Spike/s
4.5
5
5.5
6
6.5
7
-10
-20
-30
Time from OCF (s)
A
Aligned on cues
occurence
Aligned on cues
occurence
Sorted by the OCF
Aligned on the OCF
C
Reward effect (N = 45)
Force effect (N = 50)
N = 16
N = 6
N = 16
N = 12
N = 12
N = 7
N = 16
N = 10
D
Cues
OCF
Pause
9
48
48
10
9
Cues > OCF
OCF > Cues
Cues
Cues > OCF
29
Cues = OCF
Reward effect (N = 45)
N = 12
N = 7
N =
N =
Cues
Cues > OCF
Cues = OCF
Figure5. Influenceoftheonsetofthechangeinforceonthepauseandtheelevationofthedischargerateaftercueoccurrence.A,ExampleoftheactivityofaTAN(allconditionsmingled).Left,
Activityisalignedonthecueoccurrence.Center,ActivityisalignedoncueoccurrenceandsortedbythetimeoftheOCFoftheanimal(down,trialwiththeshortestRT;up,trialwiththelongestRT). Right, Activity aligned on the OCF. This neuron exhibited a more pronounced pause and a more pronounced elevation of the discharge rate. B, Population histogram aligned on the OCF (same
conventionsasinFig.3A).Thepauseoccurredbetween190and10ms,andtheelevationofthedischargeratebetween0and250msaroundtheOCF.Theperiodofpauseisnarrowerandmore
pronounced when aligned on the OCF. C, Venn diagrams reprensent the number of neurons showing a pause (top) or an elevation of the discharge rate (bottom) when the activity was aligned on
cue occurrence (left), the OCF (right), and both. For this latter case, the numbers of neurons showing a statistically significant or nonsignificant pause or elevation in discharge rate with one or the
otheralignmentareindicatedatthebottomofeachdiagram.D,Piechartsillustratethenumberofneuronsshowingaforce(top)orareward(bottom)effectduringtheelevationofthedischarge
ratewhentheactivitywasalignedonthecueoccurrence(black)orontheOCF(gray).Thehatchedblackarearepresentsthenumberofneuronswithastrongereffectwhentheactivitywasaligned
on the cue occurrence, and the hatched gray area represents the number of neurons with a stronger effect when the activity was aligned on the OCF. Nougaret and Ravel • TANs’ Modulation by Force and Reward
J. Electrophysiological results
l
f h
d For this latter case, the numbers of neurons showing a statistically significant or nonsignificant pause or elevation in discharge rate with one or the
otheralignmentareindicatedatthebottomofeachdiagram.D,Piechartsillustratethenumberofneuronsshowingaforce(top)orareward(bottom)effectduringtheelevationofthedischarge
ratewhentheactivitywasalignedonthecueoccurrence(black)orontheOCF(gray).Thehatchedblackarearepresentsthenumberofneuronswithastrongereffectwhentheactivitywasaligned
on the cue occurrence, and the hatched gray area represents the number of neurons with a stronger effect when the activity was aligned on the OCF. pattern of activity of TANs when the neuronal activity was
aligned on the OCF. We observed an influence of the perfor-
mance of the animal on the cue responses of TANs (Fig. 5A). Aligned on the time of OCF of the monkeys, the significant de-
crease of TAN activity was found between 190 and 10 ms. This pause had a shorter duration than that found when the
activity was aligned on the cues; it was also more pronounced
when the activity was aligned on the time of OCF (mean, 5.14 Hz;
minimal frequency, 4.52 Hz) than on cue occurrence (mean, 5.42
Hz; minimal frequency, 5.07 Hz; t 2.23; p 0.036; Fig. 5B). When aligned on the time of OCF, the significant increase of TAN
activity was found between 0 and 250 ms. The size of the elevation
of the discharge rate was found to be similar if aligned on the time
of OCF of the animals (mean, 6.64 Hz; maximal frequency, 7.16
Hz) or aligned on the occurrence of the cues (mean, 6.60 Hz; discharge rate following the reward were found in separate neu-
ronal populations. Only one neuron was included in both groups. In contrast, of the 26 neurons showing a reward effect during the
elevation of the discharge rate following the reward occurrence,
11 (42%) showed this modulation following the occurrence of
the cues as well (Table 2). discharge rate following the reward were found in separate neu-
ronal populations. Only one neuron was included in both groups. In contrast, of the 26 neurons showing a reward effect during the
elevation of the discharge rate following the reward occurrence,
11 (42%) showed this modulation following the occurrence of
the cues as well (Table 2). Influence of the OCF on the activity of TANs Influence of the OCF on the activity of TANs f
f
y f
Response period time locked on the onset of the change in force. Previous reports have mentioned TAN responses occurring at the
time of movement onset (Lee et al., 2006; Benhamou et al., 2014). In their tasks, the movement is either the only event leading to a
reward or plays a crucial role in succeeding in a trial. Even though
it was not the case in our task, the control of the required force
was an important parameter in performing a correct trial. Thus,
despite the fact that it has rarely been reported, we studied the 15224 • J. Neurosci., November 11, 2015 • 35(45):15214–15226 Nougaret and Ravel • TANs’ Modulation by Force and Reward maximal frequency, 6.87 Hz; t 0.54; p 0.59; Fig. 5B). How-
ever we observed that when the neuronal activity was time locked
to the OCF, the beginning of the elevation of the discharge rate
corresponded exactly to the beginning of the movement of the
animals. animal. Another group of TANs showed significant or larger
modulations when aligned to the time of OCF. Seventeen out of 29 neurons (59%) were modulated by the
amount of reward for both alignments. The activity of 16 addi-
tional neurons was modulated only when their activity was
aligned to the time of OCF. Of the 17 neurons modulated by the
amount of reward in both cases, 10 neurons showed a higher RSI
with their activity time locked to the OCF, versus a higher RSI in
7 neurons when time locked to cue occurrence (Fig. 5D). There
was no difference in the distribution of the RSIs between these
two populations. In both cases, the majority of TANs were more
modulated in the small reward conditions (time locked to cues,
mean, 0.38; time locked to OCF, mean, 0.46). As before, one
population of TANs seemed to encode the information related to
the amount of reward independently of the movement initiated
by the animal, whereas another one was very sensitive to the OCF. Number of responsive neurons time locked to the onset of the
change in force. The analysis of the activity of TANs time locked to
the OCF revealed that 57% of the recorded neurons (96 of 169)
showed a significant pause in their activity in relation to this
event. TANs respond to both cues and reward TANs respond to both cues and reward
TANs are known to respond to temporally unpredictable reward
or conditioned stimulus associated with reward and to lose their
response to reward with learning (Aosaki et al., 1994, 1995; Api-
cella et al., 1997). Compared to these findings, our results have
shown that a relatively small number of TANs were responsive to
the temporally unpredictable visual cues associated with reward. Conversely, a large number of TANs was found to be responsive
to the reward itself, even though it was predictable in time and
quantity. It could be concluded that TANs held their response
until the reward delivery because the animals did not know the
significance of the cues; however, the behavioral results clearly
demonstrate that they understood it and modulated their behav-
ior accordingly (ERs and acceptance levels). Finding neurons re-
sponding to cues and to reward in the TAN population suggests
that when there is an added complexity in the task, these neurons
will encode more than the temporal predictability of a rewarding
stimulus. Neurons responsive to the cues could encode the stim-
ulus–reward association, as described previously, neurons re-
sponsive to the reward could encode the reward value. In the
present task, the reward value can change from trial to trial. One
hypothesis is that there is an estimation of this value by the ani-
mals at the cue’s occurrence, and that its accuracy is updated by
the neurons responding to the reward. Influence of the OCF on the force and reward selectivity indices. The distributions of the FSIs and the RSIs were compared when
the elevation of the discharge rate of TANs was aligned on cue
occurrence or time locked to the OCF. Eighteen of 34 neurons
(53%) were modulated by the amount of force for both align-
ments. The activity of 16 additional neurons was modulated only
when their activity was aligned on the time of OCF. On the 18
neurons modulated by the amount of force in both cases, 12
(67%) neurons showed a higher FSI with their activity time
locked to the OCF, and 6 neurons when their activity was time
locked to cue occurrence (Fig. 5D). There were no differences in
the distribution of the FSIs between these two populations. TANs respond to both cues and reward In
both cases, the majority of TANs were more modulated in the
high force conditions (time locked to cues, mean, 0.36; time
locked to OCF, mean, 0.43). Consequently, one population of
TANs seemed to encode the information related to the effort to
develop force independently of the movement initiated by the Influence of the OCF on the activity of TANs This proportion of neurons was higher than that observed
when the activity was aligned on the occurrence of the cues (96 of
169 vs 57 of 169, 2 17.24, p 3.29.10 5). Among these
neurons, half of them (48 of 96; 50%) showed a pause in their
activity only if the electrophysiological signal was time locked to
the OCF. In contrast, among the neurons showing a pause when
their activity was aligned on the cues, 16% (9 of 57) showed a
pause only if their activity was time locked to the cues. Conse-
quently, in 48 neurons, the pause was detected in both cases. Among these neurons, the pause was more pronounced when the
activity was time locked to the OCF in 9 neurons (19%; Wilcoxon
rank sum text, p 0.05) and to cue occurrence in 10 neurons
(21%; Wilcoxon rank sum text, p 0.05; Fig. 5C). Since the pause
occurred before the initiation of the movement, it could not be
concluded that this response was triggered by the force-
generating movement. However, considering the higher number
of responses detected and the higher magnitude of the responses
when activities ware aligned on the time of OCF, we can hypoth-
esize that the characteristics of the pause of TANs were dependent
on the OCF. For example, beside the variation of magnitudes
observed, the end of the pause was time locked on the OCF. Discussion The present study revealed new features of TAN responses in an
instrumental task modulating motivation. First, we found a large
number of TANs responding to cues and/or reward occurrence
by a pause or an elevation of the discharge rate in their activity. Second, this elevation, but not the pause, was modulated by the
force required and expected reward in each trial, and was most
sensitive to high force or small reward conditions. Finally, TAN
responses appeared to be better aligned on the time of OCF than
on cue occurrence (triggered by the latter, but time locked on the
former). These data suggest that the striatal TAN population is
not as homogeneous as previously reported, and that TANs can
be involved in encoding either force or reward parameters. p
When the activity was time locked to the OCF, 39% (66 of
169) of the recorded neurons showed an elevation of the dis-
charge rate in their activity, versus 33% (58 of 169) when the
activity was aligned on the cues ( 2 0.62, p 0.43). Among
these neurons, only 13 of 66 (20%) showed an elevation of the
discharge rate in their activity only if it was time locked to the
OCF, and 9% (5 of 58) only if it was aligned on the occurrence of
the cues. Consequently, in 53 neurons, the elevation of the dis-
charge rate was detected in both cases. Among these neurons, the
elevation of the discharge rate was greater when the activity was
time locked to the OCF in 18 neurons (34%; Wilcoxon rank sum
text, p 0.05) and to cue occurrence in 26 neurons (49%; Wil-
coxon rank sum text, p 0.05; Fig. 5C). Consequently, compared
to the pause, the elevation of the discharge rate seemed to be less
influenced by the OCF. TAN responses are triggered by the cues but largely time
locked to the OCF Pauses in TAN activity triggered by the cues were more pro-
nounced when aligned on the movement onset, and elevations of
the discharge rate were better time locked to the OCF. This sug-
gests that TAN activity could be modulated by the movement of
the animals, a feature rarely reported. One hypothesis to explain
this new property could be that, in the present task, the behavioral
response required to succeed in the trial relies on movement
modulation. In monkeys, Lee et al. (2006) described modulations
around the time of the movement linked to the fact that the
movement could be self-initiated in some conditions in their
task. In rats, Benhamou et al. (2014) reported an excitation dur-
ing movement and hypothesized that the absence of such modu-
lation in monkeys could rely on the differences in the protocols
used, and thus on the behavioral context. In the present study, the
modulation of the movement and the condition in which it is
performed, as in the study by Lee et al. (2006), are prominent
features of succeeding in the task, and might explain the influence
of the OCF on TAN activity. Functionally, the pauses time locked
to the OCF could serve to potentiate the movement, when it is a
key feature of a task, by influencing striatal projection neuron
excitability and allowing the expression of thalamic and cortical
afferents. After the reward, the pause was equally modulated by force,
reward, or a force–reward interaction, suggesting that the task
parameters are integrated by the pause of the TANs at the time of
the outcome. As discussed above, TANs showing a pause after the
reward could allow a check in the accuracy of the reward value
prediction and an update of this value if necessary, via the plas-
ticity window created by the decrease in ACh release. It is also in
line with the properties of TANs to encode reward prediction
error reported previously (Apicella et al., 2011). In our data, the
elevation of the discharge rate after reward occurrence was bigger
for a smaller reward. It has been suggested that this increase in
activity is involved in maintaining the behavior in response to
external stimuli (Yarom and Cohen, 2011; Schulz and Reynolds,
2013). References
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of membrane excitability of neostriatal neurons. II. An in vitro analysis. J Neurophysiol 63:663–675. Medline Saleem K, Logothetis N (2007) A combined MRI and histology atlas of
the rhesus monkey brain in stereotaxic coordinates. San Diego:
Elsevier/Academic. Calabresi P, Centonze D, Gubellini P, Pisani A, Bernardi G (2000)
Acetylcholine-mediated modulation of striatal function. Trends Neurosci
23:120–126. CrossRef Medline Sardo P, Ravel S, Legallet E, Apicella P (2000) Influence of the predicted
time of stimuli eliciting movements on responses of tonically active neu-
rons in the monkey striatum. Eur J Neurosci 12:1801–1816. CrossRef
Medline Crutcher MD, Alexander GE (1990) Movement-related neuronal activity
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linergic signaling in the striatum. Trends Neurosci 36:41–50. CrossRef
Medline Deffains M, Legallet E, Apicella P (2010) Modulation of neuronal activity in
the monkey putamen associated with changes in the habitual order of
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the monkey putamen associated with changes in the habitual order of y p
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sequential movements. J Neurophysiol 104:1355–1369. CrossRef Medline Shimo Y, Hikosaka O (2001) Role of tonically active neurons in primate
caudate in reward-oriented saccadic eye movement. J Neurosci 21:
7804–7814. Medline DiCarlo JJ, Maunsell JH (2005) Using neuronal latency to determine
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physiol 93:2974–2986. CrossRef Medline Stern EA, Jaeger D, Wilson CJ (1998) Membrane potential synchrony of
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475–478. CrossRef Medline Hikosaka O, Sakamoto M, Usui S (1989) Functional properties of monkey
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bition of neostriatal neurons in the rat. Exp Brain Res 51:227–235. Medline Joshua M, Adler A, Mitelman R, Vaadia E, Bergman H (2008) Midbrain
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difference between reward and aversive events at different epochs of prob-
abilistic classical conditioning trials. J Neurosci, 28:11673–11684. CrossRef Yamada H, Matsumoto N, Kimura M (2004) Tonically active neurons in the
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motivational outcomes of action. J Neurosci 24:3500–3510. CrossRef
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neurons exhibit set-dependent responses. Proc Natl Acad Sci U S A 81:
4998–5001. CrossRef Medline Yarom O, Cohen D (2011) Putative cholinergic interneurons in the ventral
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alternative association task. Front Syst Neurosci 5:36. Medline 4998–5001. CrossRef Medline Lee IH, Seitz AR, Assad JA (2006) Activity of tonically active neurons in the Lee IH, Seitz AR, Assad JA (2006) Activity of tonically active neurons in the
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Physical and Mechanical Properties Analysis of Wood-waste Composite Panels
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Physical and Mechanical Properties Analysis of Wood-waste
Composite Panels IMAD ANTYPAS*, ALEXEY DYACHENKO
Don State Technical University. Gagarin square 1, Rostov-on-Don, 344000, Russian Federation Abstract: The timber industry takes an important place in our modern life, and there has been an
increased interest in it for a long time, especially in industrially developed countries. The concerns of
the environmental organization regarding the plant wealth preservation have caused scientific trends to
search for sources or industries that can achieve acceptable alternatives to natural wood. The materials
in this article are related to industrial timber manufacturing of greenhouse wood waste, low density
polyethylene and non-standard joinery waste. Moreover, panel samples made from the above materials
were briefly subjected to tests as well as tested by fresh and salt water absorption.The density calculation
results for all samples have shown the possibility of obtaining a wide range of wooden planks from the
lightest to the heaviest ones, depending on the type of filler used. Additionally, the conducted research
has shown the possibility of producing artificial wood panels made from the studied materials with good
quality and fully satisfactory physical and mechanical properties. MATERIALE PLASTICE
https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 *email: imad.antypas@mail.ru MATERIALE PLASTICE https://revmaterialeplastice.ro p
p
https://doi.org/10.37358/Mat.Plast.1964 Panels were manufactured by adding watermelon skin waste after drying, mixing and precision
separation into recycled low density polyethylene, after which its microstructure, absorption, moisture,
tensile modulus, elastic modulus, strength modulus, internal adhesion and impact strength were
determined. Research objective: industrial timber manufacturing of greenhouse wood waste, low density
polyethylene and non-standard joinery waste. 1.Introduction The timber industry takes an important place in modern life, and there has been an increased interest
in it for a long time, especially in developed industrialized countries. Timber consumption volume in the world is constantly increasing; therefore, resource-efficient
technologies developing is important for ensuring the forest resources protection by exploiting the
recycling of raw materials left over from timber processing [1-4]. Scientific studies [5-7] show the use of thermoplastic waste or mixtures of recycled plastics with new
plastics in the timber processing industry. The study [8] demonstrates the possibility of using RLDPE waste in the industrial timber panel
production; moreover, these panels were pressure formed, with an increase in the percentage of low
density polyethylene waste (30-50%) at temperatures of 140°C and 180°C, forming pressure of 30-40
kg/cm2 with a pressure time of 3-7 min. This article also investigates the mechanical properties and
stability of these wood-plastic materials panels affected by various conditions, in addition to determining
their microstructure. Chow Poo and his colleagues [9] developed thermoplastic composite panels using corn fiber mesh
as the main material. Corn stalk fibers were cropped from the western part of the United States of
America and the panels were formed in various ways. Fatih Mengel and his colleague [10] studied the thermal behavior of recycled high-density
polyethylene (RHDPE) composites as base material, to which eucalyptus timber waste was added. Nwigho Solomon and his colleague [11] studied the evaluation of industrial wood panels mechanical
properties made from recycled LDPE as base material stuffed with ‘Iroko’ sawdust as a filler. The
RLDPE was accordingly mixed with sawdust by applying molding at a pressure of 150 MPa and a
temperature of 160°C. The tests have shown a tensile strength decrease and impact resistance as well as
an increase in hardness with a sawdust increased amount; the researchers concluded that it was possible
to use sawdust to increase the RLDPE/timber composites hardness and impact resistance. Idris and his colleagues [12] investigated the possibility of watermelon skin waste to produce timber-
plastic mixtures. *email: imad.antypas@mail.ru https://doi.org/10.37358/MP.22.2.5585 Mater. Plast., 59 (2), 2022, 61-72 61 MATERIALE PLASTICE
https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 2.1. Plastic matrix Chopped plastic greenhouse crumbles, made from low density polyethylene (RLDPE) and shown in
Figure 1, were used as the base matrix material: Figure 1. Low Density Polyethylene (RLDPE) Figure 1. Low Density Polyethylene (RLDPE) Figure 1. Low Density Polyethylene (RLDPE) 2.2. Joinery waste, resulted from the processes of creating wood products in the form of organic material
having different particle sizes (powder, medium particles, large particles, long particles), is shown in
Figure 2: Figure 2. Wood waste: a) powder, b) large particles,
c) medium particles, d) long particles
Four sample groups were formed, in which sawdust of various sizes and particle sizes of organic
substance were used as a matrix:
- a wood powder set; Figure 2. Wood waste: a) powder, b) large particles,
c) medium particles, d) long particles Four sample groups were formed, in which sawdust of various sizes and particle sizes of organic
substance were used as a matrix:
- a wood powder set; Four sample groups were formed, in which sawdust of various sizes and particle sizes of organic
substance were used as a matrix:
- a wood powder set; Four sample groups were formed, in which sawdust of various sizes and particle sizes of organic
substance were used as a matrix:
- a wood powder set; Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 62 MATERIALE PLASTICE
https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 MATERIALE PLASTICE https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 - a group of medium particle sizes; - a group of long particle sizes; - a random set of particles group. Each of these groups contained five samples with the following proportions by weight - 20% organic filler + 80% RLDPE; - 30% organic filler + 70% RLDPE; - 40% organic filler + 60% RLDPE; - 50% organic filler + 50% RLDPE; - 60% organic filler + 40% RLDPE. These samples were manufactured using a semi-hydraulic molding press according to the following
system: - mold temperature T = 160°C; - initial molding pressure P = 4 kg/cm2; - initial pressure time t = 5 min; - final molding pressure P = 70 kg/cm2; - final pressure time t = 10 min. At the end of the molding cycle, the mold was cooled at a cooling rate of V = 10°C / min, while the
final pressure remained unchanged until the temperature, at which the final product could be removed
without deformation, was reached. Figure 3. 2.1. Plastic matrix an artificial wood sample made from: a) 60% wood powder + 40% RLDPE,
b) 20% large particles + 80% RLDPE, c) 50% medium particles + 50% RLDPE,
d) 30% long particles + 70% RLDPE Figure 3. an artificial wood sample made from: a) 60% wood powder + 40% RLDPE,
b) 20% large particles + 80% RLDPE, c) 50% medium particles + 50% RLDPE,
d) 30% long particles + 70% RLDPE 3. Results and discussions The samples density measurement results in this study have shown the different values, that can be
explained by a clear difference in the density of the fillers used. As it is known (3), the timber density is about 1.5 g / cm3, while the density of other different sizes
types fillers (large, medium and long) is the same as density of wood obtained therefrom, which is
generally in 0.4-0.8 g/cm3 range. If it is known that only RLDPE samples density was 0.898 g/cm3, the
obvious difference in the density of the matrix and fillers used showed a different behavior of the density
curves compared to the samples with an organic filler. Figure 4 shows the density curve of chopped LDPE samples with different percentages of wood
powder waste. Figure 4-1 shows that the density increases with an increase in the percentage of an
organic filler from 0.898 g/cm3 at 0% to 1.066 g/cm3 at 60%. This result can be divided due to the difference in density values between an organic filler and
RLDPE, showing that the compounds density enlarges with an increasing proportion of a filler. The
obtained results have revealed that all compounds in Figure 4-1 belong to high density hardwoods
according to the classification in [13]. The density measurement results, made on the samples consisting of chopped LDPE and medium-
sized wood particles, have shown that the differences in density values relative to the matrix density are
small, as shown in Figure 4-2, where a slight increase in the sample density of 0.898 g/cm3 can be
observed at a zero level particle, then the density with a composition of 40% medium wood particles
becomes 0.908 g/cm3, further it decreases and at 60% it becomes equal to 0.811 g/cm3. The results presented in Figure 4-2 show that these compounds are very close in density to industrial
hardwoods according to [13], and the addition of medium wood waste up to 60% leads to a slight
decrease in density values and it is possible to produce a wide range of mixtures approximately equal to
the density of the base material. Figure 4-3 shows the density development curve of chopped RLDPE matrix composites with various
percentages of fillers, consisting of long wood particles and at 60% filler the density value is 0.42 g/cm3. 2.3.2. Short-time absorption The test was made with samples prepared within a short period of time (2 h, 24 h and 48 h). The test
samples were cut from 2.2 cm plates. The test samples weight was measured before the immersion into
water to find the percentage absorption that occurred after the immersion for the specified time. The
absorption percentage was calculated by the ratio: 𝑊= 𝑊2 −𝑊1
𝑊1
. 100 𝑊= 𝑊2 −𝑊1
𝑊1
. 100 𝑊1 where: W1 - sample weight before immersion (g); W1 - sample weight before immersion (g); W2 - sample weight after immersion (g); W - absorption percentage (%). MATERIALE PLASTICE https://revmaterialeplastice.ro https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 p
p
https://doi.org/10.37358/Mat.Plast.1964 Charpy Test: the samples for tests were prepared in accordance with the standard specifications
(ASTM D256-93) and were 10 mm in length and 10.5 mm2 in cross section. 2.3. Experimental procedure 2.3.1. The density was calculated by subtracting samples from the prepared panels to determine whether
the formed panels are light or heavy and whether their density is close to that of natural wood or not. Density was calculated using the following formula: ,
/v
m
P =
gr/cm3 - m - sample mass, gr; - v - sample size, cm3. Mater. Plast., 59 (2), 2022, 61-72 63 63 https://doi.org/10.37358/MP.22.2.5585 Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 63 MATERIALE PLASTICE 3. Results and discussions The results shown in Figure 4-3 show a wide range of variable density panels production: from high
density panels up to 30% to low density panels, comparable to that of insulation panels [14]. Alternatively, this result presents the compositions, which are similar to those of certain light woods,
such as spruce, that is the main purpose of our study - developing of industrial mixtures with properties
as close as possible to those of wood. The search for specific particle sizes of an organic filler increases the cost of the production process
due to the production stages lack, and since the main purpose was to develop waste wood and plastic Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 64 MATERIALE PLASTICE
https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 MATERIALE PLASTICE
https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 products as an organic filler, subsequent research entailed the use of various random shapes and sizes
waste without sorting or categorizing. The experimental work has shown that it is advisable to use such random organic waste and to
produce products whose properties can be considered good to obtain the required consistency of the
studied samples after immersion them into water for more than 40 days. This percentage amounted to 60% for all types and shapes of organic fillers, because using higher
ratios, which is the main purpose of our study, it was impossible to obtain cohesive samples with
acceptable properties under the same conditions. Since experimental objective of this study was to
develop products at the lowest cost, an increase in the proportion of an organic filler to a value greater
than the declared value requires preliminary procedures to develop compounds before they are formed,
increasing the economic cost. Figure 4-4 shows the density evolution curve of the low density polyethylene chopped with a random
set of wood particles samples. In addition, the density values are almost constant up to 30% of the
composition, and a gradual decrease in the density values is due to an increase in the filling percentage
from 30 to 60%. Figure 4. The RLDPE composites density development curve containing
different proportions of a 1) wood powder, 2) medium-sized wood, 3) long wood,
4) random set of wood particles as an organic filler Figure 4. 3. Results and discussions The RLDPE composites density development curve containing
different proportions of a 1) wood powder, 2) medium-sized wood, 3) long wood,
4) random set of wood particles as an organic filler The results given in Figure 3-4 show that the use of random waste gave approximately the same
results for a long particle filler, and the density measurements of all previous samples mentioned in
Figures 3 have demonstrated the possibility of a wide range of industrial wood panels producing
comparable to light wood of (0.40 - 0.45) g/cm3, used as insulation panels, as well as high-density heavy
wood of 0.9 g/cm3, close in density to polymer - based materials. 3.1. Impact tests It can be noted that when using a medium size organic filler, the impact
resistance of the studied compositions improves and it is clear that the impact stress values have
decreased from 20 kJ / m2 to 20% to 11.2 kJ / m2 at 60%, that is 2 times less. This result confirms a marked improvement in impact resistance over the impact strength values of
compositions containing wood powder, and is due to an improvement in the distribution of the base
material due to a reduction in the surface area of an organic filler. Figure 5-2 also shows that when using
medium size fillers, the impact strength values remain almost constant until the percentage of organic
fillers reaches 30%, followed by a slight decrease in impact resistance at a value of 18.2 kJ/m2. This
result is explained by the ability of the base material for better wetting and encapsulation until this
percentage is reached, then there is a significant decrease in impact strength values to 11.21 kJ/m2 at
60%. These results are considered important from the point of view of application, as they show that the
optimal percentage of the filling process for this type of filler is up to 40% with keeping the good values
of impact resistance, and that an increase in an organic filler volume improves the impact resistance up
to 40%: when using wood powder of 10.12 kJ/m2 and when using medium-sized particles, it reaches up
to 18.2 kJ/m2, hence, there is a result importance, that showed an improvement in impact resistance using
medium-sized particles, therefore, the effect of larger particles on impact resistance has been
investigated. g
Figure 5-3 gives the impact resistance evolution curve of mixtures containing long wood particles,
and it is clear that the general behavior of the impact resistance curve is identical to the previous curves,
where it decreases with an increase in the percentage of organic filler, and the value of this stress at
20% is 31.26 kJ/m2, and at 40% it becomes 18.24 kJ/m2, i.e., it is more than the impact resistance at the
same ratio in the case of using wood powder, or when using medium-sized particles, and this result
confirms the improvement of the penetrating power of the base material used due to the reduction of
the surface area of the organic filler particles. 3.1. Impact tests The impact strength values of plastic products give an adequate representation of the final product
resistance to dynamic loads, since it is known [15] that most objects are destroyed under the influence
of dynamic loads, such as hit or regular loads, while the effect of static loads, such as creep, remains
very insignificant and lasts for a long time compared to the product life expectancy under the influence
of dynamic loads. Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 65 MATERIALE PLASTICE
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https://doi.org/10.37358/Mat.Plast.1964 MATERIALE PLASTICE
https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 Determining the RLDPE impact resistance in terms of organic loads percentage is an important point
for the future study of the resistance of products made from these materials to the dynamic loads to
which they may be subjected during the tests. Figure 5-1 shows that the impact strength values of chopped RLDPE compositions with wood
powder as a filler decrease with increasing percentage of an organic filler. This result complies with
many reference studies [15] and is predictable since, as it is known, additives generally cause a decrease
in the impact strength values, regardless of their percentage value. According to Griffith's theory [14], fillers, regardless of their composition in general, are as stress
concentrators in the final product, that in its turn causes different types of loads (static - dynamic)
concentration in certain parts of the structure. Figure 5-1 shows the development curve of the impact RLDPE resistance as a function of the
percentage of an organic filler (wood powder). A high percentage of wood powder results in a decrease
in wetting and penetration of the base material, that, in addition to an increase in agglomeration, makes
it difficult to develop the batch-to-batch uniformity final structure. It should be noted that in order to reduce the size difference between the chopped RLDPE and the
wood powder a separation process was performed using a set of sieves, with the approximately equal
particle sizes of wood powder to achieve uniformity of the components of the mixture. The curve in Figure 5-1 shows a decrease in impact resistance from 20 kJ/m2 at 20% wood powder
to 7.91 kJ/m2 at 60%, i.e., about threefold. Figure 5-2 shows that as the particle size of an organic filler increases, the impact resistance of the
tested compositions improves. 3.1. Impact tests The replacement of the polymeric material with low cost and high performance polymeric materials
aims at reducing the economic cost of the final product with obtaining satisfactory physical and
mechanical properties. A simple comparison of the impact strength values at 60% shows that the values
are generally close to each other and that the best of them are associated with the use of compounds
containing organic fillers with large or medium particles, compared to a wood powder filler. Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 66 MATERIALE PLASTICE
https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 https://revmaterialeplastice.ro https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 The effect of randomly mixed (short - medium - long) particle sizes material on the impact resistance
was studied to demonstrate the clear effect of the filler particle size and its uniformity. Figure 5-4 shows
the development curve of impact resistance due to the percentage of organic fillers with any particles
set. The results have shown that at 40% composition of this type of fillers the stress value was 24.75
kJ/m2, while at 60% it was 14.54 kJ/m2 and compared to previous results these values were higher than
when using fillers with long and medium particles. Figure 5. The LDPE impact resistance development curve depending on the percentage
of organic fillers 1) wood powder, 2) medium-sized wood, 3) long wood, 4) random particles Figure 5. The LDPE impact resistance development curve depending on the percentage
of organic fillers 1) wood powder, 2) medium-sized wood, 3) long wood, 4) random particles According to Charpy, the results of impact tests show that if there is a need for impact-resistant
products, the effective range to be investigated is in the range of 20% organic fillers, regardless of the
difference in shape of these particles, and that the intensification of the research should not depend on
the particle size of this filler and should be aimed to improve the adhesion conditions by preliminary
treating of the wood particles with matrix, they provide the group "polymer - filler" with the functional
basis, which is necessary to ensure the adhesion of the elements of this group together. Short Term absorption study p
y
The water absorption study over a short period of time focuses on the rapid assessment of the water
resistance of the samples produced. This test is considered to be one of the rapid tests performed on
industrial timber and it has been approved by the international standards as a resistance test as one of the
physical tests for this type of material [13]. These tests usually involve measurements of swelling or
thickening of the materials to determine any irreversible changes that may occur due to the size of the
wood particles. zing the results obtained, presented in the Figures 6-13, the following can be noted: p
By analyzing the results obtained, presented in the Figures 6-13, the following can 1. The high resistance of cellulose compounds to water is explained by an increase of the absorption
percentage with an increase in the percentage of a filler and a time increase in water. 1. The high resistance of cellulose compounds to water is explained by an increase of the absorption
percentage with an increase in the percentage of a filler and a time increase in water. 2. A decrease in the coating and wetting capacity of the base material with an increase in the filler
percentage is explained by an increase in the absorbency with an increase in the organic filler percentage. 3. There a low absorption capacity of salt water compared to fresh water due to the existence of high
molecular weight compounds such as sodium chloride in salt water. 2. A decrease in the coating and wetting capacity of the base material with an increase in the filler
percentage is explained by an increase in the absorbency with an increase in the organic filler percentage. p
g
p
y
y
g
p
g
3. There a low absorption capacity of salt water compared to fresh water due to the existence of high
molecular weight compounds such as sodium chloride in salt water. Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 67 MATERIALE PLASTICE https://revmaterialeplastice.ro p
p
https://doi.org/10.37358/Mat.Plast.1964 Figure 6. The fresh water absorption by RLDPE samples
depending on the percentage of wood powder
Figure 7. The salt water absorption by RLDPE samples
depending on the percentage of wood powder Figure 6. The fresh water absorption by RLDPE samples
depending on the percentage of wood powder Figure 6. Short Term absorption study The fresh water absorption by RLDPE samples
depending on the percentage of wood powder Figure 7. The salt water absorption by RLDPE samples
depending on the percentage of wood powder Figure 7. The salt water absorption by RLDPE samples
depending on the percentage of wood powder The density measurements results have shown a clear discrepancy of the density values of
compounds with different particle sizes of an organic filler, as shown in Figures 8, 9. The results of
measuring the absorption rate of fresh and salt water by medium particle compounds showed that the
values reach high levels at a filler composition of 60%, with the absorption exceeding approximately at
40% after immersion in water for 24 h and 48 h. This result shows that the RLDPE base coating capacity
is low and does not guarantee the necessary of an organic filler all particles cohesion. g
y
g
p
This result conforms with the impact tests, which have shown that the lowest impact resistance
values were for mixtures with a high composition of an organic filler. Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 https://doi.org/10.37358/MP.22.2.5585 68 MATERIALE PLASTICE https://revmaterialeplastice.ro https://revmaterialeplastice.ro p
p
https://doi.org/10.37358/Mat.Plast.1964 Figure 8. The fresh water absorption by RLDPE samples
due to the percentage of organic fillers - medium-sized particles
Figure 9. The salt water absorption by RLDPE samples
depending on the percentage of an organic filler – medium-sized particles Figure 8. The fresh water absorption by RLDPE samples
due to the percentage of organic fillers - medium-sized particles Figure 8. The fresh water absorption by RLDPE samples
due to the percentage of organic fillers - medium-sized particles Figure 9. The salt water absorption by RLDPE samples
depending on the percentage of an organic filler – medium-sized particles Figure 9. The salt water absorption by RLDPE samples g
p
y
p
depending on the percentage of an organic filler – medium-sized particles The rapid density measurements were made on the samples containing large wood particles precisely
the same way. As can be seen from two Figures 10,11, the average absorption in fresh and salt water
after 48 h is approximately 16% for a 60% filler, and Figures 12, 13 show a change in the density value
as a percentage of an organic filler represented by wood large-sized particles, differing in a random way. Short Term absorption study p
g
g
p
y
g
p
,
g
y
As can be seen from Figures 12, 13 the highest absorption values are achieved when the percentage
of a filler is 60%, with the average absorption value to be 31% in fresh and 28% in salt water after 24 h
of immersion. Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 https://doi.org/10.37358/MP.22.2.5585 69 MATERIALE PLASTICE https://revmaterialeplastice.ro p
p
https://doi.org/10.37358/Mat.Plast.1964 Mat.Plast.1964
Figure 10. The fresh water absorption by RLDPE samples
as the percentage of an organic filler - long particles
Figure 11. The salt water absorption by RLDPE samples as the
percentage of an organic filler - long particles Figure 10. The fresh water absorption by RLDPE samples
as the percentage of an organic filler - long particles
Figure 11. The salt water absorption by RLDPE samples as the
percentage of an organic filler - long particles Figure 10. The fresh water absorption by RLDPE samples
as the percentage of an organic filler - long particles 2022 61 72
70
https://doi org/1
Figure 10. The fresh water absorption by RLDPE samples
as the percentage of an organic filler - long particles
Figure 11. The salt water absorption by RLDPE samples as the
percentage of an organic filler - long particles
Figure 12. Fresh water absorption by RLDPE samples as the
percentage of organic fillers – random particles Figure 10. The fresh water absorption by RLDPE samples
as the percentage of an organic filler - long particles Figure 11. The salt water absorption by RLDPE samples as the
percentage of an organic filler - long particles Figure 11. The salt water absorption by RLDPE samples as the
percentage of an organic filler - long particles Figure 12. Fresh water absorption by RLDPE samples as the
percentage of organic fillers – random particles Figure 12. Fresh water absorption by RLDPE samples as the
percentage of organic fillers – random particles Mater. Plast., 59 (2), 2022, 61-72 https://doi.org/10.37358/MP.22.2.5585 70 https://doi.org/10.37358/Mat.Plast.1964 Figure 13. The salt water absorption by RLDPE samples as the
percentage of organic fillers - random particles Figure 13. The salt water absorption by RLDPE samples as the
percentage of organic fillers - random particles 4. Conclusions The density calculation results for all samples show a wide spectral range of panels that can be made
from these compounds, from the lightest with the density of 0.40-0.45 g/cm3 to the heaviest with the
density of 1.066-0.89 g/cm3. The impact energy decreases with an organic filler percentage increase and declines with a particles
size and volume increase up to a certain percentage due to the distribution improvement of the material
base according to a filler area decrease. The fresh and salt water absorption coefficient increases with increasing immersion in water time,
an organic filler percentage and its size. This means that these panels can be used in a wet environment. That it depends on the nature of the wood and on the initial moisture. p
The test results have shown that it is possible to produce good quality artificial wood panels without
preliminary procedures for the materials used in the study. The given studies have shown that it is possible to use wood waste of different sizes and that any
filler size allows developing a product with its own final properties. Manuscript received: 3.01.2022 Reference A., IGBOANUGO A.C., Potential of Using Recycled Low
Density Polyethylene in Wood Composite Board – Tribolgy in industry, Vol 5 (5), 2011, 389-396. 9.POO CHOW, CHARLIE T. BOWERS, JOHN. A., YOUNGQUIST, JIM H. MUEHI, ANDREZEJ M. KRZYSIK, Fasteners Resistance of Non-Woven and Melt-Blended Composite Made from Cornstalk
Fibers and Recycled Plastic - International Wood and Natural Fiber Composites Symposium April 10-
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Recycled High Density Polyethylene Polymer -Composites - Turk J Agric, 32, 2008, 537-546 11.NWIGBO SOLOMON, C. U. ATUANYA - Evaluation of the Mechanical Properties of Recycled
Polyethylene - Iroko wood Saw Dust Particulate Composite - J. Basic. Appl. Sci. Res, 1(12), 2011, 2806-
2810. 12.IDRISU. D, AIGBODION. V. S, ATUANYA. C. U, ABDULL AHI. J- Eco-Friendly Water Melon
Peels Alternative to Wood - Based Particleboard Composite - Tribology in Industry, Vol. 33(4), 2011,
173-181. 13.ABDOLLAH NAJAFI, HABIBOLLAH KHADEMI-ESLAM. Lignocelluloses Filler / Recycled
HDPE Composites Filler Type on Physical and Flexural Properties. BioResources, 2011, 6(3), 2411-
2424. 13.ABDOLLAH NAJAFI, HABIBOLLAH KHADEMI-ESLAM. Lignocelluloses Filler / Recycled
HDPE Composites Filler Type on Physical and Flexural Properties. BioResources, 2011, 6(3), 2411-
2424. 14.SUCHSLAND. O, WOODSON. G. E., Fiberboard Manufacturing in the United State, Agric-
Handbook 1987, 640. 14.SUCHSLAND. O, WOODSON. G. Reference Reference
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polyethylene blends with virgin isotactic polypropylene. Materials and Design, 46, 2013, 435-441. doi: 10.1016/j.matdes.2012.10.004
5 AZADE KIAEIFAR MOHSEN SAFFARI BEHZAD KORD C
ti
I
ti
ti
Th (
)
2.SAMSON AYELE BEKALO, HANS WOLF REINHARDT, Fiber of Coffee and Husk and Hulls for
Production of Particleboard - Materials and Structure, 43, 2010, 1049-1060. DOI 10.1617/s11527-009-9565-0
3.ANTYPAS, I.R., The influence of polyethylene processing on the plastic containers blowing. Journal
of Physics: Conference Series, 1515 (4), 2020, 1-7. doi: 10.1088/1742-6596/1515/4/042042
4.MADI, N.K. Thermal and mechanical properties of injection molded recycled high Telekom fivdensity
polyethylene blends with virgin isotactic polypropylene. Materials and Design, 46, 2013, 435-441. doi: 10.1016/j.matdes.2012.10.004 3.ANTYPAS, I.R., The influence of polyethylene processing on the plastic containers blowing. Journal
of Physics: Conference Series, 1515 (4), 2020, 1-7. doi: 10.1088/1742-6596/1515/4/042042
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Mechanical Properties of Wood Plastic Composites Made of Virgin and Recycled Plastics – World
Applied Sciences Journal, 14(5), 2011, 735-738. https://doi.org/10.37358/MP.22.2.5585 Mater. Plast., 59 (2), 2022, 61-72 71 MATERIALE PLASTICE
https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 MATERIALE PLASTICE
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https://doi.org/10.37358/Mat.Plast.1964 6.BLEZKI A. K., REIHMANE S, J. GASSAN, Thermoplastics reinforced with wood fillers - A
literature review, Polymer-Plastics Technology and Engineering, 37, 2006:451-468. https://doi.org/10.1080/03602559808001373 6.BLEZKI A. K., REIHMANE S, J. GASSAN, Thermoplastics reinforced with wood fillers - A
literature review, Polymer-Plastics Technology and Engineering, 37, 2006:451-468. https://doi.org/10.1080/03602559808001373 https://doi.org/10.1080/03602559808001373
7.YONG LEI, QINHLIN WU, FEI YAO, YANJUN XU, Preparation and properties of recycled
HDPE/natural fiber composites, 38, 2007, 1664-1667.DOI 10.1016/j.compositesa.2007.02.001
8.ATUANYA C. U., IBHADODE. A. O. Reference E., Fiberboard Manufacturing in the United State, Agric-
Handbook 1987, 640. 15.V. P. SOLOMKO - FILLED CRYSTALLINE POLMERS - Kiev - NAUKOVA DUMKA - 1980,264
16.G. KAUSHR - POLYMER DESTRUCTION - Publishing House of the World - Moscow, 1981,440 Manuscript received: 3.01.2022 Mater. Plast., 59 (2), 2022, 61-72 72 https://doi.org/10.37358/MP.22.2.5585 https://doi.org/10.37358/MP.22.2.5585
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https://projecteuclid.org/journals/communications-in-mathematical-physics/volume-172/issue-3/Vertex-IRF-correspondence-and-factorized-L-operators-for-an-elliptic/cmp/1104274318.pdf
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Vertex-IRF correspondence and factorizedL-operators for an ellipticR-operator
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Communications in mathematical physics/Communications in Mathematical Physics
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Received: 16 September 1994/in revised form: 27 March 1995 Abstract: As for an elliptic /^-operator which satisfies the Yang-Baxter equation,
the incoming and outgoing intertwining vectors are constructed, and the vertex-IRF
correspondence for the elliptic i^-operator is obtained. The Boltzmann weights of the
corresponding IRF model satisfy the star-triangle relation. By means of these inter-
twining vectors, the factorized L-operators for the elliptic /^-operator are also con-
structed. The vertex-IRF correspondence and the factorized L-operators for Belavin's
/^-matrix are reproduced from those of the elliptic /^-operator. Commun. Math. Phys. 172, 661 - 677 (1995) Commun. Math. Phys. 172, 661 - 677 (1995) Communications IΠ
Mathematical
Physics
© Springer-Verlag 1995 Vertex-IRF Correspondence and Factorized L-operators
for an Elliptic /?-operator Youichi Shibukawa
Department of Mathematics, Hokkaido University, Sapporo 060, Japan. Email: shibu@math.hokudai.ac.jp Youichi Shibukawa
Department of Mathematics, Hokkaido University, Sapporo 060, Japan. Email: shibu@math.hokudai.ac.jp Youichi Shibukawa R(ξn)(Vk(ξι) ® Vk(ξ2 + μ)) C Vk(ξ2) 0 Vk(ξλ + μ) , where ξn := ζ\ — ζi. Then we define outgoing intertwining vectors φ\{ξ)
κ
λ(z) G
Vk(ξ+ |A|k), which are the duals of Φχ\vk(ξ+\λ\ky Making use of the properties of
the incoming and outgoing intertwining vectors, we can easily construct factorized
L-operators. p
This paper is organized as follows. In Sect. 1, we review the properties of the
elliptic ^-operator R(ξ) proved in [12,13,14,4]. In Sect. 2, we shall define in-
coming intertwining vectors φλ and Boltzmann weights W
λ
ξ
V
of an IRF
K
model. Then we have the vertex-IRF correspondence for the elliptic ^-operator
(Theorem 2.1). Theorem 0.1 (Vertex-IRF Correspondence). For Λ,κ,v G Λ, φλ ® φκR(ξ) =
κ'eΛ
λ
ξ v
K
Φλ Because the elliptic i?-operator satisfies the Yang-Baxter equation, we can show
that these Boltzmann weights satisfy the star-triangle relation. This IRF model can
be regarded as the limiting case n —> oo of the A^\ face model. In Sect. 3, making
use of the results obtained by Felder and Pasquier [4], we shall construct outgoing
intertwining vectors in the same way as [6,7,10]. We can consequently define
factorized L-operators L^(ξ) (Theorem 3.4). heorem 0.2 (Factorized L-operator). For ξuξ2 $Έ + Zτ, Theorem 0.2 (Factorized L-operator). For ξuξ2 $Έ + Zτ, Theorem 0.2 (Factorized L-operator). For ξuξ2 $Έ + Zτ, In the last section, after stating the results obtained by Felder and Pasquier
[4] more precisely, we show that the vertex-IRF correspondence and the factorized
L-operators for the elliptic i?-operator imply those for Belavin's /^-matrix. 0. Introduction In [12,13,14] we have introduced an infinite-dimensional /^-matrix. It is a new
solution of the Yang-Baxter equation. By means of the Fourier transformation of the
i?-matrix, we defined an /^-operator acting on some function space. This ^-operator
also satisfies the Yang-Baxter equation. Since this operator is deeply linked to an-
alytic properties of an elliptic theta function, we call it the elliptic ί?-operator. We
have shown some properties satisfied by the elliptic i?-operator, for example, first in-
version relation, fusion procedure, etc. For the trigonometric degenerate case of the
elliptic /^-operator, we proved that the finite-dimensional, trigonometric i^-matrices
are constructed from the ^-operator through restricting the domain of the ^-operator
to some finite-dimensional subspaces. Recently Felder and Pasquier [4] showed
that Belavin's i?-matrix [3,11] can be obtained through restricting the domain
of a modified version of the elliptic ^-operator to a suitable finite-dimensional
subspace. In [1], Baxter has introduced the intertwining vectors for the eight-vertex model. Jimbo, Miwa and Okado [8] constructed the outgoing intertwining vectors between
Belavin's vertex model and the A^\
face model. We call this relation the vertex-
IRF correspondence for Belavin's i?-matrix. Hasegawa [6,7], Quano and Fujii [10]
defined the incoming intertwining vectors which are the dual vectors of the outgoing
intertwining vectors. Then they constructed the factorized L-operators for Belavin's
i^-matrix. The vertex-IRF correspondence plays a central role in their methods. 662 Y. Shibukawa Y. Shibukawa The aim of this paper is to extend the result above to the elliptic i?-oρerator. p p
p
Our strategy to construct factorized Z-operators for the elliptic ^-operator is as
follows. At first we define incoming intertwining vectors φλ of the elliptic ^-operator
R(ξ) and establish a vertex-IRF correspondence. The vertex-IRF correspondence
plays the most important role in this paper. Next we find finite-dimensional sub-
spaces with the following property (cf. Theorem 1.3); 1. Review of the Properties of an Elliptic /^-operator In this section, we review the construction and the properties of an elliptic R-
operator [4,12,13,14]. We fix τ G (C such that Im τ > 0 and define an open subset
D c C b y Imτ\
= jz G C;
D= lz G <C;|Imz|
2 J ' Vertex-IRF Correspondence for an Elliptic /^-operator Vertex-IRF Correspondence for an Elliptic /^-operator Vertex-IRF Correspondence for an Elliptic /^-operator 663 Let Y be a space of all functions / holomoφhic on D and such that Similarly let
property
be a space of all functions / holomorphic on D x D with the Similarly let
property
be a space of all functions / holomorphic on D x D with the /(zi + l,z2) = /(Z!,z2 + 1) = /(zi,z 2)
Vz1?z2 G D . Now we define an elliptic /^-operator R(ξ) on Y®Ϋ". Let μ be a complex
number such that μ $Z + Έτ and let #i(z) = i?i(z,τ) be an elliptic theta function = £exp Lemma 1.1. There is a unique function F holomorphic on D x D and such that
F(zuz2)
= (R(ξ)f)(zuz2)
for (zuz2)
e D x
D\X. The elliptic theta function ϋ\(z) satisfies the following properties. The elliptic theta function ϋ\(z) satisfies the following properties. (1) ϋ\(z) is entire, (1) ϋ\(z) is entire,
(3) ΰλ(z + τ) = - exp(-2π λ/
=Tz -
π v ^ λ
λ
(4) ϋ\{z) has simple zeros at z G Z + Zτ, (5) i?i(z) satisfies the three term equation (cf. [15] p. 461); (5) i?i(z) satisfies the three term equation (cf. [15] p. 461); - y)
- z)
+ ΰι(x + z)ϋi(x - z)i?,
+ ϋi(x + w)ΰι(x - w)ΰ (6) M-z)
= -i?!(z). Definition 1.1 (Elliptic Λ-operator). For f e
, we define z2i : = z 2 — zi, ^ί(O) = ^-(z,τ)| z =o
called a spectral parameter. G C. The complex number ξ is We set X = {(zi,z2) e D x D; z2\ £%}. By the property (4) of the elliptic theta
function ι?i(z), the function R(ξ)f
has the singularities at the points (zi,z2) G X. The lemma below tells us that all singularities are removable. Lemma 1.1. There is a unique function F holomorphic on D x D and such that
F(zuz2)
= (R(ξ)f)(zuz2)
for (zuz2)
e D x
D\X. 664 Y. Shibukawa Proof. For (zuz2)eD
x D\X and m G Z Proof. For (zuz2)eD
x D\X and m G Z, (R(ξ)f)(zuz2) (ξ)f)(zuz2)
( Q ) / ( )
ϋ{-μ-m)-ϋx{-μ)
^ z2 - zλ - m
z2-zλ-m
ϋx{z2X-m)
— z\—m
V\(z2\—m)
-ξ-m)-
1?,(-<
z2-z\-m
f(zuz2
-/w)-. z2-zx-m Thus there is a function F continuous o n D x D and such that F(z\,z2) = (R(ξ)f)
(z\,z2) for (zi,z2) G D x Z)\X. In fact, we define Thus there is a function F continuous o n D x D and such that F(z\,z2) = (R(ξ)f)
(z\,z2) for (zi,z2) G D x Z)\X. In fact, we define F(zuz2) F(zuz2)
,^2),
otherwise. otherwise. otherwise. Making use of the Riemann removable singularity theorem (cf. [5]), this function
F is holomorphic on D x D. D We also denote by R(ζ)f this holomorphic function F. It is easy to see that Ui) = for (zi,z2) £DxD. Hence Λ(ξ)/ ^ ^ Θ ^ for / G 'T&'T, and ^ ( 0 is an operator
on i^(§i^ as a result. (§
Let Y&'V&'V be a space of all functions / on D x D x D and such that
/(zi + l,Z2,z3) = /(zi,z2 + l,z3) = /(zi,z2?z3 + 1) = /(zi,z2,z3) V zi,z2,z3 eD. We have ^ I 2 ) ( ^ ~ ( ^ i ) ® F-(ξ 2 + μ)) C V-(ξ2) 0 F - ( 6 + μ) . ^ I 2 ) ( ^ ~ ( ^ i ) ® F-(ξ 2 + μ)) C V-(ξ2) 0 F - ( 6 + μ) . A basis of V~(ξ) is as follows. A basis of V~(ξ) is as follows. A basis of V~(ξ) is as follows. jez/nz Remark 1.2. Let J b e a space of the meromorphic functions on (C
2. Then we note
that the elliptic /^-operator R(ξ) can be regarded as an operator on Jt and satisfies
the Yang-Baxter equation (1.1). The elliptic theta function ϋ\(z) satisfies the following properties. Let Y&'V&'V be a space of all functions / on D x D x D and such that Let Y& V& V be a space of all functions / on D x D x D and such that
/(zi + l,Z2,z3) = /(zi,z2 + l,z3) = /(zi,z2?z3 + 1) = /(zi,z2,z3) V zi,z2,z3 eD. (zi + l,Z2,z3) = /(zi,z2 + l,z3) = /(zi,z2?z3 + 1) = /(zi,z2,z3) V zi,z2,z3 eD. By the three term equation of τ?i(z) (the property (5)), we get the following theo-
rem. Theorem 1.2 ([12,13,14]). R(ξ) satisfies the Yang-Baxter equation on Theorem 1.2 ([12,13,14]). R(ξ) satisfies the Yang-Baxter equation on orem 1.2 ([12,13,14]). R(ξ) satisfies the Yang-Baxter equation on (1 ®Λ(ξi2))(Λ(£13)® 1)(1 ®Λ(ξ23)) - (Λ(ξ23)Θ 1)(1 (8>Λ(ξi3))(Λ(ίi2)®l),
(1.1)
h
ξij
ξ
ξj (1 ®Λ(ξi2))(Λ(£13)® 1)(1 ®Λ(ξ23)) - (Λ(ξ23)Θ 1)(1 (8>Λ(ξi3))(Λ(ίi2)®l),
(1.1)
where ξij = ξt - ξj. (1 ®Λ(ξi2))(Λ(£13)® 1)(1 ®Λ(ξ23)) - (Λ(ξ23)Θ 1)(1 (8>Λ(ξi3))(Λ(ίi2)®l),
(1.1)
where ξij = ξt - ξj. where ξij = ξt - ξj. For <J G C and « = 1,2,..., let Kw(ξ) be a space of all functions / holomorphic
on <C and such that / ( z + l ) = /(z), /(z + τ) = (-l)"exp(2π>/=ϊ(£ - nz))/(z). /(z + τ) = (-l)"exp(2π>/=ϊ(£ - nz))/(z). Vertex-IRF Correspondence for an Elliptic /^-operator
665 Vertex-IRF Correspondence for an Elliptic /^-operator
665 665 It is well known that Vn(ξ) has dimension n. We easily see that It is well known that Vn(ξ) has dimension n. We easily see that (1.2)
Z/nZ (1.2) is a basis of Vn(ξ). Here ϋ \
a (z,τ) is a theta function with rational characteristics; ϋ I 11 (z, τ) = £ expfπV^Tίm + α)
2τ -f 2πv
/ΓT(m + a)(z + b)]. 1°}
mez In [4] Felder and Pasquier show the following. In [4] Felder and Pasquier show the following. Theorem 1.3 ([4]). R{ξn){Vn(ξx)
® Vn(ξ2 + μ)) C FΛ(&) ® Vn{ξx + μ). Theorem 1.3 ([4]). R{ξn){Vn(ξx)
® Vn(ξ2 + μ)) C FΛ(&) ® Vn{ξx + μ). Remark 1.1. Let Ί ^ ~ be a space of all functions / holomorphic on D and suc
that Theorem 1.3 ([4]). R{ξn){Vn(ξx)
® Vn(ξ2 + μ)) C FΛ(&) ® Vn{ξx + μ). Remark
1 1
Let Ί ^
be a space of all functions / holomorphic on D a Remark 1.1. The elliptic theta function ϋ\(z) satisfies the following properties. Let Ί ^ ~ be a space of all functions / holomorphic on D and such
that We set Ψ"~®Ϋ"~ and V~(&i
r~&i
r~
in the same way as TΓ. Then we can define
the elliptic i?-operator ^((^) on i
r~®i
r~,
which is the same as in Definition 1.1. It is easy to see that R(ξ) on Ί^~<8)^~ satisfies the Yang-Baxter equation (1.1). It is easy to see that R(ξ) on Ί
8)
satisfies the Yang Baxter equation (1.1). We denote V~(ξ) as a space of all functions / holomorphic on (C and such
that We have 2. Incoming Intertwining Vectors and Vertex-IRF Correspondence In what follows μ e 1R\Z, and let A be a set of sequences λ — (λt) (i G TL) such
that λij := λi ~ λj$ Z + Zμ
V/φy € Z . λij := λi ~ λj$ Z + Zμ
V/φy € Z . We take r e 1R such that r^ Q + Qμ, and set
ηi:=ir
( i G l ) . We take r e 1R such that r^ Q + Qμ, and set ηi:=ir
( i G l ) . ηi:=ir
( i G l ) . Y. Shibukawa 666 Y. Shibukawa Then η = (ηt) G A. Hence, for any μ, the set A is not empty. For i G Έ, we define
the sequences ε, = (βij) (j G Έ), and for λ G A, let λ -f μεz denote the sequence We note that λ 4- με, G A for all / G Έ by the definition of A Definition 2.1 (Boltzmann Weight of the IRF Model). For λ, K, K', V G Λ, Boltzmann
„ r *' i
weights W \λ
ξ
v \ e <C of an interaction-round-a-face (IRF) ratfίfe/ are g/i eH
L
K: J
as follows (cf. [1,6,7,8,10]). For /I G Λ, we put Definition 2.1 (Boltzmann Weight of the IRF Model). For λ, K, K', V G Λ, Boltzmann
„ r *' i
weights W \λ
ξ
v \ e <C of an interaction-round-a-face (IRF) ratfίfe/ are g/i eH
L
K: J
as follows (cf. [1,6,7,8,10]). For /I G Λ, we put λ 4- με. W
λ 4-
2μεi
4-
λ 4-
με7-
4- με7-
4- otherwise we set W
λ
ξ
K
: = 0 . Next we define incoming intertwining vectors of the elliptic i?-operator. Next we define incoming intertwining vectors of the elliptic i?-operator. Definition 2.2 (Incoming Intertwining Vector). For λ,κ G Λ, define an incoming
intertwining vector φλ G 'V* as follows: 3ί G TL s.t. κ =
otherwise. μεi The incoming intertwining vectors are the Dirac delta functions essentially. By
Definition 1.1 we can get a vertex-IRF correspondence for the elliptic ^-operator. Theorem 2.1 (Vertex-IRF Correspondence). For λ, κ9 v G Λ, φ
K
λ®φ
V
κR(ξ)
=
κ'eΛ
ξ
v
K
φ
K
λ
(2.1) (2.1) C
where both sides are the operators o
i j.
Since
satisfies the Yang-Baxter equation (1.1), we can show j
Since
satisfies the Yang-Baxter equation (1.1), we can show Proposition 2.2. The Boltzmann weights of the IRF model satisfy the star-triangle
relation', Σ
w
w
K
λ
ξ2i
K
a. λ
ξn
K'
K
W
K'
i
β
ξv. V
W
Γ
β
y w
A
y
ξ
X
12
K'
β
(2.2) (2.2) for λ, K, v, oc,β,ye A. for λ, K, v, oc,β,ye A. Proof Unless there exist i9j,k G Έ such that K — λ + μεt, v = λ -f μ(ε, + εy) and
y = λ + μ(εz + εy + ε^), both sides of Eq. (2.2) are zero. Then we assume that κ = λ + μεi9
v = λ + μ(ε, + ε, ),
y = A H- μ(ε,
εk) κ = λ + μεi9
v = λ + μ(ε, + ε, ),
y = A H- μ(ε,
εk) Moreover both sides of Eq. (2.2) are zero unless
α = λ + με/? A + μεy
or
A + Moreover both sides of Eq. (2.2) are zero unless and jS = A 4- μ(ε, + εy ),
A + μ(ε/ + ε^)
or A jS = A 4- μ(ε, + εy ),
A + μ(ε/ + ε^)
or A so it suffices to show Eq. (2.2) in each case. Since R(ξ) satisfies the Yang-Baxter equation (1.1), ?2, Z 3 ) . C
where both sides are the operators It is to be noted that, by Definition 2.1 and 2.2, both sides of Eq. (2.1) are zero
unless there exist ij G Έ such that K = λ 4- με/, v = λ 4- μ(εz 4- εy). The other cases Vertex-IRF Correspondence for an Elliptic /^-operator
667 Vertex-IRF Correspondence for an Elliptic /^-operator Vertex-IRF Correspondence for an Elliptic /^-operator 667 are as follows: R(ξ) =
Φx
ΦΪ
με' for i+j. for i+j. ?2, Z Putting z\ — λf, Z2 = Ay + μ(5^y and z-^ — λΛ- μ(βik Λ- δjk) in the coefficient
of
/(zi,Z2?z3)? we obtain Eq. (2.2) in the case α = A + με^ and β = A + μ(ε; + εy). We can prove the other cases in the similar way, so we omit the proof. D
Remark 2.1. We define an incoming intertwining vector φ} G (Ϋ"~)* in the same
way as Definition 2.2; for / G i^~, κ
(f(λi\
λf•=
i Λ
L 0,
otherwise. Y. Shibukawa Y. Shibukawa 668 In this case, we also get a vertex-IRF correspondence; for λ,κ,v e A, K'
λ
ξ
V
K
-K
-V
Φλ ® Φκ
f ' 3. Outgoing Intertwining Vectors and Factorized L-operators To begin with, we define outgoing intertwining vectors of the elliptic i?-operator
(cf. [6,7,10]). Let k\ and k2 be integers such that k\ ^ Aτ2, and we set k:~ (k\,k2)
and
k = k2 - k\ + 1. For λ, K e A and kx ^ j ^ k2, we define φk(ξ)f
e C by )? := Φ'. where |λ|k = Efi* 1^ Proposition
3.1. For
λ £ Λ
and
ξ tfϊ Έ + Έτ,
the
k — by — k
matrix
i
is invertible. Proof. Since λ
+μει'
J
)k{ £ijzk2 =
d i aS (
e xP πy/^Tkλkι,
, exp πλ/ it suffices to prove + 0. The Weyl-Kac denominator formula for Ak\
(cf. [9,7]) yields det [ϋ
2
k
k
i=\ det [ϋ
2
k
k i=\ Here η(τ) is Dedekind's ^-function Here η(τ) is Dedekind's ^-function Here η(τ) is Dedekind's ^-function πyfΛτ
η(τ) = exp-
12
fi (1 ~~
w=l Vertex-IRF Correspondence for an Elliptic ^-operator
669 Vertex-IRF Correspondence for an Elliptic ^-operator Vertex-IRF Correspondence for an Elliptic ^-operator Vertex-IRF Correspondence for an Elliptic ^-operator Vertex-IRF Correspondence for an Elliptic ^-operator
669 669 Then we obtain l
j-h
det ( 7? \λ\k-kλi9kτ)
1 _ L
2
k
k
2 thereby completing the proof. D thereby completing the proof. D The proposition above says that for λ, K G Λ, k\ S j S ^2 and ξ £ Z + Zτ, there
exist φk(O;, ^ ^ which are characterized by the following duality relations; (3.1)
(A:i ^ Vz ^ A:2) we set (3.1) and for K Φ λ + μεz (A:i ^ Vz ^ A:2) we set Definition 3.1 (Outgoing Intertwining Vector). For A, K G A and ξ £ ΊL + Zτ, «w
outgoing intertwining vector φ^{ξ)
κ
λ{z) e Vk(ξ + |A|k) o///ze elliptic R-operator is
defined as follows (cf (1.2)): *ω := Σ
μ|k - fe, Equation (3.1) is equivalent to Equation (3.1) is equivalent to Equation (3.1) is equivalent to ;
+ μ ε / = id on Vk(ξ
(3.2) (3.2) The outgoing intertwining vectors satisfy the following: The outgoing intertwining vectors satisfy the following: The outgoing intertwining vectors satisfy the following:
Proposition 3.2. For λ,κ,v G A and ξ\,ξι i TL + Zτ, Proposition 3.2. For λ,κ,v G A and ξ\,ξι i TL + Zτ, Proposition 3.2. For λ,κ,v G A and ξ\,ξι i TL + Zτ, K
λ
ξU V
κ
f K
λ
ξU V
κ
f Proof By Definition 2.1 and 3.1, it suffices to show λ + μ
λ
ξu
λ + μ Y. Shibukawa 670 for any λ G A and kλ <. Vι, j ^ k2. With the aid of Theorem 2.1 and Eq. (3.2), we
obtain for k\ ^ V#, b ^ k2, /I + με,
ζn /I + με, ζn Then Σ Σ
h
= Σ
w
t+εj)
λ + μεa
λ
ξl2
λ + μεi
3/) /+μ(εα+ε6)
λ
ζ\2 h
= Σ
t+εj) w
λ + μεa
λ
ξl2
λ + μεi
3/) /+μ(εα+ε6) L
/I + By virtue of Definition 3.1 and Theorem 1.3 we deduce
\λ\k + μ) By virtue of Definition 3.1 and Theorem 1.3 we deduce
\λ\k + μ) From Eq. (3.2), we are led to the desired result. D From Eq. (3.2), we are led to the desired result. D From Eq. (3.2), we are led to the desired result. D
For λ, K G A and ξ £ Έ -f Zτ, we define an operator
^
by From Eq. (3.2), we are led to the desired result. D
For λ, K G A and ξ £ Έ -f Zτ, we define an operator
^
by From Eq. (3.2), we are led to the desired result. D
For λ, K G A and ξ £ Έ -f Zτ, we define an operator
(4(£)5/)( )
(ξf ( )
κ f
(/ f)
^
by For λ, K G A and ξ £ Έ -f Zτ, we define an operator
^
by (4(£)5/)(z) := φk(ξfλ(z)φ
κ
λf (/ef). Theorem 2.1 and Proposition 3.2 say Theorem 2.1 and Proposition 3.2 say Lemma 3.3. i w A, v e A and ξu ξ2 £ TL + Zτ, Lemma 3.3. i w A, v e A and ξu ξ2 £ TL + Zτ, Vertex-IRP Correspondence for an Elliptic ^-operator Vertex-IRP Correspondence for an Elliptic ^-operator Vertex-IRP Correspondence for an Elliptic ^-operator 671 Proof. For / G T Proof. For / G T Σ
κ,κ'eΛ
λ
ξn
v
(Φλ®Φκ)f We have thus proved the lemma. D We have thus proved the lemma. D We have thus proved the lemma. D We have thus proved the lemma. D Now we are in the position to construct factorized I-operators for the elliptic
i?-operator. Let if be a space of all (C-valued functions on A, and let 'V&'Ψ (resp. i^&i^)
be a space of all functions g : D x Λ —• (C (resp. Λ x D —> (C) such that
G f
(resp. g(λ,
) G f^) for any λ £ A. We define a factorized L-operator
1T01Γ as follows [2,6,7,10]. For g e Ϋ~®ir zxAξ^Έ
+ TLτ, (Lk(ξ)g)(λ,z)
:= E (Z k(O;^(
, *))(*) . (3.3) (3.3) For λ G Λ we set <5
;v G ^
as follows: δ\κ) = δ λ ί C. We note that TT = Y[κeA^δ
κ
(cf. [6]). Then, for / G TΓ, We note that TT = Y[κeA^δ
κ
(cf. [6]). Then, for / G TΓ, We note that TT = Y[κeA^δ
κ
(cf. [6]). Then, for / G TΓ, and Eq. (3.3) is hence equivalent to We define 'V&'V&IV
(resp. I^&I^&Ψ*) by a space of all functions g :
D x D x y l - ^ C
(resp. / I X D X D ^ C )
such that g(
,
,/ί) G Ί ^ Θ ^ (resp. g(λ,
,
) G i^®i^)
for any λ e Λ. By means of Lemma 3.3, we immediately
obtain the following theorem. Theorem 3.4 (Factorized L-operator). For ζ\9ξ2 ί- % + %
τ> are ίήe operators 'V& Remark 3.1. In the same way as this section, we can construct factorized L-operators
for R(ξ) on T T - ^ T T " by using FΛ"(O instead of Fn(ξ) (cf. Remark 1.1 and 2.1). 672 Y. Shibukawa Y. Shibukawa Y. Shibukawa In this case, outgoing intertwining vectors are characterized by the following duality
relation: In this case, outgoing intertwining vectors are characterized by the following duality
relation: Here, for
λ9κ e Λ,
h <± j <L k2,
we
define
φk(ξ)f
e €
as follows
(c
Remark 1.1): Here, for
λ9κ e Λ,
h <± j <L k2,
we
define
φk(ξ)f
e €
as follows
(c
Remark 1.1): _.. /
Γ 1
j-h
Ί
\
/
ZT(^+ l)z)
. 4. Vertex-IRF Correspondence and Factorized L-operators
for Belavin's /^-matrix In this section, we apply Theorem 2.1 to the /^-matrix obtained through restricting
the domain of the elliptic i?-operator to some finite-dimensional subspace. Then
we will show that the vertex-IRJF correspondence for Belavin's /^-matrix proved by
Baxter [1], Jimbo, Miwa and Okado [8] is obtained from Theorem 2.1. Moreover
we will construct the factorized L-operators for Belavin's i?-matrix obtained by
Hasegawa [6], Quano and Fujii [10]. First let us state the results proved by Felder
and Pasquier [4] more precisely. q
p
y
For k — 1,2,..., let Vk(ζ) be a space of entire functions / of one variable such
that f(z + τ) = (-l/exp(-2πΛ/^T(^ - ξ + We note that Vk(ξ) c ^ if k is even and that Vk(ξ) c V~ if k is odd. In the same
fashion as Theorem 1.3 and Remark 1.1, we obtain + μ)) C Vk(ξi) The space Vk(ζ) is of A: dimensions and a basis is given by {ej(ξ)(z) := ΰ \j 7 * ] (ξ - kz,kτ)}jeΣ/kπ . For k — 1,2,..., define a translation operator Tk(ξ) on the space of all holomoφhic
functions on C [4] by (Tk(ξ)f)(z) ;= Tk(ξ) maps isomorphically V^ := Vk(0) onto ^t(ξ). We modify the elliptic
^-operator as Vertex-IRF Correspondence for an Elliptic i?-operator Vertex-IRF Correspondence for an Elliptic i?-operator
673 Vertex-IRF Correspondence for an Elliptic i?-operator
67 673 We note that ^(£12) is determined by the difference ξn In fact, We note that ^(£12) is determined by the difference ξn In fact, + 7>
zl ~ 7 Felder and Pasquier prove Theorem 4.1 ([4]). Rk(ζ) preserves Vk 0 Vk and obeys the Yang-Baxter equation
(1.1). Theorem 4.1 ([4]). Rk(ζ) preserves Vk 0 Vk and obeys the Yang-Baxter equation
(1.1). Let {e
j}Jez/kπ
C Vk be the dual basis of {ej := e;(0)} C Vk; e'iej) = δtj . Now we define an operator Rk(ζ)* on Vk (8) Vk , the transpose of Rk(ζ) on F^ 0 j ^ . ® ea). Proposition 4.2 (cf. [4]). The R-matrix Rk(ξ)* is Belavin's R-matrix up to
constant. Proof. Let A and B be operators on the space of all holomorphic functions on
C as (Bf)(z) = -exp (iπV^Λ (z + ^ ) ) / (z + | ) . (Bf)(z) = -exp (iπV^Λ (z + ^ ) ) / (z + | ) . The space F^ is invariant under the actions of A and B. 4. Vertex-IRF Correspondence and Factorized L-operators
for Belavin's /^-matrix In fact, ^4 and B are
expressed on Vk as - βj exp
Bej = eJ+ι . Bej = eJ+ι . We define operators A* and B* on F^ to be the transposes of A and B on F^
respectively; To prove this proposition, it is enough to show the following [3,6,7]. (1) Rk(ζ)* is an entire End(F/ (g> F/)-valued function in ξ. (1) Rk(ζ)* is an entire End(F/ (g> F/)-valued function in ξ. (2)&k(ξ) χ®x=x®x&k(ξ)*
x=A*,B*. k
k
(3) Rk{ξ + 1)* = (1 ®A*y
1Rk(ξ)*(A* ® 1) x (-1). (4) Rk(ξ + τ)* = (1 ® B*)-'Rk(ξ)*{B* ® 1) x (-exp2π^/=T(ξ + | - £))-». (4) Rk(ξ + τ)* = (1 ® B*)-'Rk(ξ)*{B* ® 1) x (-exp2π^/=T(ξ + | - £))-». 674
u/'t Y. Shibukawa
i. oiiiuuivαwα Y. Shibukawa
i. oiiiuuivαwα Y. Shibukawa
i. oiiiuuivαwα The operator Rk(ξ) on Vk 0 Vk has the properties below, which imply the properties
(2), (3), (4), and (5) above, respectively. The operator Rk(ξ) on Vk 0 Vk has the properties below, which imply the properties
(2), (3), (4), and (5) above, respectively. (2) Rk(ξ)x ® x = x ® xRk(ξ)
x=A9B. (3) Rk(ξ + 1) = (1 ®A)Rk(ξ)(A 0 I)"
1 x (-1). (Λ) Rk(ξ + τ) = (1 ® B)Rk(ξ)(B ® iyι x (- exp2πv
/zT(ξ + f - f))"
1. (5) 4 ( 0 ) = t?ί(0)id. The proof is quite straightforward, so we omit it. The proof is quite straightforward, so we omit it. To prove (1), it suffices to show that Rk(ξ) is an entire End(^
function in ξ. Let us introduce another basis of Vk (cf. [4]);
^)-valued In the same way as [4], we can calculate the matrix coefficients of Rk(ζ) on Vk 0 Vk
with respect to the basis {ey 0 ej} and can check that all matrix coefficients are
entire in ξ. This completes the proof. D In the same way as [4], we can calculate the matrix coefficients of Rk(ζ) on Vk 0 Vk
with respect to the basis {ey 0 ej} and can check that all matrix coefficients are
entire in ξ. This completes the proof. 4. Vertex-IRF Correspondence and Factorized L-operators
for Belavin's /^-matrix D For λ,ιc £ A, we put φ(ξ)« := φ
λ
κ oTk(ξ + |A|k - kμ)\Ϋk
Since For λ,ιc £ A, we put φ(ξ)« := φ
λ
κ oTk(ξ + |A|k - kμ)\Ϋk
Since 0,
1 _ L
2
k
k
2
(ξ
-
kλhkτ)
^ 3ί ^
otherwise , otherwise , otherwise , otherwise , we get we get k-\ _λ
7=0
ί To \
u
'I _ I
. 2
- kμ)(ej)e>
^ 3/ ^
otherwise. ί To \
u
'I _ I
. 2
^ 3/ ^
otherwise. otherwise. otherwise. Hence the vector φ{ζ)
κ
λ is nothing but the outgoing intertwining vector of Belavin's
7?-matrix [6,7], which was first discovered by Baxter [1], Jimbo, Miwa and
Okado [8]. Hence the vector φ{ζ)
κ
λ is nothing but the outgoing intertwining vector of Belavin's
7?-matrix [6,7], which was first discovered by Baxter [1], Jimbo, Miwa and
Okado [8]. [ ]
On the other hand, we put On the other hand, we put W
λ ξ v := W
V
ξ λ
K and then Theorem 2.1 and Remark 2.1 lead us to and then Theorem 2.1 and Remark 2.1 lead us to Vertex-IRF Correspondence for an Elliptic i?-operator Vertex-IRF Correspondence for an Elliptic i?-operator 675 Theorem 4.3 (Vertex-IRF Correspondence for Belavin's ^-matrix [1,8]). For λ,κ,
v G Λ, K
1
λ
ξn
v . Next we construct the factorized Z-operators for Belavin's 7?-matrix proved by
Hasegawa [6], Quano and Fujii [10]. To begin with, we introduce outgoing inter-
twining vectors in Vk(ζ) in the same fashion as Definition 3.1. In the sequel, we fix
k\,k2 G % such that k = k2 — k\ + 1 and assume that λ,κ,v e Λ and the ξ,ξ\,ξ2 £
Έ + Έτ. For k\ S j S k2, we define φk(ξ)f
€ <C by For k\ S j S k2, we define φk(ξ)f
€ <C by For k\ S j S k2, we define φk(ξ)f
€ <C by nd also define ψk(ζ)fj £ & by the following duality relations (cf. Proposition 3.1): ί
l-ki
λj
(
ζ
ζ l i=kι Pv\ζ)χ
For κή=λ + μsi (k\ ^ Vz ^ i 2) we set l i=kι Pv\ζ)χ
For κή=λ + μsi (k\ ^ Vz ^ i 2) we set Φk(ί)ϊy := 0 . 4. Vertex-IRF Correspondence and Factorized L-operators
for Belavin's /^-matrix Outgoing
intertwining
vectors
ψk{ξ)
κ
λ{z) G Vk(ξ + |/l|k)
of the elliptic
i?-operator are defined as ,(ξ + μ| kχ z). Then we define the operators Ly{ζ)\ as follows: Then we define the operators Ly{ζ)\ as follows: L){z)
:=
φk(ξ)t(z)φ
K
λf where / G y if k is even and / G i
r~ if k is odd. In the same way as Sect. 3,
these operators satisfy (cf. Lemma 3.3) We put Z k(O" := Tk{ξ + |κ| k - kμ)-
ιLk
(ξ - kμ)
λ
κTk(ξ + |ιc|k - kμ)\^ ,
and denote its transpose as Lγ(ξ)
κ
λ : Vk —> Vk . Thus, for Belavin's i^-matrix Rk(ξ)*,
M
l
l
= Σ Ki&l ® 1*^)1 Rk(ξn)* •
KEΛ Z k(O" := Tk{ξ + |κ| k - kμ)-
ιLk
(ξ - kμ)
λ
κTk(ξ + |ιc|k - kμ)\^ ,
and denote its transpose as Lγ(ξ)
κ
λ : Vk —> Vk . Thus, for Belavin's i^-matrix Rk(ξ)*,
M
l
l
= Σ Ki&l ® 1*^)1 Rk(ξn)* •
KEΛ Z k(O" := Tk{ξ + |κ| k - kμ)-
ιLk
(ξ - kμ)
λ
κTk(ξ + |ιc|k - kμ)\^ , note its transpose as Lγ(ξ)
κ
λ : Vk —> Vk . Thus, for Belavin's i^-matrix Rk(ξ)*,
M
l
l
= Σ Ki&l ® 1*^)1 Rk(ξn)* •
KEΛ Y. Shibukawa Y. Shibukawa 676 We define an operator l£(ξ) : Ϋl 0 iV -» IT 0 V* by The theorem below tells us that the operator Lk(ξ) is the factorized Z-operator
for Belavin's ^-matrix, which were first constructed by Hasegawa [6], Quano and
Fujii [10]. Theorem 4.4 (Factorized L-operator for Belavin's i?-matrix). For ζ\,ζι £ Z + Έ ^ 2 ) * ) ( I k ( i i ) ® 1)(1 ®Ll(ξ2)) = (tk(ξ2) ® 1)(1
£
& ^ 2 ) * ) ( I k ( i i ) ® 1)(1 ®Ll(ξ2)) = (tk(ξ2) ® 1)(1
£
& ^ 2 ) * ) ( I k ( i i ) ® 1)(1 ®Ll(ξ2)) = (tk(ξ2) ® 1)(1
£
& 57'ίfos
1 are ί/*e operators Ϋζ 0 ί^* 0 #^ —> #" Acknowledgement. The author would like to express his deep gratitude to Professor Kimio Ueno
for useful advice, to Professor Takayuki Hibi for constant encouragement, and to Professor
Michio Jimbo for pointing out to him another proof of Proposition 2.2 which is used in this paper. 4. Vertex-IRF Correspondence and Factorized L-operators
for Belavin's /^-matrix Note added in proof. We have two remarks about the incoming and outgoing intertwining vectors. Note added in proof. We have two remarks about the incoming and outgoing intertwining vectors. (1) We canaddonemore parameter to the incoming intertwining vector φ in Definition 2 2 Note added in proof. We have two remarks about the incoming and outgoing intertwining vectors. (1) We can add one more parameter to the incoming intertwining vector φλ in Definition 2.2. (1) We can add one more parameter to the incoming intertwining vector φλ in Definition 2. For α € 1R, we set ί f(
λi +
α)>
3i
e % s t. κ = λ + μεi9
Φλ(*)f
:= 1
,
. ^ 0
otherwise ,
. otherwise 0,
otherwise . These incoming intertwining vectors also satisfy the vertex-IRF correspondence (Theorem 2.1). Making use of the incoming intertwining vectors φλ(a) instead of φλ, we can construct the
factorized L-operators (Theorem 3.4). p
(
)
(2) By means of the Weyl-Kac denominator formula (cf. Proposition 3.1), we obtain the
explicit form of the outgoing intertwining vector in Definition 3.1. For k\ S i S h, References 1. Baxter, R.: Eight-vertex model in lattice statistics and one-dimensional anisotropic Heisenbe
chain. II. Equivalence to a generalized ice-type lattice model. Ann. Phys. 76, 25-47 (1973 1. Baxter, R.: Eight-vertex model in lattice statistics and one-dimensional anisotropic Heisenberg
chain. II. Equivalence to a generalized ice-type lattice model. Ann. Phys. 76, 25-47 (1973) q
g
yp
y
(
2. Bazhanov, V.V., Kashaev, R.M., Mangazeev, V.V., Stroganov, Yu. G.: (ZNx)
n~
ι
generaliz
tion of the chiral Potts model. Commun. Math. Phys. 138, 393-408 (1991) y
,
(
)
3. Belavin, A.A.: Dynamical symmetry of integrable quantum systems. Nucl. Phys. B180 [FS2],
189-200 (1981) (
)
4. Felder, G., Pasquier, V.: A simple construction of elliptic i?-matrices. Lett. Math. Phys. 32,
167-171 (1994) (
)
5. Gunning, R.C., Rossi, H.: Analytic functions of several complex variables. Englewood Cliffs,
NJ: Prentice-Hall, 1965 6. Hasegawa, K.: Crossing symmetry in elliptic solutions of the Yang-Baxter equation and a new
I-operator for Belavin's solution. J. Phys. A. Math. Gen. 26, 3211-3228 (1993) p
y
7. Hasegawa, K.: L-operator for Belavin's i?-matrix acting on the space of theta functions. J. Math. Phys. 35 (11), 6158-6171 (1994) 8. Jimbo, M., Miwa, T., Okado, M.: Local state probabilities of solvable lattice models: An A^lx
family. Nucl. Phys. B300 [FS22], 74-108 (1988) Vertex-IRF Correspondence for an Elliptic /^-operator 677 9. Kac, V.G.: Infinite dimensional Lie algebras. (3
rded.) Cambridge: Cambridge University Press,
1990 10. Quano, Y., Fujii, A.: Yang-Baxter equation for broken Z®""
1 models. Mod. Phys. Lett. A8,
1585-1597 (1993) (
)
11. Richey, M.P., Tracy, C.A.: TLn Baxter model: Symmetries and the Belavin parametrization. Stat. Phys. 42 3/4, 311-348 (1986) 11. Richey, M.P., Tracy, C.A.: TLn Baxter model: Symmetries and the Belavin parametrization. J. Stat. Phys. 42 3/4, 311-348 (1986)
12
Shib k
Y
U
K
C
l
l
Έ
i
R
i
L
M
h Ph
25
239 248 y
(
)
12. Shibukawa, Y., Ueno, K.: Completely Έ symmetric R matrix. Lett. Math. Phys. 25, 239-24
(1992) 13. Shibukawa, Y., Ueno, K.: A new solution of the Yang-Baxter equation with complete TL
symmetry. Int. J. Mod. Phys. A (Proc. Suppl.) 3A. Yang, C.N., Ge, M.L., Zhou, X.W. (eds.)
Differential geometric methods in theoretical physics. Proceedings, Tianjin 1992, Singapore:
World Scientific, 1993, pp. 309-312 14. Shibukawa, Y., Ueno, K.: Infinite-dimensional R matrix with complete TL symmetry. In: Ge,
M.L., de Vega, H.J. References (eds.) Quantum groups, integrable statistical models and knot theory. Singapore: World Scientific, 1993, pp. 302-318 g p
,
, pp
15. Whittaker, E.T., Watson, G.N.: A course of modern analysis. Fourth edition. Cambridge:
Cambridge University Press, 1927 Communicated by M. Jimbo
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https://openalex.org/W1983320158
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https://ijpam.eu/contents/2015-101-1/1/1.pdf
|
Latin
| null |
CORDIAL LABELING FOR CYCLE OF COMPLETE BIPARTITE GRAPHS AND CYCLE OF WHEELS
|
International Journal of Pure and Applied Mathematics
| 2,015
|
cc-by
| 3,250
|
PA
ijpam.eu PA
ijpam.eu ijpam.eu AMS Subject Classification:
05C78 Key Words:
cordial labeling, cycle of complete bipartite graphs and cycle of
wheels V.J. Kaneria1, Meera Meghpara2 §, H.M. Makadia3 1Department of Mathematics
Saurashtra University
Rajkot, 360005,
2Om Engineering College
Junagadh, 362001,
3Govt. Engineering College
RAJKOT, 360005, Abstract: In this paper we have obtained cordial labeling for cycle of complete
bipartite graphs and cycle of wheels. AMS Subject Classification:
05C78 CORDIAL LABELING FOR CYCLE OF COMPLETE
BIPARTITE GRAPHS AND CYCLE OF WHEELS V.J. Kaneria1, Meera Meghpara2 §, H.M. Makadia3 1. Introduction The concept of cordial labeling was introduced by Cahit [2] in 1987 as a weaker
version of graceful and harmonious labelings. Many researchers have studied
cordiality of graphs. Ho et al. [3] proved that unicyclic graph is cordial unless
it is C4k+2. Kaneria et al. [6] introduced a graph known as cycle of graphs. In
[7] Kaneria et al. proved that cycle of a cycle is cordial. c⃝2015 Academic Publications, Ltd. url: www.acadpubl.eu Received:
September 4, 2014 §Correspondence author 2 V.J. Kaneria, M. Meghpara, H.M. Makadia The recent survey on graph labeling can be found in Gallian [4], which pro-
vide vast amount of literature on graph lableling. Labelled graph have variety
of applications in coding theory. A detailed study about applications of graph
labeling is carried out in Bloom and Golomb [1]. For all terminology and no-
tations we follws Harary [5]. First of all we shall recall some definitions, which
are used in this paper. Definition 1.1. A function f : V (G) −→{0, 1} is called binary vertex
labeling of a graph G and f(v) is called label of the vertex v of G under f. For an edge e = (u, v), the induced function f ∗: E(G) −→{0, 1} defined
as f ∗(e) = |f(u) −f(v)|. Let vf(0), vf(1) be number of vertices of G having
labels 0 and 1 respectively under f and let ef(0), ef(1) be number of edges of
G having labels 0 and 1 respectively under f ∗. A binary vertex labeling f of a graph G is called cordial labeling if |vf(0) −vf(1)| ≤1 and |ef(0) −ef(1)| ≤1. A graph which admits cordial labeling is called cordial graph. A graph which admits cordial labeling is called cordial graph. Definition 1.2. For a cycle Cn, each vertices of Cn is replace by connected
graphs G1, G2, . . . , Gn is known as cycle of graphs and we shall denote it by
C(G1, G2, . . . , Gn). If we replace each vertices by a graph G i.e. G1 = G,
G2 = G, . . ., Gn = G, such cycle of a graph G, we shall denote it by C(n · G). 2. Main Results Theorem 2.1. C(t · Km,n) is cordial, ∀m, n, t ∈N −{1}. Theorem 2.1. C(t · Km,n) is cordial, ∀m, n, t ∈N −{1}. , n,
1
when t ≡0 1 (mod 4) and k = n + 1 n + 2
n or f(v1,k) =
(
0,
∀k = 1, 2, . . . , n
2 ,
1,
∀k = n
2 + 1, n
2 + 2, . . . , n; f(ut,j) =
0,
when t ≡0, 1, 2 (mod 4) and j = 1, 2, . . . , m
2 or
t ≡3 (mod 4) and j = m
2 + 1, m
2 + 2, . . . , m,
1,
when t ≡0, 1, 2 (mod 4) and j = m
2 + 1, m
2 + 2, . . . , m or
t ≡3 (mod 4) and j = 1, 2, . . . , m
2 ;
f(vt,k) =
0,
when t ≡0, 1 (mod 4) and k = 1, 2, . . . , n
2 or
t ≡2, 3 (mod 4) and k = n
2 + 1, n
2 + 2, . . . , n,
1,
when t ≡0, 1 (mod 4) and k = n
2 + 1, n
2 + 2, . . . , n or
t ≡2, 3 (mod 4) and k = 1, 2, . . . , n
2 . f(vt,k) =
0,
when t ≡0, 1 (mod 4) and k = 1, 2, . . . , n
2 or
t ≡2, 3 (mod 4) and k = n
2 + 1, n
2 + 2, . . . , n,
1,
when t ≡0, 1 (mod 4) and k = n
2 + 1, n
2 + 2, . . . , n or
t ≡2, 3 (mod 4) and k = 1, 2, . . . , n
2 . Case II. W.l.o.g. we assume that m is even and n is odd. Therefore: Case II. W.l.o.g. we assume that m is even and n is odd. Therefore: f(u1,j) =
(
0,
∀j = 1, 2, . . . , m
2 ,
1,
∀j = m
2 + 1, m
2 + 2, . . . Theorem 2.1. C(t · Km,n) is cordial, ∀m, n, t ∈N −{1}. Proof. Let G be a cycle of t copies of the complete bipartite graph Km,n. Let ui,j (1 ≤j ≤m) and vi,k (1 ≤k ≤n) be vertices of ith copy of Km,n, ∀
i = 1, 2, . . . , t. We shall join ui,m vertex of ith copy of Km,n with vi+1,1 vertex
of (i + 1)th copy of Km,n by an edge, ∀i = 1, 2, . . . , t −1. We also join ut,m
with v1,1 by an edge to form the cycle graph C(t · Km,n). ,
,
To define the labeling function f : V (C(t · Km,n)) −→{0, 1} we shall con-
sider following three cases. Case I. m and n are even. Then: f(u1,j) =
(
0,
∀j = 1, 2, . . . , m
2 ,
1,
∀j = m
2 + 1, m
2 + 2, . . . , m; CORDIAL LABELING FOR CYCLE OF COMPLETE... 3 3 f(v1,k) =
(
0,
∀k = 1, 2, . . . , n
2 ,
1,
∀k = n
2 + 1, n
2 + 2, . . . , n;
f(ui,j) =
(
f(u1,j),
when i ≡0, 1 (mod 4),
1 −f(u1,j),
when i ≡2, 3 (mod 4);
f(vi,k) =
(
f(v1,k),
when i ≡0, 1 (mod 4),
1 −f(v1,k),
when i ≡2, 3 (mod 4),
where j = 1, 2, . . . , m, k = 1, 2, . . . , n, i = 2, 3, . . . , t −1. Hence
f(ut,j) =
0,
when t ≡0, 1, 2 (mod 4) and j = 1, 2, . . . , m
2 or
t ≡3 (mod 4) and j = m
2 + 1, m
2 + 2, . . . , m,
1,
when t ≡0, 1, 2 (mod 4) and j = m
2 + 1, m
2 + 2, . . . , m or
t ≡3 (mod 4) and j = 1, 2, . . . , m
2 ;
f(vt,k) =
0,
when t ≡0, 1 (mod 4) and k = 1, 2, . . . , n
2 or
t ≡2, 3 (mod 4) and k = n
2 + 1, n
2 + 2, . . . Theorem 2.1. C(t · Km,n) is cordial, ∀m, n, t ∈N −{1}. , m;
f(v1,k) =
(
0,
∀k = 1, 2, . . . , n−1
2 ,
1,
∀k = n+1
2 , n+3
2 , . . . , n; f(ui,j) =
(
f(u1,j),
when i ≡0, 1 (mod 4),
1 −f(u1,j),
when i ≡2, 3 (mod 4);
f(vi,k) =
(
f(v1,k),
when i ≡0, 1 (mod 4),
1 −f(v1,k),
when i ≡2, 3 (mod 4),
where j = 1, 2, . . . , m, k = 1, 2, . . . , n, i = 2, 3, . . . , t −1. So, we have
f(ut,j) =
0,
when t ≡0, 1, 2 (mod 4) and j = 1, 2, . . . , m
2 or
t ≡3 (mod 4) and j = m
2 + 1, m
2 + 2, . . . , m,
1,
when t ≡0, 1, 2 (mod 4) and j = m
2 + 1, m
2 + 2, . . . , m or
t ≡3 (mod 4) and j = 1, 2, . . . , m
2 ; f(ui,j) =
(
f(u1,j),
when i ≡0, 1 (mod 4),
1 −f(u1,j),
when i ≡2, 3 (mod 4); f(ui,j) =
(
f(u1,j),
when i ≡0, 1 (mod 4),
1 −f(u1,j),
when i ≡2, 3 (mod 4);
f(vi,k) =
(
f(v1,k),
when i ≡0, 1 (mod 4),
1 −f(v1,k),
when i ≡2, 3 (mod 4),
where j = 1, 2, . . . , m, k = 1, 2, . . . , n, i = 2, 3, . . . , t −1. So, we have
f(ut,j) =
0,
when t ≡0, 1, 2 (mod 4) and j = 1, 2, . . . , m
2 or
t ≡3 (mod 4) and j = m
2 + 1, m
2 + 2, . . . , m,
1,
when t ≡0, 1, 2 (mod 4) and j = m
2 + 1, m
2 + 2, . . . , m or
t ≡3 (mod 4) and j = 1, 2, . . . , m
2 ; where j = 1, 2, . . . , m, k = 1, 2, . . . , n, i = 2, 3, . . . Theorem 2.1. C(t · Km,n) is cordial, ∀m, n, t ∈N −{1}. , t −1. So, we have
f(ut,j) =
0,
when t ≡0, 1, 2 (mod 4) and j = 1, 2, . . . , m
2 or
t ≡3 (mod 4) and j = m
2 + 1, m
2 + 2, . . . , m,
1,
when t ≡0, 1, 2 (mod 4) and j = m
2 + 1, m
2 + 2, . . . , m or
t ≡3 (mod 4) and j = 1, 2, . . . , m
2 ; 4 V.J. Kaneria, M. Meghpara, H.M. Makadia Figure 1: Cycle graph C(6 · K3,5) and its cordial labeling Figure 1: Cycle graph C(6 · K3,5) and its cordial labeling f(vt,k) =
0,
when t ≡0, 1 (mod 4) and k = 1, 2, . . . , n−1
2
or
t ≡2, 3 (mod 4) and k = n+1
2 , n+3
2 , . . . , n,
1,
when t ≡0, 1 (mod 4) and k = n+1
2 , n+3
2 , . . . , n or
t ≡2, 3 (mod 4) and k = 1, 2, . . . , n−1
2 . Case III. m and n both are odd. Hence f(ui,j) =
(
0,
∀j = 1, 2, . . . , m−1
2 , ∀i = 1, 2, . . . , t,
1,
∀j = m+1
2 , m+3
2 , . . . , m, ∀i = 1, 2, . . . , t;
f(vi,k) =
(
1,
∀k = 1, 2, . . . , n−1
2 , ∀i = 1, 2, . . . , t,
0,
∀k = n+1
2 , n+3
2 , . . . , n, ∀i = 1, 2, . . . , t. The above labeling pattern give rise cordial labeling to the given graph G,
as it satisfies |vf(0) −vf(1)| ≤1 and |ef(0) −ef(1)| ≤1 in above three cases. Thus G = C(t · Km,n) is a cordial graph, ∀t, m, n ∈N −{1}. Example 2.2. C(6·K3,5) (it is related with case-III) and its cordial labeling
shown in Figure 1. Theorem 2.3. C(t · Wn) is cordial, t, n ∈N −{1, 2}. Proof. Theorem 2.1. C(t · Km,n) is cordial, ∀m, n, t ∈N −{1}. Let G be cycle of t copies of wheel Wn. Let vi,j (0 ≤j ≤n) be
vertices of ith copy of Wn, where vi,0 is vertex of apex of the wheel Wn, ∀ CORDIAL LABELING FOR CYCLE OF COMPLETE... 5 CORDIAL LABELING FOR CYCLE OF COMPLETE... 5 i = 1, 2, . . . , t. We shall join vi,0 with vi+1,0, ∀i = 1, 2, . . . , t −1 and vt,0 with
v1,0 unless t ≡2 (mod 4), otherwise join v1,0 with vt,1 to form the cycle graph
C(t · Wn). (
)
To define the labeling function f : V (t · Wn) −→{0, 1, }, we have following
four cases. Case I. Let t ≡0, 2 (mod 4). Then Case I. Let t ≡0, 2 (mod 4). Then Case I. Let t ≡0, 2 (mod 4). Then f(vi,0) =
0,
when i ≡0, 1 (mod 4),
1,
when i ≡2, 3 (mod 4),
∀i = 1, 2, . . . , t; f(vi,j) =
1,
when i ≡0, 1 (mod 4),
0,
when i ≡2, 3 (mod 4),
∀i = 1, 2, . . . , t, ∀j = 1, 2, . . . , n. Case II. Let t ≡1, 3 (mod 4) and n ≡0, 1, 2 (mod 4). Then Case II. Let t ≡1, 3 (mod 4) and n ≡0, 1, 2 (mod 4). Then f(vi,0) =
0,
when i ≡0, 1 (mod 4),
1,
when i ≡2, 3 (mod 4),
∀i = 1, 2, . . . , t −1; f(vi,0) =
0,
when i ≡0, 1 (mod 4),
1,
when i ≡2, 3 (mod 4),
∀i = 1, 2, . . . , t −1; f(vi,j) =
1,
when i ≡0, 1 (mod 4),
0,
when i ≡2, 3 (mod 4),
∀i = 1, 2, . . . , t −1, ∀j = 1, 2, . . . , n;
f(vt,j) =
1,
when j ≡1, 2 (mod 4),
0,
when j ≡0, 3 (mod 4),
∀j = 0, 1, 2, . . . , n. 3. Concluding Remarks Cordial labeling of some cycle of graphs discussed. Here we provide cordial
labeling to C(t · Km,n) and C(t · Wn). V.J. Kaneria, M. Meghpara, H.M. Makadia 6 f(vt,j) =
0,
when j = 1, 2 or j ≡2, 3 (mod 4),
1,
when j = 0, 3, 4, 5 or j ≡0, 1 (mod 4),
∀j = 6, 7, . . . , n. Case IV. Let t ≡3 (mod 4) and n ≡3 (mod 4). Then Case IV. Let t ≡3 (mod 4) and n ≡3 (mod 4). Then f(vi,0) =
0,
when i ≡0, 1 (mod 4),
1,
when i ≡2, 3 (mod 4),
∀i = 1, 2, . . . , t −1; f(vi,j) =
1,
when i ≡0, 1 (mod 4),
0,
when i ≡2, 3 (mod 4),
∀i = 1, 2, . . . , t −1, ∀j = 1, 2, . . . , n;
f(vt,j) =
1,
when j = 1, 2 or j ≡2, 3 (mod 4),
0,
when j = 0, 3, 4, 5 or j ≡0, 1 (mod 4),
∀j = 6, 7, . . . , n. The above labeling pattern give rise cordial labeling to the given graph G,
as it satisfies |vf(0) −vf(1)| ≤1 and |ef(0) −ef(1)| ≤1 in above four cases. Thus G = C(t · Wn) is a cordial graph, ∀t, n ∈N −{1, 2}. Example 2.4. C(5W7) and its cordial labeling (it is related with Case III)
shown in Figure 2. Example 2.5. C(5W4) and its cordial labeling (it is related with Case II)
shown in Figure 3. Theorem 2.1. C(t · Km,n) is cordial, ∀m, n, t ∈N −{1}. f(vt,j) =
1,
when j ≡1, 2 (mod 4),
0,
when j ≡0, 3 (mod 4),
∀j = 0, 1, 2, . . . , n. Case III. Let t ≡1 (mod 4) and n ≡3 (mod 4). Then Case III. Let t ≡1 (mod 4) and n ≡3 (mod 4). Then f(vi,0) =
0,
when i ≡0, 1 (mod 4),
1,
when i ≡2, 3 (mod 4),
∀i = 1, 2, . . . , t −1; f(vi,j) =
1,
when i ≡0, 1 (mod 4),
0,
when i ≡2, 3 (mod 4),
∀i = 1, 2, . . . , t −1, ∀j = 1, 2, . . . , n;
1,
when i ≡0, 1 (mod 4),
1,
when i ≡0, 1 (mod 4), f(vi,j) =
0,
when i ≡2, 3 (mod 4),
∀i = 1, 2, . . . , t −1, ∀j = 1, 2, . . . , n;
∀i = 1, 2, . . . , t −1, ∀j = 1, 2, . . . , n; V.J. Kaneria, M. Meghpara, H.M. Makadia V.J. Kaneria, M. Meghpara, H.M. Makadia [7] V.J. Kaneria, H.M. Makadia, Meera Meghpara, Gracefulnes of cycle of
cycles and complete bipartite graphs, I.J.M.T.T., 12, No. 1 (2014), 19-26. V.J. Kaneria, M. Meghpara, H.M. Makadia References [1] G.S. Bloom, S.W. Golomb, Application of numbered undirected graphs,
Proc. of IEEE, 65, No. 4 (1977), 562-570. CORDIAL LABELING FOR CYCLE OF COMPLETE... 7 7 Figure 2: Cycle graph C(5 · W7) and its cordial labeling
Figure 3: Cycle graph C(5 · W4) and its cordial labeling Figure 2: Cycle graph C(5 · W7) and its cordial labeling Figure 2: Cycle graph C(5 · W7) and its cordial labeling Figure 2: Cycle graph C(5 · W7) and its cordial labeling Figure 2: Cycle graph C(5 · W7) and its cordial labeling Figure 3: Cycle graph C(5 · W4) and its cordial labeling Figure 3: Cycle graph C(5 · W4) and its cordial labeling [2] I. Cahit, Cordial graphs: A weaker version of graceful and harmonious
graphs, Ars Combin, 23 (1987), 201-207. [3] Y.S. Ho, S.M. Lee, S.C. Shee, Cordial labeling of unicyclic graphs and
generalized Petersen graphs, Congress. Numer., 68, 109-122. [4] J.A. Gallian, The Electronics Journal of Combinatorics, 19 (2013). [5] F. Harary, Graph Theory, Addition Wesley, Massachusetts, 1972. [6] V.J. Kaneria, H.M. Makadia, M.M. Jariya, Graceful labeling for cycle of
graphs, Int. J. of Math. Res., 6, No. 2 (2014), 173-178. V.J. Kaneria, M. Meghpara, H.M. Makadia 8 [7] V.J. Kaneria, H.M. Makadia, Meera Meghpara, Gracefulnes of cycle of
cycles and complete bipartite graphs, I.J.M.T.T., 12, No. 1 (2014), 19-26.
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Advanced Fabrication and Characterization of Hydrothermal Responsive Fabric from Microcrystalline Cellulose-Reinforced Shape Memory Polyurethane Filament
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Textile & Leather Review
ISSN 2623-6281 | www.tlr-journal.com | 10.31881/TLR
Advanced Fabrication and Characterization of
Hydrothermal Responsive Fabric from
Microcrystalline Cellulose-Reinforced Shape
Memory Polyurethane Filament
Direselgn Molla Semanie, Lei Zhang, Bewuket Teshome Wagaye, Buguang
Zhou, Yalin Dong, Jiansheng Guo
How to cite: Semanie DM, Zhang L, Wagaye BT, Zhou B, Dong Y, Guo J. Advanced Fabrication and
Characterization of Hydrothermal Responsive Fabric from Microcrystalline Cellulose-Reinforced
Shape Memory Polyurethane Filament. Textile & Leather Review. 2024; 7:670-687.
https://doi.org/10.31881/TLR.2024.074
How to link: https://doi.org/10.31881/TLR.2024.074
Published: 8 May 2024
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License
SEMANIE DM et al.
TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687
Advanced Fabrication and Characterization of
Hydrothermal Responsive Fabric from Microcrystalline
Cellulose-Reinforced Shape Memory Polyurethane
Filament
Direselgn Molla SEMANIE, Lei ZHANG, Bewuket Teshome WAGAYE, Buguang ZHOU, Yalin
DONG, Jiansheng GUO*
Key Laboratory of Textile Science & Technology, Ministry of Education, College of Textiles, Donghua University,
Shanghai, China
*jsguo@dhu.edu.cn
Article
https://doi.org/10.31881/TLR.2024.074
Received 2 April 2024; Accepted 30 April 2024; Published 8 May 2024
ABSTRACT
Demands for smart textiles have recently increased quickly in terms of functionality and responsiveness to
wearers and environmental changes. This paper explores the development of hydrothermal responsive shape
memory woven fabric from Microcrystalline Cellulose Reinforced Shape Memory Polyurethane Microcomposite
Filament. The concentration of microcrystalline cellulose and drawing ratio of the filaments were first optimized
according to the tensile strength and shape recovery ratio and taken as 15 wt% and 2.0 respectively.
Hydrothermal responsive shape memory micro-composite filaments were then produced from shape memory
polyurethane (SMPU) and microcrystalline cellulose (MCC) with optimized concentration and draw ratio by wet
spinning process. The physical, mechanical, thermal and shape memory performances of the filaments were
studied. The optimized filament was found to have a tensile strength of 0.91 cN/dtex and elongation of 385.2%
in which the strength is much more improved when compared to a pure SMPU filament of strength 0.72 cN/dtex.
The shape fixity and shape recovery results of the micro-composite filament were found to be 71.2% and 93.6%
respectively. A woven fabric was manufactured from pure polyester, cotton as warp and SMPU-MCC filaments as
weft and its breathability and shape memory properties were investigated. The air permeability of SMPU-PE
fabrics was found to be 172.9 mm/s and 155.1 mm/s in its fixed temporary shape and recovered shape
respectively. The water vapour permeability of SMPU-CT fabric was found to be 612.01 g/m2.h and 540.28 g/m2.h
in its fixed temporary and recovered shape respectively which shows smart breathable fabrics can be made and
adopted with enhanced properties.
KEYWORDS
cellulose, micro-composite filaments, hydrothermal responsive fabrics, shape memory polyurethane
INTRODUCTION
Shape memory materials (SMMs) have gained significant attention in recent years due to their ability
to change their shape in response to specific stimuli (temperature, water or moisture, magnetic field,
etc.), and memorize their original shape and potential applications in various fields such as smart
textiles, energy harvesting, biomedical engineering and responsive materials [1]. The two most used
https://doi.org/10.31881/TLR.2024.074
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SEMANIE DM et al.
TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687
types of SMMs are shape-memory alloys (SMAs) and shape-memory polymers (SMPs) [2]. SMAs exhibit
their shape memory effect through the transformation of austenite to martensite, a process that has
found applications in textiles in various forms such as filaments or springs [3]. On the other hand, the
shape memory effect of SMPs is due to the movement of hard and soft segments within polymer
chains. This allows a new temporary shape to be formed and later returned to its original shape [2,4].
SMPs offer several advantages over SMAs, including greater production versatility, low manufacturing
cost, high shape recovery rate, and a broad triggering temperature range. The relatively low melting
point, ability to withstand up to 800% deformation, and thermoplastic properties of SMPs also enable
them to be produced as thin filament yarns, composite, and various types of textile fabrics [5].
In the textile industry, SMPs can be used for temperature regulation and maintenance applications
such as breathable sportswear that can provide better comfort under variable temperature and
moisture environments. Since their discovery in 1980, SMPs have been developed to respond to a
variety of stimuli, including heat, moisture or water, electrical current, alternating magnetic fields and
light [6]. There are various types of temperature and water-responsive SMPs, but shape memory
polyurethane (SMPU) is suitable for the textile application since it can be applied as coating as well as
shape memory filaments used to make fabrics with a triggering or glass transition temperature (Tg)
very close to a human body temperature. However, SMPU pure filaments and yarns are not strong
enough to be woven or knitted into a textile fabric. Therefore, the incorporation of reinforcements
mainly cellulose and carbon fibres are used in different stages of textile manufacturing processes which
leads to the development of SMPU filament composites [7,8]. Korkmaz et al. produced dual moisture
and temperature-responsive shape memory nanocomposite filaments from SMPU and cellulose
nanowhiskers and achieved a shape recovery ratio of up to 100% with remarkable strength of filaments
[9]. It has been noted that the incorporation of cellulose has improved the strength and waterresponsive properties of the filaments. However, no prior studies have been conducted to produce
fabrics from microcrystalline cellulose (MCC) reinforced shape memory polyurethane filaments and
the effect of MCC on the breathability and overall properties of fabrics has not been investigated. The
combination of MCC and SMPU provides a synergistic effect, where the MCC acts as a reinforcing
agent, enhancing the mechanical strength and dimensional stability of the fabric, while the SMPU gives
the shape memory effect [10].
This paper presents an advanced fabrication of woven hydrothermal responsive shape memory fabrics
using a microcrystalline cellulose-reinforced shape memory polyurethane composite filament as weft
and either polyester or cotton as warp yarn. The physical properties, permeability and shape memory
properties of these fabrics are also examined.
https://doi.org/10.31881/TLR.2024.074
671
SEMANIE DM et al.
TEXTILE & LEATHER REVIEW | 2024 | 7 | 670-687
EXPERIMENTAL
Materials
Pellet-type SMPU granules purchased from Sigma-Aldrich, USA were used as matrix material and
Microcrystalline cellulose (MCC) (70-140μm) also purchased from Sigma-Aldrich was used as a
reinforcement. In addition, N-dimethylformamide (DMF) and Polyoxyethylene sorbitan monooleate
(Tween 80) both from Sigma-Aldrich were used as a polar solvent and nonionic surfactant respectively.
Production of Optimized Micro-composite Filament
A multivariate Response Surface Methodology (RSM) using central composite design (CCD) was used
to determine the optimal concentration of microcrystalline cellulose (MCC) and drawing ratio. Through
optimization, it was found that 15 wt% MCC (relative to the polymer mass) and a draw ratio of 2.0
were the ideal values. A spinning solution containing 25 wt% SMPU polymer, 15 wt% MCC in DMF and
a non-ionic surfactant called Tween80 at a ratio of 1:2 w/w with MCC was prepared. This solution was
prepared at 60 °C for 6 hours using an ultrasonic bath with a mechanical stirrer at a speed of 200 rpm
[11]. The spinning solution was then extruded using Sazhuo wet spinning equipment (Shanghai Sazhuo
New Material Technology CO., LTD, China) with a needle diameter of 1.05 mm with a constant flow
rate of 0.81 ml/min into a coagulation bath of distilled water and then take-up speed of 1.9 m/min.
The SMPU-MCC micro-composite filaments were then soaked in distilled water for 24 hours to remove
any residual solvent (DMF) and dried for 10 hours at 40 °C.
Production of Hydrothermal Fabrics
Plain woven fabrics were manufactured using 250 dtex polyester and 280 dtex cotton yarns as warp
yarn, while SMPU-MCC micro-composite filament in its fixed temporary shape was used as the weft.
The fabrics were produced using SGA598 semi-automatic sampling loom (TONG YUAN TEXTILE
MACHINERY CO.LTD, China) in which weft yarns were inserted manually using a bobbin. To make a
comparison, plain-weave fabrics made entirely of 100% polyester and cotton were also produced,
along with plain-weave fabric incorporating SMPU-MCC micro-composite filament. Hydrothermal
responsive fabrics at their fixed temporary shape were immersed in hot water at a temperature of
Tg+10 °C for ten minutes to recover to their original shape.
Determination of Tensile Properties
The mechanical properties of the filaments were assessed using the LLY-06E-500 Electronic Single Fiber
Strength Tester (Laizhou Electronic Instruments Co., Ltd, China). This was done in compliance with
ASTM D3822-07, using an extension rate of 200 mm/min and a gauge length of 20 mm.
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Morphological Test of Filaments
TM4000 Table Top Scanning Electron Microscope (Thermo Scientific, USA) was used to assess the
distribution of MCC in the filament structure and the overall morphological setup.
Determination of Functional Groups
The chemical structure and functional group of SMPU-MCC filaments were studied using Fourier
Transform Infrared (FTIR) spectroscopy (PerkinElmer, USA) within a spectral range of 4000 cm-1 to 400
cm-1.
Thermal Analyses
TGA 8000 Thermogravimetric analysis equipment (PerkinElmer Instruments, USA) was utilized to
evaluate the thermal properties of micro-composite filaments. The test was conducted in a nitrogen
environment, with temperatures ranging from 50 to 600 °C, and a heating rate of 10 °C/min. The
decomposition temperature (Tmax), T10% and T50% of the micro-composite filaments were calculated
based on the thermogravimetric data.
The glass transition temperature (Tg) of filaments was examined using a Differential Scanning
Calorimetry DSC 8500 (PerkinElmer Instruments, USA) according to ISO 11,357:1-7 standard.
Filaments, weighing between 5 and 10 mg, were heated from -10 to 200 °C at a rate of 10 °C/min, then
cooled to 20 °C at a rate of 20 °C/min and Tg was identified from the first heating.
Physical Tests
The linear density of both pure SMPU and SMPU-MCC filaments, in their original and fixed temporary
shape, was measured in dtex as per the ASTM D2591-07 (Reapproved 2020) standard. The warp (EPC)
and weft density (PPC) of hydrothermal responsive fabrics were determined using a thread counter.
The warp or ends per centimetre (EPC) and picks or picks per centimetre (PPC) of the fabrics were
determined using Pick glass (Nantong Hongda Laboratory Instrument Co., Ltd, China). The thickness of
the fabrics was tested using YG (B) 141G fabric thickness tester (WENZHOU DARONG TEXTILE
INSTRUMENT CO., LTD, China) according to GB/T 3820-1997 and ASTM D1777 − 96 (Reapproved 2019)
with 28.7 ± 0.02 mm foot diameter and 4.14 ± 0.21 kPa applied pressure.
Shape Memory Properties of Filaments
The shape memory effect (SME) of filaments was evaluated using a mechanical-thermo-aqueous
programming test. Initially, the filaments, with an initial length (Li) of 5 cm were stretched (100%) in
hot water at a temperature of Tg +10 °C to a temporary length (Lt) for 10 minutes. Then, the filaments
were cooled to a temperature below their glass transition (23 °C), and dried for 12 hours and a fixed
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temporary length (Lf) was recorded. The shape memory effect (SME) was then triggered by
reimmersing the filaments in water at a recovery temperature of Tg +10 °C for ten minutes and the
samples returned to their recovered length (Lr) [5]. Finally, the shape-memory properties of the
filaments were calculated using the shape-recovery ratio (Rr) and the shape-fixity ratio (Rf)(9)(12).
Figure 1 shows a schematic representation of the mechanical-thermo-aqueous programming test and
its parameters.
Figure 1. Schematic representation of mechanical-thermo-aqueous programming test
Air and Water Vapor Permeability Properties
The air-permeability of fabrics was evaluated in accordance with GB/T 5453-1997, using the YG641E
Air-permeability Tester (WENZHOU FANGYUAN INSTRUMENT CO. LTD, China). Five samples from each
fabric with a size of 20 cm2 were used and the average permeability rate was reported as the air
permeability of the fabric.
The water vapour transmission rate (WVTR) of all (pure PE, pure CT, SMPU-PE, and SMPU-CT) fabrics
was measured using a YG601H computer-type fabric moisture permeability testing apparatus (Ningbo
Textile Instrument, China). A separate balance with 0.0001 g weighing precision was used to measure
the consecutive mass of test samples. A pair of samples each with a sample size of 28.3 cm² from both
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pure and SMPU woven fabrics were used. A desiccant technique was used in which the specimen was
sealed to the open mouth of a test cup that contained a desiccant (Calcium chloride) and put in a
controlled environment. The dishes were regularly measured to establish the rate at which water
vapour passes through the specimen into the desiccant. All of the experiments were carried out
according to GB/T 12704.1-2009 and ASTM E96 at 38.0 °C and 90% relative humidity with an air velocity
of 0.5 m/s. The water vapour transmission rate (WVTR) and Water vapour permeability (WVP) of
fabrics were calculated using the formulas stated in the above standard test methods as follows:
WVTR =
WVP =
WVT
∆P
∆m−∆m′
A.t
WVT
= Pcb(R1−R2)
Water Permeability = WVP × Thickness
(1)
(2)
(3)
Where WVTR is the water vapour transmission rate of fabrics in g/m2.h, ∆m is the difference between
two weightings of the same test combination in g, ∆m' is the difference between two weightings of
the blank sample in g, A is the effective test area in m2 (0.00283 m2 was used), t is test time in h, WVP
is water vapour permeance of fabrics in g/m2.Pa.h. ∆P is the difference in water vapour pressure on
both sides of the sample in pa, Pcb is saturated water vapour pressure at test temperature in pa
(6625.056 Pa at 38.0 °C), R1 is the relative humidity of the test chamber during the test (90%) and R2
is relative humidity in the cup (0%).
RESULTS AND DISCUSSION
Mechanical Properties of Filaments
The strength of shape-memory polyurethane fibres can be improved by the incorporation of
reinforcing materials such as cellulose [3,10,11,13]. Figure 2 shows the stress-strain curves of pure
SMPU and SMPU-MCC micro-composite filaments at their original and fixed temporary shapes. In its
original shape, the SMPU-MCC micro-composite filament has 0.91 cN/dtex tenacity and 385.2%
elongation. However, these values decreased to a tenacity of 0.74 cN/dtex and an elongation of
331.8% in its fixed temporary shape. Pure SMPU filaments have a tenacity of 0.72 cN/dtex and an
elongation percentage of 519.3% in their original shape and 386.4% in their fixed temporary shape.
SMPU-MCC micro-composite filaments have higher strength due to better load transfer due to matrixcellulose chain alignment [11].
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Table 1. Tensile properties of micro-composite filaments at their original and fixed temporary shape
Original shape (OS)
Filament code
Fixed temporary shape (FTS)
Tenacity (cN/dtex)
Strain (%)
Tenacity (cN/dtex)
Strain (%)
SMPU
0.72
519.3
0.61
386.4
SMPU-MCC
0.91
385.2
0.74
331.8
1.0
SMPU-OS
SMPU-MCC-OS
SMPU-FTS
SMPU-MCC-FTS
Tenacity (cN/dtex)
0.8
0.6
0.4
0.2
0
100
200
300
400
500
600
Strain (%)
Figure 2. Tensile properties of Microcomposite filaments at their original and fixed temporary shape
Morphological Analyses
Figures 3 a & b for pure SMPU filaments and c & d for SMPU-MCC micro-composite filaments show the
longitudinal and cross-sectional views of the two types of filaments. The figure shows that the crosssection of pure SMPU is slightly irregular and has foldings, microcracks and a distinct fibre edge [14].
This was explained by Korkmaz et al. as the lack of reinforcement materials such as cellulose, which
affects viscosity and causes a low mass transfer rate difference between the coagulating solvent
diffusion into the spinning solution and the solvent extraction into the coagulation bath [11]. Hence,
the cross-sections of SMPU-MCC micro-composite filaments are comparatively regular and circular
[15-17].
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Figure 3. Longitudinal (on the left) and Cross-sectional (on the right) SEM images of a) and b) pure SMPU-2.0; c) and d)
SMPU-MCC
Determination of Functional Groups
The FT-IR spectra of SMPU-MCC and pure SMPU filaments are shown in Figure 4. A peak at 3271 cm-1
corresponds to the stretching vibrations of the (-OH-) and (-NH-) groups in SMPU polymers. The peaks
2850 cm-1 show the symmetric stretching of (-CH-) and 1627 cm-1 shows the bending vibrations of (C=O-) groups of SMPU. The particular peaks correspond to the stretching vibrations of the(-NH-) and
(-OH-) shift for SMPU-MCC filaments from 3271 cm-1 to 3346 cm-1. This shift shows the presence of
extra hydroxyl (-OH) groups from microcrystalline cellulose [15,18,19]. For SMPU-MCC, the distinctive
peaks at 2949 cm-1 show the (-CH2-) and (-C-H-) stretching vibrations of the hydrocarbon chains. The
peak at 1728 cm-1 corresponds to the (-C=C-) bending vibration of alkenes found in SMPU and cellulose,
whereas the peak absorption at 1627 cm-1 and 1540 cm-1 shows (-C=O-) stretching vibrations
[14,16,20,21].
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1206
1066
1728
Absorbance (%)
1540
3346
SMPU-MCC
16
32
4000
3500
523
2949
71
3000
28
27
50
SMPU
2500
2000
1500
1000
500
Wavenumber (cm-1)
Figure 4. FTIR spectra values of pure SMPU and SMPU-MCC micro-composite filaments
Thermal Analyses
Thermogravimetric Analysis (TGA) test results of SMPU-MCC micro-composite filaments and pure
SMPU filaments are shown in Figure 5. The TGA curve showed that the T10% temperature rose from
309.9 °C for pure SMPU filaments to 325.6 °C SMPU-MCC microcomposite filaments and the T50%
temperature increased from 336.9 °C to 380.96 °C for SMPU-MCC microcomposite filaments compared
to pure SMPU. This indicates that the presence of MCC improved the thermal stability of filaments.
The degradation temperatures at T10% can be attributed to the removal of loosely bound volatile
compounds whereas the first step degradations at around 343 °C is the most representative indicator
of the decomposition of SMPU polymer hard segment linkages [11]. The DTG graph shows a significant
increase in the maximum decomposition temperature (Tmax) of the filaments from 370 °C (SMPU) to
390 °C (SMPU-MCC), indicating better thermal stability due to interaction between the SMPU matrix
and MCC through hydrogen bonding [22]. Gan et al. indicated that strong intermolecular bonding
increases the energy needed for macromolecule chain breaking, which improves thermal stability
[8,23]. In addition, the char residue for pure SMPU and SMPU-MCC micro-composite filaments were
found to be 4.96 wt% and 10.97 wt% respectively.
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100
SMPU
SMPU-MCC
60
40
DTG (%)
Mass ()
80
SMPU
SMPU-MCC
20
100
200
300
400
500
Temperature (°C)
0
100
200
300
400
500
600
Temperature (°C)
Figure 5. TGA and DTG curves of SMPU and MCC-SMPU micro-composite filaments
As indicated in Figure 6 and Table 2, the DSC test results showed that the Tg of the pure SMPU filament
and SMPU-MCC micro-composite filaments were 41.65 °C and 41.02 °C respectively. The presence of
MCC in the SMPU filament structure resulted in slightly lower Tg and higher heat absorption than pure
SMPU micro-composite filaments [24]. This is consistent with Korkmaz et al. and Gan et al. where a
reduction in Tg of composite fibres after the incorporation of cellulose was reported [8,11]. Korkmaz
et al. studied that the plasticizing effect and micro-Brownian thermal movements of molecular chain
segments caused by CNW particles contribute to a reduction in Tg [11].
Heat Flow Endo Up (mW)
SMPU-MCC
Pure SMPU
50
100
150
Temperature (°C)
Figure 6. First heating DSC curves for pure SMPU and SMPU-MCC microcomposite filaments
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Table 1. First heating DSC results for pure SMPU and SMPU-MCC micro-composite filaments
Sample Code
Tg (°C)
SMPU
41.65
SMPU-MCC
41.02
Shape Memory Properties
The shape memory characteristics of micro-composite filaments and pure SMPU at a triggering
temperature of Tg+10 oC are shown in Figure 7. The micro-composite filament (SMPU-MCC) has a
shape fixity ratio of 71.3%, which is higher than that of pure SMPU filaments (64%) and suggests that
the SMPU-MCCC filaments will be able to retain its fixed temporary shape when the load is removed
[14]. However, the shape recovery ratio decreases when Microcrystalline Cellulose (MCC) is added to
the SMPU filaments. This is due to the reduced mobility of the soft segments as the addition of MCC
increases the strength and rigidity, which is associated with an increased hard segment portion of the
filaments [14]. For MCC-SMPU, the shape recovery ratio was found to be 93.6%, while the shape
recovery of pure SMPU filaments was 96%. These values align with Korkmaz et al., which reported a
shape recovery ratio between 91% and 100% for SMPU reinforced with cellulose nanowhiskers [11].
The moisture-responsive shape memory effect of MCC-SMPU filaments is primarily due to the
incorporation of MCC, which introduces hydrophilic properties to the shape memory polyurethanes.
Hydrogen bonds will be formed between water molecules and the hydroxyl groups in MCC when the
filaments are exposed to hot water above Tg [11].
Shape Fixity (Rf)
Shape Recovery (Rr)
100
96
93.6
80
71.2
%
64
60
40
20
0
SMPU
SMPU-MCC
Microcomposite Code
Figure 7. Shape memory properties of pure SMPU and SMPU-MCC filaments
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Physical Property of Filaments
Figure 8 shows the linear densities of SMPU-MCC micro-composite filaments and pure SMPU in their
original shape (OS) and fixed temporary shapes (FTS), ranging from 155.3 to 182 dtex. Incorporating
MCC into the SMPU matrix influences the linear density of the microcomposite filaments. Pure SMPU
filaments have a linear density of 176.6 dtex, whereas SMPU-MCC filaments exhibit a higher linear
density value of 181.4 dtex. This increase in linear density indicates that the stiffness and density of
MCC contribute to the overall mass of the composite filament [2,11]. The linear density of both
filaments in their fixed temporary shape is smaller compared to their original shape. This reduction in
linear density is related to the stretching of the shape memory filaments, which leads to a decrease in
the mass per unit length.
Original shape (OS)
Fixed temporary shape (FTS)
180
Linear Density (dTex)
150
120
90
60
30
0
SMPU
SMPU-MCC
Figure 8. Linear density of filaments at their a) original shape b) Fixed temporary shape
Physical Property of Fabrics
Table 3 shows the warp density and thickness values for pure fabrics with SMPU micro-composite
filaments in both recovered shape (RS) and fixed temporary shape (FTS). The thermodynamics of
hydrothermal shape memory woven fabric is based on the variation in pore size between yarns in the
fabric. The fabric had an open structure in fixed temporary shape (FTS), but a more closed structure in
recovered shape (RS), due to dynamic changes in the diameter of shape memory filaments. These
diameter changes affect the fabric density and thickness [3]. As a result, the warp density and thickness
of the fabrics in the fixed temporary shape for both SMPU-polyester (PE) and SMPU-cotton (CT) fabrics
are greater than that of pure polyester and cotton fabrics. This increase is related to the recovery of
the weft SMPU filaments to their original shape after the shape memory effect was triggered by
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treating the fabrics in hot water at Tg+10 oC. These results are consistent with the results reported by
Bertran et al., where polyester yarns are used as the warp yarn and pure SMPU filaments as the weft
yarn in different weft ratios and found that the fabric parameters including tightness and thickness
increase after the load was removed from weft yarns [3].
Table 2. Physical properties of different fabrics
Fabric
Ends per cm (EPC)
Picks per cm (PPC)
Thickness (mm)
Pure PE
18
46.2
0.60
Pure CT
17.6
42.6
0.69
FTS
RS
FTS
RS
FTS
RS
SMPU-PE
17.8
22
56
56.4
0.77
0.80
SMPU-CT
18.2
21.2
59
58.6
0.78
0.79
Figure 9. A simplified representation of the hydrothermal plain-weave fabric with weft SMPU-MCC filament
Air and Water Vapor Permeability Properties
Air and water vapour permeability tests were carried out to assess the breathability and shape memory
properties of hydrothermal responsive fabrics. Results from pure polyester (PE) and pure cotton fabrics
were used as references. As shown in Table 4, the air permeability of shape Memory PolyurethaneCotton (SMPU-CT) fabric in its fixed temporary shape (FTS) (213.2 mm/s) is higher than that of its
recovered shape (RS) (187.4 mm/s). Additionally, the air permeability of pure PE fabric (214.9 mm/s)
is higher than that of SMPU-PE fabrics in both shapes and the air permeability of pure cotton fabric is
also higher than SMPU-CT fabric in both shapes due to the difference in fabric density. Despite having
a slightly lower air permeability due to its high thread density, hydrothermal responsive fabrics still
exhibit comparable air permeability to pure cotton and polyester fabrics. This indicates that the use of
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shape-memory polyurethane micro-composite filaments as the weft can provide similar air circulation
and comfort as cotton and polyester fabrics, while also providing shape-memory properties for body
moisture and temperature regulation. Air permeability, which shows how porous the fabrics are,
affects both heat and sweat flow within the fabric and is affected by filament structure and properties.
This can be observed in SMPU-PE hydrothermal responsive fabrics, where the air permeability at their
fixed temporary shape (172.9 mm/s) is higher than their recovered shape (155.1 mm/s). SMPU-MCC
filaments enable the fabric to change its air permeability by adjusting the porosity. This helps the
wearer be comfortable in various body or environmental conditions [3,10].
Table 4. Air permeability properties of fabrics
Fabric
Air permeability (mm/s)
Pure PE
214.9
Pure CT
221.2
Fixed temporary shape (FTS)
Recovered shape (RS)
SMPU-PE
172.9
155.1
SMPU-CT
213.2
187.4
Figure 10 shows the water vapour transmission rate (WVTR) and water permeability (WP) of pure and
hydrothermal responsive fabrics. The WVTR of SMPU-PE and SMPU-CT hydrothermal responsive
fabrics is 541.7 g/m2.h and 612.01 g/m2.h respectively in their fixed temporary shape, and 505.65
g/m2.h and 540.28 g/m2.h respectively in their recovered shape. The WVTR of pure polyester fabrics is
lower than that of SMPU-PE fabrics in their fixed temporary shapes. This can be attributed to the
smooth fibre structure, crystallinity, hydrophobicity and the lack of -OH- groups in polyester
morphology which affects the movement or passage of water molecules through it. In addition, SMPUPE hydrothermal responsive fabrics exhibit a higher WP than pure polyester fabrics, even at higher
fabric density (as stated in Table 3). This is due to the incorporation of microcrystalline cellulose, which
improves the interaction and absorption of fabric with water and facilitates the movement of water
molecules through the fabric by creating hydrogen bonds with water molecules. This is consistent with
the findings of Jahid et al. and Korkmaz et al. where the water vapour transmission largely depends on
the water affinity of the functional group of polymers [10,25]. The WVTR of pure cotton fabrics (657.24
g/m2.h) is higher than hydrothermal responsive fabrics made from microcrystalline cellulose
incorporated shape memory polyurethane at both fixed temporary and recovered shapes. This is due
to the low water affinity and reaction property of polyurethane molecules compared to cellulose. Pure
cotton fabrics can create more hydrogen bonds with water than SMPU-CT fabrics. Both hydrothermal
responsive shape memory fabrics in their fixed temporary shape have higher WVTR than fabrics in
their recovered shape, facilitating the passage of water vapour in and out of the body related to the
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temperature and humidity of the environment. This can be attributed to the change in the pore size
of the fabrics, which occurs when shape memory filaments recover to their original shape. This is
consistent with the findings of Bertran et al. where fabric breathability increases with the incorporation
of more shape-memory polyurethane [3].
700
0.28
(a)
657.24
600
541.7
540.28
505.65
500
0.21
WP (g/m.Hg.h)
539.93
WVTR (g/m2h)
(b)
612.01
400
300
200
0.14
0.07
100
T
RS
SM
PU
-C
PU
-C
T
FT
S
RS
SM
PU
-P
E
FT
S
SM
-P
E
re
CT
SM
PU
Pu
re
P
Pu
T
-C
-C
T
PU
SM
PU
RS
FT
S
RS
SM
PU
-P
E
FT
S
SM
-P
E
PU
SM
Pu
re
CT
E
re
P
Pu
E
0.00
0
Figure 10. Water vapour transmission rate (WVTR) (a) and water permeability (WP) (b) of fabrics
CONCLUSION
This study investigated the production and characterization of plain woven, shape memory fabrics with
responsive properties to moisture and heat. The fabrics were constructed using shape memory microcomposite filaments as weft yarns, woven with either polyester or cotton warp yarns. The presence of
microcrystalline cellulose in the composite filament increased the mechanical, crystallinity and waterresponsive shape memory properties of the filaments. The shape fixity and recovery of pure SMPU and
SMPU-MCC filaments were evaluated by using a mechanical-thermo-aqueous programming test in hot
water at a temperature of Tg +10 °C and exhibited a shape recovery of 93.6%. The air permeability of
hydrothermal responsive fabrics was 172.9 mm/s and 213.2 mm/s for SMPU-PE and SMPU-CT at their
fixed temporary shape and 155.1 mm/s and 187.4 mm/s at their recovered shape respectively. In
addition, the water vapour transmission rate of hydrothermal responsive fabrics is 541.7 g/m2.h and
612.01 g/m2.h for SMPU-PE and SMPU-CT at their fixed temporary shape and 505.65 g/m2.h and
540.28 g/m2.h at their recovered shape respectively. These results show that hydrothermal responsive
fabrics can offer better breathability and maintain body thermodynamics indicating a potential
application in smart textiles such as sportswear, waterproof and breathable clothes, socks etc.
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Author Contributions
Conceptualization – Semanie DM; methodology – Semanie DM; formal analysis – Semanie DM;
investigation – Semanie DM; writing-original draft preparation – Semanie DM; writing-review and
editing – Zhang L, Wagaye BT, Zhou B; visualization – Dong YL; supervision – Guo JS. All authors have
read and agreed to the published version of the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
Funding
This research received no external funding.
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Dicarbonyls and glyoxalase in disease mechanisms and clinical therapeutics
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Glycoconj J (2016) 33:513–525
DOI 10.1007/s10719-016-9705-z Glycoconj J (2016) 33:513–525
DOI 10.1007/s10719-016-9705-z REVIEW REVIEW REVIEW * Paul J. Thornalley
P.J.Thornalley@warwick.ac.uk Dicarbonyls and glyoxalase in disease mechanisms
and clinical therapeutics Naila Rabbani1 & Mingzhan Xue2 & Paul J. Thornalley1,2 Received: 28 February 2016 /Revised: 6 June 2016 /Accepted: 9 June 2016 /Published online: 12 July 2016
# The Author(s) 2016. This article is published with open access at Springerlink.com Received: 28 February 2016 /Revised: 6 June 2016 /Accepted: 9 June 2016 /Published online: 12 July 2016
# The Author(s) 2016. This article is published with open access at Springerlink.com Abstract The reactive dicarbonyl metabolite methylglyoxal
(MG) is the precursor of the major quantitative advanced
glycation endproducts (AGEs) in physiological systems - argi-
nine-derived hydroimidazolones and deoxyguanosine-derived
imidazopurinones. The glyoxalase system in the cytoplasm of
cells provides the primary defence against dicarbonyl glycation
by catalysing the metabolism of MG and related reactive
dicarbonyls. Dicarbonyl stress is the abnormal accumulation
of dicarbonyl metabolites leading to increased protein and
DNA modification contributing to cell and tissue dysfunction
in ageing and disease. It is produced endogenously by increased
formation and/or decreased metabolism of dicarbonyl metabo-
lites. Dicarbonyl stress contributes to ageing, disease and activ-
ity of cytotoxic chemotherapeutic agents. It contributes to age-
ing through age-related decline in glyoxalase 1 (Glo-1) activity. Glo-1 has a dual role in cancer as a tumour suppressor protein
prior to tumour development and mediator of multi-drug resis-
tance in cancer treatment, implicating dicarbonyl glycation of
DNA in carcinogenesis and dicarbonyl-driven cytotoxicity in
mechanism of action of anticancer drugs. Glo-1 is a driver of
cardiovascular disease, likely through dicarbonyl stress-driven
dyslipidemia and vascular cell dysfunction. Dicarbonyl stress is
also a contributing mediator of obesity and vascular complica-
tions of diabetes. There are also emerging roles in neurological
disorders. Glo-1 responds to dicarbonyl stress to enhance
cytoprotection at the transcriptional level through stress- responsive increase of Glo-1 expression. Small molecule Glo-
1 inducers are in clinical development for improved metabolic,
vascular and renal health and Glo-1 inhibitors in preclinical
development for multidrug resistant cancer chemotherapy. Keywords Methylglyoxal . Glycation . Glyoxalase . Obesity . Diabetes, cancer . Renal failure . Cardiovascular
disease . Therapeutics Keywords Methylglyoxal . Glycation . Glyoxalase . Obesity . Diabetes, cancer . Renal failure . Cardiovascular
disease . Dicarbonyls and glyoxalase in disease mechanisms
and clinical therapeutics Therapeutics Abbreviations
ADH
Aldehyde dehydrogenase
AGE
Advanced glycation endproduct
AKR
Aldoketo reductase
ARE
Antioxidant response element
CVD
Cardiovascular disease
3-DG
3-Deoxyglucosone
DHAP
Dihydroxyacetonephosphate
ESRD
End stage renal disease
GA3P
Glyceraldehyde-3-phosphate
GdG
3-(2′-deoxyribosyl)-6,7-dihydro-6,7-
dihydroxyimidazo-[2,3-b]purin-9(8)one
Glo-1
Glyoxalase 1
HFD
High fat diet
HIF1α
Hypoxia-inducible factor-1α
HSP27
Heat shock protein 27
MDR
Multidrug resistance
MG
Methylglyoxal
MGdG
3-(2′-deoxyribosyl)-6,7-dihydro-6,7-dihydroxy-6/
7-methylimidazo-[2,3-b]purine-9(8)one
MG-H1
Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine
NAFLD
Non-alcoholic fatty liver disease
Nrf2
Nuclear factor erythroid 2–related factor 2
PBMC
Peripheral blood mononuclear cells
RAGE
Receptor for advanced glycation endproducts * Paul J. Thornalley
P.J.Thornalley@warwick.ac.uk Glycoconj J (2016) 33:513–525 514 ROS
Reactive oxygen species
SSAO
Semicarbazide amine oxidase
T2DM
Type 2 diabetes mellitus g l y c e r a l d e h y d e - 3 - p h o s p h a t e ( G A 3 P ) a n d
dihydroxyacetonephosphate (DHAP) - 0.05 – 0.1 % of flux. GA3P was ca. 8-fold more reactive than DHAP in degrading
to MG but as the concentration ratio of DHAP/GA3P in cells
in situ is ca. 9 or similar [9], both of these triosephosphates are
important sources of MG formation in physiological systems
in situ [10]. MG formation is a minor fate of triosephosphates:
early studies with red blood cells suggested only 0.089 %
glucotriose (2 x glucose consumption) was converted to MG
[11] and our subsequent studies with endothelial cells and
fibroblasts suggest a similar flux. The rate of total cellular
formation of MG was estimated to be ca. 125 μmol/kg cell
mass per day [11], which for an adult human of 70 kg body
mass and 25 kg body cell mass [12] equates to a predicted
whole body rate of formation of ca. 3 mmol MG per day (or
ca. 3 mg/kg body weight/day). MG is also formed by the
oxidation of acetone catalysed by cytochrome P450 2E1 in
the catabolism of ketone bodies [13] – which is low except
where ketone bodies are increased as in diabetic ketoacidosis,
prolonged (>3 days) fasting or low calorie diet [13–15]. MG
may also be formed from the oxidation of aminoacetone by
semicarbazide amine oxidase (SSAO) in the catabolism of
threonine [16]. Recent estimates of the concentration and rates
of metabolism of aminoacetone in the presence and absence of
SSAO inhibitor suggest this pathway has a flux of ca. 0.1 mmol MG per day in human subjects [17] or ca. Dicarbonyl stress and the glyoxalase system Dicarbonyl stress is the abnormal accumulation of dicarbonyl
metabolites leading to increased modification of protein and
DNA contributing to cell and tissue dysfunction in ageing and
disease [1]. Highly reactive dicarbonyl metabolites often mediat-
ing dicarbonyl stress in physiological systems are methylglyoxal
(MG), glyoxal, 3-deoxyglucosone (3-DG) and others. The
glyoxalase system is a cytoplasm enzymatic pathway, which
metabolises the most highly reactive acyclic dicarbonyls – main-
ly MG and glyoxal. It thereby plays a major role in suppressing
dicarbonyl stress in physiological systems, keeping dicarbonyl
metabolites at low, tolerable levels. Typical concentrations of
glyoxal and MG are 50–150 nM in human plasma and 1–
4 μM in plant and mammalian cells [2–4]. When dicarbonyl
concentrations increase beyond this there is potential for protein
and cell dysfunction leading to impaired health and disease. Examples of dicarbonyl stress are the increased MG in ageing
plants [2], increased MG-protein modification in ageing human
lens [5], increased plasma and tissue concentration of MG in
diabetes [6], and increased concentrations of MG and glyoxal
in renal failure [4]. Dicarbonyl stress is caused by an imbalance
of the formation and metabolism of dicarbonyl metabolites and
also by increased exposure to exogenous dicarbonyls – Fig. 1a. In this review there is a particular but not exclusive focus on MG
as it is a major contributor to dicarbonyl stress in physiological
systems. Other recent reviews focussing mostly on dicarbonyl
stress in obesity and diabetes have been given elsewhere [7, 8]. Dicarbonyls and glyoxalase in disease mechanisms
and clinical therapeutics 3 % of
total MG formation. Vascular adhesion protein-1 is considered
the origin of SSAO activity in mammals in vivo [18]. It is
found in plasma, endothelium, adipose tissue and smooth
muscle and increases ca. 2-fold in congestive heart failure,
diabetes and inflammatory liver diseases [19], and may relat-
edly increase MG formation in these conditions. MG is also
formed by the degradation of proteins glycated by glucose and
the degradation of monosaccharides [20]. Under physiological
conditions with low level phosphate and chelation of trace
metal ion, the predicted flux of MG formation from glycated
protein degradation is ca. 0.2 mmol MG per day or ca. 7 % of
total MG formation. Dietary contributions to MG exposure
from food are normally relatively low: sweetened soft drink, Formation of methylglyoxal In mammalian metabolism, MG is formed at relatively high
flux mainly by the trace level degradation of triosephosphates, Fig. 1 Biochemistry of dicarbonyl stress. a Metabolism of MG by the glyoxalase system. b Formation of hydroimidazolone MG-H1 from arginine
residues. c Formation of imidazopurinone MGdG in DNA. Adduct residue is shown with guanyl base only Fig. 1 Biochemistry of dicarbonyl stress. a Metabolism of MG by the glyoxalase system. b Formation of hydroimidazolone MG-H1 from arginine
residues. c Formation of imidazopurinone MGdG in DNA. Adduct residue is shown with guanyl base only Glycoconj J (2016) 33:513–525 515 330 ml – 0.1 μmol MG [21], fruit juice, 330 ml – 0.7 μmol,
bread/cakes, 100 g, 1–2 μmol and other foodstuffs [22]; that
is, combined likely <0.03 mmol MG per day or <1 % MG
exposure. MG in foodstuffs was also metabolised and/or
reacted with proteins before absorption in the gastrointestinal
tract and imposed dicarbonyl stress mainly in the gastrointes-
tinal lumen [23]. The diet may contribute markedly greater to
total exposure for other dicarbonyls where culinary heating is
a source of formation; for example, 3-DG [24]. Sources of
formation of MG and routes of metabolism are summarised
in Fig. 2. abundance in human cells [29]. In principle, ADHs may also
metabolise glyoxal and MG to glyoxylate and pyruvate, re-
spectively. In examination of ADH-linked MG dehydroge-
nase activity in human cells to date we have found very low
or undetectable activity. AKRs and ADH catalyse the metab-
olism of 3-DG whereas Glo-1 does not [4, 30]. Other proteins,
Bglyoxalase III^ and DJ1, were proposed as glyoxalases but
their low catalytic efficiency and cellular content suggests this
is unlikely [31]. Basal and inducible expression of Glo-1, AKRs and ADH
are under stress-responsive control by nuclear factor erythroid
2–related factor 2 (Nrf2) through regulatory antioxidant re-
sponse elements (AREs) [32–36]. The cytoprotective function
of Nrf2 therefore involves enhancing basal and inducible ex-
pression and activities of enzymes of dicarbonyl metabolism
and thereby prevention of dicarbonyl stress [32]. Other regu-
latory elements in the mammalian GLO-1 gene are: metal
response element, insulin response element, early gene 2 fac-
tor-isoform-4, and activating enhancer binding protein-2α, as
reviewed [31]. Glo-1 expression is negatively regulated by
hypoxia-inducible factor-1α (HIF1α) in hypoxia [37] and also
by the receptor for advanced glycation endproducts (RAGE)
[31]. Formation of methylglyoxal Hypoxia may be an important physiological driver of
dicarbonyl stress as it both increases MG formation by flux
through anaerobic glycolysis and likely decreases Glo-1 ex-
pression through activation of HIF1α. Fig. 2 Formation of
methylglyoxal, metabolism and
glycation of protein and DNA in
vivo. Tissue levels of MG and
MG-H1 adduct residues in tissues
are given for mice. PBMC DNA
AGEs are for human subjects and
flux of MG metabolised by the
glyoxalase system and urinary
excretion of MG-H1 free adduct
is for healthy adduct humans.
Data from [2, 63, 89, 122, 137]
and Masania, J, Shafie, A,
Rabbani, N and Thornalley, P.J.,
Unpublished observations Dicarbonyl metabolism by the glyoxalase system There are also minor lysine-
derived AGEs formed: Nε-carboxymethyl-lysine and Nε(1-
carboxyethyl)lysine formed from glyoxal and MG, respective-
ly, and others. The major source of CML formation, however, is
the oxidative degradation of Nε-fructosyl-lysine residues [49]. Dicarbonyl glycation is particularly insidious as it is direct-
ed to arginine – the amino acid residue with highest probabil-
ity of location in functional sites of proteins, modification
induces loss of charge of the side chain guanidino group and
functionally important arginine residues tend to be those most
reactive towards dicarbonyl glycation [50]. The extent of
glycation of proteins by dicarbonyls is low, usually 1–5 %,
but may increase in ageing and disease. Proteins modified by
glyoxal and MG in dicarbonyl stress are recognised as mis-
folded and directed to the proteasome for proteolysis. In yeast
an unfocussed gene deletion analysis showed strains deleted
for genes of ubiquitin-dependent protein degradation were
sensitive to glyoxal and MG toxicity [51]. Examples of phys-
iological dysfunction mediated by dicarbonyl glycation of ar-
ginine residues of proteins are: mitochondrial protein dysfunc-
tion and increased formation of reactive oxygen species
(ROS) [52], inflammatory protein expression (RAGE, S100
proteins and high mobility group box-1) [53], mitochondrial
pathway activated apoptosis [54] and cell detachment from
the extracellular matrix and anoikis [55]. At in situ concentrations of MG, GSH and Glo-1, the for-
mation and fragmentation of the hemithioacetal of MG and
GSH are rapid compared to the Glo-1-catalysed step [31]. A
consequence of this is that in situ activity of Glo-1 is directly
proportional to GSH concentration. So cellular GSH concen-
tration is an influential factor on in situ activity of Glo-1 and
oxidative or non-oxidative depletion of GSH leads to in-
creased glyoxal and MG [46]. The concentration of S-D-
lactoylglutathione (SLG) is also maintained at low levels. This is likely so that lactoyl-transfer to protein thiol groups
and related inactivation of enzymes with functional cysteinyl
thiols is prevented [31]. SLG is also toxic if it leaks out of cells
and is metabolised by γ-glutamyl transferase and dipeptidase
with rearrangement to N-lactoylcysteine which is an inhibitor
of pyrimidine synthesis [47]. A mathematical model of the
glyoxalase pathway predicted very low levels of MG and
SLG in cells which fitted well with experimental observation
[31]. A surprising observation was increased chaperone function
of αA-crystallin with very high modification by MG for re-
versing dithiothreitol- and heat-induced misfolding of proteins
[56]. Dicarbonyl metabolism by the glyoxalase system Glyoxal and MG are metabolised mainly by glyoxalase 1
(Glo-1) of the glutathione-dependent glyoxalase system, with
minor metabolism by aldoketo reductases (AKRs) and alde-
hyde dehydrogenases (ADHs). As total MG-derived glycation
adducts excreted in urine of healthy human subjects was typ-
ically ca. 10 μmol per day [25, 26], it can be inferred that less
than 1 % MG formed endogenously modifies the proteins. Most of the MG formed (>99 %) is metabolised by glyoxalase
1 (Glo-1) and aldoketo reductase (AKR) isozymes, which
thereby constitute an enzymatic defence against MG
glycation. From studies of the level of expression of Glo-1
and AKRs [27, 28], it can be inferred that Glo-1 activity in
situ exceeds that of AKR activity for MG metabolism by >30-
fold in all human tissues except the renal medulla where the
expression of AKR is extraordinarily high. Indeed, Glo-1 is a
highly efficient and high abundance enzyme; typically 0.02 %
of total protein [27] and is in the top 13 % of proteins by Glo-1 expression is also increased by copy number varia-
tion (CNV) of the GLO-1 gene in human and mouse genomes. Human GLO-1 is located in chromosome 6 at locus 6p21.2
[38] and mouse Glo-1 in chromosome 17 at locus 17 a3.3
[39]. Gene cloning and bioinformatics analysis showed that
human GLO-1 coding regions consists of 12 kb with introns Fig. 2 Formation of
methylglyoxal, metabolism and
glycation of protein and DNA in
vivo. Tissue levels of MG and
MG-H1 adduct residues in tissues
are given for mice. PBMC DNA
AGEs are for human subjects and
flux of MG metabolised by the
glyoxalase system and urinary
excretion of MG-H1 free adduct
is for healthy adduct humans. Data from [2, 63, 89, 122, 137]
and Masania, J, Shafie, A,
Rabbani, N and Thornalley, P.J.,
Unpublished observations Glycoconj J (2016) 33:513–525 516 separating six exons [40, 41]. CNV of human GLO-1 was
detected by Redon et al. with a prevalence of ca. 2 %. [42]. Murine Glo-1 CNV was found in inbred strains of mice which
included complete copies of Glo-1 and complete and partial
copies of other genes [43, 44], giving rise to a 2–4-fold in-
crease in Glo-1 expression. GLO-1 duplication appeared
linked to anxiety-like behaviour in mice but may rather be
due to a proximate genetic locus co-duplicated with Glo-1
[45]. physiological systems – Fig. 1b. Dicarbonyl metabolism by the glyoxalase system With lower, physiological extent of modification by MG,
however, the chaperone function of αA-crystallin was not
enhanced further [57]. The likely evolutionary pressure for development of Glo-1
was to evolve an enzyme that accepts the major solution species
of MG, the MG-GSH hemithioacetal, which is also highly ef-
ficient with kcat/KM at the diffusion limit. Glo-1 is thereby ex-
quisitely suited to its function. High reactivity of arginine and
cysteine residues in Glo-1 protein could be a problem for Glo-1
stability which would occur with relatively low microscopic
pKa. Our computations of microscopic pKa values (as previ-
ously described [48]) indicates functionally important R37,
R122 and C139 of human Glo-1 have high microscopic pKa
values (>12), which confers low reactivity towards MG. Also in
cells with 1–4 μM MG, there is a pool of ca. 20 mM cysteinyl
thiol groups and ca. 80 mM arginine residues to which MG
may bind and only 2 μM and 4 μM of these, respectively, are
functionally important residues of Glo-1 [27]. Therefore, Glo-1
is very resistant to inactivation in situ by MG. Early studies of specific proteins modified by MG used
antibodies to the trace MG-derived AGE, argpyrimidine. In
endothelial cells the heat shock protein-27 (HSP27) was de-
tected as a major MG modified protein [58]. This could not be
verified by ultrahigh resolution Orbitrap mass spectrometry
with direct examination for MG-modification in tryptic pep-
tides – even when modification of proteins was increased 10-
fold in cell lysates [59], suggesting earlier studies suffered
interference. Subsequent studies showed recombinant
HSP27 was modified by 500 μM – 5 mM MG at multiple
sites and MG-modified HSP27 was more protective than un-
modified protein against apoptotic cell death [60]. The phys-
iological significance is unclear, however, as recent studies
were unable to find evidence of MG modification of HSP27
[59] and increased MG and Glo-1 inhibitors tend to promote
rather than suppress apoptosis [61, 62]. Diabetes and diabetic vascular complications Hypotheses for the involvement of dicarbonyl stress in disease
are most advanced and critically evaluated for involvement in
the vascular complications of diabetes. Glo-1 activity is de-
creased and MG-H1 residue content of proteins is increased in
the kidney, retina and nerve of pre-clinical models of micro-
vascular complications of diabetes (nephropathy, retinopathy
and neuropathy) [79–83]. Functional genomics studies with
Glo-1 deficient mice and Glo-1 overexpressing transgenic
mice and preventive intervention such as high dose thiamine
and Benfotiamine support increased MG as a risk factor linked
to the development of diabetic microvascular complications
[79, 84–87]. Dicarbonyl stress is also linked to diabetic car-
diovascular disease – see below. Formation of MG is in-
creased in cells with GLUT1 glucose transport incubated in
high glucose concentration [11, 88]. Decreased Glo-1 activity
synergises with increased MG formation to increase cellular Biochemical consequences of dicarbonyl stress Dicarbonyl stress produces increased in situ rates of glycation
by dicarbonyls of proteins, DNA and basic phospholipids. Reaction with proteins is directed to arginine residues forming
dihydroxyimidazolidine and hydroimidazolone adducts. The
hydroimidazolone derived from MG, MG-H1, is one of the
most quantitatively and functionally important AGEs in Glyoxal and MG are important precursors of DNA adducts
in physiological systems: major adducts are imidazopurinones
GdG and MGdG – nucleotide AGEs. MGdG was the major
nucleotide AGE found physiologically – Fig. 1c. DNA con-
tent of MGdG exceeded those of the major DNA oxidative
damage adduct, 8-hydroxydeoxyguanosine. Increased 517 Glycoconj J (2016) 33:513–525 nucleotide AGEs was associated with DNA strand breaks and
mutagenesis [63]. nucleotide AGEs was associated with DNA strand breaks and
mutagenesis [63]. lens with age and this was linked to cataract formation [5, 68]
and also in skin but to markedly lower extent [68]. Decreased
Glo-1 activity was associated with age-linked impairment of
wound healing [69] and increased dicarbonyl stress is associ-
ated with several ageing-linked diseases – see below. Glo-1
activity declines with age so there is also increased stress on
cell proteolysis and compensatory gene expression to keep
AGE-modified proteins to a low tolerable level and provide
replacement unmodified proteins. Dicarbonyl stress is likely a
feature of proliferative senescence of fibroblasts in culture
where Glo-1 expression is decreased and glycolytic flux is
increased [70, 71]. It is also likely involved in senescence of
plants: dicarbonyl content of broccoli increased with age [2]
and MG-H1 was a major AGE in Arabidopsis leaves [72]. Obesity For many years a genetic linkage of Glo-1 to body weight in
mice [73] and of GLO-1 to upper-arm circumference and
supra-iliac skinfold thickness in human subjects [74] sug-
gested a role for Glo-1 in obesity. In the mouse overeating
model of obesity, leptin mutant (ob/ob) mice, Glo-1 protein
was decreased 80 % in the liver [75]. Recent conference re-
ports described increased weight gain on high fat diet (HFD)-
fed mouse with through-life expression of GLO-1 siRNA and
Glo-1 deficiency, compared to wild-type controls [76], and
decreased weight gain in Glo-1 overexpressing transgenic
mice [77], suggesting a functional role of Glo-1 and
dicarbonyl stress in obesity. HFD-fed wild-type mice had in-
creased MG-H1 content of heart and liver, as judged by im-
munoassay [78]. Dicarbonyl stress may be a mediator of obe-
sity and insulin resistance and thereby a risk factor for devel-
opment of type 2 diabetes mellitus (T2DM) and non-alcoholic
fatty liver disease (NAFLD). Physiological consequences of dicarbonyl stress Where dicarbonyl stress occurs there is potential for increased
cell dysfunction, detachment from the extracellular matrix and
anoikis, and apoptosis. Cell dysfunction is driven by the ef-
fects of protein glycation: loss of substrate protein activity –
through inactivation and/or increased rate of proteolysis and
decreased half-life (unless compensatory increased expression
is activated), or by gaining a new and damaging function – for
example, low density lipoproteins become small, dense and
atherogenic by MG modification [64]. This affects multiple
proteins – the dicarbonyl proteome - in different cell and tissue
compartments [50]. The effects span multiple compartments
by diffusion of increased MG or other dicarbonyl in
dicarbonyl stress. MG permeates cell plasma membranes by
passive diffusion of the unhydrated form. This is rate limited
by MG dehydration, giving a half-life of ~4 min [2]. The half-
life of MG for metabolism by the glyoxalase system to D-
lactate from in situ rates of D-lactate formation in cells is ca. 10 min with free MG mostly (>95 %) reversibly bound to
protein. The rate of irreversible binding to protein in plasma
was ca. 3.6 h [65]. This implies that part of the MG formed
inside cells leaks out from the site of formation and may dif-
fuse through interstitial fluid into plasma and thereafter per-
meate back into interstitial fluid and cells of other tissues. Also, MG formed from the degradation of glycated proteins
in the extracellular compartment may enter cells for metabo-
lism by Glo-1 and AKRs. The locus of dicarbonyl stress and
related pathogenesis linked to MG accumulation is therefore
likely particularly sensitive to local decrease of Glo-1 expres-
sion and activity. Kinetic considerations of the rate of
glycation of protein, similar to those for ROS [66], indicates
a diffusion distance of MG of ca. 2–3 cm before irreversible
reaction, suggesting that MG has relatively long range and
half-life to locate and modify sensitive sites of proteins, often
leading to protein inactivation and dysfunction. Cardiovascular disease and extracellular MG concentration. MG content of blood
samples was increased by up to 5–6 fold in patients with
diabetes [6]. Increased MG has been found in diabetic kidney
in vivo [89] and vascular endothelial cells in high glucose
concentration cultures in vitro [55] – including in mitochon-
dria [90]. In locations where it is difficult to excise tissues
without leaking of MG from cells and disrupting extracellular
fluid – such as retina and cellular and extracellular compart-
ment of peripheral nerve, evidence of dicarbonyl stress is in-
creased levels of dicarbonyl–derived AGEs [83, 91]. Increased MG concentration and protein content of MG-H1
was not found in liver, skeletal muscle and brain in experi-
mental models of type 1 diabetes [25, 83, 92]. MG-derived
AGEs were increased in plasma protein and skin collagen of
diabetic patients and were linked to risk of microvascular and
macrovascular complications [26, 93–95]. A recent pre-clinical and clinical integrative genomics study
revealed Glo-1 deficiency as a driver of CVD [107]. Chemical
inhibition of Glo-1 induced atherosclerosis in apoE deficient
mice [108]. MG-derived AGEs in plasma protein have been
found to be linked to risk of CVD in diabetes [94, 109]. High
levels of MG-H1 and CML were associated with rupture-
prone plaques in human carotid endarterectomy, accumulating
in macrophages surrounding the necrotic core. The expression
of Glo-1 was decreased in ruptured compared with stable
plaque segments [110]. However, overexpression of Glo-1 in
mice did not affect atherosclerotic lesion size and severity in
ApoE−/−mice with or without diabetes [111]. Unexpectedly
plasma MG and glyoxal concentration were not decreased in
Glo-1 transgenic mice. The reason for this is not clear but
dicarbonyls are predominately sourced from metabolism and
there was limited increase in Glo-1 activity of the liver in the
Glo-1 transgenic mice [112]. Dicarbonyl stress in plasma like-
ly contributes to CVD risk through induction of dyslipidaemia
and vascular cell dysfunction. MG modification of LDL in-
duced atherogenic transformation to small, dense LDL with
increased affinity for arterial walls through binding to heparan
sulfate proteoglycans [64]. MG modification of HDL induced
re-structuring of the HDL particles, increasing density, de-
creasing stability and plasma half-life in vivo [113]. Dicarbonyl stress also induces vascular cell dysfunction: si-
lencing of Glo-1 in human aortal endothelial cells changed
expression of >400 genes – including increased expression
of RAGE and associated ligands [53, 107]. Chronic renal disease Experimental models of renal failure, bilateral nephrectomy
and bilateral ureteral ligation - models of acute total loss and
partial loss of renal function, respectively, were associated
with profound dicarbonyl stress. Plasma glyoxal and MG in-
creased 5 and 15 fold within 72 h [99]. Patients with end stage
renal disease (ESRD) on hemodialysis and peritoneal dialysis
had increased plasma MG and flux of formation of
dicarbonyl-derived AGEs [4, 100]. The cause of dicarbonyl
stress is unlikely due to decreased dicarbonyl excretion as
there is little in normal health [23], although it is linked to
renal function [99]. Decreased Glo-1 expression by hypoxia
and inflammation in ESRD [101, 102], hypoxia-induced in-
creased anaerobic glycolysis [103] and decreased disposal of
triosephosphates by the reductive pentosephosphate pathway
(enzymes of which are inhibited by uremic toxins [104]) lead-
ing to increased formation of MG may produce dicarbonyl
stress in ESRD. Decreased Glo-1 activity in rare GLO-1
frameshift mutation heterozygote human subjects was associ-
ated with decreased glomerular filtration rate [67]. A patient
with ESRD and low Glo-1 activity had a high frequency of
recurrent cardiovascular disease (CVD) events [105]. Further
studies showed a high mortality rate in patients with homozy-
gous GLO-1 419CC mutation - reviewed in [106]. This sug-
gests a link of dicarbonyl stress to development of renal failure
and CVD complications of ESRD. Ageing The link of dicarbonyl stress to ageing was unequivocally
established in a functional genomics study of Glo-1 in the
nematode C. elegans [52]. Ageing-related decline in renal
function was prevented in transgenic rats overexpressing
Glo-1 [67]. Intuitively we expect this is due to AGE accumu-
lation in proteins of tissues and body fluids with related pro-
tein dysfunction. MG-derived AGEs were increased in human 518 Glycoconj J (2016) 33:513–525 Cardiovascular disease Dicarbonyl stress may also play a role in development of
T2DM through promotion of insulin resistance [77, 96] and
development of type 1 diabetes through mediation of beta-cell
toxicity [97]. Recent studies have found increased levels of
MG, glyoxal and 3-DG of subjects with impaired glucose tol-
erance and patients with T2DM in the fasting state and during
an oral glucose tolerance test challenge [98]; plasma and
glyoxal levels were ca. 3-fold higher for MG and glyoxal than
obtained using the reference assay protocol which control [2]. Other diseases Following discovery of Glo-1 gene duplication in some strains
of mice with an anxiety phenotype [43], a link of Glo-1 to
pathologic anxiety was proposed – although the anxiety phe-
notype was linked to both increased and decreased Glo-1 ex-
pression [119, 120]. In attempts to link Glo-1 metabolically to
dysfunctional brain metabolism, MG was found to agonise the
GABAA receptor in primary cerebellar granule neurons with a
median effective concentration EC50 of 10.5 μM and this was
proposed as mediator of sedation to explain increased GLO-1
CNV with an anxiety phenotype [121]. MG concentration in
mouse brain tissue is ca. 7-fold lower than this [122] and only
approached the EC50 value with dosing of 300 mg/kg MG
[121] – similar to doses producing acute toxicity [123]. Transgenic mice with 2-fold, 4-fold and 5-fold increased
Glo-1 expression had an anxiety phenotype with 4- and 5-
fold increased expression but not with 2-fold increased ex-
pression [121]. Some inconsistences remain, therefore, and
further investigation is required. A better strategy is development of Glo-1 inducers through
activation and binding of Nrf2 to the GLO-1 functional ARE
[32]. This offers an alternative that is likely safe and effective. It also addresses a key cause of dicarbonyl stress in disease –
disease-associated, tissue-specific deficiency of Glo-1. Detection of Nrf2 activators for ARE-linked induction of
Glo-1 expression requires a specific screen with the GLO-1-
ARE motif or similar as not all Nrf2 activators induce expres-
sion of Glo-1. Nrf2 activators typically change expression of a
subset of ARE-linked genes. The basis of this subset selection
is unknown but it is likely determined by the level of
functionally-active Nrf2 achieved in the cell nucleus in re-
sponse to the Nrf2 activator, recruitment of relevant accessory
proteins – such as small maf proteins [134, 135], and absence
of activation of counter-signalling effects required to induce
the ARE-linked gene of interest. Nrf2 regulates inducible ex-
pression of ca. 890 genes [136]. The advantage of Nrf2 regu-
lated, ARE-regulated genes is that they are a battery of pro-
tective genes and so where co-regulated along with Glo-1,
they tend to add to the health beneficial response. In this re-
gard, concurrent induction of GSH synthesis for increased
cellular GSH concentration to support increased in situ activ-
ity of Glo-1 is particularly beneficial [137]. formation [63]. MDR tumours are susceptible to cell perme-
able Glo-1 inhibitors [116, 118]. formation [63]. MDR tumours are susceptible to cell perme-
able Glo-1 inhibitors [116, 118]. pyridoxamine are not efficient scavengers of MG and where
associated with alleviation of dicarbonyl stress likely function
by other mechanisms [128–131]. High dose thiamine supple-
ments for prevention of T2DM and vascular complications of
diabetes may work partly by preventing the formation of MG
through increased disposal of triosephosphates in the reductive
pentosephosphate pathway [132, 133]. Other diseases Dicarbonyl stress has also been linked to severe schizo-
phrenia through a rare frameshift mutation of GLO-1 [124],
synucleinopathies such as Parkinson’s disease in experimental
pre-clinical models [122] and Alzheimer’s disease through
clinical biomarker studies [125]. Carcinogenesis, tumour growth and cancer chemotherapy Dicarbonyl stress has a duality of function in cancer develop-
ment and treatment. Glo-1 is a tumour suppressor protein and
a mediator of multidrug resistance (MDR) in cancer chemo-
therapy. The tumour suppressor function of Glo-1 was re-
vealed in a genome-wide study in p53 knockout, Ras overex-
pression model of liver carcinogenesis [114]. This is consis-
tent with mutation arising from of dicarbonyl glycation of
DNA sometimes leading to carcinogenesis. There were 14
tumour suppressor genes found and currently Glo-1 is the only
one for which a readily available strategy for increased cancer
prevention exists – dietary Glo-1 inducers in functional foods. The role of Glo-1 in MDR was revealed in a transcriptome-
wide subtraction technique of drug-sensitive and drug-
resistant tumour cell lines [115]. Increased Glo-1 expression
in tumours may be mediated through GLO-1 amplification
[116], and also by mutation and increased transcriptional ac-
tivity of Nrf2 through ARE-linked up-regulation of Glo-1
transcription [117]. High Glo-1 activity may be permissive
for growth with high glycolytic activity and flux of MG 519 Glycoconj J (2016) 33:513–525 formation [63]. MDR tumours are susceptible to cell perme-
able Glo-1 inhibitors [116, 118]. Dicarbonyl stress-based therapeutics A Glo-1 inducer formulation has been optimised and eval-
uated in Phase 1 clinical trial (Clinicaltrials.org;
NCT02095873) in overweight and obese subjects for safety
and target pharmacology at a working dose. We also did
functional assessments – Phase 2 A trials for health
improvement in obesity. The Glo-1 inducer is a binary com-
bination of trans-resveratrol and hesperetin (tRES-HESP) and
was evaluated in a randomised, placebo-controlled crossover
clinical trial in 29 overweight and obese subjects. In highly
overweight subjects (BMI >27.5 kg/m2), tRES-HESP co-for-
mulation increased expression and activity of Glo-1, de-
creased plasma methylglyoxal and total body methylglyoxal-
protein glycation. It decreased fasting and postprandial plasma References glucose, increased oral-glucose-insulin-sensitivity (OGIS) in-
dex – an assessment of insulin sensitivity, and improved arte-
rial dilatation. In all subjects, it decreased vascular inflamma-
tion marker sICAM-1. In previous clinical evaluations, tRES
and HESP individually were ineffective. tRES-HESP co-for-
mulation could be a suitable treatment for improved metabolic
and vascular health in overweight and obese populations. It
now available for evaluation in Phase 2 clinical trial against
disease targets. 1. Rabbani N., Thornalley P.J.: Glyoxalase centennial conference:
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This first-in-class Glo-1 inducer trial establishes Glo-1 tar-
get pharmacology for tRES-HESP. Whilst increased Glo-1
expression likely contributes to the observed health beneficial
effects, changes in other gene expression occurred and their
interplay may also mediate the overall health benefit achieved. Nevertheless it was pursuit and optimisation of induction of
Glo-1 expression that arrived at this synergistic combination
of bioactive compounds and achieved improved metabolic
and vascular health in overweight and obese subjects that is
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With thank research colleagues and collaborators
who have contributed to studies covered in this review. Alleviation of dicarbonyl stress by glyoxalase 1 inducers Alleviation of dicarbonyl stress by glyoxalase 1 inducers Initial attempts to alleviate dicarbonyl stress were made
through dicarbonyl scavengers and claimed for dicarbonyl
scavenging properties of existing drugs in treatments for vas-
cular complications of diabetes. A challenge in the design of
dicarbonyl scavengers is to achieve sufficient reactivity for the
low concentration of drug achieved clinically with the 1000–
10,000 fold higher concentration of arginine residues in tissues
and body fluids. Aminoguanidine and phenacylthiazolium bro-
mide showed some promise but their toxicity and instability
prohibited further development [4, 126, 127]. Metformin and Table 1
Therapeutic agents in development targeting the glyoxalase system
Therapeutic class Mechanism of action
Primary target application (secondary)
Stage of development
Reference
Glyoxalase 1
inducer
Small molecule Nrf2 activator
targeting GLO-1-ARE
transcriptional activity
Diabetic nephropathy (other microvascular
complications of diabetes; obesity –
NAFLD; cardiovascular disease)
Clinical trial Phase 2 ready
(Phase 1 complete with safety,
dose and pharmacology established). [137]
Glyoxalase 1
inhibitor
Substrate analogue inhibitor
diester (prodrug)
Cancer (GLO-1 overexpressing,
MDR tumours)
Pre-clinical in vivo models
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Patient characteristics associated with being offered take home naloxone in a busy, urban emergency department: a retrospective chart review
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O’Brien et al. BMC Health Services Research (2019) 19:632
https://doi.org/10.1186/s12913-019-4469-3 O’Brien et al. BMC Health Services Research (2019) 19:632
https://doi.org/10.1186/s12913-019-4469-3 (2019) 19:632 O’Brien et al. BMC Health Services Research
https://doi.org/10.1186/s12913-019-4469-3 Patient characteristics associated with
being offered take home naloxone in a
busy, urban emergency department: a
retrospective chart review Patient characteristics associated with
being offered take home naloxone in a
busy, urban emergency department: a
retrospective chart review Daniel C. O’Brien1, Daniel Dabbs2, Kathryn Dong3, Paul J. Veugelers4 and Elaine Hyshka1* © 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: Overdose deaths can be prevented by distributing take home naloxone (THN) kits. The emergency
department (ED) is an opportune setting for overdose prevention, as people who use opioids frequently present for
emergency care, and those who have overdosed are at high risk for future overdose death. We evaluated the
implementation of an ED-based THN program by measuring the extent to which THN was offered to patients
presenting with opioid overdose. We analyzed whether some patients were less likely to be offered THN than
others, to identify areas for program improvement. Methods: We retrospectively reviewed medical records from all ED visits between April 2016 and May 2017 with a
primary diagnosis of opioid overdose at a large, urban tertiary hospital located in Alberta, Canada. A wide array of
patient data was collected, including demographics, opioid intoxicants, prescription history, overdose severity, and
whether a naloxone kit was offered and accepted. Multivariable analyses were used to identify patient
characteristics and situational variables associated with being offered THN. Results: Among the 342 ED visits for opioid overdose, THN was offered in 49% (n = 168) of cases. Patients
were more likely to be offered THN if they had been found unconscious (Adjusted Odds Ratio 3.70; 95%
Confidence Interval [1.63, 8.37]), or if they had smoked or injected an illegal opioid (AOR 6.05 [2.15,17.0] and
AOR 3.78 [1.32,10.9], respectively). In contrast, patients were less likely to be offered THN if they had a current
prescription for opioids (AOR 0.41 [0.19, 0.88]), if they were admitted to the hospital (AOR 0.46 [0.22,0.97], or if
they unexpectedly left the ED without treatment or before completing treatment (AOR 0.16 [0.22, 0.97). Conclusions: In this real-world evaluation of an ED-based THN program, we observed that only half of
patients with opioid overdose were offered THN. ED staff readily identify patients who use illegal opioids or
experience a severe overdose as potentially benefitting from THN, but may miss others at high risk for future
overdose. We recommend that hospital EDs provide additional guidance to staff to ensure that all eligible
patients at risk of overdose have access to THN. Keywords: Take home naloxone, Emergency department, Opioids, Overdose * Correspondence: ehyshka@ualberta.ca
1University of Alberta, School of Public Health, 3-300 Edmonton Clinic Health
Academy, 11405 – 87 Ave, Edmonton, Alberta T6G 1C9, Canada
Full list of author information is available at the end of the article Aims of the study Research on THN programs has shown that THN
kits are frequently used by people who use drugs to
respond to overdoses [7, 8]. Specifically, approxi-
mately 25% of people who use drugs who are trained
and supplied with naloxone will use it to reverse an
overdose within 1 year [7]. At the population-level,
reductions in overdose mortality have been observed
following the implementation of THN programs, and
higher rates of kit distribution lead to greater reduc-
tions in mortality [9, 10]. THN distribution can be
particularly effective at reducing overdose mortality
when targeted at high risk populations, such as re-
cently released prisoners who have lost their opioid
tolerance [11]. The present study evaluates a recently-implemented
THN program in a busy urban ED to determine the
extent to which THN was offered to patients at high-
est risk of fatal overdose: those who present with a
nonfatal opioid overdose. Our specific aims are to
measure the proportion of ED visits for opioid over-
dose in which THN was offered, and identify patient
characteristics and other situational variables associ-
ated with being offered a THN kit in the ED. Ideally,
100% of individuals who present to the ED with opi-
oid overdose should have an opportunity to leave
with a THN kit. However, we predicted that even
among this high-risk population, patients with certain
demographic or clinical characteristics would have a
higher likelihood of being offered THN. Additionally,
we anticipated that certain situational variables, such
as the time of day, length of stay in the ED, or subse-
quent hospital admittance, might impact whether cli-
nicians offer THN to patients. By identifying patients
that may have been systematically missed, we aimed
to develop new insights and recommendations for
optimizing the implementation of ED-based THN dis-
tribution. Finally, while the focus of our study is
whether THN was offered to patients, we also de-
scribe the proportion of patients who accepted THN,
and any reasons that had been charted for why pa-
tients declined THN. In recent years, THN programs have been increas-
ingly incorporated into hospital Emergency depart-
ments (EDs) in an effort to reach high risk patients
[10, 12]. Hospital EDs provide a critical opportunity
to reach people at risk for overdose, because people
who use opioids frequently present for emergency
care, and those who have overdosed are at high risk
of future overdose death [13–15]. Aims of the study Indeed, ED visits
may be one of the few occasions that an individual
comes in contact with the health system before ex-
periencing a fatal overdose. Previous investigations
have demonstrated that ED-based THN distribution
is feasible [16, 17], and that the majority of clini-
cians are willing to provide THN in the ED. [18, 19]
Further, approximately 70% of at-risk ED patients
who are offered THN accept it [20]. Page 2 of 12 O’Brien et al. BMC Health Services Research (2019) 19:632 O’Brien et al. BMC Health Services Research (2019) 19:632 Background all ED patients at risk of overdose are offered nalox-
one may be challenging [22, 23]. For instance, one
evaluation found that THN was offered to only 8%
of ED patients with International Classification of
Disease codes for opioid overdose, misuse, or de-
pendence [23]. In previous qualitative studies, ED
providers who were asked to identify barriers to
providing THN reported lacking clarity about which
patients should be offered naloxone [23, 24]. This is
problematic, because correctly identifying at-risk pa-
tients to offer THN is a crucial step in providing
THN to those who need it. Despite the importance
of this process, the extent to which different patients
are offered naloxone has not yet been studied in an
ED setting. g
Opioid overdose is a leading, yet preventable cause of
death in North America and around the world [1, 2]. Naloxone, an opioid antagonist, is commonly used in
clinical settings to reverse the potentially lethal respira-
tory depression that occurs during opioid overdose [3]. “Take Home Naloxone” (THN) programs aim to prevent
deaths by distributing naloxone to people likely to wit-
ness an opioid overdose, such as people who use drugs
and their family and friends [4–6]. Typically, THN pro-
grams train people to recognize the signs of an opioid
overdose and respond appropriately by providing basic
life support and administering naloxone via either intra-
nasal spray or intramuscular injection [2, 5, 6]. The
World Health Organization has identified THN distribu-
tion as a key health intervention to prevent opioid over-
dose deaths [2]. Study design and setting We conducted a retrospective chart review of all ED
visits for which the primary diagnosis was opioid
overdose between May 1st, 2016 and April 31st 2017
at the Royal Alexandra Hospital, which is located in
Edmonton, Alberta, Canada. This large, urban, ter-
tiary hospital received 73,163 ED visits in 2016–2017
[25]. Additionally, the Royal Alexandra Hospital sees For ED-based THN programs to have the greatest
impact, THN should be offered to all patients who
are at risk of overdose, including those who report
using illegal opioids, those taking high doses of pre-
scribed opioids, and those using opioids who present
with complications other than opioid overdose (e.g. abscesses, trauma, etc.) [20–22]. However, ensuring O’Brien et al. BMC Health Services Research (2019) 19:632 Page 3 of 12 Page 3 of 12 O’Brien et al. BMC Health Services Research the highest number of ED visits related to opioids
and other substances of misuse of any hospital in
the province of Alberta, with almost 4000 substance
use-related ED visits occurring between 2014 and
2017 [26]. The hospital ED began offering and dis-
pensing THN kits in February 2016. During the
study period, patients who were identified by either
a physician or a nurse as being at risk for opioid
overdose would be offered a THN kit just before
they were discharged from the ED. Whether a THN
kit was offered was left to the discretion of ED clini-
cians, as a standardized protocol of which patients
to target had yet to be developed at the time of this
study. policy researcher). To establish inter-rater reliability of
the abstraction protocol, a second registered nurse inde-
pendently reviewed a random subset of 70 (20%) medical
records. Percent agreement and kappa statistics were
calculated for variables collected. For all variables, kappa
was above or approaching 0.8, the commonly accepted
standard for excellent inter-rater agreement [28]. In addition to reviewing patient charts, each ED visit
was linked to data from the provincial Pharmaceutical
Information Network (PIN), which tracks prescription
medication dispensations. The PIN data were obtained
for all opioids dispensed to patients in the 180 days prior
to their ED visit for opioid overdose, and included the
date each opioid was dispensed, the type of opioid dis-
pensed, and the period it was prescribed for. Our re-
search
protocol
received
ethics
approval
from
the
University of Alberta’s Health Research Ethics Board. Variables of interest Our primary outcome of interest was whether THN was
offered during their hospital ED visit, or subsequent in-
patient hospital stay if admitted. We also determined the
number of patients that accepted a THN kit, and re-
ported any reasons that were given by the patient for de-
clining a THN kit. Evidence that THN was offered and
accepted was identified in ED physician and nursing
charts, as well as inpatient hospital records. Additionally,
medication dispensation data were reviewed to confirm
cases in which THN was provided. Patient variables believed to be potentially associated
with being offered THN included demographics such as
age (per year older) and sex (male vs. female). Addition-
ally, we included several patient characteristics that indi-
cate an increased risk of overdose, such as being a
resident of Edmonton’s inner city area (Edmonton East-
wood, yes vs. no), which is the local geographic area with
the highest rate of opioid overdose in the city [26]. Other variables linked to overdose risk included having
“no fixed address”, which was charted for patients with-
out a permanent address and indicates unstable housing
or homelessness [29], (yes vs. no), mental health disorder
[13] (defined as bipolar disorder, major depressive dis-
order, psychosis, personality disorder, or schizophrenia,
yes vs. no), and public overdose location [30] (not in a
private residence, hotel, healthcare facility or prison, yes
vs. no). Study design and setting y
If the patient accepted the THN kit, a registered nurse
would dispense the kit and provide overdose response
training, with friends and family included if possible. Registered nurses working in the ED were required to
take a training module prior to distributing THN. The
kits distributed are publicly funded as part of a province
wide THN program, and are provided directly to non-
medical persons at no cost and without a prescription. They contain 3 vials of 0.4 mg/ml naloxone, safety-engi-
neered intramuscular syringes, gloves, a CPR face shield,
alcohol swabs, and an instructional pamphlet. Kits can
be dispensed 24 h per day, 7 days per week. Because
THN kits are dispensed directly to the patient from the
ED, it is not necessary for patients to fill a naloxone pre-
scription after they leave. Other services for hospital pa-
tients who use drugs are available through the Addiction
Recovery and Community Health (ARCH) team, a spe-
cialty consult service consisting of a multidisciplinary
group of physicians with expertise in addiction medicine,
nurse practitioners, social workers, addictions coun-
selors, and peer support workers [27]. The ARCH team
is available by referral to all ED and hospital inpatients,
and offers a combination of harm reduction, in-hospital
addiction treatment, health promotion activities, and
links to appropriate community health and social sup-
ports [27]. Case identification and data collection Cases were identified from the patient hospital adminis-
trative system according to ICD-10 codes, as any of the
following: T40.0-T40.4, and T40.6. We excluded cases if
the hospital chart could not be retrieved, or if the patient
died in hospital. All medical documents related to pa-
tient care were subject to review and data abstraction,
including ED physician charts, ED nursing charts, EMS
charts, inpatient hospital charts, laboratory reports, and
the medication dispensation tracking system. The data
abstraction protocol was developed by DD who is an ex-
perienced ED nurse, in consultation with KD (emer-
gency medicine specialist) and EH (health services and Additionally, we examined whether the patient was
currently prescribed opioids (yes vs. no), and the type of
opioid they had overdosed with, as these characteristics
may have impacted clinician perceptions of overdose
risk. We identified patients who had a current prescrip-
tion for any opioid medication at the time of their ED
visit by using PIN data to determine whether the pre-
scription period for any opioid dispensed to the patient O’Brien et al. BMC Health Services Research (2019) 19:632 Page 4 of 12 Page 4 of 12 in the 180 days prior to their ED visit overlapped with
their ED visit date. was offered and not offered. To determine whether
each variable was associated with offering THN, we
conducted a series of analyses using Generalized
Estimating Equations (GEE) regressions for binary
outcomes with logit link. Initially, we fitted separate
bivariate
regression
models
for
each
independent
variable, with offering THN kits as the outcome. Variables that appeared statistically significant in the
initial bivariate analyses at the 0.1-level were in-
cluded in the final multivariable analysis. The patient’s primary opioid intoxicant and route
of consumption that caused their opioid overdose
was abstracted in the chart review, and was coded
into six categories: i) pharmaceutical opioid- oral, ii)
pharmaceutical opioid- smoked, iii) pharmaceutical
opioid- injected, iv) illegal opioid- oral v) illegal
opioid- smoked or vi) illegal opioid- injected. The
patient’s primary opioid intoxicant was confirmed by
patient self-report, or else suspected by EMS or ED
staff, and classified as either illegal or pharmaceut-
ical. Illegal opioids included heroin, carfentanil, and
illegally
manufactured
fentanyl. Fentanyl
was
as-
sumed to be illegally manufactured if the patient did
not have a current prescription for fentanyl at the
time of their ED visit, which was determined using
PIN data. Results From May 1st 2016 to April 30th 2017, there were 347
visits to the ED in which the patient received a primary
diagnosis of opioid overdose. Among these visits, only
344 patient charts were reviewed, as patient records
could not be retrieved for 3 ED visits. Additionally, 2
visits were excluded because the patient died while hos-
pitalized. The remaining 342 ED visits were made by
297 unique patients, of whom 67.3% were males and the
mean age was 38.4 (standard deviation: 14.0). Repeat ED
visits for opioid overdose were made by 35 patients dur-
ing the study period, with a range of 1 to 4 ED visits per
patient. p
Several situational variables were examined that could
possibly impact whether THN was offered, including ad-
mission to hospital (yes vs. no), time of day at presentation
and discharge (0:01–8:00, 8:01–16:00, 16:01–24:00), length
of ED stay (hours), and whether the visit occurred during
a weekend (Saturday or Sunday, yes vs. no). We examined
patient disposition, including whether the patient was dis-
charged against medical advice (AMA, yes vs. no), or left
ED without treatment or before treatment completion
(yes vs. no). A patient was considered to have left the ED
AMA if they disclosed to the providers that they intended
to leave and signed an AMA form. In contrast, patients
who left without treatment or before treatment comple-
tion would have registered with triage but then left either
from the waiting room or their patient care space, typic-
ally without disclosing their intent to leave. Overall, THN was offered to the patient in 168
(49.1%) visits, and was accepted by patients in 128
(76.2%) visits (Fig. 1). Of the ED visits in which the
patient accepted THN, a friend or family member
was included during the THN training in 31 (24.2%)
cases. Among the 40 visits in which the patient de-
clined THN, the patients already possessed a kit in
11 (27.5%) cases, and left the ED prior to receiving
naloxone in 4 (10.0%) cases (Fig. 1). Additionally, in Case identification and data collection Pharmaceutical opioids included prescribed
fentanyl, and all other pharmaceutical opioids (ie. oxycodone, hydromorphone, morphine) regardless of
whether they were prescribed to the patient at the
time of their ED visit. We used GEE with logit link for all regression analyses
because a significant portion of patients had multiple ED
visits for opioid overdose within our study period, and
the data collected for these patients were potentially cor-
related. With GEE, standard errors are calculated that
adjust for multiple observations per patient, in this case
using an exchangeable correlation structure [32]. A significant portion of values were missing for
pre-hospital GCS (14.9%) and primary opioid intoxi-
cant (23.1%). Missing data for these and other vari-
ables were imputed using chained equations with
augmented regression and 30 imputations [33]. All
variables were included in the imputation regres-
sions,
and
the
outcome
was
not
imputed. We
assessed the performance of the multiple imputations
by conducting the same multivariate analysis without
imputation (Table 3 in Appendix). In this analysis,
variables
with
a
high
proportion
of
missingness
(GCS,
primary
opioid
intoxicant)
contained
an
additional category for cases with missing values. Analyses were performed using STATA 14.0 IC. We anticipated that an ED provider’s decision to
offer THN may be impacted by the patient’s over-
dose intention (self-reported by patient, intentional
vs. unintentional), as well as the severity of the pa-
tient’s
overdose. Overdose
severity
was
measured
using the patient’s pre-hospital Glasgow Coma Scale
(GCS) [3–15], which is a standard test that measures
a patient’s eye, motor, and visual responsiveness,
with higher scores signifying higher consciousness
[31]. GCS was measured by EMS on arrival at the
overdose scene, and was therefore only available for
patients who arrived at the ED via ambulance. Analysis We calculated the number and percentage of each
independent variable among ED visits in which THN O’Brien et al. BMC Health Services Research (2019) 19:632 Page 5 of 12 Fig. 1 Study flow diagram showing identification of ED visits for inclusion, and the frequencies and percentages of ED visits in which take home
naloxone was offered and accepted Fig. 1 Study flow diagram showing identification of ED visits for inclusion, and the frequencies and percentages of ED visits in which take home
naloxone was offered and accepted (OR = 0.30 [0.13,0.65]), left the ED without treatment
or before treatment completion (OR = 0.19 [0.07,0.53]),
or were admitted to hospital (OR = 0.35 [0.19,0.65]),
(Table 1). Additionally, older patients were less likely to
be offered THN in bivariate analysis (OR = 0.97 [0.96,
0.99], per year older), (Table 1). In contrast, patients
were more likely to be offered THN if they had a severe
GCS score upon EMS arrival (OR = 5.60 [2.76,11.3]) for
GCS 3–8 vs. 14–15), had smoked an illegal opioid
(OR = 9.97 [4.66,21.3] for illegal opioid-smoked vs. pharmaceutical opioid- oral), or had injected an illegal
opioid (OR = 15.1 [6.61,34.3] for illegal opioid- injected
vs. pharmaceutical opioid- oral), (Table 1). two ED visits the patient declined a THN kit be-
cause “it won’t happen again,” or “there won’t be a
next time.” In one case they stated “I can quit,” and
in one instance the patient declined because “if I hit
the dirt, I can’t use it” (Fig. 1). Table 1 shows the breakdown of patient characteris-
tics by ED visit. In 73 (21.4%) ED visits, the patient
held a current prescription for an opioid medication at
the time of their ED visit. The most common primary
opioid intoxicants were smoked illegal opioids (n = 66
(25.1%)), injected illegal opioids (n = 85 (32.2%)), and
pharmaceutical opioids taken orally (n = 77 (29.3%))
(Table 1). The patients’ primary opioid intoxicant was
confirmed by self-report in 241 (91.6%) visits, sus-
pected by EMS in 8 (3.0%) visits, suspected by ED staff
in 12 (4.6%) visits, and confirmed by toxicology
screening in 2 (0.8%) visits. Among ED visits in which
the patient arrived by ambulance, the patient’s pre-
hospital GCS was most frequently in the severe cat-
egory (GCS 3–8; n = 210, 72.2%). Table 2 shows the results of the multivariable ana-
lysis. Analysis Variables that were independently and posi-
tively associated with being offered THN included
having
a
severe
overdose
as
measured
by
GCS
(AOR = 3.70 [1.63,8.37] for GCS 3–8 vs. GCS 14–
15),
and
smoking
or
injecting
an
illegal
opioid
(AOR = 3.78 [1.32,10.9] and AOR = 6.05 [2.15,17.0]
respectively for illegal opioid- smoked vs. pharma-
ceutical opioid- oral and illegal opioid-injected vs. pharmaceutical opioid- oral), (Table 2). In contrast,
patients were less likely to receive a THN kit if they Table 1 shows the results of the initial unadjusted bi-
variate analyses. We found that patients were less likely
to be offered THN if they overdosed in a public loca-
tion
(OR = 0.62
[0.39,0.99],
intentionally
overdosed O’Brien et al. *** N = 342 for all independent variables unless stated otherwise Analysis The results of the multivariate a
out multiple imputation are sim
with imputations (Table 3 in Appe
same variables were statistically sig
with the one exception being that
be significantly associated with be
non-imputed multivariate model (T
** Odds ratio of being offered take home nalo
* Statistically significant at the level of 0.1 and
a- Overall P-value < 0.001
b- Overall P-value =0.77
c- Overall P-value = 0.26
† Postal code overlaps Local Geographical Area
†† Postal code was charted as “no fixed addre
¥ Anxiety disorder, Bipolar disorder, Major Dep
§ Methadone (liquid form, once daily ingestion
Does not include prescriptions for opioid agon
¶ Oral includes oral (97%), rectal (1%), transder
‡ disclosed to the providers that they intended
‡‡ registered with triage and was assessed, bu
Table 2 Multivariable associations of p
overdose, with multiple imputation
Visit Characteristic (n = 342)
Male sex
Age
Mental Health disorder
Opioid agonist therapy
Any current opioid prescription at time of
Primary opioid intoxicant illegal†
Pharmaceutical opioid- oral
Pharmaceutical opioid- smoked
Pharmaceutical opioid- injected
Illegal opioid- oral
Illegal opioid- smoked
Illegal opioid- injected
Overdosed in public
Overdosed intentionally
Pre-hospital GCS††
Severe (3–8)
Moderate (9–13)
Mild (14, 15)
Left without treatment/ before treatment c
Admitted to hospital
†Overall p-value = 0.009
††Overall p value
0 008 ** Odds ratio of being offered take home naloxone for each independent variable ** Odds ratio of being offered take home naloxone for each independent variable ** Odds ratio of being offered take home naloxone for each independent variable
* Statistically significant at the level of 0.1 and thus eligible for inclusion in multivariable analysis
a- Overall P-value < 0.001
b- Overall P-value =0.77
c- Overall P-value = 0.26
† Postal code overlaps Local Geographical Area of Edmonton Eastwood, includes T5B, T5G, T5H, T5J, T5K, T6W
†† Postal code was charted as “no fixed address,” indicating that the patient is likely unstably or homeless
¥ Anxiety disorder, Bipolar disorder, Major Depressive Disorder, Psychosis, Personality Disorder (Axis II), or Schizophrenia)
§ Methadone (liquid form, once daily ingestion, max period of 7 days), or Suboxone (once daily dosing, max period of 7 days)
Does not include prescriptions for opioid agonist therapy
¶ Oral includes oral (97%), rectal (1%), transdermal (1%), and Percutaneous endoscopic gastrostomy (1%)
‡ disclosed to the providers that they intended to leave and signed an AMA form
‡‡ registered with triage and was assessed, but then left without warning or disclosing their intent to leave * Statistically significant at the level of 0.1 and thus eligible for inclusion in multivariable analysis
O
ll P
l
0 001 †† Postal code was charted as “no fixed address,” indicating that the patient is likely unstably or homeless
¥ Anxiety disorder, Bipolar disorder, Major Depressive Disorder, Psychosis, Personality Disorder (Axis II), or Schizophrenia)
§ Methadone (liquid form, once daily ingestion, max period of 7 days), or Suboxone (once daily dosing, max period of 7 days)
Does not include prescriptions for opioid agonist therapy ¶ Oral includes oral (97%), rectal (1%), transdermal (1%), and Percutaneous endoscopic gastrostomy (1%)
‡ disclosed to the providers that they intended to leave and signed an AMA form
‡‡ registered with triage and was assessed, but then left without warning or disclosing their intent to leave Analysis BMC Health Services Research (2019) 19:632 Page 6 of 12 Table 1 Characteristics of ED visits for opioid overdose, and associations with offering of take home naloxone kits
Visit Characteristic (n = 342)***
Number of ED visits (%)
Unadjusted
OR [95% CI]**
P-value
Total (n = 342)
Offered THN (n = 168)
Not offered THN (n = 174)
Patient characteristics
Male sex
234 (68.4)
129 (76.8)
105 (60.3)
2.06 [1.27,3.34]
0.004*
Age (n = 341) (Mean, SD)
38.2 (14.0)
35.3 (11.1)
40.9 (15.8)
0.97 [0.96,0.99]
< 0.001*
Resident of inner city† (n = 339)
102 (30.1)
50 (30.1)
52 (30.1)
1.04 [0.66,1.65]
0.86
No fixed address (n = 339)††
23 (6.8)
12 (7.2)
11 (6.4)
1.03 [0.39,2.67]
0.96
Mental health disorder¥
97 (28.4)
37 (22.0)
60 (34.5)
0.62 [0.38,1.01]
0.054*
Current prescription medications
Opioid agonist therapy§
9 (2.6)
1 (0.6)
8 (4.6)
0.15 [0.02,0.91]
0.04*
Any current opioid prescription
73 (21.4)
21 (12.5)
52 (29.9)
0.37 [0.22,0.65]
< 0.001*
Overdose details
Primary opioid intoxicant (n = 263)a
Pharmaceutical opioid- oral¶
77 (29.3)
14 (10.5)
63 (48.8)
ref
Pharmaceutical opioid- smoked
7 (2.7)
3 (2.3)
4 (3.1)
3.60 [0.72,18.1]
0.12
Pharmaceutical opioid- injected
16 (6.1)
5 (3.8)
11 (8.5)
2.11 [0.62,7.14]
0.23
Illegal opioid- oral
12 (4.6)
5 (3.8)
7 (5.4)
3.39 [0.91,12.7]
0.070
Illegal opioid- smoked
66 (25.1)
44 (33.1)
22 (16.9)
9.97 [4.66,21.3]
< 0.001*
Illegal opioid- injected
85 (32.3)
62 (46.6)
23 (17.7)
15.1 [6.61,34.3]
< 0.001*
Overdosed in public (n = 337)
102 (30.3)
40 (24.1)
62 (36.3)
0.62 [0.39,0.99]
0.043*
Overdose intentional (n = 340)
28 (8.2)
6 (3.6)
22 (12.8)
0.30 [0.13,0.65]
0.002*
Glasgow Coma Scale Score (n = 291)a
Severe (3–8)
210 (72.2)
127 (88.2)
83 (56.5)
5.60 [2.76,11.3]
< 0.001*
Moderate (9–13)
27 (9.3)
6 (4.2)
21 (14.3)
1.11 [0.37,3.30]
0.85
Mild (14-15)
54 (18.6)
11 (7.6)
43 (29.3)
ref
EMS & ED care
Admitted to hospital
56 (16.4)
15 (8.9)
41 (23.6)
0.35 [0.19,0.65]
0.001*
Left against medical advice‡
34 (9.9)
14 (8.3)
20 (11.5)
0.72 [0.33,1.57]
0.40
Left without treatment/before treatment complete‡‡
29 (8.5)
6 (3.6)
23 (13.2)
0.19 [0.07,0.53]
0.002*
Time day at ED presentationb
0:01–8:00
94 (27.5)
47 (28.0)
47 (27.0)
ref
8:01–16:00
108 (31.6)
49 (29.2)
59 (33.9)
0.92 [0.54,1.59]
0.77
16:01–24:00
140 (40.9)
72 (42.9)
68 (39.1)
1.10 [0.65,1.84]
0.73
Time of day at dischargec
0:01–8:00
121 (35.4)
58 (34.5)
63 (36.2)
ref
8:01–16:00
94 (27.5)
53 (31.5)
41 (23.6)
1.43 [0.82,2.50]
0.21
16:01–24:00
127 (37.1)
57 (33.9)
70 (40.2)
0.94 [0.55,1.59]
0.82
ED visit during weekend
102 (29.8)
49 (29.2)
53 (30.5)
0.99 [0.61,1.59]
0.96
Length of ED stay (median, IQR)
5.5 (3.3,9.2)
4.52 (2.7,7.6)
6.45 (3.9,11.0)
0.98 [0.95,1.01]
0.20
*** N = 342 for all independent variables unless stated otherwise Table 1 Characteristics of ED visits for opioid overdose, and associations with offering of take home naloxone kits
Visit Characteristic (n = 342)***
Number of ED visits (%)
Unadjusted
OR [95% CI]**
P-value
Total (n = 342)
Offered THN (n = 168)
Not offered THN (n = 174) Page 7 of 12 O’Brien et al. O’Brien et al. BMC Health Services Research (2019) 19:632 Analysis BMC Health Services Research (2019) 19:632 O’Brien et al. BMC Health Services Research (2019) 19:632 left the ED without treatment or before treatme
completion (AOR = 0.16 [0.05,0.48]), if they were a
mitted to hospital (AOR = 0.46 [0.22,0.97]) or if th
had an opioid prescription at the time of their E
visit (AOR = 0.41 [0.19,0.88]). The results of the multivariate analysis conducted wit
out multiple imputation are similar to those generat
with imputations (Table 3 in Appendix ). In particular, t
same variables were statistically significant in both mode
with the one exception being that male sex was found
be significantly associated with being offered THN in t
non-imputed multivariate model (Table 3 in Appendix). ** Odds ratio of being offered take home naloxone for each independent v
* Statistically significant at the level of 0.1 and thus eligible for inclusion in
a- Overall P-value < 0.001
b- Overall P-value =0.77
c- Overall P-value = 0.26
† Postal code overlaps Local Geographical Area of Edmonton Eastwood, inc
†† Postal code was charted as “no fixed address,” indicating that the patien
¥ Anxiety disorder, Bipolar disorder, Major Depressive Disorder, Psychosis, P
§ Methadone (liquid form, once daily ingestion, max period of 7 days), or Su
Does not include prescriptions for opioid agonist therapy
¶ Oral includes oral (97%), rectal (1%), transdermal (1%), and Percutaneous
‡ disclosed to the providers that they intended to leave and signed an AMA
‡‡ registered with triage and was assessed, but then left without warning o
Table 2 Multivariable associations of patient characteristics w
overdose, with multiple imputation
Visit Characteristic (n = 342)
Male sex
Age
Mental Health disorder
Opioid agonist therapy
Any current opioid prescription at time of ED visit
Primary opioid intoxicant illegal†
Pharmaceutical opioid- oral
Pharmaceutical opioid- smoked
Pharmaceutical opioid- injected
Illegal opioid- oral
Illegal opioid- smoked
Illegal opioid- injected
Overdosed in public
Overdosed intentionally
Pre-hospital GCS††
Severe (3–8)
Moderate (9–13)
Mild (14, 15)
Left without treatment/ before treatment completion
Admitted to hospital
†Overall p-value = 0.009 left the ED without treatment
completion (AOR = 0.16 [0.05,0
mitted to hospital (AOR = 0.46
had an opioid prescription at
visit (AOR = 0.41 [0.19,0.88]). Discussion left the ED without treatment or before treatment
completion (AOR = 0.16 [0.05,0.48]), if they were ad-
mitted to hospital (AOR = 0.46 [0.22,0.97]) or if they
had an opioid prescription at the time of their ED
visit (AOR = 0.41 [0.19,0.88]). In this evaluation of a recently implemented ED-based
THN program, THN was offered to patients in ap-
proximately half of ED visits for opioid overdose. We
sought to determine whether a recently implemented
ED-based THN program was missing certain patients
by identifying patient characteristics and other situ-
ational variables associated with being offered THN. We found that patients were more likely to be offered
THN if they experienced a severe overdose (GCS of
3–8), or had consumed an illegal opioid. In contrast,
patients who had an active opioid prescription at the The results of the multivariate analysis conducted with-
out multiple imputation are similar to those generated
with imputations (Table 3 in Appendix ). In particular, the
same variables were statistically significant in both models,
with the one exception being that male sex was found to
be significantly associated with being offered THN in the
non-imputed multivariate model (Table 3 in Appendix). Table 2 Multivariable associations of patient characteristics with offering of take home naloxone during ED visits for opioid
overdose, with multiple imputation
Visit Characteristic (n = 342)
Adjusted OR [95% CI]**
P-value
Male sex
1.75 [0.97,3.16]
0.064
Age
1.00 [0.98,1.02]
0.92
Mental Health disorder
1.12 [0.59,2.12]
0.74
Opioid agonist therapy
0.37 [0.04,3.35]
0.37
Any current opioid prescription at time of ED visit
0.41 [0.19,0.88]
0.021*
Primary opioid intoxicant illegal†
Pharmaceutical opioid- oral
ref
Pharmaceutical opioid- smoked
1.32 [0.24,7.27]
0.75
Pharmaceutical opioid- injected
1.40 [0.36,5.51]
0.63
Illegal opioid- oral
1.39 [0.33,5.88]
0.66
Illegal opioid- smoked
3.78 [1.32,10.9]
0.014*
Illegal opioid- injected
6.05 [2.15,17.0]
0.001*
Overdosed in public
0.61 [0.33,1.11]
0.11
Overdosed intentionally
0.59 [0.21,1.62]
0.30
Pre-hospital GCS††
Severe (3–8)
3.70 [1.63,8.37]
0.002*
Moderate (9–13)
2.09 [0.61,7.21]
0.24
Mild (14, 15)
ref
Left without treatment/ before treatment completion
0.16 [0.05,0.48]
0.001*
Admitted to hospital
0.46 [0.22,0.97]
0.040*
†Overall p-value = 0.009
††Overall p-value = 0.008
*Statistically significant at the level of 0.05
**Odds ratio of being offered take home naloxone for each independent variable, adjusted for all other variables in the model ns of patient characteristics with offering of take home naloxone during ED visits for opioid Admitted to hospital Similarly, only about 13% of patients with chronic
non-cancer pain report that they would be offended if
offered THN [41]. Our results showed that even among patients who
have recently overdosed, certain patients were more
likely to be offered than others. It is likely that in the
context of a busy ED, a clinician’s decision to offer
THN may be driven by their perceptions of which pa-
tients are most at risk for overdose death. For instance,
ED staff may more readily offer THN to patients who
have experienced a severe overdose because they are
easily recognized as being at risk for future overdose
death. Similarly, clinicians may more readily recognize
people who smoke or inject illegal drugs as being more
vulnerable for future overdose. This tendency is likely
shaped by both clinical experience and media reports,
as it is true that in the Western Canadian provinces of
Alberta and British Columbia, over 80% of accidental
opioid overdose deaths involved illegally manufactured
fentanyl in 2017 [26, 35]. Additionally, clinicians may
believe that THN is predominately meant for people
who use illegal drugs, given that THN was originally
developed to serve people who inject heroin [36]. Indeed, the initial rationale for THN programs was in-
part to empower people who use heroin who were
reluctant to call an ambulance in cases of overdose for
fear of criminal prosecution [37]. Other primary care providers have expressed reluc-
tance to co-prescribe THN with opioids because they
felt that THN should not be necessary if opioids are
prescribed properly [38]. However, offering THN to
patients prescribed opioids can facilitate important
conversations about overdose risk, and may even
change behavior in ways that reduce the risk of fu-
ture overdose and ED visits [40, 42]. For instance, at
least some patients who received THN and overdose
education through primary care report safer dosing,
safer timing, and increased knowledge of opioids and
overdose [40]. Additionally, a non-randomized trial
of THN co-prescriptions in primary care found that
patients who received THN had 63% less ED-related
visits at 12-months compared to those that did not
receive THN [42]. It was not surprising that patients who unexpectedly
left the ED without treatment or before treatment
completion were less likely to be offered THN. At the
time of this study, ED staff typically waited until dis-
charge to offer THN to patients. Admitted to hospital Statistically significant at the level of 0.05
**Odds ratio of being offered take home naloxone for each independent variable, adjusted for all other variables in the model Page 8 of 12 Page 8 of 12 O’Brien et al. BMC Health Services Research (2019) 19:632 time of their ED visit, who left the ED without treat-
ment of before treatment completion, or who were
admitted to the hospital were less likely to be offered
THN. found that almost all ED staff who were interviewed
agreed certain patients should receive THN—such as
those who are have overdosed in the past, are opioid
dependent, or who inject opioids [23, 24]. However,
other staff disagreed on whether it was appropriate,
necessary, or realistic to offer THN to all patients
prescribed opioids [23, 24]. In some cases, this ap-
peared be due to their perceived lower risk of over-
dose [23, 24]. The proportion of patients offered THN (49%) is
significantly higher than in previous studies which
have
evaluated
the
implementation
of
ED-based
THN programs [23, 34]. This higher rate is likely
attributable to our decision to only include individ-
uals who were discharged with a diagnosis of opioid
overdose. This decision was made based on our de-
sire to examine whether the THN program was
reaching the highest risk patients. These patients are
likely to be readily identifiable by ED providers, and
thus more likely to be offered THN compared to
other patient groups at risk of overdose. In previous
qualitative studies about ED-based THN programs,
ED providers frequently acknowledged that it is im-
portant not to miss patients who have visited the ED
for an opioid overdose [23, 24]. Specific reasons as to why clinicians may be less
likely to provide THN to patients taking prescription
opioids have been explored in studies of primary care
patients with chronic pain [38, 39]. For instance, in
one qualitative study, primary care providers believed
that co-prescribing THN may offend patients due to
the stigma associated with substance use disorders and
THN [38]. Previous research from primary care has
generally refuted this perception. Indeed, the majority
of patients on long-term opioid prescriptions give ei-
ther positive or neutral reactions to being offered a
naloxone prescription, report wanting naloxone pre-
scriptions in the future, and agree that THN should be
available to patients prescribed opioids for pain [40]. Admitted to hospital Therefore, patients
who left the ED without disclosing their intent to leave
may have been missed. Patients who have been treated
for opioid overdose may be experiencing symptoms of
withdrawal and be eager to leave the ED to address
these symptoms. Consequently, they may be less will-
ing to complete discharge paperwork or THN training
[23]. These patients may be especially vulnerable to a Our finding that patients who were taking pre-
scription opioids were less likely to be offered THN
is consistent with previous reports. In a survey of
Canadian ED physicians, it was found that while the
large
majority
of
participants
(> 90%)
agreed
or
strongly agreed that patients with a history of emer-
gency care for opioid overdose would benefit from
THN programs, fewer physicians (69%) agreed that
patients
prescribed
high
doses
of
opioids
would
benefit [19]. Similarly, previous qualitative studies Page 9 of 12 O’Brien et al. BMC Health Services Research (2019) 19:632 O’Brien et al. BMC Health Services Research (2019) 19:632 We attempted to account for the uncertainty created
by missing data with multiple imputation. We ob-
tained similar results from the multivariate analysis
with and without the use of multiple imputation
(Table 3 in Appendix). subsequent overdose immediately following their ED
visit, given the relatively short half-life of naloxone
[43], and the additional risk posed by consuming fur-
ther doses of opioids after leaving the ED. Equipping
these patients with THN (and offering opioid agonist
treatment and other supports) is especially critical. Finally, our findings showed that ED patients who
were admitted to the hospital were more likely to be
missed. Further efforts are needed to expand THN
distribution
to
at-risk
hospital
inpatients
in
this
setting. subsequent overdose immediately following their ED
visit, given the relatively short half-life of naloxone
[43], and the additional risk posed by consuming fur-
ther doses of opioids after leaving the ED. Equipping
these patients with THN (and offering opioid agonist
treatment and other supports) is especially critical. Finally, our findings showed that ED patients who
were admitted to the hospital were more likely to be
missed. Further efforts are needed to expand THN
distribution
to
at-risk
hospital
inpatients
in
this
setting. The data may not always be accurate for variables
that were partially or fully based on patient self-re-
port or clinician suspicion, such as the patient’s pri-
mary intoxicant. Admitted to hospital Patients may have been reticent to
disclose the complete details related to their sub-
stance use, or they may have consumed a different
substance from what they believed. However, given
that the aim of the study was to examine clinician
behavior, measuring the clinicians’ perception of the
patients’ opioid intoxicant is likely more useful than
the actual intoxicant. The majority (82%) of patients in our study who
were offered a THN kit either accepted it or already
possessed one, which confirms previous reports show-
ing that ED-based naloxone distribution is acceptable
to patients. This THN acceptance rate was slightly
higher than the 68% reported in a previous study by
Kestler et al., in which THN was offered to patients
who had reported illegal drug use, were prescribed a
high dose of prescription opioids, were receiving opi-
oid agonist therapy, or had any clinical presentation
suggestive of opioid use [20]. The higher acceptance
rate among our population may suggest that people
who have recently experienced an opioid overdose are
more accepting of THN than other THN-eligible pa-
tients. In a minority of cases, the patient’s reason for
declining a THN kit had been recorded. The reasons
included feeling that they were no longer at risk,
intending to stop using drugs, and believing that THN
is not useful if opioids are used alone. Several of these
reasons are consistent with a previous evaluation of
ED patient acceptance of THN, which found that the
patient beliefs of being “not at risk” and “done with
drugs” were the two most common rationales given
for declining THN [44]. Other variables that were not collected may have
also impacted a clinician’s decision to offer THN. For instance, we were unable to obtain reliable data
on whether patients had been prescribed or had con-
sumed any benzodiazepines. It is possible that ED
providers were more likely to offer THN to patients
who used opioids in combination with benzodiaze-
pines, given that this combination is a strong risk
factor for overdose among people receiving opioid
analgesics [45, 46]. The data abstracters were not blinded to the pur-
pose of the study. However, it is unlikely this affected
the results significantly because there were no specific
a priori hypotheses regarding which variables would
be associated with THN being offered. Admitted to hospital Additionally,
because our sample was limited to a single hospital
site, it is possible that our results may not generalize
to other geographic locations. While we have reported on reasons that patients gave
for declining THN, these reasons were only recorded for
a minority of patients. Consequently, it was not possible
to draw conclusions based on this data. Finally, this
study only captured a small subpopulation of individuals
visiting the ED who are potentially at risk for overdose. Future research is needed to evaluate whether ED pro-
viders are able to reach other patient groups at risk for
overdose. Limitations There are several limitations to this study that are
inherent
to
retrospective
chart
reviews. Because
medical records are created for clinical purposes,
some information may not be charted consistently. In particular, we were unable to ensure that every
instance of THN being offered was documented. We
attempted
to
ameliorate
this
issue
by
reviewing
medication dispensation data to confirm cases in
which THN was both offered and accepted by the
patient. However, for some cases in which THN was
offered but declined by the patient, ED providers
may not have charted that they offered THN. There-
fore, the proportion of ED visits in which THN was
offered may have been underestimated. Appendix to identify high risk patients, ED providers may con-
sciously or unconsciously rely on their own percep-
tions as to who is at risk and overlook certain patients,
such as those who use prescription opioids. To ensure
optimal implementation of ED-based THN programs,
ED staff should be provided with information on the
importance of offering THN widely, and clear guide-
lines regarding which patients should receive THN. Further, administrators or clinicians implementing
ED-based THN programs should establish a reliable
inpatient pathway to provide THN for eligible patients
prior to discharge. As we have shown, patients admit-
ted to the hospital after receiving acute care for an
opioid overdose can be missed by ED-based THN
programs. Another way for ED-based THN programs to im-
prove their coverage to at-risk patients is to avoid
waiting until discharge to offer THN. As discussed,
our evaluation of an ED-based THN program showed
that patients who left the ED unexpectedly were more
likely to be missed by providers. Where possible, THN
should be offered earlier in the patient’s ED visit, to
ensure they are not missed should they leave the ED
unexpectedly. p
y
At minimum, all patients presenting to an ED with
an
opioid
overdose
should
be
offered
THN. However,
several
other
groups
would
likely
also
benefit from THN distribution and training in an ED
setting. In particular, the Center for Disease Control
and Prevention recommends that THN be offered to
patients taking higher doses of prescription opioids
(≥50 OME/day), patients with concurrent opioid and
benzodiazepine use, and patients with a history of a
substance use disorder [47]. We suggest that in
addition to targeting people who use opioids, ED
providers should consider offering THN to people
who use any illegal substance, including stimulants. People who use non-opioid illegal substances may be
exposed to opioids through contamination [48], or
may witness an opioid overdose among their peers. Future evaluations are needed to examine the extent
to which these different patients can be targeted,
and to develop systematic protocols to identify vari-
ous individuals at risk of overdose. One such inter-
vention
that
holds
promise
is
electronic
health
record prompts, which can be triggered by terms en-
tered into the patient’s initial assessment [49, 50]. Such
interventions
have
been
previously
demon-
strated to increase the distribution of THN to ED
patients discharged after opioid overdoses [49, 50]. Authors’ contributions EH and KD conceived and designed the study, and obtained research
funding. DD designed the data abstraction protocol in consultation with EH
and KD. DD abstracted the data. DO planned and conducted the data
analysis in consultation with PV, KD and EH. DO drafted the manuscript, and
all authors contributed substantially to its revision. EH takes responsibility for
the paper as a whole. All authors read and approved the final manuscript. Acknowledgements Not applicable. Appendix As evidenced by our study, without such systematic
approaches, attaining complete coverage of even the
highest risk patients is difficult when implementing
ED-based THN programs. Abbreviations Abbreviations
AMA: Against Medical Advice; AOR: Adjusted Odds Ratio; ARCH: Addiction
Recovery and Community Health; ED: Emergency Department;
EMS: Emergency Medical Service; GCS: Glasgow Coma Scale;
GEE: Generalized Estimating Equation; OR: Odds Ratio; PIN: Pharmaceutical
Information Network; THN: Take Home Naloxone Conclusions This study is the first to identify patient characteristics
associated with being offered THN among a high risk
population of ED patients. Our findings add to the
current literature by demonstrating that implementa-
tion of an ED-based THN program and accompanying
staff training does not necessarily guarantee that even
patients presenting with opioid overdose will be of-
fered THN. In the absence of a standardized protocol Other variables were missing values for a signifi-
cant percentage of ED visits, including pre-hospital
GCS (14.9%), and primary opioid intoxicant (23.1%). Page 10 of 12 O’Brien et al. BMC Health Services Research (2019) 19:632 O’Brien et al. BMC Health Services Research (2019) 19:632 Appendix
Table 3 Multivariable associations of patient characteristics with
offering of take home naloxone during ED visits for opioid
overdose, without multiple imputation
Visit Characteristic (n = 334)
Adjusted OR [95% CI]** P-value
Male sex
1.94 [1.10,3.41]
0.022*
Age (n = 341)
1.00 [0.98,1.02]
0.93
Mental Health disorder
1.15 [0.61,2.15]
0.67
Opioid agonist therapy
0.30 [0.04,2.44]
0.26
Any current opioid prescription at
time of ED visit
0.44 [0.21,0.92]
0.029*
Primary opioid intoxicant (n = 263)†
Pharmaceutical opioid- oral
ref
Pharmaceutical opioid- smoked
1.14 [0.25,5.26]
0.87
Pharmaceutical opioid-injected
1.43 [0.34,6.01]
0.63
Illegal opioid- oral
1.26 [0.33,4.83]
0.74
Illegal opioid- smoked
3.65 [1.35,9.88]
0.011*
Illegal opioid- injected
5.47 [2.07,14.5]
0.001*
Missing
1.61 [0.63,4.08]
0.32
Overdosed in public (n = 337)
0.64 [0.35,1.15]
0.13
Overdosed intentionally (n = 340)
0.47 [0.17,1.31]
0.15
Pre-hospital GCS (n = 291)††
Severe (3–8)
3.57 [1.62,7.83]
0.002*
Moderate (9–13)
1.39 [0.39,4.98]
0.62
Mild (14, 15)
Ref
Missing
3.64 [1.40,9.42]
0.008*
Left without treatment/ before
treatment completion
0.18 [0.06,0.57]
0.003*
Admitted to hospital
0.40 [0.19,0.83]
0.014*
†Overall P-value 0.003
††Overall p-value 0.006
*Statistically significant at the level of 0.05
**Odds ratio of being offered take home naloxone for each independent
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& Grants & studentship funding:
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Canadian Research Initiative on Substance Misuse – Prairie Node
KD receives a stipend from Alberta Health Services as director of the Inner
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naloxone programme for reducing opioid-related deaths: a before (2006-10)
versus after (2011-13) comparison. Addiction. 2016;111(5):883–91. The above funding bodies played no role in any of the following: The above funding bodies played no role in any of the following: The design of the study The design of the study 12. Verdier M, Routsolias JC, Aks SE. Naloxone prescriptions from the
emergency department: an initiative in evolution. Am J Emerg Med. 2019;37(1):164–5. The collection, analysis, or interpretation of data The writing the manuscript. The writing the manuscript. 13. Brady JE, DiMaggio CJ, Keyes KM, Doyle JJ, Richardson LD, Li G. Emergency
department utilization and subsequent prescription drug overdose death. Ann Epidemiol. 2015;25(8):619.e2. Availability of data and materials The datasets analyzed during the current study are available from the
corresponding author on reasonable request. 14. Caudarella A, Dong H, Milloy MJ, Kerr T, Wood E, Hayashi K. Non-fatal
overdose as a risk factor for subsequent fatal overdose among people who
inject drugs. Drug Alcohol Depend. 2016;162:51–5. 14. Caudarella A, Dong H, Milloy MJ, Kerr T, Wood E, Hayashi K. Non-fatal
overdose as a risk factor for subsequent fatal overdose among people who
inject drugs. Drug Alcohol Depend. 2016;162:51–5. Received: 11 February 2019 Accepted: 27 August 2019 22. Pedigo JR, Seifert CF. Rate of patients at elevated risk of opioid overdose
visiting the emergency department. Am J Emerg Med. 2018;36(12):2161–5. 23. Drainoni M, Koppelman EA, Feldman JA, Walley AY, Mitchell PM, Ellison J, et
al. Why is it so hard to implement change? A qualitative examination of
barriers and facilitators to distribution of naloxone for overdose prevention
in a safety net environment. BMC Research Notes. 2016;9:465. Consent for publication
Not applicable. 17. Samuels E. Emergency department naloxone distribution: a Rhode Island
department of health, recovery community, and emergency department
partnership to reduce opioid overdose deaths. R I Med J. 2014;97(10):38. Competing interests 18. Samuels EA, Dwyer K, Mello MJ, Baird J, Kellogg AR, Bernstein E, et al. Emergency department-based opioid harm reduction: moving physicians
from willing to doing. Acad Emerg Med. 2016;23(4):455–65. 18. Samuels EA, Dwyer K, Mello MJ, Baird J, Kellogg AR, Bernstein E, et al. Emergency department-based opioid harm reduction: moving physicians
from willing to doing. Acad Emerg Med. 2016;23(4):455–65. The authors declare that they have no competing interests. Author details
1 19. Lacroix L, Thurgur L, Orkin AM, Perry JJ, Stiell IG. Emergency physicians’
attitudes and perceived barriers to the implementation of take-home naloxone
programs in Canadian emergency departments. CJEM. 2018;20(1):46–52. 1University of Alberta, School of Public Health, 3-300 Edmonton Clinic Health
Academy, 11405 – 87 Ave, Edmonton, Alberta T6G 1C9, Canada. 2University
of Alberta, Faculty of Medicine and Dentistry, 2J2.00 WC Mackenzie Health
Sciences Centre, 8440 112 St. NW, Edmonton, Alberta T6G 2R7, Canada. 3University of Alberta, Faculty of Medicine and Dentistry, 790 University
Terrace Building, 8303 112 St. NW, Edmonton, Alberta T6G 2T4, Canada. of Alberta, Faculty of Medicine and Dentistry, 2J2.00 WC Mackenzie Health
Sciences Centre, 8440 112 St. NW, Edmonton, Alberta T6G 2R7, Canada. 3University of Alberta, Faculty of Medicine and Dentistry, 790 University
Terrace Building, 8303 112 St. NW, Edmonton, Alberta T6G 2T4, Canada. 20. Kestler A, Buxton J, Meckling G, Giesler A, Lee M, Fuller K, et al. Factors
associated with participation in an emergency department–based take-home
naloxone program for at-risk opioid users. Ann Emerg Med. 2017;69(3):340. 21. Ellison J, Walley AY, Feldman JA, Bernstein E, Mitchell PM, Koppelman EA, et
al. Identifying patients for overdose prevention with ICD-9 classification in
the emergency department, Massachusetts, 2013-2014. Public Health Rep. 2016;131(5):671–5. 4University of Alberta, School of Public Health, 33-50 University Terrace, 8303
- 112 Ave, Edmonton, Alberta T6G 2T4, Canada. 4University of Alberta, School of Public Health, 33-50 University Terrace, 8303
- 112 Ave, Edmonton, Alberta T6G 2T4, Canada. Received: 11 February 2019 Accepted: 27 August 2019 Ethics approval and consent to participate 15. Wagner KD, Liu L, Davidson PJ, Cuevas-Mota J, Armenta RF, Garfein RS. Association between non-fatal opioid overdose and encounters with
healthcare and criminal justice systems: identifying opportunities for
intervention. Drug Alcohol Depend. 2015;153:215–20. Our research protocol received ethics approval from the University of
Alberta’s Health Research Ethics Board. The Research Ethics Board agreed
that it was not necessary or feasible to obtain individual consent because
our study used secondary use of extracted and anonymized administrative
health data. Therefore, there was no contact with human subjects. 16. Dwyer K, Walley AY, Langlois BK, Mitchell PM, Nelson KP, Cromwell J, et al. Opioid education and nasal naloxone rescue kits in the emergency
department. West J Emerg Med. 2015;16(3):381–4. References 2016;6(3):34–41. 6. Mueller SR, Walley AY, Calcaterra SL, Glanz JM, Binswanger IA. A review of
opioid overdose prevention and naloxone prescribing: implications for
translating community programming into clinical practice. Subst Abus. 2015;36(2):240–53. 6. Mueller SR, Walley AY, Calcaterra SL, Glanz JM, Binswanger IA. A review of
opioid overdose prevention and naloxone prescribing: implications for
translating community programming into clinical practice. Subst Abus. 2015;36(2):240–53. 28. McHugh ML. Interrater reliability: the kappa statistic. Biochemia medica. 2012;22(3):276–82. 29. Briggs D, Rhodes T, Marks D, Kimber J, Holloway G, Jones S. Injecting drug
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al. Naloxone use among overdose prevention trainees in new York City: a
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Available from: https://www.stata.com/manuals13/mi.pdf. 34. Barbour K, McQuade M, Somasundaram S, Chakravarthy B. Emergency
physician resistance to a take-home naloxone program led by community
harm reductionists. Am J Emerg Med. 2018;36(11):2110–2. 35. British Columbia Coroners Service. References CDC guideline for prescribing opioids for
chronic pain - United States, 2016. MMWR Recomm Rep. 2016;65(1):1–49. 48. Tupper KW, McCrae K, Garber I, Lysyshyn M, Wood E. Initial results of a drug
checking pilot program to detect fentanyl adulteration in a Canadian
setting. Drug Alcohol Depend. 2018;190:242–5. 48. Tupper KW, McCrae K, Garber I, Lysyshyn M, Wood E. Initial results of a drug
checking pilot program to detect fentanyl adulteration in a Canadian
setting. Drug Alcohol Depend. 2018;190:242–5. 49. Devries J, Rafie S, Ajayi TA, Kreshak A, Edmonds KP. Results of a naloxone
screening quality-improvement project in an academic emergency
department. J Emerg Med. 2019;56(4):378–85. 49. Devries J, Rafie S, Ajayi TA, Kreshak A, Edmonds KP. Results of a naloxone
screening quality-improvement project in an academic emergency
department. J Emerg Med. 2019;56(4):378–85. 50. Marino R, Landau A, Lynch M, Callaway C, Suffoletto B. Do electronic health
record prompts increase take-home naloxone administration for emergency
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death-marriage-and-divorce/deaths/coroners-service/statistical/illicit-drug.pdf 36. McDonald R, Campbell ND, Strang J. Twenty years of take-home naloxone
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prescribing in primary care settings: a systematic review. Prev Med. 2018;114:79–87. 40. Behar E, Rowe C, Santos GM, Murphy S, Coffin PO. Primary care patient
experience with naloxone prescription. Ann Fam Med. 2016;14(5):431–6. 41. Nielsen S, Peacock A, Lintzeris N, Bruno R, Larance B, Degenhardt L. Knowledge of opioid overdose and attitudes to supply of take-home
naloxone among people with chronic noncancer pain prescribed opioids. Pain Med. 2018;19(3):533–40. 42. Coffin PO, Behar E, Rowe C, Santos G, Coffa D, Bald M, et al. Nonrandomized intervention study of naloxone coprescription for primary
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evidence and clinical implications. Ther Adv Drug Saf. 2018;9(1):63–88. 44. Kestler A, Giesler A, Buxton J, Meckling G, Lee M, Hunte G, et al. Yes, not
now, or never: an analysis of reasons for refusing or accepting emergency
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prescribing patterns and deaths from drug overdose among US veterans
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chronic pain - United States, 2016. MMWR Recomm Rep. 2016;65(1):1–49. 47. Dowell D, Haegerich TM, Chou R. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
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Didaktisches zur Aberration
|
Astronomische Nachrichten
| 1,920
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public-domain
| 3,023
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Didaktisches zur Aberration. Von F. Hayn. I
I1
111
x’/u = c cos a. cos a0 + 2 or4 7 sin Q
- or3 2 cosy sin e
y‘/v = csinorocosdO -20.47
COSQCOSE +0.3zcos~cost1
z’/v = c sin So
-20.47 cosOsins. I
I1
111
x’/u = c cos a. cos a0 + 2 or4 7 sin Q
- or3 2 cosy sin e
y‘/v = csinorocosdO -20.47
COSQCOSE +0.3zcos~cost1
z’/v = c sin So
-20.47 cosOsins. Das Folgende mag vielleicht vielen als langst bekanntes
oder selbstverstandliches erscheinen; dafi ich es trotzdem dep
Lesern der A. N. unterbreite, dam haben mich Mifiverstand-
nisse veranlaflt, die ihren Grund in der Behandlungsweise
zu haben scheinen, die das Problem der Aberration in ver-
schiedenen Lehrbuchern der Astronomie und Physik und auch
anderswo erfahren hat und noch erfahrt. Dabei Ieitete mich
die Ansicht, dafl vieles dem Manne der Praxis als selbst-
verstandlich erscheint, dem Lernenden jedoch nicht, und dafl
dieser beim Studium der Aberration in den verschiedenen
Bucliern und Schriften zu der Anslcht kommen kann, als
bote das Problem der Aberration gewisse Schwierigkeiten
und enthielte Widerspruche, die noch zu losen waren. Den absoluten Betrag des von I abhangigen Teils der
Aberration werden wir nie bestimmen konnen, da sich kein‘
im Raume festes System definieren lant. Man pflegt die von
I, 11, I11 abhangigen Teile der Aberration der Reihe nach
rnit sakularer, jahrlicher, taglicher Aberration zu bezeichnen. In jedem Zeitpunkte ist demnach gleich die sakulare Aberration fur alle Bewohner des Sonnensystems,
n jahrliche
))
>)
))
))
der Erde,
)) tagliche
))
)>
)>
))
eines gewissen Be-
zirkes auf der Erde. Ich mufl zur Vervollstandigung des Bildes meinen Be-
trachtungen in aller Kurze die Berechnungsweise der Aber-
ration vorausschicken. Die Aberration wird bedingt durch die relative Be-
wegung gegen den Ort, von dem das Licht ausging, aber
nicht durch die relative Bewegung Gestirn -Erde. Deshalb
mussen auch alle Sterne dieselbe Aberrationskonstante er-
geben. Wir werden aus dem Folgenden sehen, dafl wir
nirgends irgend eine Lichttheorie vorauszusetzen brauchen ;
das ganze Problem la& sich rein geometrisch behandeln. Zwei Erfahrungstatsachen miissen vorausgesetzt werden : I . Das Licht pflanzt sich im leeren Raume geradlinig
rnit gleichformiger Geschwindigkeit (n) fort. 2. Die Fortpflanzung ist unabhangig von der Ge-
schwindigkeit der Lichtquelle, wenigstens wenn diese klein
ist gegenuber der Lichtgeschwindigkeit. Wir betrachten der
Einfachheit halber Be-
wegungsvorgange in einer
Ebene. ASTRONOMISCHE NACHRICHTEN.
Band 211.
Nr. 5042. Band 211. Didaktisches zur Aberration. Von F. Hayn. Gegeben in einein
ruhenden System 0 zwei
Punkte A und B, deren
Verbindungslinie rnit der
Nullrichtung den Winkel
0
y’ bildet. Geht vom
Punkte B Licht aus, so
~ trifft es in A ein nach
BA/v Sek. Die schein-
A
A‘
bare Richtung von AB
ist gleich der wahren y‘. Wird jetzt das System in gleichformige Bewegung in
der Richtung der Nullinie rnit der Geschwindigkeit (I ver-
setzt, dann trifft ein Lichtblitz voni Punkte B den Punkt A
zwar im Orte A’, aber der Strahl ist gewandert auf der sich
gleichzeitig parallel verschiebenden Geraden AB. In eineni
engen Rohre, das diese Gerade einhullt, wiirde man also
das Licht B erblicken, ganz gleich, ob das ganze System
ruht oder sich bewegt; d. h. die scheinbare Richtung im
ruhenden und bewegten System ist dieselbe. AbsoIut im
Raume hat das Licht, das in das Auge des Beobachters A
gelangt, den Weg BA’ zuruckgelegt, relativ aber, d. h. fur
den Beobachter, der an der Bewegung des Systems teilnimmt,
den Weg BA. 0
A
A‘ 0
A
A‘ Angenommen werde ein rechtwinkliges, im Raume
festes Koordinatensystem, das nach dem Aquator orientiert
ist. Die Koordinaten des Punktes, von dem das Licht aus-
ging, seien X,
1; 2, die des Beobachters x, y, 3. Sind nun
die Koordinaten der Sonne xo, yo, zo, die der Erde gegen
die Sonne xl, yl, zL und die des Beobachters gegen den
Erdmittelpunkt xz, y2, z2, dann ist x = xo+x,+x*
y =yo+y1+yz
z = zo+z,+z,
und die 3 Geschwindigkeitskomponenten desBeobachtungsortes
x’ = x’o +x’, +x’,
y’ = y’o +y’, +y’2
2’ = z’o +z’, +z’* . x = xo+x,+x*
y =yo+y1+yz
z = zo+z,+z, x = xo+x,+x*
y =yo+y1+yz
z = zo+z,+z,
und die 3 Geschwindigkeitskomponenten desBeobachtungsortes und die 3 Geschwindigkeitskomponenten desBeobachtungsortes x’ = x’o +x’, +x’,
y’ = y’o +y’, +y’2
2’ = z’o +z’, +z’* . Der Rurze halber werde die Fortpflanzungsrichtung des
Lichtes mit ))Strahl(( bezeichnet. Der vom Punkte (X’YZ)
ausgehende Strahl trifft den Beobachter im Punkte (xyz) ;
die 3 Richtungskosinus dieses absoluten Strahles (wahre
Richtung) seien cos S cos a
cos 6 sin a
sin 6 die Richtungskosinus des relativen Strahles (scheinbare Rich-
tung)
cos d’ cos a’
cos 6’ sin a’
sin d’ . die Richtungskosinus des relativen Strahles (scheinbare Rich-
tung)
cos d’ cos a’
cos 6’ sin a’
sin d’ . Didaktisches zur Aberration. Von F. Hayn. 2 . Beziehen sie sich auf den Erdmittelpunkt, dann ist
die Richtung frei von sakularer und jahrlicher Aberration. 3. Alle auf der Erdoberflache beobachteten Richtungen
sind frei von jeder Aberration. Diese letzte, bekannte und naturgeniatl als selbstver-
standlich angesehene Tatsache sagt . aber nichts anderes aus
als der bekannte Interferenzversuch von Michelson, daD nam-
lich, wie oben ausgesprochen, die Lichtvorgange zwischen
z Punkten auf der Erde fur den mitbewegten Beobachter
sich genau so abspielen, als waren die Punkte in Ruhe. Aus
den Uberlegungen, die Michelson. zu seinem beruhmten Ver-
such veranlanten, mufite man ganz ebenso folgern, daO irdische
Azimute alle Arten von Aberration zeigen mufiten. Die
obigen Betrachtungen aber beweisen, daf3 das ein arger Fehl-
schluD ware; damit sind auch die Jfichelsanschen Voraus-
setzungen wideriegt. Man sieht aus diesem Beispiele, daO
Selbstverstandlichkeit ein sehr relativer Begriff ist. Von zwei
Folgerungen, die auf genau denselben Pramissen aufgebaut
sind, wird die eine ohne weiteres als unrichtig 'nicht in den
Kreis der Erwagung gezogen, wahrend die andere groOes
Aufsehen erregt hat und AnlaD zu allerlei Hypothesen ge-
wesen ist. Diese Umrechnung der Zeit oder der Richtung ist
gleichbedeutend mit einer 'Aberration, hervorgerufen durch
die relative Bewegung der Erde gegen ein System, das sich
auf den lichtaussendenden Ort bezieht. Daher stamint wohl
auch der Name Planetenaberration. Wenn geozentrische
Koordinaten fur die Zeit t2 gegeben sind, wie es bei Sonne
und Mond iiblich ist, dann ist nur die tagliche Aberration
zu berucksichtigen. Der Fall der Mondephemeride wird
ubrigens in keinem Buche erwahnt. Mein Bestreben ging dahin, in dieser kurzen Darlegung
zu zeigen, rnit wie einfachen Uberlegungen man auf alle
Fragen des Aberrationsproblems bestimmte Antworten geben
kann, ohne 211 irgend welchen Theorien und ohne zum
schwersten Rustzeug moderner Wissenschaft greifen zu mussen. Ich habe in der Einleitung dieses Aufsatzes von MiDver-
standnissen gesprochen, die durch die Behandlungsweise des
vorliegenden Problems herbeigefuhrt werden konnten. Zur
Rechtfertigung meiner Ansicht sei noch folgendes angefuhrt. Aus dem obigen Beispiele sehen wir: Licht, das im
bewegten System vom Punkte R ausgeht, verhalt sich geradeso,
als kame es von einem Fixsterne, dessen wahre Richtung y
gefunden wird aus y ' - y = -(.I.)
s h y ; d. h. es besteht
kein Unterschied zwischen diesen beiden relativen Strahlen. Didaktisches zur Aberration. Von F. Hayn. Dann ist nach bekannten Formeln, wenn man sich auf die
Hauptglieder beschrankt, Dann ist nach bekannten Formeln, wenn man sich auf die
Hauptglieder beschrankt, a’-a = (~/n)[y’cosa-x’sina] secS
8-8 = -(~/v)[.r’cosasinS+y’sinas a’-a = (~/n)[y’cosa-x’sina] secS
8 8 a’-a = (~/n)[y’cosa-x’sina] secS
8-8 = -(~/v)[.r’cosasinS+y’sinasin~-z’cosd]. 8-8 = -(~/v)[.r’cosasinS+y’sinasin~-z’cosd]. Wir musen vorlaufig annehmen,, die Sonne bewege
sich geradlinig und gleichformig nach eineni Punkte (ao do);
wenn wir uns auf die Hauptglieder beschranken, ist g
Man kann also den Satz aussprechen: D i e L i c h t -
v o r g a n g e z w i s c h e n 2 P u n k t e n e i n e s b e w e g t e n 2 5042 I 9 2 0 S y s t e m s spielen sich fur d e n m'itbewegten Beob-
a c h t e r genau so ab, als ware das System in Ruhe. Dieser Satz gilt auch fur den Fall, wenn das System sich
in gleichformiger Rotation befindet, vorausgesetzt, daO die
Bewegung der Punkte A und.R innerhalb der Zeit 2x1. als geradlinig betrachtet werden darf. S y s t e m s spielen sich fur d e n m'itbewegten Beob-
a c h t e r genau so ab, als ware das System in Ruhe. Dieser Satz gilt auch fur den Fall, wenn das System sich
in gleichformiger Rotation befindet, vorausgesetzt, daO die
Bewegung der Punkte A und.R innerhalb der Zeit 2x1. als geradlinig betrachtet werden darf. Zu den obigen Behauptungen I . und 2. ist noch einiges
hinzuzufugen. Das Licht geht aus vom (XI'Z) zur Zeit t,
und trifft zur Zeit tz in ( x y z ) . ein. Die Richtung dieses
Strahls war gegeben durch a und 6, die beobachtete schein-
bare Rlchtung durch a' und 8'. Bell den groOen Entfernungen
der Fixsterne interessiert die Zeit tl nicht; wir rechnen bei
Fixsternen nicht rnit Raumkoordinaten, sondern nur mit Rich-
tungen, die fur die Zeit t2 gelten. Didaktisches zur Aberration. Von F. Hayn. g
g
Den gefundenen Satz konnen wir auch in der Form
aussprechen: Richtungen in einem b e w e g t e n System
s i n d f r e i v o n d e r A b e r r a t i o n , d i e d u r c h d i e B e -
we g u n g e r ze u g t wird. 1. Im Falle heliozentrischer Koordinaten von Licht-
quelle und Beobachtungsort unterscheiden sich die beiden
Richtungen um den Betrag : jahrl. ttagl. Aberration. Alle
Erscheinungen in diesem Bezugssystem sind von diesen
beiden Aberrationen beeinfluat. g
g
g
Hieraus ergeben sich weiter folgende Tatsachen : I . Beziehen sich die Koordinaten von Lichtquelle und
Beobachter auf die Sonne, so ist die Richtung des Strahles
frei von der sakularen Aberration. I . Beziehen sich die Koordinaten von Lichtquelle und
Beobachter auf die Sonne, so ist die Richtung des Strahles
frei von der sakularen Aberration. 2 . Geozentrische Koordinaten .sind gegeben. Die Ephe-
meride gibt die wahre Richtung von der r u h e n d e n Erde
fur die Zeit tl. Sol1 die Reobachtung zur Zeit f2 mit der
Ephemeride verglichen werden, so ist t2 rnit der Lichtzeit
und derjenigen Entfernung, die fur die Zeit 1/z(tl+t2) gilt,
auf t l zu reduzieren. Oder man kann auch rnit den aus der
Ephemeride folgenden Geschwindigkeiten der geozentrischen
Koordinaten und der Zeit tz -tl
den Ephemeridenort auf
die Zeit tz bringen. Beides kommt auf dasselbe hinaus. Bei
Sonne und Mond ist i2 - tl nahezu konstant, dem entspricht
eine konstante Richtungsanderung, die in der Ephemeride
schon enthalten ist ; die Reduktion unterbleibt daher. 2 . Geozentrische Koordinaten .sind gegeben. Die Ephe-
meride gibt die wahre Richtung von der r u h e n d e n Erde
fur die Zeit tl. Sol1 die Reobachtung zur Zeit f2 mit der
Ephemeride verglichen werden, so ist t2 rnit der Lichtzeit
und derjenigen Entfernung, die fur die Zeit 1/z(tl+t2) gilt,
auf t l zu reduzieren. Oder man kann auch rnit den aus der
Ephemeride folgenden Geschwindigkeiten der geozentrischen
Koordinaten und der Zeit tz -tl
den Ephemeridenort auf
die Zeit tz bringen. Beides kommt auf dasselbe hinaus. Bei
Sonne und Mond ist i2 - tl nahezu konstant, dem entspricht
eine konstante Richtungsanderung, die in der Ephemeride
schon enthalten ist ; die Reduktion unterbleibt daher. Ioo(p-pFLO)/,uO
= -0!~04sina-0~14 cosa Ioo(p-pFLO)/,uO
= -0!~04sina-0~14 cosa Ioo(p-pFLO)/,uO
= -0!~04sina-0~14 cosa
wo mit a die vom Jahresanfang in BogenmaD gerechnete
Zeit, niit po der mittlere und mit p der momentane Wert
der Refraktionskonstante bezeichnet wird. wo mit a die vom Jahresanfang in BogenmaD gerechnete
Zeit, niit po der mittlere und mit p der momentane Wert
der Refraktionskonstante bezeichnet wird. Zwischen den beobachteten Zenitdistanzen und den zu
bestimmenden GroDen besteht. folgende Beziehung : Es schien wunschenswert eine ahnliche Untersuchung
fur Pulkovo auszufuhren, um festzustellen, ob wir es hier mit
einer lokalen Anomalie oder mit einer allgemeingultigen
Refraktionserscheinung zu tun haben. Zu diesem Zwecke
wurde eine Bearbeitung folgender groi3en Beobachtungsreihen
mit dem Pulkovoer Vertikalkreise vorgenommen : Die Beob-
achtungen der absoluten DekIinationen fur die Epoche 1845
von GyZa'Ln, die fur die Epochen 1865, 1885 und 1900 von
Nyrkn, sowie die fur 1905 von Kma'vawtzef. Alle Beob-
achtungen mit Z> 30' wurden mitaufgenommen. Von je z Be-
obachtungen eines Sternes wurde eine Gleichung zur gleich-
zeitigen Ableitung des Ausdehnungskoeffizienten der Luft,
sowie der als Sinussoide angenomnienen Jahresperiode der Re-
fraktion gebildet. Ich erhielt folgende Refraktionsperiode
(siehe Publ. d. 1'Obs. Centr. Nic. XXVII): g
g
AZ = ~ / l o o ~ z ~ (
A ~ K + A ( ~ ,
- ti).i+dz .p +Asinor. a+LIcosa.b)
wo
k = I O O ( ~ - ~ , ) / ( I + Y ' . ~ ~ Z ~ )
i = ioo.m-s und to = ti+s(t,-ti)
p = roox/8B wenn Bo = B-'/sxrr
~ o o ( p - p ~ ) / p ~
= -asina-bcosa. k = I O O ( ~ - ~ , ) / ( I + Y ' . ~ ~ Z wo p = roox/8B wenn Bo = B-'/sxrr ( p - p ~ ) / p ~
= -asina-bcosa. Mit a, z, q,
Y,, p und m werden bezeichnet: die in
Winkelma8 angegebene Zeit der Beobachtung, die Zenit-
distanz, die absolute Feuchtigkeit der Luft, die Refraktion
in Zenitdistanz z, die Refraktionskonstante und der Aus-
dehnungskoeffizient der Luft. Alle Sterne rnit 2130' wurden in die Untersuchung
mithineingezogen. ' Beobachtungen mit noch kleineren Zenit-
distanzen wurden nicht beriicksichtigt, weil bei diesen die
kleinen Refraktionsglieder von unbedeutenden systematischen
Fehlern anderen Ursprungs leicht iibertroffen werden und
dadurch eine Verzerru,ng der Resultate zu befiirchten ist. Didaktisches zur Aberration. Von F. Hayn. Wir wissen, daD ein Strahl von B aus seine Richtung bei-
behalt, gleichviel ob man ein
)) wassergefulltes Fernrohra in
den Strahlengang AB einschaltet oder nicht; demnach gilt
dasselbe auch fur den relativen Strahl vom Fixsterne. Beide
Strahlen folgen in gleicher Weise allen optischeh Gesetzen
uber Rrechung, Reflexion usw., die ja aus den auf der Erde
allein moglicheh Beobachtungen von relativen Strahlen ab-
geleitet worden sind. Nunmehr la& sich ohne weiteres an-
geben, bis zu welchem Betrage die beobachtete Aberration
von der Atmosphare der Erde abhangig sein kann. Man
braucht nur den Punkt B an die Grenze der Atmosphare zu
verlegen und fur diesen Punkt die Aberration zu berechnen. So interessant Beweise, die auf dem Umwege uber irgend
eine Lichttheorie dartun, daO die Aberration vom bewegten
Medium unabhangig ist, sein mogen, notwendig sind sie nicht. Alle unsere optischen Gesetze beziehen sich auf bewegte
Medien, an deren Bewegung der Beobachter teilninimt. Die Darstellungsweise, die zu der SchluDfolgerung ver-
leitete, die Aberration sei vom Fernrohrinnern abhangig, ist
noch nicht verschwunden. 1st doch in einer mir vorliegen-
den Vorlesung die Frage offen gelassen, warum die Aber-
ration vom Medium am Ende des Lichtwegs unabhangig sei. Es ist qn mehr als einer Stelle zu lesen, unsere Vorsteliungen
reichten noch nicht aus, um die Aberration vollig zu erklaren. Es ist verstandlich, wenn in speziellen Lehrbuchern nur so-
vie1 von der Aberration gegeben wird, wie zur regelrechten
Reduktion der in Frage komnienden Beobachtungen not-
wendig ist. Aber in Lehrbuchern der allgemeinen und spha-
rischen Astronoinie und in denen der Physik sucht man
doch eine erschop€ende Erklarung der Erscheinungen. So-
lange einzelne Punkte noch nicht vollig geklart sind oder
von anderer Seite unrichtig dargestellt werden, erscheint es 2 2 5042 5042 2 1 voraussetzte und deren Ausbleiben nicht erklart werden
konnte. mir didaktisch nicht richtig, stillschweigend solche Punkte
zu ubergehen ; eine Widerlegung ist dann notwendig. Uber
das Fehlen jeder Aberration bei terrestrischen Richtungen
habe ich nirgends eine Bemerkung gefunden, trotzdem es
in vollem Widerspruch steht zu Erscheinungen, die man Die Vorschriften zur Berucksichtigung der Aberration
werden durch meine Darlegungen naturgemat3 nicht beruhrt. .E Nayn. Leipzig, 1920 Jan. zy. Leipzig, 1920 Jan. zy. Die jahrlichen Refraktionsanderungen in Odessa und Pulkovo. 2. Beobachtungen mit dem Ertelschen Kreise im Me-
ridiansaale, 2. Beobachtungen mit dem Ertelschen Kreise im Me-
ridiansaale, Die in Odessa 1908-1910 mit dem Repsoldschen
Vertikalkreise ausgefuhrten Deklinationsbeobachtungen fuhren
zu einer ausgesprochenen Jahresperiode der Refraktionskon-
stante. Die empirisch ohne jegliche Voraussetzungen uber
den Ausdehnungskoeffizienten der Luft oder uber den ana-
lytischen Ausdruck
der Refraktionsanderungen erhaltene
Jahreskurve (siehe Publ. d. 1'Obs. Centr. Nic. XXIV) kann
durch folgende Sinussoide gut dargestellt werden : 3. Beobachtungen mit dem Ertelschen Kreise in der
Siidsternwarte. 3. Beobachtungen mit dem Ertelschen Kreise in der
Siidsternwarte. Aus den Beobachtuegen sollten gleichzeitig folgende
Grohen abgeleitet werden : die Korrektion des angewandten
Ausdehnungskoeffizienten der Luft (m,= 0.003689), die fur
die Refraktion mafigebende Lufttemperatur (to) als Funktion
der Temperatur ram Instrument (ti) und in freier T,uft (ta),
der Einflui3 der Feuchtigkeit auf die Refraktion, die Ko-
effizienten der als Sinussoide angenomrnenen Jahresperiode
der Refraktion. Ioo(p-pFLO)/,uO
= -0!~04sina-0~14 cosa ~ o o ( p - p ~ ) / p ~
= -or01 sinac-oYIocosa
f o . 0 2
-+0.*03 Bei der Bearbeitung der Deklinationsbeobachtungen in
Pulkovo 19 I 1-16 habe ich die Untersuchung der jahrlichen
Refraktionsaidderungen
wieder aufgenommen. Diese uber
I 3 ooo Beobachtungen von zum Teil tief kulminierenden
Sternen enthaltende Reihe ist zu derartigen Untersuchungen
besonders geeignet, weil mit zwei Instrumenten (dem Repsold-
schen und dem Ertelschen Vertikalkreis) und in zwei sehr
verschiedenen Beobachtungsraumen (Pulkovoer grofier Meri-
diansaal und der Westturm der Sudsternwarte) beobachtet
worden ist. I In jeder der 3 obengenannten Gruppen wurde aus den
Beobachtungen je eines Katalogsternes eine Gleichung ge-
bildet. Auf diese Weise. wurden 6 Beobachtungen von jedem
Sterne verwertet. In solchen Fallen, wo mehrere Beobach-
tungen von dem Sterne vorhanden waren, wurden die beiden
gewahlt, die in nioglichst verschiedenen Temperaturen beob-
achtet worden waren Von den tief kulminierenden Sternen,
die speziell fur Refraktionsuntersuchung beobachtet worden
sind, wurden alle Beobachtungen berucksichtigt. Die Gewichtseinheit entspricht dem mittleren Fehler
=t oY42 eines Unterschieds Az. Wir teilen die Untersuchung in drei Gruppen: I . Beobachtungen mit dem Repsoldschen Kreise im
Meridiansaale, Die Koeffizienten der Normalgleicbungen fur die Be-
stimmung voni R, i, p, a und b sind folgende:
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https://openalex.org/W3172840272
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https://www.researchsquare.com/article/rs-540987/latest.pdf
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English
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Sodium Glucose Co-Transporter 2 (SGLT2) Inhibitor Dapagliflozinstabilizes Diabetes-Induced Atherosclerotic Plaque Instability
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Research Square (Research Square)
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cc-by
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Methodology Sodium glucose co-transporter 2 (SGLT2) inhibitor dapagliflozin
stabilizes diabetes-induced atherosclerotic plaque instability
Yung Chih Chen1,2,3, Karin Jandeleit-Dahm3,4,5*, Karlheinz Peter1,2,3,6*
1Baker Heart & Diabetes Institute, Melbourne, Australia
2Central Clinical School, Monash University, Melbourne, Australia
3Department of Cardiomentabolic Health, University of Melbourne, Melbourne, Australia
4Department of Diabetes, Monash University, Melbourne, Australia
5German Diabetes Centre, Leibniz Centre for Diabetes Research at Heinrich Heine
University, Duesseldorf, Germany
6Department of Cardiology, The Alfre Hospital, Melbourne, Australia
*equally contributing senior authors
Corresponding authors:
Karlheinz Peter, MD PhD
Atherothrombosis & Vascular Biology,
Baker Heart and Diabetes Institute
PO Box 6492, St Kilda Road Central
Melbourne, VIC 8008, Australia
Phone: +613 8532 1490; Fax: +613 8532 1100
Email: karlheinz.peter@baker.edu.au
Karin Jandeleit-Dahm, MD, PhD
Department of Diabetes Results: Diabetic mice showed an increase in the size of unstable atherosclerotic plaques in the TS
model. Plaque instability markers such as MCP-1, CD68, and necrotic core (NC) size were
significantly increased. Mice treated with the sodium glucose co-transporter 2i (SGLT2i)
dapagliflozin demonstrated attenuated glucose levels. Importantly, these mice demonstrated
plaque stabilization with enhanced collagen accumulation, increased fibrosis, increased cap-
to-lesion ratios, and significant upregulation of plaque NADPH oxidase 4 (NOX4) expression. Aims/hypothesis: Diabetes is known to accelerate the progression of atherosclerosis and increase plaque
instability. However, there has been a lack of suitable animal models to study the effect of
diabetes on plaque instability. We hypothesized that the tandem stenosis (TS) mouse model,
which reflects plaque instability and rupture as seen in patients, can be applied to study the
effects of diabetes and its respective therapeutic approaches on plaque instability/rupture. Methods: ApoE-/- mice at 7 weeks of age were injected with streptozotocin (STZ) for 5 consecutive days. 5 weeks after STZ injection, mice were surgically subjected to TS in the right carotid artery
and fed with a high-fat diet for an additional 7 weeks. To validate this newly developed animal
model, administration of the interventional drug dapagliflozin was provided via drinking water
(25 mg/kg) 3 days after TS surgery. Conclusions/interpretation: The TS mouse model in combination with the application of STZ represents a highly suitable
and unique mouse model for studying plaque destabilization under diabetic conditions. Furthermore, for the first time, we provide evidence of plaque-stabilizing effects of SGLT2i. Our data also suggest that this newly developed mouse model is an attractive preclinical tool for testing anti-diabetic drugs for their highly sought-after potential to stabilize atherosclerotic
plaques. Keyword: Animal model, Streptozotocin; Sodium glucose co-transporter 2 inhibitor; Plaque
instability. Background Myocardial infarction (MI) is a major cause of mortality/morbidity in patients with diabetes
mellitus and typically atherosclerosis is the underlying pathology [1]. Diabetes has been
postulated to accelerate the development of atherosclerosis and increase plaque
instability/vulnerability, ultimately causing plaque rupture and MI. Therapeutic approaches to
stabilizing vulnerable plaques in diabetic patients are thus highly sought-after. Until recently
there was no conclusive proof that any of the antidiabetic drugs provided cardiovascular
protection. However, the advent of new antidiabetic agents, such as the sodium glucose co-
transporter 2 inhibitor (SGLT2i) and GLP-1 receptor agonists, has substantially changed this
scenario, adding certain antidiabetic drugs to the standard repertoire of cardiologists. This
paradigm shift is based on several randomized, controlled clinical trials demonstrating that
SGLT-2 inhibitors confer beneficial effects on cardiovascular outcomes in patients with type
2 diabetes mellitus [2]. There is clinical evidence that SGLT2i protects against heart failure;
however, although postulated as a mechanism, there is no direct clinical proof of anti-
atherosclerotic and/or plaque-stabilizing effects of SGLT2i. We have previously validated a unique mouse model of plaque instability/rupture by
introducing a tandem stenosis (TS) to the carotid artery [3]. This TS model closely reflects
plaque instability/rupture as seen in patients and has recently been successfully used to define
the plaque-stabilizing effects of a myeloperoxidase inhibitor [4] and anti-CD47 antibodies [5]. We now systematically use this model to investigate whether diabetes promotes plaque
instability and, most importantly, whether SGLT2i exhibits plaque-stabilizing effects in this
newly developed animal model. Animal studies ApoE-/- mice of C57/Bl6 background (Australia Resource Centre, Western Australia) at 7
weeks of age were injected with streptozotocin (STZ) at a dose of 55 mg/kg for 5 consecutive
days. At 12 weeks of age, 5 weeks after STZ, mice were anesthetized intraperitoneally by a
mixture of ketamine (100 mg/kg) and xylazine (20 mg/kg). An incision was made in the neck
and the right common carotid artery was dissected from circumferential connective tissues. TS
with 150 µm outer diameters was introduced, with the distal point 1 mm from the carotid artery
bifurcation and the proximal point 3 mm from the distal stenosis. The stenosis diameter was
obtained by placing a 6-0 blue braided polyester fiber suture around the carotid artery together
with a 150 µm needle that was tied to it and later removed as described previously [3, 4]. Animals were sacrificed at 7 weeks after surgery. Histology and immunohistochemistry Histology and immunohistochemistry The left and right common carotid arteries and brachiocephalic trunk were sectioned as
described previously [3]. Serial cryosections were obtained at 100 µm intervals at 1,500–
2,000 µm proximal to the proximal suture in the right common carotid artery and from the
brachiocephalic trunk and stored at −80 °C until staining was performed. Picrosirius red
staining was performed to highlight collagen within the atherosclerotic plaque. Hematoxylin
and eosin (H&E) staining was used for necrotic core determination and atherosclerotic plaque
burden. Oil red o staining was used for lipid content quantification in the plaque. Images were
obtained using a light microscope (Olympus BX50). Sections were incubated with rat anti-
mouse TER-119 biotin-labeled antibody (eBioscience 13-5921) 1:400; rat anti-mouse CD68
(AbD Serotech MCA1957GA) 1:100 or MOMA-2 (AbD Serotech MCA519G); rabbit polyclonal anti-NOX4 (Sigma-Aldrich ABC459) 1:100; and anti-monocyte chemoattractant
protein-1 antibody (Abcam ab7202) 1:200 at 4 °C overnight. After washing steps and
incubation in ABC reagent, DAB reagent was applied to each section. Conversion of the DAB
substrate into a colored product was monitored and slides were immersed in dH2O to stop the
reaction. Sections were dehydrated and covered with coverslips using DPX mounting media. Quantification of images was performed on Fiji Image J[6]. polyclonal anti-NOX4 (Sigma-Aldrich ABC459) 1:100; and anti-monocyte chemoattractant
protein-1 antibody (Abcam ab7202) 1:200 at 4 °C overnight. After washing steps and
incubation in ABC reagent, DAB reagent was applied to each section. Conversion of the DAB
substrate into a colored product was monitored and slides were immersed in dH2O to stop the
reaction. Sections were dehydrated and covered with coverslips using DPX mounting media. Quantification of images was performed on Fiji Image J[6]. Statistical analyses Quantitative results are expressed as mean±SEM. Comparisons of parameters among two
groups were made by the unpaired Student’s t-test or Mann–Whitney test. Comparisons of
table statistics were made by Fisher's exact test. Comparisons of parameters among three
groups were made by one-way ANOVA followed by post hoc analysis using the Newman–
Keuls test. A value of P<0.05 was considered to be statistically significant. Results We induced insulin-deficient diabetes using the beta-cell toxin STZ in ApoE-/- mice on a high-
fat diet who underwent TS surgery after confirming diabetes. The diabetic mice showed
significantly elevated glucose and glycated hemoglobin levels (Figure 1A, 1B). Under diabetic
conditions, the locally defined unstable atherosclerotic plaque area in the TS model showed a
significant increase (Figure 1C). Importantly, several typical markers of plaque instability,
such as the monocyte chemoattractant protein-1 (MCP-1; Figure 1D), macrophages (CD68;
Figure 1E), and necrotic core size (Figure 1F), were significantly increased as quantified as a
percentage of plaque area. As a direct proof of plaque instability, intraplaque hemorrhage (IH)
increased from 52% of untreated mice to 81% in STZ-treated mice with diabetes (Figure 1G)
and plaque rupture increased from 32% to 56%, respectively (Figure 1H) as measured in a total of 226 mice, whereby 116 were untreated mice with TS and 100 were STZ-treated mice with
TS. These data confirm that diabetic conditions cause plaque instability and demonstrate the
suitability of the TS mouse model for detecting diabetes-associated changes in plaque
instability. Next, we examined the potential of the SGLT2 inhibitor dapagliflozin to stabilize plaques. Repeated STZ protocol as previously described, dapagliflozin was given to ApoE-/- mice via
drinking water 3 days after TS surgery. 2 weeks after the start of dapagliflozin treatment, STZ-
induced hyperglycemia was modestly attenuated (Figure 2). The effect on lowering glucose
was sustained for another 6 weeks; however, normoglycemia was never achieved. We focused
on investigating markers of plaque instability. Interestingly, we did not see changes in
inflammatory markers such as the macrophage markers (CD68 and MOMA-2) or MCP-1 levels
(data not shown). Although the strongest markers of plaque instability such as plaque rupture
and IH (data not shown) were unchanged, early signs of plaque stabilization such as an overall
reduction in lipid content (Figure 3A) and increases in collagen content and the cap-to-core
ratio were significant in the dapagliflozin-treated group (Figure 3B, 3C). These data are of
substantial translational relevance as the cap-to-core ratio is a particularly important clinical
marker that reflects plaque stability and is closely linked to improved clinical outcomes in
patients. Increased formation of reactive oxygen species (ROS) is associated with plaque instability in
diabetes. Discussion SGLT2i has emerged as a new therapeutic target for lowering glucose. Several clinical trials
have demonstrated the efficacy of the glucose lowering of SGLT-2 inhibitors (EMPA-REG
OUTCOME, CANVAS, DECLARE-TIMI) in association with improved cardiovascular
outcomes [8-10]. The EMPA-REG OUTCOME trial of empagliflozin demonstrated a
reduction in cardiovascular outcomes and death from other causes [10]. Nevertheless, direct
evidence that SGLT2i stabilizes atherosclerotic plaques is still lacking, partly due to the fact
that animal models of atherosclerosis typically do not exhibit plaque instability and rupture
[11]. Currently, only a few experimental studies have investigated the effect of SGLT2i in
classical mouse models of atherosclerosis. Al-Sharea et al. treated LDLR-/- mice on a high-fat
diet and after STZ application with dapagliflozin and reported a reduction in peripheral
monocyte count and a lower degree of atherosclerosis, postulating a change in plasma
lipoproteins as the major cause [12]. In ApoE-/- mice on a high-fat diet, empagliflozin reduced
blood glucose, aortic plaque area, insulin resistance, and inflammation[13]. These studies
provide the first experimental indication that SGLT2i exhibits anti-atherosclerotic effects. Our
studies go beyond and demonstrate the plaque-stabilizing capability of SGLT2i and suggest
that changes in the pro-oxidant enzyme NOX4 are mechanistically involved in plaque
stabilization. Results NADPH oxidases, particularly increased expression of NADPH oxidase1 (NOX1)
and reduced expression of the vasculoprotective NOX4 isoform in advanced plaques, have
been shown to contribute to plaque development and remodeling in diabetes [7]. Interestingly,
while expression of pro-atherosclerotic NOX-1 (data not shown) was not upregulated, the expression of anti-atherosclerotic NOX-4 (Figure 3D) was upregulated in the dapagliflozin
group. Therefore, the regulation of certain NOX isoforms by SGLT2i therapy represents a
potential mechanistic explanation for the beneficial effect seen in patients treated with SGLT2i. Consent for publication Not applicable Authors' contributions YCC collected and analysed and interpreted the data. YCC performed the histological
examination of the atherosclerotic plaques. YCC, KP, KJD co-designed the animal experiment
and discussed the data, and jointly wrote the manuscript. All authors have read and have
approved the final manuscript. Funding Yung-Chih Chen is supported by the Heart Foundation Future leader fellowship (No:102068). Karlheinz Peter is supported by an NHMRC L3 investigator fellowship. The work was partially
supported by an NHMRC project grant (APP1163233, K Jandeleit-Dahm). Ethics approval and consent to participate All experimental protocols and animal experiments have been approved by the Animal Ethics
Committees of the Alfred Research Alliance approved number (E/1838/2018/B). Availability of data and materials The datasets used and/or analysed during the current study are available from the
corresponding authors on reasonable request. Conclusion In conclusion, the tandem stenosis mouse model in conjunction with STZ-induced diabetes
reflects plaque destabilization as seen in patients with diabetes and importantly provides initial proof of the plaque-stabilizing effects of SGLT2i. Indeed, these effects on plaque stability
could explain at least in part the reduction of cardiovascular events seen in diabetic patients
treated with SGLT2i and as such bears direct translational relevance. Our data also establish
the TS mouse model in combination with STZ-treatment as a preclinical tool for testing of anti-
diabetic drugs for highly sought-after plaque-stabilizing effects. proof of the plaque-stabilizing effects of SGLT2i. Indeed, these effects on plaque stability
could explain at least in part the reduction of cardiovascular events seen in diabetic patients
treated with SGLT2i and as such bears direct translational relevance. Our data also establish
the TS mouse model in combination with STZ-treatment as a preclinical tool for testing of anti-
diabetic drugs for highly sought-after plaque-stabilizing effects. Competing interests All authors declare no competing interests. Acknowledgments Not applicable Not applicable Figures Figures Figure 1: (A) The TS+STZ group shows upregulation of blood glucose levels compared
to non-diabetic TS mice. (B) STZ-treated mice show an increase in the glycation of
hemoglobin (GHb). Glycated hemoglobin A1c was measured by HPLC. (C) Diabetic mice
show an increase in plaque area in the right carotid artery of the TS model. Quantification
was performed on H&E stained sections, where the plaque was measured from the lumen to
the internal elastic lamina. Each data point represents the mean of 6 sections per mouse at
200 µm intervals. (D) MCP-1 is upregulated in diabetic mice with TS. The percentage of
positive MCP-1 staining per atherosclerotic plaque area was obtained. Each data point
represents the mean of 2 sections per mouse. (E) Unstable plaques in diabetic mice show an
increase in macrophage staining. Macrophages were detected by staining for CD68 and
quantified as described in D. (F) Unstable carotid plaques in diabetic mice show an increase
in necrotic core size. The size of the NC is a major characteristic of plaque vulnerability in Figure 1: (A) The TS+STZ group shows upregulation of blood glucose levels compared Figure 1: (A) The TS+STZ group shows upregulation of blood glucose levels compared Figure 1: (A) The TS+STZ group shows upregulation of blood glucose levels c Figure 1: (A) The TS+STZ group shows upregulation of blood glucose levels compared
to non-diabetic TS mice. (B) STZ-treated mice show an increase in the glycation of
hemoglobin (GHb). Glycated hemoglobin A1c was measured by HPLC. (C) Diabetic mice
show an increase in plaque area in the right carotid artery of the TS model. Quantification
was performed on H&E stained sections, where the plaque was measured from the lumen to
the internal elastic lamina. Each data point represents the mean of 6 sections per mouse at
200 µm intervals. (D) MCP-1 is upregulated in diabetic mice with TS. The percentage of
positive MCP-1 staining per atherosclerotic plaque area was obtained. Each data point
represents the mean of 2 sections per mouse. (E) Unstable plaques in diabetic mice show an
increase in macrophage staining. Macrophages were detected by staining for CD68 and
quantified as described in D. (F) Unstable carotid plaques in diabetic mice show an increase
in necrotic core size. The size of the NC is a major characteristic of plaque vulnerability in humans and we found that diabetes increases NC size. Not applicable Quantification was performed as
described in D. (G) Intraplaque hemorrhage (IH) is increased in diabetic mice. IH was
defined by immunostaining for the red blood cell marker TER-119 or by visible hemorrhage. IH in the TS and TS+STZ groups were 60 out of 116 and 81 out of 100, respectively. Non-IH
in the TS and TS+STZ groups were 56 out of 116 and 19 out of 100, respectively. P<0.0001
by Fisher’s exact test. (H) Plaque rupture (PR) was increased in diabetic mice. PR was
defined by visible defects of the fibrous cap with or without luminal thrombi. PR in the TS and
TS+STZ groups were 25 out of 96 and 48 out of 87, respectively. Non-PR in the TS and
TS+STZ groups were 71 out of 96 and 39 out of 87, respectively. P<0.0001 by Fisher’s exact
test. All other data are shown; N=13–16, mean±SEM, *P<0.05 by Student t-test. Figure 2: The SGLT2 inhibitor dapagliflozin reduces hyperglycemia in TS mice treated
with STZ. Dapagliflozin was given via drinking water at 25 mg/kg after TS. 2 weeks after the
initiation of daily dapagliflozin, glucose levels were significantly reduced to
24.66±1.43 mmol/L compared to vehicle-treated diabetic TS mice 28.61±1.13 mmol/L and
remained steady until the endpoint. N=20–23, mean±SEM, *P<0.05 by multiple t-test. ure 2: The SGLT2 inhibitor dapagliflozin reduces hyperglycemia in TS mice treate with STZ. Dapagliflozin was given via drinking water at 25 mg/kg after TS. 2 weeks after the
initiation of daily dapagliflozin, glucose levels were significantly reduced to
24.66±1.43 mmol/L compared to vehicle-treated diabetic TS mice 28.61±1.13 mmol/L and
remained steady until the endpoint. N=20–23, mean±SEM, *P<0.05 by multiple t-test. Figure 3: (A) Dapagliflozin significantly reduces the lipid content and (B) increases the
collagen in the unstable atherosclerotic plaque area in the TS model. Oil Red O staining
and Picrosirius red staining were used to quantify the total lipid and collagen content,
respectively. (C) Diabetes significantly reduces the cap-to-core ratio, whereas
dapagliflozin restores it, indicating a more stable plaque phenotype. The cap-to-core ratio
was determined by dividing the total fibrous cap area by the maximal lesion height in every
cross-section. N=13–16, mean±SEM, *P<0.05 by ANOVA with Dunnett’s multiple
comparisons test. (D) Dapagliflozin increases NOX4 expression in unstable plaques of the
TS model. The NOX4 expression in plaques was investigated by immunohistochemistry and
each data point represents the mean of 2 sections per mouse. N=13–16, mean±SEM, *P<0.05
by Student’s t-test. Not applicable Figure 3: (A) Dapagliflozin significantly reduces the lipid content and (B) increases the Figure 3: (A) Dapagliflozin significantly reduces the lipid content and (B) increases th Figure 3: (A) Dapagliflozin significantly reduces the lipid content and (B) increases the
collagen in the unstable atherosclerotic plaque area in the TS model. Oil Red O staining
and Picrosirius red staining were used to quantify the total lipid and collagen content,
respectively. (C) Diabetes significantly reduces the cap-to-core ratio, whereas
dapagliflozin restores it, indicating a more stable plaque phenotype. The cap-to-core ratio
was determined by dividing the total fibrous cap area by the maximal lesion height in every
cross-section. N=13–16, mean±SEM, *P<0.05 by ANOVA with Dunnett’s multiple
comparisons test. (D) Dapagliflozin increases NOX4 expression in unstable plaques of the
TS
d l Th NOX4
i
i
l
i
i
d b i
hi
h
i
d Figure 3: (A) Dapagliflozin significantly reduces the lipid content and (B) increases the TS model. The NOX4 expression in plaques was investigated by immunohistochemistry and
each data point represents the mean of 2 sections per mouse. N=13–16, mean±SEM, *P<0.05
by Student’s t-test. References [1]
Cavallari I, Bhatt DL, Steg PG, et al. (2021) Causes and Risk Factors for Death in
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60(2): 364-376. References 10.1007/s00125-016-4158-2 Figures Figures Figure 1
(A) The TS+STZ group shows upregulation of blood glucose levels compared to non-diabetic TS m
(B) STZ-treated mice show an increase in the glycation of hemoglobin (GHb). Glycated hemoglobin
was measured by HPLC. (C) Diabetic mice show an increase in plaque area in the right carotid arter
the TS model. Quanti¦cation was performed on H&E stained sections, where the plaque was measu
from the lumen to the internal elastic lamina. Each data point represents the mean of 6 sections pe
mouse at 200 μm intervals (D) MCP-1 is upregulated in diabetic mice with TS The percentage of p Figure 1
(A) The TS+STZ group shows upregulation of blood glucose levels compared to non-diabetic TS mice. (B) STZ-treated mice show an increase in the glycation of hemoglobin (GHb). Glycated hemoglobin A1c
was measured by HPLC. (C) Diabetic mice show an increase in plaque area in the right carotid artery of
the TS model. Quanti¦cation was performed on H&E stained sections, where the plaque was measured
from the lumen to the internal elastic lamina. Each data point represents the mean of 6 sections per
mouse at 200 μm intervals. (D) MCP-1 is upregulated in diabetic mice with TS. The percentage of positive
MCP-1 staining per atherosclerotic plaque area was obtained. Each data point represents the mean of 2
sections per mouse. (E) Unstable plaques in diabetic mice show an increase in macrophage staining. Macrophages were detected by staining for CD68 and quanti¦ed as described in D. (F) Unstable carotid
plaques in diabetic mice show an increase in necrotic core size. The size of the NC is a major
characteristic of plaque vulnerability in humans and we found that diabetes increases NC size. Quanti¦cation was performed as described in D. (G) Intraplaque hemorrhage (IH) is increased in diabetic
mice. IH was de¦ned by immunostaining for the red blood cell marker TER-119 or by visible hemorrhage. IH in the TS and TS+STZ groups were 60 out of 116 and 81 out of 100, respectively. Non-IH in the TS and
TS+STZ groups were 56 out of 116 and 19 out of 100, respectively. P<0.0001 by Fisher’s exact test. (H) Figure 1 (A) The TS+STZ group shows upregulation of blood glucose levels compared to non-diabetic TS mice. (B) STZ-treated mice show an increase in the glycation of hemoglobin (GHb). Glycated hemoglobin A1c
was measured by HPLC. (C) Diabetic mice show an increase in plaque area in the right carotid artery of
the TS model. Quanti¦cation was performed on H&E stained sections, where the plaque was measured
from the lumen to the internal elastic lamina. Each data point represents the mean of 6 sections per
mouse at 200 μm intervals. (D) MCP-1 is upregulated in diabetic mice with TS. The percentage of positive
MCP-1 staining per atherosclerotic plaque area was obtained. Each data point represents the mean of 2
sections per mouse. (E) Unstable plaques in diabetic mice show an increase in macrophage staining. Macrophages were detected by staining for CD68 and quanti¦ed as described in D. (F) Unstable carotid
plaques in diabetic mice show an increase in necrotic core size. The size of the NC is a major
characteristic of plaque vulnerability in humans and we found that diabetes increases NC size. Quanti¦cation was performed as described in D. (G) Intraplaque hemorrhage (IH) is increased in diabetic
mice. IH was de¦ned by immunostaining for the red blood cell marker TER-119 or by visible hemorrhage. IH in the TS and TS+STZ groups were 60 out of 116 and 81 out of 100, respectively. Non-IH in the TS and
TS+STZ groups were 56 out of 116 and 19 out of 100, respectively. P<0.0001 by Fisher’s exact test. (H) (A) The TS+STZ group shows upregulation of blood glucose levels compared to non-diabetic TS mice. (B) STZ-treated mice show an increase in the glycation of hemoglobin (GHb). Glycated hemoglobin A1c
was measured by HPLC. (C) Diabetic mice show an increase in plaque area in the right carotid artery of
the TS model. Quanti¦cation was performed on H&E stained sections, where the plaque was measured
from the lumen to the internal elastic lamina. Each data point represents the mean of 6 sections per
mouse at 200 μm intervals. (D) MCP-1 is upregulated in diabetic mice with TS. The percentage of positive
MCP-1 staining per atherosclerotic plaque area was obtained. Each data point represents the mean of 2
sections per mouse. (E) Unstable plaques in diabetic mice show an increase in macrophage staining. Macrophages were detected by staining for CD68 and quanti¦ed as described in D. Figure 1 All other data are shown; N=13–16, mean±SEM, *P<0.05 by Student t-
test. Plaque rupture (PR) was increased in diabetic mice. PR was de¦ned by visible defects of the ¦brous cap
with or without luminal thrombi. PR in the TS and TS+STZ groups were 25 out of 96 and 48 out of 87,
respectively. Non-PR in the TS and TS+STZ groups were 71 out of 96 and 39 out of 87, respectively. P<0.0001 by Fisher’s exact test. All other data are shown; N=13–16, mean±SEM, *P<0.05 by Student t-
test. Figure 2
The SGLT2 inhibitor dapagli§ozin reduces hyperglycemia in TS mice treated with STZ. Dapagli§ozin was
given via drinking water at 25 mg/kg after TS. 2 weeks after the initiation of daily dapagli§ozin, glucose
levels were signi¦cantly reduced to 24.66±1.43 mmol/L compared to vehicle-treated diabetic TS mice
28.61±1.13 mmol/L and remained steady until the endpoint. N=20–23, mean±SEM, *P<0.05 by multiple t-
test. Figure 1 (F) Unstable carotid
plaques in diabetic mice show an increase in necrotic core size. The size of the NC is a major
characteristic of plaque vulnerability in humans and we found that diabetes increases NC size. Quanti¦cation was performed as described in D. (G) Intraplaque hemorrhage (IH) is increased in diabetic
mice. IH was de¦ned by immunostaining for the red blood cell marker TER-119 or by visible hemorrhage. IH in the TS and TS+STZ groups were 60 out of 116 and 81 out of 100, respectively. Non-IH in the TS and
TS+STZ groups were 56 out of 116 and 19 out of 100, respectively. P<0.0001 by Fisher’s exact test. (H) Quanti¦cation was performed as described in D. (G) Intraplaque hemorrhage (IH) is increased in diabetic
mice. IH was de¦ned by immunostaining for the red blood cell marker TER-119 or by visible hemorrhage. IH in the TS and TS+STZ groups were 60 out of 116 and 81 out of 100, respectively. Non-IH in the TS and
TS+STZ groups were 56 out of 116 and 19 out of 100, respectively. P<0.0001 by Fisher’s exact test. (H) Quanti¦cation was performed as described in D. (G) Intraplaque hemorrhage (IH) is increased in diabetic
mice. IH was de¦ned by immunostaining for the red blood cell marker TER-119 or by visible hemorrhage. IH in the TS and TS+STZ groups were 60 out of 116 and 81 out of 100, respectively. Non-IH in the TS and
TS+STZ groups were 56 out of 116 and 19 out of 100, respectively. P<0.0001 by Fisher’s exact test. (H) Plaque rupture (PR) was increased in diabetic mice. PR was de¦ned by visible defects of the ¦brous cap
with or without luminal thrombi. PR in the TS and TS+STZ groups were 25 out of 96 and 48 out of 87,
respectively. Non-PR in the TS and TS+STZ groups were 71 out of 96 and 39 out of 87, respectively. P<0.0001 by Fisher’s exact test. All other data are shown; N=13–16, mean±SEM, *P<0.05 by Student t-
test. Plaque rupture (PR) was increased in diabetic mice. PR was de¦ned by visible defects of the ¦brous cap
with or without luminal thrombi. PR in the TS and TS+STZ groups were 25 out of 96 and 48 out of 87,
respectively. Non-PR in the TS and TS+STZ groups were 71 out of 96 and 39 out of 87, respectively. P<0.0001 by Fisher’s exact test. Figure 2 The SGLT2 inhibitor dapagli§ozin reduces hyperglycemia in TS mice treated with STZ. Dapagli§ozin was
given via drinking water at 25 mg/kg after TS. 2 weeks after the initiation of daily dapagli§ozin, glucose
levels were signi¦cantly reduced to 24.66±1.43 mmol/L compared to vehicle-treated diabetic TS mice
28.61±1.13 mmol/L and remained steady until the endpoint. N=20–23, mean±SEM, *P<0.05 by multiple t-
test. Figure 3 Figure 3 (A) Dapagli§ozin signi¦cantly reduces the lipid content and (B) increases the collagen in the unstable
atherosclerotic plaque area in the TS model. Oil Red O staining and Picrosirius red staining were used to
quantify the total lipid and collagen content, respectively. (C) Diabetes signi¦cantly reduces the cap-to-
core ratio, whereas dapagli§ozin restores it, indicating a more stable plaque phenotype. The cap-to-core
ratio was determined by dividing the total ¦brous cap area by the maximal lesion height in every cross-
section. N=13–16, mean±SEM, *P<0.05 by ANOVA with Dunnett’s multiple comparisons test. (D)
Dapagli§ozin increases NOX4 expression in unstable plaques of the TS model. The NOX4 expression in
plaques was investigated by immunohistochemistry and each data point represents the mean of 2
sections per mouse. N=13–16, mean±SEM, *P<0.05 by Student’s t-test. (A) Dapagli§ozin signi¦cantly reduces the lipid content and (B) increases the collagen in the unstable
atherosclerotic plaque area in the TS model. Oil Red O staining and Picrosirius red staining were used to
quantify the total lipid and collagen content, respectively. (C) Diabetes signi¦cantly reduces the cap-to-
core ratio, whereas dapagli§ozin restores it, indicating a more stable plaque phenotype. The cap-to-core
ratio was determined by dividing the total ¦brous cap area by the maximal lesion height in every cross-
section. N=13–16, mean±SEM, *P<0.05 by ANOVA with Dunnett’s multiple comparisons test. (D)
Dapagli§ozin increases NOX4 expression in unstable plaques of the TS model. The NOX4 expression in
plaques was investigated by immunohistochemistry and each data point represents the mean of 2
sections per mouse. N=13–16, mean±SEM, *P<0.05 by Student’s t-test. Dapagli§ozin increases NOX4 expression in unstable plaques of the TS model. The NOX4 expression in
plaques was investigated by immunohistochemistry and each data point represents the mean of 2
sections per mouse. N=13–16, mean±SEM, *P<0.05 by Student’s t-test.
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Employing plant functional groups to advance seed dispersal ecology and conservation
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Employing plant functional groups to advance seed
dispersal ecology and conservation Lowell Street, Tucson, AZ 85721, USA
5Dynamic Macroecology, Landscape Dynamics, Swiss Federal Research Institute WSL, Zürcherstrasse 111, CH-8903 Birmensdorf,
Switzerland 6Faculty of Biology and Pre-Clinical Medicine, University of Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany
7Department of Biology, Pennsylvania State University, 208 Mueller Laboratory, University Park, PA 16802, USA
8Department of Fish, Wildlife and Conservation Biology, Colorado State University, 1474 Campus Delivery, Fort Collins, CO 80523, USA
9Biology Department Woods Hole Oceanographic Institution 266 Woods Hole Road Woods Hole MA 02543 USA Faculty of Biology and Pre Clinical Medicine, University of Regensburg, Universitätsstraße 31, D 93053 Regensburg, Germany
7Department of Biology, Pennsylvania State University, 208 Mueller Laboratory, University Park, PA 16802, USA gy
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10Nicholas School of the Environment, Duke University, 9 Circuit Drive, Durham, NC 27708, USA y
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las School of the Environment, Duke University, 9 Circuit Drive, Durham, NC 27708, USA gy
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10Nicholas School of the Environment, Duke University, 9 Circuit Drive, Durham, NC 27708, USA 11Department of Biology, University of Washington, Seattle, WA 98195, USA epartment of Biology, University of Washington, Seatt 12Department of Evolution, Ecology and Organismal Biology, The Ohio State University, 318 W 12th Avenue, Columbus, OH 43210,
USA 12Department of Evolution, Ecology and Organismal Biology, The Ohio State University, 318 W 12th Aven
USA rtment of Wildlife Ecology and Conservation, University of Florida, Gainesville, FL 32611, USA
C
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i 13Department of Wildlife Ecology and Conservation, University of Florida, Gainesville, FL 32611, USA
14CRS F
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i 13Department of Wildlife Ecology and Conservation, University of Florida, Gainesville, FL 32611, USA
14CRS Forestry Commission, Calabar, Nigeria Department of Wildlife Ecology and Conservation, University of Florida, Gainesville, FL 32611, USA
14CRS Forestry Commission, Calabar, Nigeria 4CRS Forestry Commission, Calabar, Nigeria y
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15National Socio-Environmental Synthesis Center, 1 Park Place, Annapolis, MD 21401, USA y
p
andschaftsökologie, Albert-Ludwigs-Universität Freiburg, Tennenbacher Str. 4, D-79106 Freiburg, Germany 16Naturschutz & Landschaftsökologie, Albert-Ludwigs-Universität Freiburg, Tennenbacher Str. 4, D-79106 Freiburg, Germany
17Department of Geography, Texas A&M University, College Station, TX 77843, USA
18Centre for Ecology & Hydrology, Wallingford, UK
19Wildlife Biology Program, University of Montana, 32 Campus Drive, Missoula, MT 59812, USA
20Department of Mathematics, University of Miami, 1365 Memorial Drive, Coral Gables, FL 33146, USA
21University of California-Davis, Davis, CA 95616, USA
22Department of Mathematics, Howard University, Washington, DC 20059, USA
23Physics Department, California Polytechnic State University, 1 Grand Avenue, San Luis Obispo, CA 93407, USA
24Department of Biology, College of Charleston, Charleston, SC 29424, USA
25Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT 06269, USA
26Environmental and Plant Biology, Ohio University, Athens, OH 45701, USA
27Department of Mathematics, University of Tennessee, Knoxville, TN 37996, USA
28Department of Mathematics, Lafayette College, 730 High Street, Easton, PA 18042, USA
Received: 15 August 2018 Editorial decision: 24 January 2019 Accepted: 5 February 2019 Published: 7 February 2019
Associate Editor: Kim McConkey
Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 Point of View
SPECIAL ISSUE: The Role of Seed Dispersal in Plant
Populations: Perspectives and Advances in a Changing World
Employing plant functional groups to advance seed
dispersal ecology and conservation
Clare Aslan1*, Noelle G. Beckman2, Haldre S. Rogers3, Judie Bronstein4, Damaris Zurell5, Florian
Hartig6, Katriona Shea7, Liba Pejchar8, Mike Neubert9, John Poulsen10, Janneke HilleRisLambers11,
Maria Miriti12, Bette Loiselle13, Edu Effiom14, Jenny Zambrano15, Geno Schupp2, Gesine Pufal16,
Jeremy Johnson17, James M. Bullock18, Jedediah Brodie19, Emilio Bruna13, Robert Stephen Cantrell20,
Robin Decker21, Evan Fricke3, Katie Gurski22, Alan Hastings21, Oleg Kogan23, Onja Razafindratsima24,
Manette Sandor25, Sebastian Schreiber21, Rebecca Snell26, Christopher Strickland27 and Ying Zhou28
1Landscape Conservation Initiative, Northern Arizona University, Flagstaff, AZ 86011, USA
2Department of Biology, Utah State University, 5305 Old Main Hill, Logan, UT 84322, USA
3Department of Ecology, Evolution, and Organismal Biology, Iowa State University, 251 Bessey Hall, Ames, IA 50011, USA
4Department of Ecology and Evolutionary Biology, University of Arizona, 1041 E. Lowell Street, Tucson, AZ 85721, USA
5Dynamic Macroecology, Landscape Dynamics, Swiss Federal Research Institute WSL, Zürcherstrasse 111, CH-8903 Birmensdorf,
Switzerland
6Faculty of Biology and Pre-Clinical Medicine, University of Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany
7Department of Biology, Pennsylvania State University, 208 Mueller Laboratory, University Park, PA 16802, USA
8Department of Fish, Wildlife and Conservation Biology, Colorado State University, 1474 Campus Delivery, Fort Collins, CO 80523, USA
9Biology Department, Woods Hole Oceanographic Institution, 266 Woods Hole Road, Woods Hole, MA 02543, USA
10Nicholas School of the Environment, Duke University, 9 Circuit Drive, Durham, NC 27708, USA
11Department of Biology, University of Washington, Seattle, WA 98195, USA
12Department of Evolution, Ecology and Organismal Biology, The Ohio State University, 318 W 12th Avenue, Columbus, OH 43210,
USA
13Department of Wildlife Ecology and Conservation, University of Florida, Gainesville, FL 32611, USA
14CRS Forestry Commission, Calabar, Nigeria
15National Socio-Environmental Synthesis Center, 1 Park Place, Annapolis, MD 21401, USA
16Naturschutz & Landschaftsökologie, Albert-Ludwigs-Universität Freiburg, Tennenbacher Str. Employing plant functional groups to advance seed
dispersal ecology and conservation Clare Aslan1*, Noelle G. Beckman2, Haldre S. Rogers3, Judie Bronstein4, Damaris Zurell5, Florian
Hartig6, Katriona Shea7, Liba Pejchar8, Mike Neubert9, John Poulsen10, Janneke HilleRisLambers11,
Maria Miriti12, Bette Loiselle13, Edu Effiom14, Jenny Zambrano15, Geno Schupp2, Gesine Pufal16,
Jeremy Johnson17, James M. Bullock18, Jedediah Brodie19, Emilio Bruna13, Robert Stephen Cantrell20,
Robin Decker21, Evan Fricke3, Katie Gurski22, Alan Hastings21, Oleg Kogan23, Onja Razafindratsima24,
Manette Sandor25, Sebastian Schreiber21, Rebecca Snell26, Christopher Strickland27 and Ying Zhou28 1Landscape Conservation Initiative, Northern Arizona University, Flagstaff, AZ 86011, USA 1Landscape Conservation Initiative, Northern Arizona University, Flagstaff, AZ 86011, USA
2Department of Biology Utah State University 5305 Old Main Hill Logan UT 84322 USA 1Landscape Conservation Initiative, Northern Arizona University, Flagstaff, AZ 86011, USA
2Department of Biology, Utah State University, 5305 Old Main Hill, Logan, UT 84322, USA p
,
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g
,
,
2Department of Biology, Utah State University, 5305 Old Main Hill, Logan, UT 84322, USA 3Department of Ecology, Evolution, and Organismal Biology, Iowa State University, 251 Bessey Hall, Ames, IA 50011, USA
4Department of Ecology and Evolutionary Biology, University of Arizona, 1041 E. Aslan et al. – Defining functional groups relevant to the importance of seed dispersal Citation: Aslan C, Beckman NG, Rogers HS, Bronstein J, Zurell D, Hartig F, Shea K, Pejchar L, Neubert M, Poulsen J, HilleRisLambers
J, Miriti M, Loiselle B, Effiom E, Zambrano J, Schupp G, Pufal G, Johnson J, Bullock JM, Brodie J, Bruna E, Cantrell RS, Decker R, Fricke
E, Gurski K, Hastings A, Kogan O, Razafindratsima O, Sandor M, Schreiber S, Snell R, Strickland C, Zhou Y. 2019. Employing plant
functional groups to advance seed dispersal ecology and conservation. AoB PLANTS 11: plz006; doi: 10.1093/aobpla/plz006 Abstract. Seed dispersal enables plants to reach hospitable germination sites and escape natural enemies. Understanding when and how much seed dispersal matters to plant fitness is critical for understanding plant
population and community dynamics. At the same time, the complexity of factors that determine if a seed will
be successfully dispersed and subsequently develop into a reproductive plant is daunting. Quantifying all fac-
tors that may influence seed dispersal effectiveness for any potential seed-vector relationship would require an
unrealistically large amount of time, materials and financial resources. On the other hand, being able to make
dispersal predictions is critical for predicting whether single species and entire ecosystems will be resilient to
global change. Building on current frameworks, we here posit that seed dispersal ecology should adopt plant
functional groups as analytical units to reduce this complexity to manageable levels. Functional groups can
be used to distinguish, for their constituent species, whether it matters (i) if seeds are dispersed, (ii) into what
context they are dispersed and (iii) what vectors disperse them. To avoid overgeneralization, we propose that
the utility of these functional groups may be assessed by generating predictions based on the groups and then
testing those predictions against species-specific data. We suggest that data collection and analysis can then
be guided by robust functional group definitions. Generalizing across similar species in this way could help us
to better understand the population and community dynamics of plants and tackle the complexity of seed dis-
persal as well as its disruption. Keywords: dependency; directed dispersal; dispersal vectors; generalization; mutualism; seed dispersal
effectiveness. Employing plant functional groups to advance seed
dispersal ecology and conservation 4, D-7 17Department of Geography, Texas A&M University, College Station, TX 77843, USA 17Department of Geography, Texas A&M University, College Station, TX 77843, USA 7Department of Geography, Texas A&M University, Co 18Centre for Ecology & Hydrology, Wallingford, UK 19Wildlife Biology Program, University of Montana, 32 Campus Drive, Missoula, MT 59812, USA 19Wildlife Biology Program, University of Montana, 32 Campus Drive, Missoula, MT 59812, USA 19Wildlife Biology Program, University of Montana, 32 gy
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p
20Department of Mathematics, University of Miami, 1365 Memorial Drive, Coral Gables, FL 33146, USA 0Department of Mathematics, University of Miami, 13 20Department of Mathematics, University of Miami, 1365 Memorial Drive, Coral Gab 21University of California-Davis, Davis, CA 95616, USA 22Department of Mathematics, Howard University, Washington, DC 20059, USA 22Department of Mathematics, Howard University, Washington, DC 20059, USA 22Department of Mathematics, Howard University, Wa 22Department of Mathematics, Howard University, Washington, DC 20059, USA 24Department of Biology, College of Charleston, Charleston, SC 29424, USA 24Department of Biology, College of Charleston, Charleston, SC 29424, USA 25Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT 06269, USA 25Department of Ecology and Evolutionary Biology, University of Connecticut, Stor 26Environmental and Plant Biology, Ohio University, Athens, OH 45701, USA 26Environmental and Plant Biology, Ohio University, Athens, OH 45701, USA gy
y
27Department of Mathematics, University of Tennessee, Knoxville, TN 37996, USA 27Department of Mathematics, University of Tennessee, Knoxville, TN 37996, USA 28Department of Mathematics, Lafayette College, 730 High Street, Easton, PA 18042, USA Received: 15 August 2018 Editorial decision: 24 January 2019 Accepted: 5 February 2019 Published: 7 February 2019
Associate Editor: Kim McConkey *Corresponding author’s e-mail address: clare.aslan@nau.edu This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommo
licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work i
© The Author(s) 2019. Published by Oxford University Press on behalf of the Annals of Botany Company. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) 2019. Published by Oxford University Press on behalf of the Annals of Botany Company. 1 © The Author(s) 2019 AoB PLANTS https://academic.oup.com/aobpla Aslan et al. – Defining functional groups relevant to the importance of seed dispersal Introduction: Seed Dispersal Is
Fundamental to Populations and
Communities, Yet Complex – Defining functional groups relevant to the importance of seed dispersal behaviours and physiology of biotic dispersers amplify
the complexity of seed dispersal. Seed handling, for
example, can affect the condition of the seed and change
the likelihood of germination and subsequent survival
and growth after seed deposition (Ladley and Kelly
1996; Traveset and Verdú 2002; Fricke et al. 2013). Some
plant species exhibit extreme specialization in micro-
habitats and require dispersers to move seeds to these
locations (e.g. desert mistletoe requires dispersal to the
branches of a very limited range of host trees; Aukema
2004). The preferences and physiology of dispersers may
influence the direction and distance of seed dispersal
(Beckman and Rogers 2013) (Fig. 1). Stochastic events
may include rare, long-distance dispersal events, which
are difficult to observe and measure but can be critical
for colonization of new geographic regions and provide
connectivity among habitat patches across a landscape
(Muller-Landau et al. 2003; Jordano et al. 2007; Shea
2007; Auffret et al. 2017). Behavioral aspects of biotic
dispersers, such as local aggregation, social organiza-
tion, mating system, competition and territoriality, can
influence both spatial and temporal dispersal of seeds,
with potential ramifications for seed aggregation and
competition between seeds (reviewed in Karubian and
Durães 2009). A given disperser may also disperse seeds
of certain shapes or sizes, depending on disperser body
or gape sizes (McConkey and Drake 2006; Muñoz et al. 2017). An extensive literature has explored the dispersal
syndromes, or seed and fruit traits (e.g. size, shape, col-
our, chemistry, dormancy) that appear predictive of the
primary dispersers of a given plant species, with investi-
gation into the roles of co-evolution, secondary disper-
sal and specialization (e.g. Vander Wall and Beck 2012; much it matters by what vector they are dispersed. We
list such functional groups and discuss their potential
value in achieving general insights. We close by consid-
ering key knowledge gaps that this proposed functional
group approach may address. Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 Introduction: Seed Dispersal Is
Fundamental to Populations and
Communities, Yet Complex It is evident that a quantitative understanding of dis-
persal is key for predicting how environmental changes,
and consequent changes in dispersal vectors, will impact
plant populations and communities. Operationalizing
this goal and moving seed dispersal ecology towards a
predictive science, however, requires confronting a wide
array of interacting factors and stochastic elements
(Robledo-Arnuncio et al. 2014). Here, we discuss how a
functional group approach may help simplify the com-
plexity of seed dispersal ecology and boost our predic-
tive capacity. Plants rely on dispersal vectors—for example,
animals, wind and water—to move across the
landscape. We focus here on the dispersal of
seeds, although many of the arguments we make
could be generalized to other forms of dispersal. Dispersal occurs when a seed is moved from its
origin and deposited elsewhere (Schupp et al. 2010). Through dispersal, plants may experience
reduced exposure to competition, predation and
parasitism (Janzen 1970; Connell 1971; Howe and
Miriti 2004); colonize open habitats after distur-
bance (Wunderle 1997; Puerta-Piñero et al. 2013);
reach potential suitable microsites in otherwise
unsuitable landscapes (Wenny 2001); track cli-
mate fluctuations and environmental change
(Corlett and Westcott 2013); and contribute to
gene flow within and between populations (Bacles
et al. 2006). As a result of these processes, seed
dispersal is a fundamental driver of the diversity,
structure, composition and spatial arrangement
of plant communities. Seed dispersal ecology thus
elucidates mechanisms of species coexistence,
implications of species extinctions and impacts of
global environmental change. Functional group frameworks, in which species are
categorized by ecological functions and the result-
ing groups treated as analytical units, have helped
researchers confront complexity in other ecological sub-
disciplines and have been tentatively explored in seed
dispersal (e.g. Dennis and Westcott 2006; Brodie et al. 2009b; Bastazini et al. 2017). However, they have not yet
been developed sufficiently to link empirical patterns
of seed dispersal with theoretical predictions. In this
Viewpoint, we discuss the complexity of seed dispersal
and the need to reach generalities about it. We propose
that to better understand the importance of seed dis-
persal in plant populations and communities, it would
be useful to identify functional groups that distinguish
plant species based on (i) how much it matters if their
seeds are dispersed at all, (ii) how much it matters into
what ecological context they are dispersed and (iii) how 2 © The Author(s) 2019 AoB PLANTS https://academic.oup.com/aobpla Aslan et al. Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 Where and when a seed is deposited are clearly influ-
enced by many sources of variability (Robledo-Arnuncio
et al. 2014). Additionally, the spatial pattern of seed
deposition on the landscape can then influence subse-
quent interspecific interactions (e.g. pollination, mycor-
rhizal associations, competition, predation, herbivory). Such interactions are important to the fitness of the
newly established plant and determine the likelihood
of survival and growth, access to limiting resources, the
likelihood of mortality due to natural enemies and the
probability of successful reproduction (Beckman and
Rogers 2013). As a result of these interactions, the re-
sulting plant community may more or less closely reflect
the initial template established by seed deposition. ;
)
A full utilization of the SDE framework involves quanti-
fying the effectiveness of dispersal for interacting pairs of
seed and disperser species, taking into account pre-, mid-
and post-dispersal factors that might affect seed survival
and germination and the growth and fecundity of the re-
sulting plant (e.g. Fig. 1). However, parameterization of
SDE requires immense investment of empirical resources
and includes up to 15 different measurable quantities for
a given seed-disperser pair (Schupp et al. 2010). In one
study, plant species were dispersed by an average of just
over seven different disperser species (Aslan et al. 2013);
parameterization of SDE for such a plant would therefore
require a minimum of 7 × 15 = 105 separately measured
parameters—a degree of complexity that would exhaust
the resources of most scientific endeavours. Nevertheless,
SDE has guided impressive efforts to measure subsets
of these parameters, generating important insights. For
example, McConkey et al. (2014) measured disperser ef-
fectiveness as a combination of the percent of monitored
fruit dispersed by each disperser species combined with
the distance of dispersal and survival of seedlings at each
distance. Nogales et al. (2017) compared the number of
seeds dispersed and effect of gut treatment by reptile vs. bird frugivores in the Galápagos. González-Castro et al. (2015) combined the number of seeds dispersed with con-
dition of seeds after dispersal and seedling emergence/
survival probabilities to compare SDE for birds and lizards. As these studies illustrate, different dispersers contribute
in different ways to the template constraining an even-
tual plant community. Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 To understand these roles across
many more sites and for many more species, we require
approaches that build off the SDE framework while sim-
plifying the complexity inherent in biologically diverse
systems. The complexity of seed dispersal Due to their complexity, seed dispersal processes are dif-
ficult to quantify empirically (Fig. 1). Since the quantifica-
tion of these processes forms the basis for understanding
plant population and community dynamics, methods
to reduce this complexity are essential. Both biotic and
abiotic dispersal vectors can influence which seeds are
dispersed, the risks and costs of dispersal, the spatial
direction and distance that seeds travel, the probability
that seeds will encounter specific microhabitats and the
probability of seed aggregation (Howe and Miriti 2004;
Côrtes and Uriarte 2013; Morales et al. 2013). For seeds
transported by abiotic vectors, wind and water speeds
and turbulence determine the distance and direction of
seed movement (Katul et al. 2005; Nathan et al. 2011):
not only are these factors intrinsically variable, but that
variation interacts with the physical structure of the envi-
ronment and the size and shape of the seed. In biotic
dispersal, the set of disperser animals interacting with
a seed may dictate its survival, growth and eventual
reproduction (García and Martínez 2012). Dispersal vec-
tors vary in their interactions with landscape structure,
implying that the mechanism of dispersal may dictate
the composition and arrangement of a plant community
(Metzger 2000; Albrecht et al. 2012; Effiom et al. 2013;
Razafindratsima and Dunham 2016; Chen et al. 2017). We largely focus on biotic seed dispersal because the Figure 1. Seed dispersal exemplifies ecological complexity. Survival to adulthood and the fitness of individual adults are influenced by pre-,
mid- and post-dispersal variables including the availability of abiotic and biotic vectors; the behaviours, preferences, morphology and physi-
ology of dispersers; the spatio-temporal heterogeneity in seed deposition locations; and the probability of encountering other mutualists,
facilitators, predators, pathogens and competitors following dispersal. Figure 1. Seed dispersal exemplifies ecological complexity. Survival to adulthood and the fitness of individual adults are influenced by pre-,
mid- and post-dispersal variables including the availability of abiotic and biotic vectors; the behaviours, preferences, morphology and physi-
ology of dispersers; the spatio-temporal heterogeneity in seed deposition locations; and the probability of encountering other mutualists,
facilitators, predators, pathogens and competitors following dispersal. 3 3 © The Author(s) 2019 AoB PLANTS https://academic.oup.com/aobpla Aslan et al. – Defining functional groups relevant to the importance of seed dispersal Howe 2016). Below, we briefly touch on the importance
of dispersal syndromes as a form of functional group-
ing that categorizes dispersal adaptations. The complexity of seed dispersal However, our
proposed framework focuses instead on functional group
delineations that distinguish the importance of dispersal
in plant populations and communities.l combined with measures of habitat quality across the
landscape (Schupp et al. 2010). The SDE framework
examines the complexity of variation within and among
seeds, dispersers and other interactors determining
the likelihood that a seed grows into a seedling (and,
ultimately, reproductive adult), given a specific vector
moving that seed to a specific location (e.g. Alvarez-
Buylla and Martinez-Ramos 1990; Godinez-Alvarez and
Jordano 2007; Escribano-Avila 2014; Rey and Alcántara
2014; Rother et al. 2016). Seeking predictive capacity in light of global
change: adapting current frameworks for
functional groups As established above, the plant community in a given lo-
cation is constrained by the template established by seed
deposition, but the post-deposition interactions within the
seedscape (i.e. the full environmental context into which
the seed is dispersed) determine which subset of those
seeds succeeds. Empirically quantifying all relevant pre-
and post-dispersal variables is a complex task for even one
plant-disperser pair, and impossible for the thousands of
species pairs that participate in seed dispersal mutualisms
worldwide (Howe and Smallwood 1982; Aslan et al. 2013;
Beckman and Rogers 2013). Nevertheless, without an at-
tempt to understand these processes, their variability and
the drivers of that variability, our understanding of system
dynamics is hamstrung. The Seed Dispersal Effectiveness (SDE) framework is
a comprehensive framework to summarize the full suite
of variables affecting the dispersal service provided to
any particular plant species by any particular vector
(Schupp et al. 2010). The SDE framework summarizes
the contribution of each dispersal vector (whether bi-
otic or abiotic) to the production of new adult plants by
evaluating variables influencing the quantity of seeds
dispersed and the quality of the seed dispersal event. Quantity metrics within SDE include, for example, the
frequency of visits from the disperser to the plant spe-
cies and the number of seeds dispersed per visit. Quality
metrics include, for example, the condition of the de-
posited seed (which depends, e.g., on an animal’s seed-
handling behaviour) or a disperser’s movement patterns To achieve this goal, we propose using plant func-
tional groups in place of individual species in the SDE
framework (Table 1). Functional groups are employed
in many fields of ecology and have proven to be use-
ful (e.g. functional group classifications yielded insights
into plant species responses to climate change in Africa,
Scheiter and Higgins 2009; successional dynamics in a
Costa Rican forest, Chazdon et al. 2010; and global vege-
tation patterns, Sato et al. 2007). By identifying relevant 4 AoB PLANTS https://academic.oup.com/aobpla © The Author(s) 2019 Aslan et al. – Defining functional groups relevant to the importance of seed dispersal Table 1. Functional groups relevant to the importance of seed dispersal for seed survival and thereby plant population and community
dynamics. We propose that researchers and conservation planners determine whether target plant species belong to functional groups
for which dispersal disruption is likely to significantly decrease fitness vs. have only minor effects on fitness. Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 Functional group
category
Characteristics of functional
groups likely significantly affected
by seed dispersal disruption
Characteristics of functional
groups likely less affected by
seed dispersal disruption
Sample references
Groups for which
plant fitness is
affected by whether
seeds are dispersed. High colonization ability
High competitive ability
Coomes and Grubb (2003)
Long-distance dispersal adaptations
Local dispersal adaptations
Muller-Landau et al. (2003)
Density-dependent survival
Density-independent survival
Rey and Alcántara (2014)
Groups for which
plant fitness is
affected by where
or when dispersal
occurs. Thin/vulnerable seed coats
Thick/hard/spiky seed coats
Notman and Gorchov (2001)
Shade-intolerant
Shade-tolerant
Alvarez-Clare and Kitajima
(2007)
Fire-intolerant
Fire-tolerant
Wenny (2001)
Self-incompatible
Self-compatible
Bond (1994)
Reproduction by seed only
Reproduces asexually
Bond (1994)
Intolerant of low nutrients
Tolerant of low nutrients
Wenny (2001)
Low phenotypic plasticity
High phenotypic plasticity
Goh et al. (2013)
Metapopulation-dependent
Continuous population distribution
Bohrer et al. (2005)
Negative distance-dependent
mortality
No negative distance-dependent
mortality
Beckman et al. (2012)
Inability to seed bank
Seed banking
Gutterman (2000)
Seasonal dispersal
Low dispersal seasonality
Ruggera et al. (2015)
Groups for which
plant fitness is
affected by the
vector of dispersal
Seed size*
Seed size*
Tamme et al. (2014)
Intraspecific competitor/
non-facilitator
Intraspecific facilitator
Martorell and Freckleton (2014)
Seed coat with germination
inhibitors
No seed coat germination inhibitors
Traveset and Verdú (2002)
aded
o
ps //acade
c oup co
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c e abs ac /
/ /p 006/5308909 by U a
S a e U
e s y traits, functional group frameworks unite species shar-
ing those traits under a common lens allowing gener-
alization across diverse organisms. Because functional
groups by definition describe the ecological functions
present in a given site, functional group diversity has
in some cases been found to predict whole-ecosystem
function almost as well (or better than) species diver-
sity (Dı́az and Cabido 2001). At the same time, func-
tional groups are conceptual constructs and thereby
subject to the perspective of ecologists identifying traits
they deem important to particular questions. Beginning
with SDE allows us to anchor our functional group rec-
ommendations in a robust and established guiding traits, functional group frameworks unite species shar-
ing those traits under a common lens allowing gener-
alization across diverse organisms. Seeking predictive capacity in light of global
change: adapting current frameworks for
functional groups These groups are categorized
based on how much it matters whether a seed is dispersed (shown in red; groups 1–3); how much it matters where or when dispersal occurs
(shown in blue; groups 4–14); and how much it matters what vector disperses the seed (shown in green; groups 15–17). Applying vulnerability
assessments and SDE calculations at the level of these functional groups may enable us to achieve a predictive understanding of seed
dispersal ecology in the face of combined global change and complexity. *For species exhibiting a measurable fitness boost from dispersal,
seed size may dictate which abiotic or biotic vectors are effective. Aslan et al. – Defining functional groups relevant to the importance of seed dispersal Aslan et al. – Defining functional groups relevant to the importance of seed dispersal Previous uses of functional groups in seed dispersal
ecology have been narrow in scope (focusing on single
systems or a small number of focal functions) but are
indicative of the usefulness of this approach. For ex-
ample, Dennis and Westcott (2006) distilled 26 detailed
measurements of seed disperser traits into 10 trait di-
mensions. They used these dimensions to identify 15
functional groups in a suite of 65 Australian seed dis-
perser species; their mathematical approach could be
more broadly applied to reduce complexity in other sys-
tems (Dennis and Westcott 2006). Rodríguez-Rodríguez
et al. (2017) categorized plant–animal interactions into
typologies and evaluated links between these typolo-
gies and plant fitness. Zamora (2000) explored how
the consistency of fitness benefits offered by seed dis-
persal across systems and groups of species permits
generalization within functional groups. A key benefit of
a functional group approach is that it could provide an
understanding of the functions that may be lost when
extinctions occur (Blondel 2003; Bastazini et al. 2017). This conservation-oriented conceptual application was
highlighted by Schleuning et al. (2014) in their call for
more work examining the linkage between trait-based
approaches such as functional group delineation and
structural approaches such as network analysis (Ruggera
et al. 2015). Functional groups can be used to predict
the role of suites of species in an ecosystem and the re-
sponse of those species to drivers of global change. Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 Because functional
groups by definition describe the ecological functions
present in a given site, functional group diversity has
in some cases been found to predict whole-ecosystem
function almost as well (or better than) species diver-
sity (Dı́az and Cabido 2001). At the same time, func-
tional groups are conceptual constructs and thereby
subject to the perspective of ecologists identifying traits
they deem important to particular questions. Beginning
with SDE allows us to anchor our functional group rec-
ommendations in a robust and established guiding comprehensive framework. Thus, the three broad cat-
egories of functional groups described below have been
selected to distil the comprehensive SDE framework into
straightforward conceptual bins. We acknowledge that
other approaches to simplifying matters are possible,
perhaps based on different criteria, or on different quan-
tifications of the same criteria. However, as we demon-
strate here, considerable insights can be obtained with
our approach. It is also important to acknowledge that,
compared with a species-level SDE analysis, a functional
group-level SDE analysis carries a risk of overgeneraliza-
tion, whereby meaningful sources of variation are dis-
missed due to limited understanding. 5 © The Author(s) 2019 AoB PLANTS https://academic.oup.com/aobpla Functional groups distinguishing how much it
matters if a seed is dispersed at all Species may be categorized based on the importance
of dispersal for their survival and reproduction. Well-
established frameworks examining fitness benefits that
may be derived from seed dispersal can guide functional
group determination in this arena, since functional
groups can be defined by traits linked to such fitness
benefits. The escape hypothesis states that seeds will
experience fitness boosts as a result of removal from
the neighbourhood of natural enemies, including patho-
gens, parasites, herbivores and competitors (Howe and
Smallwood 1982; Howe and Miriti 2004). Escape from be-
neath the canopy of a parent tree reduces the chances
of pathogens and herbivores finding a seed (Janzen
1970; Connell 1971), as well as the chance that a seed
will be deposited immediately adjacent to a close rela-
tive and thus compete for necessary resources. A logical
extension of this hypothesis suggests that species can
be assembled into functional groups by traits indicating
dependence upon such escape (i.e. susceptible to infec-
tion or herbivory; exhibiting negative density-depend-
ence in survival and growth) vs. those less dependent
upon escape (i.e. exhibiting thick seed coats or other
protections against infection and herbivory; exhibiting
low negative density-dependence). As an example, in a
study of olive (Olea europaea) regeneration in human-
altered vs. unaltered landscapes in Spain, proximity to
maternal trees was associated with elevated seedling
mortality; O. europaea thus appears to occupy a func-
tional group characterized by escape dependence and
negative density-dependence (Rey and Alcántara 2014)
(Table 1). Similarly, fungal pathogens led to strong den-
sity-dependent mortality in Pleradenophora longicuspis
in Belize, evidence that functional group categorization
based on density-dependence is appropriate for this
species (Bagchi et al. 2010). By contrast, species with low
density-dependence, and thus likely to be classified into
functional groups with reduced dispersal-dependence,
include a suite of common species in a Panamanian
rainforest, where density-dependence varies consider-
ably among tree species (Comita et al. 2010). Species
with greater seed mass exhibited reduced negative
density-dependence on Barro Colorado Island (Lebrija-
Trejos et al. 2016). Previous species-specific studies have examined
density dependent damage and mortality in seeds and Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 Functional groups distinguishing how much it
matters where or when a seed is dispersed Downloaded from https://academic.oup.com/aobpla/article-abstract/11/2/plz006/5308909 by Utah State University Libraries user on 24 June 2019 A large fraction of the ‘quality’ element of the SDE frame-
work centres on where and when a seed is dispersed. Fundamentally, this will dictate which abiotic and bi-
otic resources and threats are encountered by the seed
and subsequent plant (Schupp et al. 2010; Beckman and
Rogers 2013). Abiotic resources may include nutrients,
moisture, space and light. Abiotic threats could include
drought, nutrient deficiencies, frost and fire. On the bi-
otic side, resources could include mutualists such as soil
mycorrhizae, pollinators, seed dispersers and facilita-
tors, and threats could include herbivores, competitors,
predators and pathogens. Functional groups that cat-
egorize species by whether deposition setting matters
to a seed may include (i) groups of plants that are par-
ticularly susceptible to abiotic stressors/disturbances or
natural enemies (e.g. plants with low competitive ability
or thin seed coats) vs. (ii) those tolerant of threats (e.g. shade-tolerant, fire-tolerant, drought-tolerant, etc.). Other relevant functional groups would include spe-
cies dependent on mutualists or facilitators (Calvo and
Horvitz 1990; Onguene and Kuyper 2002; Hoehn et al. 2008; Teste et al. 2009), frost-intolerant species that re-
quire nurse plants, species dependent on forest gaps to
escape shading and species that require a narrow range
of soil nutrient content. Interspecific interactions can affect parameters of
matrix population models, enabling their effect on fit-
ness to be examined using elasticity and sensitivity
analyses (McGraw and Caswell 1996; Horvitz et al. 1997;
Benton and Grant 1999; Mills et al. 1999; Carslake et al. 2009; Jongejans et al. 2011). The effect of seed dispersal
failure can be explored via elasticity analyses simulating
loss of dispersers and resulting failure to escape from
natural enemies or encounter recruitment sites (Howe
and Miriti 2004; Brodie et al. 2009a; Rodríguez-Pérez
and Traveset 2012; Traveset et al. 2012; Caughlin et al. 2015; Pérez-Méndez et al. 2015). Applying such analyses
to functional groups that enable generalization beyond
a few carefully measured surrogates to other species
within a group might greatly expand the predictive cap-
acity of such analyses across systems.ii Plant species with plastic phenotypes may be rela-
tively generalized with regard to their interspecific inter-
action requirements, suggesting that functional groups
defined by plasticity may be appropriate. Plasticity may
influence dependence upon certain abiotic conditions or
interspecific interactions. Meaningful Functional Groups in Seed
Dispersal Ecology We define plant functional groups based on traits influ-
encing the importance of seed dispersal for plants (Table
1). Our proposed functional groups categorize plants
based on: (i) how important it is to plant recruitment
if seeds are dispersed at all, (ii) how important the lo-
cation and timing of seed deposition are and (iii) how
much vector identity matters. These groups thus define
important points in the dispersal process at which seed
fate may be influenced, with a focus on the fitness bene-
fits derived from dispersal events. Applying SDE to these
functional groups will enable researchers to predict how
populations of the species within a group will be af-
fected by total or partial dispersal disruption, changes in
phenology or habitat conditions, or entry of non-native
species into dispersal networks. A given plant species
may display traits that make them likely vulnerable to
dispersal disruption based on one functional group cat-
egory and less vulnerable based on another; in such a
case, these categories will help to pinpoint sources of
such vulnerability. Previous species-specific studies have examined
density-dependent damage and mortality in seeds and
seedlings encountering abundant herbivores, patho-
gens and predators in close proximity to parent trees
(the Janzen–Connell effect) (e.g. Petermann et al. 2008; Bagchi et al. 2010; Liu et al. 2012). Study results
have been mixed, but largely show increased success 6 © The Author(s) 2019 AoB PLANTS https://academic.oup.com/aobpla Aslan et al. – Defining functional groups relevant to the importance of seed dispersal at greater distances from one another (Snyder and
Chesson 2003; Snyder 2011). of seeds and seedlings after removal from the parent,
with various explanatory mechanisms (e.g. Thomas
1990; Blundell and Peart 1998; Packer and Clay 2000;
Petermann et al. 2008; Bagchi et al. 2014). These studies
suggest that assigning seeds to functional groups based
on whether they exhibit negative density-dependence is
important. For those groups that do exhibit such den-
sity-dependence (Table 1), loss of dispersers may be
expected to affect plant fitness significantly. Assigning
plants to functional groups may in some cases be pos-
sible through observational studies, generalizing from
what we know about similar species, and in other cases
may require experimental assessments—which are still
far less extensive than a traditional species-specific SDE
assessment (Table 1).i Functional groups distinguishing how much it
matters where or when a seed is dispersed Gutterman 2000). In
other cases, the timing of dispersal interacts with char-
acteristics that determine habitat quality (e.g. ephem-
eral environmental conditions or seasonally migratory
dispersers) (Ruggera et al. 2015). Timing can matter on
the plant side, too: in a study of Pistacia lentiscus dis-
persal in Spain, seed viability was found to vary during
the fruiting season, such that dispersers interacting with
the species when viability is high were more effective
than those handling fruits at other times (González-Varo
et al. 2018). as seed size sets a lower limit on the type and size of
dispersal vector that can lift (e.g. wind) or ingest (e.g. animals) the seed (Wheelwright 1985; Ganeshaiah and
Shaanker 1991; McConkey and Drake 2002). In some
cases, dispersal syndromes explain some variation in
dispersal distances and can be used to predict dispersal
distances (Tamme et al. 2014), but the variation within
dispersal syndromes can be very high (Clark et al. 2005;
Muller-Landau et al. 2008). Dispersal syndromes tend
to be broad categories (e.g. large mammal vs. small
mammal vs. wind). Even within these categories, spe-
cies may be dispersed by a diversity of vectors, and in
some cases secondary dispersal is performed by an al-
together different class of vector than primary dispersal
(Böhning-Gaese et al. 1999; Vander Wall and Beck 2012). Whether the identity of the vector matters to the even-
tual success of the seed is an important component of
understanding the role of dispersal in eventual plant
population and community dynamics. In spite of these successful attempts to achieve gen-
eral insights, there are certain risks associated with
generalizing across vectors (or dispersers). Identifying
a dispersal syndrome may suggest that a broad cate-
gory of vector is the likely disperser, but such categories
could include many potential disperser species varying
in effectiveness (Jordano et al. 2007; Howe 2016). Thus,
dispersal syndromes are not sufficient to predict the
effects of losing certain vectors. Nor do dispersal syn-
dromes give us information on the likelihood of being
dispersed by a ‘non-standard’ dispersal vector—that is,
a vector other than the most common vector or vec-
tors interacting with a particular plant—which might be
more influential than ‘standard’ vectors in long-disper-
sal events (Higgins et al. 2003; Jordano et al. 2007) and
therefore exert larger effects on plant populations (Kot
et al. 1996; Neubert and Caswell 2000). Functional groups distinguishing how much it
matters where or when a seed is dispersed As an important
lesson for dispersal ecology, the concept of syndromes
has faced substantial criticism in pollination ecology
(Ollerton et al. 2009). Careful empirical study has dem-
onstrated that in most cases both plants and pollinators
are much more opportunistic and interact with a much
broader suite of partners than morphological pollination
syndromes would suggest (Waser et al. 1996; Fenster
et al. 2004; Ollerton et al. 2009; Waser et al. 2018). If
syndromes are similarly uninformative in dispersal, this
carries implications for conservation and management,
since incorrect generalization stemming from syn-
dromes could lead to fallacious assumptions about the
redundancy of dispersers within interaction networks
and, consequently, about restoration and conservation
needs (Howe 2016). In theory, the functional groups most dependent
upon dispersal include species in patchy habitats, those
with strong density-/distance-dependent mortality,
those lacking the ability to maintain a seed bank and
those with specific requirements for the timing and lo-
cation of the dispersal event (Table 1). Functional groups distinguishing how much it
matters where or when a seed is dispersed For example, mycorrhizal asso-
ciations could provide critical assistance to plant individ-
uals with delicate or small root systems, but individuals
with plastic growth (e.g. those able to divert resources
towards robust root growth as required) might be less
affected by an absence of root symbionts (Valladares
et al. 2007; Goh et al. 2013). Other fitness benefits of seed dispersal may arise from
colonization of unpredictable and newly available germi-
nation sites and directed dispersal to hospitable micro-
sites located within a non-hospitable matrix (Wenny
2001; Howe and Miriti 2004). Dispersal is likely to mat-
ter most to functional groups of species with specialized
spatio-temporal germination and growth site require-
ments or low competitive ability and thus high depend-
ence on vacant establishment sites. Identification of
such species may be informed by competition/coloniza-
tion trade-off theory, which predicts that species exhibit
a trade-off between dispersal ability and competitive
ability (for example, plant species may trade off the
production of a few large, well-provisioned seeds for
the production of many smaller seeds) (e.g. Bolker and
Pacala 1999; Dalling and Hubbell 2002; but see Coomes
and Grubb 2003). Similarly, theoretical ecologists have
investigated when long-distance dispersal vs. local dis-
persal is evolutionarily advantageous, given the fitness
advantages of colonizing new sites and the lower prob-
ability of finding habitats sharing specific characteristics Dispersal also matters for plants living in habitats
that are temporally or spatially variable. Important
functional groups include those with specific habitat
requirements that are spatially heterogeneous (e.g. species dependent on metapopulation processes for
persistence; Bohrer et al. 2005) vs. general habitat re-
quirements that are widespread and homogeneous. For example, in a human-disturbed, patchy landscape,
affinity of dispersers for seedling habitat leads to in-
creased germination of the relic Chinese yew (Taxus 7 AoB PLANTS https://academic.oup.com/aobpla © The Author(s) 2019 Aslan et al. – Defining functional groups relevant to the importance of seed dispersal chinensis), indicating that directed dispersal matters
for this endangered plant species (Li et al. 2016) (Table
1). In another example, seeds of the shrub Daphne rod-
riguezii dispersed to sites below nurse plants exhibit
higher seedling survival (Rodríguez-Pérez and Traveset
2010) (Table 1). Functional groups distinguishing how much it
matters where or when a seed is dispersed Lower location specificity can be found
in, for example, shade-tolerant species that exhibit
physical defence mechanisms and are thus able to sur-
vive and reproduce in conditions of high competition;
eight such species were studied in Panama and their
physical traits documented (Alvarez-Clare and Kitajima
2007). This indicates that material characteristics can be
identified to classify such species into functional groups
with less dependence on dispersal (Table 1). Some func-
tional groups may be affected by positive or negative
distance- or density-dependent mortality (e.g. if seeds
must be dispersed in clumps to germinate and grow;
Beckman et al. 2012), or may require rare micro-condi-
tions (Pufal and Garnock-Jones 2010). Temporally, some
species can protect themselves against poor dispersal
years by living many years as adults or remaining viable
in a seed bank for a long time (e.g. Gutterman 2000). In
other cases, the timing of dispersal interacts with char-
acteristics that determine habitat quality (e.g. ephem-
eral environmental conditions or seasonally migratory
dispersers) (Ruggera et al. 2015). Timing can matter on
the plant side, too: in a study of Pistacia lentiscus dis-
persal in Spain, seed viability was found to vary during
the fruiting season, such that dispersers interacting with
the species when viability is high were more effective
than those handling fruits at other times (González-Varo
et al. 2018). chinensis), indicating that directed dispersal matters
for this endangered plant species (Li et al. 2016) (Table
1). In another example, seeds of the shrub Daphne rod-
riguezii dispersed to sites below nurse plants exhibit
higher seedling survival (Rodríguez-Pérez and Traveset
2010) (Table 1). Lower location specificity can be found
in, for example, shade-tolerant species that exhibit
physical defence mechanisms and are thus able to sur-
vive and reproduce in conditions of high competition;
eight such species were studied in Panama and their
physical traits documented (Alvarez-Clare and Kitajima
2007). This indicates that material characteristics can be
identified to classify such species into functional groups
with less dependence on dispersal (Table 1). Some func-
tional groups may be affected by positive or negative
distance- or density-dependent mortality (e.g. if seeds
must be dispersed in clumps to germinate and grow;
Beckman et al. 2012), or may require rare micro-condi-
tions (Pufal and Garnock-Jones 2010). Temporally, some
species can protect themselves against poor dispersal
years by living many years as adults or remaining viable
in a seed bank for a long time (e.g. Functional groups enabling us to distinguish how
much the identity of the dispersal vector matters 2007; Bueno et al. 2013; Escribano-Avila et al. 2014; González-Varo et al. 2017). Plant functional groups of interest when deter-
mining whether a specific vector is important include
groups defined by seed size, seed coat thickness (e.g. groups of species with thick coats requiring substantial
gut treatment for germination), presence of germin-
ation inhibitors, and intraspecific facilitation or positive
density-dependence. scale, straightforward functional groups founded on
seed morphology and physiology (e.g. determined by
seed size and shape) may dictate potential disperser
suites and indicate how important different dispersal
vectors may be, relative to one another, for a given
plant species (Table 1). Identifying plant species at risk
from dispersal disruption (e.g. McConkey et al. 2018)
may be possible when the importance of vector iden-
tity is understood. Vectors may differ in the number of
seeds dispersed, the condition of dispersed seeds, dis-
persal distances and dispersal spatial arrangements. Wind, for example, is most likely to move seeds that are
small in mass (Shea 2007; Nathan et al. 2011). Disperser
animals with large gape sizes are more likely than small
dispersers to disperse greater numbers of larger seeds
over longer distances (Cox et al. 1991). Since large dis-
persers with low reproductive rates are often most
threatened by direct human exploitation coupled with
low reproductive rates (Farwig and Berens 2012), the
plant functional group that includes large-seeded spe-
cies is of particular interest in seed dispersal conserva-
tion. Losses of key large dispersers can threaten plant
species and functional group diversity in seed dispersal
networks (Donoso et al. 2017). Dispersers with special-
ized habitat requirements may aggregate seeds by re-
turning frequently to a limited number of sites (Howe
1989). Because different vectors may provide dispersal
services in different ways, plant species may experience
complementary dispersal services from them, with a
greater diversity of vectors maximizing the success of a
plant (Levin et al. 2003; Jordano et al. 2007; Bueno et al. 2013; Escribano-Avila et al. 2014; González-Varo et al. 2017). Plant functional groups of interest when deter-
mining whether a specific vector is important include
groups defined by seed size, seed coat thickness (e.g. groups of species with thick coats requiring substantial
gut treatment for germination), presence of germin-
ation inhibitors, and intraspecific facilitation or positive
density-dependence. to the functional groups we propose (Table 1) (McConkey
et al. 2018). Functional groups enabling us to distinguish how
much the identity of the dispersal vector matters By contrast, SDE of a suite of bird species
was studied for two Miconia species in Brazil (Santos
et al. 2017). Although the birds varied in the quantity
of seeds they dispersed, they did not vary in quality of
dispersal (Santos et al. 2017). Miconia species with their
small seeds and large disperser suites (e.g. Levey and
Byrne 1993) therefore appear to fall into a proposed
functional group for which vector identity is less impor-
tant (Table 1). Dispersal vector identity has been shown to af-
fect population growth rates for some but not all of
the few vertebrate-dispersed plant species that have
been studied (e.g. Godinez-Alvarez and Jordano 2007;
Brodie et al. 2009b; Loayza and Knight 2010). However,
the importance of different vectors is unknown for
most plant species, and that lack of clarity ham-
pers our ability to predict the outcomes of changes
in vectors. Predictions are better-developed for bal-
listic- and wind-mediated dispersal than for animal-
mediated dispersal (Skarpaas and Shea 2007; Nathan
et al. 2011; Bullock et al. 2012), in large part because
of the complexity of animal behaviour and movement
and the diffuse nature of most seed dispersal systems,
wherein multiple animals disperse any given plant
(Shea 2007). Even when detailed information about
the role of specific vectors has been obtained for a
given plant species, studies are often narrow in spa-
tial and temporal extent and thus context-dependent
(i.e. information is specific to a particular time and
place, given a particular disturbance history), and the
importance of individual vectors may change under
different contexts. Functional groups enabling us to distinguish how
much the identity of the dispersal vector matters Dispersal syndromes are used to categorize plants by
the type of vector known or assumed to best disperse
their seeds. Syndromes are the most common functional
group classifications used in seed dispersal ecology. Illustrating the potential value of generalization across
similar dispersers, Tamme et al. (2014) successfully used
plant traits to predict dispersal distances for over 500
species. Dispersal distances could then be related to dis-
persal syndromes, growth form and other plant traits,
such as plant height and seed size (Thomson et al. 2011;
Tamme et al. 2014). Previous studies have reported an
interaction between seed size and dispersal vector size, Although the use of dispersal syndromes per se thus
carries a risk of drawing conclusions at too crude a 8 AoB PLANTS https://academic.oup.com/aobpla © The Author(s) 2019 Aslan et al. – Defining functional groups relevant to the importance of seed dispersal scale, straightforward functional groups founded on
seed morphology and physiology (e.g. determined by
seed size and shape) may dictate potential disperser
suites and indicate how important different dispersal
vectors may be, relative to one another, for a given
plant species (Table 1). Identifying plant species at risk
from dispersal disruption (e.g. McConkey et al. 2018)
may be possible when the importance of vector iden-
tity is understood. Vectors may differ in the number of
seeds dispersed, the condition of dispersed seeds, dis-
persal distances and dispersal spatial arrangements. Wind, for example, is most likely to move seeds that are
small in mass (Shea 2007; Nathan et al. 2011). Disperser
animals with large gape sizes are more likely than small
dispersers to disperse greater numbers of larger seeds
over longer distances (Cox et al. 1991). Since large dis-
persers with low reproductive rates are often most
threatened by direct human exploitation coupled with
low reproductive rates (Farwig and Berens 2012), the
plant functional group that includes large-seeded spe-
cies is of particular interest in seed dispersal conserva-
tion. Losses of key large dispersers can threaten plant
species and functional group diversity in seed dispersal
networks (Donoso et al. 2017). Dispersers with special-
ized habitat requirements may aggregate seeds by re-
turning frequently to a limited number of sites (Howe
1989). Because different vectors may provide dispersal
services in different ways, plant species may experience
complementary dispersal services from them, with a
greater diversity of vectors maximizing the success of a
plant (Levin et al. 2003; Jordano et al. AoB PLANTS https://academic.oup.com/aobpla Acknowledgements Our discussion emerged from a National Science
Foundation-funded Seed Dispersal Workshop held at
the National Socio-Environmental Synthesis Center in
May 2016. All workshop participants are co-authors on
this paper and have contributed to the development of
these ideas. Contributions by the authors C.A. led the development of the concepts, writing, and
revising of the manuscript with input from N.G.B. and
H.S.R. All authors contributed to the development of
concepts and are listed in order of contribution and
alphabetical order within each level of contribution. Sources of funding Ideas for this manuscript initiated during the Seed
Dispersal Workshop held in May 2016 at the Socio-
Environmental Synthesis Center in Annapolis, MD and
supported by the US National Science Foundation
Grant DEB-1548194 to N.G.B. and the National Socio‐
Environmental Synthesis Center under the US National
Science Foundation Grant DBI‐1052875. D.Z. received
funding from the Swiss National Science Foundation
(SNF, grant: PZ00P3_168136/1) and from the German
Science Foundation (DFG, grant: ZU 361/1- 1). Compiling large amounts of data from multiple
systems to test functional group approaches can
be resource-intensive, although increasingly global
databases are becoming available and serving as
common data hubs (e.g. COMPADRE Plant Matrix
Database and COMADRE Animal Matrix Database;
Max Planck Institute for Demographic Research
(Germany); available at www.compadre-db.org). One promising approach is to standardize data col-
lection by many research groups working across
many systems (e.g. NutNet; Borer et al. 2014). If
groups collect the same data across systems, those
data can be introduced to a common modelling plat-
form to explore patterns that hold across systems. By assembling data sets in this way, the costs are
spread across research groups, and data collection
can be useful even when sample sizes within a spe-
cific system are limited. As an example, standard-
ized data collection across systems has enabled
researchers to identify consistent patterns of grass-
land responses to land use change (Garnier et al. 2007). We thus recommend that research teams join
forces to collect standardized data exploring vary-
ing effectiveness of dispersal by different agents,
the role of spatio-temporal dynamics and the influ-
ence of interspecific interactions pre-, mid- and
post-dispersal. Once data are collected in many sys-
tems, it will be necessary to bring them together to
make them available for broad analysis. Depositing
seed dispersal data into public-access repositories
is therefore important. Useful repositories include
Dryad (http://datadryad.org) and KNB (http://knb. ecoinformatics.org). AoB PLANTS https://academic.oup.com/aobpla Using Functional Groups to Close Our
Knowledge Gaps The use of functional groups defined by dispersal-
related traits can reduce the amount of data needed to
parameterize models (Mokany et al. 2014). The digital
availability of trait data is increasing (e.g. via publicly ac-
cessible databases such as TRY; Kattge et al. 2011) but
continued empirical research is needed to relate those
data to dispersal processes. Even so, certain functional
groups can now be defined and used to distinguish spe-
cies that are relatively more or less strongly dispersal-
dependent (Table 1). If a particular plant species belongs
to a group for which fitness is strongly linked to dispersal
(Table 1, column 2), we can predict that this species is
likely to be vulnerable in the face of dispersal disruption,
based strictly on functional group. Exemplifying the importance of this functional group
delineation, different behaviours of large mammal dis-
persers resulted in differential contributions to dispersal
of the large-seeded Platymitra macrocarpa in Thailand,
with some species dispersing higher quantities of seeds
with poor survival outcomes and others dispersing fewer
seeds with greater success per seed (McConkey et al. 2018). In this case, dispersers contributed differentially
to the dispersal of the plant but overall plant regenera-
tion was poor, leading researchers to speculate that
there may be important dispersers that are missing or
rare (McConkey et al. 2018). In that context, the large
seed size of the plant suggests that the identity of the
dispersal vector in this example is important according While functional groups may enable us to generalize
across full plant communities, overgeneralization could 9 AoB PLANTS https://academic.oup.com/aobpla © The Author(s) 2019 Aslan et al. – Defining functional groups relevant to the importance of seed dispersal plant populations and communities. We invite the eco-
logical community to join us in this effort. cause us to lose sight of meaningful sources of varia-
tion. It is thus necessary to test the value of functional
group approaches in seed dispersal ecology by generat-
ing predictions based on them and then testing those
predictions against species-specific data. As functional
groups ‘pass’ these tests, our ability to generalize by
constructing our models and predictions around func-
tional groups will help us manage the broad variability
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The main directions in the implementation of organic agriculture
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* Corresponding author: zuev_1960_nikolai@mail.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons
Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). The main directions in the implementation of
organic agriculture
N.P. Zuev*, N.N. Shvetsov, Y.P. Breslavets, V.A. Shumsky, and Y.P. Masalykina
Belgorod State Agrarian University named after V. Y. Gorin, Belgorod, Russia N.P. Zuev*, N.N. Shvetsov, Y.P. Breslavets, V.A. Shumsky, and Y.P. Masalykina
Belgorod State Agrarian University named after V. Y. Gorin, Belgorod, Russia Abstract. Measures for the introduction of organic agriculture, along with
the use of appropriate techniques in agriculture, imply the rejection of the
use of chemical-synthetic growth regulators or hormones; limited, strictly
tied to the area, livestock; feeding animals, if possible, with their own feed,
minimum purchase of feed and avoiding antibiotics. However, there is
currently no way to permanently stop using antibiotics. Therefore, a
gradual decrease in doses, frequency and timing of their application is
relevant. The work is devoted to the implementation of this direction. A
synergistic relationship of gentamicin and tylosin was established with an
increase in the efficiency of using the created composite preparation with a
decrease in the dose and the possibility of the formation of drug-resistant
populations of bacteria - causative agents of animal diseases. BIO Web of Conferences 39, 05003 (2021)
MTSITVW 2021 BIO Web of Conferences 39, 05003 (2021)
MTSITVW 2021 https://doi.org/10.1051/bioconf/20213905003 2 Materials and methods In relation to organic livestock are needed [6]: - description of premises in which animals are raised, areas for outdoor walking,
pastures, outdoor trails, etc., as well as premises for slaughter of animals and premises for
packaging and storage of animal products, incoming materials and raw materials; - description of premises in which animals are raised, areas for outdoor walking,
pastures, outdoor trails, etc., as well as premises for slaughter of animals and premises for
packaging and storage of animal products, incoming materials and raw materials; - a complete description of the manure warehouses or storage areas that are obtained
from the breeding of animals; - a complete description of the manure warehouses or storage areas that are obtained
from the breeding of animals; - contracts concluded with other producers (if necessary), when applying manure,
i h
b i
d d
i
h b
di
f
i
l - contracts concluded with other producers (if necessary), when applying manure,
which was obtained during the breeding of animals; - contracts concluded with other producers (if neces
which was obtained during the breeding of animals; - information on breeding or origin of livestock; - information about the purchase or sale of livestock; - a veterinary care plan used to prevent and treat disease, injury and reproductive
problems; - information on all medical procedures and medicines used for any purpose, including - information on all medical procedures and medicines used for any purpose, including
for the purposes of ensuring the quarantine period and processing of animals; information on all medical procedures and medicines used for any purpose, inclu
for the purposes of ensuring the quarantine period and processing of animals; e purposes of ensuring the quarantine period and processing of animals; - information about purchased feed and its sources, animal rations; - information on the movement of livestock within the production unit; - information on the transportation, slaughter and sale of animals outside the
production unit. 1 Introduction The main idea of organic farming is to manage the farming in accordance with the laws of
nature. At the same time, an agricultural enterprise is considered, first of all, as an organism
with its constituent parts - man, animal, plant and earth. It is planned to increase the
consumption of organic products in the Belgorod region. At the same time, the speed of this
process is not sufficient and does not correspond to the needs of society. In 2020, this
economic and social direction accounted for 1-3% of the total volume of livestock
production of all types of property. Purpose: development and implementation of the main elements of organic agriculture
in animal husbandry in the system of organic-rational relationship of its constituent parts:
land, plants, animals and humans. The main directions of the implementation of organic agriculture include: rejection of
plant protection products with chemical-synthetic means; the use of slow-acting natural
fertilizers (encapsulated and nanostructured pesticides of various directions); refusal to use
chemical-synthetic growth regulators or hormones; livestock associated with land;
production of animal feed directly on the farm; development of antibiotic substitution
measures and minimization of their harmful effect on the earth-plant-animal-human system
(the main components of organic agriculture). https://doi.org/10.1051/bioconf/20213905003 BIO Web of Conferences 39, 05003 (2021)
MTSITVW 2021 2 Materials and methods An important component of the relationship in the earth-plant-animal-human system is the
production of micronutrient-enriched food products: the production of fortified human
food products up to 25-35% (by 2030); the fortification of dairy products, flour and bread
with minerals and vitamins [6] An important social moment is the possibility of
consumption of food products enriched with micronutrients by adults with low and low
incomes and children in social institutions, as well as their use in the prevention of
pathologies associated with a lack of biologically active substances in humans (vitamins,
vitamin-like substances, macro- and microelements). Organizational and economic support for the implementation of organic agriculture
necessarily provides for the associated and coordinated activities in agricultural
technology, fodder production, plant and animal protection, processing industry
technologies at the regional, municipal level of action [6]. In the presence of livestock on the farm, the necessary need for feed (coarse, juicy,
green, concentrated) is determined, and how much feed will be obtained from natural
lands. The missing amount of forage must be produced on arable land [6]. Determine which crops will cover the need for hay, silage, haylage, green fodder and
concentrates. 2 Materials and methods As an example, the specific implementation of one of the areas of organic farming,
and organic agriculture in general, we present the technology for the development of the
production and use of composite antimicrobial drugs, which determine their use in lower
doses and thereby reduce their intake into the body of animals during their treatment and
prevention of diseases, people with food, together with manure into the soil, reducing the
possibility of the formation of drug-resistant populations of bacteria - causative agents of
diseases of animals and humans and their harmful effect on the biological components of
the soil. The use of such funds helps to increase the rate of elimination (excretion) of them
from the body of animals. BIO Web of Conferences 39, 05003 (2021)
MTSITVW 2021 The proposed invention relates to animal husbandry and can be used for therapy with
Escherichiosis gastroenteritis of piglets. The proposed invention relates to animal husbandry and can be used for therapy with
Escherichiosis gastroenteritis of piglets. Known drugs for therapy with gastroenteritis of pigs using various antibacterial
agents: tanibel, phosphopag, combinations of tylosin, nitrofurans, and nitazole [7, 8, 9,
12]. Therapeutic efficacy in gastroenteritis of piglets has fradizin itylosin tartrate [1, 2, 3,
4]. Tylosin tartrate is one of the preparations for microbiological synthesis. The
disadvantages of tylosin-containing preparations and other above-mentioned preparations,
including in relation to E. coli, are: a narrow spectrum of activity in relation to
microorganisms that cause gastroenteritis in pigs, the rapid emergence of pathogens of
resistance to them. Based on the foregoing, the task of the invention is set: the creation of
a drug for therapy for Escherichiosis gastroenteritis of pigs by increasing the prophylactic
activity of tylosin for gastroenteritis of pigs by increasing its antimicrobial activity and the
creation of its composite forms. p
When solving the task, it was necessary: 1. Expansion of the range of antimicrobial drugs in severe infections against
microbial pathogens of gastroenteritis in piglets that require immediate initiation of
treatment before bacteriological diagnosis, or in mixed infection, when pathogens of
microbial association have different susceptibility to antibacterial drugs. 2. To prevent the development of toxic effects by achieving a rapid and more
complete effect with the simultaneous introduction of two or more drugs in smaller than
usual, course doses. This is important when using antibiotics that cause nephrotoxic
(aminoglycosides, polymyxin, griseofulvin), hepatotoxic (tetracycline, erythromycin,
novobicin) effects, adverse effects on the gastrointestinal tract (tetracycline
(chloracycline, erythioramine, erythiram). 3. Achieving a potential antibacterial effect. 4. Reduction or elimination of the possibility of the acquired drug resistance of
pathogenic microorganisms. Based on the above requirements, macrolide, nitrofuran and aminoglycoside
(gentamicin) drugs are promising as means for creating composite antibacterial drugs
with a wide spectrum of action. Based on the above requirements, macrolide, nitrofuran and aminoglycoside
(gentamicin) drugs are promising as means for creating composite antibacterial drugs
with a wide spectrum of action. Tylosin-containing drugs have high antimicrobial activity against gram-positive and
relatively low - gram-negative microflora. 3 Results and discussion Example No. 1
The drug for therapy with Escherichiosis gastroenteritis of piglets 2 https://doi.org/10.1051/bioconf/20213905003 BIO Web of Conferences 39, 05003 (2021)
MTSITVW 2021 BIO Web of Conferences 39, 05003 (2021)
MTSITVW 2021 Nitrofuran compounds exhibit a broad range
of antimicrobial activity Previously, we found that a synergistic combination of tylosin tartrate with furazonal
in a ratio of 3: 1 is effective in treating sick animals and preventing the development of
gastroenteritis in piglets. This combination is effective when administered with feed at a
dose of 10 mg / kg body weight [5, 6, 7]. However, due to the limited use of nitrofuran
drugs and the insufficient effectiveness of the above drugs, as well as the need to prevent
the formation of bacterial resistance, the use of antibiotics from the aminoglycoside
group (gentamicin) is more promising. We have carried out laboratory studies that determine the optimal ratio of
components in the created composite agent while studying their antimicrobial action
against bacteria that play an etiological role in the occurrence of gastroenteritis in pigs. The results of the studies are presented in table 1. 3 3 https://doi.org/10.1051/bioconf/20213905003 BIO Web of Conferences 39, 05003 (2021)
MTSITVW 2021 Table 1. Antimicrobial activity of the composition of tylosin tartrate with gentamicin. Type of micro-
organisms
Minimum bacteriostatic concentration (MBK, µg/ml)
Fractional inhibitory
concentration (μg /
ml) in a 1: 1 ratio
FIC-
index of
the
compoun
d
tylosin
tartrate
gentami
cin
compositions of tylosin tartrate with
gentamicin
tylosin
tartrate
Gentam
icin
3:1
2:1
1:1
Escherichia coli
15±0,17
10±0,12
7,03,1±0,7
6,0±0,8
5,0±0,4
0,15
0,25
0,40 Table 1. Antimicrobial activity of the composition of tylosin tartrate with gentamicin. The information presented in Table 1 on the search for the most effective proportions of
the constituent ingredients of the composite preparation indicate the potentiation of the
antimicrobial activity of the combination of tylosin tartrate with gentamicin in a 1: 1 ratio. The fractional inhibitory concentration (FIC) of tylosin tartrate in relation to E. coli when
interacting with gentamicin was 0.15, and that of gentamicin - 0.25, and the fractional
inhibitory index of the composite compound in relation to a microorganism was
respectively equal to 0.40, which predetermined the prospects for further drug
development. Prevention of adaptation in causative agents of gastroenteritis to tylosin tartrate, which
occurs after 30 passages, 30 was studied in a separate experiment, the results of which are
reflected in table 2. Table 2. Prevention of adaptation of microorganisms to causative
agents of gastroenteritis in piglets. BIO Web of Conferences 39, 05003 (2021)
MTSITVW 2021 The disadvantages of
tylosin-containing drugs are: a narrow spectrum of activity in relation to microorganisms
that cause gastroenteritis in piglets, the rapid emergence of pathogens of resistance to them. The objective of the invention: increasing the therapeutic activity of tylosin in dysentery
gastroenteritis of pigs. Based on the above requirements, the macrolide preparations tylosin
and erythromycin are promising for the creation of complex antibacterial agents with a
wide spectrum of action. Tylosin-containing drugs have high antimicrobial activity against
gram-positive and relatively low - against gram-negative microflora. Erythromycin exhibits
a wide range of antimicrobial activity [1, 2, 3, 4, 5]. A synergistic combination of tylosin
tartrate with erythromycin in a 1: 1 ratio is effective in treating sick animals. This
combination is effective when administered with feed at a dose of 10 mg / kg body weight. Laboratory studies were carried out to determine the optimal ratios of the components in
the created composite agent while studying their antimicrobial action against bacteria that
play an etiological role in the occurrence of gastroenteritis in pigs. The studies carried out
to find the most effective proportions of the constituent ingredients of the composite
preparation indicate the potentiation of the antimicrobial activity of the combination of
tylosin tartrate with erythromycin in a 1: 1 ratio. The fractional inhibitory concentration
(FIC) of tylosin tartrate in relation to Brachispira hyodysenteriae when interacting with
erythromycin was 0.4, for erythromycin, respectively, 0.1 μg / ml. FIC - the index of the
composite compound for the above microorganisms, respectively, equal to 0.2; 0.3; 0.3. In an experiment to study the therapeutic efficacy of a composite preparation at a
dosage of 5 mg / kg of body weight (animals received the preparation with food for 10
days), its high therapeutic efficacy (90%) was established in comparison with its
components (80%) and in control - 45%. The data obtained indicate that the therapeutic efficacy of the composite preparation
increases due to the synergistic interaction of tylosin tartrate and erythromycin. The present invention is a composite preparation for the prevention and treatment of
piglet dysentery by administering to animals a composition of tylosin tartrate and
erythromycin in a 1: 1 ratio. The compositional preparation is effective for piglets from 2
weeks to 4 months of age. The drug is effective at a dose of 5 mg / kg of body weight of
piglets for 10 days in the treatment of dysentery. BIO Web of Conferences 39, 05003 (2021)
MTSITVW 2021 Bacteriostatic concentration of tylosin tartrate, μg / ml nutrient medium
Background (MBK after the
first passage)
Аfter 30 passages on culture media
containing gentamicin
Colibacillus
Colibacillus
15±0.14
15±0.14 Table 2. Prevention of adaptation of microorganisms to causative
agents of gastroenteritis in piglets. From the data in Table 2, it can be seen that the antimicrobial activity of tylosin tartrate
against Escherichia coli did not change even after 30 passages. In an experiment to study the therapeutic effect of a composite drug at a dosage of 10
mg / kg of body weight (animals received the drug with food for 10 days), the results of
which are presented in Table 3, its high therapeutic efficacy (90%) was established,
compared with its components (80%). Table 3. Comparative therapeutic efficacy of drugs for gastroenteritis of pigs. Table 3. Comparative therapeutic efficacy of drugs for gastroenteritis of pigs. Group of animals
Composite
preparation
Positive control
Control
Tylosin
Gentamicin
The number of sick pigs at the
beginning of the experiment
(heads)
20
20
20
20
Recovered
- piglets (head)
18
16
16
15
- therapeutic effectiveness, %
90
80
80
75 The present invention is a compositional preparation for therapy for colorectal
gastroenteritis in pigs by administering to piglets a composition of tylosin tartrate and
gentamicin in a 1: 1 ratio. The compositional preparation is effective for piglets from 2 4 BIO Web of Conferences 39, 05003 (2021)
MTSITVW 2021 https://doi.org/10.1051/bioconf/20213905003 weeks to 4 months of age. The use of a developed and tested composite preparation with
feed is the most technologically advanced and economically feasible method. Example No. 2 weeks to 4 months of age. The use of a developed and tested composite preparation with
feed is the most technologically advanced and economically feasible method. Example No. 2 Composite preparation for the treatment of gastroenteritis in dysentery. The present invention relates to veterinary medicine and can be used for the treatment of
piglet gastroenteritis caused by brachispira. Known drugs for the treatment of
gastroenteritis of pigs using various antibacterial agents: tetracyclines, oxamycins,
macrolides and fluoroquinolones, as well as immunoglobulin-C. It is known that fradizin
and other tylosin preparations have therapeutic efficacy in piglet gastroenteritis [1. 2, 3, 4,
5, 10, 11]. However, they have a narrowly targeted antibacterial effect [1]. Tylosin is one of the preparations for microbiological synthesis. BIO Web of Conferences 39, 05003 (2021)
MTSITVW 2021 The introduction of the composite
preparation can be started when the first signs of gastroenteritis appear in piglets. The use
of a developed and tested composite preparation with feed is the most technologically
advanced and economically feasible method. 4 Conclusions Thus, the development of composite prophylactic and therapeutic agents realizes the
possibility of reducing the doses of individual pharmacological pharmacological agents and
thereby reduces the drug load on the main components of organic farming and agriculture:
soil - plants - feed - animals - humans. 5 5 https://doi.org/10.1051/bioconf/20213905003 BIO Web of Conferences 39, 05003 (2021)
MTSITVW 2021 References 1. V.A. Antipov, B.C. Grishkevich, Technical preparations of antibiotics - effective
therapeutic and prophylactic means for veterinary medicine and animal husbandry,
Collection of scientific works Pharmacology and toxicology of new drugs and feed
additives in veterinary medicine, 171-173 (L., 1990) 2. V.D. Bukhanov, Therapy and prevention of pig dysentery, 24 (Author. Dis ... Cand. vet. n. M., 1986) 3. N.P. Zuev, V.D. Bukhanov, Preparation and development of antimicrobial
compositions based on tylosin-containing preparations, Materials of the first congress
of veterinary pharmacologists of Russia, 311-316 (Voronezh, RAAS, VNIVIPF and T. - S., 2007) 4. N.P. Zuev, V.D. Bukhanov, Therapeutic efficacy of composite tylosin-containing
drugs in acute experience, Materials of the first congress of veterinary pharmacologists
of Russia, 307-311 (Voronezh, RAAS, VNIVIPF, 2007) 5. N.P. Zuev, V.A. Shumsky, A.M. Kovalenko, V.Yu. Kovaleva, E.E. Zueva, V.V. Aristov, V.V. Kontsevenko, The use of tylosin preparations in animal husbandry and
veterinary medicine, (Monograph Belgorod, 2018) 6. Concept on organic agriculture, (Belgorod, 2013) 7. RU, 2 033 172, C1, A61K 35/60 (1995.01), (20.04.1995) 8. RU 2 412 702, C1, A61K31 / 351 (2006.01), A61K 31/70 (2006.01), A61 K31 / 341
(2006.01), A61 K31 / 4196 (2006.01), A61 P31 / 09 (2006.01) (
),
(
),
(
)
9. RU 2 337 670 C2, A61 K31 / 00 (2006.01), A61 9/48 (2006.01), A61D 7/00 (2006.01) 9. RU 2 337 670 C2, A61 K31 / 00 (2006.01), A61 9/48 (2006.01), A61D 7/00 (2006.0 10. I.Ye. Mozgov, Pharmacology, 416 (M.: Agropromizdat, 1985) 11. A.M. Makukhina, A.G. Rezvykh, О.А. Gavrilova et al., Description of the invention to
the copyright certificate (11), 681598 (1981) 12. Z. Sevryuk, Environmental problems of pathology, pharmacology and therapy of
animals. - International coordination meeting, 346-348 (Voronezh) 12. Z. Sevryuk, Environmental problems of pathology, pharmacology and therapy of
animals. - International coordination meeting, 346-348 (Voronezh) 6
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Mehrheitsfähige Klimapolitik
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sui generis
Atila Stocker
Mehrheitsfähige Klimapolitik
Ein Kommentar zu «Whistleblowing für das Weltklima» von
Marc Thommen und Jascha
Mattmann, erschienen im Januar
2021 in sui generis
Am 13. Januar 2020 hat das Bezirksgericht Lausanne ein
aufsehenerregendes Urteil gefällt, indem es zwölf Klimaaktivisten, die in einer Credit-Suisse-Filiale Tennis gespielt hatten, in allen Anklagepunkten freigesprochen
hat. Das Gericht wurde scharf dafür kritisiert, dass es einen rechtfertigenden Notstand (Art. 17 StGB) angenommen hat. Einige Autoren beliessen es jedoch nicht bei
der blossen Kritik, sondern brachten gleich eine alternative Begründung vor, um von der Bestrafung der Aktivisten abzusehen. Der Autor geht hier auf den Rechtfertigungs- bzw. Schuldausschlussgrund der Wahrung
berechtigter Interessen ein, welcher von Marc Thommen
und Jascha Mattmann ins Feld geführt wurde.
I.
Zugrunde liegendes Urteil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
II. Gegenstand des Kommentars .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
III. Wahrung berechtigter Interessen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Berechtigtes Ziel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Eignung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Erforderlichkeit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV. Fazit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
104
104
104
104
105
105
106
Zitiervorschlag:
Atila Stocker, Mehrheitsfähige Klimapolitik,
sui generis 2021, S. 103
Atila Stocker, stud. iur., juristischer Mitarbeiter Switzerlend AG
(atila.stocker@uzh.ch).
URL: sui-generis.ch/173
DOI: https://doi.org/10.21257/sg.173
Dieses Werk ist lizenziert unter einer Creative Commons Namens
nennung — Weitergabe unter gleichen Bedingungen 4.0 International
Lizenz.
103
I.
1
Zugrunde liegendes Urteil
Durch das Urteil des Bezirksgerichts Lausanne
PE19.000742/PCL/llb vom 13. Januar 2020 wurde ein
Dutzend Klimaaktivisten, die in einer Credit-Suisse-
Filiale Tennis gespielt hatten, vom Vorwurf des Hausfriedensbruchs, der Hinderung von Amtshandlungen
und der unbewilligten Demonstration freigesprochen.1
Die Aktivisten wollten mit ihrer Aktion auf die Beteiligung der Banken an der Klimaerwärmung aufmerksam
machen. Der Richter nahm an, dass sämtliche Taten
durch Notstand gerechtfertigt waren. Er begründete
diesen Schluss damit, dass der Klimawandel eine imminente Gefahr für die Menschheit und somit auch für die
Beschuldigten darstelle und der Tennis-Match das mildest mögliche geeignete Mittel war, um diese Gefahr
abzuwenden. Der Entscheid wurde vielfach kritisiert.2
Das Urteil wurde durch die Folgeinstanz aufgehoben.3
II. Gegenstand des Kommentars
2
Am 20. Januar 2021 haben Marc Thommen und Jascha
Mattmann eine Besprechung des Lausanner Bezirksgerichtsurteils unter dem Titel «Whistleblowing für das
Weltklima» publiziert. Die Autoren gehen darin zuerst
auf den Vorwurf des Ungehorsams gegenüber der Polizei
ein und besprechen anschliessend die möglichen Rechtfertigungs- und Schuldausschlussgründe für die Tat. Sie
stellen richtigerweise fest, dass entgegen dem Bezirksgericht nicht von einem rechtfertigenden Notstand ausgegangen werden kann, da die begangene Tat nicht geeignet war, das Leben der Aktivisten oder Dritter zu retten.4 Auch die Voraussetzungen eines übergesetzlichen
Notstands sehen sie als nicht erfüllt an, weil die Aktion
auch nicht geeignet war, kollektive Rechtsgüter wie das
1
2
Urteil des Bezirksgerichts Lausanne PE19.000742/PCL/llb vom
13. Januar 2020; grosses Medienecho in den darauffolgenden Tagen
vgl. NZZ vom 14. Januar 2020, S. 1 (Freispruch für Klimaaktivisten)
und Tages-Anzeiger vom 15. Januar 2020, S. 4 («Das Lausanner Urteil
lässt aufhorchen»).
Vgl. Lucien Scherrer, Richter im Aktivistenmodus, NZZ vom
15. Januar 2020, S. 11; Hansueli Schöchli, «Das würde zum Chaos
führen», NZZ vom 15. Januar 2020, S. 13; Katharina Fontana, Freipass für den Rechtsbruch, Weltwoche vom 16. Januar 2020, S. 31;
Markus Melzl, Rechtsprechung mit Zeitgeist verwechselt, Basler
Zeitung vom 20. Januar 2020, S. 2; Grace Schild Trappe / Felix
Schöbi, Not kennt kein Gebot beim Hausfriedensbruch?, Jusletter
vom 11. Mai 2020. Mit abweichender Argumentation kamen einige
Autoren jedoch ebenfalls zur Straflosigkeit: Andrés Payer, Klimawandel als strafrechtlicher Notstand, sui generis 2020, S. 226; Marc
Thommen / Jascha Mattmann, Whistleblowing für das Weltklima,
sui generis 2021, S. 13; Martino Mona, in: Watson vom 15. Januar
2020 (Weshalb sich Experten nach dem Freispruch der Klimaaktivisten an den Kopf fassen).
Weltklima zu retten.5 In der Folge prüfen die Autoren die
Wahrung berechtigter Interessen sowohl als Rechtfertigungs- als auch als Schuldausschlussgrund und kommen
zum Schluss, dass die Aktivisten kein Schuldvorwurf
trifft.6 Diese Argumentation soll im Folgenden näher beleuchtet und deren Schwachstellen aufgezeigt werden.
III. Wahrung berechtigter Interessen
Das Bundesgericht hat die Wahrung berechtigter Interessen als Rechtfertigungsgrund anerkannt, wenn die
Tat das mildest mögliche Mittel darstellt, ein berechtigtes Ziel zu erreichen (Eignung, Erforderlichkeit) und sie
offenkundig weniger schwer wiegt als die gewahrten
Interessen (Verhältnismässigkeit).7
1.
Berechtigtes Ziel
Die Autoren führen aus, dass ein berechtigtes Interesse
vorliegt, wenn das angestrebte Ziel entweder sozial erwünscht ist oder in der Ausübung verfassungsmässiger
Freiheitsrechte liegt.8 Es wird argumentiert, dass sich aus
der Ratifizierung des Pariser Klimaübereinkommens9
ergibt, dass es sich beim Klimaschutz um ein sozial erwünschtes Ziel handelt. Dem ist grundsätzlich nicht zu
widersprechen, jedoch ist die Formulierung des Ziels
derart allgemein gefasst, dass wohl kaum singuläre, und
nicht unmittelbar themenbezogene Handlungen direkt
darauf hinstreben können. Vielmehr stellt sich die Frage, auf welchem Weg dieses grossmehrheitlich anerkannte Ziel umgesetzt wird. Die Klimaaktivisten im vorliegenden Fall möchten jegliche Investitionen in fossile
Energien durch Schweizer Banken unterbinden.10 Ein
solch radikales Vorgehen wird von der Mehrheit der Bevölkerung nicht unterstützt und es ist somit fraglich, ob
es noch als sozial erwünscht betrachtet werden kann.11
Sozial erwünscht bedeutet von der Gesellschaft gewollt.
5
Thommen/Mattmann (Fn. 2), N 28.
6 Thommen/Mattmann (Fn. 2), N 36 in fine.
7
BGE 134 IV 216 E. 6.1.
8 Thommen/Mattmann (Fn. 2), N 29; so auch Payer (Fn. 2), N 23.
9 Übereinkommen von Paris vom 12. Dezember 2015 (SR 0.814.012), in
Kraft getreten für die Schweiz am 5. November 2017.
10 «Ensemble pour […] pousser CS à cesser ses investissements dans
les énergies fossiles !» Lausanne Action Climat auf Facebook am
23. November 2018.
4 Thommen/Mattmann (Fn. 2), N 27; so auch Trappe/Schöbi (Fn. 2),
N 17; Payer (Fn. 2), N 18; Urteil des Kantonsgerichts Waadt Nr. 371
PE19.000742/PCL vom 22. September 2020 E. 6.3.
11 Auf eine staatliche Regulierung des Finanzplatzes Schweiz in Bezug
auf klimaschädliche Investitionen wurde im neulich verabschiedeten CO2 -Gesetz (Bundesgesetz über die Verminderung von Treibhausgasemissionen vom 25. September 2020, BBl 2020 7847) bewusst zugunsten der Selbstregulierung verzichtet (Ablehnung
Antrag Levrat, AB S 2019 925 ff.). Auch der Bundesrat zeigt in seinem
Bericht «Nachhaltigkeit im Finanzsektor Schweiz: Eine Auslegeordnung und Positionierung mit Fokus auf Umweltaspekte» vom 24. Juni 2020 (insb. Ziff. 4) verschiedene Massnahmen auf, die auf die Eigenverantwortung der Finanzinstitute und der Anleger setzen und
verzichtet klar auf staatliche Investitionsverbote.
Atila Stocker, Mehrheitsfähige Klimapolitik, sui generis 2021, S. 103
104
3
Urteil des Kantonsgerichts Waadt Nr. 371 PE19.000742/PCL vom
22. September 2020.
3
4
Der Wille einer Gesellschaft bestimmt sich aus dem Willen der Einzelnen, welcher sich in den politischen Mehrheiten widerspiegelt.
5
Den Autoren zufolge ist das Vorgehen der Aktivsten zudem grundrechtlich geschützt durch Art. 16 und 22 BV12,
sowie Art. 10 und 11 EMRK13.14 Sie verkennen dabei die
bundesgerichtliche Rechtsprechung, aus der klar hervorgeht, dass die Versammlungsfreiheit nicht das Recht beinhaltet, sich auf fremdem Boden zu versammeln.15 Dies
ergibt sich bereits daraus, dass die Credit Suisse mangels
Drittwirkung nicht an die genannten Grundrechte gebunden ist.16 Auch Art. 10 und 11 EMRK sehen ein solches
Recht nicht vor.17
6
Das von den Aktivisten verfolgte Ziel scheint somit weder sozial erwünscht, noch liegt es in der Ausübung verfassungsmässiger Freiheitsrechte.
2.
7
Die Rechtfertigung der Aktivisten scheitert zusätzlich an
der Eignung ihrer Handlungen, denn nur Handlungen,
die geeignet sind, das berechtigte Interesse zu wahren,
können gerechtfertigt sein. Es steht fest, dass der unbe
willigte Aufenthalt in der Credit-Suisse-Filiale weder geeignet ist, die Klimaerwärmung zu stoppen, noch geeignet ist, einen Investitionsstopp für fossile Energien herbeizuführen. Die Autoren umgehen diese Frage, indem
sie prüfen, ob der Tennis-Match geeignet war, die Aufmerksamkeit der Öffentlichkeit auf die Klimapolitik zu
richten.18 Sie legen jedoch nicht dar, inwieweit es sich
beim Aufmerksammachen der Öffentlichkeit um ein sozial erwünschtes oder grundrechtlich geschütztes Ziel handelt. Dies geht nicht aus dem Pariser Klimaabkommen
hervor, das darauf abzielt, dass die Mitgliedstaaten einen direkten Beitrag zum Klimaschutz leisten und nicht
bloss öffentlich darauf aufmerksam machen.
3.
8
Eignung
Erforderlichkeit
Bei der Frage nach der Erforderlichkeit der Handlungen
ist zu prüfen, ob mildere, insbesondere legale Mittel hät12 Bundesverfassung der Schweizerischen Eidgenossenschaft vom
18. April 1999 (BV; SR 101).
13 Konvention zum Schutze der Menschenrechte und Grundfreiheiten, abgeschlossen in Rom am 4. November 1950 (EMRK; SR 0.101).
14 Thommen/Mattmann (Fn. 2), N 30.
15 «La liberté de réunion [...] ne comprend en tout cas pas le droit de
s’assembler sur le fonds d’autrui.» (BGE 97 I 911 E. 3a).
16 Ulrich Häfelin / Walter Haller / Helen Keller / Daniela
Thurnherr, Schweizerisches Bundesstaatsrecht, 10. Auflage, Zürich 2020, N 279.
ten gewählt werden können, um das berechtigte Ziel zu
erreichen.19 «In einem demokratischen Rechtsstaat sind
politische und ideelle Anliegen grundsätzlich auf politischem Wege bzw. auf dem Rechtsweg zu verfolgen»20,
hält das Bundesgericht zutreffend fest. Die Autoren folgen hier der Argumentation des Bezirksrichters, dass
die Aktivisten bereits alle legalen Mittel, insbesondere
den Weg über die Politik, ausgeschöpft hätten und diese
erfolglos geblieben seien.21 Es ist jedoch fraglich, ob dies
in Bezug auf einen Investitionsstopp in fossile Energien
tatsächlich zutrifft. Das Bezirksgericht führt aus, dass
der Bundesrat ungenügend auf entsprechende Vorstösse von Bundesparlamentariern eingegangen ist.22 Es wurden etliche Interpellationen zu diesem Thema eingereicht, welche allesamt vom Bundesrat ordnungsgemäss
beantwortet wurden.23 Das einzige eingereichte Postulat
wurde vom Nationalrat abgelehnt und zwei Motionen
wurden abgeschrieben, weil sie nicht innert zwei Jahren
behandelt wurden, und von den entsprechenden Parlamentsmitgliedern nicht wieder aufgegriffen wurden,
wodurch sich der Bundesrat nie damit beschäftigen
musste.24 Zusätzlich hätte das Anliegen auf dem Weg der
parlamentarischen Initiative oder der Volksinitiative
durchgesetzt werden können. Es ist falsch, der Regierung
hier einen Vorwurf zu machen, vielmehr muss man zum
Schluss gelangen, dass die politischen Mittel nicht genügend ausgeschöpft wurden.
Selbst wenn man davon ausgeht, dass alle politischen
Möglichkeiten erfolglos ausgeschöpft wurden, «gibt
[dies] Letzteren [den Aktivisten] kein Recht, ihre Anliegen mit strafbaren Methoden zu verfolgen.»25 Wenn radikale Klimaschutzmassnahmen, wie sie die Aktivisten
fordern, auf dem Weg der politischen Mitsprache nicht
19 BGE 134 IV 216 E. 6.1.
20 BGE 129 IV 6 E. 3.1.
21 Thommen/Mattmann (Fn. 2), N 32.
22 Urteil des Bezirksgerichts Lausanne PE19.000742/PCL/llb vom
13. Januar 2020, S. 54.
23 Interpellation Thorens (15.3613) «CO2 -Fussabdruck von Investitionen senken (Desinvestition aus fossilen Energieträgern oder ‹Divestment›). Handeln im Hinblick auf die Klimakonferenz in Paris»
vom 17. Juni 2015; Interpellation Girod (15.4109) «Klimaschutz und
Finanzplatzrisiken. Wie setzt die Schweiz die Empfehlungen um?»
vom 8. Dezember 2015; Interpellation Chevalley (17.3120) «Was tut
die Schweiz für nachhaltige Finanzen?» vom 15. März 2017; Interpellation Jans (17.3914) «Wann wird der Finanzsektor in die Pflicht
genommen?» vom 29. September 2017; Interpellation Jans (17.3915)
«Wann kontrolliert die Finma die Klimarisiken?» vom 29. September 2017; Interpellation Thorens (17.3946) «Ein Nachhaltigkeitstest
für die künftigen Regulierungen der Finanzmärkte?» vom 29. September 2017; Interpellation Landolt (17.4104) «Zukunftsorientiertes
Investieren» vom 13. Dezember 2017.
17 Vgl. Guide on Article 11 of the European Convention on Human
Rights, herausgegeben vom Europäischen Gerichtshof für Menschenrechte am 31. Dezember 2020, N 21.
24 Postulat Chevalley (18.3589) «Instrumente zur Überprüfung nachhaltiger Finanzen» vom 14. Juni 2018; Motion Masshardt (18.3918)
«Klimastrategie für den Schweizer Finanzmarkt» vom 27. September 2018; Motion Jans (18.3974) «Den grössten Klimaheizern den
Geldhahn zudrehen» vom 27. September 2018.
18 Thommen/Mattmann (Fn. 2), N 31.
25 BGE 129 IV 6 E. 3.1.
Atila Stocker, Mehrheitsfähige Klimapolitik, sui generis 2021, S. 103
105
9
durchgesetzt werden können, so zeigt dies, dass sie nicht
von der Mehrheit der stimm- und wahlberechtigten Bevölkerung unterstützt werden. Dies wiederum lässt darauf schliessen, dass diese Massnahmen nicht sozial erwünscht sind. Die Argumentation der Autoren ist widersprüchlich, wenn sie die Forderungen der Aktivisten
einerseits als sozial erwünscht, aber andererseits als demokratisch nicht durchsetzbar, also nicht mehrheitsfähig betrachten.
Die Wahrung berechtigter Interessen ist im vorliegenden Fall weder als Rechtfertigungs-, noch als Schuldausschlussgrund anwendbar, da die Aktivisten (1) kein
berechtigtes Interesse verfolgen, (2) ihre Aktion nicht
Die Argumentation nach Thommen und Mattmann würde darauf hinauslaufen, dass zu illegalen Mitteln gegriffen werden darf, wenn ein Ziel auf politischem Wege
nicht erreicht werden kann. Dahinter versteckt sich der
Gedanke, dass es erstrebenswerte Ziele gibt, die von der
Mehrheit der Bevölkerung nicht erkannt würden. Dies
steht dem Konzept der Demokratie entgegen, da es sich
in einer Demokratie gerade aus der Mehrheit ergibt, was
die erstrebenswerten Ziele und der richtige Weg sind, um
diese zu erreichen. Es bleibt unumstritten, dass auch dem
Volkswillen Grenzen gesetzt sind, namentlich durch das
ius cogens. Eine lasche Klimapolitik steht damit jedoch
nicht im Widerspruch.
Atila Stocker, Mehrheitsfähige Klimapolitik, sui generis 2021, S. 103
106
IV. Fazit
10
geeignet war, das verfolgte Interesse zu erreichen, und (3)
der Grundsatz der Erforderlichkeit nicht erfüllt wurde,
da mildere Mittel hätten gewählt werden können.
11
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Os Arquivos Brasileiros de Nutrição: uma revisão sobre produção científica em nutrição no Brasil (1944 a 1968)
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3 0 3
A RTIGO A RT I C L E 3 0 3
A RTIGO A RT I C L E 3 0 3
A RTIGO A RT I C L E Os A rquivos Brasileiros de Nutrição:
uma revisão sobre produção científica
em nutrição no Brasil (1944 a 1968) Os A rquivos Brasileiros de Nutrição:
uma revisão sobre produção científica
em nutrição no Brasil (1944 a 1968) A rquivos Brasileiros de Nutrição:
a review of scientific re s e a rch on nutrition
in Brazil from 1944 to 1968 A rquivos Brasileiros de Nutrição:
a review of scientific re s e a rch on nutrition
in Brazil from 1944 to 1968 Francisco de Assis Guedes de Vasconcelos 1 Abstract This study re v i ews 209 original articles published in the journal Arq u i vos Bra s i l e i ro s
de Nutrição (1944/1968), a periodical edited by Josué de Ca s t ro, p h y s i c i a n , specialist in nutri-
t i o n , and founder- d i rector of the Institute of Nutrition at the Un i versity of Brazil (now the Fe d e r-
al Un i versity of Rio de Ja n e i ro ) . Our methodology was based on quantitative and qualitative
a n a l y s e s , aimed at summarizing both the topic itself and the authors’ b a c k g ro u n d s . Re s u l t s
s h owed that 134 of the articles (64%) adopted a biological perspective to nutrition, mostly focus-
ing on labora t o ry re s e a rch concerning the chemical composition and nutritional value of Bra z i l-
ian foodstuffs. On the other hand, 75 articles (36%) took a social perspective , testifying to the
first efforts by Brazilian nutritional ex p e rts to create and improve specific methodological tools
for investigating our population’s nutritional conditions, t h e re by helping to consolidate the field
of nutrition in the country. 1 De p a rtamento de Nu t r i ç ã o,
Ce n t ro de Ciências da Saúde,
Un i versidade Fe d e ral
de Santa Ca t a r i n a .
Campus Un i versitário –
Tr i n d a d e , Fl o r i a n ó p o l i s ,S C
8 8 0 4 0 - 9 0 0 , Bra s i l . Francisco de Assis Guedes de Vasconcelos 1 I n t ro d u ç ã o En t re as investigações citadas acima, consi-
d e ramos que apenas duas, apesar de suas limi-
t a ç õ e s, re a l i z a ram revisões críticas específicas
s o b re os Arq u ivos Bra s i l e i ros de Nu t r i ç ã o. O es-
tudo de Co i m b ra et al. (1982) sobre a Hi st ór i a
da Alimentação Escolar no Bra s i l, uma das pri-
m e i ras e mais completas das sistematizações
s o b re este tema específico, apresenta impor-
tantes informações e comentários a re s p e i t o
daquele peri ó d i c o. L’ Abbate (1982, 1988), por
sua vez, ao estudar as Pol ít icas de Al i m e n t a ç ã o
e Nutrição no Bra s i l, também dedica uma part e
de sua atenção à revisão bibliográfica narra t i va
do saber científico que foi produzido pelos in-
telectuais do campo da nutrição e divulgado
a t ravés dos Arq u ivo s. Nos últimos vinte anos, embora muito limita-
d a s, foram realizadas importantes inve s t i g a-
ções abordando diferentes aspectos acerca do
p rocesso de constituição do campo da nutri ç ã o
no Brasil, considerada como disciplina, política
social e/ou profissão (Ca s t ro, 1977; Co i m b ra et
al., 1982; L’ Ab b a t e, 1982, 1988; Natal, 1982; Bo s i ,
1988, 1995; Sa n t o s, 1988; Va s c o n c e l o s, 1988;
Pra d o, 1993; Lima, 1997; Ma g a l h ã e s, 1997). Nessas distintas inve s t i g a ç õ e s, são sempre
o b s e rvadas referências ao periódico Arq u ivo s
Bra s i l e i ros de Nu t r i ç ã o, considerado uma das
p rincipais fontes documentais da história desse
campo específico do saber no Brasil. I n t ro d u ç ã o A publica-
ção dessa revista científica, que teve início em
maio de 1944, ainda no transcurso do chama-
do Estado Novo e da Segunda Gu e r ra Mu n d i a l ,
constitui uma das inúmeras idealizações da
t ra j e t ó ria político-intelectual do eminente mé-
dico-nutrólogo pernambucano Josué de Ca s t ro
(1908-1973) (Co i m b ra et al., 1982; L’ Ab b a t e,
1988). Por meio de um convênio estabelecido
e n t re o Se rviço Técnico da Alimentação Na c i o-
nal (STAN) – órgão da Co o rdenação da Mo b i l i-
zação Econômica, instituída pelo Pre s i d e n t e
Getúlio Va rg a s, em setembro de 1942, em de-
corrência da Segunda Gu e r ra Mundial – e a Nu-
t r ition Fo u n d a t i o n , de Nova Io rque – órg ã o
n o rt e - a m e ricano re s p o n s á vel pela publicação
da conceituada Nut r ition Re v i ew s –, Josué de
Ca s t ro criou os Arq u ivos Bra s i l e i ros de Nu t r i ç ã o
com os seguintes objetivos: a) “contribuir com
a difusão de conhecimentos seguros da Ci ê n c i a
da Nutrição para a formação deste corpo de nu-
t r i c i o n i s t a s , especializados em quaisquer dos
d i f e rentes setores da matéria” e b) “dar um co-
nhecimento mais efetivo e mais amplo dos re-
sultados dos trabalhos e dos estudos re a l i z a d o s
no Brasil no campo da Nu t r i ç ã o” ( Ca s t ro, 1944:
5-6). Ainda em agosto de 1944, a publicação
dos Arq u ivo s, cuja periodicidade deve ria ser
mensal, foi interrompida após a edição do q u a r-
to número do seu pri m e i ro vo l u m e, em ra z ã o
da extinção do STAN e da Co o rdenação de Mo-
bilização Econômica. I n t ro d u ç ã o Em nove m b ro de 1946,
os Arq u ivo s vo l t a ram a ser editados sob a re s-
ponsabilidade do então Instituto de Nu t ri ç ã o
da Un i versidade do Brasil (Inub), atual In s t i t u-
to de Nu t rição Josué de Ca s t ro da Un i ve r s i d a d e
Fe d e ral do Rio de Ja n e i ro (Arq u i vos Bra s i l e i ro s
de Nu t ri ç ã o, 1946; Ca s t ro, 1946). Segundo algu-
mas análises, até deze m b ro de 1968, mês de
c i rculação do último volume do peri ó d i c o, d e n-
t ro e fora deste espaço acadêmico-institucio-
nal, os Arq u ivo s c o n s t i t u í ram-se no mais im-
p o rtante veículo de difusão do saber científico Assim sendo, o presente estudo tem como
o b j e t i vo geral realizar uma reflexão acerca da
p rodução científica no campo da nutrição no
Brasil, enfocando os artigos originais publica-
dos nos Arq u ivos Bra s i l e i ros de Nu t r i ç ã o, n o
período de 1944 a 1968. Quanto aos objetivo s
e s p e c í f i c o s, a proposta desse trabalho é: a) re a-
lizar uma análise quantitativa dos artigos pu-
blicados; b) traçar um perfil dos autores dos ar-
tigos no que se re f e re a suas identidades pro f i s-
s i o n a i s, seus vínculos institucionais e seus
campos específicos de conhecimento; c) iden-
tificar os conteúdos temáticos abordados pelos
a rtigos e d) construir subsídios para uma re f l e-
xão sobre a tra j e t ó ria do campo da nutrição em
saúde pública no Bra s i l . 3 0 3
A RTIGO A RT I C L E em alimentação produzido no País (Ca s t ro,
1946; Co i m b ra et al., 1982; L’ Ab b a t e, 1988). 3 0 3
A RTIGO A RT I C L E Key words Nut r it i o n ; Hi s t o ry of Nu t r i t i o n ; Serial Pu b l i c a t i o n s ; Pe r i o d i c a l s Resumo Este trabalho consiste em uma revisão de 209 artigos originais publicados pelos A rq u i-
vos Bra s i l e i ros de Nu t ri ç ã o ( 1 9 4 4 / 1 9 6 8 ) , um periódico editado pelo médico-nutrólogo Josué de
Ca s t ro, d i re t o r-fundador do Instituto de Nutrição da Un i versidade do Brasil (atual Un i ve r s i d a d e
Fe d e ral do Rio de Ja n e i ro ) . A metodologia utilizada baseou-se na realização de uma análise
q u a n t i - q u a l i t a t i va , buscando sumariar tanto os conteúdos temáticos abord a d o s , como o perf i l
dos autores dos art i g o s . Os resultados deste estudo re ve l a m , por um lado, que 134 destes art i g o s
(64%) foram inseridos dentro da perspectiva biológica da nutrição e corre s p o n d e m , na maioria,
a pesquisas laboratoriais sobre a composição química e o valor nutricional de alimentos nacio-
n a i s . Por outro lado, a análise dos 75 artigos (36%) incluídos na perspectiva social atesta o esfor-
ço dos primeiros nutrólogos bra s i l e i ros em construir e aperfeiçoar os instrumentos metodológi-
cos específicos para investigar as condições nutricionais de nossa população, bem como em con-
tribuir para a consolidação do campo da nutrição no Bra s i l . Palavras-chave Nut r iç ã o ; História da Nu t r i ç ã o ; Publicações Se r i a d a s ; Pe r i ó d i c o s Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 3 0 4 VASCONCELOS, F. A. G. em alimentação produzido no País (Ca s t ro,
1946; Co i m b ra et al., 1982; L’ Ab b a t e, 1988). Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 M e t o d o l o g i a De forma semelhante, os
a rtigos da perspectiva social foram classifica-
dos em outros cinco campos disciplinares ou
temáticos da nutrição: avaliação nutricional de
populações; política e planejamento em ali-
mentação e nutrição; epidemiologia nutri c i o-
nal; determinantes do estado nutricional de
populações e educação nutricional. Tal classifi-
cação corresponde a um constructo metodoló-
gico que estabelecemos baseados na atual con-
f o rmação desses campos disciplinares ou te-
máticos no interior da nutri ç ã o. A opção por investigar apenas a seção Art i-
gos Originais dos Arq u ivo s re p resenta, antes de
t u d o, um re c o rte metodológico, uma via de
a p roximação para melhores aprofundamento e
c o m p reensão sobre a produção científica em
n u t ri ç ã o, ao longo do extenso período analisa-
d o. Cada artigo original (nossa unidade de aná-
lise) foi examinado conforme um ro t e i ro de in-
vestigação construído para possibilitar tanto a
análise quantitativa e de categorização dos ar-
tigos por volume editado, como a análise das
c a racterísticas dos autores envo l v i d o s. De s s a
f o rma, além da elaboração de listagem dos 209
a rt i g o s, conforme normas bibliográficas (nome
de autore s, ano, título, nome do peri ó d i c o, vo-
l u m e, número, páginas inicial e final e mês),
cada artigo foi categorizado de acordo com os
seguintes atributos: número de autores; núme-
ro de páginas; apresentação de referências bi-
bliográficas e outras condições de publicação
definidas pelo periódico; vínculo institucional
e identidade profissional do(s) autor(es); natu-
reza da pesquisa que serviu de base para o art i-
go e conteúdo temático abord a d o. Os resultados desse estudo são apre s e n t a-
dos sob a forma de tabelas de distribuição de
f reqüência dos atributos inve s t i g a d o s. M e t o d o l o g i a a revista pre s t a va homenagem a uma pessoa
n o t á vel dentro do cenário universal ou nacio-
nal da nutrição; 2a) Ed i t o rial, onde os diri g e n-
tes da revista apre s e n t a vam abordagens con-
j u n t u rais sobre os mais diversificados temas;
3a) Artigos Ori g i n a i s, que foram objetos de nos-
sa reflexão; 4a) Recentes Aquisições da Nu t ro-
logia, composta por resenhas dos artigos pu-
blicados na Nut r ition Re v i ew s, quase sempre a
seção mais extensa do periódico; 5a) Re s e n h a
Nacional de Nu t ri ç ã o, composta por re s e n h a s
de artigos e de outras modalidades de publica-
ções editadas no Brasil; 6a) Tra n s c ri ç õ e s, com-
posta por artigos publicados anteri o rmente em
o u t ros periódicos; 7a) Cursos e Co n f e r ê n c i a s,
onde são apresentadas bre ves inform a ç õ e s, re-
s u m o s, pro g ra m a s, agendas e/ou currículos de
e ventos científicos, e 8a) Notas e In f o rm a ç õ e s,
a p resentação de rel e as e s s o b re uma miscelâ-
nea de temas relacionados ao campo da nutri-
ç ã o. Tais seções, com exceção das três últimas,
m a n t i ve ram-se sem alteração do pri m e i ro ao
último volume do peri ó d i c o. Em seguida, na tentativa de garantir inteli-
gibilidade à nossa análise, os artigos da pers-
p e c t i va biológica foram classificados em qua-
t ro campos disciplinares ou temáticos da nu-
t rição: nutrição básica e experimental; ciência
e tecnologia de alimentos; nutrição clínica ( i n-
c l u i n d o f is i ol ogia, patologia e dietoterapia) e
n u t rição e dietética. M e t o d o l o g i a O universo do nosso estudo foi constituído por
um total de 209 trabalhos publicados na seção
de artigos originais dos 24 volumes (76 fascícu-
los) dos Arq u ivos Bra s i l e i ros de Nu t r i ç ã o, edita-
dos no período de maio de 1944 a deze m b ro de
1968. Cabe ressaltar que, de acordo com as
p rincipais condições de publicação dos Arq u i-
vo s ( i n s t ruções que não se altera ram ao longo
da tra j e t ó ria do periódico), naquela seção se-
riam aceitos para publicação trabalhos inédi-
tos de autores nacionais sobre nutrologia, que
d e ve riam ve r s a r, de modo original, sobre os te-
mas a que se pro p u s e ram; deve riam, ainda,
a p resentar resumo em português e, se possíve l ,
em inglês e espanhol, seguindo as normas bi-
bliográficas do Ind ex Me d i c u s. De s t a c a m o s,
também, que os Arq u ivo s se subdividiam nas
seguintes seções seqüenciais: 1a) Grandes Vu l-
tos da Nu t rologia, na qual, a cada número, por
meio de fotografia e de uma síntese biográfica, Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 PRODUÇÃO CIENTÍFICA EM NUTRIÇÃO NO BRASIL 3 0 5 m o d o, os artigos categorizados dentro da pers-
p e c t i va biológica originam-se e/ou atendem às
finalidades de três das quatro seções org a n i z a-
t i vas do então Inub: a Seção de Pesquisas Bi o-
l ó g i c a s, a Seção de Patologia da Nu t rição e, de
c e rta forma, a Seção de Educação Alimentar. Ig u a l m e n t e, os artigos categorizados dentro da
p e r s p e c t i va social originam-se e/ou atendem
às finalidades da Seção de Pesquisas Ec o n ô m i-
c o - Sociais da re f e rida instituição (Arq u i vo s
Bra s i l e i ros de Nu t ri ç ã o, 1946:71-73). M e t o d o l o g i a Assim,
p a ra cada um destes atri b u t o s, foi constru í d o
um índice descri t i vo (número de artigos por
volume; número de artigos por natureza da
pesquisa e volume; número de páginas por ar-
tigo; número de autores por artigo; apre s e n t a-
ção de referências bibliográficas por naturez a
da pesquisa; vínculo institucional do(s) au-
tor(es) por artigo; identidade profissional do
autor principal por artigo etc.), que será anali-
sado na seqüência de apresentação dos re s u l-
t a d o s. A trajetória dos A rq u ivos Brasileiros de
N u t r i ç ã o: volumes, número de art i g o s ,
periodicidade e dire ç ã o Na seqüência dos procedimentos metodo-
lógicos adotados, os artigos foram categori z a-
dos dentro de duas modalidades de estudos:
i n vestigações de natureza biológica (que cha-
m a remos de perspectiva biológica) e inve s t i g a-
ções de natureza social (que chamaremos de
p e r s p e c t i va social). A ri g o r, tal pro c e d i m e n t o
foi adotado pro c u rando-se seguir tanto as fina-
l i d a d e s, como a estru t u ra de organização do
In u b, idealizadas por Josué de Ca s t ro. De s s e A Tabela 1 apresenta a distribuição dos 209 ar-
tigos originais analisados conforme o vo l u m e,
bem como a indicação do ano e o cri t é rio de
p e riodicidade de circulação da revista. Me-
diante a análise do índice número de art i g o s
por vo l u m e, observa-se que, ao longo do eixo
longitudinal de edição da revista (1944 a 1968),
f o ram publicados, em média, 8,7 artigos por Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 VASCONCELOS, F. A. G. 3 0 6 Tabela 1
Distribuição dos artigos originais publicados nos A rquivos Brasileiros de Nutrição
segundo o volume (ano/periodicidade) do periódico, 1944-1968. A trajetória dos A rq u ivos Brasileiros de
N u t r i ç ã o: volumes, número de art i g o s ,
periodicidade e dire ç ã o Volume (ano/periodicidade)
No de art i g o s
%
% acumulado
1 (1944/mensal)
8
3 , 8
3 , 8
2 (1946/mensal)
3
1 , 4
5 , 2
3 (1947/mensal)
6
2 , 9
8 , 1
4 (1947/mensal)
1 0
4 , 8
1 2 , 9
5 (1948/bimestral)
6
2 , 9
1 5 , 8
6 (1949/bimestral)
1 4
6 , 7
2 2 , 5
7 (1950/bimestral)
1 1
5 , 3
2 7 , 8
8 (1951/bimestral)
1 0
4 , 8
3 2 , 6
9 (1953/trimestral)
5
2 , 4
3 5 , 0
10 (1954/trimestral)
4
1 , 9
3 6 , 9
11 (1955/semestral)
2
1 , 0
3 7 , 9
12 (1956/semestral)
2
1 , 0
3 8 , 9
13 (1957/semestral)
3
1 , 4
4 0 , 3
14 (1958/semestral)
6
2 , 9
4 3 , 2
15 (1959/semestral)
9
4 , 3
4 7 , 5
16 (1960/semestral)
1 2
5 , 7
5 3 , 2
17 (1961/semestral)
9
4 , 3
5 7 , 5
18 (1962/anual)
1 1
5 , 3
6 2 , 8
19 (1963/semestral)
1 8
8 , 6
7 1 , 4
20 (1964/semestral)
1 0
4 , 8
7 6 , 2
21 (1965/semestral)
1 5
7 , 1
8 3 , 3
22 (1966/semestral)
1 2
5 , 7
8 9 , 0
23 (1967/semestral)
1 3
6 , 2
9 5 , 2
24 (1968/anual)
1 0
4 , 8
1 0 0 , 0
To t a l
2 0 9
1 0 0 , 0
– de publicações, com uma média de 12 art i g o s
por vo l u m e, tendo sido publicados 58,3% do
total de artigos neste período. Tabela 1 Distribuição dos artigos originais publicados nos A rquivos Brasileiros de Nutrição
segundo o volume (ano/periodicidade) do periódico, 1944-1968. Em seu último vo l u m e, re l a t i vo ao ano de
1968, a revista teve como dire t o r- re s p o n s á ve l
o médico-nutrólogo Hélio Vecchio Alves Ma u-
r í c i o, que, desde 1958, ocupava o cargo de re-
d at o r- s e c re t á ri o. Os artigos originais: natureza
da pesquisa, tamanho do artigo
e normas bibliográficas A Tabela 2 reúne a apresentação de dois dos ín-
dices construídos para a análise dos artigos dos
Arq u ivo s: o número de artigos por natureza da
pesquisa e volume e número de páginas por
n a t u reza da pesquisa e vo l u m e. Em relação ao
n ú m e ro de artigos por natureza da pesquisa e
vo l u m e, observa-se que 134 (64,0%) dos 209 ar-
tigos foram incluídos na perspectiva biológica,
c o n t ra 75 (36,0%) associados à perspectiva so-
cial. Quanto ao número de páginas por nature-
za da pesquisa e vo l u m e, os resultados são se-
m e l h a n t e s, apontando um total de 61,0% de
páginas para a perspectiva biológica, contra
39,0% para a perspectiva social. vo l u m e, entretanto podem ser observados cor-
tes tra n s versais que denotam marcantes osci-
lações desse índice. No corte inicial (1944 a 1954), corre s p o n-
dente à direção Josué de Ca s t ro, a perspectiva
social detém apenas 30,0% dos artigos e 27,5%
do número de páginas publicadas. Já no cort e
final (1955 a 1968), apesar da perspectiva bio-
lógica continuar com sua hegemonia quantita-
t i va, observa-se um aumento re l a t i vo das pu-
blicações da perspectiva social, a qual passou a
deter 39,4% dos artigos e 48,3% do número de
páginas publicadas. Assim, por exemplo, no período de 1944 a
1954, época em que os Arq u ivo s e ram diri g i d o s
por Josué de Ca s t ro, tendo como secre t á ri o s /
re d a t o res os médicos nutrólogos Pe d ro Bo rg e s
(1946 a 1947) e Rubens de Si q u e i ra (1947 a
1953), foram publicados 36,9% do total de art i-
g o s, com uma média de 7,7 artigos por vo l u m e. A trajetória dos A rq u ivos Brasileiros de
N u t r i ç ã o: volumes, número de art i g o s ,
periodicidade e dire ç ã o Neste seu último ano de cir-
c u l a ç ã o, assim como em 1962, a sua peri o d i c i-
dade foi anual, com dez artigos publicados. A l i á s, é preciso destacar que, em seus últimos
vo l u m e s, conforme atestam algumas notas ex-
p l i c a t i vas contidas em artigos dos volumes 21,
23 e 24, o periódico circulou com bastante
a t ra s o. Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 Os artigos originais: natureza
da pesquisa, tamanho do artigo
e normas bibliográficas Nesse interva l o, a revista, inicialmente com pe-
riodicidade mensal, passou a ter uma circ u l a-
ção bimestral (1948 a 1951) e depois tri m e s t ra l
(1953 a 1954), enquanto re g i s t rou-se a inter-
rupção de sua circulação nos anos de 1944 (as-
sociada à extinção do STAN) e em 1952 (justifi-
cada por motivo ss up eri ores não explicitados). A análise desses dois índices demonstra
q u e, no decorrer de toda a série histórica de
c i rculação do peri ó d i c o, a perspectiva biológi-
ca esteve à frente da produção e difusão do sa-
ber em nutri ç ã o. A perspectiva social, além de
vir a reboque ao longo da séri e, não aparece no
d e c o r rer de pelo menos cinco volumes da re-
vista (3, 5, 12, 13 e 14), com exceção do período
1959-1964, momento em que, quantitativa-
m e n t e, supera a perspectiva biológica. Co i n c i-
d e n t e m e n t e, este período de auge da perspec-
t i va social, conforme discutiremos adiante,
c o r re s p o n d e, em termos históri c o - c o n j u n t u- En t re 1955 a 1967, sob a direção de Pe d ro
Bo rg e s, que desde 1954 ocupava o cargo de re-
d a t o r, passou a ter circulação semestral e, após
s u p e rar a crise dos anos 1952-1958 (quando
a p resentou os menores índices de publicação
de artigos por volume), entrou na fase de auge Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 PRODUÇÃO CIENTÍFICA EM NUTRIÇÃO NO BRASIL 3 0 7 Tabela 2 Distribuição dos artigos originais publicados nos A rquivos Brasileiros de Nutrição segundo o número de artigos
e o número de páginas por natureza da pesquisa (biológica ou social) e volume (ano) do periódico, 1944-1968. Os artigos originais: natureza
da pesquisa, tamanho do artigo
e normas bibliográficas Volume (ano)
No de art i g o s
No de páginas
b i o l ó g i c a
s o c i a l
t o t a l
b i o l ó g i c a
s o c i a l
t o t a l
1 (1944)
5
3
8
4 9
2 8
7 7
2 (1946)
1
2
3
1 6
5 5
7 1
3 (1947)
6
–
6
1 4 9
–
1 4 9
4 (1947)
9
1
1 0
1 4 9
7
1 5 6
5 (1948)
6
–
6
2 1 6
–
2 1 6
6 (1949)
9
5
1 4
1 5 2
6 0
2 1 2
7 (1950)
6
5
1 1
1 2 2
96
2 1 8
8 (1951)
5
5
1 0
1 7 8
1 4 6
3 2 4
9 (1953)
4
1
5
7 8
2 2
1 0 0
10 (1954)
3
1
4
7 4
3 5
1 0 9
11 (1955)
1
1
2
8
3 6
4 4
12 (1956)
2
–
2
4 3
–
4 3
13 (1957)
3
–
3
3 4
–
3 4
14 (1958)
6
–
6
9 4
–
9 4
15 (1959)
4
5
9
3 6
9 9
1 3 5
16 (1960)
5
7
1 2
6 8
1 1 8
1 8 6
17 (1961)
5
4
9
6 3
8 5
1 4 8
18 (1962)
5
6
1 1
6 0
1 2 1
1 8 1
19 (1963)
1 2
6
1 8
1 5 0
1 2 2
2 7 2
20 (1964)
5
5
1 0
5 6
1 1 1
1 6 7
21 (1965)
1 0
5
1 5
1 1 7
6 5
1 8 2
22 (1966)
6
6
1 2
7 5
9 3
1 6 8
23 (1967)
9
4
1 3
1 4 0
4 2
1 8 2
24 (1968)
7
3
1 0
7 2
5 7
1 2 9
To t a l
1 3 4
7 5
2 0 9
2 . 1 9 9
1 . 3 9 8
3 . Os artigos originais: natureza
da pesquisa, tamanho do artigo
e normas bibliográficas 5 9 7
%
( 6 4 , 0 )
( 3 6 , 0 )
( 1 0 0 , 0 )
( 6 1 , 0 )
( 3 9 , 0 )
( 1 0 0 , 0 ) Distribuição dos artigos originais publicados nos A rquivos Brasileiros de Nutrição segundo o número de artigos
e o número de páginas por natureza da pesquisa (biológica ou social) e volume (ano) do periódico, 1944-1968. c), que perf a zem um total de 150 páginas; os de
Mo u ra - Campos et al. (1951a e b), que perf a ze m
um total de 104 páginas; e os de Orsini ( 1 9 4 7 a ,
b), com 92 páginas. ra i s, a um momento de grande mobilização so-
cial no País (movimento das Ligas Ca m p o n e-
sas; movimentos urbanos pelas re f o rmas de ba-
s e, incluindo a re f o rma agrária etc.) e que c u l-
mina no golpe militar de março de 1964 (Alen-
car et al., 1979; Aze v ê d o, 1982; Vi e i ra, 1983). Em relação à distribuição do índice apre-
sentação de referências bibliográficas por na-
t u reza da pesquisa, observamos que 68 (32,5%)
dos 209 artigos publicados não apresentam re-
ferências bibliográficas; destes, 40 (58,8%) são
da perspectiva social e 28 (41,2%), da perspec-
t i va biológica. Além dessas evidências, obser-
vamos que muitos dos artigos com re f e r ê n c i a s
bibliográficas não as apresentam dentro das
n o rmas indicadas pelos Arq u ivo s (Ind ex Me d i-
c u s). Os artigos originais: natureza
da pesquisa, tamanho do artigo
e normas bibliográficas Na seqüência de análise dos aspectos das
condições re d a t o riais que contribuem para a
d e m o n s t ração do rigor metodológico da re v i s-
ta, observamos que os artigos da perspectiva
biológica, em sua ampla maioria, encontra m -
se dentro da estru t u ra básica tradicional de pu-
blicações periódicas (intro d u ç ã o, metodologia, Na Tabela 3, encontra-se a distribuição do
índice número de páginas por art i g o, utilizado
aqui como um dos demonstra t i vos da inexis-
tência de ri g i d ez nos cri t é rios ou condições de
publicação do peri ó d i c o. Na distribuição desse
í n d i c e, observou-se uma média de cerca de 17,
um mínimo de quatro e um máximo de 64 pá-
ginas por art i g o. Apesar de a grande maiori a
dos artigos estar abaixo de 25 páginas, obser-
vamos que o não-estabelecimento de um limi-
te máximo de páginas por artigo fez com que
os Arq u ivo s publicassem ve rd a d e i ros tra t a d o s
de fisiopatologia nutricional ou de ciência e
tecnologia de alimentos, a exemplo de alguns
a rt i g o s, como os de Si q u e i ra & Vogel (1948a, b, Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 3 0 8 VASCONCELOS, F. A. G. a rtigo (considerada aqui formação básica), to-
mamos como referência apenas os 88 autore s
p rincipais (excluídos os três autores institucio-
nais). Os autores dos A rq u iv o s: identidade
p rofissional, vínculo institucional
e outras características Na Tabela 4, apresentamos os dados da distri-
buição do índice número de autores por art i g o,
onde se observa que 123 (58,9%) dos art i g o s
são de autoria individual; 50 (23,9%) são de au-
t o ria dupla; 33 (15,8%) são assinados por três
ou mais autores e 3 (1,4%), por autores institu-
c i o n a i s. Os 209 artigos originais publicados nos Ar-
q u ivo s são assinados por um total de 134 dife-
rentes autore s, 91 dos quais foram considera-
dos autores principais (pri m e i ro nome, no ca-
so de mais de um autor) e 43, autores secundá-
rios ou colabora d o re s. Pa ra a construção do ín-
dice identidade profissional do(s) autor(es) por De acordo com o índice vínculo institucio-
nal do(s) autor (es) por art i g o, os resultados ob-
s e rvados para os 209 artigos originais foram os
seguintes: 95 (45,5%) são assinados por autore s
vinculados ao Inub; 38 (18,2%), por autores vin-
culados a outras instituições localizadas no Rio
de Ja n e i ro, quase sempre com forte art i c u l a ç ã o
com o grupo de pesquisadores dos Arq u ivo s ( a
exemplo do Se rviço de Alimentação da Pre v i-
dência Social – Saps – e da Comissão Na c i o n a l
de Alimentação – CNA –, instituições também
idealizadas por Josué de Ca s t ro) (Co i m b ra et al.,
1982; L’ Ab b a t e, 1988); 45 (21,5%), por autore s
vinculados a órgãos da Un i versidade de São
Paulo (USP) e de outras instituições deste Esta-
do; e 31 (14,8%), por autores vinculados a insti-
tuições de outros estados bra s i l e i ros (Ba h i a ,
Minas Ge ra i s, Alagoas e Pe rnambuco), a insti-
tuições internacionais (Organização para a Ali-
mentação e Agri c u l t u ra – FAO – e Org a n i z a ç ã o
Mundial da Saúde – OMS) e alguns não identifi-
c a d o s. Os artigos originais: natureza
da pesquisa, tamanho do artigo
e normas bibliográficas Apesar da falta de informações acerca da
f o rmação básica de boa parcela destes autore s,
o que limita bastante a análise desse índice, a
d i s t ribuição dos dados obtidos é a seguinte: 39
(44,3%) dos autores são médicos; oito (9,1%)
são químicos ou farm a c ê u t i c o - b i o q u í m i c o s ;
cinco (5,7%) são nutricionistas; três (3,4%) são
e n g e n h e i ros agrônomos; três (3,4%) são médi-
cos ve t e ri n á rios; um é assistente social; um é
economista; seis são autores estra n g e i ros (con-
vidados especiais dos dire t o res da revista) e de
22 (25,0%) não obtivemos inform a ç õ e s. Po d e -
se afirm a r, port a n t o, que o perfil do autor dos
Arq u ivo s a p resenta as seguintes cara c t e r í s t i c a s :
homem, médico-nutrólogo, inserido na pers-
p e c t i va biológica da nutri ç ã o. Ca ra c t e r í s t i c a s
q u e, apesar da incorporação de novos elemen-
t o s, permanecem hegemônicas ao longo da
t ra j e t ó ria da re v i s t a . re s u l t a d o s, conclusões, re f e r ê n c i a s, re s u m o s ) ,
enquanto os artigos da perspectiva social ape-
nas em sua minoria seguem essa estru t u ra . Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 Os autores dos A rq u iv o s: identidade
p rofissional, vínculo institucional
e outras características Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 PRODUÇÃO CIENTÍFICA EM NUTRIÇÃO NO BRAS Tabela 5 Relação dos vinte principais autores dos artigos originais publicados nos A rquivos Brasileiros de Nutrição segundo
f o rmação básica, vínculo institucional, campo disciplinar e número absoluto e percentual de artigos publicados
em relação ao total, 1944-1968. No de ord e m / n o m e
F o rm a ç ã o
I n s t i t u i ç ã o *
C a m p o
No de artigos
% do total
1 Pechnik, E. Q u í m i c a
I n u b
B i o l ó g i c o
42
2 0 , 2
2 Guimarães, L. R. Q u í m i c o
I n u b
B i o l ó g i c o
21
1 0 , 1
3 Borges, P. M é d i c o
I n u b
S o c i a l
17
8 , 2
4 Siqueira, R. M é d i c o
I n u b
B i o l ó g i c o
15
7 , 2
5 Maurício, H. V. A. M é d i c o
I n u b
S o c i a l
10
4 , 8
6 Silva, W. M é d i c o
I n u b / C N A
S o c i a l
9
4 , 3
7 Moura-Campos, F. A. M é d i c o
U S P
B i o l ó g i c o
9
4 , 3
8 Mucciolo, P. Med. Ve t e r i n á r i a
U S P
B i o l ó g i c o
7
3 , 4
9 Fioravanti, I. M é d i c a
I n u b
S o c i a l
7
3 , 4
10 Chaves, J. M. Q u í m i c o
I n u b
B i o l ó g i c o
7
3 , 4
11 Schneider, I. S. Med. Ve t e r i n á r i a
USP
B i o l ó g i c o
6
2 , 9
12 Castro, J. Os autores dos A rq u iv o s: identidade
p rofissional, vínculo institucional
e outras características M é d i c o
Inub
S o c i a l
5
2 , 4
13 Dutra-de-Oliveira, J. E. M é d i c o
USP
B i o l ó g i c o
5
2 , 4
14 Maldonado, G. F. M é d i c o
Inub/Saps
B i o l ó g i c o
5
2 , 4
15 Ferreira Lima, J. M é d i c o
Ascofam/PE
S o c i a l
4
1 , 9
16 Luz, H. S. M é d i c o
Inub
B i o l ó g i c o
4
1 , 9
17 Azevedo, T. M é d i c o
DS/Bahia
S o c i a l
3
1 , 4
18 Parahim, O. M é d i c o
DSP/PE
S o c i a l
3
1 , 4
19 Lopes, N. M é d i c o
Ufal/AL
S o c i a l
3
1 , 4
20 Santos, W. M é d i c o
Inub/CNA
S o c i a l
3
1 , 4
* Inub: Instituto de Nutrição da Universidade de Brasília; CNA: Comissão Nacional de Alimentação; USP: Universidade
de São Paulo; Saps: Serviço de Alimentação da Previdência Social; Ascofam: Associação Mundial de Luta contra
a Fome; DS: Departamento de Saúde; DSP: Departamento de Saúde Pública; Ufal: Universidade Federal de Alagoas. * Inub: Instituto de Nutrição da Universidade de Brasília; CNA: Comissão Nacional de Alimentação; USP: Universidade
de São Paulo; Saps: Serviço de Alimentação da Previdência Social; Ascofam: Associação Mundial de Luta contra
a Fome; DS: Departamento de Saúde; DSP: Departamento de Saúde Pública; Ufal: Universidade Federal de Alagoas. A Tabela 5 apresenta a relação dos vinte
p rincipais autores de acordo com o número de
a rtigos publicados, constituindo-se em uma
síntese do perfil de autoria discutido nesta se-
ç ã o. Observa-se que os químicos Emília Pe c h-
nik e Luiz Ribeiro Gu i m a r ã e s, técnicos da Se-
ção de Pesquisas Biológicas do In u b, lidera m
em pri m e i ro e segundo lugare s, re s p e c t i va-
m e n t e, a lista de publicações. Os autores dos A rq u iv o s: identidade
p rofissional, vínculo institucional
e outras características Os médicos nu-
trólogos do Inub Pe d ro Bo rg e s, Rubens de Si-
q u e i ra e Hélio Vecchio Alves Ma u r í c i o, que no
d e c o r rer do corte longitudinal 1944-1968 re ve-
z a ram-se nos cargos de secre t á ri o, redator e di-
retor do peri ó d i c o, ocupam os postos de terc e i-
ro, quarto e quinto lugare s, re s p e c t i va m e n t e. Os cientistas da USP Franklin A. de Mo u ra
Ca m p o s, Paschoal Mu c c i o l o, I. S. Schneider e
José Ed u a rdo Du t ra de Ol i ve i ra, todos vincula-
dos à perspectiva biológica, assumem os pos-
tos de sétimo, oitavo, 11o e 13o l u g a re s, re s p e c-
t i va m e n t e. Josué de Ca s t ro, em razão de seu
papel na criação e direção da revista e na tra j e-
t ó ria de constituição do campo da nutrição no
Brasil, neste caso, ostenta apenas a 12a p o s i-
ç ã o. De s t a c a m o s, ainda, a presença dos médi- cos nutrólogos nordestinos Jamesson Fe r re i ra
Lima (Associação Mundial de Luta Co n t ra a Fo-
me – Ascofam/PE), Thales de Aze vedo (De p a r-
tamento de Saúde – DS/Bahia), Orlando Pa ra-
him (De p a rtamento de Saúde Pública – DSP/
PE) e Nabuco Lopes (Un i versidade Fe d e ral de
Alagoas – Ufal/AL), todos vinculados à pers-
p e c t i va social. O fato de a cientista Emília Pechnick (na
condição de mulher e de química) ocupar, de
f o rma isolada, o pri m e i ro lugar da lista de pu-
blicações é tão surpreendente e esclare c e d o r,
quanto contrário a algumas constatações que
temos apontado até aqui. Os autores dos A rq u iv o s: identidade
p rofissional, vínculo institucional
e outras características Dessa forma, às características do perf i l
do autor dos Arq u ivo s apontadas acima, acre s-
centamos: estar vinculado ao Inub e residir no
Rio de Ja n e i ro. Ca racterísticas estas que se jus-
tificam tanto pelo papel desempenhado pela ci-
dade do Rio de Ja n e i ro (Di s t rito Fe d e ral na épo-
ca; centro político-intelectual; centro concen-
t rador de três das seis escolas de nutrição exis-
tentes até então no País e sede das pri n c i p a i s
agências estatais da política de alimentação e
n u t rição), quanto pelo papel desempenhado
pelo grupo de médicos nutrólogos do Inub na-
quele período da tra j e t ó ria de consolidação do
campo da nutrição no Brasil (Co i m b ra et al.,
1982; L’ Ab b a t e, 1982; Bosi, 1988). Tabela 3 Distribuição dos artigos originais publicados nos A rquivos Brasileiros de Nutrição
segundo o número de páginas por artigo, 1944-1968. No de páginas
n
%
% acumulado
4 a 10
7 7
3 6 , 8
3 6 , 8
11 a 20
7 9
3 7 , 8
7 4 , 6
21 a 30
2 3
1 1 , 0
8 5 , 6
31 a 40
2 0
9 , 6
9 5 , 2
41 a 50
5
2 , 4
9 7 , 6
51 a 64
5
2 , 4
1 0 0 , 0
To t a l
2 0 9
1 0 0 , 0
–
Tabela 4
Distribuição dos artigos originais publicados nos A rquivos Brasileiros de Nutrição
segundo o número de autores por artigo, 1944-1968. No de autore s
n
%
1
1 2 3
5 8 , 9
2
5 0
2 3 , 9
3
2 4
1 1 , 5
4
5
2 , 4
5
3
1 , 4
1 0
1
0 , 5
I n s t i t u c i o n a l
3
1 , 4
To t a l
2 0 9
1 0 0 , 0 Distribuição dos artigos originais publicados nos A rquivos Brasileiros de Nutrição
segundo o número de páginas por artigo, 1944-1968. Cad. Os autores dos A rq u iv o s: identidade
p rofissional, vínculo institucional
e outras características Por outro lado, reforça as teses da
i m p o rtância da química, considerada como
uma das disciplinas-matriz na conform a ç ã o
deste campo, tanto no cenário universal, co-
mo no Brasil ( C h a ve s, 1978; Lima, 1997; Ma g a-
l h ã e s, 1997). Além dos vinte autores listados, é pre c i s o
f a zer referência a um grupo que emerge ape-
nas a partir dos anos 60, denotando uma cert a
a b e rt u ra dos Arq u ivo s p a ra a publicação da
p rodução científica de outras especialidades
que entra vam em jogo na contra d i t ó ria dinâ-
mica de competição e interação que se estabe-
lecia na tra j e t ó ria de consolidação do campo
da nutrição no Brasil. Assim, Enilda Lins da
Cruz Go u veia (Saps/RJ) e Ma ria de Lourd e s
Mello (Sesi/MG) foram as duas pri m e i ras nu-
t ricionistas a publicarem nos Arq u ivo s, cada
uma assinando um artigo dentro da perspecti-
va social (Go u veia, 1960; Me l l o, 1960). A part i r
de então, outras nutricionistas passaram a as-
sinar artigos dentro das duas perspectivas que
i n ve s t i g a m o s, a exemplo de Lieselotte H. Or-
nellas (Escola de En f e rmagem da Un i ve r s i d a d e
do Brasil – UB), de Neuza T h e rezinha Reze n d e
Ca valcante (Inub), de Mi rtila C. Araújo (Inub) e
de Gilda Linhares Mello (Inub). Re f e rimos ain-
da a entrada em cena do médico pern a m b u c a-
no Nelson Chave s, fundador do Instituto e do
Curso de Nu t rição da Un i versidade Fe d e ral de
Pernambuco, que, entre 1963 e 1964, assina dois
a rtigos naquele peri ó d i c o. Fato que, a nosso
ve r, significa mais um reforço à perspectiva bio-
lógica, part i c u l a rmente às disciplinas fisiologia
e endocrinologia, também consideradas disci-
plinas-matriz na conformação do campo da nu-
t rição no Brasil (Lima, 1997; Ma g a l h ã e s, 1997). Os autores dos A rq u iv o s: identidade
p rofissional, vínculo institucional
e outras características Por um lado, indica
um certo rompimento com as teses de que a
constituição do campo da nutrição no Bra s i l
tenha sido “d em a rc ada por um espaço de luta
c o m p e t i t i va pelo monopólio da autoridade
c i e n t í f i c a” (Bo u rdieu, 1994:131) entre os gêne-
ros masculino e feminino (Sa n t o s, 1988; Pra-
d o, 1993; Bosi, 1995; Lima, 1997). Sem dúvida,
a existência dos 42 artigos assinados pela quí-
mica Emília Pechnik nos volumes 2 ao 24 da
revista indica que, apesar de majori t á ria, a
p resença do gênero masculino na tra j e t ó ria da Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 VASCONCELOS, F. A. G. 3 1 0 c a d o s. Estes art i g o s, frutos de pesquisas labo-
ra t o riais (análises químico-bromatológicas ou
ensaios biológico-experimentais), têm como
ponto comum a utilização de métodos de de-
t e rminação do valor nutricional dos alimentos
n a c i o n a i s. Sendo assim, abordam a composi-
ção químico-nutricional, o valor calóri c o, o t e o r
vitamínico e/ou o teor mineral dos mais dive r-
sificados alimentos bra s i l e i ro s, dos mais tra d i-
cionais (feijão pre t o, farinha de mandioca, tri-
g o, milho, leite, lara n j a s, manga etc.) aos mais
exóticos (buriti, tucumã, pupunha, óleo de pa-
tauá, pinhão, mucunã, macambira, sururu e t c. ) . Josué de Ca s t ro assina dois artigos do campo
temático em questão, um no qual são re ve l a-
dos os resultados químico-analíticos de ali-
mentos bárbaros do sertão nordestino (Ca s t ro
et al., 1947), e outro que apresenta o valor nu-
t ri t i vo da mistura de milho com leite (Ca s t ro &
Pechnik, 1951). constituição deste campo não é uma condição
e xc l u s i va. Os autores dos A rq u iv o s: identidade
p rofissional, vínculo institucional
e outras características Esse fato re p resenta um dos exemplos que
nos ajudam a atestar que não existiam fro n t e i-
ras rígidas entre as duas perspectivas inve s t i-
g a d a s, nem tampouco entre campos temáticos
ou disciplinares dentro de uma mesma pers-
p e c t i va. V á rios autores dos Arq u ivo s c i rc u l a m
e n t re as duas perspectivas e entre os distintos
campos temáticos existentes no interior de ca-
da uma delas, ocorre n d o, inclusive, tentativa s
c o n c retas de conexão entre o biológico e o so-
cial, a exemplo de artigo incluído nessa temáti-
ca (Pechnik et al., 1950), no qual os autore s
a p resentam os resultados de uma inve s t i g a ç ã o
s o b re a castanha-do-pará, investigação esta
composta por um detalhado estudo econômi-
co-social da produção e por um ensaio bioló-
g ic o - e x p e rimental do valor nutricional deste
p ro d u t o. A temática tecnologia dos alimentos apare-
ce em segundo lugar dentro da perspectiva
biológica, em 35 (26,1%) dos artigos publica-
d o s. Trata-se da única temática alheia aos pes-
q u i s a d o res do In u b, uma vez que quase a tota-
lidade destes artigos são oriundos de pesquisa-
d o res do Estado de São Pa u l o. Paschoal Mu c-
ciolo e I. S. Schneider, da Faculdade de Me d i c i-
na Ve t e ri n á ria da USP, são os dois pri n c i p a i s
a u t o res dessa linha, cujos artigos abordam os
mais diversificados temas ligados à industri a l i-
zação de alimentos (métodos de pro c e s s a m e n-
t o, de enri q u e c i m e n t o, de conserva ç ã o, de em-
balagem, de controle de qualidade etc.). Em-
b o ra apareça, de forma bastante tímida, desde
1947, esta temática só teve uma melhor expre s-
são numérica a partir de 1954. Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 A perspectiva biológica: dos métodos
de determinação do valor nutricional
aos métodos de industrialização
dos alimentos A temática nutrição e dietética aparece em
último lugar, em 13 (9,7%) dos artigos publica-
d o s. Os artigos desta linha abordam temas re-
lacionados a aspectos do planejamento e ad-
m i n i s t ração de serviços de alimentação, da téc-
nica dietética e culinária e da dietética pro p ri a-
mente dita. Quase a totalidade destes art i g o s
(92,0%) foram publicados entre 1959 a 1968 e
versam, em sua maioria, sobre o planejamento
da alimentação de grupos militares (exérc i t o,
m a rinha e aeronáutica). En t re os seus autore s,
destacamos os médicos nutrólogos Pe d ro Bo r-
g e s, Hélio Vecchio A. Maurício e Irma Fi o ra va n-
ti (Seção de Pesquisas Ec o n ô m i c o - Sociais do
Inub); Ge raldo Francisco Maldonado (Saps) e a
n u t ricionista Lieselotte H. Ornellas (Escola de
En f e rmagem da UB). Em síntese, através dos conhecimentos ob-
j e t i vos produzidos por estas pesquisas labora-
t o riais e ensaios biotecnológicos sobre a com-
posição e o metabolismo dos alimentos; sobre
os métodos de industrialização e enri q u e c i-
mento dos alimentos e sobre os modern o s
p rincípios da dietética e da dietotera p i a , o s
médicos nutrólogos vinculados aos Arq u ivo s
c o n s t ru í ram uma importante base cognitiva
p a ra a legitimidade da tese, atribuída a Jo s u é
de Ca s t ro, de que “a partir de uma alimentação
ra c i o n a l , seria possível valorizar a ra ç a ,c o n s-
truir o homem bra s i l e i ro e forjar a nação” (Li-
ma, 1997; Ma g a l h ã e s, 1997:79). No campo temático Pol ít ica e Pl a n e j a m e n-
to em Alimentação e Nu t r i ç ã o, foram incluídos
20 (26,7%) dos art i g o s. A perspectiva biológica: dos métodos
de determinação do valor nutricional
aos métodos de industrialização
dos alimentos Tais art i g o s, publicados do pri m e i ro
ao último volume da revista, demonstram que,
após o inquérito pioneiro: As condições de vida
das classes operárias no Re c i f e, realizado por Jo-
sué de Ca s t ro em 1933 (Ca s t ro, 1935), esta mo-
dalidade de inve s t i g a ç ã o, além de apri m o ra r-
se enquanto instrumental básico de pesquisa
d e n t ro da perspectiva social da nutri ç ã o, ga-
nhou adeptos em todo o país, a exemplo das
pesquisas realizadas pelos médicos nutrólogos
Orlando Pa rahim e Jamesson Fe r re i ra Lima, em
Pe rnambuco; Thales de Aze ve d o, na Bahia; Os-
waldo Costa e Rubem de Si q u e i ra, no Rio de Ja-
n e i ro; Domingos Lopes e Benedito Ph i l a d é l p h o
Si q u e i ra, em Minas Ge rais; Nabuco Lopes, em
A l a g o a s, e Walter Si l va, no Rio Grande do No rt e. •
Oito artigos sobre avaliação nutricional ba-
seada em métodos antro p o m é t ri c o s, clínicos e
s ó c i o - e c o n ô m i c o s. Ressalte-se que tais ava l i a-
ções clínico-antro p o m é t ricas tive ram como di-
re t ri zes comuns: a população em idade esco-
l a r; algum cri t é rio de estratificação sócio-eco-
nômica desta população; a utilização de pro c e-
dimentos estatísticos na amostragem e análise
dos dados e resultados apontando melhore s
p e rfis antro p o m é t ri c o - n u t ricionais entre os es-
c o l a res com maior poder aquisitivo. Estes art i-
g o s, publicados entre 1949 a 1968, são assina-
dos pelos médicos nutrólogos Josué de Ca s t ro,
Walter Si l va, Pe d ro Bo rg e s, Thales de Aze ve d o,
Irma Fi o ra vanti, Nabuco Lopes e Ge raldo Fra n-
cisco Ma l d o n a d o. A perspectiva biológica: dos métodos
de determinação do valor nutricional
aos métodos de industrialização
dos alimentos Esta temática emerg e
em setembro de 1947, a partir de um artigo de
Walter Si l va que apresenta uma abord a g e m
t e ó rica sobre os três grupos de fatores a sere m
c o n s i d e rados na formulação de uma política
alimentar para o Brasil (disponibilidade de ali-
m e n t o s, poder aquisitivo e capacidade de sele-
ção de alimentos). A articulação destes três
g rupos de fatores apontados por Si l va (1947),
no nosso entender, caminhará para a form u l a-
ção da matriz teórico-conceitual sobre a pro-
blemática alimentar e nutricional bra s i l e i ra ,
que unificará os intelectuais do campo da nu-
t rição em torno do seu cri a d o r, Josué de Ca s t ro
(Lima, 1997; Ma g a l h ã e s, 1997). Ainda, no nosso
e n t e n d e r, implicitamente, através do re f e ri d o
a rtigo (Si l va, 1947), os médicos nutrólogos vin- A perspectiva biológica: dos métodos
de determinação do valor nutricional
aos métodos de industrialização
dos alimentos uso de métodos antro p o m é t ri c o s, clínicos, la-
b o ra t o ri a i s, dietéticos, demográficos e sócio-
econômicos para a realização de diagnósticos
n u t ricionais de comunidades), foram incluídos
21 (28,0%) dos art i g o s, destacando-se: •
Nove artigos sobre consumo alimentar, uti-
lizando distintas modalidades de inquéri t o s
d i e t é t i c o s. Tais art i g o s, publicados do pri m e i ro
ao último volume da revista, demonstram que,
após o inquérito pioneiro: As condições de vida
das classes operárias no Re c i f e, realizado por Jo-
sué de Ca s t ro em 1933 (Ca s t ro, 1935), esta mo-
dalidade de inve s t i g a ç ã o, além de apri m o ra r-
se enquanto instrumental básico de pesquisa
d e n t ro da perspectiva social da nutri ç ã o, ga-
nhou adeptos em todo o país, a exemplo das
pesquisas realizadas pelos médicos nutrólogos
Orlando Pa rahim e Jamesson Fe r re i ra Lima, em
Pe rnambuco; Thales de Aze ve d o, na Bahia; Os-
waldo Costa e Rubem de Si q u e i ra, no Rio de Ja-
n e i ro; Domingos Lopes e Benedito Ph i l a d é l p h o
Si q u e i ra, em Minas Ge rais; Nabuco Lopes, em
A l a g o a s, e Walter Si l va, no Rio Grande do No rt e. •
Nove artigos sobre consumo alimentar, uti-
lizando distintas modalidades de inquéri t o s
d i e t é t i c o s. A perspectiva biológica: dos métodos
de determinação do valor nutricional
aos métodos de industrialização
dos alimentos Nesta seção, passaremos à análise do conteúdo
temático dos 134 artigos incluídos na perspec-
t i va biológica da nutri ç ã o, dentro dos quatro
campos temáticos ou disciplinares em que es-
tes foram categori z a d o s. A temática nutrição básica e experi m e n t a l ,
l i d e rada pelos químicos Emília Pechnik, Lu i z
R i b e i ro Guimarães e José Ma ria Chave s, da Se-
ção de Pesquisas Biológicas do In u b, e pelo
médico Franklin A. de Mo u ra Ca m p o s, do De-
p a rtamento de Fisiologia da Faculdade de Me-
dicina da USP, detém a hegemonia dos tra b a-
lhos desta linha, com 61 (45,5%) artigos publi- A temática nutrição clínica aparece em ter-
c e i ro lugar, com 25 (18,7%) artigos publicados. Os médicos Rubens de Si q u e i ra, Hélio de So u- Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 PRODUÇÃO CIENTÍFICA EM NUTRIÇÃO NO BRASIL 3 1 1 za Luz, Clementino Fraga Filho (da Seção de
Patologia da Nu t rição do Inub), José Ed u a rd o
Du t ra de Ol i ve i ra e Demosthenes Orsini (da Fa-
culdade de Medicina da USP) são os pri n c i p a i s
a u t o res dessa linha, cujo auge de produção ve-
rificou-se no período de 1947 a 1953, com 16
(64,0%) art i g o s. Os artigos desta área abord a m
aspectos de fisiologia, patologia e dietotera p i a ;
o ri g i n a ram-se de pesquisas do tipo experi m e n-
tal (ensaios biológicos) e estudos de casos (en-
saios clínicos), tratando dos mais diversos te-
m a s, tais como semiótica das hipov i t a m i n o s e s ;
metabolismo basal; dietoterapia em diabetes,
disfunções hepáticas e gastrectomias e padro-
nização de dietas hospitalare s. A perspectiva social: da eugenia através
da alimentação racional à indústria
alimentar no combate à fome No campo temático Ed uc ação Nu t r i c i o n a l,
f o ram classificados sete (9,3%) dos art i g o s, en-
t retanto apenas três são especificamente fiéis a
este campo disciplinar: uma extensa re v i s ã o
bibliográfica sobre a temática educação nutri-
cional no Brasil (à época denominada Ed u c a-
ção Al i m e n t a r), publicada em 1951 pelo oficial
médico do exército e nutrólogo Walter José dos
Santos; um relato da experiência da nutri c i o-
nista Ma ria de Lourdes Mello (Sesi/MG) em
educação alimentar para menores tra b a l h a d o-
res e um esboço de projeto de pesquisa sobre
tabus e hábitos alimentares no Brasil, de auto-
ria do economista Souza Ba r ros (Se s i / A s c o-
fam). Os quatro demais estão relacionados à
f o rmação de recursos humanos (nutri c i o n i s-
t a s, médicos, tecnólogos de alimentos e ve t e ri-
n á rios), sendo incluídos neste grupo por ser
esta a temática mais semelhante. Nesse aspec-
t o, é importante registar que esses tra b a l h o s
a c e rca da formação de recursos humanos, pu-
blicados a partir de 1963, parecem datar o acir-
ramento da discussão em torno da demarc a ç ã o
dos campos de saber, de competência e de po-
der entre os profissionais (especialidades) do
campo da nutri ç ã o. Nesse sentido, em 1963, foi
publicado um artigo sobre tecnologia de ali-
mentos na formação do ve t e ri n á rio (Mu c c i o l o,
1963); a revista re l a t i va ao pri m e i ro semestre
de 1966 publicou um artigo sobre o ensino de
n u t rição para estudantes de Medicina (Du t ra -
d e - Ol i ve i ra, 1966) e, ainda no segundo semes-
t re de 1966 e em 1967, foram publicados dois
a rtigos sobre a formação do profissional nutri-
cionista (Ca va l c a n t e, 1966, 1967). A perspectiva social: da eugenia através
da alimentação racional à indústria
alimentar no combate à fome De s t a c a-
mos a publicação, no período 1961-1963, de
cinco artigos apresentando propostas de pro-
d u ç ã o, abastecimento de alimentos e de re f o r-
ma agrária (Ma u r í c i o, 1961; Fe r re i ra-Lima &
Ba n c ov s k y, 1962 e 1963; Fe r re i ra Lima et al.,
1962; Salomão & Gomes da Si l va, 1963), quan-
do o Brasil inteiro, part i c u l a rmente a re g i ã o
No rd e s t e, vive n c i a va uma intensa mobilização
dos tra b a l h a d o res ru rais com a criação das Li-
gas Camponesas e dos Sindicatos Ru rais (Aze-
v ê d o, 1982). De s t a c a m o s, ainda, no período
1951-1968, a publicação de quatro artigos a b o r-
dando a questão da mere n d a e sc ol a r; a nosso
ve r, foram importantes veículos de difusão das
idéias em torno da u t i l i d a d e da política nacio-
nal de alimentação escolar, implantada no Bra-
sil, no decorrer dos anos 50, a partir dos pro g ra-
mas internacionais de ajuda alimentar (Co i m-
b ra et al., 1982). E, finalmente, os artigos que
p re n u n c i a vam o futuro envolvimento que teri a
a indústria de alimentos na formulação e con-
dução da política de alimentação e nutrição do
País no pós-1964. A perspectiva social: da eugenia através
da alimentação racional à indústria
alimentar no combate à fome Os 75 artigos incluídos na perspectiva social da
n u t rição foram categorizados dentro dos cinco
campos temáticos ou disciplinares que discuti-
remos a seguir. No campo temático Aval i ação Nu t r i c i o n a l
de Po p u l a ç õ e s (que compreendemos como o Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 VASCONCELOS, F. A. G. 3 1 2 3 1 2 VASCONCELOS, F. A. G. t rição pro t é i c o - c a l ó rica (DPC), de autoria dos
médicos Irma Fi o ra vanti e Nelson Chave s, que
t ratam, re s p e c t i va m e n t e, de um estudo de pre-
valência sobre DPC hospitalar e de uma pre l e-
ção conferida pelo pro f e s s o r, abordando as-
pectos epidemiológicos desta carência nutri-
cional no No rdeste; c) um artigo sobre hipov i-
taminose A, de autoria de Thales de Aze ve d o ;
t rata-se de uma curta revisão bibliográfica,
a p resentando alguns dados sobre a pre va l ê n-
cia desta carência nutricional na Ba h i a . culados aos Arq u ivo s b u s c a vam gerar consen-
so e legitimidade para a regulamentação da
Comissão Nacional de Alimentação (CNA),
agência criada em 1945, mas que só passaria a
o p e ra r, efetiva m e n t e, a partir de 1951, com o
re t o rno de Getúlio Va rgas à Presidência da Re-
pública (L’ Ab b a t e, 1982). Aliás, a maioria des-
tes artigos são de natureza teóri c o - c o n c e i t u a l ,
a p resentando enfoques e propostas políticas
s o b re temas emergentes em determinadas c o n-
j u n t u ra s. A perspectiva social: da eugenia através
da alimentação racional à indústria
alimentar no combate à fome Como exemplo, podemos apontar o
a rtigo Plano de política alimentar na Amazônia
( Ca s t ro, 1951), um re l a t ó rio técnico, no qual Jo-
sué de Ca s t ro, dentro da abordagem geográfi-
co-ecológica que cara c t e rizará a sua obra (Ma-
g a l h ã e s, 1997; Lima, 1997), já àquela época, su-
g e ria a implantação de medidas de interve n ç ã o
a l i m e n t a r- n u t ricional visando ao desenvo l v i-
m e n t o e c on ôm ic o s u st e nt ável para aquela re-
gião bra s i l e i ra, que estava em evidência desde
o início da Segunda Gu e r ra Mundial. De s t a c a-
mos a publicação, no período 1961-1963, de
cinco artigos apresentando propostas de pro-
d u ç ã o, abastecimento de alimentos e de re f o r-
ma agrária (Ma u r í c i o, 1961; Fe r re i ra-Lima &
Ba n c ov s k y, 1962 e 1963; Fe r re i ra Lima et al.,
1962; Salomão & Gomes da Si l va, 1963), quan-
do o Brasil inteiro, part i c u l a rmente a re g i ã o
No rd e s t e, vive n c i a va uma intensa mobilização
dos tra b a l h a d o res ru rais com a criação das Li-
gas Camponesas e dos Sindicatos Ru rais (Aze-
v ê d o, 1982). De s t a c a m o s, ainda, no período
1951-1968, a publicação de quatro artigos a b o r-
dando a questão da mere n d a e sc ol a r; a nosso
ve r, foram importantes veículos de difusão das
idéias em torno da u t i l i d a d e da política nacio-
nal de alimentação escolar, implantada no Bra-
sil, no decorrer dos anos 50, a partir dos pro g ra-
mas internacionais de ajuda alimentar (Co i m-
b ra et al., 1982). A perspectiva social: da eugenia através
da alimentação racional à indústria
alimentar no combate à fome E, finalmente, os artigos que
p re n u n c i a vam o futuro envolvimento que teri a
a indústria de alimentos na formulação e con-
dução da política de alimentação e nutrição do
País no pós-1964. culados aos Arq u ivo s b u s c a vam gerar consen
so e legitimidade para a regulamentação da
Comissão Nacional de Alimentação (CNA),
agência criada em 1945, mas que só passaria a
o p e ra r, efetiva m e n t e, a partir de 1951, com o
re t o rno de Getúlio Va rgas à Presidência da Re-
pública (L’ Ab b a t e, 1982). Aliás, a maioria des-
tes artigos são de natureza teóri c o - c o n c e i t u a l ,
a p resentando enfoques e propostas políticas
s o b re temas emergentes em determinadas c o n-
j u n t u ra s. Como exemplo, podemos apontar o
a rtigo Plano de política alimentar na Amazônia
( Ca s t ro, 1951), um re l a t ó rio técnico, no qual Jo-
sué de Ca s t ro, dentro da abordagem geográfi-
co-ecológica que cara c t e rizará a sua obra (Ma-
g a l h ã e s, 1997; Lima, 1997), já àquela época, su-
g e ria a implantação de medidas de interve n ç ã o
a l i m e n t a r- n u t ricional visando ao desenvo l v i-
m e n t o e c on ôm ic o s u st e nt ável para aquela re-
gião bra s i l e i ra, que estava em evidência desde
o início da Segunda Gu e r ra Mundial. Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 A perspectiva social: da eugenia através
da alimentação racional à indústria
alimentar no combate à fome Além disso, é preciso re g i s t rar que: a) a par-
tir de 1959, com a adesão de novos médicos
m i l i t a res à nutrologia (Jair de Matos Mo n t e-
d ôn i o, José João Barbosa e Ge raldo Fra n c i s c o
Maldonado), começaram a ser publicados art i-
gos sobre nutrição nas forças armadas e b) em
1961 foi publicado um re l a t ó rio técnico sobre a
a t u a ç ã o, no Brasil, do pro g rama Al im e ntos pa-
ra a Pa z, desenvolvido pelo gove rno nort e - a m e-
ri c a n o, prenunciando o assistencialismo ali-
mentar que vigora ria no País no pós-64 (Co i m-
b ra et al., 1982; L’ Ab b a t e, 1982). Por fim, entre 1964 e 1968, a discussão cen-
t ral girou em torno dos temas desenvo l v i m e n-
to econômico, população, alimentação, tecno-
logia e, pri n c i p a l m e n t e, sobre o papel da in-
d ú s t ria alimentar no combate à fome (Orn e l-
l a s, 1965). p
q
Nesse sentido, é preciso atentar para a
e m e rgência de temas específicos em determ i-
nados cortes tra n s versais da tra j e t ó ria da re v i s-
ta. En t re 1944 a 1955, permanecia em evidên-
cia a tese da ignorância alimentar do povo bra-
s i l e i ro, presente em todas as camadas sociais,
e m b o ra com certas distinções de classe social
( Pa rahim, 1944; Si q u e i ra, 1950). Po rt a n t o, tor-
n a va-se fundamental a sedutora proposta da
“a l im e nt ação ra c i o n a l , como fundamento da
vida e condição precípua de saúde, eugenia e
p ro g re s s o” (Pa rahim, 1944:23), legitimadora da
constituição do campo da nutrição e da educa-
ção alimentar como um dos seus instru m e n-
tais básicos (Lima, 1997). A perspectiva social: da eugenia através
da alimentação racional à indústria
alimentar no combate à fome s é rie histórica 1944-1968, a nosso ve r, pode ser
a t ribuída à articulação de distintos elementos
na tra j e t ó ria dos intelectuais vinculados aos
Arq u ivo s, tais como a maior liberdade de ex-
p ressão que, gra d a t i va m e n t e, foi sendo am-
pliada no País a partir da queda do Estado No-
vo; a incorporação de novas concepções cientí-
ficas e filosóficas, a exemplo, entre outra s, da
t e o ria do círculo vicioso da pobreza e da doen-
ça no início dos anos 50 (Braga & Paula, 1981);
das teorias do subdesenvo l v i m e n t o - d e s e n vo l-
vimento a partir de meados dos anos 50 (Ma n-
tega, 1985) e do discurso pre ventivista dos anos
60 (Arouca, 1975) e, finalmente, a maior auto-
nomia que o campo da nutrição foi conquis-
tando no interior do espaço acadêmico (nas
u n i versidades e em outras instituições de ensi-
no e pesquisa), do espaço político-institucio-
nal (nas agências e instituições estatais vincu-
ladas à política s ocial de alimentação e nutri-
ção) e no seio da sociedade civil (Co i m b ra et
al., 1982; L’ Ab b a t e, 1982; Natal, 1982). Assim
como nos demais, este campo temático tam-
bém reflete aspectos das conjunturas específi-
cas em que os artigos foram produzidos e di-
fundidos pelos Arq u ivo s. (
)
En t re 1959 a 1963, a discussão assumiu no-
vos contorn o s, ampliando-se o debate em tor-
no dos determinantes demográficos, econômi-
co-sociais e estru t u ra i s, part i c u l a rmente sobre
re f o rma agrária, produção e abastecimento de
a l i m e n t o s, conforme apontamos anteri o rm e n-
t e. A perspectiva social: da eugenia através
da alimentação racional à indústria
alimentar no combate à fome No campo temático Ep id em i ol ogia Nu t r i-
c i o n a l (que compreendemos como o estudo
das doenças nutricionais em uma determ i n a d a
população), foram identificados apenas sete
(9,3%) dos art i g o s, sendo: a) quatro sobre bó-
cio endêmico, publicados entre 1944 a 1959,
o riginados das atividades desenvolvidas pelo
então De p a rtamento Nacional de Sa ú d e, que
relatam estudos de pre valência entre escola-
res ou apresentam revisões sobre pre va l ê n c i a ,
á reas e n d ê m i c a s, profilaxia e tratamento da
doença. Seus autores são os médicos Álva ro
Lobo Leite Pe re i ra, Amílcar Ba rca Pellon, Wa l-
ter Si l va e Pe d ro Bo rges; b) dois sobre desnu- No campo temático Det e rm in a ntes do Es t a-
do Nutricional de Po p u l a ç õ e s, foram incluídos
20 (26,7%) dos art i g o s. Apesar de implícito nos
a rtigos da perspectiva social desde 1944, este
campo temático só ganhou especificidade a
p a rtir de 1950 e expressão numérica nos anos
60. Os artigos nele incluídos, de natureza teóri-
co-conceitual, tratam das concepções ou pon-
tos de vista de distintos autores acerca da gê-
n e s e, re p rodução e conseqüências da pro b l e-
mática alimentar e nutricional no Brasil. Esta
politização observada nos artigos ao longo da Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 PRODUÇÃO CIENTÍFICA EM NUTRIÇÃO NO BRASIL 3 1 3 p ro m ovam atividades que visem aumentar as
disponibilidades de alimentos de alto valor nu-
t r i t i vo” (Costa & Si l va, 1946:16-17). A perspectiva social: da eugenia através
da alimentação racional à indústria
alimentar no combate à fome Observa-se que a te-
se do mal de fome e não de raça, formulada por
Josué de Ca s t ro nos anos 30 – sua forma de in-
terlocução com os cientistas de outros campos
d i s c i p l i n a res que, à época, pro c u ra vam desfo-
car da questão biológica para a questão sócio-
c u l t u ral o preconceito de clima (meio) e de ra-
ça que se tinha sobre o povo bra s i l e i ro (Lima,
1997; Ma g a l h ã e s, 1997) –, continuava, nos anos
44-55, ecoando entre os intelectuais do campo
da nutri ç ã o. En t re t a n t o, nesse mesmo contex-
t o, outras teses foram sobrepondo-se a essa an-
t e ri o r, a exemplo da linha que concluía que a
questão alimentar estava “mais na dependên-
cia do poder aquisitivo que de outro fator qual-
quer e, na impossibilidade de agir sobre o salá-
r i o, impõe-se que os serviços de saúde pública,
ao lado das atividades de educação alimentar, Considerações finais Fase esta liderada por um grupo de médicos
nutrólogos que, ao mesmo tempo em que pro-
c u ra ram estabelecer as conexões entre o bioló-
gico e o social, com base na interd i s c i p l i n a ri-
dade e na complexidade inerentes ao novo c a m-
po do saber que se constituía, contra d i t o ri a-
m e n t e, cri a ram a demarcação de outras fro n-
t e i ra s, definidoras dos limites de competência,
de autonomia e de poder – na concepção teóri-
ca de Bo u rdieu (1987 e 1994) –, entre as espe-
cialidades do campo da nutri ç ã o, part i c u l a r-
mente entre médicos nutrólogos e nutri c i o n i s-
t a s. Assim, em 1968, formou-se um hiato para
a (re ) c o n s t rução de uma nova escola bra s i l e i ra
de nutri ç ã o. Mas isso é uma outra históri a . q u ivos de Ga s t ro e n t e ro l o g i a, os Arq u ivos de
Bro m a t o l o g i a e t c.) não nos autoriza a genera l i-
zar as conclusões deste estudo. Sendo assim,
deixamos acesa a chama para o trilhar de no-
vas investigações neste campo. A análise dos 209 artigos originais publica-
dos pelos Arq u ivo s nos possibilitou a identifi-
cação não apenas do elenco de intelectuais
que part i c i p a ram do processo de constituição
do campo da nutri ç ã o, mas também de como
as temáticas por estes abordadas foram sendo
t ra n s f o rmadas ao longo dos 24 anos de circ u l a-
ção da revista. Considerações finais A opção metodológica adotada neste estudo –
a realização de uma investigação sistemati-
zada baseada em princípios da meta-análise
( Greenland, 1987; Jenicek, 1989; Riegelman &
Hirsch, 1992; Jo n e s, 1993; T h a c k e r, 1993; Esco-
b a r, 1994), buscando sumariar aspectos quan-
t i - q u a l i t a t i vos dos artigos originais publicados
nos Arq u ivos Bra s i l e i ros de Nutrição –, apesar
de demonstra r-se eficiente na consecução dos
o b j e t i vos a que nos pro p u s e m o s, apre s e n t a
também as suas limitações. Em pri m e i ro lugar,
é preciso destacar que o nosso olhar debru ç o u -
se apenas sobre uma das seções da estru t u ra
i n t e rna de organização do periódico (a seção
de artigos originais), o que não nos autoriza a
t i rar conclusões sobre a tra j e t ó ria desta publi-
cação científica como um todo. Em segundo
l u g a r, é preciso re g i s t rar que, embora acre d i t e-
mos que os artigos publicados nos Arq u ivo s s e-
jam re p re s e n t a t i vos do que foi produzido e di-
fundido no campo da nutrição ao longo da sé-
rie histórica 1944-1968, a inexistência de estu-
dos acerca da produção difundida em outra s
publicações científicas contemporâneas ao pe-
riódico (os Arq u ivos Bra s i l e i ros de Me d i c i n a, a
Revista Bra s i l e i ra de Me d i c i n a, os Arq u ivos Bra-
s i l e i ros de Endocrinologia e Me t a b o l o g i a, os Ar- Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 3 1 4 VASCONCELOS, F. A. G. tos idealizados por seu dire t o r-fundador de
c o n t ribuir com a difusão dos modernos conhe-
cimentos científicos sobre nutrição para a for-
mação de novas gerações de nutrólogos, nutri-
cionistas e outros especialistas deste campo
( Ca s t ro, 1944). Considerações finais Além desse mérito técnico-cien-
t í f i c o, é preciso reconhecer a importância dos
Arq u ivo s como instrumento político-ideológi-
co a serviço de interesses específicos dos cien-
tistas que os conduziram. A análise da séri e
h i s t ó rica 1944-1968 nos faz concluir que, mes-
mo com o afastamento oficial de Josué de Ca s-
t ro da direção da revista, quando o mesmo pas-
sou a exercer sua militância política no perío-
do de 1955-1964 (ocupando os cargos de depu-
tado federal pelo estado de Pe rnambuco e de
embaixador bra s i l e i ro junto à Organização das
Nações Unidas – ONU) (Co i m b ra et al., 1982;
L’ Ab b a t e, 1982), sob a direção dos seus discípu-
los mais diletos (Pe d ro Bo rges e Hélio Ve c c h i o
A. Maurício), os Arq u ivo s m a n t i ve ram a mesma
d i re t riz e até dinamizaram quantitativa m e n t e
o seu número de publicações. Após o golpe mi-
litar de 1964, com o exílio de seu dire t o r- f u n-
d a d o r, os Arq u ivo s c o n s e g u i ram circular até
1968, encerra n d o, assim, uma importante fase
da nossa história, quando “d e nt ro das pare d e s
fechadas dos labora t ó r i o s , no campo aberto da
pesquisa social, na cátedra e nos livro s , se criou
a escola bra s i l e i ra de Nu t r i ç ã o” (Ca s t ro, 1 9 4 4 : 3 ) . Considerações finais Grosso modo, podemos dize r
q u e, no período 1944-1954, correspondente à
d i reção Josué de Ca s t ro, os estudos sobre a
composição química e o valor nutricional de
alimentos nacionais, desenvolvidos pelos inte-
lectuais vinculados à perspectiva biológica,
aliados aos estudos sobre consumo alimentar e
estado nutricional da população bra s i l e i ra, de-
s e n volvidos pelos intelectuais vinculados à
p e r s p e c t i va social, constituíram a base cogniti-
va gera d o ra da especificidade do campo da nu-
t rição no Brasil, considerada como disciplina,
política social e/ou pro f i s s ã o. A matriz teóri c a
que implícita ou explicitamente unificou tais
i n t e l e c t u a i s, “a tese do valor eugênico da ali-
mentação na construção da nação e do homem
brasileiro” (Lima, 1997; Magalhães, 1997), a nos-
so ve r, sintetiza a ressonância das concepções
de cunho nacionalista que cara c t e ri z a ram os
anos 30-40, no campo da nutri ç ã o, no Brasil (Mi-
celi, 1979; Pecaut, 1990; Go m e s, 1996). Por ou-
t ro lado, no período 1955-1968, corre s p o n d e n-
te à direção Pe d ro Bo rges/Hélio Vecchio Ma u-
r í c i o, a maior politização dos intelectuais vin-
culados aos Arq u ivo s em suas concepções acer-
ca da gênese, re p rodução e enfrentamento da
f o m e / s u b n u t rição da população bra s i l e i ra, si-
naliza, em última instância, a comunhão destes
intelectuais com as teorias do nacional desen-
volvimentismo dos anos 50-60 (Mantega, 1985). Fi
li
d
di í
ó Fi n a l i z a n d o, diríamos que, para nós, ao
longo dos seus 24 anos de circ u l a ç ã o, os Arq u i-
vo s p a recem ter cumprido os objetivos explíci- Cad. Saúde Pública, Rio de Janeiro, 15(2):303-316, abr-jun, 1999 R e f e r ê n c i a s ALENCAR, F.; CARPI, L. & RIBEIRO, M. V., 1979. Hi s-
tória da Sociedade Bra s i l e i ra. Rio de Ja n e i ro: Ao
L i v ro T é c n i c o. C H AVES, N., 1978. Nutrição Básica e Ap l i c a d a. Rio de
Ja n e i ro: Gu a n a b a ra - Ko o g a n . DUTRA-DE-OLIVEIRA, J. E., 1966. Ensino de nutri ç ã o
p a ra estudantes de Medicina. Arq u i vos Bra s i l e i ro s
de Nu t r i ç ã o, 22:21-36. A ROUCA, A. S. S., 1975. O Dilema Pre ve n t i v i s t a :C o n-
tribuição para a Compreensão e Crítica da Me d i-
cina Pre ve n t i va. Tese de Doutora d o, Ca m p i n a s :
Faculdade de Ciências Médicas, Un i versidade Es-
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n i a : Uma Me t a - An á l i s e. Di s s e rtação de Me s t ra d o,
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ca Cu l t u ral do Estado Novo. Rio de Ja n e i ro: Ed i t o-
ra da Fundação Getúlio Va rg a s. BOURDIEU, P., 1987. A Economia das Trocas Si m b ó l i-
c a s. São Paulo: Pe r s p e c t i va . BOURDIEU, P., 1994. El campo científico. Revista de
Estudios Sociales de la Ciencia (Re d e s ), 1:131-160. GOUVEIA, E. L. C., 1960. O papel dos inquéritos ali-
m e n t a res nos pro g ramas educacionais e assisten-
ciais de coletividades. Arq u i vos Bra s i l e i ros de Nu-
t r i ç ã o, 16:69-78. B R AGA, J. C. S. & PAU LA, S. G., 1981. Saúde e Pre v i-
d ê n c i a : Estudos de Política Social. São Pa u l o :
Ce b e s / Hu c i t e c. G R E E N LA N D, S., 1987. Qu a n t i t a t i ve methods in the
review of epidemiological litera t u re. A g r a d e c i m e n t o s Aos pro f e s s o res Ricardo Ve n t u ra Santos e Carlos E. A. Co i m b ra Jr. (Escola Nacional de Saúde Pública, Fu n-
dação Oswaldo Cruz), pela prestimosa orientação e
l e i t u ra crítica do texto ori g i n a l . PRODUÇÃO CIENTÍFICA EM NUTRIÇÃO NO BRASIL 3 1 5 R e f e r ê n c i a s Arq u i vos Bra s i l e i ros de Nu t r i ç ã o, 3:6-
3 6 . S I QUEIRA, R. & VOGEL, J., 1948b. Aspectos semióti-
cos das carências vitamínicas no homem e nos
animais domésticos (continuação). Arq u i vos Bra-
s i l e i ros de Nu t r i ç ã o, 5:5-58. ORSINI, D., 1947b. O metabolismo de base de jove n s
b ra s i l e i ras (continuação). Arq u i vos Bra s i l e i ros de
Nu t r i ç ã o, 3:6-66. PARAHIM, O., 1944. O problema alimentar no Se rt ã o
No rdestino: a eugenia através da alimentação ra-
cional. Arq u i vos Bra s i l e i ros de Nu t r i ç ã o, 1:23-36. S I QUEIRA, R. & VOGEL, J., 1948c. Aspectos semióti-
cos das carências vitamínicas no homem e nos
animais domésticos (conclusão). Arq u i vos Bra-
s i l e i ros de Nu t r i ç ã o, 5:5-58. PE C AU T, D., 1990. Os Intelectuais e a Política no
Bra s i l : En t re o Povo e a Na ç ã o. São Paulo: Ed i t o ra
Á t i c a . T H ACKER, S. B., 1993. Metanálisis: un enfoque cuan-
t i t a t i vo para la integración de inve s t i g a c i o n e s. Boletín de la Oficina Sanitaria Pa n a m e r i c a n a,
1 1 5 : 3 2 8 - 3 3 9 . PECHNIK, E.; BORGES, P. & SIQUEIRA, R., 1950. Estu-
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menor tra b a l h a d o r. Arq u i vos Bra s i l e i ros de Nu-
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cos das carências vitamínicas no homem e nos
animais domésticos. Arq u i vos Bra s i l e i ros de Nu-
t r i ç ã o, 5:5-46. ORSINI, D., 1947a. O metabolismo de base de jove n s
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Self-organization of swimmers drives long-range fluid transport in bacterial colonies
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Nature communications
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ARTICLE Self-organization of swimmers drives long-range
fluid transport in bacterial colonies Haoran Xu1, Justas Dauparas2, Debasish Das2, Eric Lauga
2 & Yilin Wu
1 Motile subpopulations in microbial communities are believed to be important for dispersal,
quest for food, and material transport. Here, we show that motile cells in sessile colonies of
peritrichously flagellated bacteria can self-organize into two adjacent, centimeter-scale motile
rings surrounding the entire colony. The motile rings arise from spontaneous segregation of a
homogeneous swimmer suspension that mimics a phase separation; the process is mediated
by intercellular interactions and shear-induced depletion. As a result of this self-organization,
cells drive fluid flows that circulate around the colony at a constant peak speed of ~30 µm s−1,
providing a stable and high-speed avenue for directed material transport at the macroscopic
scale. Our findings present a unique form of bacterial self-organization that influences
population structure and material distribution in colonies. 1 Department of Physics and Shenzhen Research Institute, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, People’s Republic of
China. 2 Department of Applied Mathematics and Theoretical Physics, University of Cambridge, Cambridge CB3 0WA, UK. Correspondence and requests for
materials should be addressed to Y.W. (email: ylwu@cuhk.edu.hk) NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications 1 TURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/ ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 M M
icrobes commonly live in structured communities that
play important roles in human health and ecology1–5,
such as biofilm aggregates during chronic infections6,
commensal microbiome in animal guts, microbial mats in river
beds or ocean floors, and bacterial colonies in soils or food pro-
ducts5. The multicellular lifestyle is key to the prospering of
microbes in diverse habitats as it confers high resistance to var-
ious environmental stresses7–11. Understanding the physiology of
these structured microbial communities is not only essential to
the treatment of chronic infections, but also important to
industrial applications, such as bioremediation, anti-biofouling
and food hygiene. In addition, as a type of active matter,
microbial communities have provided model systems for the
research of biological self-organization and complex fluids12–16. colony-scale motile rings is present in colonies of Proteus mir-
abilis, Escherichia coli, and Bacillus subtilis, suggesting that the
phenomenon is conserved among bacteria species with peri-
trichous flagella. Results
S lf 1e, Supplementary Fig. 1; Supplementary Movie 2). None-
theless, the motion of cells in the inner ring shows a weak CCW
bias [defined as NCCWVCCW/NCWVCW, where NCCW (or NCW)
and VCCW (or VCW) denote the number and the mean speed of
CCW (or CW) moving cells, respectively] ranging from ~1.1 to
~1.2, and the inner motile ring collectively circles CCW around
the colony at a mean speed of ~1 µm s−1 (Fig. 1d). Our phe-
nomenon is distinct from the self-organized vortex reported in
microscale circular confinement31,32, which did not display
chirality bias and nematic order in cells’ moving directions. Note
that hereinafter in the paper CW and CCW refer to the sense of
chirality with respect to the entire colony viewed from above,
unless otherwise indicated. Colony mode of bacterial growth on solid substrates (e.g., food
products and solidified nutrient agar) is a common experimental
approach to study structured microbial communities11 and is
closely related to biofilm development23,24. Cells in a bacterial
colony move in confined space surrounded by boundaries
between gas, liquid, and solid phases. Interfaces and spatial
confinement are known to affect motile behavior of bacteria in
more artificial settings such as microfluidic systems. Bacteria
swimming near liquid–solid interfaces have the tendency moving
in parallel to the wall due to hydrodynamic trapping25–30, but it
was
less
clear
how
swimming
bacteria
behave
at
the
gas–liquid–solid three phase interface, such as the edge of bac-
terial colonies. Also it was reported that confined bacterial sus-
pension droplets of size less than ~100 micron31,32 can self-
organize into vortices, in which two concentric, counter-rotating
regions of bacteria are present near the edge of the droplet. This
type of bacterial self-organization has not yet been observed in
the context of naturally developed colonies. Understanding how
bacterial motility adapts to the physical environment in colonies
may
therefore
have
implications
for
active
matter
self-
organization and for the engineering of self-assembled micro-
fluidic systems that perform fluid pumping and cargo trans-
port33–38. Here we sought to examine the behavior of motile cell popu-
lations in bacterial colonies and to explore their potential phy-
siological functions. In routinely cultured bacterial colonies where
most cells have transitioned into a sessile state, we discover that
motile cells can self-organize into two adjacent, centimeter-scale
motile rings that surround the entire colony. Results
S lf Heterogeneous populations, including motile and non-motile
populations, often coexist in bacteria communities17. Motile
subpopulations in microbial communities are believed to be
important to dispersal18,19, quest for food20, and material trans-
port21. However, except in circumstances where motile cells drive
colony expansion (e.g., bacterial swarming8,10,22), the physiolo-
gical functions of motile subpopulations in bacterial communities
are largely unclear. For example, in many occasions bacterial
colonies are sessile and their expansion is driven by growth rather
than by cell motility, but these sessile colonies normally preserve a
subpopulation of motile cells for reasons that are not well
understood17. Self-organization of colony-scale motile rings. Our organism of
choice was P. mirabilis, a Gram-negative rod-shaped bacterium
well known for its swarming behavior on hard agar surfaces39,40. P. mirabilis is widely distributed in the natural environment and
as a human pathogen with clinical importance, it is often asso-
ciated with urinary tract infections41. When grown on soft agar
plates in humid environment (~85% relative humidity) for ~24 h
(Methods; Supplementary Table 1), P. mirabilis colonies have not
initiated swarming yet and most cells have become sessile,
although a sub-population of motile planktonic cells (ranging
from 2 to 10 µm in length) are present. Such sessile colonies of P. mirabilis provide a model system for studying the behavior and
physiological function of motile sub-populations. We discovered
that, surprisingly, motile cells in such colonies self-organize into
two adjacent motile rings that surround the entire colony
(Fig. 1a–d; Supplementary Movie 1). The outer motile ring is
located at the outmost rim of the colony; it measures ~10 µm in
width and ~1 µm in height. Cells in this ring are well aligned with
each other, with the average orientation making an angle of ~30°
with colony edge, presumably due to steric repulsion between
densely packed cells42; they circle exclusively clockwise (CW)
around the colony (viewed from above; observed in >100 P. mirabilis colonies on tens of agar plates) at a uniform, constant
speed (mean: 27.8 µm s−1, S.D.: 2.7 µm s−1, N = 10), i.e., their
collective motion is polarly ordered. On the other hand, the inner
motile ring is ~20–40 µm in width and ~2–3 µm in height; it is
bounded by the outer ring and the sessile part of the colony, and
cells in this ring tend to swim in parallel to colony edge bi-
directionally with nematic order in cells’ moving directions
(Fig. Self-organization of swimmers drives long-range
fluid transport in bacterial colonies As a result of this self-organization, cells in the
outer motile ring drive fluid flows in the inner motile ring to
circulate counterclockwise (CCW) around the colony at a con-
stant peak speed of ~30 µm s−1, providing a stable and high-
speed avenue for directed material transport at the macroscopic
scale. Our findings reveal a unique form of colony-scale self-
organization and active transport in bacterial colonies, which may
shape the population structure and material distribution of bac-
terial communities in widespread environments. NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunicatio NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 2 and Supplementary Movie 3), albeit
less robust against environmental perturbations. B. subtilis
displays vigorous swarming due to the secretion of surfactin, a
lipoprotein having potent surfactant activities43. Nonetheless, B. subtilis colonies prior to the onset to swarm expansion (~3-h post
inoculation; “Methods”) displayed self-organization of motile
rings as well (Supplementary Fig. 2 and Supplementary Movie 4). These findings suggest that the phenomenon is conserved among
bacteria species with peritrichous flagella. We note that smooth- inoculation, at which time a highly organized outer motile ring
very rapidly emerges at the colony edge. The full development is
completed within 10 min, as shown by the sharp transition of
mean tangential speed at T = 0 min in Fig. 1f. Since the motility
and the density of cells at colony edge is similar before and after
this transition point, we suggest that the physicochemical
conditions of the colony, presumably water content (Supplemen-
tary Table 1) and surface tension, may just reached an
appropriate state at the transition point permitting the formation
of a thin wetting film ~1–2 µm in thickness at the colony edge
that can support 2D motion of cells. Once emerged, the motile
rings remain stable for many hours until the initiation of
swarming. Environmental humidity is key to the formation of
motile rings of Proteus mirabilis. Motile rings cannot be observed
if the relative humidity is below 47% and they appear earlier on
plates with higher agar concentrations (Supplementary Table 1). Motile ring self-organization arises from physical interactions. Next we sought to understand the self-organization process of
motile rings. A recent study reported that concentrated swim-
ming bacteria confined in 1D microfluidic channels can display
unidirectional collective motion due to flagella-driven fluid flows
generated by cells at channel edge44. The geometry in our phe-
nomenon is similar to a 1-D channel, since the two motile rings
are bounded by colony edge and the sessile part of the colony. In
contrast to the phenomenon reported earlier44, cells in the inner
motile ring only display weakly directed collective motion
(Fig. 1), suggesting a different mechanism at work. Nonetheless,
we decided to examine whether channel-like confinement is
necessary for motile ring self-organization. We collected motile
cells from swarming colonies of P. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 Time = 0 min is chosen at the onset of collective motion
with high polar order and it corresponds to ~20 h colony growth 1e–1.0
1e–0.0
c
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Vring (µm/s) f e Fig. 1 Self-organization of two adjacent colony-scale motile rings at the edge of a P. mirabilis colony. a Phase contrast image of P. mirabilis colony. Scale bar,
0.5 mm. b Enlarged view of the area enclosed by magenta box in panel A. Scale bar 10 μm. Also see Supplementary Movie 1. c Time-averaged collective
velocity field of cells in the region of panel b computed by optical flow analysis (“Methods”) based on phase contrast images. The collective velocity field
was averaged over a duration of 10 s. Arrows represent velocity direction, and colormap represents velocity magnitude in log scale (with the color bar
provided to the right, in μm s−1). d The mean tangential speed of collective cellular motion (based on optical flow data) plotted against the distance from
colony edge. Positive value of speed indicates motion along CW direction, i.e., along + Y axis of the coordinate system specified in panel b, and X = 0 is set
at the position of colony edge. e Speed distribution of individual bacteria at the inner motile ring moving in CCW direction (gray) and in CW direction
(brown). Cell speed was computed by tracking fluorescent cells seeded into the colony (“Methods”) and located near the center of inner motile ring where
the magnitude of mean tangential speed was larger than 1/3 of the maximum CCW velocity magnitude in panel d. Inset: Probability distribution of velocity
direction, with 3/2π and 1/2π corresponding to −Y and +Y directions, respectively. See Supplementary Fig. 1 and Supplementary Movie 2. f Dynamics of
motile ring emergence during colony growth. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 1e–1.0
1e–0.0
a
b
d
X
Y
c
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20
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80
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0
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0.1
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0.05
e
2
60
−300 −200 −100
0
100
0
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8
12
f
Time (min)
1e+1.5
1e+0.5
1e+1.0
1e+0.0
(µm/s)
X (µm)
Vring (µm/s)
Vring (µm/s)
Vcell (µm/s)
Fig. 1 Self-organization of two adjacent colony-scale motile rings at the edge of a P. mirabilis colony. a Phase contrast image of P. mirabilis colony. Scale bar,
0.5 mm. b Enlarged view of the area enclosed by magenta box in panel A. Scale bar 10 μm. Also see Supplementary Movie 1. c Time-averaged collective
velocity field of cells in the region of panel b computed by optical flow analysis (“Methods”) based on phase contrast images. The collective velocity field
was averaged over a duration of 10 s. Arrows represent velocity direction, and colormap represents velocity magnitude in log scale (with the color bar
provided to the right, in μm s−1). d The mean tangential speed of collective cellular motion (based on optical flow data) plotted against the distance from
colony edge. Positive value of speed indicates motion along CW direction, i.e., along + Y axis of the coordinate system specified in panel b, and X = 0 is set
at the position of colony edge. e Speed distribution of individual bacteria at the inner motile ring moving in CCW direction (gray) and in CW direction
(brown). Cell speed was computed by tracking fluorescent cells seeded into the colony (“Methods”) and located near the center of inner motile ring where
the magnitude of mean tangential speed was larger than 1/3 of the maximum CCW velocity magnitude in panel d. Inset: Probability distribution of velocity
direction, with 3/2π and 1/2π corresponding to −Y and +Y directions, respectively. See Supplementary Fig. 1 and Supplementary Movie 2. f Dynamics of
motile ring emergence during colony growth. The mean tangential speed of bacteria (computed by optical flow analysis with phase contrast images) in the
10-μm-wide outmost rim of colonies (i.e., the region of outer motile ring) is plotted against time. Results
S lf Located at the
outmost rim of the colony, the outer motile ring measures about
ten microns in width; cells in this ring circle clockwise around the
colony (viewing from above) with high polar order. On the other
hand, the inner motile ring is several tens of microns in width;
cells in this ring tend to swim in parallel to colony edge bi-
directionally with nematic ordering in cells’ moving directions. We demonstrate that the motile rings arise from spontaneous
segregation of a homogeneous swimmer suspension that mimics a
phase separation; the process is mediated by intercellular inter-
actions and by a shear-induced depletion that concentrates bac-
teria in sheared regions. The remarkable self-organization of To characterize the dynamics of motile ring development, we
measured the collective speed of bacteria at colony edge over a
time course of 7 h starting from ~16 h after colony inoculation. At
the early stage of colony development, cells adapt to the surface
environment, extract water from the substrate, and become able
to move on agar surface. At this stage the motion of cells
displayed certain degree of ordering (as shown by the non-zero
mean tangential speed of ~4 µm s−1 prior to time = 0 min in
Fig. 1f). This stage lasts for several hours until ~20 h after colony 2 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 The mean tangential speed of bacteria (computed by optical flow analysis with phase contrast images) in the
10-μm-wide outmost rim of colonies (i.e., the region of outer motile ring) is plotted against time. Time = 0 min is chosen at the onset of collective motion
with high polar order and it corresponds to ~20 h colony growth swimming mutants of E. coli and B. subtilis do not display the self-
organization of motile rings, at least under our experimental
conditions. Smooth-swimming cells are not able to switch flagellar
rotation direction autonomously. Consequently they tend to get
stuck to each other during collisions and form jammed clusters. Indeed, jammed clusters frequently formed at the edge of such
colonies (Supplementary Movie 5). Due to these jammed clusters,
the orientation of cells at the colony edge remained disordered
(Supplementary Fig. 3, panel A), and unidirectional motion could
not develop there, despite a weak CW bias in the average speed of
cells (Supplementary Fig. 3, panel B). inoculation, at which time a highly organized outer motile ring
very rapidly emerges at the colony edge. The full development is
completed within 10 min, as shown by the sharp transition of
mean tangential speed at T = 0 min in Fig. 1f. Since the motility
and the density of cells at colony edge is similar before and after
this transition point, we suggest that the physicochemical
conditions of the colony, presumably water content (Supplemen-
tary Table 1) and surface tension, may just reached an
appropriate state at the transition point permitting the formation
of a thin wetting film ~1–2 µm in thickness at the colony edge
that can support 2D motion of cells. Once emerged, the motile
rings remain stable for many hours until the initiation of
swarming. Environmental humidity is key to the formation of
motile rings of Proteus mirabilis. Motile rings cannot be observed
if the relative humidity is below 47% and they appear earlier on
plates with higher agar concentrations (Supplementary Table 1). To see whether this remarkable self-organization phenomenon
was due to any physiological properties specific to P. mirabilis, we
examined colonies of another two model flagellated bacteria, E. coli and B. subtilis. E. coli colonies were grown on LB agar plates
that do not support swarm expansion (“Methods”), and we
observed the same self-organization of motile rings described
above (Supplementary Fig. ARTICLE Assuming that the flagella bundle is rotating about cell body
long axis with angular speed Ω, then the torque balance at low
Reynolds number gives: Gdrive ¼ D1Ω;
ð3Þ
Gdrag ¼ D2ω;
ð4Þ
ω ¼ D1
D2
Ω;
ð5Þ Gdrive ¼ D1Ω;
ð3Þ
Gdrag ¼ D2ω;
ð4Þ
ω ¼ D1
D2
Ω;
ð5Þ ð3Þ ð4Þ ω ¼ D1
D2
Ω;
ð5Þ ð5Þ where D1 is the constant reorientation coefficient relating the
driving torque to the rotation rate Ω of the flagella bundle, and
the constant coefficient D2 relates the drag torque to the angular
speed ω of cell body rotation about the cell pole in contact with
drop edge. We derived D1 and D2 based on slender body
theory47,48 and resistive force theory49, respectively (“Methods”),
which allowed us to compute ω as a function of the flagellar
bundle distance to the bottom surface h. Our simplified model is
more appropriate for E. coli than for P. mirabilis, since P. mirabilis has 2–3 fold higher flagellar density on cell surface than
E. coli and its flagellar filaments may not form a single bundle as
modeled here12. Therefore we chose to verify our model with E. coli. Using the reorientation angular speed ω ≈2.1 rad s−1
measured in our experiment as well as other known parameters
of E. coli, we found the flagellar bundle distance to substrate as
h ≈0.4 μm (“Methods” and Supplementary Fig. 4), i.e., approxi-
mately half of cell body width. This result is reasonable, since cells
are supposed to sit just above the substrate surface at the edge of
liquid drop. CW bias is a result of cell-substrate hydrodynamic interaction. To understand the origin of CW bias of collective motion in the
outer motile ring, we examined the motion pattern of individual
cells at low densities. P. mirabilis colony edge was diluted by
adding external liquid, creating a liquid drop dispersed with
isolated cells that mimicked the fluid environment of colonies
(“Methods”). We found that cells colliding with the edge of such a
diluted colony tended to turn CW with respect to the contact
point about an axis normal to the underlying substrate and
subsequently move to the right along the edge (Fig. 3; Supple-
mentary Movie 8). In fact, cells moved exclusively CW after
collision with the diluted colony edge for colliding angles between
0 and 1.75 rad (Fig. 3e). As expected, isolated cells of both E. ARTICLE We
varied cell speed by tuning environmental temperature (“Meth-
ods”) and found that the width of inner motile ring increases
linearly with cell speed at the outer motile ring (Fig. 2e). This
result suggests that physical interaction between cells, mediated
by either steric repulsion or hydrodynamic forces or both,
controls the formation of inner motile ring. We also tuned the cell
density in suspension drops and found that the order of cellular
motion in the 10-μm-wide outmost rim of the drop depends on
cell density (Fig. 2f): At low densities, cellular motion in this rim
does not display polar order, despite a weak CW bias (mean
tangential speed ~2 μm s−1); beyond a critical cell density (cell
occupation ratio ~0.4), cellular motion experiences a sharp
transition to maximal polar order (mean tangential speed ~12
μm s−1), and a highly ordered CW motile ring emerges. This
result demonstrates that the emergence of a highly ordered outer
motile ring is mediated by intercellular interactions. The results
presented in Fig. 1f and Fig. 2f reveals the two essential
requirements of forming a highly ordered outer motile ring: (1)
Appropriate physicochemical conditions of the colony edge that
support 2D motion of cells; (2) Sufficiently high cell density that
allows for intensive cell-cell interactions. As suggested earlier, the
first requirement was not satisfied in naturally developed P. mirabilis colonies prior to T = 0 min in Fig. 1f, although the
second requirement had already been met. By contrast, the first
requirement was always satisfied in suspension drops of P. mirabilis with the help of exogenously supplied surfactant. We
concluded that the self-organization of motile rings near the edge
of the colony is a collective effect arising from physical
interactions between swimmers under 2D confinement. X
i
Gi ¼ 0
ð1Þ
Gdrive ¼ Gdrag
ð2Þ ð1Þ Gdrive ¼ Gdrag
ð2Þ ð2Þ In our model, the driving torque Gdrive arises in the system
because the bacterium rotates its flagella bundle CCW around
flagellar axis (when looked from behind the cell body) next to the
edge of the drop, where the bottom surface (agar) induces more
friction than the top air-liquid interface46. Gdrive depends on the
height of flagellar bundle above bottom surface. As the body with
flagellar bundle reorients, the driving torque balances a resisting
drag torque Gdrag due to rotation of the whole cell in the viscous
fluid. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 ARTICLE three regions with different types of order, mimicking a phase
separation: a dense and polarly ordered layer at the edge
undergoing CW collective motion (~10 µm in width), a dense
layer with nematic order in cells’ moving directions just adjacent
to the polar-order layer (~30 µm in width), and a low-density
disordered phase in the bulk where cells swim randomly. The two
densely-packed ordered layers resemble the colony-scale motile
rings observed in naturally grown colonies (Fig. 2a–c, Supple-
mentary Fig. 1; Supplementary Movies 6, 7). Our results suggest
that channel-like confinement is not required for motile ring self-
organization in colonies. The results also exclude the possibility
that the sessile part of the colony may have orchestrated the
collective motion in both motile rings through chemical signaling. We note that the speed distribution of cells in the nematic layer is
Gaussian like (Fig. 2d), which is different from the speed dis-
tribution in the inner motile ring of colonies (Fig. 1e); this is
because cells in the inner motile ring of colonies occasionally
collide with sessile cells lying underneath and get stuck tran-
siently, thus contributing to a large number of low-speed traces. contact point (Supplementary Movie 9, 10). Interestingly, by
imaging fluorescently labeled flagellar filaments of GFP-tagged E. coli cells45, we found that the flagellar bundle of cells colliding
with the edge of a suspension drop did not change its orientation
with respect to the cell body throughout the turning process
(Supplementary Movie 9; “Methods”). Presumably the CW bias
of cell reorientation upon contact with colony edge gives rise to
the exclusively CW bias of the collective motion in the outer
motile ring of colonies. g
To understand the mechanism of CW turning around contact
point upon collision, we measured the time dependence of cell
orientation and found that cells rotated at a constant angular
speed ω during CW turning around contact point (for P. mirabilis, ω = 8.3 (3.3) rad s−1, mean (SD), N = 56; for E. coli,
ω = 2.1 (0.9) rad s−1, mean (SD), N = 15). This constant angular
speed of reorientation suggests the balance of torque about the
cell pole in contact with drop edge. We build a simplified
swimmer model to understand this torque balance, as illustrated
in Fig. 3f: We
further
used
suspension
drops
of
P. mirabilis
to
characterize the dynamics of motile ring self-organization. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 mirabilis, re-suspended them
in a medium containing surfactant Tween 20, and deposited them
at appropriate densities on agar surface, forming a non-spreading
liquid drop with motile cells alone that resembles a colony
(referred to as an artificial colony; “Methods”). We found that
motile cells in the artificial colony spontaneously separated into To see whether this remarkable self-organization phenomenon
was due to any physiological properties specific to P. mirabilis, we
examined colonies of another two model flagellated bacteria, E. coli and B. subtilis. E. coli colonies were grown on LB agar plates
that do not support swarm expansion (“Methods”), and we
observed the same self-organization of motile rings described
above (Supplementary Fig. 2 and Supplementary Movie 3), albeit
less robust against environmental perturbations. B. subtilis
displays vigorous swarming due to the secretion of surfactin, a
lipoprotein having potent surfactant activities43. Nonetheless, B. subtilis colonies prior to the onset to swarm expansion (~3-h post
inoculation; “Methods”) displayed self-organization of motile
rings as well (Supplementary Fig. 2 and Supplementary Movie 4). These findings suggest that the phenomenon is conserved among
bacteria species with peritrichous flagella. We note that smooth- TURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications 3 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 8
a
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Fig. 2 Self-organization of two adjacent motile rings at the edge of a P. mirabilis suspension drop (artificial colony). a Fluorescent image of the drop edge
(“Methods”). Scale bar, 10 μm. See Supplementary Movie 6 (phase contrast) and Supplementary Movie 7 (fluorescence). b Time-averaged collective
velocity field of cells in the region of panel a computed by optical flow analysis (“Methods”) based on phase contrast images. The collective velocity field
was averaged over a duration of 10 s. Arrows represent velocity direction, and colormap represents velocity magnitude (with the color bar provided to the
right, in μm s−1). c The mean tangential speed of collective cellular motion (based on optical flow data) plotted against the distance from suspension-drop
edge. Following the coordinate system specified in panel a, positive value of speed indicates motion along +Y axis, i.e., along CW direction viewed from
above the suspension drop, and X = 0 is set at the position of drop edge. d Speed distribution of individual bacteria obtained by single-cell tracking at the
inner motile ring where the magnitude of mean tangential speed was larger than 1/3 of the maximum CCW velocity magnitude in panel c. Brown and
orange columns represent statistics for cells moving in CCW and CW direction, respectively. Inset: Distribution of velocity direction of individual bacteria;
3/2π and 1/2π correspond to −Y and +Y directions, respectively. Also see Supplementary Fig. 1. e The width of inner motile ring plotted against the
mean tangential speed of collective motion at the outer motile ring. Dashed line is a linear fit with R2 = 0.84. Error bars indicate standard error of the mean
(n = 1200 measurements in three biologically independent samples). f The mean tangential speed of cells plotted against cell occupation ratio in the
10-μm-wide outmost rim of the suspension drop (corresponding to the region of outer motile ring in naturally developed colonies). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 Horizontal error bars
indicate standard error of the mean (n = 300 measurements in three biologically independent samples); vertical error bars indicate standard deviation
(n = 10 cells)
|
p
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Vcell (µm/s) f f e Fig. 2 Self-organization of two adjacent motile rings at the edge of a P. mirabilis suspension drop (artificial colony). a Fluorescent image of the drop edge
(“Methods”). Scale bar, 10 μm. See Supplementary Movie 6 (phase contrast) and Supplementary Movie 7 (fluorescence). b Time-averaged collective
velocity field of cells in the region of panel a computed by optical flow analysis (“Methods”) based on phase contrast images. The collective velocity field
was averaged over a duration of 10 s. Arrows represent velocity direction, and colormap represents velocity magnitude (with the color bar provided to the
right, in μm s−1). c The mean tangential speed of collective cellular motion (based on optical flow data) plotted against the distance from suspension-drop
edge. Following the coordinate system specified in panel a, positive value of speed indicates motion along +Y axis, i.e., along CW direction viewed from
above the suspension drop, and X = 0 is set at the position of drop edge. d Speed distribution of individual bacteria obtained by single-cell tracking at the
inner motile ring where the magnitude of mean tangential speed was larger than 1/3 of the maximum CCW velocity magnitude in panel c. Brown and
orange columns represent statistics for cells moving in CCW and CW direction, respectively. Inset: Distribution of velocity direction of individual bacteria;
3/2π and 1/2π correspond to −Y and +Y directions, respectively. Also see Supplementary Fig. 1. e The width of inner motile ring plotted against the
mean tangential speed of collective motion at the outer motile ring. Dashed line is a linear fit with R2 = 0.84. Error bars indicate standard error of the mean
(n = 1200 measurements in three biologically independent samples). ARTICLE coli
and B. subtilis displayed similar CW bias of reorientation around Colony-scale directed flows are present in the inner motile
ring. Fluid pumping due to flagellar rotation of cells in the outer
motile ring may generate strong unidirectional fluid flows, as
suggested by previous studies50,51. To examine this idea, we NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 f The mean tangential speed of cells plotted against cell occupation ratio in the
10-μm-wide outmost rim of the suspension drop (corresponding to the region of outer motile ring in naturally developed colonies). Horizontal error bars
indicate standard error of the mean (n = 300 measurements in three biologically independent samples); vertical error bars indicate standard deviation
(n = 10 cells) visualized fluid flows in P. mirabilis colonies by adding 0.1-μm
fluorescent microspheres (Life Technologies, Cat. No. F8820) as
tracers. We found that tracers were transported in a CCW
manner around the entire colony at a peak constant speed of ~30
µm s−1 (Fig. 4a–d; Supplementary Movie 11), along a narrow
channel of width ~20–30 µm that coincides with the inner motile
ring. A similar stream of fluid flows was found with a much
smaller tracer, fluorescently labelled dextran of a size ~10 nm
(FITC–Dextran, mol. wt. 2000 kDa; Sigma, FD2000S) that can
move freely in between cells (Supplementary Fig. 5). The peak
speed and the spatial range of fluid flows we uncovered are comparable to those of the flagella-driven flows measured near
individual cells whose motion were restricted in quasi-2D liquid
films50,51. The CCW directed flows in the inner motile ring cause
a shear rate up to ~10 s−1 in the plane perpendicular to substrate
surface; this shear rate falls within the range of intermediate shear
rates (~9–12 s−1) for slight downstream motion bias of flagellated
bacteria52, which may account for the weak CCW bias (i.e.,
downstream bias) of collective motion in the inner motile ring. Our flow measurement revealed a stable, high-speed avenue for
directed material transport in bacterial colonies at the macro-
scopic scale. In addition, we also found that microspheres and TURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 0.00 s
0.30 s
0.24 s
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−50
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/2
/4
3/4
f
CCW
CW
CCW
CW
Drop edge
Gdrag=D2
Gdrive=D1
x
Lb
Lf
y
y′
x′
Fig. 3 Single-cell motion pattern of P. mirabilis near diluted colony edge. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 a A sequence of phase contrast images recording the CW turning of an isolated cell
colliding with the diluted colony edge (“Methods”). The dark area in upper portion of images is virgin agar. Scale bar, 5 μm. b Trajectory of the cell in panel
a generated by overlaying a series of phase contrast images. Magenta arrows by the side of trajectory (white dotted line) indicate instantaneous moving
direction of the cell. See Supplementary Movie 8. θ denotes interaction angle, defined as the angle between the diluted colony edge and the cell orientation
just before collision. Scale bar, 20 μm. c Representative trajectories (N = 31) of cells that collided with diluted colony edge. Following the coordinate system
specified in panel b, X = 0 μm is set at the edge of liquid drops. The starting point of all trajectories are aligned and set as Y = 0 μm and X = 55 μm. Blue (or
magenta) lines represent the trajectory of cells that turned CW (or CCW) around the contact point during collision. d Tangential position of cells (whose
trajectories were shown in panel e) plotted against time. The collision positions of all trajectories are aligned and set as Y = 0 μm, and T = 0 is set at the
time of collision. Positive slope at T > 0 indicates that the cell moved in CW sense around the colony after collision. e Probability distribution of cells’
motion bias after collision with diluted colony edge plotted against interaction angle (Blue column: CW bias; Magenta column: CCW bias). f Model of a
swimming bacterium in contact with liquid drop edge (top view). The torque driving reorentation Gdrive and the drag torque Gdrag balance, which leads to
rotation at a constant angular speed ω about the x = 0; y = 0 point (green dot). The flagellar bundle is rotating at an angular speed Ω. The length of cell
body and flagellar bundle is Lb and Lf, respectively. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 Also see “Methods” 0.00 s
0.30 s
0.24 s
0.18 s
0.06 s
0.12 s
a b a
Y
X −1
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1
2
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0
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/2
/4
3/4 f ( )
f
Drop edge
Gdrag=D2
Gdrive=D1
x
Lb
Lf
y
y′
x′ e Fig. 3 Single-cell motion pattern of P. mirabilis near diluted colony edge. a A sequence of phase contrast images recording the CW turning of an isolated cell
colliding with the diluted colony edge (“Methods”). The dark area in upper portion of images is virgin agar. Scale bar, 5 μm. b Trajectory of the cell in panel
a generated by overlaying a series of phase contrast images. Magenta arrows by the side of trajectory (white dotted line) indicate instantaneous moving
direction of the cell. See Supplementary Movie 8. θ denotes interaction angle, defined as the angle between the diluted colony edge and the cell orientation
just before collision. Scale bar, 20 μm. c Representative trajectories (N = 31) of cells that collided with diluted colony edge. Following the coordinate system
specified in panel b, X = 0 μm is set at the edge of liquid drops. The starting point of all trajectories are aligned and set as Y = 0 μm and X = 55 μm. Blue (or
magenta) lines represent the trajectory of cells that turned CW (or CCW) around the contact point during collision. d Tangential position of cells (whose
trajectories were shown in panel e) plotted against time. The collision positions of all trajectories are aligned and set as Y = 0 μm, and T = 0 is set at the
time of collision. Positive slope at T > 0 indicates that the cell moved in CW sense around the colony after collision. e Probability distribution of cells’
motion bias after collision with diluted colony edge plotted against interaction angle (Blue column: CW bias; Magenta column: CCW bias). f Model of a
swimming bacterium in contact with liquid drop edge (top view). Discussion of supply from upstream, microspheres became barely visible in
the inner motile ring downstream of the motility-deactivation
region (Fig. 5f). Flagellated bacteria grown on solid substrates often develop into
structured communities, in which most cells have transitioned
into a non-motile state while a small population remains motile. In this paper, we investigated the behavior of motile cell popu-
lation in sessile colonies of flagellated bacteria. Our major dis-
covery is that motile cells in routinely cultured bacterial colonies
can self-organize into two adjacent motile rings surrounding the
entire colony via spontaneous segregation that mimics a phase
separation. Cells in the outer motile ring circle CW around the
colony (viewing from above) with high polar order, while cells in
the inner ring swim in parallel to colony edge bi-directionally
with nematic order in cells’ moving directions. The self-
organization of motile cells is mediated by intercellular interac-
tions and shear-induced depletion. Flagellar rotation of cells in
the outer motile ring generates directed fluid flows in the inner
motile ring that circulate CCW around the colony at a constant
speed of ~30 µm s−1. Overall, our findings reveal that motile sub-
populations in sessile colonies of flagellated bacteria can self-
organize in a remarkable and heretofore unnoticed manner. Shear-induced depletion facilitates inner motile ring forma-
tion. Here we introduce a conceptual model to explain the for-
mation of inner motile ring. The results presented above provide
strong evidence that the CCW directed flows in inner motile ring
are driven by flagellar motility of cells in the outer motile ring. The directed fluid flows can cause shear-induced depletion to aid
the formation of inner motile ring; shear-induced depletion is a
phenomenon recently reported in bacterial suspensions that leads
to the accumulation of swimmers in high-shear regions55. To
examine this idea, we used fluorescent microspheres as tracers to
map the flow speed profile generated by the CW outer motile ring
of P. mirabilis suspension drops in the absence of the inner motile
ring (thus avoiding the influence of cells in the inner motile ring
on tracer motion, as microspheres tend to stick to cells) (Sup-
plementary Fig. 8). We found that the region with horizontal
shear rate coincided with the expected range of inner motile ring
(ranging from ~10 to ~40 μm in X-axis of Supplementary Fig. 8). We further measured the radial profile of bacterial density in
suspension drops (Supplementary Fig. 9). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 The torque driving reorentation Gdrive and the drag torque Gdrag balance, which leads to
rotation at a constant angular speed ω about the x = 0; y = 0 point (green dot). The flagellar bundle is rotating at an angular speed Ω. The length of cell
body and flagellar bundle is Lb and Lf, respectively. Also see “Methods” motile ring, we used patterned illumination modulated by a
digital mirror device (Andor Mosiac; “Methods”) to optically
deactivate the flagellar motility of cells in outer motile ring, while
measuring how the transport of fluorescent microspheres in inner
motile ring was affected. We took advantage of the photosensitiz-
ing effect of membrane stain FM 4–64 (Life Technologies, Cat. No. T13320; “Methods”) for motility deactivation; upon excita-
tion, FM4–64 disrupts flagellar motility, most likely due to the
release of reactive oxygen species53,54. When cell motility was
deactivated in a selected area of outer motile ring, we found rapid
accumulation of microspheres in the region of inner motile ring
just adjacent to the deactivation area (Fig. 5a–g) and the
disappearance of CCW bias of collective motion in this region
(Fig. 5h–i), indicating that CCW directed fluid flows were
substantially halted in this region. Due to the substantial decrease FITC–Dextran get transported into the sessile, interior part of the
colony along some crack-like conduits (Fig. 4e–h; Supplementary
Movie 12; Supplementary Fig. 5). The conduits typically formed
at ~15–20 h after inoculation and became fully developed within
1 h (Supplementary Fig 6, panel A); they remained stable for
many hours until the colony started to swarm, and then they
gradually disappeared within ~3 h (Supplementary Fig. 6, panel
B). The conduits have a mean radial extension of ~65 µm, with
the longest ones up to ~200 µm (Supplementary Fig. 7). The
inwards moving flows along the conduits are presumably driven
by flagellar rotation of motile cells confined along the boundary of
these conduits, and the flows may increase nutrient availability
for the interior part of the colony. y
To further verify that the observed CCW directed flows in the
inner motile ring are driven by flagellar rotation of cells in outer NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 0 s
200 s
100 s
300 s
e
g
h
f
a
0 s
c
120 s
b
60 s
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180 s
Fig. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 4 Long-range, colony-scale directed transport in a P. mirabilis colony. a–d Image sequence showing the directed transport of 0.1-μm fluorescent
microspheres in the inner motile ring around colony edge (“Methods”). Fluorescent images of microspheres were overlaid with phase contrast images of
the colony (at low light intensity). Bright blob in the upper part of the images is where the fluorescent microspheres were deposited. Arrows indicate the
position of microspheres transported for the longest distance at indicated times; these foremost microspheres are only visible when enlarging the figure or
when viewing the associated Supplementary Movie 11. Scale bar, 500 μm. e–h Image sequence showing the directed transport of 0.1-μm fluorescent
microspheres inwards along crack-like conduits present near colony edge. The conduits are inside the rectangular area showed in the images. Also see
Supplementary Movie 12. Scale bar, 50 μm 100 s
f 0 s
e 300 s
h 200 s
g g 200 s
g 0 s h Fig. 4 Long-range, colony-scale directed transport in a P. mirabilis colony. a–d Image sequence showing the directed transport of 0.1-μm fluorescent
microspheres in the inner motile ring around colony edge (“Methods”). Fluorescent images of microspheres were overlaid with phase contrast images of
the colony (at low light intensity). Bright blob in the upper part of the images is where the fluorescent microspheres were deposited. Arrows indicate the
position of microspheres transported for the longest distance at indicated times; these foremost microspheres are only visible when enlarging the figure or
when viewing the associated Supplementary Movie 11. Scale bar, 500 μm. e–h Image sequence showing the directed transport of 0.1-μm fluorescent
microspheres inwards along crack-like conduits present near colony edge. The conduits are inside the rectangular area showed in the images. Also see
Supplementary Movie 12. Scale bar, 50 μm Discussion CCW bias of cellular motion changed from ~1.19
to ~1.04 in this experiment b
20 s a
16 s b 24 s
c a c d
28 s e
32 s f
84 f
84 s d f e 0.10
0.15
0.20
Probability
h
10
20
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30
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Vcell (µm/s)
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Probability
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Vcell (µm/s) Vcell (µm/s)
0.10
0.15
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i
0
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30
50 g 0
i i h Fig. 5 Optical deactivation of flagellar motility in the outer motile ring. A region of the outer motile ring (enclosed by solid white rectangles in panels
a–f was chosen for motility deactivation with intense light (“Methods”). a–e Fluorescent image sequence of 0.1-μm microspheres in the same field of view
before (a–b) and after (c–e) the deactivation of flagella motility. Motility-deactivation light at the selected region was turned on at time = 24 s (panel c). Scale bars, 50 μm. f Fluorescent image of 0.1-μm microspheres overlaid with the phase-contrast image of colony edge at time = 84 s. Microspheres were
barely visible in the inner motile ring downstream of the motility-deactivation region. Scale bar, 50 μm. g Mean fluorescence intensity of microspheres
averaged over a selected area of the inner motile ring (enclosed by blue rectangles in panels a–f) plotted against time. The shaded area starting at time =
24 s indicates the duration of motility-deactivation. Before motility deactivation (from 0 to 24 s), the slope of fluorescence intensity given by linear fit
is 0.013 ± 0.002 a.u. s−1 (±sign indicates fitting error; R2 = 0.94); the slope increased to 0.041 ± 0.002 a.u. s−1 (R2 = 0.95) after motility deactivation. h–i Speed distribution of individual cells in the inner motile ring before (panel h) and after (panel i) motility deactivation, respectively. The speed
distribution of cells moving in CCW or CW direction were represented by columns filled in magenta or yellow color, respectively, and both types of
columns were plotted together in the same histogram with the overlapping portion filled with white color. Discussion Near the transition
region between the dilute phase and the inner motile ring (at ~40
micron from the edge), the surface cell density experienced a
sharp increase when moving towards the edge. This increase of
surface cell density cannot be attributed to the variation of the
height of liquid film, since the fluid height in the dilute phase is
comparable or greater than that in the inner motile ring; indeed,
the increase of surface cell density reflects the increase of volume
cell density and proves that cells accumulate towards the edge
from the dilute phase. The region with cell accumulation (~10–40
micron from the edge) corresponds to the region of inner motile
ring (Fig. 2a), and it coincides with the region with horizontal
shear shown in Supplementary Fig. 8. These results (Supple-
mentary Figs. 8, 9), together with the fact that the width of inner
motile ring increases linearly with cell speed (Fig. 2e), supports
the idea that shear-induced depletion helps attract cells to colony
edge and contributes to the formation of inner motile ring in
colonies. g
The
mechanism
underlying
motile-ring
self-organization
deserves further study in the context of flagellar hydrodynamics
and active matter self-organization. Shear-induced depletion may
account for the accumulation of cells that leads to the formation
of inner motile ring. As the inner motile ring is densely packed
with swimming cells, would the modification of flow field by these
cells affect the width or the stability of the motile rings? We note
that these cells are moving bi-directionally and therefore the flows
they generated would cancel one another out; consequently the
flows generated by the outer motile ring would be largely pre-
served, as demonstrated by the strong CCW flows present in the
inner motile ring in colonies (Fig. 4). Nonetheless, the weak CCW
bias of collective motion in the inner motile ring would compli-
cate the issue. Moreover, hydrodynamic attraction between cells,
similar to that responsible for cohesive swimming of cells in 2D
confinement56, may help to reduce the orientational noise and
thereby stabilize the motile rings. On the other hand, accumu-
lation of cells via the shear-depletion mechanism is not sufficient
to account for the nematic order in cells’ moving directions in the
inner motile ring. Discussion CCW bias of cellular motion changed from ~1.19
to ~1.04 in this experiment The unique mechanism of bacterial self-organization we
uncovered here may be important to the physiology and stress
response of bacterial communities in widespread environments. Self-segregation of motile subpopulations to the colony edge
apparently reshapes the population structure in bacterial com-
munities, which could facilitate the initiation of dispersal or range
expansion (e.g., via swarming process). In addition, the self-
organization of swimmers generates highly directed fluid flows,
providing a stable high-speed avenue for long-range directed
material transport in bacterial communities. Diffusion has been
assumed to dominate material transport within bacterial com-
munities58–60, and only a few examples of directed transport have
been found so far51,61–63. It was reported that B. subtilis colonies
can make use of evaporation-driven fluid flows to aid colony-
scale material transport;62 however, the process is not driven by stabilize unidirectional collective motion (in which cells align
their orientations nematically but move unidirectionally)32, and
nematically ordered collective motion (in which cells move bi-
directionally in parallel with each other as seen in our phenom-
enon) was not reported. In a recent study, computational mod-
eling predicted that self-propelled polar particles interacting
locally by nematic alignment could self-segregate into high-
density bands with nematic order in particles’ moving directions
in the midst of low-density disordered regions57. It will be
intriguing to examine the relevance of these model results to
our findings. However, the model did not include hydrodynamic
interaction between cells. The dynamics of motile rings may be
better understood by modeling dense suspensions of swimmer
that
takes
into
account
both
hydrodynamic
and
steric
interactions. stabilize unidirectional collective motion (in which cells align
their orientations nematically but move unidirectionally)32, and
nematically ordered collective motion (in which cells move bi-
directionally in parallel with each other as seen in our phenom-
enon) was not reported. In a recent study, computational mod-
eling predicted that self-propelled polar particles interacting
locally by nematic alignment could self-segregate into high-
density bands with nematic order in particles’ moving directions
in the midst of low-density disordered regions57. It will be
intriguing to examine the relevance of these model results to
our findings. However, the model did not include hydrodynamic
interaction between cells. The dynamics of motile rings may be
better understood by modeling dense suspensions of swimmer
that
takes
into
account
both
hydrodynamic
and
steric
interactions. Discussion Advection due to fluid flows generated by
flagellated bacteria at confinement boundary was shown to 7 TURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 24 s
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16 s
20 s
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Vcell (µm/s)
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i
0
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10
20
40
30
50
Fig. 5 Optical deactivation of flagellar motility in the outer motile ring. A region of the outer motile ring (enclosed by solid white rectangles in panels
a–f was chosen for motility deactivation with intense light (“Methods”). a–e Fluorescent image sequence of 0.1-μm microspheres in the same field of view
before (a–b) and after (c–e) the deactivation of flagella motility. Motility-deactivation light at the selected region was turned on at time = 24 s (panel c). Scale bars, 50 μm. f Fluorescent image of 0.1-μm microspheres overlaid with the phase-contrast image of colony edge at time = 84 s. Microspheres were
barely visible in the inner motile ring downstream of the motility-deactivation region. Scale bar, 50 μm. g Mean fluorescence intensity of microspheres
averaged over a selected area of the inner motile ring (enclosed by blue rectangles in panels a–f) plotted against time. The shaded area starting at time =
24 s indicates the duration of motility-deactivation. Before motility deactivation (from 0 to 24 s), the slope of fluorescence intensity given by linear fit
is 0.013 ± 0.002 a.u. s−1 (±sign indicates fitting error; R2 = 0.94); the slope increased to 0.041 ± 0.002 a.u. s−1 (R2 = 0.95) after motility deactivation. h–i Speed distribution of individual cells in the inner motile ring before (panel h) and after (panel i) motility deactivation, respectively. The speed
distribution of cells moving in CCW or CW direction were represented by columns filled in magenta or yellow color, respectively, and both types of
columns were plotted together in the same histogram with the overlapping portion filled with white color. Methods
B
t
i l t Bacterial strains. The following strains were used: wildtype P. mirabilis BB2000,
and a fluorescent P. mirabilis KAG108 (BB2000 background with constitutive
expression of Green Fluorescent Protein (GFP);69 from Karine Gibbs, Harvard
University, Cambridge, MA); E. coli HCB1737 (a derivative of E.coli AW405 with
wildtype flagellar motility but with FliC S219C mutation for fluorescent labeling;
from Howard Berg, Harvard University, Cambridge, MA); E. coli HCB1737 GFP
(HCB1737 transformed with pAM06-tet plasmid with constitutive GFP expres-
sion70 from Arnab Mukherjee and Charles M. Schroeder, University of Illinois at
Urbana-Champaign); wildtype B. subtilis 3610 and a smooth-swimming mutant B. subtilis DK2178, which is a cheB-deleted derivative of B. subtilis 3610 (from Daniel
B. Kearns, Indiana University at Bloomington). Single-colony isolates were grown
overnight (~13–14 h) with shaking in LB medium (1% Bacto tryptone, 0.5% yeast
extract, 0.5% NaCl) at 30 °C to stationary phase. For E. coli HCB1737 GFP,
kanamycin (50 μg per mL) was added to the growth medium for maintaining the
plasmid. Single-cell motion pattern at the edge of liquid drops. For P. mirabilis BB2000,
1 μL motility buffer was deposited near the edge of a P. mirabilis colony. The
diluted colony edge formed a liquid drop dispersed with isolated cells. The motion
pattern of isolated cells near the diluted colony edge was observed in phase contrast
with the 20× dark phase objective and was recorded at 30 fps for 10 min (Fig. 3). For B. subtilis, overnight culture was diluted with motility buffer 10−4, and 1-μL
drops of the diluted suspension were deposited onto 2.0% LB agar plates. The
motion pattern of isolated B. subtilis cells near the edge of the suspension drop was
observed in phase contrast with the 20× dark phase objective and was recorded at
30 fps for 10 min (Supplementary Movie 10). For E. coli (HCB1737 GFP), flagellar
filaments were fluorescently labeled prior to observation of single-cell motion
pattern, following procedures established in ref. 45 and modified in ref. 56. Briefly,
0.1 μL overnight culture of E. coli (HCB1737 GFP) was diluted 10−4 to a volume of
1 mL in a glass test tube. Labeling dye stock solution (Alexa Flour 546 C5- Preparation of agar plates for colony growth. LB agar (Difco Bacto agar at
specified concentrations infused with 1% Bacto tryptone, 0.5% yeast extract, 0.5%
NaCl) was autoclaved and stored at room temperature. ARTICLE mirabilis occurs under our experimental conditions following the emergence of
motile rings (>24 h after inoculation, with large variability of the exact initiation
time of swarming). Observations were made in phase contrast with the 40× dark
phase objective (Nikon CFI Super Plan Fluor ELWD ADM 40XC, N.A. 0.60, W.D. 3.6–2.8 mm) and were recorded at 30fps. For single-cell tracking at the edge of P. mirabilis colonies, overnight culture of the GFP-tagged P. mirabilis KAG108 was
mixed with wildtype P. mirabilis at 1:2000 before inoculating the agar plates. P. mirabilis KAG108 cells were imaged with an FITC single band filter set (excitation
482/35 nm, emission 536/40 nm, dichroic: 506 nm; FITC-3540C-000, Semrock
Inc.) and were recorded at 30 fps for 2 min. To monitor the development of motile
rings and crack-like conduits during colony growth, the edge of P. mirabilis
colonies was recorded at 30 fps for 10 s duration every 20 min with the 20× dark
phase objective (Nikon CFI Super Plan Fluor ELWD ADM 20XC, N.A. 0.45, W.D. 8.2–6.9 mm). Bacterial self-organization at the edge of cell suspension drops. To observe
self-organization at the edge of cell suspension drops (artificial colonies) (Fig. 2),
overnight culture of the GFP-tagged P. mirabilis KAG108 was either used alone
(Fig. 2a, e, f, Supplementary Movies 6, 7) or mixed with wildtype P. mirabilis
BB2000 at 1:50 (Fig. 2b–d), and 1-μL drops of the pure culture or the mixture were
inoculated on 0.6% LB agar plates. The plates were incubated at 30 °C and ~85%
relative humidity for 36 h; at this time the colony had started to expand. Cells were
collected from the leading edge of the colony with motility buffer (0.01 M potas-
sium phosphate, 0.067 M NaCl, 10−4 M EDTA, pH 7.0). The collected suspension
was washed by mobility buffer for three times and resuspended in motility buffer to
reach appropriate cell densities. Tween 20 (Sigma-Aldrich, Cat. No. P7949) was
then added to the cell suspension at a final concentration of 0.002% (wt/wt). 1-μL
drops of this suspension were deposited onto 2.0% LB agar plates supplemented
with 0.002% (wt/wt) Tween 20. Bacterial self-organization at the edge of such
suspension drops was observed in phase contrast and in fluorescence (via the FITC
single band filter set described above) with the 40× dark phase objective. Images
were recorded at 30 fps. ARTICLE On the other hand, we wonder whether the long-range directed
flows could be exploited to perturb the behavior of bacterial
communities; for example, drugs and chemical effectors loaded in
vesicles could be supplied locally and transported by the flows to
manipulate distant parts of a bacterial colony. plates were dried for another 10 min without a lid inside the Plexiglas box. The plates
were then covered and incubated at 30 °C and ~85% relative humidity in an incubator
with water tray for specified durations of time (see below). To vary the environmental
humidity for colony growth, the plates were incubated at 30 °C in a custom-built
incubator in which different kinds of saturated salt solutions (NaBr: 47.4%RH; KI:
55.0%RH; NaCl: 60.4%RH) or DI water (~97% RH) were supplemented, or incubated
in a regular incubator without water tray (~42% RH) at 30 °C. Phase contrast and fluorescence imaging. Imaging was performed on a
motorized inverted microscope (Nikon TI-E). The following objectives were used
in different experiments for both phase contrast and fluorescence imaging: Nikon
CFI Plan Fluor DL4X, N.A. 0.13, W.D. 16.4 mm; Nikon CFI Achromat DL 10×, N. A. 0.25, W.D. 7.0 mm; Nikon CFI Super Plan Fluor ELWD ADM 20XC, N.A. 0.45,
W.D. 8.2–6.9 mm; and Nikon CFI Super Plan Fluor ELWD ADM 40XC, N.A. 0.60,
W.D. 3.6–2.8 mm. Fluorescence imaging was performed in epifluorescence using
filter sets specified below, with the excitation light provided by a mercury pre-
centered fiber illuminator (Nikon Intensilight). All recordings were made with a
sCMOS camera (Andor ZYLA 4.2 PLUS USB 3.0) at full frame size (2048 × 2048
pixels) using the software NIS-Elements AR (Nikon). In all experiments the petri
dishes were covered with lid to prevent evaporation and air convection, and the
sample temperature was maintained at 30 °C using a custom-built temperature
control system installed on microscope stage, unless otherwise stated. Bacterial self-organization at the edge of sessile colonies. To observe self-
organization at the edge of sessile colonies (Fig. 1 and Supplementary Fig. 2), P. mirabilis BB2000 or E. coli HCB1737 colonies were grown on 0.6% LB agar plates
at ~85% relative humidity (as described above) for 24 h, and B.subtilis colonies
were grown on 2.0% LB agar plates ~85% relative humidity for 2 h. Swarming of P. ARTICLE To study the velocity dependence of the width of inner
motile ring, we made use of the fact that the swimming speed of flagellated bacteria
depends on temperature71, and we varied cell speed in P. mirabilis suspension
drops by changing environmental temperature from 15 to 37 °C with a
temperature-controlled water bath. ARTICLE On the other hand, we wonder whether the long-range directed
flows could be exploited to perturb the behavior of bacterial
communities; for example, drugs and chemical effectors loaded in
vesicles could be supplied locally and transported by the flows to
manipulate distant parts of a bacterial colony. autonomous cellular activities. Expanding swarms of flagellated
bacteria can generate CW directed flows at colony edge at a
similar magnitude (~10 µm s−1)51, but it is not stable due to
constant swarm expansion (lasting for <1 min) and material
transport can only persist for up to ~500 µm; these time and
length scales are two orders of magnitude smaller than that
associated with the CCW flows in inner motile ring, which last for
hours and persist for centimeters. Moreover, the long-range active
transport we report here is enabled by bacterial self-organization,
whereas cells at the edge of bacterial swarms do not display stable
ordering of orientations or moving directions despite the pre-
sence of transient jets and vortices lasting for a fraction of a
second. The unique means of long-range active transport in
bacterial colonies we uncovered here is reminiscent of mucus flow
driven by motile cilia of epithelial cells in various organ systems,
such as the respiratory tract and oviduct64. We speculate that this
long-range active transport may have profound effect on the
physiology of bacterial communities. Bacteria communities in
natural and clinical settings normally grow in anisotropic,
structured environments with heterogeneous nutrient or chemical
distribution. In such environments, bacterial communities do not
have rotational symmetry and the self-organization of motile
subpopulation may occur in various locations not limited to the
colony edge; thus the long-range directed flows enabled by
motile-cell self-organization may efficiently redistribute slowly-
diffusing substances or molecules with high binding affinity to
cell surface within bacterial communities. For example, high-
molecular-weight polymeric metabolites65 and membrane vesicles
that encapsulate nutrients or signaling molecules66 could travel
for 1 cm in ~5 min via the long-range directed flows, which is
much more efficient than diffusion process alone (either passive
diffusion driven by thermal energy or active diffusion driven by
active forces in a bath of motile cells61,67,68). In fact, the 0.1-μm
fluorescent microspheres we used to track the flows are com-
parable to the size of membrane vesicles secreted by many bac-
terial species, and thus the motion of microspheres should closely
resemble the transport of membrane vesicles in situ in colonies. ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 autonomous cellular activities. Expanding swarms of flagellated
bacteria can generate CW directed flows at colony edge at a
similar magnitude (~10 µm s−1)51, but it is not stable due to
constant swarm expansion (lasting for <1 min) and material
transport can only persist for up to ~500 µm; these time and
length scales are two orders of magnitude smaller than that
associated with the CCW flows in inner motile ring, which last for
hours and persist for centimeters. Moreover, the long-range active
transport we report here is enabled by bacterial self-organization,
whereas cells at the edge of bacterial swarms do not display stable
ordering of orientations or moving directions despite the pre-
sence of transient jets and vortices lasting for a fraction of a
second. The unique means of long-range active transport in
bacterial colonies we uncovered here is reminiscent of mucus flow
driven by motile cilia of epithelial cells in various organ systems,
such as the respiratory tract and oviduct64. We speculate that this
long-range active transport may have profound effect on the
physiology of bacterial communities. Bacteria communities in
natural and clinical settings normally grow in anisotropic,
structured environments with heterogeneous nutrient or chemical
distribution. In such environments, bacterial communities do not
have rotational symmetry and the self-organization of motile
subpopulation may occur in various locations not limited to the
colony edge; thus the long-range directed flows enabled by
motile-cell self-organization may efficiently redistribute slowly-
diffusing substances or molecules with high binding affinity to
cell surface within bacterial communities. For example, high-
molecular-weight polymeric metabolites65 and membrane vesicles
that encapsulate nutrients or signaling molecules66 could travel
for 1 cm in ~5 min via the long-range directed flows, which is
much more efficient than diffusion process alone (either passive
diffusion driven by thermal energy or active diffusion driven by
active forces in a bath of motile cells61,67,68). In fact, the 0.1-μm
fluorescent microspheres we used to track the flows are com-
parable to the size of membrane vesicles secreted by many bac-
terial species, and thus the motion of microspheres should closely
resemble the transport of membrane vesicles in situ in colonies. Discussion NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications 8 NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunicatio ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09818-2 maleimide, 5 mg per ml in DMSO; Life Technologies) was added to the cell sus-
pension in test tube at a final concentration of 25 μg per ml. Labeling was allowed
to proceed for 7 min in a shaker (30 °C and 180 rpm). When labeling was com-
pleted, the cell suspension was transferred to 1.5 ml Eppendorf tubes, brought to a
volume of 10 mL with mobility buffer, and washed free of unreacted dyes by
centrifugation and resuspension for three times (10,000 × g for 4 min in the first
wash, 2000 × g for 10 min in the second and third washes). After washing, cells
were resuspended in mobility buffer to 1 mL, and 1-µl drops of this cell suspension
were deposited onto 2.0% LB agar plates supplemented with 0.002% (wt/wt) Tween
20. The motion pattern of cell body and flagellar filaments of isolated E. coli cells
near the edge of the suspension drop was imaged in fluorescence with the 20× dark
phase objective, via an EGFP/mCherry dual band filter set (excitation: 471/38 nm
and 571/34 nm, emission: 522/42 nm and 634/62 nm, dichroic: 522/52 nm and 636/
84 nm; Part No. 59022, Chroma), and was recorded at 30 fps (Supplementary
Movie 9). different ways for the outer motile ring and for the inner motile ring, respectively. In the outer motile ring, cells move at an almost uniform speed, so we randomly
chose 10 cells there and computed their mean speeds over ~10 s. The speed of cells
in the inner motile ring has a broad distribution, so we tracked ~1000 cells in ~5-
min movies and divided their trajectories into ~10,000 segments, each segment
with a duration of 1 s. The mean speed of these 1-s segments were computed to
yield the velocity statistics of cells. The velocity statistics of microspheres were
obtained in a similar way. To study the cell density dependence of collective motion
in outer motile ring, the cell occupation ratio at the 10-μm-wide outmost rim of
suspension drops was defined as the area occupied by cells divided by total area of
the rim. To determine the width of inner motile ring in P. mirabilis suspension
drops, GFP-tagged P. Model of cell reorientation upon collision with colony edge. The driving
ll
d
h
l
d
d
h Model of cell reorientation upon collision with colony edge. The driving
reorientation torque Gdrive is numerically estimated using the Slender Body Theory Model of cell reorientation upon collision with colony edge. The driving
reorientation torque Gdrive is numerically estimated using the Slender Body Theory
(SBT)47,48 for the helix rotating above the no-slip wall ignoring the top air/fluid
Interface. The SBT model takes into account full hydrodynamic interactions with
the surface (except for lubrication when the helix is all but touching the surface). Firstly the force distribution f on the stationary helix rotating next to the no-slip
wall is calculated. From this force distribution we calculate the instantaneous
reorientation torque about the tip of the cell (x = 0; y = 0 point in Fig. 3f): Visualization of fluid flows in colonies and suspension drops. P. mirabilis
BB2000 colonies were grown on 0.6% LB agar plates as described above for 24 h. To
visualize fluid flows (Fig. 4, Supplementary Figs. 5 and 8), fluorescent microspheres
(Fluo-Spheres, carboxylate-modified, 0.1 μm diameter; Life Technologies Cat. No. F8820) or fluorescently labeled dextran (FITC–Dextran, mol. wt. 2000 kDa; Sigma,
FD2000S) were deposited near the colony edge. Fluorescence of microspheres was
excited via a Cy3/TRITC single band filter set (excitation 535/50 nm, emission 610/
75 nm, dichroic: 565 nm; Part No. 41007, Chroma), and FITC–Dextran was excited
via the FITC single band filter set. Phase contrast images of colony edge (low-
intensity illumination from above the sample provided by the built-in halogen
lamp of the microscope) and fluorescent images of microspheres were taken with
the 4× phase-contrast objective (Nikon CFI Plan Fluor DL4X, N.A. 0.13, W.D. 16.4
mm) and recorded simultaneously at 5 fps (Supplementary Movie 11). The directed
transport of fluorescent microspheres inwards along crack-like conduits near col-
ony edge was observed with the 10× objective (Nikon CFI Achromat DL 10X, N.A. 0.25, W.D. 7.0 mm), and was recorded at 5 fps simultaneously in phase contrast
(illumination from above the sample provided by the built-in halogen lamp of the
microscope) and in fluorescence (illumination provided by Nikon Intensilight via
the Cy3/TRITC single band filter set) (Supplementary Movie 12). The transport of
FITC–Dextran near colony edge was observed with the 10× objective and recorded
at 5 fps in fluorescence (illumination provided by Nikon Intensilight via the FITC
single band filter set). To measure the flow speed profile generated by CW outer
motile ring (Supplementary Fig. Model of cell reorientation upon collision with colony edge. The driving
ll
d
h
l
d
d
h 8), P. mirabilis BB2000 suspension drops were
deposited onto 2.0% LB agar as described above, and fluorescent microspheres
were deposited near the suspension drop edge. Fluorescence of microspheres was
excited via the Cy3/TRITC single band filter set and recorded with the 20×
objective at 30 fps. GdriveðtÞ ¼
ZLf
0
Lb þ x′
ð
ÞfY′ðtÞsec Ψdx′; tan Ψ ¼ 2πR
P
ð6Þ ð6Þ where fy′ (t, h, r, x′) is the force density in the y′ direction which is driving the
reorientation (Fig. 3f), h the height above the wall of the helical axis, r the radius of
flagellar bundle, Lf the length of the flagellum along its axis, Lb the length of the cell
body, x′ the length parameter x′ ∈[0, Lf], R the radius of flagellar helix, and P the
pitch of flagellar helix. The contribution due to the force component fx will be small
because of the geometry. The instantaneous torque is averaged over one period T of
the flagellar rotation: 0.25, W.D. 7.0 mm), and was recorded at 5 fps simultaneously in phase contrast
(illumination from above the sample provided by the built-in halogen lamp of the
microscope) and in fluorescence (illumination provided by Nikon Intensilight via
the Cy3/TRITC single band filter set) (Supplementary Movie 12). The transport of
FITC–Dextran near colony edge was observed with the 10× objective and recorded
at 5 fps in fluorescence (illumination provided by Nikon Intensilight via the FITC
single band filter set). To measure the flow speed profile generated by CW outer
motile ring (Supplementary Fig. 8), P. mirabilis BB2000 suspension drops were
deposited onto 2.0% LB agar as described above, and fluorescent microspheres
were deposited near the suspension drop edge. Fluorescence of microspheres was
excited via the Cy3/TRITC single band filter set and recorded with the 20×
objective at 30 fps. Gdrive ¼ 1=T
ZT
0
GdriveðtÞdt
ð7Þ ð7Þ The parameters for the helix are: the pitch P = 2.3 μm73, radius R = 0.3 μm73,
the axial length Lf = 7.3 ± 2.4 μm73, the length of the cell body Lb = 1.9 μm74,
radius of the bundle r = 24 nm (double radius of a single flagellum)75, frequency of
rotation 1/T = Ω/2π = 110 Hz73. The driving torque as a function of height is
computed as shown in Supplementary Fig. 4. Model of cell reorientation upon collision with colony edge. The driving
ll
d
h
l
d
d
h The closer the flagellar bundle is to
the bottom wall, the larger reorientation torque is created. Optical deactivation of motility via patterned illumination. P. mirabilis BB2000
colonies were grown on 0.6% LB agar plates supplemented with FM 4–64 (final
concentration 1.0 μg per mL) as described above for 24 h. Fluorescent microspheres
were deposited near the colony edge (described above) and got transported along
the colony edge. A selected region of the outer motile ring (length: 150 μm; width:
20 μm) was chosen for motility deactivation. A beam of intense green-orange light
provided by an LED illuminator (Thorlabs 530 nm Mounted High-Power LED L3,
item No. M530L3, installed on X-Cite XLED1; Excelitas Technologies Corp) was
modulated by a digital mirror device (Andor Mosiac) to form a spatially defined
illumination pattern, and the modulated light passed through the 40× objective via
the Cy3/TRITC filter set (described above) to excite FM4–64 in the selected region
for motility deactivation. Meanwhile, the entire field of view was illuminated at low
light intensity for imaging in phase contrast (illumination from above the sample
provided by the built-in halogen lamp of the microscope) and/or in fluorescence
(illumination provided by Nikon Intensilight via the Cy3/TRITC filter set). Recordings were made at 30 fps. We use the Resitive Force Theory (RFT)49 to estimate the drag torque Gdrag due
to the rotation of the body with flagellar bundle around the tip of the cell. The
majority of the drag torque comes from the flagellar bundle because it is much
longer than the body and the flagellar bundle is further away from the rotation
point. Therefore we approximate the cell-flagella system as a slender rod of length
L = Lb + Lf (which is a sum of the body length and flagellum length) and of radius
r (i.e., the radius of flagellar bundle). The force per unit length f on a slender
straight filament moving with velocity u is given by: f ¼ ðξ?nn þ ξkttÞ u
ð8Þ ð8Þ where t is the vector along the axis of filament and n perpendicular to it. In this
case the body-flagella system is rotating about the tip of the cell. The point which is
l distance away from the tip is moving at the linear speed ωl. Methods
B
t
i l t Before use, the agar was
melted in a microwave oven, cooled to ~60 °C, and pipetted in 10-mL aliquots into
90-mm polystyrene petri plates. For optical deactivation of flagellar motility, the
membrane stain FM 4–64 (Life Technologies, Cat. No. T13320) was dissolved in
deionized water and added at a final concentration of 1.0 μg per mL to the liquefied
agar before pipetting. The plates were swirled gently to ensure surface flatness, and
then cooled for 10 min without a lid inside a large Plexiglas box. Drops of 1 μL
overnight bacterial cultures were inoculated onto the LB agar plates. The inoculated 9 TURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications ARTICLE ARTICLE mirabilis (KAG108) was used alone and imaged in fluores-
cence (as described above); the width of inner motile ring was taken as the region
with fluorescence intensity greater than half of the maximum fluorescence at the
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ll
d
h
l
d
d
h The drag torque is
given by:76 Gdrag ¼
ZL
0
f n
ð
Þldl ¼
ZL
0
ξ?ωl
ð
Þldl ¼ ξ?ω L3
3 Image processing and data analysis. Images were processed using the open
source Fiji (ImageJ) software (http://fiji.sc/Fiji) and custom-written programs in
MATLAB R2016b (The MathWorks; Natick, Massachusetts, United States). The
velocity field of cells’ collective motion was obtained by performing optical flow
analysis based on microscopy movies using the built-in functions of MATLAB. Prior to computing the optical flow fields, the images were first smoothed to reduce
noise by convolution with a Gaussian kernel of standard deviation 1. The optical
flow field for any two consecutive video frames was computed using the Horn-
Schunck algorithm72 (maximum iteration number, 128; smoothness parameter, 1)
and then smoothed by local averaging. The grid size of the optical flow field was 1
pixel × 1 pixel and the initial value of optical flow vectors was set to zero. The
obtained optical flow fields were used to compute the collective speed profile in
Figs. 1d and 2c. To visualize the collective velocity field (Figs. 1c and 2b), the
optical flow fields were coarsened to a grid size of 5.2 μm × 5.2 μm. The results were
insensitive to different parameters of smoothing. For single-cell and single-
microsphere tracking, bacteria and microsphere trajectories were obtained by a
custom-written particle-tracking program in Matlab (The MathWorks, Inc) based
on phase-contrast or fluorescence movies. Single-cell speed calculation was done in ξ? ¼
4πμ
1=2 þ lnð0:18Psec Ψ=rÞ ; tan Ψ ¼ 2πR
P
ð9Þ ð9Þ which means that the reorientation angular speed is which means that the reorientation angular speed is ω ¼ 3Gdrive
ξ?L3
ð10Þ ð10Þ We then use the result from the SBT for the driving torque Gdrive to calculate
the reorientation angular speed ω as a function of h, as shown in Supplementary
Fig. 4. Reporting summary. Further information on research design is available in
the Nature Research Reporting Summary linked to this article. NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications 10 Competing interests: The authors declare no competing interests. Competing interests: The authors declare no competing interests. 69. Gibbs, K. A., Urbanowski, M. L. & Greenberg, E. P. Genetic determinants of
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adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative
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
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68. Sokolov, A., Goldstein, R. E., Feldchtein, F. I. & Aranson, I. S. Enhanced
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019-09818-2. Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467-
019-09818-2. Competing interests: The authors declare no competing interests. © The Author(s) 2019 Acknowledgements We thank Howard C. Berg, Karine Gibbs, Daniel B. Kearns, Arnab Mukherjee, and
Charles M. Schroeder for their kind gifts of bacterial strains, and Howard C. Berg for
helpful comments. The work of H.X. and Y.W. was supported by the National Natural
Science Foundation of China (NSFC 21473152; to Y.W.) and by the Research Grants
Council of Hong Kong SAR (RGC Ref. No. GRF 14322316&14301915, CUHK Direct © The Author(s) 2019 NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunications 12 NATURE COMMUNICATIONS | (2019) 10:1792 | https://doi.org/10.1038/s41467-019-09818-2 | www.nature.com/naturecommunicatio
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Assessment of Coral Reef Life-Form Classification Scheme using Multiresolution Images on Parang Island, Indonesia
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Abstract Spatial information on the varying composition of coral reefs is beneficial for the
management and preservation of natural resources in coastal areas. Its availability is
inseparable from environmental management goals; however, it can also be used as a means
of supporting tourism activities and predicting the emergence of certain living species. A
satellite image is one of the effective and efficient data sources that provide spatial
information on coral reef variations. This study aimed to evaluate the classification scheme of
coral reef life-form using images with different spatial resolutions on Parang Island,
Karimunjawa Islands, Central Java. These images were from PlanetScope (3m), PlanetScope
resampling (6m), and Sentinel-2A MSI (10m), whose spatial resolutions functioned as the
base for building the 3m, 6m, and 10m classification schemes producing 12, 11, and 9
classes, respectively. As for the classification method, it integrated both object-based and
pixel-based approaches. The results showed that the highest overall accuracy (60%) was
obtained using Sentinel-2A MSI image (10m), followed by PlanetScope (3m) with 48%
accuracy, and PlanetScope resampling (6m) with 40% accuracy. This finding indicates that
multiresolution images can be used to produce complex coral reef life-form maps with
different levels of information details. Keywords: Coral reef; Life-form; Planetscope; Spatial resolution; Classification scheme Assessment of Coral Reef Life-Form Classification Scheme using
Multiresolution Images on Parang Island, Indonesia Wahyu Lazuardi1, Pramaditya Wicaksono2,*
1Geospatial Information Agency, Bogor, 16911, Indonesia
2Department of Geographic Information Science, Faculty of Geography, Universitas Gadjah Mada,
Yogyakarta, 55281, Indonesia Wahyu Lazuardi1, Pramaditya Wicaksono2,*
1Geospatial Information Agency, Bogor, 16911, Indonesia
2Department of Geographic Information Science, Faculty of Geography, Universitas Gadjah Mada,
Yogyakarta, 55281, Indonesia Received 26 October 2021/Revised 14 December 2021/Accepted 21 December 2021/Published 30 December 2021 Received 26 October 2021/Revised 14 December 2021/Accepted 21 December 2021/Published 30 December 2021 *Corresponding author.
Email address : prama.wicaksono@ugm.ac.id (Pramaditya Wicaksono) Geosfera Indonesia Geosfera Indonesia Vol. 6 No. 3, December 2021, 377-397
p-ISSN 2598-9723, e-ISSN 2614-8528
https://jurnal.unej.ac.id/index.php/GEOSI
DOI : 10.19184/geosi.v6i3.27592 377
*Corresponding author.
Email address : prama.wicaksono@ugm.ac.id (Pramaditya Wicaksono) *Corresponding author. Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 management and monitoring of coral reefs are paramount to improve the sustainability and
usefulness of these shallow-water ecosystems, especially when under a significant amount of
environmental stress. In this situation, spatial information on the characteristics of benthic
compositions and coral substrates becomes necessary (Hoegh-Guldberg et al., 2007). When
available, this information is not only beneficial for detecting and monitoring the impact of
environmental management and changes in the living patterns of marine biota due to altered
environmental conditions, but it can also be used as support facilities in tourism (Dudgeon et
al., 2010). Most importantly, spatial information on coral reef life-forms is acquired for its close
relationship with coastal environment dynamics, especially with several coastal environment
aspects. Complex geomorphological characteristics and diverse life-form compositions can
be used in biodiversity assessment. Certain life-forms are also known to affect the growth and
development of organisms, particularly the abundance of fish (Madduppa et al., 2019), and
some are highly sensitive to the coastal environment dynamics. Therefore, their spatial
information can be the basis for monitoring, explaining the potential of and threats to coastal
resources, and ultimately creating opportunities to build and grow the blue economy (Voyer
et al., 2018). Large-scale mapping and monitoring are a stepin coral reefs management and
conservation (Green et al., 2000). To meet these requirements means, among others, to utilize
multispectral remote sensing technology, that is, satellite imagery that can provide an
effective and efficient alternative source of data for coral reef mapping (Hedley et al., 2005). Since multispectral satellite images with visible to near-infrared (VNIR) channels can be used
to remotely characterize benthic habitats, such as coral reefs, seagrass beds, and macroalgae
(Goodman et al., 2013), they have been widely used to map ones in optically shallow waters
(Phinn et al., 2012; Wicaksono et al. 2019a). The specifications of multispectral satellite
imagery continue to evolve, including increased spatial and spectral resolution and higher
data acquisition capacity. Straightforward integration and access to multispectral satellite
imagery enable its further development, in particular, applications for mapping, monitoring,
and modeling for coral reef studies. Such advancement can contribute to increasing the
accuracy of benthic habitats classification (Roelfsema et al., 2013). 1. Introduction Coral reefs are one of the natural resources in coastal areas that play fundamental
ecological roles for the life of marine flora and fauna (Larkum et al., 2006). If managed,
preserved, and utilized optimally, coral reefs can provide many benefits for coastal
communities. Their exceptional socio-economic and cultural values and interests spread
beyond their function as fish habitat, including abundant sources of seafood, coastal
protection, and marine tourism (Giyanto et al., 2017). Therefore, effective and efficient 377
*Corresponding author. Email address : prama.wicaksono@ugm.ac.id (Pramaditya Wicaksono) 377 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 used (Roelfsema et al., 2013). Therefore, each remote sensing image needs to be assessed for
its capability to produce spatial information on a particular complexity of benthic habitats. used (Roelfsema et al., 2013). Therefore, each remote sensing image needs to be assessed for
its capability to produce spatial information on a particular complexity of benthic habitats. Several studies related to coral reefs use a classification scheme at the life-form level. For example, Wicaksono (2016) mapped 28 classes of coral life-forms around Kemujan
Island, Zhang et al. (2013) used two classification schemes—namely group level (3 classes)
and code level (12 classes), Phinn et al. (2012) mapped benthic habitat communities with 15
class schemes, and Roelfsema et al. (2013) also mapped coral reefs with a 20-class scheme. In some of these studies, the classification results do not cover all existing classes of coral
reef life-forms, indicating limitations in the ability of the used images to discriminate
between them. In addition, each coastal environment has different characteristics of coral
reefs, meaning that any classification scheme and model developments must be first adjusted
to them. Mapping coral reefs at the life-form level is one of the challenges several researchers
remain to experience, mainly because the model designed for a coastal environment depends
on the complexity of the coral reef composition and, thus, may very well be inapplicable to
other coasts. Therefore, it is imperative that the role of multiresolution imagery in producing
spatial information of coral reefs with various levels of detail be examined. Therefore, this study aimed to evaluate the classification scheme of coral reef life-
form using images with different spatial resolutions on Parang Island, Karimunjawa Islands,
Central Java. The multispectral satellite images used in this study are Planet Scope (3m),
PlanetScope resulted from the resampling process (6m), and Sentinel-2A MSI (10m). Despite
their easy access and fitting spatial resolution for mapping the individual variation of a class
of coral reefs, these images are still rarely used in detailed benthic habitat studies, underlying
their uses in this research. The study was designed to assess the classification scheme for
coral reef life-form mapping using satellite images with different spatial resolutions on
Parang Island, Karimunjawa Islands, Central Java Province (Figure 1). Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Using different images can produce detailed information regarding the existence,
diversity, and type of coral reefs, the composition of the geomorphological zone of coral
reefs, benthic habitats, and coral life-form composition (Goodman et al., 2013). The level of
detail of coral life-form information offers depends on the spatial resolution of the image 378 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Parang is one of many
islands in Indonesia that has highly diverse coastal ecosystems and is part of the
Karimunjawa National Park Region. Compared with the other regions in Karimunjawa
Islands, Parang is the least visited destination because it is located somewhat far from the
main island of Karimunjawa. Low tourism activity has little to no effect on the condition of
shallow water ecosystems on Parang Island. Accordingly, the coral reef cover on the island
remains widely diverse and preserved. 379 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Figure 1. The location of Parang Island Figure 1. The location of Parang Island 2.1 Image Preprocessing The multispectral images used in the research were PlanetScope (3m spatial
resolution), PlanetScope Resampling (6m) recorded on April 30, 2018, and Sentinel-2A MSI
(10m) on April 17, 2018. These images were corrected to minimize any biases caused by
sensory and atmospheric conditions. In this study, the Sentinel-2A MSI image (10m) had the
level 1C, and its Digital Number (DN) was converted to Top-Of-Atmosphere Reflectance
(RTOA). Furthermore, this image was subjected to atmospheric, sunglint, and water column
corrections. The PlanetScope image was the SR Product that had been corrected to Bottom-
of-Atmosphere Reflectance (RBOA) and subjected to water column correction. However,
sunglint correction was not applied to the PlanetScope image because of a missed
coregistration between visible and NIR bands, entirely removing the possibility of performing
this correction.The specifications of both images can be seen in Table 1. Table 1. Specification of the PlanetScope and Sentinel-2A MSI Images (Suhet, 2014;
PlanetLabs, 2017)
Image Spesification
PlanetScope
Sentinel-2A MSI
Spatial Resolution (m)
3
10
Radiometric Resolution
12-bit
12-bit
Temporal Resolution
1 day
15‒30 days
Spectral Resolution (µm)
-
-
Blue Band
0.42‒0.53
0.45‒0.52
Green Band
0.50‒0.59
0.54‒0.58
Red Band
0.61‒0.70
0.65‒0.68
near-infrared Band
0.77‒0.90
0.78‒0.90
Note : For the Sentinel-2A MSI image, only the bands used in the study are presented, including
Visible – NIR bands Specification of the PlanetScope and Sentinel-2A MSI Images (Suhet, 2014;
s, 2017) 380 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 2.3 Sunglint Correction Sunglint is a mirror-like specular reflection on the water surface that can produce
noise in remote sensing applications for mapping in the optically shallow and optically
deepwater regions. This study used a sunglint correction method by Hedley et al. (2005). Kay
et al. (2009) compared various sunglint correction methods and showed that the Hedley et al. (2005) and Lyzenga et al. (2006) has the most constant results compared to other methods. Kay et al. (2009) also explained that the use of the Hedley et al. (2005) is more suitable for
mapping benthic habitats, whereas for the correction method Lyzenga et al. (2006) is more
suitable for use in bathymetry mapping. 2.2 Atmospheric Correction Dark-Object Subtraction (DOS) was applied in this research because although it is
one of the simplest atmospheric correction methods (Chavez, 1996 in Nazeer et al., 2014), it
produces better results than other methods (Nazeer et al., 2014). DOS was carried out by
analyzing the reflectance of dark objects in the image, and then the reflectance value was
used to remove the atmospheric offset in the Sentinel-2A MSI image at RTOA level (Chavez,
1996 in Nazeer et al., 2014). 2.5 Field Data Collection The field data were collected by the photo-transect method, which is very efficient in
terms of cost, time, and energy (Roelfsema et al., 2006). Photos of benthic habitat were
captured using an underwater camera by the surveyors while snorkeling in the optically
shallow water. A GPS 78s was attached to the surveyor and set to the tracking mode to record
coordinates at an interval of two seconds, with a UTM coordinate system in the 49M zone
and the datum WGS84. The time indicators on both GPS and underwater cameras were
synchronized (up to seconds) to geotag the taken benthic habitat photos with GPS coordinates
accurately (Phinn et al., 2012). The compositions of benthic habitat and coral reef life-forms
were obtained by interpreting the photo samples taken during the photo-transect survey in
Coral Point Count Excel (CPCe) software. 2.4 Water Column Correction The water column correction of sunglint-free images aimed to minimize changes in
the spectral response of benthic habitats due to depth variations and the attenuation of
electromagnetic energy in the water column (Maritorena, 1996). Lyzenga (1978) developed
one of the most widely used algorithms for water column correction (Zhang et al., 2013) and
is one of the simplest methods of water column correction (Zoffoli et al., 2014). This water
column correction method utilizes the water column attenuation coefficient or ratio at two
different wavelengths to eliminate the need for depth information and the water column
attenuation coefficient at each wavelength (Lyzenga, 1978). The attenuation coefficient value
for each band pairis presented in Table 2. Table 2. The ratio of water column attenuation coefficient value for each band pair
Image
Attenuation Coefficient (ki/kj)
DII 12
DII 13
DII 23
Planet (3 m)
0.822
0.600
0.733
Planet Resampling (6 m)
0.854
0.601
0.710
Sentinel 2 MSI (10 m)
0.7195
0.518
0.722 Table 2. The ratio of water column attenuation coefficient value for each band pair 381 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 2.6 Image Classification Images that had been subjected to water column correction functioned as the input to
the two-level classification process. In the first level, the major classifier was types of benthic
habitats, namely coral reefs, seagrass, macroalgae, and bare substratum. In the second level,
the minor classifier was the coral life-form, which was built according to the spatial
resolution and segments of the used satellite images and based on the dominant life-form
samples in each segment. In this level, the cross-classification of coral life-forms was carried
out in each spatial resolution to assess whether the classification results were consistently
accurate. After applying all classification schemes, the most suitable one for each satellite
image was determined based on the highest accuracy produced (Table 4). As an attempt to improve mapping accuracy, this study integrated two different
approaches in the classification scheme, namely object-based and pixel-based (Ma et al.,
2017). Several studies have proven that using the hybrid OBIA-Supervised classification
method can improve mapping accuracy (Ma et al., 2017). The object-based approach adopted
in this study was limited to the segmentation process in IDRISI Selva 17. Meanwhile, the
pixel-based supervised classification involved the use of algorithms, such as Support Vector
Machine (SVM) and Classification Tree Analysis (CTA), both of which possess the capacity
to provide accurate classification of remote sensing images (Zambon et al., 2006; Wicaksono
et al., 2019a). Compared with other algorithms in supervised classification, SVM can produce
higher mapping accuracy than Maximum Likelihood (ML) and Artificial Neural Network
(ANN) (Pal & Mather, 2005; Wahidin et al., 2015). CTA can analyze large amounts of data
with complex structures (Cappelli et al., 2002 in Zambon et al., 2006). In terms of 382 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 classification, CTA algorithm has been reported to increase classification accuracy in several
previous studies (Wicaksono et al., 2019b) and provide better results than ML. In addition, the Object-Based Image Analysis (OBIA) relies on object characters or
aspects to create classification, namely shape, texture, and relationship between objects
(Mafanya et al., 2017). Although it can provide good mapping results, this study did not
solely use OBIA because it lacks quantitative verification (Ma et al., 2017) and is highly
dependent on the developed rules when applied to remote sensing data. 2.6 Image Classification Furthermore,
designing and generating the rules requires adequate knowledge and experience, and even
then, it is not easy to ascertain the error or truth of said rules. In addition, the development of
OBIA rules strongly depends on the number of classes to be used in the classification. For
instance, a high class diversity requires the developed rules to pay attention to each class,
which is a lengthy process where repeated experiments are conducted to obtain a good rule
for each step. 2.7 Accuracy Assessment Accuracy assessment is an approach used to determine the extent to which a study
result can be trusted. In the case of benthic habitat classification, its accuracy was assessed
using a confusion matrix analysis. Samples for validation were used as reference data. Confusion matrix analysis offers information about user's accuracy (UA), producer's accuracy
(PA), overall accuracy (OA), and kappa coefficient (Congalton & Green, 2009). We
determined the most appropriate classification scheme for each image based on the confusion
matrix results. 3.1 Field Data Collection The field survey of benthic habitat was carried out on April 17-19, 2018, and the
location of the transects for data collection is presented in Figure 2. During this field survey,
1,658 samples were obtained. Based on the life-form variations at the study location, the
dominant coral reef life-forms obtained by analyzing the photo samples were branching, stag
horn, massive, pillar, foliage, encrusting, tabular, and dead coral; thereby, only these classes
were used as the training areas for object classification. However, not all life forms can be
used as a single class for training areas in the classification process, except for the frequently
encountered and homogeneous ones. In some locations, the life-forms of coral reefs were not
always homogeneous but consisted of multiple types. 383 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Figure 2. Field data collection using the photo-transect method Figure 2. Field data collection using the photo-transect method 3.2 Coral Reef Life-Forms Classification Scheme The classification scheme of coral reef life-forms was obtained through the
dominance analysis of each segment in the three satellite images (Phinn et al., 2012). However, should the existing sample consist of different classes and has the same percentage,
it is classified based on the number of classes in the image segment (mixed class). Based on
Table 3, the classification schemes of coral reef life-forms, built from each image, have
different numbers of classes. 384 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Table 3. The classification schemes of benthic habitats and coral reef life-forms at each
spatial resolution
Benthic Habitat Classification Scheme
Classes
Classification
Validation
Bare Substratum
105
119
Macroalgae
69
55
Seagrass
219
194
Coral Reef
412
437
Coral Life-formClassification Scheme
PlanetScope (3m)
Classification
Validation
PlanetScope Resampling
(6m)
Classification
Validation
Sentinel-2A MSI (10m)
Classification
Validation
Branching
55
65
Branching
45
57
Branching
78
95
Branching Staghorn
15
10
Branching Staghorn
33
21
Branching Tabular
62
51
Branching Tabular
79
74
Branching Tabular
79
85
Foliage Staghorn
23
16
Foliage
19
9
Foliage
22
15
Massive
54
68
Massive
54
64
Massive
24
30
Massive Branching
72
65
Massive Branching
36
41
Massive Branching
85
80
Massive Staghorn
47
41
Massive Foliage
11
6
Massive Branching Tabular
29
22
Massive Tabular
24
30
Massive Staghorn
50
69
Massive Staghorn
19
26
Staghorn
29
38
Massive Tabular
45
36
Massive Tabular
46
41
Tabular
48
32
Staghorn
27
16
Staghorn
31
39
Staghorn Tabular
14
8
Tabular
24
20
Tabular
32
38 e classification schemes of benthic habitats and coral reef life-forms at each
i 3. The classification schemes of benthic habitats and coral reef life-forms at each
resolution 3.3 Benthic Habitat Mapping Benthic habitat mapping sought to obtain the best spatial distribution of coral reef
(coral reef mask image), which was used as a basis for the classification of coral reef life-
forms. In the classification process, the benthic habitat was a major classifier that defined
four types of habitat, namely coral reef, seagrass, macroalgae, and bare substratum (Figure 3). The classification scheme of benthic habitat has an OA of 57-63% (Table 4). PlanetScope
Resampling (6m) images produced the highest accuracy, with an OA of 63.10% and a kappa
coefficient of 0.38, which was obtained using the CTA classification algorithm and was
higher than using the SVM algorithm. The accuracies of benthic habitat mapping using the
CTA and SVM algorithms differed by approximately 5%. The overall accuracies of benthic
habitat classification from different algorithms and images are compared in Table 4. Table 4. Summary of the assessment of benthic habitat mapping accuracy
Image
CTA
SVM
OA (%)
Kappa
OA (%)
Kappa
PlanetScope (3 m)
57.26
0.49
61.86
0.32
PlanetScope Resampling (6 m)
63.10*
0.38
58.75
0.22
Sentinel-2A MSI (10 m)
57.26
0.16
57.14
0.08
Note : *indicates the highest overall accuracy Table 4. Summary of the assessment of benthic habitat mapping accuracy Judging from the overall classification schemes of benthic habitat, CTA algorithm
produced a spatial distribution of benthic habitats more consistently—in which all types of 385 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 habitats were classified—and with a higher OA for each satellite image than SVM algorithm,
even though the accuracy difference is merely 5%. SVM algorithmprovided less accurate
results because several types of benthic habitats, such as macroalgae and sand were not
applicable for classification. Another reason was inter-class misclassification; for example,
sand was misclassified into seagrass and macroalgae. Sand has a spectral response that is
similar to low seagrass and macroalgae due to the contribution of sand reflectance as the
substrate of seagrass and macroalgae (Wicaksono, 2016). Also, coral reefs were misclassified
into macroalgae because both objects have comparable spectral response due to similar
pigmentation (Hochberg & Atkinson, 2000; Wicaksono, 2016). Figure 3. A comparison of benthic habitat classification resulted from different classification
algorithms and satellite images Figure 3. 3.3 Benthic Habitat Mapping A comparison of benthic habitat classification resulted from different classification
algorithms and satellite images Figure 3. A comparison of benthic habitat classification resulted from different classification
algorithms and satellite images able 5. Comparison of user’s accuracy (UA) and producer’s accuracy (PA) of coral
asses Table 5. Comparison of user’s accuracy (UA) and producer’s accuracy (PA) of co
classes Table 5. Comparison of user’s accuracy (UA) and producer’s accuracy (PA) of coral reef
classes Table 5. Comparison of user’s accuracy (UA) and producer’s accuracy (PA) of coral reef
classes
Image
CTA-based
SVM-based
PA (%)
UA (%)
Resulted
Accuracy
(%)
PA
(%)
UA
(%)
Resulted
Accuracy
(%)
PlanetScope (3 m)
80.55
73.79
59.43
89.93
59.10
53.14
PlanetScope resampling
(6m)
83.07
73.19
60.79
87.87
61.84
54.33
Sentinel-2A MSI (10 m)
85.35
68.69
58.62
99.77
56.26
56.13 386 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Based on the overall results of the benthic habitat classification, the individual
accuracy (UA and PA) of the coral reef life-forms classification ranged from 56 - 99% (Table
5). The CTA-based classification from PlanetScope (3m) was used in the masking of coral
reef images or to exclude non-coral reef pixels in the mapping. Although the OA of the CTA-
based classification from PlanetScope (3m) image was slightly lower than PlanetScope
Resampling (6m), the spatial resolution was higher than PlanetScope Resampling (6m) and
Sentinel-2A MSI (10m), and producing more precise coral reef masking delineation. 3.4 Coral Reef Life-Form Mapping The coral reef life-forms were mapped by applying the classification schemes built on
each satellite image (see Table 3). The coral reef life-form classification (Figure 4) with the
highest OA (60.78%) was obtained from Sentinel-2A MSI image using CTA algorithm and
the 10m scheme, and then followed by PlanetScope (48.39%), and PlanetScope Resampling
(40.83%) (Table 6). Although the CTA algorithm could consistently produce maps of coral
reef life-forms with higher OA than SVM, the generated classification was relatively low, in
particular, because of a large number of misclassifications as a result of similar statistics
between the classes of coral reef life-forms. For this reason, both CTA and SVM
classification algorithms could not separate one life-form from another straightforwardly. The coral reef life-form classifications built from the three satellite images showed
different spatial distribution (Figure 4). PlanetScope (3m) and Sentinel-2A MSI (10m)
images presented similar distributions of life-forms in some locations, and different spread
patterns are found in some others. This similarity indicates consistency in the classification
results produced using different images. The spatial distribution of several coral reef life-
forms corresponds to the theory proposed by English et al. (1994), that is, coral reef life-
forms spread following the locations of optimal environmental conditions for their growth. The opposite is true for the classification scheme built from the PlanetScope Resampling
(6m), which only has one class, i.e., the tabular branching that dominated the study area. It is
caused by the effects of the image pixel resampling process from 3m to 6m. 387 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Figure 4. Comparison of coral reef life-form maps with the highest OA Figure 4. Comparison of coral reef life-form maps with the highest OA Table 6. Comparison of the overall accuracies of the resulted coral reef life-form
classifications Images
3m Scheme
6m Scheme
10m Scheme
CTA (%) SVM (%) CTA (%) SVM (%) CTA (%) SVM (%)
PlanetScope (3m)
38.76
30.05
35.32
27.52
48.39
39.45
PlanetScope resampling
(6m)
40.83
31.65
39.91
27.52
36.01
40.60
Sentinel-2A MSI (10m)
39.91
26.38
46.56
30.28
60.78
42.20 The resampling process generalizes pixel values by considering the surrounding
pixels (i.e., mean values). Therefore, variations in the pixel values of the analyzed objects
canbe very similar, and as a result, pixels that should be classified as different objects are
categorized as the same object. 3.4 Coral Reef Life-Form Mapping Accordingly, the image resampling process is not
recommended for addressing issues of unavailable images with a particular spatial resolution
due to its poor results. Different distributions of coral reef life-forms obtained from the
multiresolution images were potentially caused by the size of the spatial resolution itself. As
evidence, different spatial resolutions have different spectral responses from the object
composition. Accordingly, the distributions of coral reef life-forms are different from one
satelliteimage to another (Figure 4 and Table 7). The classification results of the satellite
image with 6m spatial resolution were not included in the comparison because the 388 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 generalization ofthe spatial resolution was believed to produce less representative
distribution. Table 7. The classification schemes of coral reef life-forms
3m Scheme
6m Scheme
10m Scheme
CORAL REEF LIFE-FORM
CLASSES
Branching
Branching
Branching
Branching Staghorn
Branching Staghorn
Branching Tabular
Foliage
Foliage
Foliage Staghorn
Branching Tabular
Branching Tabular
Massive
Massive
Massive
Massive Branching
Massive Branching
Massive Branching
Tabular
Massive Staghorn
Massive Foliage
Massive Branching
Massive Tabular
Massive Staghorn
Massive Staghorn
Staghorn
Massive Tabular
Massive Tabular
Tabular
Staghorn
Staghorn
Staghorn Tabular
Tabular
Tabular Table 7. The classification schemes of coral reef life-forms Based on the highest accuracy of the maps produced from each satellite image and
classification scheme (Table 6), the coral reef life-forms captured in PlanetScope (3m) and
Sentinel-2A MSI (10m) were best mapped using the 10m scheme. Unlike in the two satellite
images, the coral reef life-forms featured in PlanetScope Resampling (6m) were best mapped
using the 3m scheme. However, not all life-forms were applicable for classification. Eight
classes of coral reef life-forms were consistently classified in all images and schemes, namely
branching, tabular branching, massive, massive branching, massive staghorn, massive
tabular, staghorn, and tabular. As seen in Tables 8, 9, and 10, each life-form was properly
classified for each image with high UA and PA. Nevertheless, overestimate and
underestimate could occur. Therefore, the classes above can be used in the classification
schemes of coral reef life-forms in various images (Table 7). Table 8. Confusion matrix of PlanetScope (3m) using the 10m scheme based on the CTA-
based classification result 389
Table 8. Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397
Table 9. Confusion matrix of PlanetScope (3m) using the 3m scheme based on the CTA-
based classification result
Classes
References
1
2
3
4
5
6
7
8
9
Total
UA %
7
0
0
3
0
0
0
15
0
6
24
62.50
8
0
0
1
0
0
0
0
3
0
4
75.00
9
1
8
0
0
2
0
0
0
4
15
26.67
Total
95
51
16
68
65
41
30
38
32
436
PA (%)
65.26
72.55
0.00
63.24
66.15
9.76
50.00
7.89
12.50
OA (%)
48.39
Legend:
1 :Branching
4 :Massive
7: Massive Tabular
2: Branching Tabular
5 :Massive Branching
8 :Staghorn
3: Foliage Staghorn
6: Massive Staghorn
9: Tabular Table 9. Confusion matrix of PlanetScope (3m) using the 3m scheme based on the CTA-
based classification result
Classes
References
1
2
3
4
5
6
7
8
9
Total
UA %
7
0
0
3
0
0
0
15
0
6
24
62.50
8
0
0
1
0
0
0
0
3
0
4
75.00
9
1
8
0
0
2
0
0
0
4
15
26.67
Total
95
51
16
68
65
41
30
38
32
436
PA (%)
65.26
72.55
0.00
63.24
66.15
9.76
50.00
7.89
12.50
OA (%)
48.39
Legend:
1 :Branching
4 :Massive
7: Massive Tabular
2: Branching Tabular
5 :Massive Branching
8 :Staghorn
3: Foliage Staghorn
6: Massive Staghorn
9: Tabular Table 9. Confusion matrix of PlanetScope (3m) using the 3m scheme based on the CTA-
based classification result Table 9. Confusion matrix of PlanetScope (3m) using the 3m scheme based on the CTA-
based classification result Table 9. Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Confusion matrix of PlanetScope (3m) using the 3m scheme based on the CTA-
based classification result
Class
References
1
2
3
4
5
6
7
8
9
10
11
12
Total
UA (%)
1
46
5
6
0
14
19
0
23
0
0
8
4
125
36.80
2
0
0
0
2
0
0
0
0
0
0
0
0
2
0.00
3
16
1
48
1
20
1
6
13
6
9
0
11
132
36.36
5
0
0
1
1
16
0
0
0
1
3
0
0
22
72.73
6
0
4
0
5
3
21
0
3
0
0
0
0
36
58.33
8
2
0
14
0
4
0
0
25
26
0
0
8
79
31.65
9
0
0
0
0
0
0
0
0
3
0
0
0
3
100
10
0
0
0
0
0
0
0
0
0
4
0
0
4
100
12
1
0
5
0
7
0
0
5
0
0
0
15
33
45.45
Total
65
10
74
9
64
41
6
69
36
16
8
38
436
PA (%)
70.77
0
64.86
0
25.00
51.22
0
36.23
8.33
25.00
0
39.47
OA (%)
41.00
Legend:
1 :Branching
4 :Foliage
7: Massive Foliage
10 :Staghorn
2: Branching Staghorn
5 :Massive
8 :Massive Staghorn
11 :Staghorn Tabular
3: Branching Tabular
6: Massive Branching
9: Massive Tabular
12 :Tabular Table 10. Confusion matrix of Sentinel-2A MSI (10m) using the 10m scheme based on the
CTA-based classification result
Class
References
1
2
3
4
5
6
7
8
9
Total
UA %
1
83
0
5
28
17
20
0
3
0
156
53.21
2
7
46
0
12
2
0
0
0
14
81
56.79
3
0
0
11
0
0
0
0
0
0
11
100
4
0
5
0
28
8
0
0
5
3
49
57.14
5
2
0
0
0
38
6
17
0
5
68
55.88
6
0
0
0
0
0
10
0
0
0
10
100
7
0
0
0
0
0
5
13
0
0
18
72.22
8
3
0
0
0
0
0
0
30
4
37
81.08
9
0
0
0
0
0
0
0
0
6
6
100
Total
95
51
16
68
65
41
30
38
32
436
PA (%)
87.36
90.19
68.75
41.17
58.46
24.39
43.33
78.94
18.75
OA (%)
60.78 Table 10. 3.4 Coral Reef Life-Form Mapping Confusion matrix of PlanetScope (3m) using the 10m scheme based on the CTA-
based classification result
Classes
References
1
2
3
4
5
6
7
8
9
Total
UA %
1
62
0
7
22
13
12
0
2
0
118
52.54
2
3
37
2
3
6
0
7
6
10
74
50.00
3
0
0
0
0
0
1
0
0
0
1
0.00
4
18
4
3
43
1
7
7
19
1
103
41.75
5
11
0
0
0
43
17
1
8
11
91
47.25
6
0
2
0
0
0
4
0
0
0
6
66.67 389 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Confusion matrix of Sentinel-2A MSI (10m) using the 10m scheme based on the
CTA-based classification result 390 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Legend:
1: Branching
4: Massive
7: Massive Tabular
2: Branching Tabular
5: Massive Branching
8: Staghorn
3: Foliage Staghorn
6: Massive Staghorn
9: Tabular The classification schemes of coral reef life-forms are constructed from three satellite
images with different spatial resolutions. Three schemes of classification with varying
numbers of classes have been built in this study (Table 3). Some classes in these schemes
depict similar types of coral reef life-forms, such as branching, tabular branching, massive,
massive branching, massive staghorn, massive tabular, staghorn, and tabular. Nevertheless,
some classes are only found in a particular scheme because their generation depends on the
uniquely formed image segments (Phinn et al., 2012). Each satellite images of which indeed
produces classification schemes with different suitability for coral reef life-form mapping. Here, suitability is defined by the highest OA in each image. Table 6 shows that PlanetScope
(3m) and Sentinel-2A MSI (10m) images are suitable using the 10m scheme, whereas
PlanetScope Resampling (6m) image using the 3m scheme. PlanetScope (3m) is not suitable using the 3m and 6m schemes because the statistical
value of each pixel in its segment is too precise. Consequently, the same objects have varying
pixel values, causing too many misclassifications (Purkis, 2018). Meanwhile, the 10m
scheme produces higher accuracy than the 3m and 6m schemes because the classification
wasbuilt from images with a lower spatial resolution (general information) (Zhang et al.,
2013). As a result, variations of pixel values in a segment are considered one life-form class
so that the said segment can be classified according to the input sample (Phinn et al., 2012). PlanetScope Resampling (6m) is more suitable using the 3m scheme because the
generalization during the spatial resolution resampling results in similarities. In this case, the
10m scheme is not suitable for use because the effect of generalization increases the
homogeneity of image statistics to PlanetScope (3m) instead of the Sentinel-2A MSI (10m),
even though the level of precision is different. Several studies have shown that the higher the
number of classes used, the lower the accuracy (Wicaksono, 2016; Wicaksono et al., 2019a). However, the contrary is true for this study, where the findings correspond to Zhang et al. (2013) instead. Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Sentinel-2A MSI (10m) is more suitable using the 10m scheme because it produces
classes that are more general than the 3m and 6m schemes. The classified types of coral reef
life-forms in the Sentinel-2A MSI image will not be optimal if the used classification scheme
is built from images with more detailed spatial resolutions. For example, if two different
classes from the 3m or 6m scheme occupy a segment, one that appears dominantly will be
selected over the other, creating low accuracy. On the contrary, if the used classification
scheme is built from the same image, each sample of each class undoubtedly corresponds to
the previously formed segments. Consequently, each segment can be classified more
accordingly to the input sample, leading to a higher OA than the other schemes. The selected satellite images can map the life-forms of coral reefs with overall
accuracies of 48.39% (PlanetScope 3m, CTA, 10m scheme), 40.83% (PlanetScope
Resampling 6m, CTA, 3m scheme), and 60.78% (Sentinel-2A MSI 10 m, CTA, 10m
scheme). Multiresolution images produce classifications with multilevel accuracy (Mumby &
Edward, 2002), and satellite imagery with a more detailed spatial resolution can yield higher
accuracy (Goodman et al., 2013). However, the highest accuracy in this study is shown by
images with a lower spatial resolution (10m) because the more detailed ones, i.e., 3m and 6m,
cannot represent a community or a dominant class of coral reef life-forms. In addition,
Sentinel 2 MSI also has a fairly consistent image quality (Hedley et al., 2018; Wicaksono et
al., 2021) compared with PlanetScope, whose pixels between scenes are inconsistent and
SNR is of poor quality (Wicaksono & Lazuardi, 2018). Based on the percentage, the coral
reef life-forms are covered with diverse objects, meaning that the resulted spectral response is
a function of various classes. Sentinel-2A MSI (10m) has successfully produced the highest accuracy, followed by
PlanetScope (3m) and PlanetScope Resampling (6m). The resampling process generalizes the
spectral response value of the image, and the results are less suitable for representing a
variety of objects because the spectral response is the mean value of the surrounding pixels. In other terms, the spectral response produced in the resampling process shows similarities
among different objects. These numerous cases of misclassifications contribute to the overall
accuracy of PlanetScope Resampling (6m), which is lower than that of PlanetScope (3m). Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 They used two classification schemes consisting of group level (3 classes) and
code level (12 classes) and found that the latter produces a higher accuracy (86.7%) than the
former (84.3%). 391 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 When the accuracies are compared, the CTA algorithm evidently produces the best
and most consistent accuracy (Zambon et al., 2006) of life-form classification (Wahidin et al.,
2015). It allows the proper classification of coral reef life-forms in each of the three satellite
images. The CTA and SVM algorithms produce different OA by averagely 12%, which, in
terms of OA, is significant. The SVM algorithm provides a low accuracy because not all
coral reef life-forms are classified. On the contrary, the CTA algorithm has a better capability
to classify coral reef life-forms, especially in schemes that create a large number of classes. Although the accuracy derived in this study is merely 40‒60%, the results are in line with
previous similar studies (Phinn et al., 2012; Roelfsema et al., 2013; Wahidin et al., 2015;
Wicaksono, 2016). Although Sentinel-2A MSI (10m) can create a coral reef life-form map
with higher accuracy than other images, the level of information obtained from it is
somewhat general. Meanwhile, PlanetScope (3m) has lower accuracy, but it can map coral
reef life-forms in more detail and involve precise information compared with Sentinel-2A
MSI (10m). Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Furthermore, the difference in OA can be caused by the quality of the used satellite image. For instance, despite the high spatial resolution, the pixel sharpness of the PlanetScope (3m)
image is still lower than Sentinel-2A MSI (10m) that has a lower resolution. Furthermore, the
results obtained from the three satellite images have different UA and PA. 392 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Conflict of interest There is no conflict of interest with any financial, personal, or other relationships with
other people or organizations related to the material discussed in the article. 4. Conclusion Our results have shown that both PlanetScope (3m) and Sentinel-2A MSI (10m) can
be used to map coral reef with higher complexity. This finding indicates that multiresolution
images can be used to produce complex coral reef life-form maps with different levels of
information details. In our case, the classification scheme on multiresolution images shows
that the coral reef life-forms in PlanetScope and Sentinel-2A MSI images can be mapped
using the 10m scheme, whereas the ones in PlanetScope Resampling (6m) image are mapped
according to the 3m scheme. Parang Island has eight types of coral reef life-forms that can be
classified consistently in each satellite image, namely branching, branching staghorn,
tabular branching, massive branching, massive staghorn, massive tabular, staghorn, and
tabular. Based on the statistical analysis and accuracy assessment, the maximum overall
accuracies of the coral reef life-form maps produced from PlanetScope (3m), PlanetScope
Resampling (6m), and Sentinel-2A MSI (10m) images are 48.39% (10m scheme), 40.83%
(3m scheme), and 60.78% (10m scheme), respectively. The accuracy obtained in our work
can be used as a reference for future works of coral reef life-forms mapping. Possibly,
variations in accuracy is expected since coral reefs life-forms may vary significantly between
areas. Finally, the future availability of detailed coral reef map at regular basis will be
beneficial to assist coastal manager in determining and monitoring the effective action to
managed their area sustainably. 393 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Wahyu Lazuardi & Pramaditya Wicaksono/ Geosfera Indonesia 6 (3), 2021, 377-397 Acknowledgements This research is funded by “Direktorat Riset dan Pengabdian Masyarakat – Direktorat
Jenderal Penguatan Riset dan Pengembangan – Kementerian Riset, Teknologi, dan
Pendidikan Tinggi Republik Indonesia” via Penelitian Dasar Unggulan Perguruan Tinggi –
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Measuring Biases of Word Embeddings: What Similarity Measures and Descriptive Statistics to Use?
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Proceedings of the First Workshop on Trustworthy Natural Language Processing, pages 8–14
June 10, 2021. ©2021 Association for Computational Linguistics Abstract gate biases of word embeddings (Liang et al., 2020;
Ravfogel et al., 2020). Word embeddings are widely used in Natural
Language Processing (NLP) for a vast range
of applications. However, it has been consis-
tently proven that these embeddings reflect the
same human biases that exist in the data used
to train them. Most of the introduced bias in-
dicators to reveal word embeddings’ bias are
average-based indicators based on the cosine
similarity measure. In this study, we examine
the impacts of different similarity measures
as well as other descriptive techniques than
averaging in measuring the biases of contex-
tual and non-contextual word embeddings. We
show that the extent of revealed biases in word
embeddings depends on the descriptive statis-
tics and similarity measures used to measure
the bias. We found that over the ten categories
of word embedding association tests, Maha-
lanobis distance reveals the smallest bias, and
Euclidean distance reveals the largest bias in
word embeddings. In addition, the contextual
models reveal less severe biases than the non-
contextual word embedding models with GPT
showing the fewest number of WEAT biases. Different approaches have been used to present
and quantify corpus-level biases of word embed-
dings. Bolukbasi et al. (2016) proposed to mea-
sure the gender bias of word representations in
Word2Vec and GloVe by calculating the projections
into principal components of differences of embed-
dings of a list of male and female pairs. Basta et al. (2019) adapted the idea of "gender direction" of
(Bolukbasi et al., 2016) to be applicable to contex-
tual word embeddings such as ELMo. In (Basta
et al., 2019) first, the gender subspace of ELMo
vector representations is calculated and then, the
presence of gender bias in ELMo is identified. Go-
nen and Goldberg (2019) introduced a new gender
bias indicator based on the percentage of socially-
biased terms among the k-nearest neighbors of a
target term and demonstrated its correlation with
the gender direction indicator. Caliskan et al. (2017) developed Word Embed-
ding Association Test (WEAT) to measure bias by
comparing two sets of target words with two sets of
attribute words and documented that Word2Vec and
GloVe contain human-like biases such as gender
and racial biases. May et al. (2019) generalized the
WEAT test to phrases and sentences by inserting
individual words from WEAT tests into simple sen-
tence templates and used them for contextual word
embeddings. 2
Method Implicit Association Test (IAT) was first intro-
duced by Greenwald et al. (1998a) in psychology to
demonstrate the enormous differences in response
time when participants are asked to pair two con-
cepts they deem similar, in contrast to two con-
cepts they find less similar. For example, when
subjects are encouraged to work as quickly as pos-
sible, they are much likely to label flowers as pleas-
ant and insects as unpleasant. In IAT, being able
to pair a concept to an attribute quickly indicates
that the concept and attribute are linked together
in the participants’ minds. The IAT has widely
been used to measure and quantify the strength of
a range of implicit biases and other phenomena,
including attitudes and stereotype threat (Karpinski
and Hilton, 2001; Kiefer and Sekaquaptewa, 2007;
Stanley et al., 2011). d = |s(x, A, B) −s(y, A, B)|
std-devw∈X∪Y s(w, A, B) It is worth mentioning that d is a measure used to
calculate how separated two distributions are and is
basically the standardized difference of the means
of the two distributions (Cohen, 2013). Controlling
for the significance, a larger effect size reflects a
more severe bias. WEAT and almost all the other studies inspired
by it (Garg et al., 2018; Brunet et al., 2018; Gonen
and Goldberg, 2019; May et al., 2019) use the fol-
lowing approach to measure the association of a
single target word with the two sets of attributes
(equation 1). First, they use cosine similarity to
measure the target word’s similarity to each word
in the attribute sets. Then they calculate the average
of the similarities over each attribute set. Inspired by IAT, Caliskan et al. (2017) intro-
duced WEAT to measure the associations between
two sets of target concepts and two sets of attributes
in word embeddings learned from large text cor-
pora. A hypothesis test is conducted to demon-
strate and quantify the bias. The null hypothesis
states that there is no difference between the two
sets of target words in terms of their relative dis-
tance/similarity to the two sets of attribute words. A permutation test is performed to measure the
null hypothesis’s likelihood. where: where: s(w, A, B) = fa∈A(s(−→
w , −→a )) −fb∈B(s(−→
w , −→b ))
(1) In other words, s(w, A, B) quantifies the associ-
ation of a single word w with the two sets of at-
tributes, and s(X, Y, A, B) measures the differen-
tial association of the two sets of targets with the
two sets of attributes. Denoting all the partitions
of X ∪Y with (Xi, Yi)i, the one-sided p-value of
the permutation test is: Pri(s(Xi, Yi, A, B) > s(X, Y, A, B)) The magnitude of the association of the two target
sets with the two attribute sets can be measured
with the effect size as: 1
Introduction Word embedding models including Word2Vec
(Mikolov et al., 2013), GloVe (Pennington et al.,
2014), BERT (Devlin et al., 2018), ELMo (Peters
et al., 2018), and GPT (Radford et al., 2018) have
become popular components of many NLP frame-
works and are vastly used for many downstream
tasks. However, these word representations pre-
serve not only statistical properties of human lan-
guage but also the human-like biases that exist in
the data used to train them (Bolukbasi et al., 2016;
Caliskan et al., 2017; Kurita et al., 2019; Basta
et al., 2019; Gonen and Goldberg, 2019). It has
also been shown that such biases propagate to the
downstream NLP tasks and have negative impacts
on their performance (May et al., 2019; Leino et al.,
2018). There are studies investigating how to miti- Kurita et al. (2019) proposed a new method to
quantify bias in BERT embeddings based on its
masked language model objective using simple
template sentences. For each attribute word, us-
ing a simple template sentence, the normalized
probability that BERT assigns to that sentence for
each of the target words is calculated, and the dif-
ference is considered the measure of the bias. Ku-
rita et al. (2019) demonstrated that this probability-
based method for quantifying bias in BERT was
more effective than the cosine-based method. 8 Motivated by these recent studies, we compre-
hensively investigate different methods for bias ex-
posure in word embeddings. Particularly, we inves-
tigate the impacts of different similarity measures
and descriptive statistics to demonstrate the degree
of associations between the target sets and attribute
sets in the WEAT. First, other than cosine similarity,
we study Euclidean, Manhattan, and Mahalanobis
distances to measure the degree of association be-
tween a single target word and a single attribute
word. Second, other than averaging, we investigate
minimum, maximum, median, and a discrete (grid-
based) optimization approach to find the minimum
possible association to report between a single tar-
get word and the two attribute sets in each of the
WEAT tests. We consistently compare these bias
measures for different types of word embeddings
including non-contextual (Word2Vec, GloVe) and
contextual ones (BERT, ELMo, GPT, GPT2). observed difference. Let X and Y be two sets
of target word embeddings and A and B be two
sets of attribute embeddings. 1
Introduction The test statistics is
defined as: s(X, Y, A, B) = | P
x∈X s(x, A, B) −P
y∈Y s(y, A, B)| 2
Method This test computes
the probability that target words’ random permuta-
tions would produce a greater difference than the In this paper we investigate the impacts of other
functions such as min(·), mean(·), median(·), or
max(·) for function f(·) in equation (1) (originally
only mean(·) has been used). Also, in this paper in
addition to cosine similarity, we consider Euclidean
and Manhattan distances as well as the following
measures for the s(−→
w, −→a ) in equation (1). Mahalanobis distance:
introduced by P. C. Ma-
halanobis (Mahalanobis, 1936) this distance mea-
sures the distance of a point from a distribution:
s(−→
w, −→a ) = ((−→
w −−→a )T Σ−1
A (−→
w −−→a ))
1
2 . It is 9 in Table 2. We used publicly available pre-trained
models. For contextual word embeddings, we used
single word sentences as input instead of using
simple template sentences used in other studies
(May et al., 2019; Kurita et al., 2019). The sim-
ple template sentences such as "this is TARGET"
or "TARGET is ATTRIBUTE" used in other stud-
ies do not really provide any context to reveal the
contextual capability of embeddings such as BERT
or ELMo. This way, the comparisons between the
contextual embeddings and non-contextual embed-
dings are fairer as both of them only get the target
or attribute terms as input. For each model, we
performed the WEAT tests using four similarity
metrics mentioned in section 2: cosine, Euclidean,
Manhattan, Mahalanobis. For each similarity met-
ric, we also used min(·), mean(·), median(·), or
max(·) as the f(·) in equation (1). Also, as ex-
plained in section 2, we discretely optimized the
association measure and found the minimum asso-
ciation in equation (1). In these experiments (Table
3 and Table 4), the larger and more significant ef-
fect sizes imply more severe biases. worth noting that the Mahalanobis distance takes
into account the distribution of the set of attributes
while measuring the association of the target word
w with an attribute vector. Discrete optimization of the association mea-
sure:
In equation (1), s(w, A, B) quantifies the
association of a single target word w with the two
sets of attributes. Model Median is close to the mean with
(µ = 0.74, σ = 0.48) among all its effect sizes. The effect sizes for minimum (µ = 0.68, σ = 0.48)
and maximum (µ = 0.65, σ = 0.48) are close
to each other, but smaller than mean and median. The discretely optimized association measure (Eq. 2) provides the smallest effect sizes (µ = 0.39,
σ = 0.3) and reveals the least number of implicit
biases. These differences as the result of apply-
ing different descriptive statistics in the association
measure (Eq. (1)) show that the revealed biases
depend on the applied statistics to measure the bias. 6
Male vs female names with Career vs family 7
Math vs arts with male vs female terms 8
Science vs arts with male vs female terms 9
Mental vs physical disease with temporary vs permanent 10
Young vs old people’s name with pleasant vs unpleasant
Table 1: The associations studied in the WEAT As described in section 2, we need each attribute
set’s covariance matrix to compute Mahalanobis
distance. To get stable covariance matrix estima-
tion due to the high dimension of the embeddings
we first created larger attribute sets by adding syn-
onym terms. Next, we estimated the sparse covari-
ance matrices as the number of samples in each
attribute set is smaller than the number of features. To enforce sparsity, we estimated the l1 penalty
using k-fold cross validation with k=3. The discretely optimized association measure (Eq. 2) provides the smallest effect sizes (µ = 0.39,
σ = 0.3) and reveals the least number of implicit
biases. These differences as the result of apply-
ing different descriptive statistics in the association
measure (Eq. (1)) show that the revealed biases
depend on the applied statistics to measure the bias. 2
Method To quantify the minimum pos-
sible association of a target word w with the two
sets of attributes, we first calculate the distance of
w from all attribute words in A and B, then calcu-
late all possible differences and find the minimum
difference. s(w, A, B) =
min
a∈A,b∈B |s(−→
w , −→a ) −s(−→
w , −→b )|
(2) (2) Model WEAT
Association
1
Flowers vs insects with pleasant vs unpleasant
2
Instruments vs weapons with pleasant vs unpleasant
3
Eur.-American vs Afr.-American names with Pleasant vs
unpleasant (Greenwald et al., 1998b)
4
Eur.-American vs Afr.-American names (Bertrand and Mul-
lainathan, 2004) with Pleasant vs unpleasant (Greenwald
et al., 1998b)
5
Eur.-American vs Afr.-American names (Bertrand and Mul-
lainathan, 2004) with Pleasant vs unpleasant (Nosek et al.,
2002)
6
Male vs female names with Career vs family
7
Math vs arts with male vs female terms
8
Science vs arts with male vs female terms
9
Mental vs physical disease with temporary vs permanent
10
Young vs old people’s name with pleasant vs unpleasant
Table 1: The associations studied in the WEAT 2
Instruments vs weapons with pleasant vs unpleasant 3
Eur.-American vs Afr.-American names with Pleasant vs
unpleasant (Greenwald et al., 1998b) 4
Eur.-American vs Afr.-American names (Bertrand and Mul-
lainathan, 2004) with Pleasant vs unpleasant (Greenwald
et al., 1998b) Table 2: Word embedding models, used representa-
tions, and their dimensions. 5
Eur.-American vs Afr.-American names (Bertrand and Mul-
lainathan, 2004) with Pleasant vs unpleasant (Nosek et al.,
2002) Impacts of different descriptive statistics:
Our
first goal was to report the changes in the mea-
sured biases when we change the descriptive statis-
tics. The range of effect sizes was from 0.00 to
1.89 (µ = 0.65, σ = 0.5). Our findings show
that mean has a better capability to reveal biases
as it provides the most cases of significant effect
sizes (µ = 0.8, σ = 0.52) across models and dis-
tance measures. Median is close to the mean with
(µ = 0.74, σ = 0.48) among all its effect sizes. The effect sizes for minimum (µ = 0.68, σ = 0.48)
and maximum (µ = 0.65, σ = 0.48) are close
to each other, but smaller than mean and median. Impacts of different descriptive statistics:
Our
first goal was to report the changes in the mea-
sured biases when we change the descriptive statis-
tics. The range of effect sizes was from 0.00 to
1.89 (µ = 0.65, σ = 0.5). Our findings show
that mean has a better capability to reveal biases
as it provides the most cases of significant effect
sizes (µ = 0.8, σ = 0.52) across models and dis-
tance measures. 3
Biases studied We studied all ten bias categories introduced in IAT
(Greenwald et al., 1998a) and replicated in WEAT
to measure the biases in word embeddings. The ten
WEAT categories are briefly introduced in Table 1. For more detail and example of target and attribute
words, please check Appendix A. Although WEAT
3 to 5 have the same names, they have different
target and attribute words. Model
Embedding
Dim
GloVe (840B tokens, web corpus) -
300
Word2Vec (GoogleNews-negative) -
300
ELMo (original)
First hidden layer
1024
BERT (base, cased)
Sum of last 4 hidden
layers in [CLS]
768
GPT
Last hidden layer
768
GPT2
Last hidden layer
768
Table 2: Word embedding models, used representa-
tions, and their dimensions. 4
Results of experiments p
We examined the 10 different types of biases in
WEAT (Table 1) for word embedding models listed 10 biases in 10 WEAT categories (Table 3). Using
mean of Euclidean, our results confirm all the re-
sults by Caliskan et al. (2017), which used mean
of cosine in all WEAT tests. The difference is that
with the mean of Euclidean measure, the biases are
revealed as being more severe. (smaller p-values). Using mean of Euclidean, GPT and ELMo show
the fewest number of implicit biases. GPT model
shows bias in WEAT 2, 3, and 5. ELMo’s signifi-
cant biases are in WEAT 1, 3, and 6. Using mean
Euclidean, almost all models (except for ELMo)
confirm the existence of a bias in WEAT 3 to 5. Moreover, all contextualized models found no bias
in associating female with arts and male with sci-
ence (WEAT 7), mental diseases with temporary
attributes and physical diseases with permanent at-
tributes (WEAT 9), and young people’s name with
pleasant attribute and old people’s name with un-
pleasant attributes (WEAT 10). For example, in the cosine distance of Word2Vec,
if we change the descriptive statistic from mean to
minimum, the biases for WEAT 3 and WEAT 4 will
become insignificant (no bias will be reported). In
another example, in GPT model, while the result
of mean cosine is not significant for WEAT 3 and
WEAT 4, they become significant for median co-
sine. Moreover, almost for all models, the effect
size of the discretely optimized minimum distance
is not significant. Our intention for considering
this statistic was to report the minimum possible
association of a target word with the attribute sets. If this measure is used for reporting biases, one can
misleadingly claim that there is no bias. Impacts of different similarity measures:
The
effect sizes for cosine, Manhattan, and Euclidean
are closer to each other and greater than the Ma-
halanobis distance (cosine: (µ = 0.72, σ = 0.49),
Euclidean: (µ = 0.67, σ = 0.5), Manhattan:
(µ = 0.63, σ = 0.48), Mahalanobis: (µ = 0.58,
σ = 0.45)). Mahalanobis distance also detects the
fewest number of significant bias types across all
models. As an example, while mean and median
effect sizes for WEAT 3 and WEAT 5 in GloVe
and Word2Vec are mostly significant for cosine,
Euclidean, and Manhattan; the same results are
not significant for the Mahalanobis distance. 4
Results of experiments That
means with the Mahalanobis distance as the mea-
sure of the bias, no bias will be reported for WEAT
3 and WEAT 5 tests. This emphasizes the impor-
tance of chosen similarity measures in detecting
biases of word embeddings. More importantly, as
the Mahalanobis distance considers the distribution
of attributes in measuring the distance, it may be a
better choice than the other similarity measures for
measuring and revealing biases with GPT showing
fewer number of biases. Model
mean cosine mean Euc mean Maha Maha Eq.2
GloVe
9
9
3
0
(1.39, 0.21)
(1.41, 0.2)
(0.79, 0.53) (0.34, 0.27)
Word2Vec
7
7
5
0
(1.13, 0.54)
(1.13, 0.55) (0.84, 0.52) (0.32, 0.33)
ELMo
3
3
3
0
(0.64, 0.51)
(0,65, 0.52) (0.61, 0.42) (0.36, 0.23)
BERT
5
5
2
2
(0.74, 0.5)
(0.74, 0.48)
(0.47, 0.5)
(0.55, 0.52)
GPT
2
3
4
0
(0.61, 0.48)
(0.65, 0.42) (0.59, 0.35) (0.29, 0.27)
GPT2
3
4
3
3
(0.73, 0.46)
(0.71, 0.46) (0.69, 0.49) (0.66, 0.49)
Table 3: Number of revealed biases out of the 10
WEAT bias types for the studied word embeddings
along with the (µ, σ) of their effect sizes. The larger
the effect size the more severe the bias. 5
Conclusions We studied the impacts of different descriptive
statistics and similarity measures on association
tests for measuring biases in contextualized and
non-contextualized word embeddings. Our find-
ings demonstrate that the detected biases depend
on the choice of association measure. Based on
our experiments, mean reveals more severe biases
and the discretely optimized version reveals fewer
number of severe biases. In addition, cosine dis-
tance reveals more severe biases and the Maha-
lanobis distance reveals less severe ones. Report-
ing biases with mean of Euclidean/Mahalanobis
distances identifies more/less severe biases in the
models. Furthermore, contextual models show less
biases than the non-contextual ones across all 10
WEAT tests with GPT showing the fewest number
of biases. Biases in different word embedding models:
Using any combination of descriptive statistics and
similarity measures, all the contextualized mod-
els have less significant biases than GloVe and
Word2Vec. In Table 3 the number of tests with
significant implicit biases out of the 10 WEAT tests
along with the mean and standard deviation of the
effect sizes for all embedding models have been
reported. The complete list of effect sizes along
with their p-value are provided in Table 4. Following our findings in the previous sections,
we choose mean of Euclidean to reveal biases. 5
Conclusions By
doing so, GloVe and Word2Vec show the most num-
ber of significant biases with 9 and 7 significant 11 Cosine
Euclidean
Manhattan
Mahalanobis
Model
WEAT
Mean
Median
Min
Max
Eq.2
Mean
Median
Min
Max
Eq.2
Mean
Median
Min
Max
Eq.2
Mean
Median
Min
Max
Eq.2
GloVe
1
1.50∗∗∗∗
1.34∗∗∗∗
1.35∗∗∗
1.41∗∗∗∗
0.27
1.52∗∗∗∗
1.47∗∗∗∗
1.31∗∗∗∗
1.23∗∗∗∗
0.03
1.50∗∗∗∗
1.46∗∗∗∗
1.32∗∗∗∗
0.90∗∗
0.15
1.53∗∗∗∗
1.54∗∗∗∗
1.19∗∗∗∗
1.61∗∗∗∗
0.00
2
1.53∗∗∗∗
1.37∗∗∗∗
0.83∗
1.57∗∗∗∗
0.08
1.53∗∗∗∗
1.42∗∗∗∗
1.42∗∗∗∗
0.03
0.13
1.51∗∗∗∗
1.43∗∗∗∗
1.44∗∗∗∗
0.27
0.24
1.61∗∗∗∗
1.63∗∗∗∗
1.49∗∗∗∗
0.98∗∗∗
0.28
3
1.41∗∗∗∗
1.13∗∗∗∗
1.53∗∗∗∗
1.41∗∗∗∗
0.60∗
1.37∗∗∗∗
0.98∗∗∗∗
1.51∗∗∗∗
0.09
0.31
0.82∗∗
0.37
1.24∗∗∗∗
0.69∗
0.21
0.57
0.66∗
0.37
0.89∗∗
0.13
4
1.50∗∗∗∗
1.02∗
1.55∗∗∗∗
1.47∗∗∗∗
0.17
1.51∗∗∗∗
0.40
1.58∗∗∗∗
0.32
0.06
0.93∗
0.36
1.14∗∗∗
0.80∗
0.05
0.30
0.57
0.04
0.67
0.31
5
1.28∗∗∗
1.39∗∗∗
0.45
1.29∗∗∗
0.57
1.30∗∗∗∗
1.62∗∗∗∗
1.13∗∗
0.36
0.61
0.54
1.03∗
0.17
0.11
0.37
0.17
0.36
0.01
0.69
0.35
6
1.81∗∗∗
1.83∗∗∗
1.70∗∗∗
1.67∗∗∗
1.01
1.80∗∗∗∗
1.75∗∗∗∗
1.75∗∗∗∗
1.56∗∗∗
0.17
1.78∗∗∗∗
1.78∗∗∗∗
1.71∗∗∗∗
1.46∗∗
0.86
1.17∗
0.83
1.27∗
0.60
0.43
7
1.06
0.85
0.61
1.05
0.18
1.10
0.65
0.26
0.70
0.16
0.70
0.03
0.55
0.63
0.50
0.20
0.80
0.02
0.23
0.10
8
1.24∗
0.93
1.29∗
1.16∗
0.36
1.23∗
1.07
1.12
0.92
0.21
1.03
0.81
0.99
0.83
0.13
0.92
0.71
0.86
0.26
0.26
9
1.38∗
0.83
0.37
1.47∗
1.03
1.47∗
1.04
1.20
1.32∗
0.90
1.50∗
0.26
1.18
1.42∗
0.61
0.99
0.93
1.20
0.55
0.85
10
1.21∗
1.05
1.01
0.75
0.99
1.26∗
1.42∗
0.84
0.64
0.41
0.70
0.90
0.34
0.46
0.25
0.47
0.83
0.45
0.60
0.71
word2vec
1
1.54∗∗∗∗
1.34∗∗∗∗
0.55
1.49∗∗∗∗
0.16
1.50∗∗∗∗
1.30∗∗∗∗
1.31∗∗∗∗
0.95∗∗
0.31
1.49∗∗∗∗
1.34∗∗∗∗
1.38∗∗∗∗
0.75∗
0.26
0.84∗
1.06∗∗∗
0.79∗
0.34
0.13
2
1.63∗∗∗∗
1.49∗∗∗∗
1.19∗∗∗∗
1.60∗∗∗∗
0.22
1.58∗∗∗∗
1.36∗∗∗∗
1.37∗∗∗∗
0.68∗
0.10
1.44∗∗∗∗
1.24∗∗∗∗
1.19∗∗∗∗
0.70∗
0.36
1.39∗∗∗∗
0.99∗∗∗
0.39
0.15
0.05
3
0.58∗
0.46
0.10
0.81∗∗
0.38
0.78∗∗
0.46
0.82∗∗
0.62∗
0.19
0.82∗∗
0.56
0.68∗
0.63∗
0.17
0.24
0.41
0.98∗∗∗∗
0.68∗
0.19
4
1.31∗∗∗∗
1.21∗∗∗
0.44
1.27∗∗∗∗
0.09
1.49∗∗∗∗
0.80∗
1.66∗∗∗∗
0.60
0.35
1.44∗∗∗∗
1.13∗∗∗
1.37∗∗∗
0.55
0.86∗
0.55
0.16
1.30∗∗∗∗
0.49
0.28
5
0.72
0.68
0.58
0.41
0.19
0.43
0.38
0.41
0.08
0.25
0.27
0.23
0.11
0.05
0.09
0.02
0.61
0.11
0.12
0.24
6
1.89∗∗∗
1.87∗∗∗
1.76∗∗∗
1.65∗∗∗
0.91
1.88∗∗∗∗
1.88∗∗∗∗
1.63∗∗
1.70∗∗∗∗
0.85
1.89∗∗∗∗
1.87∗∗∗∗
1.39∗
1.76∗∗∗∗
0.39
1.21∗
0.24
1.49∗∗
0.29
0.01
7
0.97
0.98
0.52
0.71
0.67
0.92
0.45
1.11∗
1.27∗
0.70
1.06
0.87
1.04
1.27∗
1.29∗
0.97
0.90
0.55
1.35∗
0.08
8
1.24∗
1.23∗
1.18∗
0.99
0.59
1.25∗
1.09
1.21∗
1.49∗∗
0.60
1.47∗
1.36∗
1.33∗
1.67∗∗∗
0.00
0.40
0.30
0.48
0.52
0.88
9
1.30∗
0.69
0.14
1.31
0.42
1.32∗
1.18
1.07
0.92
0.55
1.08
0.92
0.92
0.46
0.09
1.55∗∗∗∗
1.23
0.59
0.41
0.94
10
0.09
0.01
0.19
0.66
0.76
0.15
0.01
0.39
0.14
0.43
0.24
0.12
0.36
0.34
0.05
1.20∗
1.24∗
1.60∗∗∗
0.03
0.44
ELMo
1
1.25∗∗∗∗
1.15∗∗∗
0.77∗
0.68∗
0.03
1.25∗∗∗∗
1.03∗∗∗
0.51
0.35
0.48
1.24∗∗∗∗
1.04∗∗∗
0.50
0.27
0.19
0.28
0.17
0.28
0.26
0.57
2
1.46∗∗∗∗
1.37∗∗∗∗
0.87∗∗
1.37∗∗∗∗
0.08
1.46∗∗∗∗
1.28∗∗∗∗
1.14∗∗∗∗
0.71∗
0.51
1.50∗∗∗∗
1.22∗∗∗∗
1.25∗∗∗∗
0.75∗
0.27
0.67∗
0.11
0.79∗
0.11
0.15
3
0.19
0.19
0.06
0.10
0.30
0.12
0.30
0.20
0.06
0.14
0.16
0.02
0.15
0.12
0.19
0.24
0.29
0.37
0.07
0.27
4
0.29
0.22
0.66
0.44
0.02
0.39
0.07
0.03
0.35
0.19
0.39
0.00
0.02
0.35
0.34
0.33
0.29
0.25
0.08
0.48
5
0.11
0.01
0.27
0.57
0.43
0.14
0.12
0.46
0.14
0.09
0.03
0.04
0.55
0.20
0.85∗
0.40
0.04
0.71
0.52
0.34
6
1.24∗
0.95
0.61
0.10
1.00
1.27∗
0.50
0.02
0.44
0.53
0.30
0.59
0.51
0.20
0.49
1.34∗
1.10
0.06
0.50
0.22
7
0.32
0.30
0.56
0.25
0.81
0.29
0.48
0.02
0.62
0.81
0.24
0.25
0.41
0.36
0.03
1.34∗
1.49∗∗
0.72
0.95
0.88
8
0.28
0.42
0.00
0.38
0.29
0.37
0.14
0.14
0.86
0.66
0.64
0.38
0.61
0.99
0.35
0.18
1.06
0.15
0.06
0.15
9
0.91
0.24
0.67
1.28∗
0.68
0.93
0.59
1.04
0.69
0.10
1.06
0.65
0.98
0.77
0.38
0.71
0.55
0.94
0.23
0.38
10
0.37
0.81
0.53
0.56
0.23
0.33
0.93
0.36
0.13
0.62
0.28
0.74
0.49
0.06
0.62
0.61
0.73
0.26
0.48
0.20
BERT
1
0.00
0.21
0.12
0.05
0.72∗
0.01
0.21
0.09
0.11
0.18
0.02
0.32
0.05
0.18
0.46
0.10
0.11
0.24
0.27
0.06
2
0.62
0.39
0.90∗∗
0.55
0.32
0.63
0.45
0.56
1.02∗∗
0.24
0.58
0.45
0.23
0.79∗
0.27
1.31∗∗∗∗
1.33∗∗∗∗
1.35∗∗∗∗
1.25∗∗∗∗
1.21∗∗∗∗
3
1.04∗∗∗
1.02∗∗∗
0.75∗
0.83∗∗
0.58∗
1.05∗∗∗∗
1.07∗∗∗∗
0.77∗∗
0.72∗
0.21
1.04∗∗∗∗
1.09∗∗∗∗
0.82∗∗
0.76∗∗
0.01
0.24
0.30
0.23
0.29
0.17
4
1.19∗∗∗
1.19∗∗∗
1.06∗∗∗
1.08∗∗
0.26
1.23∗∗∗
0.97∗
1.00∗∗
1.16∗∗∗∗
0.65
1.20∗∗∗
1.07∗∗
0.88∗
1.10∗∗∗
0.13
0.27
0.31
0.44
0.17
0.05
5
0.94∗
0.93∗
0.30
0.77∗
0.06
0.88∗
0.95∗
0.58
0.11
0.41
0.85∗
0.98∗
0.71
0.02
0.45
0.29
0.23
0.04
0.34
0.19
6
1.36∗
1.20∗
1.32∗
0.13
0.22
1.30∗
1.15∗
0.20
1.45∗∗
0.96
1.12
0.83
0.16
1.34∗
0.82
0.03
0.15
0.24
0.61
0.38
7
1.14∗
0.85
0.75
1.01
0.07
1.18∗
0.75
1.03
0.95∗
0.40
1.20∗
0.90
1.09
1.02∗
0.85
0.29
0.09
0.49
0.18
0.77
8
0.24
0.37
0.11
0.55
0.17
0.24
0.02
0.50
0.73
0.37
0.12
0.14
0.13
0.34
0.04
0.47
0.42
0.61
0.27
0.60
9
0.02
0.16
0.03
1.04
0.69
0.12
0.32
0.97
0.00
0.25
0.17
0.34
0.72
0.16
0.66
1.48∗
1.38∗
1.52∗
1.54∗
1.61∗
10
0.83
0.76
0.89
0.50
1.28∗
0.80
0.89
0.40
0.90
0.22
0.91
1.16∗
0.57
0.72
0.53
0.24
0.28
0.09
0.54
0.47
GPT
1
0.47
0.29
0.57
0.08
0.24
0.58
0.39
0.10
0.10
0.50
0.57
0.25
0.11
0.01
0.10
0.40
0.00
0.54
0.45
0.13
2
1.11∗∗∗
0.99∗∗
0.94∗∗
0.53
0.38
1.15∗∗∗∗
0.74∗
0.13
0.23
0.01
1.16∗∗∗∗
0.82∗
0.01
0.16
0.28
0.84∗∗
0.69∗
0.06
1.01∗∗
0.05
3
0.09
0.97∗∗∗
0.64∗
1.24∗∗∗∗
0.20
0.70∗
0.99∗∗∗
1.21∗∗∗∗
0.27
0.02
0.06
1.05∗∗∗∗
1.17∗∗∗∗
0.56
0.01
0.69∗
0.97∗∗∗
1.24∗∗∗∗
0.90∗∗∗
0.13
4
0.33
1.54∗∗∗∗
0.88∗
1.48∗∗∗∗
0.30
0.51
1.51∗∗∗∗
1.37∗∗∗∗
0.28
0.10
0.31
1.52∗∗∗∗
1.33∗∗∗∗
0.50
0.29
0.91∗∗
1.36∗∗∗∗
1.26∗∗∗
1.07∗∗∗
0.42
5
1.65∗∗∗∗
1.40∗∗∗∗
1.57∗∗∗∗
1.58∗∗∗∗
0.69
1.57∗∗∗∗
1.14∗∗∗∗
1.49∗∗∗∗
1.65∗∗∗∗
0.26
1.54∗∗∗∗
1.23∗∗∗∗
1.49∗∗∗∗
1.50∗∗∗∗
0.57
1.26∗∗∗∗
1.23∗∗∗∗
0.98∗∗∗
1.40∗∗∗∗
0.06
6
0.67
0.02
0.75
0.89
0.20
0.50
0.25
0.23
0.66
0.08
0.49
0.04
0.34
0.45
0.09
0.66
0.01
1.14∗
0.27
0.19
7
0.24
0.11
0.02
0.09
0.70
0.20
0.30
0.00
0.15
0.45
0.28
0.16
0.32
0.03
0.18
0.29
0.63
0.22
0.57
0.06
8
0.10
0.16
0.35
0.13
0.40
0.08
0.10
0.32
0.03
0.93∗
0.12
0.14
0.37
0.08
0.47
0.19
0.13
0.22
0.58
0.43
9
0.70
0.92
1.01
0.01
0.18
0.58
0.63
0.17
0.07
0.42
0.59
0.62
0.40
0.01
0.44
0.50
0.59
0.66
0.03
0.62
10
0.72
0.39
0.73
0.68
0.67
0.61
0.25
0.55
1.03
0.27
0.52
0.34
0.48
0.76
0.77
0.19
0.17
0.07
0.27
0.81
GPT2
1
0.11
0.06
0.20
0.20
0.19
0.06
0.21
0.14
0.04
0.01
0.08
0.05
0.05
0.07
0.04
0.27
0.27
0.26
0.23
0.28
2
0.64
0.28
0.50
0.24
0.04
0.51
0.63
0.21
0.61∗
0.02
0.47
0.69∗
0.18
0.53
0.56
0.44
0.45
0.45
0.41
0.34
3
1.27∗∗∗∗
0.70∗
1.07∗∗∗∗
1.15∗∗∗∗
0.30
1.25∗∗∗∗
1.21∗∗∗∗
0.29
1.30∗∗∗∗
0.48
1.34∗∗∗∗
1.24∗∗∗∗
0.39
1.12∗∗∗∗
0.11
1.25∗∗∗∗
1.25∗∗∗∗
1.27∗∗∗∗
1.22∗∗∗∗
1.24∗∗∗∗
4
1.19∗∗
0.64
1.28∗∗∗
0.83∗
0.56
1.17∗∗∗
1.24∗∗∗∗
0.39
1.13∗∗
0.57
1.17∗∗∗
1.10∗∗
0.28
1.05∗∗
0.44
1.29∗∗∗∗
1.29∗∗∗∗
1.28∗∗∗∗
1.28∗∗∗
1.31∗∗∗∗
5
1.17∗∗
1.15∗∗
1.31∗∗∗
1.02∗∗
0.06
1.17∗∗∗
1.21∗∗∗∗
0.92∗
1.13∗∗
0.77∗
1.14∗∗
1.18∗∗∗
0.13
1.15∗∗
0.42
1.29∗∗∗
1.29∗∗∗∗
1.28∗∗∗∗
1.30∗∗∗∗
1.29∗∗∗∗
6
0.79
1.06
0.94
0.11
0.20
0.86
0.90
0.28
0.94
0.54
0.66
0.74
0.19
0.11
0.63
0.94
0.55
0.52
0.89
0.71
7
0.03
0.49
0.21
0.69
0.17
0.12
0.10
0.24
0.00
0.07
0.23
0.42
0.34
0.01
0.16
0.16
0.17
0.16
0.16
0.14
8
0.42
1.08
0.79
0.42
0.36
0.44
0.37
0.08
0.59
0.32
0.30
0.78
0.22
0.21
0.23
0.36
0.35
0.36
0.37
0.38
9
0.57
0.68
0.16
0.17
0.73
0.36
0.41
0.39
0.64
0.24
0.04
0.10
1.19
0.32
0.05
0.06
0.05
0.14
0.21
0.04
10
1.14
1.12
0.77
0.63
0.03
1.13∗
1.11
0.09
1.12
0.14
1.12
1.12
0.24
1.13
0.71
0.80
0.83
0.82
0.65
0.86
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and the implicit association test. Journal of person-
ality and social psychology, 81(5):774. A
The studied associations: 10 WEAT categories A
The studied associations: 10 WEAT categories
WEAT
Association
NT
NA
1
Flowers vs insects with pleasant vs unpleasant
Example: {aster, clover} vs {ant, caterpillar} with {caress, freedom} vs { abuse, crash}
25 × 2
25 × 2
2
Instruments vs weapons with pleasant vs unpleasant
Example: {bagpipe, cello} vs {arrow, club} with {caress, freedom} vs { abuse, crash}
25 × 2
25 × 2
3
Eur.-American vs Afr.-American names with Pleasant vs unpleasant
Example: {Adam, Harry} vs {Alonzo, Jamel} with {caress, freedom} vs {abuse, crash}
32 × 2
25 × 2
4
Eur.-American vs Afr.-American names with Pleasant vs unpleasant
Example: {Brad, Brendan} vs {Darnell, Hakim} with {caress, freedom} vs {abuse, crash}
16 × 2
25 × 2
5
Eur.-American vs Afr.-American names with Pleasant vs unpleasant
Example: {Brad, Brendan} vs {Darnell, Hakim} with {joy, love} vs {agony, terrible}
16 × 2
8 × 2
6
Male vs female names with Career vs family
Example: {John, Paul} vs {Amy, Joan} with {executive, management} vs {home, parents}
8 × 2
8 × 2
7
Math vs arts with male vs female terms
Example: {math, algebra} vs {poetry, art} with {male, man} vs {female, woman}
8 × 2
8 × 2
8
Science vs arts with male vs female terms
Example: {science, technology} vs {art, Shakespeare} with {brother, father} vs {sister, mother}
8 × 2
8 × 2
9
Mental vs physical disease with temporary vs permanent
Example: {sad, hopeless} vs {sick, illness} with {impermanent, unstable} vs {stable, always}
6 × 2
7 × 2
10
Young vs old people’s name with pleasant vs unpleasant
Example: {Tiffany, Michelle} vs {Ethel, Bernice} with {love, peace} vs {agony, terrible}
8 × 2
8 × 2 References 13 A
The studied associations: 10 WEAT categories
WEAT
Association
NT
NA
1
Flowers vs insects with pleasant vs unpleasant
Example: {aster, clover} vs {ant, caterpillar} with {caress, freedom} vs { abuse, crash}
25 × 2
25 × 2
2
Instruments vs weapons with pleasant vs unpleasant
Example: {bagpipe, cello} vs {arrow, club} with {caress, freedom} vs { abuse, crash}
25 × 2
25 × 2
3
Eur.-American vs Afr.-American names with Pleasant vs unpleasant
Example: {Adam, Harry} vs {Alonzo, Jamel} with {caress, freedom} vs {abuse, crash}
32 × 2
25 × 2
4
Eur.-American vs Afr.-American names with Pleasant vs unpleasant
Example: {Brad, Brendan} vs {Darnell, Hakim} with {caress, freedom} vs {abuse, crash}
16 × 2
25 × 2
5
Eur.-American vs Afr.-American names with Pleasant vs unpleasant
Example: {Brad, Brendan} vs {Darnell, Hakim} with {joy, love} vs {agony, terrible}
16 × 2
8 × 2
6
Male vs female names with Career vs family
Example: {John, Paul} vs {Amy, Joan} with {executive, management} vs {home, parents}
8 × 2
8 × 2
7
Math vs arts with male vs female terms
Example: {math, algebra} vs {poetry, art} with {male, man} vs {female, woman}
8 × 2
8 × 2
8
Science vs arts with male vs female terms
Example: {science, technology} vs {art, Shakespeare} with {brother, father} vs {sister, mother}
8 × 2
8 × 2
9
Mental vs physical disease with temporary vs permanent
Example: {sad, hopeless} vs {sick, illness} with {impermanent, unstable} vs {stable, always}
6 × 2
7 × 2
10
Young vs old people’s name with pleasant vs unpleasant
Example: {Tiffany, Michelle} vs {Ethel, Bernice} with {love, peace} vs {agony, terrible}
8 × 2
8 × 2 A
The studied associations: 10 WEAT categories Association Table 1: The 10 WEAT categories. 14 14
|
https://openalex.org/W2999310251
|
https://www.combinatorics.org/ojs/index.php/eljc/article/download/v27i1p10/pdf
|
English
| null |
Ear Decomposition and Balanced Neighborly Simplicial Manifolds
|
The Electronic journal of combinatorics/The journal of combinatorics
| 2,020
|
cc-by
| 9,535
|
Abstract We find the first non-octahedral balanced 2-neighborly 3-sphere and the bal-
anced 2-neighborly triangulation of the lens space L(3, 1). Each construction has
16 vertices. We show that there exists a balanced 3-neighborly non-spherical 5-
manifold with 18 vertices. We also show that the rank-selected subcomplexes of a
balanced simplicial sphere do not necessarily have an ear decomposition. Mathematics Subject Classifications: 05E45 Ear decomposition and balanced
neighborly simplicial manifolds Hailun Zheng
Department of Mathematics
University of Michigan
Ann Arbor, MI, 48109, USA
hailunz@umich.edu Hailun Zheng
Department of Mathematics
University of Michigan
Ann Arbor, MI, 48109, USA
hailunz@umich.edu Submitted: Nov 26, 2018; Accepted: Dec 22, 2019; Published: Jan 10, 2020
c⃝The author. Released under the CC BY-ND license (International 4.0). Submitted: Nov 26, 2018; Accepted: Dec 22, 2019; Published: Jan 10, 2020
c⃝The author. Released under the CC BY-ND license (International 4.0). 1
Introduction A simplicial complex is called k-neighborly if every subset of vertices of size at most
k is the set of vertices of one of its faces. Neighborly complexes, especially neighborly
polytopes and spheres, are interesting objects to study. In the seminal work of McMullen
[12] and Stanley [19], it was shown that in the class of polytopes and simplicial spheres of
a fixed dimension and with a fixed number of vertices, the cyclic polytope simultaneously
maximizes all the face numbers. The d-dimensional cyclic polytope is ⌊d
2⌋-neighborly. Since then, many other classes of neighborly polytopes have been discovered. We refer to
[4], [16] and [18] for examples and constructions of neighborly polytopes. Meanwhile, the
notion of neighborliness was extended to other classes of objects: for instance, neighborly
cubical polytopes were defined and studied in [8], [9], and [17], and neighborly centrally
symmetric polytopes and spheres were studied in [1], [3], [7] and [14]. In this paper we discuss a similar notion for balanced simplicial complexes. Balanced
complexes were defined by Stanley in [20], where they were called completely balanced. A (d−1)-dimensional simplicial complex is called balanced if its graph is d-colorable. For
instance, the barycentric subdivision of regular CW complexes and order complexes are 1 the electronic journal of combinatorics 27(1) (2020), #P1.10 balanced. We say that a balanced simplicial complex is balanced k-neighborly if every set
of k or fewer vertices with distinct colors forms a face. For example, if ∆1 and ∆2 are
balanced k-neighborly spheres, then the join ∆= ∆1 ∗∆2 is also a balanced neighborly
k-sphere, and we call ∆join-decomposable. However, apart from the cross-polytopes,
it is not known whether other join-indecomposable balanced k-neighborly polytopes or
spheres exist. To the best of our knowledge, no examples of such objects appear in the
current literature, even for k = 2. As for balanced 2-neighborly manifolds, one such
construction that triangulates the sphere bundle is given in [11]; it is also a minimal
balanced triangulation of the underlying topological space. This more or less explains why so far there is even no plausible sharp upper bound
conjecture for balanced spheres or manifolds. The goal of this paper is to partially rem-
edy this situation by searching for balanced neighborly spheres and manifolds of lower
dimensions. 1
Introduction It turns out that even in the lower dimensional cases balanced neighborly
spheres or manifolds with a given number of vertices do not always exist. • The octahedral 3-sphere is the only balanced 2-neighborly 3-sphere with less than
16 vertices. • The octahedral 3-sphere is the only balanced 2-neighborly 3-sphere with less than
16 vertices. • There is a unique balanced 2-neighborly 4-sphere with 15 vertices, known as 4155
2 in
[10]. • There is a unique balanced 2-neighborly 4-sphere with 15 vertices, known as 4155
2 in
[10]. • There exists a balanced 3-neighborly non-spherical 5-manifold with 18 vertices • There are two constructions of balanced 2-neighborly 3-manifolds with 16 vertices;
one triangulates the sphere, and the other triangulates the lens space L(3, 1). • There are two constructions of balanced 2-neighborly 3-manifolds with 16 vertices;
one triangulates the sphere, and the other triangulates the lens space L(3, 1). In a different direction, it is also interesting to ask whether every rank-selected sub-
complex of a balanced simplicial polytope or sphere has a convex ear decomposition. This statement, if true, would imply that rank-selected subcomplexes of balanced simpli-
cial polytopes possess certain weak Lefschetz properties, see Theorem 3.9 in [22]. As a
consequence, it would also provide an alternative proof of the balanced Generalized Lower
Bound Theorem, see Theorem 3.3 and Remark 3.4 in [13]. We present an example giving
a negative answer to this question for 3-dimensional spheres. The structure of this manuscript is as follows. In Section 2, after reviewing basic
definitions, we establish basic properties of balanced neighborly spheres; in particular, we
prove that for some values of f0, such spheres cannot exist. In Section 3 we discuss how
to find balanced k-neighborly (2k −1)-manifolds from less neighborly balanced (2k −2)-
spheres, for k = 2, 3. In Section 4, we construct a balanced 2-neighborly 3-sphere with
16 vertices. In Section 5, we present the balanced 2-neighborly triangulation of L(3, 1)
with 16 vertices. In Section 6 we provide a way to construct balanced spheres whose
rank-selected subcomplex does not have an ear decomposition. 2
Basic properties of balanced neighborly spheres A simplicial complex ∆with vertex set V is a collection of subsets σ ⊆V , called faces,
that is closed under inclusion, and such that for every v ∈V , {v} ∈∆. For σ ∈∆, let the electronic journal of combinatorics 27(1) (2020), #P1.10 2 dim σ := |σ|−1 and define the dimension of ∆, dim ∆, as the maximum dimension of the
faces of ∆. A facet is a maximal face under inclusion. We say that a simplicial complex
∆is pure if all of its facets have the same dimension. If ∆is a simplicial complex and σ is a face of ∆, the star of σ in ∆is st∆σ := {τ ∈
∆: σ ∪τ ∈∆}. We also define the link of σ in ∆as lk∆σ := {τ −σ ∈∆: σ ⊆τ ∈∆},
and the deletion of a subset of vertices W from ∆as ∆\W := {σ ∈∆: σ ∩W = ∅}. If
∆1 and ∆2 are simplicial complexes on disjoint vertex sets, then the join of ∆1 and ∆2,
denoted ∆1 ∗∆2, is the simplicial complex with vertex set V (∆1) ∪V (∆2) whose faces
are {σ1 ∪σ2 : σ1 ∈∆1, σ2 ∈∆2}. If ∆is a pure (d −1)-dimensional complex such that every (d −2)-dimensional face
of ∆is contained in at most two facets, then the boundary complex of ∆consists of all
(d −2)-dimensional faces that are contained in exactly one facet, as well as their subsets. A simplicial complex ∆is a simplicial sphere (resp. simplicial ball) if the geometric
realization of ∆is homeomorphic to a sphere (resp. ball). The boundary complex of a
simplicial d-ball is a simplicial (d −1)-sphere. A simplicial sphere is called polytopal if it
is the boundary complex of a convex polytope. For instance, the boundary complex of an
octahedron is a polytopal sphere; we will refer to it as an octahedral sphere. For a fixed field or group k, we say that ∆is a (d −1)-dimensional k-homology sphere
if ˜Hi(lk∆σ; k) ∼= ˜Hi(Sd−1−|σ|; k) for every face σ ∈∆(including the empty face) and
i ⩾−1. A homology d-ball (over k) is a d-dimensional simplicial complex ∆such that (i)
∆has the same homology as the d-dimensional ball, (ii) for every face F, the link of F
has the same homology as the (d−|F|)-dimensional ball or sphere, and (iii) the boundary
complex ∂∆is a homology (d −1)-sphere. 2
Basic properties of balanced neighborly spheres The classes of simplicial (d −1)-spheres and
homology (d −1)-spheres coincide when d ⩽3. From now on all homology are computed
with coefficients in Z and we will omit it from our notation. Next we define a special structure that exists in some pure simplicial complexes. Definition 1. An ear decomposition of a pure (d −1)-dimensional simplicial complex ∆
is an ordered sequence ∆1, ∆2, · · · , ∆m of pure (d −1)-dimensional subcomplexes of ∆
such that: 1. ∆1 is a simplicial (d −1)-sphere, and for each j = 2, 3, · · · , m, ∆j is a simplicial
(d −1)-ball. 1. ∆1 is a simplicial (d −1)-sphere, and for each j = 2, 3, · · · , m, ∆j is a simplicial
(d −1)-ball. 2. For 2 ⩽j ⩽m, ∆j ∩(∪j−1
i=1∆i) = ∂∆j. 3. ∪m
i=1∆i = ∆. We call ∆1 the initial complex, and each ∆j, j ⩾2, an ear of this decompostion. No-
tice that this definition is more general than Chari’s original definition of a convex ear
decomposition, see [2, Section 3.2], where the ∆i’s are required to be subcomplexes of
the boundary complexes of polytopes. In particular, if a complex has no ear decompo-
sition, then it has no convex ear decomposition. However, by the Steinitz theorem, all
simplicial 2-spheres are polytopal, and hence also all simplicial 2-balls can be realized as
subcomplexes of the boundary complexes of 3-dimensional polytopes. So for 2-dimensional 3 the electronic journal of combinatorics 27(1) (2020), #P1.10 simplicial complexes, the notion of an ear decomposition coincides with that of a convex
ear decomposition. A (d −1)-dimensional simplicial complex ∆is called balanced if the graph of ∆is
d-colorable, or equivalently, there is a coloring map κ : V →[d] such that κ(x) ̸= κ(y)
for any edge {x, y} ∈∆. Here [d] = {1, 2, · · · , d} is the set of colors. We denote by
Vi the set of vertices of color i. A balanced simplicial complex is called balanced k-
neighborly if every set of k or fewer vertices with distinct colors forms a face. We say e
is a missing colored edge if e /∈∆and the vertices of e have distinct colors. For S ⊆[d],
the subcomplex ∆S := {F ∈∆: κ(F) ⊆S} is called the rank-selected subcomplex of ∆. 2
Basic properties of balanced neighborly spheres We also define the flag f-vector (fS(∆) : S ⊆[d]) and the flag h-vector (hS(∆) : S ⊆[d])
of ∆, respectively, by letting fS(∆) := #{F ∈∆: κ(F) = S}, where f∅(∆) = 1, and
hS(∆) := P
T⊆S(−1)#S−#TfT(∆). The usual f-numbers and h-numbers can be recovered
from the relations fi−1(∆) = P
#S=i fS(∆) and hi(∆) = P
#S=i hS(∆). #
#
In the remainder of this section, we establish some restrictions on the possible size of
color sets of balanced neighborly spheres. Lemma 2. Let ∆be a balanced k-neighborly homology (2k −1)-sphere. Then ∆has the
same number of vertices of each color. In particular, f0(∆) = 2kℓfor some ℓ⩾2. Proof. Let W ⊆[2k] be an arbitrary subset of the set of the colors with |W| = k. Since
∆is balanced k-neighborly, ∆W is also balanced k-neighborly, and hence ∆W is the join
of k color sets of colors in W, each considered as a 0-dimensional complex. By the
definition of the join and the flag h-numbers, we have fU∪{i}(∆) = fU(∆)f{i}(∆) and
hence hU∪{i}(∆) = hU(∆)h{i}(∆) for all i ∈W, U ⊂W and i /∈U. Therefore, Y
i∈W
(|Vi| −1) =
Y
i∈W
h{i}(∆) = hW(∆)
(∗)
= h[2k]\W(∆) =
Y
i∈[2k]\W
h{i}(∆) =
Y
i∈[2k]\W
(|Vi| −1), where (∗) follows from the Dehn-Sommerville relations. Since W is an arbitrary k-subset
of [2k], it follows that each color set in ∆must have the same size. Remark 3. Lemma 2 not only holds for homology (2k −1)-spheres but also for orientable
homology (2k −1)-manifolds. Indeed by replacing the flag h-numbers with the flag h′′-
numbers (see [6] for definition), Theorem 4.1 in [6] gives h′′
W(∆) = h′′
[2k]\W(∆), which
further implies that hW(∆) = h[2k]\W(∆) since both W and [2k]\W are of size k. The
rest of the proof is the same. Unfortunately, the above lemma is not sufficient to tell whether a balanced k-neighborly
homology (2k −1)-sphere or manifold with 2kℓvertices can exist for given k, ℓ⩾2. 3
Balanced neighborly (d −1)-manifolds with 3d vertices In this section, we consider balanced ⌊d
2⌋-neighborly (d −1)-manifolds (for d = 3, 4, 5)
with each color set of size 3. We begin with the following lemma. In this section, we consider balanced ⌊d
2⌋-neighborly (d −1)-manifolds (for d = 3, 4, 5)
with each color set of size 3. We begin with the following lemma. 4 4 the electronic journal of combinatorics 27(1) (2020), #P1.10 w3
z3
u2
w2
z2
u3
z1
w1
u1
u3
w3
z3
z2
u2
w2
z1
u1
w1
Figure 1: Left: triangulation of the vertex link lk∆vi for vi ∈V4, where {u1, u2, u3},
{w1, w2, w3} and {z1, z2, z3} are the three other color sets. Right: the complex Σ. w3
z3
u2
w2
z2
u3
z1
w1
u1 u3
w3
z3
z2
u2
w2
z1
u1
w1 u3 u3 Figure 1: Left: triangulation of the vertex link lk∆vi for vi ∈V4, where {u1, u2, u3},
{w1, w2, w3} and {z1, z2, z3} are the three other color sets. Right: the complex Σ. Lemma 4. Let d ⩾4. If ∆is a balanced homology (d −1)-sphere and Vd = {v1, v2, v3}
is the set of vertices of color d, then lk∆vi ∩lk∆vj is a homology (d −2)-ball for any
1 ⩽i < j ⩽3, and ∩3
k=1 lk∆vk is a homology (d −3)-sphere. Lemma 4. Let d ⩾4. If ∆is a balanced homology (d −1)-sphere and Vd = {v1, v2, v3}
is the set of vertices of color d, then lk∆vi ∩lk∆vj is a homology (d −2)-ball for any
1 ⩽i < j ⩽3, and ∩3
k=1 lk∆vk is a homology (d −3)-sphere. Proof. Let {i, j, k} = [3] be distinct, Σ = lk∆vi ∩lk∆vj and Γ = ∩3
k=1 lk∆vk. We first
prove that Σ and Γ have the same homology as a simplicial (d −2)-ball and simplicial
(d −3)-sphere respectively. Since each (d −2)-face of ∆is contained in exactly 2 facets,
it follows that lk∆vi ∪lk∆vj = ∆[d−1]. By the Mayer-Vietoris sequence, for any n ⩾0, · · →Hn+1(∆[d−1]) →Hn(Σ) →Hn(lk∆vi) ⊕Hn(lk∆vj) →Hn(∆[d−1]) →· · · . ( (1) Note that ∆[d−1] is a deformation retract of ∆minus three points, hence βd−2(∆[d−1]) = 2
and βk(∆[d−1]) = 0 for 0 ⩽k ⩽d −3. We conclude from (1) that βk(Σ) = 0 for all k ⩾0. 3
Balanced neighborly (d −1)-manifolds with 3d vertices Hence all vertex links in ∆are combinatorially
isomorphic. p
Since fi(∆[3]) = fi(∆)−P3
j=1 fi−1(lk∆vj) for all i ⩽2, we have f(∆[3]) = (1, 9, 27, 21). Let i, j, k ∈[3] be distinct and Σ := lk∆vi∩lk∆vj. Any facet F of Σ are 2-dimensional, for
otherwise if F is an edge, then lk∆F is either a 4-cycle or 6-cycle, where in both cases vi
and vj share at least one common neighbor w, i.e., F ∪{w} ∈Σ, a contradiction. Similarly,
the facet cannot be 0-dimensional. Also the facets of Σ do not belong to lk∆vk. Hence Σ
is a pure 2-dimensional subcomplex of lk∆vi with 9 vertices and f2(∆[3]) −f2(lk∆vk) = 7
triangles. On the other hand, for any u ∈Σ, the vertex link lk∆u is isomorphic to Figure 1 and
vi, vj ∈lk∆u. The intersection of links of arbitrary two vertices of the same color has the
following property (∗):
lkΣ u = lklk∆u vi ∩lklk∆u vj is either an edge, or a path of length 3. Since Σ is 2-dimensional, each connected component of Σ has at least 3 vertices. If
there are three components, then Σ is the disjoint union of three triangles, contradicting
that f2(Σ) = 7. Otherwise, if Σ is connected, then by observation (∗) we have that Σ is
a triangulated 2-manifold (with boundary). Since 21 = 3f2(Σ) = P
u∈Σ f1(lkΣ u) and by
observation (∗) f1(lkΣ u) ∈{1, 3}, the links of three vertices in Σ are single edges, while
the rest are paths of length 3. However, enumeration based on observation (∗) yields that
there is no such complex Σ. The last case is that Σ has two connected components. From observation (∗) we
see that each component cannot have 4 or 5 vertices. If one component is the triangle,
then the other component (as a 6-triangle subcomplex of the 9-vertex balanced 2-sphere)
must be the triangulated annulus as shown in Figure 1. By symmetry lk∆vi ∩lk∆vk
and lk∆vj ∩lk∆vk are also isomorphic to Σ. In this way we determine ∆[3] = lk∆vi ∪
lk∆vj: it is the union of three octahedral 2-spheres, each having a pair of antipodal facets
(F1, F2), (F2, F3) and (F3, F1), respectively. This also determines ∆, which is the balanced
triangulation of the nonorientable S2-bundle over S1 known as 31283
2 in [10]. Remark 7. 3
Balanced neighborly (d −1)-manifolds with 3d vertices Since lk∆vk ∪Σ = ∆[d−1] and lk∆vk ∩Σ = Γ, by the Mayer-Vietoris sequence we obtain Note that ∆[d−1] is a deformation retract of ∆minus three points, hence βd−2(∆[d−1]) = 2
and βk(∆[d−1]) = 0 for 0 ⩽k ⩽d −3. We conclude from (1) that βk(Σ) = 0 for all k ⩾0. Since lk∆vk ∪Σ = ∆[d−1] and lk∆vk ∩Σ = Γ, by the Mayer-Vietoris sequence we obtain · · · →Hn+1(∆[d−1]) →Hn(Γ) →Hn(lk∆vk) ⊕Hn(Σ) →Hn(∆[d−1]) →· · · . Hence βd−3(Γ) = 1 and βk(Γ) = 0 for 0 ⩽k ⩽d −4. βd
3( )
βk( )
⩽
⩽
Next, for any τ ∈Γ, we have lkΣ τ = lklk∆τ vi ∩lklk∆τ vj and lkΓ τ = ∩3
i=1 lklk∆τ vi. Since lk∆τ is a balanced homology (d−1−|τ|)-sphere, using the same argument as above,
we may show that lkΣ τ and lkΓ τ have the same homology as a (d −2 −|τ|)-ball and
(d−3−|τ|)-sphere respectively. Therefore Γ is a homology (d−3)-sphere. Finally, for any
interior face σ of Σ, lkΣ σ = lklk∆vi σ = lklk∆vj σ, and hence lkΣ σ is a homology sphere. By definition we conclude that Σ is a homology (d −2)-ball. Remark 5. The complex Γ in Lemma 4 is not balanced, since Γ is (d−1)-colorable instead
of being (d −2)-colorable. Remark 5. The complex Γ in Lemma 4 is not balanced, since Γ is (d−1)-colorable instead
of being (d −2)-colorable. Proposition 6. The only balanced 2-neighborly 3-manifold with 12 vertices triangulates
the nonorientable S2-bundle over S1. Proof. Let ∆be a balanced 2-neighborly 3-manifold with 12 vertices. Its f-vector is
f(∆) = (1, 12, 54, 84, 42). By Lemma 2 and Remark 3, each color set of ∆has three the electronic journal of combinatorics 27(1) (2020), #P1.10 5 vertices. We let V4 = {v1, v2, v3} be the set of vertices of color 4. Since ∆is balanced
2-neighborly, each lk∆vi is a 2-sphere with 9 vertices, its f-vector is (1,9,21,14). Further-
more, the balancedness of ∆implies that every vertex u ∈lk∆vi has deglk∆vi u = 4 or 6. If x is the number of vertices of degree 6 in lk∆vi, then 4(9 −x) + 6x =
X
u∈lk∆vi
deg(lklk∆vi u) = 2f1(lk∆vi) = 42, and hence x = 3. A balanced 2-sphere with 9 vertices, 3 of which have degree 6, is unique
up to isomorphism, as shown in Figure 1. 3
Balanced neighborly (d −1)-manifolds with 3d vertices The balanced 2-neighborly manifold 31283
2 is also known as BM4 defined in
[11]. In particular in [11, Proposition 6.9] it is shown that 31283
2
is the only balanced
12-vertex 3-manifold with β1 ̸= 0. See [11] and [23] for extension in higher dimensional
cases. Next we characterize all balanced 3-spheres with each color sets of size 3. 6 the electronic journal of combinatorics 27(1) (2020), #P1.10 Lemma 8. Up to an isomorphism, there are four triangulations of balanced 3-spheres
with each color set of size 3. Lemma 8. Up to an isomorphism, there are four triangulations of balanced 3-spheres
with each color set of size 3. Lemma 8. Up to an isomorphism, there are four triangulations of balanced 3-spheres
with each color set of size 3. Proof. Let ∆be such a sphere and let V4 = {v1, v2, v3}. Each vertex link of ∆is a
balanced 2-sphere with at most 9 vertices, hence it is either the octahedral sphere, the
suspension of a 6-cycle, or the connected sum of two octahedral spheres. We denote these
three 2-spheres as Σ1, Σ2 and Σ3 respectively. By Lemma 4, ∆[3] is the union of three
triangulated 2-balls Bi = lk∆vj ∩lk∆vk, where {i, j, k} = [3], glued along their common
boundary complex c. Assume that f0(lk∆vi) ⩽f0(lk∆vj) when i ⩽j. An easy counting
leads to f0(∆[3]) = f0(c) +
3
X
i=1
f0(Bi\c) = 9,
f0(lk∆vi) = f0(c) + f0(Bj\c) + f0(Bk\c) ∈{6, 8, 9},
(2) f0(c) +
3
X
i=1
f0(Bi\c) = 9,
f0(lk∆vi) = f0(c) + f0(Bj\c) + f0(Bk\c) ∈{6, 8, 9},
(2) (2) ( )
where f0(Bi\c) counts the number of interior vertices of Bi. By the Dehn-Sommerville
relations, the f-vector of any triangulated 3-manifold satisfies that f1 = f3 + f0. Since
every facet of ∆contains exactly one vertex from V4, we have that f3(∆) = P3
i=1 f2(lk∆vi)
and hence f1(∆) =
3
X
i=1
f2(lk∆vi) + f0(∆) =
3
X
i=1
(2f0(lk∆vi) −4) + 12 = 2
3
X
i=1
f0(lk∆vi) ⩽54, we enumerate the combinatorial type of each f0(lk∆vi) as follows: Case 1: lk∆v1 ∼= Σ1. It follows that lk∆v3 is obtained from lk∆v2 by a cross flip (see
[5] for a reference). Since f0(∆[3]) = 9, either lk∆v2 ∼= Σ1, lk∆v3 ∼= Σ3, and the cross flip
replaces a 2-face of lk∆v2 with its complement in the octahedral sphere. 3
Balanced neighborly (d −1)-manifolds with 3d vertices Or lk∆v2 ∼= Σ2,
lk∆v3 ∼= Σ3, and the cross flip replaces the union of three 2-faces of lk∆v2 with its
complement in the octahedral sphere. In the first case the 3-sphere is the connected sum
of two octahedral 3-spheres, which we denote as S1. In the second case we obtain a 3-
sphere S2 (with lkS2 vi ∼= Σi for i ∈[3]). Their f-vectors are f(S1) = (1, 12, 42, 60, 30) and
f(S2) = (1, 12, 46, 68, 34). (
)
(
)
Case 2: lk∆v1 ∼= Σ2. In this case the number of missing colored edges in ∆[3] is
9·6
2 −f1(∆[3]) = 27 −P3
i=1 f0(lk∆vi), which equals either 1,2 or 3. 2
[ ]
i
1
Subcase 1: lk∆v2 ∼= lk∆v3 ∼= Σ2. By conditions (2), c is a 6-cycle and ∆[3]\c consists
of three disjoint vertices of degree either 4 or 6. Note that in Σ2 every pair vertices of
degree 4 and degree 6 forms an edge. Since ∆[3] has only three missing edges between
vertices of different colors, the vertices in ∆[3]\c are of the same color and has degree 6. Hence ∆[3] is the join of c and three disjoint vertices and ∆is the join of two 6-cycles. Denote this sphere as S3; its f-vector is (1, 12, 48, 72, 36). 2
[ ]
i
Subcase 1: lk∆v2 ∼= lk∆v3 ∼= Σ2. By conditions (2), c is a 6-cycle and ∆[3]\c consists
of three disjoint vertices of degree either 4 or 6. Note that in Σ2 every pair vertices of
degree 4 and degree 6 forms an edge. Since ∆[3] has only three missing edges between
vertices of different colors, the vertices in ∆[3]\c are of the same color and has degree 6. Hence ∆[3] is the join of c and three disjoint vertices and ∆is the join of two 6-cycles. Denote this sphere as S3; its f-vector is (1, 12, 48, 72, 36). (
)
Subcase 2: lk∆vi ∼= Σi for i = 2, 3. Then c is a 7-cycle; furthermore, B3 has no
interior vertices and B1, B2 have a unique interior vertex b1, b2 respectively. Since ∆[3]
has two missing colored edges, and three vertices of degree 6 form an empty triangle in
lk∆v3 ∼= Σ3, WLOG assume that deg b1 = 4 and deg b2 = 6. the electronic journal of combinatorics 27(1) (2020), #P1.10 3
Balanced neighborly (d −1)-manifolds with 3d vertices The only vertex b3 not
connected to b2 in B2 must be the vertex of degree 6 in lk∆v1 ∼= Σ2. Hence B3 is the join Subcase 2: lk∆vi ∼= Σi for i = 2, 3. Then c is a 7-cycle; furthermore, B3 has no
interior vertices and B1, B2 have a unique interior vertex b1, b2 respectively. Since ∆[3]
has two missing colored edges, and three vertices of degree 6 form an empty triangle in
lk∆v3 ∼= Σ3, WLOG assume that deg b1 = 4 and deg b2 = 6. The only vertex b3 not
connected to b2 in B2 must be the vertex of degree 6 in lk∆v1 ∼= Σ2. Hence B3 is the join 7 the electronic journal of combinatorics 27(1) (2020), #P1.10 of b3 with the path of length 5, which is c\b3. But then b3 is also the degree-6 vertex in
lk∆v2 ∼= Σ2, and there is no way to triangulate B1 such that it shares no common interior
edge with B2. of b3 with the path of length 5, which is c\b3. But then b3 is also the degree-6 vertex in
lk∆v2 ∼= Σ2, and there is no way to triangulate B1 such that it shares no common interior
edge with B2. Subcase 3: lk∆v2 ∼= lk∆v3 ∼= Σ3. Then c is an 8-cycle and only B1 has an interior
vertex a. Also ∆[3] has one missing colored edge, so a is of degree 6. Three vertices of
degree 6 in Σ3 are of different colors, hence in lk∆v2 and lk∆v3 the other two vertices of
degree 6 must be two pairs of antipodal vertices in lk∆[3] a (the other pair of antipodal
vertices in lk∆[3] a is the missing edge in ∆[3]). In this way we recover lk∆v2 = B1 ∪
B3, lk∆v3 = B1 ∪B2, where B1, B2, B3 are the 2-balls shown in the figure below (from
left to right); in particular, labels of the vertices represent the color and the blue (red
resp.) edges form the missing triangle in lk∆v2 (lk∆v3 resp.). We call this 3-sphere S4. Its f-vector is (1, 12, 52, 80, 40). (
)
Case 3: lk∆v1 ∼= Σ3. This is not possible by Proposition 6. the electronic journal of combinatorics 27(1) (2020), #P1.10 3
Balanced neighborly (d −1)-manifolds with 3d vertices 3
1
2
3
2
1
3
2 3
1
2
3
2
1
3
2
1
3
1
2
3
2
1
3
2
3
1
2
3
2
1
3
2
Theorem 9. There exists a unique balanced 2-neighborly homology 4-sphere 4155
2 with
each color set of size 3. Theorem 9. There exists a unique balanced 2-neighborly homology 4-sphere 4155
2 with
each color set of size 3. Theorem 9. There exists a unique balanced 2-neighborly homology 4-sphere 4155
2 with
each color set of size 3. Proof. Let ∆be such a sphere and let its color set V5 = {v1, v2, v3}. By Alexander
Duality, ˜Hi(∆{4,5}) ∼= ˜H3−i(∆[3]). In particular, since ∆{4,5} is balanced 2-neighborly,
β2(∆[3]) = β1(∆{4,5}) = 4 and β1(∆[3]) = 0. Hence f2(∆[3]) = (f1 −f0 + χ)(∆[3]) = 9 · 6
2
−9 + 5 = 23. By double counting, P3
i=1 f1(lk∆vi) = P
W={i,j,5}⊆[5] f2(∆W) =
4
2
f2(∆[3]) = 138. By
Proposition 6 and Lemma 8, f1(lk∆vi) ∈{42, 46, 48, 52}, it follows that either 138 =
42 + 48 · 2, that is, lk∆v1 ∼= S1 and lk∆v2, lk∆v3 ∼= S3; or 138 = 46 · 3 and lk∆vi ∼= S2 for
all i. By double counting, P3
i=1 f1(lk∆vi) = P
W={i,j,5}⊆[5] f2(∆W) =
4
2
f2(∆[3]) = 138. By
Proposition 6 and Lemma 8, f1(lk∆vi) ∈{42, 46, 48, 52}, it follows that either 138 =
42 + 48 · 2, that is, lk∆v1 ∼= S1 and lk∆v2, lk∆v3 ∼= S3; or 138 = 46 · 3 and lk∆vi ∼= S2 for
all i. Consider the first case above. S1 is the connected sum of two octahedral 3-spheres. For any 2-subset W ⊂[4], the induced subcomplex (S1)W is the union of two 4-cycles
glued along an edge, so f1((S1)W) = 7. Similarly, S3 is the join of two 6-cycles, so we
have (S3)W is either a 6-cycle or the bipartite graph K3,3, i.e., f1((S3)W) = 6 or 9. Hence 23 = f2(∆W∪{5}) =
3
X
i=1
f1((lk∆vi)W) ∈{19, 22, 25}, the electronic journal of combinatorics 27(1) (2020), #P1.10 8 a contradiction. Now we consider the second case, where all vertex links in ∆are isomorphic to S2. Let Γ = lk∆v1 ∩lk∆v2 ∩lk∆v3. 3
Balanced neighborly (d −1)-manifolds with 3d vertices The proof of Lemma 8 implies that for any vertex p /∈V5,
lkΓ p = lklk∆p v1 ∩lklk∆p v2 ∩lklk∆p v3 is a 5-cycle (as the boundary of the union of three
2-faces, where we apply the cross-flip). Hence Γ must be the boundary of the icosahedron. Since all lk∆vi are isomorphic, by Lemma 4 Γ divides the 3-sphere lk∆v1 into two 3-balls,
each having the same number of facets. If the facets of lk∆v1 are labeled as in the link of
vertex 1 in 4155
2 (this is a vertex-transitive triangulation of 4-sphere whose vertex links are
isomorphic to S2, see [10]), then one such Γ is the intersection of vertices 1, 6, 8 in 4155
2;
we rename it to Γ1. In this case, lk∆v1 = B ∪Γ1 B′, where B, B′ are isomorphic 3-balls. We check by sage [21] that all other subcomplexes in lk∆v1 that are isomorphic to Γ1 are
of the form σ(Γ1), where σ is an element in the permutation group of lk∆v1 (of order 8). So it suffices to consider just Γ1. To reconstruct lk∆v2 and hence ∆, note that lk∆u2 has
the decomposition lk∆u2 = B′ ∪Γ1 B′′ ∼= S2 for some 3-ball B′′, and furthermore B′′ ∼= B′. To decide B′′ it is equivalent to finding a balanced simplicial isomorphism f : B′ →B′′
with B′ ∩B′′ = B ∩B′′ = Γ1 and f(Γ1) = Γ1; in other words, f is a permutation in
Aut(Γ1). We check by sage [21] that the links of vertex 6,8 in 4155
2 are the only candidates
for lk∆v2. Hence ∆= 4155
2. Indeed ∆is balanced: the color sets are {1, 6, 8}, {2, 4, 9},
{3, 7, 11}, {5, 10, 15} and {12, 13, 14}. Theorem 10. There exists a balanced 3-neighborly non-spherical 5-manifold with each
color set of size 3. Theorem 10. There exists a balanced 3-neighborly non-spherical 5-manifold with each
color set of size 3. Proof. By Theorem 9, if such 5-manifold exists, then all vertex links are isomorphic to
4155
2, which we denote as Γ. Based on the list of facets of Γ in [15], we take a color-
preserving permutation σ = (1, 6, 8)(2, 4, 9)(11, 3, 7)(10, 15, 5)(13, 14, 12). 3. The f-vector of ∆is (1, 18, 135, 540, 1035, 918, 306). 3. The f-vector of ∆is (1, 18, 135, 540, 1035, 918, 306). Furthermore by the Dehn-Sommerville relations, any balanced 3-neighborly 5-manifold
having 3 vertex in each color set also has the f-vector (1, 18, 135, 540, 1035, 918, 306). Let
Σ be such a complex, {v1, v2, v3} a color set, Mi = lkΣ vj ∩lkΣ vk for {i, j, k} = [3] and
N = M1 ∩M2 ∩M3. If F is an interior face of Mi, then F ∪{vi} /∈Σ. Since Σ is
balanced 3-neighborly, lkΣ v1 = M2 ∪M3 ∼= M1 ∪M3 = lkΣ v2 and they have 102 facets,
it follows that each Mi has no interior vertices or edges and with 51 facets. Also since all
vertex links in Σ are isomorphic to 4155
2, the same argument as in the proof of Theorem
9 implies that N is a 3-manifold whose vertex links are isomorphic to the boundary of
the icosahedron; indeed 31515
1
is one such example. We haven’t checked if there exist
other balanced 3-neighborly 5-manifolds. (It is not known if there exist 15-vertex non-
vertex-transitive 3-manifolds whose vertex links are all isomorphic to the boundary of the
icosahedron.) 3
Balanced neighborly (d −1)-manifolds with 3d vertices We choose σ
in such a way that σ /∈Aut(Γ) and furthermore, Γ ∩σ(Γ), Γ ∩σ2(Γ) and σ(Γ) ∩σ2(Γ)
are isomorphic homology manifolds with no interior faces of dimension < 2 and with a
common boundary C. By computer we check that Γ∪σ(Γ)∪σ2(Γ) is balanced 3-neighborly
and C is the vertex-transitive 3-manifold 31515
1 that triangulates S3/Q as in [15]. Finally
let ∆= (Γ ∗{16}) ∪(σ(Γ) ∗{17}) ∪(σ2(Γ) ∗{18}), where {16, 17, 18} are the vertices of
color 6. By sage [21] one verifies that all vertex links of ∆is isomorphic to Γ, which is
known as a combinatorial 4-sphere (we say a simplicial complex is a combinatorial sphere
if it is PL homeomorphic to the boundary of the simplex). Hence ∆is a combinatorial
manifold that is balanced 3-neighborly. Remark 11. The following properties of the balanced 3-neighborly 5-manifold found in
the proof of Theorem 10 are verified by sage: Remark 11. The following properties of the balanced 3-neighborly 5-manifold found in
the proof of Theorem 10 are verified by sage: 1. It is vertex-transitive and has the following generators of the automorphism group
(of order 1080): 1. It is vertex-transitive and has the following generators of the automorphism group
(of order 1080): (2, 15)(4, 5)(9, 10)(12, 17)(13, 18)(14, 16), (1, 2)(3, 15)(4, 6)(5, 7)(8, 9)(10, 11),
(1, 3)(6, 7)(8, 11)(12, 17)(13, 18)(14, 16), (1, 13)(3, 18)(6, 14)(7, 16)(8, 12)(11, 17),
(1, 6, 8)(2, 3, 17)(4, 7, 18)(5, 15, 10)(9, 11, 16). 9 9 the electronic journal of combinatorics 27(1) (2020), #P1.10 2. The homology groups of ∆are given by (Z, 0, Z2, 0, 0, Z). 4
Balanced 2-neighborly 3-sphere with 16 vertices In this section we provide a balanced 2-neighborly triangulation of the 3-sphere. The
construction is motivated by Lemma 4. w3
u3
v3
w1
u2
v1
u1
w2
v4
u4
w4
v2
w3
u3
v3
w1
u2
v1
u1
w2
v4
u4
w4
v2
w3
u3
v3
w1
u2
v1
u1
w2
v4
u4
w4
v2
Figure 2: Discs A, B and C (from left to right) w3
u3
v3
w1
u2
v1
w2
v4
u4
w4
v2
w3
u3
v3
w1
u2
v1
u1
w2
v4
u4
w4
v2
w3
u3
v3
w1
u2
v1
u1
w2
v4
u4
w4
v2
Figure 2: Discs A, B and C (from left to right) w3
u3
v3
w1
u2
v1
u1
w2
v4
u4
w4
v2 w3
u3
v3
w1
u2
v1
u1
w2
v4
u4
w4
v2 v3 u2 v1 u3 u3 u1 w3
u3
v3
w1
u2
v1
u1
w2
v4
u4
w4
v2 v3 Figure 2: Discs A, B and C (from left to right) 10 10 the electronic journal of combinatorics 27(1) (2020), #P1.10 w3
u3
v3
w1
u2
v1
u1
w2
v4
u4
w4
v2
w3
u3
v3
w1
u2
v2
u1
w2
v4
u4
w4
v1
Figure 3: Left: disc D′. Right: disc D obtained after rearranging the boundary of D′. w3
u3
v3
w1
u2
v1
u1
w2
v4
u4
w4
v2 w3
u3
v3
w1
u2
v2
u1
w2
v4
u4
w4
v1 u1 gure 3: Left: disc D′. Right: disc D obtained after rearranging the boundary of D′ w3
u3
v3
w1
u2
v2
u1
w2
v4
u4
w4
v1
w3
u3
v3
w1
u2
v2
u1
w2
v4
u4
w4
v1
Figure 4: Left: disc A′. Right: disc B′. Notice that ∂A′ = ∂B′ = ∂D. w3
u3
v3
w1
u2
v2
u1
w2
v4
u4
w4
v1 w3
u3
v3
w1
u2
v2
u1
w2
v4
u4
w4
v1 w4 v4 Figure 4: Left: disc A′. Right: disc B′. Notice that ∂A′ = ∂B′ = ∂D. Construction 12. Assume V1 = {u1, u2, u3, u4}, V2 = {v1, v2, v3, v4}, V3 = {w1, w2, w3, w4}
and V4 = {z1, z2, z3, z4} are the four color sets of a balanced 3-sphere Γ. 4
Balanced 2-neighborly 3-sphere with 16 vertices We let lkΓ z1 =
A ∪∂A∼∂C C and lkΓ z3 = B ∪∂B∼∂C C, where A, B and C are triangulated 2-balls sharing
the same boundary as shown in Figure 2. All possible edges that do not appear in A, B
and C are shown in Figure 3 as solid red edges in disc D′. Notice that the dashed edges
in D′ are edges in discs A and B, so we may rearrange the boundary of D by switching
the positions of vertices v1 and v2, and then replacing the edges containing v1 or v2 in
∂D′ by the dashed edges. In this way, we obtain a triangulation of a 12-gon D as shown
in Figure 3. Furthermore, ∂D ⊆A ∪B, and ∂D divides the sphere = A ∪∂A∼∂B B into
two discs A′ and B′ as shown in Figure 4. Construction 12. Assume V1 = {u1, u2, u3, u4}, V2 = {v1, v2, v3, v4}, V3 = {w1, w2, w3, w4}
and V4 = {z1, z2, z3, z4} are the four color sets of a balanced 3-sphere Γ. We let lkΓ z1 =
A ∪∂A∼∂C C and lkΓ z3 = B ∪∂B∼∂C C, where A, B and C are triangulated 2-balls sharing
the same boundary as shown in Figure 2. All possible edges that do not appear in A, B
and C are shown in Figure 3 as solid red edges in disc D′. Notice that the dashed edges
in D′ are edges in discs A and B, so we may rearrange the boundary of D by switching
the positions of vertices v1 and v2, and then replacing the edges containing v1 or v2 in
∂D′ by the dashed edges. In this way, we obtain a triangulation of a 12-gon D as shown
in Figure 3. Furthermore, ∂D ⊆A ∪B, and ∂D divides the sphere = A ∪∂A∼∂B B into
two discs A′ and B′ as shown in Figure 4. We let lkΓ z2 = A′ ∪∂A′∼∂D D and lkΓ z4 = B′ ∪∂B′∼∂D D. Since both stΓ z1 ∩stΓ z3 = C
and stΓ z2 ∩(stΓ z1 ∪stΓ z3) = A′ are simplicial 2-balls, it follows that Σ = ∪3
i=1 stΓ zi is a
simplicial 3-ball. Furthermore, the boundary of Σ is exactly lkΓ z4. Hence Γ = Σ ∪stΓ z4
is indeed a balanced 2-neighborly 3-sphere. Remark 13. 2. The automorphism group of Γ has two generators (The second generator is given by switching vertices of color 1 and 3, and color 2
and 4, but with the same subscript.) Hence Aut(Γ) has 8 elements. (The second generator is given by switching vertices of color 1 and 3, and color 2
and 4, but with the same subscript.) Hence Aut(Γ) has 8 elements. 3. The complex Γ given in Construction 12 is shellable. For lkΓ z1 = A∪∂A∼∂C C, there
exist two shellings c1, . . . , c10, a1, . . . , a10 and a′
1, . . . , a′
10, c′
1, . . . , c′
10 such that for any
1 ⩽i ⩽10, ci, c′
i are facets from C and ai, a′
i are facets from A. Similarly, there exist
two shellings c1, . . . , c10, b1, . . . , b10 and b′
1, . . . , b′
10, c′
1, . . . , c′
10 for lkΓ z3 = B∪∂B∼∂CC,
where bi, b′
i are facets from B. Then 3. The complex Γ given in Construction 12 is shellable. For lkΓ z1 = A∪∂A∼∂C C, there
exist two shellings c1, . . . , c10, a1, . . . , a10 and a′
1, . . . , a′
10, c′
1, . . . , c′
10 such that for any
1 ⩽i ⩽10, ci, c′
i are facets from C and ai, a′
i are facets from A. Similarly, there exist
two shellings c1, . . . , c10, b1, . . . , b10 and b′
1, . . . , b′
10, c′
1, . . . , c′
10 for lkΓ z3 = B∪∂B∼∂CC,
where bi, b′
i are facets from B. Then a′
1 ∗z1, . . . , a′
10 ∗z1, c′
1 ∗z1, . . . , c′
10 ∗z1, c1 ∗z3, . . . , c10 ∗z3, b1 ∗z3, . . . , b10 ∗z3 gives a shelling of stΓ z1∪stΓ z3. We may extend this shelling into a complete shelling
of Γ by constructing two similar shellings of lkΓ z2 and lkΓ z4. However, we tried
some computer tests and failed to prove either polytopality or non-polytopality. Remark 14. 2. The automorphism group of Γ has two generators It is easy to see that if ∆1 is a balanced 2-neighborly (d1 −1)-sphere and
∆2 is a balanced 2-neighborly (d2 −1)-sphere, then ∆1 ∗∆2 is a balanced 2-neighborly
(d1+d2−1)-sphere. Hence by taking joins, we find balanced 2-neighborly (4k−1)-spheres
with 16k vertices for any k ⩾1. Question 15. Let d ⩾4 and m ⩾5 be arbitrary integers. Is there a balanced 2-neighborly
simplicial (d−1)-sphere all of whose color sets have the same size m? Is there a polytopal
sphere with these properties? the electronic journal of combinatorics 27(1) (2020), #P1.10 4
Balanced 2-neighborly 3-sphere with 16 vertices Here we provide some properties of Γ in Construction 12. 1. (A ∪B, C, D) is an ear decomposition of Γ[3]. 1. (A ∪B, C, D) is an ear decomposition of Γ[3]. 1. (A ∪B, C, D) is an ear decomposition of Γ[3]. 11 the electronic journal of combinatorics 27(1) (2020), #P1.10 5
Balanced 2-neighborly L(3, 1) with 16 vertices In this section we present our first construction of a balanced 2-neighborly lens space
L(3, 1) with 16 vertices. We denote it by ∆. Each color set of ∆has four vertices. Construction 16. Denote the color sets of ∆by V1 = {u1, u2, u3, u4}, V2 = {v1, v2, v3, v4},
V3 = {w1, w2, w3, w4} and V4 = {z1, z2, z3, z4}. In Figure 5 we illustrate the construction of the vertex links lk∆zi for i = 1, . . . , 4. All
these links are realized as cylinders. Two links lk∆z1 and lk∆z2 share the same top and
bottom, which are triangulated hexagons spanned by vertices {ui, vi, wi : i = 1, 3} and
{ui, vi, wi : i = 2, 4}, respectively. To construct lk∆z3 from lk∆z1, we switch the positions
of vertices u3, v3, w3 with vertices u4, v4, w4 respectively and form a new cylinder. The
new top and bottom hexagons contain the 2-faces {u1, v1, w1} and {u2, v2, w2}. Similarly,
we construct the link lk∆z4 from lk∆z2 by switching the positions of vertices u3, v3, w3
with vertices u4, v4, w4 and letting {u1, v1, w1} and {u2, v2, w2} be the 2-faces that appear
in the triangulation of the top and bottom hexagons. It follows that lk∆z3 and lk∆z4 also
share the same top and bottom. 12 the electronic journal of combinatorics 27(1) (2020), #P1.10 u4
v2
w4
u2
v4
w2
w1
u3
v1
w3
u1
v3
(a) lk∆z1
u2
v4
w2
u4
v2
w4
w1
u3
v1
w3
u1
v3
(b) lk∆z2
u2
v3
w2
u3
v2
w3
w1
u4
v1
w4
u1
v4
(c) lk∆z3
u3
v2
w3
u2
v3
w2
w1
u4
v1
w4
u1
v4
(d) lk∆z4
Figure 5: Four vertex links of ∆ u4
v2
w4
u2
v4
w2
w1
u3
v1
w3
u1
v3
(a) lk∆z1 u2
v4
w2
u4
v2
w4
w1
u3
v1
w3
u1
v3
(b) lk∆z2 (b) lk∆z2 u2
v3
w2
u3
v2
w3
w1
u4
v1
w4
u1
v4
(c) lk∆z3 u3
v2
w3
u2
v3
w2
w1
u4
v1
w4
u1
v4
(d) lk∆z4 (d) lk∆z4 Figure 5: Four vertex links of ∆ Now since ∆is balanced 2-neighborly, by our construction, it only remains to show
that ∆triangulates the lens space L(3, 1). position 18. The complex ∆is a balanced vertex minimal triangulation of L(3, 1) Proof. By Proposition 6.1 in [11], each color set of ∆is of size at least 3. If there are
exactly three vertices v1, v2, v3 of color 1 in ∆, apply the Mayer-Vietoris sequence on the
triple (st∆v1 ∪st∆v2, st∆v3, ∆) and we obtain that 0 = H1(lk∆v3) →H1(st∆v1 ∪st∆v2) ⊕H1(st∆v3) →H1(∆) →H0(lk∆v3) = 0. Hence H1(st∆v1 ∪st∆v2) ∼= H1(∆) = Z/3Z. However, this is impossible since H1(st∆v1 ∪
st∆v2) ∼= H0(st∆v1 ∩st∆v2), which cannot be Z/3Z. Hence H1(st∆v1 ∪st∆v2) ∼= H1(∆) = Z/3Z. However, this is impossible since H1(st∆v1 ∪
st∆v2) ∼= H0(st∆v1 ∩st∆v2), which cannot be Z/3Z. The same argument as above also shows that the balanced triangulation of any lens
space L(p, q) with p > 1 must have at least 16 vertices. 5
Balanced 2-neighborly L(3, 1) with 16 vertices The geometric realizations of st∆z1 and st∆z2
are filled cylinders that share top and bottom. So their union A := st∆z1 ∪st∆z2 is a
filled torus (that is, a genus-1 handlebody); so is the union B := st∆z3 ∪st∆z4. Note that
these two handlebodies have identical boundary complexes, thus they provide a Heegaard
splitting of a lens space. To identify which lens space ∆triangulates, we need to determine the homeomorphism
φ : ∂A →∂B. Consider two generators γ, δ of π1(A ∩B) = π1(∂A), where γ is the 6-
cycle (u3, v1, w3, u1, v3, w1) and δ is the 4-cycle (u1, w2, u4, w3). In particular, δ is also a
generator of π1(A). From the construction we see that φ(γ) is a loop running around the
equator of ∂B thrice and the meridian of ∂B once. Also φ(δ) runs around the equator of
∂B twice and the meridian of ∂B once. Hence it is indeed the lens space L(3, 1). Remark 17. Our construction ∆has the following properties: 1. All vertex links are combinatorially equivalent. 2. From Figure 5 we see lk∆zi ∩lk∆zj has two connected components when {i, j} =
{1, 2} or {3, 4} (they are the top and bottom hexagons as shown in Figure 2); and it 2. From Figure 5 we see lk∆zi ∩lk∆zj has two connected components when {i, j} =
{1, 2} or {3, 4} (they are the top and bottom hexagons as shown in Figure 2); and it 13 the electronic journal of combinatorics 27(1) (2020), #P1.10 has three connected components when i ∈{1, 2} and j ∈{3, 4} (each component is
the union of two facets along the side of the cylinders). In general, the intersection
of two vertex links, where the vertices are of the same color, always has at least two
connected components. 3. There are three group actions on the vertices of ∆: 3. There are three group actions on the vertices of ∆: (a) Fix the subscript and rotate the corresponding vertices of color 1, 2 and 3
respectively. The generator is given by (u1v1w1)(u2v2w2)(u3v3w3). (a) Fix the subscript and rotate the corresponding vertices of color 1, 2 and 3
respectively. The generator is given by (u1v1w1)(u2v2w2)(u3v3w3). (b) Rotate vertices of the same color. The generator is The automorphism group of ∆is of size 96. The automorphism group of ∆is of size 96. Proposition 18. The complex ∆is a balanced vertex minimal triangulation of L(3, 1). (u1u3u2u4)(v1v3v2v4)(w1w3w2w4)(z1z3z2z4). (c) Exchange lk∆z1 and lk∆z2, lk∆z3 and lk∆z4, by exchanging vi and wi (or ui
and wi, ui and vi) for all i ∈[4]. The generators are (c) Exchange lk∆z1 and lk∆z2, lk∆z3 and lk∆z4, by exchanging vi and wi (or ui
and wi, ui and vi) for all i ∈[4]. The generators are (z1z2)(z3z4)(v1w1)(v2w2)(v3w3)(v4w4),
(z1z2)(z3z4)(u1w1)(u2w2)(u3w3)(u4w4) and
(z1z2)(z3z4)(u1v1)(u2v2)(u3v3)(u4v4). (z1z2)(z3z4)(v1w1)(v2w2)(v3w3)(v4w4),
(z1z2)(z3z4)(u1w1)(u2w2)(u3w3)(u4w4) and
(z1z2)(z3z4)(u1v1)(u2v2)(u3v3)(u4v4). The automorphism group of ∆is of size 96. 6
Balanced spheres and ear decomposition In this section our goal is to construct a balanced 3-sphere whose rank-selected sub-
complexes do not have ear decompositions. The motivation is from the balanced 2-
neighborly construction of L(3, 1) in Section 5. Indeed, we want to construct a balanced
3-dimensional complex ∆so that 1) each vertex link is a 2-sphere; 2) for a fixed color set
V4 = {v1, · · · , vk}, the intersection of any two vertex links lk∆vi ∩lk∆vj always has at
least two connected components (as the property listed in Remark 17); and 3) ∪4
i=1 st∆vi
is 3-ball, which together with the condition 1) guarantees that ∆is a 3-sphere. 14 the electronic journal of combinatorics 27(1) (2020), #P1.10 In the following we take k = 5 and give such a construction. Figure 6 illustrates
the links lk∆v1, · · · , lk∆v4. Every label represents the color of the vertex. Also each
connected component of lk∆v1 ∩lk∆v2 is colored in green, lk∆vi ∩lk∆v3 is colored in blue
for i = 1, 2, and lk∆vj ∩lk∆v4 is colored in pink for j = 1, 2, 3. Immediately we check
that all these intersections of vertex links have 2 or 3 connected components. 3
2
3
2
3
2
1
1
1
1
2
1 3
1
2
3
1
3
2
3
2
3
2
1
1
1
1
2
1
2
2
3 2
3
3
1
(a) lk∆v1 and lk∆v2
3
3 1
3
1
3
1 3
2
1
2
1
2
2
1
2
1
2
1
3
1
3
1
(b) lk∆v3
2
3
3
2
1
2
1
2
1
3
1
3
3
1
1
(c) lk∆v4
Figure 6: Four vertex links as triangulated 2-spheres. For simplicity’s sake, we omit some
diagonal edges in the quadrilaterals in (b), and some labels of vertices in (c). 6
Balanced spheres and ear decomposition 2
3
3
2
1
2
1
2
1
3
1
3
3
1
1
(c) lk∆v4 3
3 1
3
1
3
1 3
2
1
2
1
2
2
1
2
1
2
1
3
1
3
1
(b) lk∆v3 3
2
3
2
3
2
1
1
1
1
2
1 3
1
2
3
1
3
2
3
2
3
2
1
1
1
1
2
1
2
2
3 2
3
3
1
(a) lk∆v1 and lk∆v2 (a) lk∆v1 and lk∆v2 (b) lk∆v3 (c) lk∆v4 Figure 6: Four vertex links as triangulated 2-spheres. For simplicity’s sake, we omit some
diagonal edges in the quadrilaterals in (b), and some labels of vertices in (c). (a) lk∆v1 ∪lk∆v2
(b) ∪3
i=1 lk∆vi
(c) ∪4
i=1 lk∆v4
Figure 7: how the links are glued together. (b) ∪3
i=1 lk∆vi (c) ∪4
i=1 lk∆v4 (c) ∪4
i=1 lk∆v4 (b) ∪3
i=1 lk∆vi (a) lk∆v1 ∪lk∆v2 Figure 7: how the links are glued together. Figure 7 shows how ∆\V4 is formed from these links. First we glue lk∆v1 and lk∆v2
along two green triangles. The resulting complex lk∆v1 ∪lk∆v2 is shown in Figure 7a. Then we place lk∆v3 on top of lk∆v1 ∪lk∆v2. As we see from Figure 7b, the boundary
complex of ∪3
i=1 st∆vi is a triangulated torus. Finally, we place lk∆v4 on top of ∪3
i=1 lk∆vi
so that st∆v4 “covers the 1-dimensional hole” in ∪3
i=1 st∆vi, see Figure 7c. We denote
the subspace of R3 enclosed by lk∆vi as Si for 1 ⩽i ⩽4, and let S5 := ∪i⩽4Si. From our
construction it follows that the boundary complex of S5 is a 2-sphere; we let it be lk∆v5. 15 the electronic journal of combinatorics 27(1) (2020), #P1.10 Indeed ∆is a 3-sphere since ∆is the union of two 3-balls S5 and st∆v5 glued along their
common boundary lk∆v5. Since each lk∆vi ∩lk∆vj has at least two connected components for 1 ⩽i ̸= j ⩽4,
the Mayer-Vietoris sequence implies that Si ∪Sj is not contractible for all 1 ⩽i ̸= j ⩽4. A similar inspection of lk∆vi ∪lk∆vj ∪lk∆vk also implies that the boundary complexes
of Si ∪Sj ∪Sk’s cannot be triangulated 2-spheres for distinct 1 ⩽i, j, k ⩽4. Proposition 19. Not all rank-selected subcomplexes of balanced simplicial spheres have
ear decompositions. Proof. Consider the complex ∆constructed above. 6
Balanced spheres and ear decomposition We denote the union of interior faces
of a complex τ by int τ. Suppose ∆\V4 has an ear decomposition (Γ1, Γ2, · · · , Γk). Since
|V4| = 5 and β2(∆\V4) = 4, k must be 4. Notice first that ∪i⩽4 lk∆vi divides R3 into
five subspaces, namely, S1, · · · , S4 and the complement of S5, each having lk∆vi as the
boundary complex for 1 ⩽i ⩽5 respectively. The complex Γ1 is the union of 2-balls
B1, B2 with ∂B1 = ∂B2 = Γ1 ∩Γ2. By the Jordan theorem, B1 ∪Γ2 is a triangulated 2-
sphere that separates R3 into two connected components. Hence the bounded component
must be either Si ∪Sj or Si ∪Sj ∪Sk for some 1 ⩽i, j, k ⩽4. (We may assume that
it is not Si, since otherwise we may consider the 2-sphere ∪i⩽3Γi −∪1⩽i̸=j⩽3 int(Γi ∩Γj)
instead of Γ1 ∪Γ2 −int(Γ1 ∩Γ2), where the subset enclosed by this sphere in R3 cannot
be Si anymore.) This contradicts the fact that the boundaries of Si ∪Sj or Si ∪Sj ∪Sk
are not 2-spheres. Remark 20. One can think of all the figures illustrated above as projections of a subcom-
plex of ∆−st∆v5 onto R3. However, we do not know whether the complex provided in
this section can be realized as the boundary of a 4-polytope. Acknowledgements The author was partially supported by a graduate fellowship from NSF grant DMS-
1361423. I thank Moritz Firsching for pointing out the automorphism groups of the
constructions in Section 3 and 4 and running some computational tests to decide whether
the constructions are polytopal. Many thanks to Lorenzo Venturello and the anonymous
referees for pointing out mistakes in an earlier version and contributing to a few remarks,
improvement of the proofs in this paper. [2] M. K. Chari. Two decompositions in topological combinatorics with applications to
matroid complexes. Trans. Amer. Math. Soc., 349:3925–3943, 1997. [3] D. L. Donoho and J. Tanner. Counting the faces of randomly-projected hypercubes
and orthants, with applications. Discrete Comput. Geom., 43:522–541, 2010. [1] G. Burton. The non-neighbourliness of centrally symmetric convex polytopes having
many vertices. J. Combin. Theory Ser. A, 58:321–322, 1991. [1] G. Burton. The non-neighbourliness of centrally symmetric convex polytopes having
many vertices. J. Combin. Theory Ser. A, 58:321–322, 1991.
[2] M. K. Chari. Two decompositions in topological combinatorics with applications to
matroid complexes. Trans. Amer. Math. Soc., 349:3925–3943, 1997.
[3] D. L. Donoho and J. Tanner. Counting the faces of randomly-projected hypercubes
and orthants, with applications. Discrete Comput. Geom., 43:522–541, 2010. References [1] G. Burton. The non-neighbourliness of centrally symmetric convex polytopes having
many vertices. J. Combin. Theory Ser. A, 58:321–322, 1991. [2] M. K. Chari. Two decompositions in topological combinatorics with applications to
matroid complexes. Trans. Amer. Math. Soc., 349:3925–3943, 1997. [3] D. L. Donoho and J. Tanner. Counting the faces of randomly-projected hypercubes
and orthants, with applications. Discrete Comput. Geom., 43:522–541, 2010. 16 the electronic journal of combinatorics 27(1) (2020), #P1.10 [4] B. Gr¨unbaum. . Convex polytopes, Interscience, London, 1967. [5] I. Izmestiev, S. Klee and I. Novik. Simplicial moves on balanced complexes. Advances
in Mathematics, 320:82–114, 2017. [6] M. Juhnke-Kubitzke, S. Murai, I. Novik and C. Sawaske. A generalized lower bound
theorem for balanced manifolds. Mathematische Zeitschrift, 289:921–942, 2018. [7] W. Jockusch. An infinite family of nearly neighborly centrally symmetric 3-spheres. J. Combin. Theory. Ser. A, 72:318–321, 1995. [8] M. Joswig and T. R¨orig. Neighborly cubical polytopes and spheres. Israel J. Math.,
159:221–242, 2007. [9] M. Joswig and G. M. Ziegler. Neighborly cubical polytopes. Discrete Comput. Geom.,
24:325–344, 2000. [10] E. G. K¨ohler and F. Lutz. Triangulated Manifolds with Few Vertices: Vertex-
Transitive Triangulations I. arXiv:math/0506520v1, 2005. [11] S. Klee and I. Novik. Lower Bound Theorems and a Generalized Lower Bound
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Hereditary Angioedema: The Economics of Treatment of an Orphan Disease
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Frontiers in medicine
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Citation: Lumry WR (2018) Hereditary
Angioedema: The Economics of
Treatment of an Orphan Disease. Front. Med. 5:22. doi: 10.3389/fmed.2018.00022 Keywords: health economics, orphan disease, hereditary angioedema, burden, treatment Hereditary angioedema (HAE) is a rare autosomal-dominant genetic disease (1, 2). It occurs in
approximately 1:30,000–80,000 individuals and affects less than 8,000 individuals in the United
States (USA), 15,000 in Europe, and 200,000 worldwide (3, 4) Disease-specific therapy for HAE,
although available in other countries since 1979 (5), only recently became available in the USA. With the Food and Drug Administration (FDA) approval of a human nano-filtered human plasma-
derived C1 inhibitor administered intravenously for routine prevention of HAE attacks on October
10, 2008, a new era in therapy for HAE began (6). Since 2008, four therapies to treat HAE attacks have
received FDA approval, including nano-filtered pasteurized human plasma-derived C1 inhibitor and
ecallantide in 2009, icatibant in 2011, and recombinant C1 inhibitor in 2014 (7–11). These therapies
are currently registered in many countries around the world. A concentrated human plasma-derived
C1 inhibitor administered subcutaneously for routine prevention of HAE attacks was approved on
June 22, 2017 by the FDA and other preventive therapies are currently in development (12).hi Reviewed by: Reviewed by:
Coen Maas,
University Medical Center Utrecht,
Netherlands
Allen P. Kaplan,
Medical University of South Carolina,
United States y
y
This burst of drug development and approvals has greatly benefited individuals with HAE, their
families, caregivers, physicians, and healthcare providers (13, 14). The burden of HAE has been
reduced, quality of life improved and utilization of urgent care, emergency facilities, and hospitals
decreased significantly (14, 15). Concerns raised by healthcare payers in the USA and healthcare sys-
tems in other countries about the financial impact of these newly approved therapies on healthcare
payment systems have led to barriers to and limitation of access to these life changing and potentially
life saving therapies (16–20).hh *Correspondence:
William Raymond Lumry
lumrymd@allergyspecialists.us Specialty section:
This article was submitted to
Hematology,
a section of the journal
Frontiers in Medicine Specialty section:
This article was submitted to
Hematology,
a section of the journal
Frontiers in Medicine
Received: 15 September 2017
Accepted: 23 January 2018
Published: 16 February 2018 This review will discuss the cost burden of HAE on patients, healthcare systems, and society. The
impact of availability of and access to novel and specific therapies on morbidity, mortality, and the
overall burden of disease will be explored along with potential changes in treatment paradigms to
improve effectiveness and reduce cost of treatment. The prevalence of orphan diseases, legislative
incentives to encourage development of orphan disease therapies, and the impact of orphan disease
treatment on healthcare payment systems will be discussed. Received: 15 September 2017
Accepted: 23 January 2018
Published: 16 February 2018 Review Review
published: 16 February 2018
doi: 10.3389/fmed.2018.00022 Hereditary Angioedema:
The Economics of Treatment
of an Orphan Disease William Raymond Lumry* This review will discuss the cost burden of hereditary angioedema on patients, health-
care systems, and society. The impact of availability of and access to novel and specific
therapies on morbidity, mortality, and the overall burden of disease will be explored along
with potential changes in treatment paradigms to improve effectiveness and reduce cost
of treatment. The prevalence of orphan diseases, legislative incentives to encourage
development of orphan disease therapies and the impact of orphan disease treatment
on healthcare payment systems will be discussed. Keywords: health economics, orphan disease, hereditary angioedema, burden, treatment Edited by: Edited by:
Alvin H. Schmaier,
Case Western Reserve University,
United States BURDEN OF HAE In addition to the direct cost of medical care, indirect costs must
be considered. Missed time from work or school, decreased
productivity at work and loss of opportunity are significant
costs to the patient and society. Patients report a decrease of
productivity on the job due to HAE (26). A national audit in the
United Kingdom of patients with HAE and acquired angioedema
revealed patients lost on average 9 days of work per year rang-
ing from 0 to 43 days (29). A European Union (EU) study of
the socioeconomic burden of HAE revealed both patients and
caregivers were affected with each losing an average of 20 days
from work or school per year (24). In addition to the expense of specific HAE therapies, other
costs of caring for an individual with HAE are high. These costs
include the direct cost of providing medical care to these patients
and the indirect costs of the disease on patients, their families,
and on society. Patients with HAE consume more health care and
are more costly to the health care system. In a United Kingdom
study, the cost of care for an HAE patient was 160% more for
primary care and 447% more for secondary care than a patient
without HAE even if the costs of specialist care and medications
were excluded (23). During swelling attacks, patients with HAE
and their caregivers miss a significant amount of time from
work and school, and when “well” are often less productive at
work. Many report being unable to achieve their educational and
career goals, or even maintain employment as a result of their
disease (24, 25). Loss of educational and career opportunity is commonly
reported. In the HAEA survey conducted in 2007, 57% of HAE
patients reported having career advancement hindered, 69%
felt that they could not consider certain types of jobs because of
their disease, 100% felt that educational advancement had been
hindered, 55% had to limit their educational choices, and 48%
had not achieved the level of education that they desired (25). Even after disease-specific therapies became available, decreased
opportunity remains a problem. IMPACT OF NEW THERAPIES FOR HAE Patients with HAE have benefited from approval of novel, disease-specific drugs that treat and prevent
swelling attacks. The benefits include improvement in health and quality of life (QoL), increased February 2018 | Volume 5 | Article 22 Frontiers in Medicine | www.frontiersin.org 1 HAE Treatment Economics Lumry Lumry ability to work and pursue educational and career goals, reduced
disability and reduction of costly urgent care visits, and hospitali-
zations and longer survival (13, 14, 15).h ability to work and pursue educational and career goals, reduced
disability and reduction of costly urgent care visits, and hospitali-
zations and longer survival (13, 14, 15).h cost of having and treating HAE in the USA was also assessed in
this survey (26). The average annual cost per patient was $44,597. Cost varied depending on the severity of the disease ranging from
$11,587 for mildly affected patients to $28,764 and $104,857,
respectively, for those moderately or severely affected. The total
mean annual expenditure per HAE patient of $44,597 included
direct medical costs of $29,177 comprised of $17,381 for hospital
admissions, $2,827 for emergency department (ED) visits, $3777
for outpatient care, and $5,194 for medications and indirect costs
of $15,420, including $5,157 for reduced productivity, $6,417 for
reduced income, $3,402 for missed work, and $4,444 for travel
and childcare (26). Accounting for inflation, the average annual
cost per patient in 2017 would be more than $65,000. The economic cost of these therapies is high. In 2012, the
annual cost of nano-filtered C1 inhibitor (Cinryze®, Shire,
Lexington, MA, USA) indicated for routine prevention of HAE
attacks, when used at approved dosage and interval, was $487,000
USD per patient, the most expensive drug for any orphan disease
treated in the USA (21). In 2015, sales of Cinryze® generated the
second highest revenue per patient treated to a pharmaceutical
company, $210,000 USD, of all orphan drugs (22). Current USA
average wholesale prices (AWP) for treatments of swelling attacks
range from $5,000 to more than $12,000 per attack treated. The
cost of these therapies is generally less outside of the USA with
some national health systems able to acquire them at a significant
discount to USA prices. Emergency department usage and hospitalization before 2008
was high as only supportive treatment, including intravenous
fluids, anti-emetics, analgesics, and airway support was available. BURDEN OF HAE In an EU socioeconomic burden
of disease report published in 2014 after current therapies became
available, 42% patients reported their educational advancement
was hindered, 40% were prevented from applying for certain
jobs and 36% felt that their career advancement was diminished,
9% switched positions within their company, and 10% left their
position permanently because of this disabling disease (24). HAE
patients also suffer from anxiety and depression at much higher
rates than the normal population. Results from two independ-
ent studies suggest that between 14 and 43% of patients of HAE IMPACT OF NEW THERAPIES FOR HAE In 2007, there were 2,705 ED visits in the USA with HAE as the
primary diagnosis with 40.9% resulting in hospitalization. At a
mean cost of $1,479 per ED visit $3,727,080 was spent for urgent
care (27). Between 2004 and 2007, there were 10,125 hospitaliza-
tions where HAE was the primary or secondary diagnosis. The
average length of stay was 5.1 days and average cost per hospi-
talization was $8383 resulting in $21,220,000 annual expenditure
for hospital services (28). Although not commonly seen in the orphan disease drug
market the increasing number of approved therapies for HAE
may result in competition between pharmaceutical companies
decreasing the cost of these therapies. The first example of this
occurred recently with the pricing of Haegarda®, a subcutane-
ous C1 inhibitor concentrate (C1INH) for routine prevention of
HAE attacks approved in June, 2017. Despite approximately four
times the amount of C1 inhibitor being required to treat a patient
with Haegarda® compared to Cinryze®, the AWP per treatment
with Haegarda® is 15% less. As other drugs are developed and
approved for this indication hopefully competitive pricing will
continue to push costs down.i INNOVATIVE TREATMENT PARADIGMS Although disease-specific treatments for HAE are costly, appro-
priate and timely treatment decreases ED visits, hospitalizations,
lost time from school and work, and prevents death lowering the
overall cost of the disease to the healthcare system and to soci-
ety. ED visits and hospitalization for supportive treatment were
the norm before availability of home and self-administration of
treatment (25). Self-administration for treatment and preven-
tion of attacks is safe, effective and encouraged. Patients can
accurately recognize and safely self-treat HAE attacks leading
to earlier treatment, earlier resolution of symptoms, decreased
ED and hospital visits, and improved quality of life and cost
savings (33–39). An example of this problem is found in Argentina. Members
of the Argentine HAE Patient Association completed question-
naires about the availability and their access to HAE treatments
in 2009, 2013, and 2016. Despite C1INH being registered and
approved for treatment of HAE attacks before 2009, C1INH was
available to only 26% of those responding to the 2009 survey. This
increased to 55% by 2016. Despite being approved only 10% had
access to icatibant in 2016. Reimbursement for these medications
in 2016 was also a challenge with only 64% reimbursed at 100%
with 19% having no reimbursement available at all. The majority
of the patients received treatment from health personnel or at the
hospital and over 50% reported not receiving treatment until their
attack was severely painful. Reordering and resupply of medica-
tion was difficult for 66% of patients with only 20 per reporting
a fast replacement and 53% being able to obtain replacement
within 10 days (44). Innovative treatment paradigms may lower the cost and
the burden of disease further. Italian HAE patients who self-
administered C1INH decreased their mean annual number of
hospitalizations from 16.8 to 2.1, time to administration of treat-
ment from 3.2 to 1.9 h, time to beginning of symptom improve-
ment from 84 to 54 min, time to complete symptom resolution
from 12.8 to 10.8 h and number of missed days of work or school
from 23.3 to 7.1 compared to hospital or ED administered therapy. The total cost of therapy, including both direct and indirect cost,
was approximately 30,010 Euros ($36,000 USD) per patient when
the therapy was administered in a hospital or clinic setting and
26,621 Euros ($32,000 USD) when treatment was administered at
home. COST OF HAE CARE BEFORE SPECIFIC
THERAPIES WERE AVALIABLE
IN THE USA As disease-specific therapies for HAE were not available in the
USA before 2008, there is an opportunity to review costs and
impact of the disease before current therapies became available. An internet survey completed by members of the USA HAE
Association (HAEA) in 2007 found patients experienced an aver-
age of 26.9 swelling attacks per year lasting 61.3 h each. Over 80%
were considered moderate to severe in intensity (25). Total annual February 2018 | Volume 5 | Article 22 Frontiers in Medicine | www.frontiersin.org 2 HAE Treatment Economics Lumry experience clinically significant depression further adding to
their cost of treatment and disability (25, 30). with 84% reduction in ED visits in this group. In the self-treated
patients, there was no need for tracheotomy, no deaths reported
and an improved QoL in all physical and psychological domains. Despite a 300% increase in use of newer “high cost” treatments,
the cost to treat an average of 36 attacks per patient per year was
a manageable at $16,766 (35). A fatal HAE attack with loss of an individual both from the
work force and from society is the ultimate burden of this disease
and results in significant hardship both socially and economically
to the patient’s family and society. Unfortunately, patients with
HAE continue to succumb to this treatable disease. In a review of
all USA death certificates from 1999 to 2010, HAE was considered
a contributing factor or the underlying cause of death in 600
people with 270 of these deaths directed attributed to HAE (31). In a German study which included 728 patients from 182 families
with HAE, 214 deaths were recorded. The mean age of death from
an HAE attack was 40.6 years (32). Unfortunately in many countries, implementation of a home
or self-administration policy is not possible. In Japan, Greece and
most of Eastern Europe acute therapies are only available at the
hospital or in specialty clinics, if available at all. In Brazil and
Mexico, home therapy is available but is not reimbursed.i Specific treatments for HAE attacks are available in many
countries as shown in Table 1. Treatments to prevent attacks
are less widely available. What is not obvious from this table is
although these treatments may be registered and approved for
marketing in a particular country, they may not be accessible to
the patients that need them. INNOVATIVE TREATMENT PARADIGMS This represented savings of 11% or 57,619 Euros ($69,000
USD) for the 17 patients reported in the study (40). Despite the advances in the treatment for HAE with availabil-
ity of new effective on demand and attack preventing treatments,
these medications are not approved in many countries around
the world. Even if approved, they are often not accessible to the
patients who need them. Despite improvement of treatment out-
comes and cost savings, when the treatment is self-administered
or given in the home setting, in many countries treatment of
attacks is only available in hospital and clinic settings. Spanish investigators estimated patients who self-treated
their HAE attacks with icatibant compared to health care set-
ting administration would save an average of 121 Euros ($145)
per attack in direct and indirect costs, a 9.2% decrease in costs. Reduction in direct costs accounted for 74% of the savings. This
would achieve an annual health system savings of 551,371 Euros
($661,600) (41). An USA study reported a $650,000 savings when
249 HAE attacks over 5 months were treated with ecallantide
given at home by an infusion nurse compared to the cost of
treating these attacks in the ED or hospital (42). In the United
Kingdom, home administration of icatibant compared to hospital
administration of C1INH saved $861–$1167/attack (43). COST OF HAE CARE BEFORE SPECIFIC
THERAPIES WERE AVALIABLE
IN THE USA Barriers to access include require-
ment for health care system or judicial approval, availability only
in specialized treatment centers or hospitals, limits on reimburse-
ment, and limits on number of treatments allowed or resupply of
medication to providers or patients. Frontiers in Medicine | www.frontiersin.org ORPHAN DRUG DEVELOPMENT
POLICIES AND REIMBURSEMENT ISSUES Individuals with HAE and other rare and burdensome diseases
have benefited greatly from the development of therapies made
economically feasible by the orphan drug policies. Orphan drug
policies have been established in the USA, the EU and Japan to
encourage the development of safe and effective therapies for rare
diseases. The USA Orphan Drug Act was enacted in 1983 (45). In Denmark, 80 HAE patients were followed prospectively for
10 years. By 2012, 49% were self-administering C1NH or icatibant February 2018 | Volume 5 | Article 22 Frontiers in Medicine | www.frontiersin.org 3 SUMMARY Orphan drug policies are in place to encourage development of
safe and effective therapies for orphan diseases. With a limited
number of patients to treat, the cost per patient of orphan drugs to
recover research and development costs is high. Orphan diseases
affect approximately 8–10% of the population. Overall expendi-
tures for drugs to treat orphan diseases remain proportional to
the incidence of these diseases in the population. Expenditures
for orphan drugs are currently less than 10% of pharmaceutical
expenditures and 1% of the total healthcare costs in the USA. Despite these facts, payers in the USA and healthcare authorities
around the world perceive treatment of orphan diseases puts an
inordinate burden on their payment systems and have put barri-
ers in place to limit use of new disease specific therapies.i These policies have been successful in encouraging companies
to develop therapies for a wide variety of rare conditions. In
January, 1983 there were 38 drugs approved in the USA for orphan
disorders (22). On July 1, 2017, 635 approved drugs were listed in
the Orphan Disease Therapeutic Registry (47). and 98 designated
orphan drugs were approved for marketing in the EU (48).h There are 25–30 million individuals, 8–10% of the population
of the USA, affected by one of the 7,000 diseases designated as
orphan, who may benefit from provisions of the Orphan Drug
Act (16, 49). Although treatments for these rare diseases have
provided great benefits to affected individuals and their families,
the high cost of these therapies has led to the perception by pay-
ers and some in society that treatment of orphan diseases places
an inordinate burden on healthcare payment systems (16, 17). This concern about high cost of these treatments has resulted in
barriers to limit access to them, including formulary approval,
high coinsurance and copayment rates, prior authorization and
multiple reauthorizations, step therapy, and limits on supply and
resupply of medication (16, 17, 20). Orphan drugs benefit many people with previously under-
served orphan diseases. These drugs offer significant value to
patients and society in terms of improvements in health, reduced
disability, increased productivity, including the ability to continue
working, reduced healthcare utilization, and improved quality of
life and survival. Patients with HAE are not an exception and
have benefited greatly. Availability of new and novel therapies to
treat and prevent swelling attacks has dramatically decreased the
burden of this disease. ute
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Oral HAE Treatment Economics Lumry Under this act, a drug is given orphan designation if the disease it
treats affects less than 200,000 (approximately 1:1,650) individuals
or if there is no reasonable expectation of profitability for the drug. Incentives provided include tax credits for research costs, grants
to aid in clinical research, and a 7-year marketing exclusivity for
approved orphan drugs.i Under this act, a drug is given orphan designation if the disease it
treats affects less than 200,000 (approximately 1:1,650) individuals
or if there is no reasonable expectation of profitability for the drug. Incentives provided include tax credits for research costs, grants
to aid in clinical research, and a 7-year marketing exclusivity for
approved orphan drugs.i them registered and approved. In 2007, $13.3 billion was spent
in the USA accounting for 4.3% of $311 billon total expenditures
for pharmaceuticals that year. By 2013, this had increased to $25.8
billion, 7.7% of $337 billion pharmaceutical spend. IMS Health
Market Prognosis has forecasted USA total drug expenditures of
$465.0 billion in 2018 with orphan drugs accounting for $44.10
billion, 9.5% of this amount, representing an increase of 0.7%
over four years (52, 53). In 1999, the EU enacted its orphan drug policy that defines
an orphan disease as one with 5 or less patients per 100,000
(1:20,000) individuals. Research incentives are available within
the EU and its member States and fees are waived for approval of
the marketing application and a 10-year marketing exclusivity is
provided (46).h Frontiers in Medicine | www.frontiersin.org SUMMARY As the number of approved therapies for
HAE increases the cost of therapy may decrease in response to
competition. The first example of this occurred with the pricing
of Haegarda®, a subcutaneous C1INH for routine prevention of
HAE attacks approved in June, 2017. Prudent therapeutic choices,
utilization of new treatment paradigms, and improvement in
availability and accessibility to these new therapies will continue
to improve the lives of patients with HAE and other orphan
diseases. Coverage by payers and healthcare systems of these life
altering and potentially life saving therapies should continue with
barriers to access being appropriately addressed.hi Drug research and development are time-consuming and
expensive activities. The average cost of developing and win-
ning market approval for a new prescription drug was recently
estimated to be $2.6 billion with $1.4 billion of this amount
spent on research and development (50, 51). Orphan drugs are
no exception. With many fewer patients to treat, the cost per
patient treated must be higher in order to recover research and
development expenditures. For the top 100 drugs in 2016, the
annual median drug cost per patient in the USA for treatment of
non-orphan conditions was $27,756 compared to $140,443 per
patient treated with an orphan disease (49). Although the cost per patient to treat an orphan disease is
often high, the perception that cost of treatment of rare diseases
as a whole has an inordinate impact on total pharmaceutical
expenditures and health care costs is inaccurate. Analyses in
the USA and EU have shown this impact is minimal and in line
with the 8–10% prevalence of these diseases in the population. In 2014, total expenditures for pharmaceuticals in the USA
accounted for only 9.8% of total healthcare expenditures of $3.0
trillion. Expenditures on orphan drugs for orphan indications
was approximately $33.5 billion representing less than 10% of
pharmaceutical expenditures and only 1% of the total healthcare
expenditures (22, 52). The conversation about the high cost for benefit obtained
of orphan drugs with payers, society, and the patients needs to
change to recognition of the benefits of timely and appropriate
care. We need to solve the access problem by working with health
systems, patients, and their advocates on the most cost-effective
and efficient ways to deliver this care. We need to continue to
disseminate knowledge regarding benefits to patients and to
society of effective and safe treatments for HAE and all rare
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SignificantAmendmentstotheFDCAct/OrphanDrugAct/ 25. Lumry WR, Castaldo AJ, Vernon MK, Blaustein MB, Wilson DA, Horn PT. The humanistic burden of hereditary angioedema: impact on health-related February 2018 | Volume 5 | Article 22 Frontiers in Medicine | www.frontiersin.org 6 Lumry Lumry HAE Treatment Economics 46. Frontiers in Medicine | www.frontiersin.org February 2018 | Volume 5 | Article 22 REFERENCES Regulation (EC) No 141/2000 of the European Parliament and of the Council
of 16 December 1999 on orphan medicinal products Official Journal L 018,
22/01/2000 P. 0001 – 0005. Available from: http://eur-lex.europa.eu/legal-
content/EN/TXT/?uri=CELEX%3A32000R0141 52. Divino V, Dekoven M, Kleinrock M, Wade RL, Kaura S. Orphan drug expendi-
tures in the United States: a historical and prospective analysis 2007-18. Health
Aff (2016) 35(9):1588–94. doi:10.1377/hlthaff.2016.0030 ff
53. Health IMS. Market Prognosis 2014–2018, USA. London: IMS Health
(2014). 30 p. 47. Food and Drug Administration. Orphan Drug Approval List. MD: Silver
Spring FDA (2017). Available from: http://www.accessdata.fda.gov/scripts/
opdlisting/oopd/listResult.cfm Conflict of Interest Statement: WL is a consultant for biocryst, pharming, CSL
Behring, and shire. He speaks on behalf of CSL Behring, pharming and shire and
has received research grants from biocryst, pharming, dyax, lev, jerini, viropharma,
CSL Behring, and shire. He is a member of the medical advisory board of the us
hereditary angioedema association. 48. Orphanet. Orphanet: List of Orphan Drugs in Europe. (2017). Available from:
http://www.orpha.net/orphacom/cahiers/docs/GB/list_of_orphan_drugs_
in_europe.pdf 49. EvaluatePharma Orphan Drug Report. 4th ed. (2017). Available from: http://
info.evaluategroup.com/rs/607-YGS-364/images/EPOD17.pdf Copyright © 2018 Lumry. 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 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. 50. DiMasi JA, Grabowski HG, Hansen RW. Innovation in the Pharmaceutical
Industry: New Estimates of R&D Costs. Boston: Tufts Center for the Study
of Drug Development (2014). Available from: http://csdd.tufts.edu/news/
complete_story/cost_study_press_event_webcast 51. Avorn J. The $2.6 billion pill – methodologic and policy considerations. N Engl
J Med (2015) 372:1877–9. doi:10.1056/NEJMp1500848 51. Avorn J. The $2.6 billion pill – methodologic and policy considerations. N Engl
J Med (2015) 372:1877–9. doi:10.1056/NEJMp1500848 February 2018 | Volume 5 | Article 22 Frontiers in Medicine | www.frontiersin.org
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Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids
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Generation of a novel transgenic
rat model for tracing extracellular
vesicles in body fluids Aya Yoshimura1,2, Masaki Kawamata3, Yusuke Yoshioka3, Takeshi Katsuda3, Hisae Kikuchi4,
Yoshitaka Nagai4,5, Naoki Adachi2,6, Tadahiro Numakawa2,7, Hiroshi Kunugi2,
Takahiro Ochiya3 & Yoshitaka Tamai1 received: 24 March 2016
accepted: 13 July 2016
Published: 19 August 2016 Extracellular vesicles (EVs) play an important role in the transfer of biomolecules between cells. To
elucidate the intercellular transfer fate of EVs in vivo, we generated a new transgenic (Tg) rat model
using green fluorescent protein (GFP)-tagged human CD63. CD63 protein is highly enriched on EV
membranes via trafficking into late endosomes and is often used as an EV marker. The new Tg rat line
in which human CD63-GFP is under control of the CAG promoter exhibited high expression of GFP in
various body tissues. Exogenous human CD63-GFP was detected on EVs isolated from three body fluids
of the Tg rats: blood serum, breast milk and amniotic fluid. In vitro culture allowed transfer of serum-
derived CD63-GFP EVs into recipient rat embryonic fibroblasts, where the EVs localized in endocytic
organelles. These results suggested that this Tg rat model should provide significant information for
understanding the intercellular transfer and/or mother-child transfer of EVs in vivo. Extracellular vesicles (EVs) are small membrane vesicles (40–200 nm in diameter) that are secreted from many
different cell types in the body. EVs are originated from the plasma membrane and endosomes1. The former are
vesicles called as microvesicles generated by outward budding from plasma membrane. The latter are formed
by invagination and budding of early endosomes and the endosomes containing a number of vesicles inside the
lumen are called multivesicular bodies (MVBs). The MVBs release vesicles called exosomes into the extracel-
lular environment by fusion with the plasma membrane. EVs can contain lipids, proteins and RNAs, including
microRNAs (miRs), and the compositions depend on the cellular origin. EVs collected from the cerebrospinal
fluid (CSF) of brain-injured patients include inflammation-related proteins2, and EVs from the CSF of glioblas-
toma patients exhibits increased miR-21 levels, which is a highly overexpressed miR in glioblastoma cells3. These
results have suggested that EVs may offer important information on pathological conditions as a novel biomarker. The EVs released into the extracellular space can be incorporated into the internal components to target cells;
alternatively, the receptors and antigens (e.g., proteins, ligands) on EVs can activate cell surface receptors of target
cells4. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Generation of a novel transgenic
rat model for tracing extracellular
vesicles in body fluids The ability of EVs to affect gene expression extends not only to neighbour cells but also to distant cells,
because EVs circulate through the bloodstream and other body fluids5,6. g
yl
To facilitate further investigation of EV functions in cell-cell communication in vivo, we generated a new
transgenic (Tg) rat model using EVs labelled with green fluorescent protein (GFP). Whereas most relevant studies
have focused on EV functions in vitro, its roles in vivo such as EV transfer among tissues in the body of an indi-
vidual or from mother to child via breast milk or placenta are still uncertain. Several studies have suggested that
EVs target specific cells depending on the type of cell from which the EVs are released. Frühbeis and colleagues
have reported that oligodendroglial-derived EVs are mainly uptaken by microglia and neurons but not by astro-
cytes and oligodendrocytes7. It is essential to identify the releasing cells (i.e., donor cells) and/or the target cells 1Division of Laboratory Animals Resources, National Institute of Neuroscience, National Center of Neurology and
Psychiatry (NCNP), Tokyo, Japan. 2Department of Mental Disorder Research, National Institute of Neuroscience,
NCNP, Tokyo, Japan. 3Division of Molecular and Cellular medicine, National Cancer Center Research Institute (NCC),
Japan. 4Department of Degenerative Neurological Diseases, National Institute of Neuroscience, NCNP, Tokyo, Japan. 5Department of Neurotherapeutics, Osaka University Graduate School of Medicine, Osaka, Japan. 6Department of
Biomedical Chemistry, School of Science and Technology, Kwansei Gakuin University, Sanda, Japan. 7Department
of Cell Modulation, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan. Correspondence and requests for materials should be addressed to T.O. (email: tochiya@ncc.go.jp) Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 1 www.nature.com/scientificreports/ (i.e., recipient cells) of EVs circulating in the body not only to reveal the transfer mechanism of EVs among cells
but also to use EVs as carriers for drug delivery. g
y
We constructed a human CD63-copGFP gene regulated by the CAG promoter (CAG/human CD63-GFP). The CD63 protein is a member of the transmembrane 4 superfamily (tetraspanin), and is known as an EV marker. CD63 is highly enriched in late endosomes (MVBs) via an intracellular pathway from the trans-Golgi network or
via endocytosis from the cell surface8. Within MVBs, CD63 is incorporated into intraluminal vesicles, through a
trafficking pathway that requires ceramide in some types of cells9,10; then, the vesicles rich in CD63 are released
as exosomes by fusion with the plasma membrane. Results
P
d
i Production of CD63-GFP rats and phenotypic analysis. CAG/human CD63-GFP transgenic rats
expressing copGFP-fusion protein to human CD63 under the control of the CAG promoter (Fig. 1a) were gener-
ated by gene transfection into rat embryonic stem cells (rESCs) (Fig. 1b) and injection of transfected rESCs into
blastocysts (Fig. 1c). We selected the rESC colonies expressing GFP fluorescence, and further selected the rESC
lines showing highly fluorescent signals that were comparatively stable after several passages. To generate coat-col-
our chimaeras, the transfected rESCs, which originated from a Wistar strain (white-coated), were injected into
blastocysts from LEA x LEA mating (brown-coated) or LEA x Wistar mating (brown-coated). We observed that the
rESCs were incorporated into the inner cell mass of the blastocysts (arrow in Fig. 1c). The white coat colour from
the rESCs appeared mainly in the face (arrowhead in Fig. 1d). The chimaeric rats produced white-coated newborns
after mating with Wistar males, showing the successful germline transmission of the rESCs (arrows in Fig. 1d). Furthermore, the transgenic offspring were genotyped using PCR analysis (Fig. 1e). The germline chimaera in this
study was confirmed in one of four rESC lines, and it was named the Wistar-esTgN(CAG/CD63-GFP)3NCCRI
strain. Tg male offspring were not viable after birth. Tg male foetuses, compared with Tg female foetuses,
showed relatively high-level expression of human CD63-GFP in fluorescence microscopy observations (GFP+
+; Supplementary Fig. 1a) and western blotting analysis (anti-human CD63 and anti-copGFP; Supplementary
Fig. 1b,c). There were no apparent developmental defects during gestation, and nontransgenic and transgenic
male litters showed equal Mendelian ratios by genotyping: six males were GFP-negative (No. 2–7), and six males
were GFP-positive (No. 1–6) in one litter (Supplementary Fig. 1b); three males were GFP-negative (No. 3–5),
and three males were GFP-positive (No. 3, 5, 6) in another litter (Supplementary Fig. 1c). The Tg female rats were
fertile, but they were likely to die sooner than usual, after reaching an age of four to six months. We also gener-
ated other Tg rats with a human CD63-GFP gene regulated by the Sox2 promoter activated in neural stem cells
because the CAG promoter is ubiquitously expressed. The tissue-specific human CD63-GFP Tg rats gave birth to
both sexes; these rats were fertile and did not tend to die early (unpublished). y
GFP-positive neonatal rats expressed exogenous human CD63-GFP throughout their bodies (Fig. 2a,b). Generation of a novel transgenic
rat model for tracing extracellular
vesicles in body fluids In previous studies, a GFP-tagged CD63 gene (CD63-GFP)
has been transfected into cultured cell lines to detect EV transfer and incorporation into recipient cells11,12. In
addition, CD63-GFP in vivo transfection into the fruit fly (Drosophila melanogaster) has been reported; this study
has shown that CD63-GFP-labelled EVs from the male reproductive glands mediate the interaction between
sperm and the female reproductive tract after mating13. The present study is the first report of EVs labelled with
CD63-GFP in mammals. We showed that exogenous human CD63-GFP expression was detected in the tissues of
Tg rats by immunostaining and biochemical detection. Furthermore, the human CD63-GFP labels were detected
in EVs isolated from the blood serum, breast milk, and amniotic fluid (AF) of the Tg rats. Our animal model may
potentially provide a new approach to study the intraindividual transfer and/or mother-child transfer of EVs
in vivo. Results
P
d
i The endogenous rat CD63 was ubiquitously expressed in main organs except for the thymus and liver, which
showed expression signals that were lower and indistinct relative to those of other tissues (Fig. 2b). The heart,
kidney and stomach expressed particularly high levels of human CD63-GFP (Fig. 2a xiii” and Fig. 2b). At embry-
onic day 18–19 (E18–19), GFP fluorescence was also observed in placentas (arrows in Supplementary Fig. 1a). Interestingly, the placentas of GFP-negative foetuses expressed GFP signals only on the maternal side (arrowhead
in Supplementary Fig. 1a). CD63-GFP signals are located in endosomes, and extracellular secretion was attenuated by
GW4869. To analyse the human CD63-GFP localization inside cells, primary fibroblast cells were pre-
pared from caudal vertebrae of Wistar wild type (Wt) and Tg rats. Human CD63-GFP expression was located
close to nuclei (upper panels in Fig. 3a), and GFP signals overlapped with endogenous rat CD63-positive sig-
nals (lower panels in Fig. 3a). The cells with low GFP expression showed no human CD63-positive signals, only
rat CD63-positive signals (arrows in Fig. 3a). Time-lapse imaging of cells from Tg rats showed the movement
of small GFP-positive organelles between plasma membranes and around nuclei (Supplementary Fig. 2). We
isolated EVs from the conditioned medium (EV-free FBS) of the cultured fibroblast cells by ultracentrifuga-
tion. Both EVs from Wt and Tg cells showed a peak in particle size between 100 and 200 nm (Wt; 152 nm and
Tg; 137 nm) by measurement using a NanoSight system (Fig. 3b). It has been suggested that there are multiple
molecular machineries involved in the generation of MVB and the sorting in endosomes have different manners
(e.g. ceramide-dependent or -independent), and which mechanism predominates depends on the cell type1,14. The tetraspanin CD63 plays in the MVB formation and the endosomal sorting14,15. Next, to examine whether the
extracellular secretion of human CD63-GFP proteins was regulated via the same biogenesis pathway as endoge-
nous rat CD63-containing exosomes, the fibroblast cells were treated with a neutral sphingomyelinase (nSMase)
inhibitor, GW4869, which is known to block ceramide biosynthesis and reduce EV secretion16. The rat CD63
signals inside the Wt and Tg cells increased after incubation with 10 μM GW4869 for 24 hours, and the GFP
fluorescence signals were also increased in Tg cells after GW4869 treatment (Fig. 3c). Results
P
d
i The expression levels of
rat CD63 and human CD63-GFP proteins in the cell lysates remained unchanged after GW4869 treatment (right Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 2 www.nature.com/scientificreports/ Figure 1. Generation of CAG/human CD63-GFP transgenic (Tg) rats. (a) Structure of the transgene
construction. The transgene was constructed using human CD63-copGFP under control of the CAG prom
(b) Image of rat embryonic stem cells (rESCs) transfected with the CAG/human CD63-GFP gene. The cul
rESCs expressed GFP. (c) Blastocysts after microinjection of the transfected rESCs. The arrow indicates rE
adherence to the inner cell mass (ICM). BF: bright field. Scale bars = 100 μm. (d) Adult female chimaeric r
from Wister-derived rESC (white-coated) injection into LEA blastocysts (brown-coated). White patches w
present in the face (arrowhead). Two Tg offspring (white-coated) from mating a female chimaeric rat with
Wistar wild type (Wt) male (arrows). (e) Genotyping by PCR analysis of the extracted DNA from ear snips
of the offspring. B: brown coat colour, W: white coat colour, V: CAG/human CD63-GFP vector, and M: siz
marker. Figure 1. Generation of CAG/human CD63-GFP transgenic (Tg) rats. (a) Structure of the transgene
construction. The transgene was constructed using human CD63-copGFP under control of the CAG promoter. (b) Image of rat embryonic stem cells (rESCs) transfected with the CAG/human CD63-GFP gene. The cultured
rESCs expressed GFP. (c) Blastocysts after microinjection of the transfected rESCs. The arrow indicates rESC
adherence to the inner cell mass (ICM). BF: bright field. Scale bars = 100 μm. (d) Adult female chimaeric rat
from Wister-derived rESC (white-coated) injection into LEA blastocysts (brown-coated). White patches were
present in the face (arrowhead). Two Tg offspring (white-coated) from mating a female chimaeric rat with a
Wistar wild type (Wt) male (arrows). (e) Genotyping by PCR analysis of the extracted DNA from ear snips
of the offspring. B: brown coat colour, W: white coat colour, V: CAG/human CD63-GFP vector, and M: size
marker. in Fig. 3d). In contrast, the extracellular rat CD63 and human CD63-GFP levels in the EVs collected by ultra-
centrifugation decreased with GW4869 in a dose-dependent manner (left in Fig. 3d). The levels of another EV
marker, flotillin-1, were also reduced in Wt (10 μM GW4869) and Tg cells (1 and 10 μM GW4869) compared
with cells that did not receive GW4869. These results suggested that GW4869 blocked EV release into the con-
ditioned medium but did not affect protein expression. Results
P
d
i These results also suggested that the exogenous human
CD63-GFP in fibroblast cells was secreted through the ceramide-dependent biogenesis pathway, as was endoge-
nous rat CD63. in Fig. 3d). In contrast, the extracellular rat CD63 and human CD63-GFP levels in the EVs collected by ultra-
centrifugation decreased with GW4869 in a dose-dependent manner (left in Fig. 3d). The levels of another EV
marker, flotillin-1, were also reduced in Wt (10 μM GW4869) and Tg cells (1 and 10 μM GW4869) compared
with cells that did not receive GW4869. These results suggested that GW4869 blocked EV release into the con-
ditioned medium but did not affect protein expression. These results also suggested that the exogenous human
CD63-GFP in fibroblast cells was secreted through the ceramide-dependent biogenesis pathway, as was endoge-
nous rat CD63. The EVs labelled with CD63-GFP from rat body fluids. EVs were isolated from blood serum, breast
milk and AF of Wt and Tg rats using ultracentrifugation methods. Nanoparticle tracking analysis showed a peak
between 100 and 200 nm in each sample (Fig. 4a–c). The particles imaged by electron microscopy (EM) were
observed to be mainly between 50–150 nm in size (Fig. 4d). Western blot analysis revealed that the EVs of the three Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 3 www.nature.com/scientificreports/ Figure 2. Human CD63-GFP expression analysis in Tg rats (Wister-esTgN(CAG/CD63-GFP)3NCCRI). (a) Pictures of main organs from Tg offspring (i–xiii: bright field, i’–xiii’: GFP, and xiii”: merged). GFP-negative
(i and i’) and GFP-positive (ii and ii’) offspring were littermate. The heart, kidneys and stomach showed
especially high fluorescent signals (xiii”). (b) Western blotting for endogenous rat CD63 and exogenous
human CD63 in tissue lysates from GFP-negative (GFP−) and GFP-positive (GFP+) offspring. Ctx: cortex, cbl:
cerebellum, and hip: hippocampus. Figure 2. Human CD63-GFP expression analysis in Tg rats (Wister-esTgN(CAG/CD63-GFP)3NCCRI). (a) Pictures of main organs from Tg offspring (i–xiii: bright field, i’–xiii’: GFP, and xiii”: merged). GFP-negative
(i and i’) and GFP-positive (ii and ii’) offspring were littermate. The heart, kidneys and stomach showed
especially high fluorescent signals (xiii”). (b) Western blotting for endogenous rat CD63 and exogenous
human CD63 in tissue lysates from GFP-negative (GFP−) and GFP-positive (GFP+) offspring. Ctx: cortex, cbl:
cerebellum, and hip: hippocampus. body fluids from Wt and Tg rats were positive for EV markers, flotillin-1 and endogenous rat CD63 (Fig. 5a,b). Results
P
d
i Furthermore, signals of antibodies to human CD63 and copGFP in the EVs were detected in the three body fluids
of Tg rats but not in those of Wt rats (Fig. 5a,b). To visually confirm the presence of EVs labelled with human
CD63-GFP, immunoelectron microscopy analysis was performed using serum-derived EVs. The EVs from Wt
rats were not labelled with anti-human CD63 antibody (Fig. 5c, upper and Supplementary Fig. 3a). In contrast,
some of the EVs from Tg rats were immunolabelled with anti-human CD63 antibody, and the immunolabelling
for human CD63 was detected on EVs 50–100 nm in sizes (Fig. 5c, lower and Supplementary Fig. 3b–d). The
negative staining without the first antibody showed no immunoreactivity on the Tg EVs (Supplementary Fig. 3e). These results suggested that human CD63 was enriched in the subpopulation of EVs in Tg rats. Transfer of the labelled EVs with CD63-GFP into recipient cells in vitro. To evaluate the utility of
the labelled EVs from the body fluids of Tg rats for monitoring EV transfer between cells, the serum-derived EVs
were incubated with primary rat embryonic fibroblast cells (REFs) in vitro for 10–11 hours (Fig. 6a,b). Although
it was difficult to chase the incorporated Tg EVs by GFP fluorescent directly, the Tg EVs in the recipient cells were
detectable by immunostaining with antibodies to human CD63 and copGFP (Fig. 6a and Supplementary Fig. 4). In general, the EVs collected by ultracentrifugation consists of microvesicles and exosome subpopulations, and
the immunoelectron microscopy analysis in this study indicated human CD63-immunopositive and –immunon-
egative subpopulations of the EVs in the serum of Tg rats. To examine how the EVs with human CD63-GFP were
transferred into REFs, Wt and Tg EVs were prelabelled with PKH67, a green fluorescent lipid dye that stains all
EV subpopulations. We did not observe green fluorescent signals in the cells that were incubated with vehicle PBS
(−) labelled with PKH67. In contrast, PKH67-labelled EVs showed highly intense fluorescent signals around cells
nuclei (Fig. 6a). The human CD63 immunosignals were detected only in Tg EVs and a substantial proportion of
EVs incorporated into the recipient cells were human CD63-immunnopositive (Fig. 6a). Furthermore, late endo-
some/lysosome marker LAMP1 was colocalized to both PKH67 and human CD63 in the recipient REFs (arrows
in Fig. 6b). These results demonstrate a substantial uptake of CD63-enriched EVs by the recipient cells through
the endocytic pathway. Results
P
d
i Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Human CD63-GFP localization and extracellular vesicle (EV) analysis in the primary fibroblast
cells obtained from the caudal vertebrae of Tg rats. (a) Localization of human CD63-GFP in the cultured
Tg rat cells. Immunostaining indicated the co-localization of GFP with human CD63 (upper panels) and with
rat CD63-positive signals (lower panels) around nuclei (blue). Scale bars = 50 μm. (b) Size distribution of the
EVs isolated from the conditioned medium of Wt and Tg rat cells was determined using a NanoSight system. (c,d) The relationship between ceramide and the secretion of EVs. The intracellular rat CD63-positive signals
and GFP signals were increased after treatment with 10 μM GW4869, a neutral sphingomyelinase (nSMase)
inhibitor, for 24 hours (c). Western blotting showed a GW4869-dependent decrease of EV markers (rat CD63
and flotillin-1) and human CD63-GFP in the isolated EVs (d; left). However, the expression levels of rat CD63
and human CD63-GFP in the cell lysates were not changed by GW4869 (d; right). β-actin was used as a loading
control. Both generation and protein composition of the EVs did not show an apparent change by CD63-GFP
overexpression (Supplementary Fig. 5). Figure 3. Human CD63-GFP localization and extracellular vesicle (EV) analysis in the primary fibroblast
cells obtained from the caudal vertebrae of Tg rats. (a) Localization of human CD63-GFP in the cultured
Tg rat cells. Immunostaining indicated the co-localization of GFP with human CD63 (upper panels) and with
rat CD63-positive signals (lower panels) around nuclei (blue). Scale bars = 50 μm. (b) Size distribution of the
EVs isolated from the conditioned medium of Wt and Tg rat cells was determined using a NanoSight system. (c,d) The relationship between ceramide and the secretion of EVs. The intracellular rat CD63-positive signals
and GFP signals were increased after treatment with 10 μM GW4869, a neutral sphingomyelinase (nSMase)
inhibitor, for 24 hours (c). Western blotting showed a GW4869-dependent decrease of EV markers (rat CD63
and flotillin-1) and human CD63-GFP in the isolated EVs (d; left). However, the expression levels of rat CD63
and human CD63-GFP in the cell lysates were not changed by GW4869 (d; right). β-actin was used as a loading
control. Both generation and protein composition of the EVs did not show an apparent change by CD63-GFP
overexpression (Supplementary Fig. 5). Discussion EVs are released into the extracellular environment, and they have been found in various body fluids, such as
CSF17, breast milk18, saliva18, urine19, AF20, and blood serum. The results of proteome and miR profiling have
shown that the EVs from body fluids carry many immune- or development-related components. Furthermore,
the analysis of EVs from the blood of mothers during pregnancy21 and from breast milk during lactation22 have
provided data showing time-dependent changes in the number of EVs or RNA contents of EVs. However, the
studies of EV transfer using EVs isolated from body fluids have been very few in number compared with the
experiments using EVs isolated from in vitro culture. Most of these studies injected labelled EVs that were col-
lected from conditioned medium of cultured cells into a body to examine the transfer of EV contents23. PKH67
and PKH26 are the most common reagents used to stain EVs. Because they are membrane dyes with a high
affinity for lipids, all EVs are labelled with PKH reagents. In addition, body fluids include many more lipid com-
ponents than conditioned medium, and these lipids may also be stained by PKH reagents. In this study, we gen-
erated and analysed a novel Tg rat expressing human CD63-GFP under the CAG promoter. Human CD63-GFP
expression was detected by western blotting analysis in the EVs collected from three body fluids: blood serum,
breast milk and AF. CD63 proteins are highly enriched particularly on exosome membranes via trafficking into
late endosomes (MVBs)8. We expected that, in combination with other EV markers, human CD63-GFP might be
helpful to distinguish EVs from other vesicles and lipid components as well as provide more precise identification
of the EVs endocytosed into recipient cells.hf y
p
This study suggested that the overexpression of CD63 had strong functional effects in vivo, which resulted in
embryonic lethality for males and premature death for females. It has been reported that CD63 associates with
many protein, and has a key role in intracellular trafficking and cell signalling activity, which indicates that CD63
binding to other membrane proteins regulates various cellular processes, such as cell migration, adhesion and
differentiation, via altering complex formation at the plasma membrane8,24–26. In mast cells (MCs), the absence
of CD63 results in significantly decreased MC degranulation, which led to a reduction of allergic reactions27. Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 5 www.nature.com/scientificreports/ Figure 4. Discussion Characterization of the EVs isolated from three body fluids of Wt and Tg rats. (a–c) Size
distribution of EVs from serum (a), breast milk (b) and amniotic fluid (AF) (c) determined using a NanoSight
system. (d) Electron microscopy (EM) images of EVs. Scale bars = 200 nm. AF samples were collected from
pregnant Tg rats at embryonic day 16–17 (E16–17) after mating with Wt males. GFP−: GFP-negative foetuses. GFP+: GFP-positive foetuses. Figure 4. Characterization of the EVs isolated from three body fluids of Wt and Tg rats. (a–c) Size
distribution of EVs from serum (a), breast milk (b) and amniotic fluid (AF) (c) determined using a NanoSight
system. (d) Electron microscopy (EM) images of EVs. Scale bars = 200 nm. AF samples were collected from
pregnant Tg rats at embryonic day 16–17 (E16–17) after mating with Wt males. GFP−: GFP-negative foetuses. GFP+: GFP-positive foetuses. Figure 4. Characterization of the EVs isolated from three body fluids of Wt and Tg rats. (a–c) Size
distribution of EVs from serum (a), breast milk (b) and amniotic fluid (AF) (c) determined using a NanoSight
system. (d) Electron microscopy (EM) images of EVs. Scale bars = 200 nm. AF samples were collected from
pregnant Tg rats at embryonic day 16–17 (E16–17) after mating with Wt males. GFP−: GFP-negative foetuses. GFP+: GFP-positive foetuses. Figure 5. Analysis of the EVs labelled with human CD63-GFP in the three body fluids. (a,b) Western
blotting analysis of the EVs isolated from serum (a), breast milk and AF (b) of Wt and Tg rats for flotillin-1,
rat CD63, human CD63 and copGFP. AF samples were collected from pregnant Tg rats at E16–17 after mating
with Wt males. GFP−: GFP-negative foetuses. GFP+: GFP-positive foetuses. (c) Immunoelectron microscopy
images of serum-derived EVs from Wt and Tg rats using anti-human CD63 antibody (10 nm gold particles). Scale bars = 200 nm. Figure 5. Analysis of the EVs labelled with human CD63-GFP in the three body fluids. (a,b) Western
blotting analysis of the EVs isolated from serum (a), breast milk and AF (b) of Wt and Tg rats for flotillin-1,
rat CD63, human CD63 and copGFP. AF samples were collected from pregnant Tg rats at E16–17 after mating
with Wt males. GFP−: GFP-negative foetuses. GFP+: GFP-positive foetuses. (c) Immunoelectron microscopy
images of serum-derived EVs from Wt and Tg rats using anti-human CD63 antibody (10 nm gold particles). Scale bars = 200 nm. Discussion Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 6 www.nature.com/scientificreports/ Figure 6. Uptake of serum-derived EVs by rat embryonic fibroblast cells (REFs). (a) EVs of Wt rats, Tg
rats and vehicle PBS (−) as a control were prelabelled with PKH67. Then, REFs were incubated with PKH67-
labelled EVs for 11 hours. The incorporated EVs in the recipient REFs were detected by PKH67 (green) and
anti-human CD63 (red). The Tg EVs are also shown as magnified views (white square region) with different
colours (PKH67: blue, human CD63: red, nuclei: gray). Scale bars = 20 μm. (b) Images of co-localization of EVs
with LAMP1 in REFs. EVs of Wt (with PKH67) and Tg rats were incubated with REFs for 10 hours, and the
EVs from Tg rats were detected using an antibody to human CD63. Magnified views (arrows) are shown in the
insets. Scale bars = 25 μm. Nuclei were counterstained with Hoechst 33342 (blue). Figure 6. Uptake of serum-derived EVs by rat embryonic fibroblast cells (REFs). (a) EVs of Wt rats, Tg
rats and vehicle PBS (−) as a control were prelabelled with PKH67. Then, REFs were incubated with PKH67-
labelled EVs for 11 hours. The incorporated EVs in the recipient REFs were detected by PKH67 (green) and
anti-human CD63 (red). The Tg EVs are also shown as magnified views (white square region) with different
colours (PKH67: blue, human CD63: red, nuclei: gray). Scale bars = 20 μm. (b) Images of co-localization of EVs
with LAMP1 in REFs. EVs of Wt (with PKH67) and Tg rats were incubated with REFs for 10 hours, and the
EVs from Tg rats were detected using an antibody to human CD63. Magnified views (arrows) are shown in the
insets. Scale bars = 25 μm. Nuclei were counterstained with Hoechst 33342 (blue). The Tg rats in this study often exhibited inflammation around the eyes as animals grew (data not shown). Therefore, we considered the possibility that the Tg rats might overexpress CD63 in MCs, which in turn might
causing allergic inflammation by increased MC degranulation. We generated another line of Tg rats expressing
human CD63-GFP under the Sox2 promoter, which expressed it in the kidney in addition to the brain; a large
amount of urine output was observed, but no lethality or infertility was observed in either gender (unpublished). Methods After being blocked with 5% skim milk, the mem-
branes were probed with primary antibodies specific to the following proteins: rat CD63 (1:250; AbD Serotec,
CA, USA); human CD63 (1:250; Becton Dickinson); copGFP (1:10000; Evrogen, Moscow, Russia); and flotillin-1
(1:500; Becton Dickinson). β-actin (1:5000; Sigma-Aldrich) was used as a loading control. The membranes were
subsequently incubated with secondary antibodies, and the signals were detected using chemiluminescent rea-
gents (ImmunoStar and ImmunoStar LD, Wako, Tokyo, Japan). Immunocytochemistry. Cultured cells were fixed with 4% paraformaldehyde (PFA) for 15–20 min at room
temperature. After being washed, cells were incubated with primary antibodies in blocking solution containing
0.2% Triton X-100 (Sigma-Aldrich) and 10% FBS in PBS at 4 °C overnight. Primary antibodies specific to the
following proteins were used: rat CD63 (1:200; AbD Serotec) and human CD63 (1:200; Becton Dickinson). After
the cells were stained with Alexa Fluor 546 Goat anti-Mouse IgG1 antibody (1:2000; Life Technologies), they were
imaged with BIOREVO BZ-9000 (Keyence, Osaka, Japan) and Axiovert 200 (Carl Zeiss, Westlar, Germany) fluo-
rescence microscopes. DAPI was used to stain cell nuclei. Fibroblast culture and inhibition of EV secretion. Primary fibroblast cells were prepared from caudal
vertebrae of Wt and Tg rats (CAG/human CD63-GFP) at postnatal day 21 (P21). The primary cells were main-
tained in DMEM (Life Technologies) with 10% FBS and antibiotic-antimycotic solution (Life Technologies) at
37 °C in 5% CO2.i For inhibition of EV secretion, the fibroblast cells were plated on glass-bottom dishes and 10 cm culture dishes;
after expansion, GW4869 (Sigma-Aldrich) treatments were performed for 24 hours. The 35 mm glass-bottom
dishes were used to observe intracellular rat CD63 as an EV marker and human CD63-GFP expression, and
cells on the 10 cm dishes were stored at −80 °C until protein extraction. Conditioned medium from the 10 cm
dishes was used for the isolation of EVs. To isolate EVs, conditioned medium from the 10 cm dishes was replaced
with the EV-free FBS (System Biosciences) medium before GW4869 treatment. Culture supernatant was centri-
fuged at 1,400 rpm for 10 min and then filtered through a 0.22 μm membrane filter (Millipore) to remove cellular
debris. EVs were collected by ultracentrifugation at 35,000 rpm for 70 min at 4 °C using a Beckman SW41Ti rotor
(Beckman, CA, USA). The pellets were washed with PBS by re-ultracentrifugation and were resuspended in PBS. Methods Animals. All animals (Wistar, LEA and Tg (Wistar-esTgN(CAG/CD63-GFP)3NCCRI) rat strains) used in
this study were treated in accordance with the institutional guidelines of the Animal Ethics Committee for the
care and use of animals in the National Institute of Neuroscience, National Centre of Neurology and Psychiatry,
and National Cancer Centre Research Institute, Japan. Production of CAG/human CD63-GFP Tg rats using rat ES cells. A DNA fragment encoding human
CD63-copGFP (human CD63-GFP) from a pCT-CMV-CD63-GFP vector (System Biosciences, CA, USA) was
subcloned into a pECFP-1 plasmid (Clontech, Shiga, Japan) containing CAG promoter. The plasmid CAG/
human CD63-GFP was linearized by SalI and was then transfected into Wistar rESCs using a Mouse ES Cell
Nucleofector Kit (Lonza, Basel, Swiss) and an Amaxa Nucleofector (A-013 program) as described previously33. pCAG/human CD63-GFP contained a neomycin/kanamycin selection cassette. The rESCs were seeded onto
mitomycin-C-treated neomycin-resistant MEFs (Millipore, MA, USA) in YPAC medium33 with 2% Matrigel
(Becton Dickinson, NJ, USA). Then, G418 (Geneticin) (Sigma-Aldrich, MO, USA) was added to the culture
medium at day 1 for the selection of transfected cells. Colonies showing GFP fluorescence were selected and
expanded. The rESCs in this study were derived from the cell lines established by Kawamata and Ochiya37. l
ll
d
d
(d
) f
d
d h
Blastocysts were collected at 4.5 days post coitus (dpc) from LEA x LEA or Wistar x LEA mating and injected
with approximately 12 transfected rESCs. The injected blastocysts were surgically transferred into the uterine
horns of 3.5 dpc pseudopregnant Wistar rats. Chimaeric rats were identified by coat colour. Germline transmis-
sion was confirmed by the coat colour of F1 rats resulting from mating with Wistar rats. Transgenic offspring were
genotyped by GFP fluorescence and PCR analysis. Sex determination in foetuses was performed by PCR using the
male-specific Sry gene as a marker. Immunoblotting. For total protein extraction, the tissues and cultured cells were solubilized in lysis buffer
(1% SDS, 10 mM Tris-HCl pH 7.5, 5 mM EDTA, 10 mM sodium pyrophospate, 10 mM NaF, 1 mM PMSF and
2 mM Na3VO4). The samples were sonicated on ice, and then the lysates were cleared by centrifugation. Protein
concentration was measured using a Pierce BCA Protein Assay Kit (Life Technology, CA, USA). Cell lysates and EVs were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were trans-
ferred onto Immobilon-P Transfer membranes (Millipore). Discussion A previous report has found that, although CD63-deficient mice display no lethality or infertility due to com-
pensation by other tetraspanins, they exhibit morphologic changes in the kidney, and increased urine output28,
similarly to that of our Tg rats. These results suggest that CD63-GFP will be further useful for visualizing EVs
using tissue- or stage-specific expression constructs, with consideration of the potential effects on phenotype.l g
g
pi
p
pf
p
yp
While the present experiments did not show that serum-derived EVs from the Tg rats retained GFP fluores-
cence after transfer into recipient cells via in vitro assay, the EV signals in recipient cells were clearly detectable by
using an antibody to human CD63. The immunoelectron microscopy analysis revealed that the immunolabelling
with human CD63-specific antibody was not detected on all isolated EVs. In addition, the transfer of EVs into
recipient cells in vitro showed the human CD63 immunoreactivity in many EVs. These results suggested that
human CD63-GFP labelling would provide a helpful tool for tracing of incorporated exosomes although more
detailed experiments are required. This animal model is intended to contribute to research on the transition path-
way of EVs using body fluids. Several studies on breast milk have suggested that the composition of milk-derived
EV may be a potential immune-regulator for infants29,30 and an activator of postnatal growth31. Immune-related
miRs have been identified in the milk-derived EVs29,32. Other than miRs, the milk-derived EVs carry bioactive
TGF-β33, which is known to differentiate naïve T cells into regulatory T cells34 and to promote development
of the intestinal barrier35. Nevertheless, the transfer pathway and recipient cells of milk-derived EVs in infants
remain poorly understood. A previous report has found that mouse intestinal stem cells show uptake of food
(grape)-derived EV-like vesicles that were stained by PKH dye in vivo, and the targeted cells exhibit increased
proliferation after taking up the grape-derived vesicles36. Our Tg rats produced the EVs labelled with human
CD63-GFP, which were detected only in the tissues of Tg rats and not in those of Wt rats. We expect that study
of in vivo uptake of the milk-derived EVs from Tg rats will reveal interesting targets in the intestinal tissues of
infants. Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 7 www.nature.com/scientificreports/ The novel rat model that we generated allowed studies of the movement of endosomes before EV release and
the detection of EVs released. Discussion Our new animal model shows promise for facilitating further studies to reveal the
communication between cells/tissues via EVs. Methods The isolated EVs were quantified by size and count using a NanoSight system (NanoSight, Amesbury, UK) and by
measuring their protein concentration using a Pierce BCA Protein Assay Kit. EV isolation from serum, breast milk and AF body fluids. Blood samples were collected from Wt
and Tg adult female rats. The blood serum samples were centrifuged at 1,400 rpm for 10 min and then at twice
at 10,000 g for 15 min to remove blood cells; this was followed by ultracentrifugation at 35,000 rpm for 70 min
at 4 °C. Breast milk samples were collected from the stomachs of GFP-negative newborns (P4-5) taking milk
from Wt or Tg mother rats. Collected solid milk samples were homogenized and resuspended in PBS; then, they
were centrifuged at 2,000 g for 10 min to remove fat and cell debris. Non-transparent supernatants were filtered
through a 0.22 μm filter to remove residual fat, producing a clear supernatant (milk whey). Finally, the EVs were Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 8 www.nature.com/scientificreports/ collected by ultracentrifugation. AF samples were collected from pregnant Tg rats at E16-17 that had mated with
Wt males. Tg foetuses were identified by the fluorescence of GFP under a fluorescence stereomicroscope (Leica,
Wetzlar, Germany). AF samples were centrifuged at 2,000 g for 10 min and then at 10,000 g for 10 min, and EVs
were collected by ultracentrifugation. collected by ultracentrifugation. AF samples were collected from pregnant Tg rats at E16-17 that had mated with
Wt males. Tg foetuses were identified by the fluorescence of GFP under a fluorescence stereomicroscope (Leica,
Wetzlar, Germany). AF samples were centrifuged at 2,000 g for 10 min and then at 10,000 g for 10 min, and EVs
were collected by ultracentrifugation. EM analyses. The body fluid-derived EVs were plated on collodion-carbon-coated grids and fixed with 2%
PFA. For immunoelectron microscopy, the serum-derived EVs were permeabilized with 0.1% saponin, stained
with anti-human CD63 (1:50, Becton Dickinson), and then incubated in secondary anti-mouse antibody con-
jugated with 10-nm gold particles (Amersham, Buckinghamshire, UK). Finally, the EVs were post-fixed in 0.2%
glutaraldehyde and negatively stained with uranyl acetate. The images were captured using a transmission elec-
tron microscope (Tecnai Spirit; FEI, OR, USA). Transfer of serum EVs into REFs. REFs were prepared from Wt rats at E14.5. Methods Embryos were dissoci-
ated by trypsin treatment (Sigma-Aldrich), and the single cells were cultured in DMEM including 10% FBS and
antibiotic-antimycotic solution at 37 °C in 5% CO2. Serum-derived EVs from Wt or Tg rats were isolated using
ultracentrifugation. The EVs were visualized using a PKH67 fluorescence labelling kit (Sigma-Aldrich). Serum
EVs were incubated with REFs on 24-well glass bottom plates for 10–11 hours at 37 °C in 5% CO2. After being
washed, the cultured cells were fixed with 4% PFA. The EVs from Tg rats were detected using anti-human CD63
antibody after having been transferred into REFs. To analyse localization of EVs in REFs, anti-LAMP1 antibody
(1:250, Sigma-Aldrich) was used. After the cells were stained with Alexa Fluor 546 Goat anti-Mouse IgG1 (1:2000;
Life Technologies), Alexa Fluor 546 Goat anti-Rabbit IgG (1:2000; Life Technologies) and Alexa Fluor 488 Goat
anti-Rabbit IgG (1:200; Life Technologies) antybodies, internalization of EVs were captured with a FV1000 confo-
cal microscope (OLYMPUS, Tokyo, Japan) and a BIOREVO BZ-9000 fluorescence microscopy (Keyence, Osaka,
Japan). Hoechst 33342 was used to stain cell nuclei. J p
)
All experimental protocols were approved by the National Institute of Neuroscience, National Center of
Neurology and Psychiatry, and National Cancer Centre Research Institute, Japan. p
)
All experimental protocols were approved by the National Institute of Neuroscience, National Center o
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We thank Dr. Nobuyoshi Kosaka for expert advice on analysis of extracellular vesicles. This work was supported
by grants from the Grant-in-Aid for Young Scientists (B) (A.Y.) in the Ministry of Education, Culture, Sports,
Science and Technology of Japan, a Grant in Aid for the Japan Science and Technology Agency (JST) through
the Center of Open Innovation Network for Smart Health (COINS) initiated by the Council for Science and
Technology Policy, and a Grant in Aid for the Japan Agency for Medical Research and Development (A-MED)
through the Basic Science and Platform Technology Program for Innovative Biological Medicine. References Generation of genetically modified rats from embryonic stem cells. Proc. Natl. Acad. Sci. USA 107,
14223–14228 (2010). Author Contributions A.Y., M.K., Y.T. and T.O. designed the study. A.Y. performed the experimental work, analysed the data and wrote
the manuscripts. M.K. assisted in the generation of Tg rats using ES cells. Y.Y. provided technical assistance
and helpful discussion on the analysis of extracellular vesicles. H. Kikuchi performed the electron microscopy
experiments. T.K. assisted in the culture and provided helpful comments. N.A. performed time-lapse imaging and
provided helpful comments. Y.N., T.N. and H. Kunugi provided helpful discussion. The manuscript was finalized
by T.O. References 26 L
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26. Lee, S. Y. et al. TIMP-1 modulates chemotaxis of human neural stem cells through CD63 and integrin signaling. Biochem. J. 459,
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5. Hering, N. A. et al. Transforming growth factor-β, a whey protein component, strengthens the intestinal barrier by upregulating
claudin-4 in HT-29/B6 cells. J. Nutr. 141, 783–789 (2004).h 35. Hering, N. A. et al. Transforming growth factor-β, a whey protein component, strengthens th
claudin-4 in HT-29/B6 cells. J. Nutr. 141, 783–789 (2004). 35. Hering, N. A. et al. Transforming growth factor-β, a whey pro 36. Ju, S. et al. Grape exosome-like nanoparticles induce intestinal stem cells and protect mice from DSS-induced colotis. Mol. Ther. 21,
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37. Kawamata, M. & Ochiya, T. Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 Additional Information upplementary information accompanies this paper at http://www.nature.com/srepi Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Yoshimura, A. et al. Generation of a novel transgenic rat model for tracing extracellular
esicles in body fluids. Sci. Rep. 6, 31172; doi: 10.1038/srep31172 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2016 Scientific Reports | 6:31172 | DOI: 10.1038/srep31172 10
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English
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Significant reduction in errors associated with nonbonded contacts in protein crystal structures: automated all-atom refinement with<i>PrimeX</i>
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Acta crystallographica. Section D, Biological crystallography
| 2,012
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cc-by
| 18,307
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research papers Jeffrey A. Bell, Kenneth L. Ho
and Ramy Farid* All-atom models are essential for many applications in
molecular modeling and computational chemistry. Non-
bonded atomic contacts much closer than the sum of the van
der Waals radii of the two atoms (clashes) are commonly
observed in such models derived from protein crystal
structures. A set of 94 recently deposited protein structures
in the resolution range 1.5–2.8 A˚ were analyzed for clashes
by the addition of all H atoms to the models followed by
optimization and energy minimization of the positions of just
these H atoms. The results were compared with the same set of
structures after automated all-atom refinement with PrimeX
and with nonbonded contacts in protein crystal structures at
a resolution equal to or better than 0.9 A˚ . The additional
PrimeX refinement produced structures with reasonable
summary geometric statistics and similar Rfree values to the
original structures. The frequency of clashes at less than 0.8
times the sum of van der Waals radii was reduced over
fourfold compared with that found in the original structures,
to a level approaching that found in the ultrahigh-resolution
structures. Moreover, severe clashes at less than or equal to 0.7
times the sum of atomic radii were reduced 15-fold. All-atom
refinement with PrimeX produced improved crystal structure
models with respect to nonbonded contacts and yielded
changes in structural details that dramatically impacted on the
interpretation of some protein–ligand interactions. Schro¨dinger, 120 West 45th Street, 17th Floor,
New York, NY 10036, USA Correspondence e-mail:
ramy.farid@schrodinger.com Acta Cryst. (2012). D68, 935–952
# 2012 International Union of Crystallography
Printed in Singapore – all rights reserved Significant reduction in errors associated with
nonbonded contacts in protein crystal structures:
automated all-atom refinement with PrimeX Acta Crystallographica Section D
Biological
Crystallography
ISSN 0907-4449 Acta Crystallographica Section D
Biological
Crystallography ISSN 0907-4449 ISSN 0907-4449 Received 15 February 2012
Accepted 19 April 2012 # 2012 International Union of Crystallography
Printed in Singapore – all rights reserved research papers angle conformation of the residue backbone have recently
been demonstrated (Tronrud et al., 2010). Brunger and
coworkers (Fenn et al., 2010, 2011; Schnieders et al., 2011) have
combined the all-atom force field AMOEBA with a new
refinement scheme and have described the advantages of a
more complex molecular description that includes the calcu-
lation of electrostatic interactions between protein atoms. Additional recent innovations in the use of geometric infor-
mation in refinement include the use of deformable elastic
network refinement (Schro¨der et al., 2010), hydropathic force-
field terms (Koparde et al., 2011) and jelly-body restraints
(Murshudov et al., 2011). space refinement techniques developed for use with crystal
structures of small molecules at atomic resolution. They
recognized the necessity of using additional chemical infor-
mation combined with reciprocal-space refinement to accu-
rately determine atomic positions in this situation. A complete system of geometric restraints was devised for
the first widely used protein reciprocal-space refinement
program, PROLSQ (Konnert, 1976; Konnert & Hendrickson,
1980; Hendrickson, 1985). H atoms were not explicitly
considered in this system. The introduction of simulated-annealing refinement led to
the widespread adoption of the program X-PLOR (Bru¨nger,
1992). This program featured geometric restraints based on
the CHARMM force field (Bru¨nger et al., 1986, 1989; Bru¨nger
& Karplus, 1988). Originally, use of this force field required an
all-atom model. CHARMM-based restraints evolved in a way
that removed the requirements for hydrogen coordinates. This
change was associated with an alteration in the representation
of nonbonded contacts from a Lennard–Jones potential to a
much simpler repulsive function and the elimination of the use
of electrostatic potentials. These modifications were partially
motivated by electrostatic artifacts that were introduced into
the structural results owing to the lack of an implicit solvent
model. In addition, the long time required for computation of
the complete set of nonbonded interactions was a significant
impediment to the refinement of large crystal structures
(Nilges et al., 1988; Weis et al., 1990). By the time that X-PLOR
was superseded by the program CNS (Bru¨nger et al., 1998),
any requirement for explicit H-atom coordinates for protein
crystallographic refinement had been eliminated. However,
the capability to apply an electrostatic model and more
complete nonbonded interactions in an all-atom model
remained an essential part of CNS for the determination of
structures from NMR data (Linge et al., 2003). (Chen, Arendall et al., 2010). At this point, the question of the source of all-atom models
needed for computational work can be addressed more clearly. Currently, such all-atom models are produced by adding H
atoms to the united-atom models produced by crystallography. For water molecules and for protein H atoms whose position
is subject to some degree of freedom, i.e. non-riding H atoms,
either a force-field-dependent or a rule-based method is
employed to determine the positions of these H atoms in order
to avoid close nonbonded contacts and to form hydrogen
bonds as appropriate. Nevertheless, when H atoms are added
in this way to a very large majority of protein crystal structures
deposited in the Protein Data Bank (Berman et al., 2000),
multiple close nonbonded contacts between atoms are
observed. One goal of this work is to document this obser-
vation and to try to understand why such interactions occur,
the recent focus on protein structure validation with H atoms
present notwithstanding. Engh & Huber (1991) brought important additional infor-
mation to the definition of the geometry for protein crystal
structures. Their survey of bond lengths and angles observed
in small peptide crystal structures at high resolution has been
uniformly adopted as a standard against which protein crystal
structure models are judged. It has also become the basis for
the restraint system in all of the major refinement programs. Recent developments indicate an interest among crystallo-
graphers in the application of more complex descriptions of
molecular geometry in refinement to aid in producing better
models. The refinement programs REFMAC (Murshudov
et al., 2011) and PHENIX (Afonine et al., 2005) may be
employed with ‘riding H atoms’, even though the ultimate
result to be deposited is a united-atom model. research papers Structure-validation tools for protein geometry, partially
based on the Engh & Huber standard, are available in several
widely used computer programs, most notably PROCHECK
(Laskowski et al., 1993), WHAT_CHECK (Hooft, Vriend
et al., 1996), NUCheck (Feng et al., 1998) and SFCHECK
(Vaguine et al., 1999). These programs address close non-
bonded contacts largely from a united-atom perspective. More
recently,
the
structure-validation
programs
Reduce
and
MolProbity (Davis et al., 2007; Chen, Arendall et al., 2010)
have become important and popular additions to the toolkit of
protein crystallographers. They are based on the concept that
better judgments can be made as to the correct positioning of
certain groups in the model after the addition of H atoms to a
united-atom protein crystal structure and after observing their
interactions. Within their software system, interpenetration of
van der Waals molecular surfaces by 0.4 A˚ or more constitutes
a clash. The authors flatly state that Such large overlaps cannot occur in the actual molecule, but
mean that at least one of the two atoms is modeled incorrectly 1. Introduction The majority of protein crystal structures are solved in the
resolution range 1.7–2.8 A˚ , a resolution range in which the
diffraction experiment does not present sufficient information
to accurately place individual atoms without additional
chemical information. Electron-density peaks specifically for
H atoms are not observed in this resolution range owing to a
low signal-to-noise ratio. Therefore, H atoms are usually not
explicitly included in molecular models of protein crystal
structures. A molecular model without explicit coordinates for
H atoms is denoted as an united-atom model, in contrast to an
all-atom model. United-atom models are frequently insuffi-
cient for molecular modeling and computational chemistry
applications (such as structure-based virtual screening or lead
optimization). How is the gap bridged between current best
crystallographic practices and the requirements of these other
disciplines for all-atom structures that include hydrogen
coordinates? A brief history of the use of H atoms and chemical restraints
in protein crystal structure refinement is useful before
answering this question. Jensen and coworkers (Watenpaugh
et al., 1973) first demonstrated that moderate-resolution
protein crystal structures could benefit from the reciprocal- 935 Acta Cryst. (2012). D68, 935–952 doi:10.1107/S0907444912017453 Acta Cryst. (2012). D68, 935–952 research papers model is still consistent with the experimental data. In other
words, one does not know how far is too far. This procedure
could be especially dangerous if the original clashes were
caused by atoms that were significantly misplaced. those deposited in 2010 to ensure that refinement followed
modern practices and that sufficient time had passed for
structures to be withdrawn if found to contain gross errors. Reflection data were required to have been deposited with the
coordinates. Each entry was restricted to contain one or more
protein chains but no DNA or RNA. The reported Rfree values
were limited to 0.28 or lower. Structures were required to have
a high-resolution limit between 1.5 and 2.8 A˚ . The molecular
mass of protein within the asymmetric unit was limited to
be between 10 and 300 kDa. Proteins with homologous
sequences were removed at 30% identity. The refinement program PrimeX was implemented partially
in response to these issues. It applies well established methods
of protein crystal structure refinement (Bell et al., 2012)
combined with the all-atom OPLS force field (Jorgensen et al.,
1996; Kaminski et al., 2001; Banks et al., 2005) for geometric
restraints. Aside from the presence or absence of H atoms in
the model, these OPLS-based restraints differ in two specific
respects from what have become the traditional restraint
systems: (i) a Lennard–Jones description of both the attractive
and repulsive components of van der Waals interactions
replaces the simpler repulsive term of most Engh and Huber-
based restraints and (ii) electrostatic interactions are treated,
including a Surface Generalized Born model to account for
implicit solvent effects (Ghosh et al., 1998; Gallicchio et al.,
2002; Zhu et al., 2007; Li, Abel et al., 2011). The net effect of
these differences is very significant. In a simpler restraint
system, the bond-length targets are each a function of a single
parameter according to the atom types involved in the bond. A similar situation occurs for bond angles. However, the bond-
length and bond-angle targets specified by OPLS are a func-
tion of several parameters that can all affect a single bond
length or bond angle. In other words, the restraint target for a
particular bond length (or angle) is contingent on the local
environment of the atoms involved. 2.2. PrimeX crystallographic calculations and refinement The key features of PrimeX crystal structure refinement
have been described in some detail elsewhere (Bell et al.,
2012). Only details relevant to this work are described below. 2.2.1. Restrained reciprocal-space minimization. Reciprocal-
space coordinate minimization is applied in PrimeX with a
maximum-likelihood target, using the formulation of Pannu &
Read (1996). The PrimeX implementation follows the general
concepts developed by Bru¨nger and coworkers (Bru¨nger, 1989;
Bru¨nger et al., 1998). A maximum-likelihood target has been
shown to improve the convergence of refinement and to reduce
the effects of model bias (Murshudov et al., 1997). Refinement of protein crystal structures with an all-atom
model and a complete force field does much more than avoid
errors whose remediation may seriously degrade the accuracy
of the coordinates. The more detailed accounting for non-
bonded interactions within the protein used in PrimeX can
also produce a direct positive effect during refinement. While
even small changes in the structure near a ligand-binding site
can be critical for structure-based drug discovery, examples
are presented to show how refinement with an all-atom model
can result in large coordinate improvements at such sites. OPLS 2005 (Jorgensen et al., 1996; Kaminski et al., 2001;
Banks et al., 2005) is a general-purpose force field for modeling
proteins, nucleic acids and small molecules. PrimeX applies
this consistent molecular description as geometric restraints
during the refinement of the atomic positions for all molecular
components of large biological crystal structures. Restrained
isotropic B-factor refinement is applied in PrimeX using an
approach similar to that used in the program CNS (Bru¨nger et
al., 1989). research papers Touw & Vriend (2010)
have shown that at least one type of protein bond angle is a
complex function of the local environment and is not well
described by a single Engh & Huber (1991) target angle. The
target geometric values in the well characterized restraint
system of Karplus and coworkers depend on the local back-
bone conformation of the protein (Tronrud et al., 2010). That
any particular force field can reproduce all such dependencies
remains to be demonstrated, but potentially a force-field-
based restraint system can more effectively adapt to local
environments than current protein crystallography restraint
systems. In addition, the PrimeX-calculated Rfree was required to
exceed the R value by at least 0.008. This requirement ensured
that the test set deposited in the PDB was likely to be the one
that was actually used in the refinement of the deposited
coordinates. (The Rfree calculated using the deposited test set
was found to actually be lower than the calculated R factor in
several cases, strongly indicating that the deposited test set
was not used in the final refinement of the deposited coordi-
nates.) Others have made similar observations about test-set
entries in the PDB (Joosten et al., 2009; Afonine et al., 2010). A meaningful comparison of Rfree values was not possible
without using a single consistent test set. The high-resolution reference data set used in this study was
selected from the PDB with the following restrictions: (i) the
deposition of diffraction data was required, (ii) coordinate sets
were selected from entries containing protein but no DNA or
RNA and with a high-resolution limit of 0.9 A˚ or better, (iii)
proteins with homologous sequences were removed at 30%
identity and (iv) only proteins refined with coordinates for H
atoms were included in this set. (Chen, Arendall et al., 2010). (Riding H
atoms are those H atoms whose positions can be determined
unambiguously from the positions of the non-H atoms; for
example, the H atom attached to the O of a serine residue is
not a riding H atom since its position depends on the torsion
angle of the C—O bond, while the H atom on the C atom
of an amino acid is a riding H atom, since all torsion angles
affecting its position are determined by non-H atom coordi-
nates.) The advantages of a restraint scheme in which
geometric target values for a residue depend on the torsion- The usual remedy in computational chemistry to these high-
energy close contacts is to minimize the coordinates of the all-
atom model against a force field, with non-H atoms restrained
to their positions in the crystallography-derived model so that
they do not deviate too far from their experimentally deter-
mined positions. This solution is less than ideal, because the
method produces no feedback as to whether the all-atom 936 Bell et al. PrimeX Acta Cryst. (2012). D68, 935–952 research papers research papers H-atom positions are a
function of the force field acting on all atoms, while the
positions of non-H atoms are refined under the joint influence
of crystallographic and force-field gradients. Thus, the H-atom
coordinates are not biased towards the centre of mass of the
electron-density distribution, as may occur in some forms of
all-atom refinement (Coulson & Thomas, 1971). For H atoms bonded to more electronegative atoms, elec-
trostatic forces are a major determinant of nonbonded inter-
actions and they must be evaluated during the calculation of
geometric gradients in the refinement. Thus, PrimeX employs
the complete molecular-mechanics description of atomic
interactions embodied in OPLS, including electrostatic terms
(Jorgensen et al., 1996; Kaminski et al., 2001; Banks et al.,
2005). Also included in the current PrimeX calculations was
an optional implicit solvation term (Ghosh et al., 1998;
Gallicchio et al., 2002; Zhu et al., 2007). An overview of the OPLS force field, and a description of
the details of the second-generation Surface Generalized Born
model used to implicitly account for solvation effects, have
been provided by Li, Abel et al. (2011). Both the electrostatic
and solvation calculations employ residue-based cutoffs of
15 A˚ for long-range interactions between neutral residues, of
30 A˚ between charged residues and of 20 A˚ between mixed
charged and neutral residues. Such approximations and model
features should be considered in the context of a trade-off
between computational time and rigorous calculations, as
discussed by Moulinier et al. (2003), who pioneered the use of
the Generalized Born approach in refinement, and by Fenn et
al. (2011), who have advocated the use of an alternate elec-
trostatic model in refinement. Refinement incorporating a
complete electrostatic description has been shown to lead to
lower Rfree values compared with refinement excluding these
interactions (Knight et al., 2008; Fenn et al., 2010, 2011;
Schnieders et al., 2011). 2.2.5. The polish refinement workflow. The all-atom
structures produced by PrimeX refinement, as described in
Table 4, were the result of application of the ‘polish’ workflow
to the united-atom models obtained from the PDB without
any human intervention during the refinement process. The
refined coordinates that were the result of this process are
archived at http://www.schrodinger.com/primex. The purpose of the polish workflow is to produce the best
all-atom model possible that is consistent with the diffraction
data, starting with an already well refined crystal structure. 2.1. Data-set selection The members of the moderate-resolution protein data set
used in this study were selected from the Protein Data Bank
(Berman et al., 2000). Candidate structures were limited to Because of its dependence on the OPLS force field, PrimeX
operates on all-atom models at all stages of refinement. H-atom coordinates do not participate in crystallographic 937 Bell et al. PrimeX Acta Cryst. (2012). D68, 935–952 research papers the locations of hydrogen-bond donors and acceptors to define
clusters of such sites that might be connected through
hydrogen bonds. Within each cluster, hydrogen bonding is
evaluated using a rule-based method to find an optimal
combination of the variable components of these systems. The
structural features adjusted during hydrogen-bond optimiza-
tion are (i) alcoholic H-atom positions; (ii) sulfhydryl H-atom
positions; (iii) phenolic H-atom positions; (iv) charge and
tautomeric states of aspartic acid and glutamic acid side
chains; (v) charge states, tautomeric states and orientation
(flip) of histidine side chains; (vi) orientation (flip) of aspar-
agine and glutamine side chains; and (vii) positions of H atoms
in water molecules. The goal of this procedure is to minimize
the energy of the system by maximizing the number of
hydrogen bonds while avoiding close high-energy nonbonded
interactions. This is accomplished by enumerating plausible
orientations for each rotatable hydrogen and water molecule
by identifying nearby hydrogen-bond donors and acceptors. Initial solutions for the overall local hydrogen-bond network
are then generated by iteratively choosing the optimal state
for each species in turn until convergence, starting from a
variety of random starting conditions. These initial solutions
are then recombined with each other and further optimized
via simulated annealing. The best solution obtained overall is
then chosen. The hydrogen-bond optimization tool in PrimeX
performs essentially the same tasks as a number of other
hydrogen-bond
optimization
tools
such
as
NETWORK
(Hooft, Sander et al., 1996) and Reduce (Word et al., 1999). calculations and are not influenced directly by the diffraction
data. The advantage of this approach is that the under-
determined nature of the crystallographic refinement calcu-
lation is not made worse by the many more parameters for
H-atom coordinates and B factors. H-atom positions are a
function of the force field acting on all atoms, while the
positions of non-H atoms are refined under the joint influence
of crystallographic and force-field gradients. Thus, the H-atom
coordinates are not biased towards the centre of mass of the
electron-density distribution, as may occur in some forms of
all-atom refinement (Coulson & Thomas, 1971). calculations and are not influenced directly by the diffraction
data. The advantage of this approach is that the under-
determined nature of the crystallographic refinement calcu-
lation is not made worse by the many more parameters for
H-atom coordinates and B factors. research papers Although this equation is the default method for
assigning B-factor restraint weights in the polish workflow,
stepwise optimization of this value is provided as an option. Also, minimization performed during weight optimization
may optionally be applied towards progress in the refinement
of the structure. The current work focuses specifically on interactions among
atoms within the proteins since this issue is the central concern
for computational chemistry applications. Close interactions
with water and solvent molecules will be the focus of future
work. Clashes generated from symmetry considerations were
not counted for observations on the moderate-resolution data
set since they are not optimized by the version of the Protein
Preparation Wizard used in this study. Interatomic
contacts
were
calculated
after
removing
hydrogen bonds from consideration. Because of the lack of
certainty regarding the positions of H atoms, all donor–
acceptor atom pairs that could potentially be involved in a
hydrogen bond were also excluded from the list of close
contacts, even if an H atom was not found directly between
them. This conservative approach avoided over-reporting as
clashes any interactions that might actually be hydrogen
bonds. Where alternate conformations were found, only the
conformation with the higher occupancy was considered for
the calculation of clashes. An initial optimization of hydrogen-bond orientation is
applied and is followed by separate coordinate and B-factor
reciprocal-space minimization steps. The model is then refined
with a defined set of operations comprised of reciprocal-space
coordinate
minimization,
hydrogen-bond
optimization,
reciprocal-space coordinate/B-factor minimization, simulated
annealing and a final reciprocal-space coordinate/B-factor
minimization. The
optimization
of
X-ray
and
B-factor
restraint weights as described above is repeated after this first
refinement round. The same defined set of refinement
procedures is then repeated twice more but without simulated
annealing. The definition of a clash most commonly used in protein
crystallography derives from the work of Jane Richardson,
David Richardson and coworkers (Word et al., 1999; Davis et
al., 2007; Chen, Arendall et al., 2010). It is simply the overlap
of two van der Waals surfaces by 0.4 A˚ or more. It is employed
with an atomic radius of 1.00 A˚ for polar and aromatic H
atoms and a radius of 1.17 A˚ for all other H atoms, resulting in
clashes between two H atoms with the same radii at separa-
tions of 1.60 and 1.94 A˚ , respectively. research papers corresponds to the weight employed when the minimum Rfree
value is observed. The weights selected for this set of proteins
ranged between 0.25 and 1.73. Issues surrounding the selection
of restraint weights when using an all-atom force field have
been discussed by Fenn & Schnieders (2011). B-factor
restraint weights (wB) are estimated from the high-resolution
limit (r) of the diffraction data according to the equation was employed in this study. It was based on an analysis of
28 403 structures in the Cambridge Structural Database
(Allen, 2002). Their results agreed well with the frequently
cited van der Waals radii derived by Bondi (1964) when the
available solid-state structural data were not nearly so
extensive. The largest difference between the two studies was
that the radius of the H atom was determined to be 1.1 A˚
rather than 1.2 A˚ as in the older work. wB ¼ wA102ðr2:2Þ:
ð1Þ ð1Þ As observed by Rowland & Taylor (1996), atoms may at
times have nonbonded interactions somewhat less than the
sum of their van der Waals radii. For the purposes of this work,
a center-to-center distance of less than or equal to 0.8 times
the sum of the van der Waals radii was defined as a ‘clash’. A
reasonable conclusion from the selection of data presented by
Rowland and Taylor is that interatomic distances of less than
or equal to 0.7 times the sum of van der Waals radii are rare. Such interactions were denoted as ‘severe clashes’ in this
work. The functional form of this equation was chosen based on the
known B-factor restraint-weight requirements in PrimeX at
the bounds of the usual resolution range for refinement. For a
low-resolution structure of 2.7 A˚ or worse, a weight of at least
ten times wA is required. For refinement at high resolution
(better than 1.7 A˚ ), a very low B-factor restraint weight (<0.1
times wA) is required. The values of the two constants in the
equation were varied while observing the R factors from
refinement in a broad resolution range. The current equation
was observed to be as effective within PrimeX as stepwise
optimization of wB as described for wA above and required
much less computational time. Continued development of this
method, such as the exploration of any effect of noncrys-
tallographic symmetry restraints, will be reported in future
work. research papers Clashes between two H
atoms in the current work occur at a separation of 0.8 times
the sum of their van der Waals radii, i.e. 1.76 A˚ . For most other
atoms the definition applied here is less strict than that applied
by the Richardson group. The one exception is for O atoms,
which have a smaller radius in the Richardson system,
resulting in clashes between two O atoms at 2.40 A˚ separation
compared with 2.53 A˚ in the current work. The Richardson
atomic parameters were designed from various theoretical and
practical considerations (Word et al., 1999) to yield a system
in which all observed clashes were exceptional. The approach
in the current work was to use the values for atomic radii 2.3. Direct generation of all-atom models from selected
structures To generate the all-atom models described in Table 3, H
atoms were added to united-atom models from the PDB and
the positions of the H atoms were optimized using the
hydrogen-bond network optimization function described
above (x2.2.4) as implemented in the Protein Preparation
Wizard (Maestro v.9.2; Schro¨dinger LLC). The positions of all
H atoms were then also optimized through energy minimiza-
tion against the OPLS force field, with the positions of all
heavier atoms held fixed. research papers The workflow applies reciprocal-space optimization of co-
ordinates and thermal factors, simulated-annealing refinement
and hydrogen-bond optimization in an automated manner as
described below. It does not have as a purpose the remedia-
tion of more serious errors in crystal structure fitting such as
the choice of the wrong side-chain rotamer, mis-identification
of protein electron density as part of the solvent model or the
rebuilding of misplaced side chains, which would require the
application of additional fitting functions. 2.2.2. Simulated-annealing refinement. Simulated-annealing
refinement within PrimeX is implemented through the general-
purpose molecular-modeling package IMPACT (Banks et al.,
2005), employing concepts for simulated-annealing refinement
validated in the program CNS (Adams et al., 1997). PrimeX
simulated annealing provides two alternative energy models
for dynamic simulation refinement. In the complete energy
model, all molecular-mechanics terms are evaluated during the
simulation. In the approximate method, the electrostatic and
implicit solvation terms are not evaluated, a method similar to
that employed in CNS (Adams et al., 1997). All calculations in
this work involved the complete energy model. Bond orders are first assigned throughout the structure and
H atoms are added. Initial analysis of the input structure
provides basic crystallographic statistics for the structure using
the bulk water correction in PrimeX (the flat model of Jiang &
Bru¨nger, 1994) and overall anisotropic scaling. A detailed
analysis of close nonbonded contacts for the input structure is
also provided. 2.2.3. Electron-density map calculations. Map calculations
in PrimeX are based on the SIGMAA weighting scheme of
Read (1986), a data treatment that has been shown to
decrease the bias in electron-density maps. As a next step, reciprocal-space minimization is applied at
increasing weight on the X-ray terms (wA) in order to optimize
this weight for subsequent refinement. The value selected 2.2.4. Hydrogen-bond network optimization. An addi-
tional hydrogen-bond optimization tool in PrimeX analyzes 938 Bell et al. PrimeX Acta Cryst. (2012). D68, 935–952 research papers Table 1
Ch † A clash occurs when two atoms approach to within less than or equal to 0.8 times the sum of their van der Waals radii but greater than 0.7 times that sum. ‡ A severe clash occurs
when two atoms approach to within less than or equal to 0.7 times the sum of their van der Waals radii. Table 2
Classification of clashes and severe clashes requiring correction in Table 1. Classification
No. of clashes
and severe
clashes
Hydroxyl (or sufhydryl) group: rotation around carbon–
oxygen (sulfur) bond relieves clash
27
Mistake in positioning of non-H atoms in the electron
density
15
Flip of asparagine or glutamine residue relieves clash
4
Flip or alternate tautomer of histidine residue relieves
clash
5 (Rowland & Taylor, 1996) unadjusted and to observe from
ultrahigh-resolution protein structures how frequently clashes
might reasonably be expected to occur owing to the local
molecular environment. 2.4.2. Calculation of summary geometry statistics. Bond-
length, bond-angle and torsion-angle statistics were calculated
in NUCheck (Feng et al., 1998). Side-chain group planarity
deviation was calculated using the Protein Reports utility in
PrimeX. 3. Results In 37% of the clashes a chemically implausible interaction was
observed in which a hydroxyl or sulfhydryl H atom pointed
directly at another H atom. The positions of these H atoms
were not supported in any obvious way by the observed
electron density. The most likely explanation is that these
hydrogen positions were oversights in the model-building
process. In addition, 21% of the close interactions identified
occurred at positions where the heavy atoms to which the H
atoms were attached did not fit the electron density well and
appeared to be incorrectly positioned. Clashes were included
in this category only if the electron-density map provided
reasonable doubt as to the correctness of the structure and
suggested a more attractive alternate position. A further 12%
of close contacts could be removed by flipping or changing the
tautomer of an asparagine, glutamine or histidine side chain. 2.4. Structure-validation calculations 2.4.1. Clash detection and van der Waals radii. The
Rowland & Taylor (1996) compilation of van der Waals radii 939 Bell et al. PrimeX Acta Cryst. (2012). D68, 935–952 Acta Cryst. (2012). D68, 935–952 research papers Table 1
Characteristics of ultrahigh-resolution protein structures. Table 1
Ch Table 1
Characteristics of ultrahigh-resolution protein structures. ‘Corrected’
PDB
code
Resolution
(A˚ )
No. of
residues
Bond-
length
r.m.s.d. (A˚ )
Bond-
angle
r.m.s.d. ()
Side-chain
planarity
r.m.s.d. (A˚ )
!-Angle
standard
deviation
()
Clashes†
Severe
clashes‡
Clashes†
Severe
clashes‡
Reference
1byz
0.90
52
0.019
2.0
0.006
2.9
0
1
0
0
Prive´ et al. (1999)
1dy5
0.87
248
0.020
2.6
0.013
6.6
8
2
2
0
Esposito et al. (2000)
1i1w
0.89
303
0.041
2.9
0.016
6.4
1
2
0
0
Natesh et al. (2003)
1m40
0.85
263
0.014
2.6
0.009
6.1
1
0
0
0
Minasov et al. (2002)
1muw
0.86
386
0.016
2.5
0.010
8.2
18
3
7
0
Fenn et al. (2004)
1p9g
0.84
41
0.017
2.4
0.009
6.7
0
0
0
0
Xiang et al. (2004)
1ucs
0.62
64
0.014
2.2
0.010
5.3
0
1
0
0
Ko et al. (2003)
1vyr
0.90
364
0.017
2.3
0.012
5.8
0
0
0
0
Khan et al. (2004)
1yk4
0.69
52
0.022
2.7
0.011
5.7
2
3
0
0
Bo¨nisch et al. (2005)
2b97
0.75
142
0.028
2.8
0.016
7.6
2
0
0
0
Hakanpa¨a¨ et al. (2006)
2h5c
0.82
198
0.025
2.4
0.015
7.3
2
0
2
0
Fuhrmann et al. (2006)
2vb1
0.65
129
0.021
3.1
0.012
7.1
1
0
0
0
Wang et al. (2007)
2wur
0.90
236
0.034
3.3
0.016
6.6
10
5
7
0
Shinobu et al. (2010)
2xu3
0.90
220
0.011
1.4
0.007
5.7
1
1
1
1
Hardegger et al. (2011)
3a38
0.70
83
0.028
2.9
0.010
6.8
0
0
0
0
Takeda et al. (2010)
3g63
0.88
381
0.014
2.1
0.009
6.7
2
0
1
0
Liebschner et al. (2009)
3ip0
0.89
158
0.012
1.5
0.007
6.1
1
0
0
0
Blaszczyk et al. (2003)
3mi4
0.80
223
0.028
2.5
0.015
6.8
3
3
1
0
A. Brzuszkiewicz, M. Dauter &
Z. Dauter (unpublished work)
Total
3543
52
21
21
1
Mean
0.021
2.5
0.011
6.4
Count per 100 residues
1.5
0.6
0.6
0.03
† A clash occurs when two atoms approach to within less than or equal to 0.8 times the sum of their van der Waals radii but greater than 0.7 times that sum. ‡ A severe clash occurs
when two atoms approach to within less than or equal to 0.7 times the sum of their van der Waals radii. research papers the close contacts actually present in the structures. Thus, the
frequency of bona fide ‘clashes’ and ‘severe clashes’ was 0.6
and 0.03 per 100 residues, respectively. Note that the second
value is based on just a single observation (one severe clash in
2xu3). respectively. Overall, clashes in the moderate-resolution
structure set were observed at a frequency of 4.0 per 100
residues, over six times the rate of bona fide clashes in the
ultrahigh-resolution set. Severe clashes were observed at a
frequency of 0.5 per 100 residues, compared with 0.03 for bona
fide severe clashes for the reference ultrahigh-resolution data
set (Table 1). respectively. Overall, clashes in the moderate-resolution
structure set were observed at a frequency of 4.0 per 100
residues, over six times the rate of bona fide clashes in the
ultrahigh-resolution set. Severe clashes were observed at a
frequency of 0.5 per 100 residues, compared with 0.03 for bona
fide severe clashes for the reference ultrahigh-resolution data
set (Table 1). )
3.1.2. Summary geometry statistics. The average r.m.s. deviations (r.m.s.d.s) of bond lengths and angles with respect
to the Engh & Huber (1991) standard were 0.021 A˚ and 2.5,
respectively (Table 1). The significance of the former number
might be questioned since at this resolution the refined bond
lengths are likely to reflect the effects of restraints. However,
atomic positions are observed accurately enough at this
resolution such that bond angles can be precisely determined
from the crystallographic results. Thus, the latter value may
be significant. However, two observations must be considered
in interpreting this bond-angle value. Firstly, the program
SHELX (Sheldrick & Schneider, 1997) may apply bond-angle
restraints (as 1,3-atom distance restraints) during refinement,
although published information rarely allows one to deduce
the effect of these possible restraints on bond angles. Normally, one might expect the net effect of restraints would
be to narrow the distribution of values observed. At the same
time, a critical observation is that high-resolution protein
crystal structures very frequently have r.m.s. Z scores (Spronk
et al., 2004) greater than 1, i.e. the standard deviation of bond
angles for these structures are greater than what would be
predicted from the work of Engh & Huber (1991). 3.3. All-atom refinement with PrimeX 3.3.1. Clashes and severe clashes. The additional all-atom
refinement in PrimeX applied to the moderate-resolution data
set produced the structures characterized in Table 4. The
frequency of regular clashes overall was 0.9 per 100 residues,
which is well below the frequency originally observed for the
ultrahigh-resolution set (1.5 per 100 residues; Table 1), but
somewhat higher than the corrected value of 0.6 per 100
residues. The frequency of clashes overall was decreased more
than fourfold from all-atom models derived from the coordi-
nates as originally deposited. The frequency of severe clashes
overall was 0.03 per 100 residues, the same value as obtained
for the corrected ultrahigh-resolution structures (Table 1) and
17-fold lower than the frequency in the otherwise remediated
moderate-resolution structures (Table 3). Seven of the 94
structures had neither type of clashes after all-atom refine-
ment. All structures without clashes were solved at 2.2 A˚
resolution or better. 39 of the 94 structures had both no severe
clashes and a lower frequency of clashes than the corrected
ultrahigh-resolution structures. The average r.m.s.d. from planarity for side-chain groups
was 0.011 A˚ and the average standard deviation of the ! angle
was 6.4. For similar reasons, these two values may be
considered to be reference points for the geometry of models
at moderate resolution. Each of the residual clashes in the PrimeX-refined set was
inspected with reference to a 2Fo Fc composite OMIT map. Clear evidence of a better alternate interpretation of the
electron density was present for 14% of the close contacts,
owing to either large problems with the main-chain fit or to
the need for a substantially different side-chain rotamer. Subtracting the number of clashes attributable to these issues
from the total clashes provided an estimate of the frequency
of bona fide regular clashes as 0.7 clashes per 100 residues,
approaching the corrected frequency found in the ultrahigh-
resolution structure set (0.6 clashes per 100 residues). Within
this structural survey, many situations were observed to be
ambiguous and were not counted. Thus, the level of clashes
owing to model errors might actually have been somewhat
higher. A very time-intensive comprehensive re-refinement of
the structures would be required to confirm this suspicion,
which is beyond the scope of the present work. research papers This
observation (Joosten et al., 2009) is generally interpreted to
mean that the bond angles are too widely distributed in very
high resolution structures, possibly because these restraints
are faulty owing to variations in bond lengths at high resolu-
tion. The possibility that the Engh and Huber parameters
predict too narrow a distribution owing to biases in the small-
molecule structures from which the parameters were derived
is generally discounted. 3.2.2. Summary geometry statistics. As shown in Table 3,
the bond-length r.m.s.d.s for the set of proteins varied over a
wide range, from 0.004 A˚ for 3phe to 0.031 A˚ for 3lje, with
an average of 0.014 A˚ . Bond-angle r.m.s.d.s varied from a
minimum of 0.6 (3ni0) to a maximum of 2.5 (3nof), with an
average of 1.4. The average r.m.s.d. for side-chain group
planarity was 0.005 A˚ and the average peptide torsion-angle
standard deviation was 5.1. A more detailed examination and
comparison of these summary statistics follows in x3.3.2. 3.1. Ultrahigh-resolution structures Before exploring the close contacts (clashes and severe
clashes as defined above) and structural geometry at moderate
resolution, some perspective can be obtained on summary
geometric statistics and the occurrence of clashes from ultra-
high-resolution protein structures. 3.1.1. Clashes and severe clashes. Table 1 shows observa-
tions from 18 X-ray crystal structures with at least 0.9 A˚
resolution which were refined (by the authors of the respective
structures) with H atoms present. (At this resolution hydrogen
positions may have been guided by electron density, although
electron density need not have been observed for all H atoms.)
Over the entire set of structures, ‘clashes’ and ‘severe clashes’
were observed with a frequency of 1.5 and 0.6 occurrences per
100 residues, respectively. Ultimately, only 30% of the observed clashes withstood
critical examination and avoided being included in Table 2. In Table 1, the ‘corrected’ columns offer a better estimate of All of the observed clashes were examined individually to
determine their origin. The results are presented in Table 2. 940
Bell et al. PrimeX Acta Cryst. (2012). D68, 935–952 research papers research papers 942
Bell et al
PrimeX
Acta Cryst (2012) D68 935–952
Table 3
Statistics for structures of moderate-resolution data in the PDB. PDB
code
Resolution
(A˚ )
No. of
residues
Refinement
program
Bond-
length
r.m.s.d. (A˚ )
Bond-
angle
r.m.s.d. ()
Side-chain
planarity
r.m.s.d. (A˚ )
!-Angle
standard
deviation
()
R
factor† Rfree† Clashes‡
Severe
clashes§ Reference
2x3k
2.50
1145
REFMAC
0.015
1.5
0.004
6.7
0.216
0.266
58
7
Schmelz (2010)
2xda
1.85
150
REFMAC
0.014
1.3
0.005
5.7
0.189
0.216
2
1
Paz et al. (2011)
2xn8
1.64
409
REFMAC
0.023
1.8
0.009
6.2
0.181
0.220
6
0
Ouellet et al. (2011)
2xpp
1.74
161
REFMAC
0.018
1.6
0.005
4.8
0.208
0.252
5
0
Diebold et al. (2010)
2xs6
2.09
166
REFMAC
0.007
1.0
0.002
4.9
0.219
0.258
4
0
}
2xsn
2.68
1341
BUSTER
0.013
1.6
0.008
2.8
0.224
0.260
13
2
J. R. C. Muniz, C. D. O. Cooper, W. W. Yue,
E. Krysztofinska, F. Vondelft, S. Knapp,
O. Gileadi, C. H. Arrowsmith,
A. M. Edwards, J. Weigelt, C. Bountra,
K. L. Kavanagh & U. Oppermann
(unpublished work)
2xsq
1.72
179
REFMAC
0.009
1.2
0.004
5.4
0.173
0.191
0
1
}
2xsw
1.90
666
PHENIX
0.011
1.3
0.004
5.6
0.184
0.209
8
0
}
2xsx
1.70
869
REFMAC
0.015
1.5
0.007
5.9
0.177
0.207
11
3
M. Vollmar, E. Krysztofinska, A. Chaikuad,
T. Krojer, R. Cocking, F. Vondelft,
C. Bountra, C. H. Arrowsmith, J. Weigelt,
A. Edwards, W. W. Yue & U. Oppermann
(unpublished work)
2xu7
1.90
752
REFMAC
0.023
1.8
0.008
7.1
0.211
0.245
14
1
Lejon et al. (2011)
2xul
2.20
645
REFMAC
0.029
2.0
0.005
6.5
0.196
0.235
20
1
Fokina et al. (2010)
2xvs
1.80
166
REFMAC
0.016
1.5
0.007
5.8
0.200
0.249
0
0
Adams et al. (2012)
2xvv
2.40
582
CNS
0.007
1.2
0.005
1.0
0.211
0.252
35
1
Ryan et al. (2011)
2xxj
1.96
1239
PHENIX
0.007
1.0
0.003
9.0
0.190
0.237
27
3
J. Tickle, E. De Mendoza Barbera &
F. M. D. Vellieux (unpublished work)
3acw
1.63
284
REFMAC
0.007
1.5
0.006
4.9
0.228
0.252
5
0
Lin et al. (2010)
3aey
1.92
698
CNS
0.005
1.2
0.004
1.2
0.192
0.214
29
5
Murakawa et al. (2011)
3ajx
1.60
828
CNS
0.004
1.2
0.004
1.3
0.198
0.218
8
0
Orita et al. (2010)
3ale
2.50
1460
CNS
0.007
1.3
0.004
1.3
0.227
0.279
117
15
Morita et al. research papers (2010)
3am9 2.17
2584
REFMAC
0.022
1.9
0.008
6.9
0.182
0.248
99
12
Matsumoto et al. (2010)
3l9w
1.75
695
REFMAC
0.018
1.6
0.007
5.6
0.212
0.239
16
1
Roosild et al. (2010)
3lb4
1.56
274
REFMAC
0.011
1.3
0.005
4.2
0.253
0.281
10
0
Thompson et al. (2010)
3lfl
2.10
686
PHENIX
0.007
1.0
0.002
5.2
0.207
0.266
43
8
Zhou et al. (2011)
3lje
1.75
121
CNS
0.031
2.4
0.010
3.4
0.185
0.217
14
3
Pizzo et al. (2010)
3ljq
1.90
570
CNS
0.012
1.3
0.006
7.3
0.155
0.197
17
1
Wang & Guo (2010)
3lju
1.70
373
REFMAC
0.016
1.4
0.007
6.0
0.203
0.238
1
0
Tong et al. (2010)
3lpf
2.26
1180
REFMAC
0.011
2.2
0.004
8.6
0.275
0.300
241
34
Wallace et al. (2010)
3lre
2.20
589
REFMAC
0.020
1.7
0.006
6.4
0.231
0.278
28
3
Peters et al. (2010)
3lrp
2.50
181
CNS
0.006
1.2
0.004
1.2
0.197
0.263
7
1
Cook et al. (2010)
3lt3
2.10
404
REFMAC
0.006
0.9
0.002
4.3
0.242
0.293
5
0
Biswas et al. (2010)
3m0e 2.63
1729
PHENIX
0.010
1.2
0.003
5.3
0.216
0.250
90
11
Chen, Sysoeva et al. (2010)
3m0h 1.58
1685
CNS
0.004
1.2
0.004
1.3
0.162
0.182
28
6
Yoshida et al. (2010)
3m4z 1.94
309
REFMAC
0.012
1.2
0.004
4.9
0.177
0.202
2
0
Heldman et al. (2010)
3m5o 1.60
410
REFMAC
0.009
1.2
0.004
5.9
0.240
0.270
10
3
Romano et al. (2010)
3m67 1.80
257
REFMAC
0.027
2.0
0.010
7.0
0.178
0.230
9
0
Cˇ apkauskaite˙ et al. (2010)
3mbv 2.00
222
REFMAC
0.016
1.4
0.007
5.1
0.270
0.282
2
1
Borshchevskiy et al. (2010)
3mfa
1.63
194
REFMAC
0.013
1.3
0.004
6.9
0.198
0.223
8
0
Morin et al. (2011)
3mif
2.00
310
REFMAC
0.009
1.1
0.002
6.1
0.261
0.279
11
1
Chufa´n et al. (2010)
3mk9 2.08
173
REFMAC
0.006
1.1
0.005
1.0
0.216
0.242
7
0
Compton et al. (2011)
3mke 1.75
265
REFMAC
0.012
1.3
0.005
5.8
0.158
0.188
1
0
Ke et al. (2011)
3mvi
1.60
698
REFMAC
0.012
1.3
0.004
5.4
0.174
0.211
10
2
Niu et al. (2010)
3mxe 1.85
198
REFMAC
0.009
1.2
0.003
6.1
0.202
0.241
1
0
Ali et al. (2010)
3n2v
1.55
158
REFMAC
0.025
1.8
0.011
6.1
0.187
0.204
1
0
Attolino et al. 3.2. Moderate-resolution crystal structures as deposited 94 crystal structures with a broad range of sizes from several
different refinement programs were examined (Table 3). Their
most important common characteristics were recent deposi-
tion in the PDB and falling into the most highly populated
resolution range typical for protein crystal structures (see x2
for further details on the selection of this data set). 3.2.1. Clashes and severe clashes. The occurrence of close
contacts was enumerated after addition of H atoms and after
careful optimization of H-atom positions without changing the
coordinates of any non-H atoms. Table 3 shows the frequency
of clashes and severe clashes for each protein. Their rates of
occurrence per 100 residues were observed in a very broad
range from 20.4 (3lpf) to 0.0 (three instances) and from 2.9
(3lpf) to 0.0 (35 instances) for clashes and severe clashes, Clash frequencies derived from Tables 1, 3 and 4 are
compared in Table 5, as well as with respect to the various 941 Bell et al. PrimeX Acta Cryst. (2012). D68, 935–952 942
Bell et al.
PrimeX research papers (2010)
3nfy
1.94
498
REFMAC
0.019
1.8
0.007
6.3
0.187
0.279
23
6
Patterson et al. (2010)
3ni0
1.60
182
PHENIX
0.003
0.6
0.001
3.2
0.230
0.257
2
1
Swiecki et al. (2011)
3nk4
2.00
581
PHENIX
0.010
1.2
0.003
5.7
0.234
0.242
14
1
Han et al. (2010)
3nl6
2.61
1545
PHENIX
0.007
1.2
0.002
5.2
0.238
0.252
174
22
Paul et al. (2010)
3nm8 2.00
570
REFMAC
0.010
1.4
0.005
2.4
0.230
0.278
53
6
Sendovski et al. (2010)
3nmi
2.01
636
REFMAC
0.008
0.9
0.014
6.9
0.207
0.245
30
1
Radford et al. (2011)
3nof
1.60
213
REFMAC
0.029
2.5
0.011
5.9
0.197
0.225
19
3
Hall et al. (2011)
3nok 1.65
466
REFMAC
0.023
1.9
0.009
7.0
0.199
0.243
11
0
Carrillo et al. (2010)
3nv6
2.20
404
REFMAC
0.016
1.5
0.005
5.9
0.200
0.264
14
2
Yang et al. (2010)
3nxg
1.95
1291
REFMAC
0.008
1.1
0.002
5.9
0.180
0.213
21
0
Neu et al. (2010)
3nxp
2.20
363
REFMAC
0.011
1.3
0.003
6.1
0.213
0.239
11
2
Chen, Pelc et al. (2010)
3o0a
1.77
425
REFMAC
0.009
1.2
0.003
9.0
0.211
0.250
6
1
V. Cura, N. Olieric, E.-D. Wang, D. Moras, G
Eriani & J. Cavarelli (unpublished work)
3o3p
1.70
635
PHENIX
0.007
1.1
0.002
5.2
0.221
0.255
39
4
Empadinhas et al. (2011)
3o4h
1.82
2302
REFMAC
0.019
1.7
0.007
6.2
0.244
0.267
71
19
Harmat et al. (2011)
3o79
1.60
202
REFMAC
0.022
1.7
0.009
5.3
0.210
0.237
4
2
Khan et al. (2010) Table 3
Statistics for structures of moderate-resolution data in the PDB. 942
Bell et al. PrimeX Acta Cryst. (2012). D68, 935–952 research papers refinement programs. Unfortunately, refinement results from
BUSTER (Bricogne et al., 2011) were found to be relatively
rare and only two instances were found in our data set. However
the statistics for these two proteins suggest
0.6–0.7 clashes per 100 residues that might be considered a
reasonable target considering the results above. 3.3.2. Summary
molecular
geometry
and
refinement
statistics Table 5 also provides a summary of the measures
Table 3 (continued)
PDB
code
Resolution
(A˚ )
No. of
residues
Refinement
program
Bond-
length
r.m.s.d. (A˚ )
Bond-
angle
r.m.s.d. ()
Side-chain
planarity
r.m.s.d. (A˚ )
!-Angle
standard
deviation
()
R
factor† Rfree† Clashes‡
Severe
clashes§ Reference
3o86
1.60
709
PHENIX
0.010
1.3
0.004
5.8
0.173
0.198
17
0
Eidam et al. research papers (2010)
3oae
2.80
2105
CNS
0.011
1.5
0.006
1.4
0.272
0.288
238
40
Dasgupta et al. (2011)
3oag
2.30
669
REFMAC
0.009
1.2
0.003
7.8
0.208
0.262
13
2
Corminboeuf et al. (2010)
3oc2
1.97
495
PHENIX
0.007
1.1
0.002
5.4
0.181
0.223
21
3
Sainsbury et al. (2010)
3occ
1.70
1424
REFMAC
0.015
1.5
0.010
6.6
0.169
0.197
29
8
New York SGX Research Center for
Structural Genomics (unpublished work)
3oep
1.75
485
REFMAC
0.006
1.1
0.003
5.4
0.191
0.215
0
0
Jacques et al. (2011)
3oi7
2.40
1028
REFMAC
0.012
1.3
0.003
5.9
0.237
0.269
19
8
Clasquin et al. (2011)
3oia
1.65
405
REFMAC
0.011
1.3
0.004
5.7
0.191
0.241
6
0
Lee et al. (2011)
3olz
2.75
743
PHENIX
0.006
0.9
0.002
5.0
0.211
0.265
26
1
Kumar & Mayer (2010)
3om1 1.68
740
PHENIX
0.006
1.0
0.002
5.0
0.193
0.224
14
1
Kumar & Mayer (2010)
3onw 2.38
702
REFMAC
0.009
1.1
0.002
5.1
0.251
0.280
13
1
Bosch et al. (2011)
3orv
1.91
1707
REFMAC
0.025
1.9
0.009
6.4
0.171
0.212
22
1
Abu Tarboush et al. (2010)
3oux
2.40
550
PHENIX
0.008
1.1
0.002
4.7
0.219
0.285
35
3
Sun & Weis (2011)
3p10
1.70
471
REFMAC
0.012
1.3
0.004
5.2
0.184
0.218
7
0
Begley et al. (2011)
3p14
2.51
1608
REFMAC
0.019
1.8
0.005
6.5
0.201
0.266
108
22
T. T. N. Doan, P. Prabhu, J. K. Lee, L. W. Wang, J. K. Kim, M. Jeya & Y. J. Ahn
(unpublished work)
3p1a
1.70
281
REFMAC
0.015
1.5
0.006
5.1
0.169
0.211
4
0
Structural Genomics Consortium
(unpublished work)
3p1m 2.54
1017
REFMAC
0.013
1.3
0.003
5.8
0.240
0.270
28
2
Structural Genomics Consortium
(unpublished work)
3p2e
1.68
402
CNS
0.005
1.2
0.005
1.4
0.207
0.244
16
0
Husain et al. (2011)
3p32
1.90
306
REFMAC
0.015
1.3
0.004
5.6
0.226
0.254
3
0
Seattle Structural Genomics Center for
Infectious Disease (unpublished work)
3p4i
2.35
760
REFMAC
0.016
1.5
0.003
5.7
0.200
0.244
11
0
Seattle Structural Genomics Center for
Infectious Disease (unpublished work)
3p4l
1.80
198
CNS
0.010
1.6
0.008
2.2
0.205
0.240
7
2
Yang et al. (2011)
3p5o
1.60
127
REFMAC
0.007
1.0
0.003
4.9
0.179
0.207
1
0
Nicodeme et al. (2010)
3p5t
2.70
1653
REFMAC
0.010
1.3
0.003
6.1
0.225
0.272
179
33
Li, Tong et al. research papers (2011)
3p77
1.60
371
REFMAC
0.012
1.4
0.005
6.1
0.194
0.226
2
0
Hee et al. (2010)
3p7h
2.30
520
REFMAC
0.110
1.2
0.004
6.5
0.204
0.245
16
2
Chatwell et al. (2008)
3p8s
2.00
302
REFMAC
0.010
1.2
0.003
6.2
0.192
0.226
3
0
U. Sharma, N. Ahmed, M. V. Krishnasastry
& C. G. Suresh (unpublished work)
3paj
2.00
599
REFMAC
0.009
1.2
0.007
2.0
0.213
0.259
20
2
Center for Structural Genomics of Infectious
Diseases (unpublished work)
3pde
1.75
1149
REFMAC
0.013
1.6
0.004
5.0
0.177
0.209
17
0
New York SGX Research Center for
Structural Genomics (unpublished work)
3pdt
1.80
251
REFMAC
0.011
1.3
0.003
5.9
0.202
0.247
4
0
Crawley et al. (2011)
3peh
2.75
523
REFMAC
0.013
1.7
0.007
2.5
0.236
0.279
8
1
Structural Genomics Consortium
(unpublished work)
3pgj
2.49
1075
REFMAC
0.007
1.4
0.004
0.9
0.238
0.279
40
1
Center for Structural Genomics of Infectious
Diseases (unpublished work)
3pgy
1.92
1609
REFMAC
0.017
1.5
0.006
5.7
0.190
0.227
29
2
Center for Structural Genomics of Infectious
Diseases (unpublished work)
3ph7
2.50
1376
BUSTER
0.014
1.7
0.008
2.4
0.249
0.291
11
0
Artz et al. (2011)
3phe
2.20
2232
CNX
0.004
0.7
0.003
5.1
0.222
0.265
131
15
Kumar et al. (2011)
3pj9
2.10
549
REFMAC
0.017
1.5
0.005
6.2
0.206
0.221
16
2
Center for Structural Genomics of Infectious
Diseases (unpublished work)
3pjp
1.60
389
PHENIX
0.006
1.0
0.003
5.6
0.214
0.251
5
0
Sun et al. (2010)
3pk0
1.74
1043
REFMAC
0.017
1.5
0.007
5.5
0.184
0.210
5
0
Seattle Structural Genomics Center for
Infectious Disease (unpublished work)
Total
66891
2639
349
Mean
0.014
1.4
0.005
5.1
0.207
0.243
No. per 100 residues
4.0
0.5
† As calculated in PrimeX without TLS scaling. ‡ A clash occurs when two atoms approach to within less than or equal to 0.8 times the sum of their van der Waals radii but greater
than 0.7 times that sum. § A severe clash occurs when two atoms approach to within less than or equal to 0.7 times the sum of their van der Waals radii. } L.Tresaugues, M. Welin, C. H. Arrowsmith, H. Berglund, C. Bountra, R. Collins, A. M. Edwards, S. Flodin, A. Flores, S. Graslund, M. Hammarstrom, I. Johansson, T. Karlberg, S. Kol, T. Kotenyova, E. Kouznetsova,
M. Moche, T. Nyman, C. Persson, H. Schuler, P. Schutz, M. I. Siponen, A. G. Thorsell, S. Table 4 944
Bell et al
PrimeX
Acta Cryst (2012) D68 9
Table 4
Statistics for structures in the moderate-resolution data set as refined in PrimeX. PDB code
Resolution (A˚ )
No. of
residues
Bond-length
r.m.s.d. (A˚ )
Bond-angle
r.m.s.d. ()
Side-chain planarity
r.m.s.d. Acta Cryst. (2012). D68, 935–952 research papers Van der Berg, E. Wahlberg, J. Weigelt & P. Nordlund (unpublished work). Table 3 (continued) † As calculated in PrimeX without TLS scaling. ‡ A clash occurs when two atoms approach to within less than or equal to 0.8 times the sum of their van der Waals radii but greater
than 0.7 times that sum. § A severe clash occurs when two atoms approach to within less than or equal to 0.7 times the sum of their van der Waals radii. } L.Tresaugues, M. Welin, C. H. Arrowsmith, H. Berglund, C. Bountra, R. Collins, A. M. Edwards, S. Flodin, A. Flores, S. Graslund, M. Hammarstrom, I. Johansson, T. Karlberg, S. Kol, T. Kotenyova, E. Kouznetsova,
M. Moche, T. Nyman, C. Persson, H. Schuler, P. Schutz, M. I. Siponen, A. G. Thorsell, S. Van der Berg, E. Wahlberg, J. Weigelt & P. Nordlund (unpublished work). refinement programs. Unfortunately, refinement results from
BUSTER (Bricogne et al., 2011) were found to be relatively
rare and only two instances were found in our data set. However, the statistics for these two proteins suggest
improved results from this program regarding close contacts. None of the other programs even approached the values of 0.6–0.7 clashes per 100 residues that might be considered a
reasonable target considering the results above. 3.3.2. Summary
molecular
geometry
and
refinement
statistics. Table 5 also provides a summary of the measures
of molecular geometry over the three data sets in this study. The average bond-length r.m.s.d. for the PrimeX-refined 943 Bell et al. PrimeX Acta Cryst. (2012). D68, 935–952 944
Bell et al.
PrimeX research papers Z score of
0.87 (0.49–1.23), values that are very similar to those of the
PrimeX-refined structures. Individual bond-length r.m.s. Z
scores are available as Supplementary Material1. The
average
side-chain
group
planarity
r.m.s.d. for
PrimeX was 0.006 A˚ , which is a fairly typical value for this
quantity among the refinement programs. Side-chain planarity
deviations were all small and not very different between these
two data sets, nor did they differ much by refinement program
(Table 5). The average !-angle standard deviation for PrimeX, 7.1,
was larger than for any of the refinement programs used to
produce the original moderate-resolution data set, but com-
pared well with the value of 6.4 obtained for the ultrahigh-
resolution set (Table 5). The values among all the refinement
programs could be described as a range of values from 4.8 to
5.7, with two outliers near 2 for BUSTER and CNS/CNX. The number of examples of BUSTER-refined proteins was too
small to draw a conclusion. However, CNS and CNX clearly
often restrain the ! angle very tightly. This issue was originally
observed by Priestle (2003). Note that the overall average for
the ! angles did not represent the situation well, since two
of the 12 structures in the CNS/CNX subgroup had standard
deviations in the normal range (Table 3). These two unchar-
acteristic CNS/CNX structures indicate that at least a few
users of CNS/CNX have taken steps to loosen these peptide-
bond planarity restraints. Ten of the 12 members of the CNS/
CNX subgroup had standard deviations for ! of 1.5 or less,
implying flattened peptide bonds throughout these crystal
structures. The !-angle standard deviation did not vary much
among the other refinement programs. The average value for
REFMAC did not stand out as the one from CNS/CNX does. Table 5 also allows comparison among the four programs
originally used to refine the moderate-resolution data set. PHENIX and CNS clearly restrained bond lengths more
tightly than did PrimeX. The bond-length r.m.s.d. for the
REFMAC-refined set was not very different from the PrimeX-
refined set. The average bond-angle r.m.s.d. for PrimeX was 2.2, which
is somewhat larger than the average value of 1.4 over the
original data set. The r.m.s. Z scores for bond angles changed
from an average value of 0.71 (0.37–1.25) as deposited to an
average value of 1.17 (1.00–1.49) after PrimeX refinement. The average bond-angle r.m.s.d. research papers research papers Table 4 (continued) Table 4 (continued)
PDB code
Resolution (A˚ )
No. of
residues
Bond-length
r.m.s.d. (A˚ )
Bond-angle
r.m.s.d. ()
Side-chain planarity
r.m.s.d. (A˚ )
!-Angle standard
deviation ()
R factor Rfree
Clashes Severe clashes
3p10
1.70
471
0.018
2.1
0.006
6.3
0.173
0.222
4
0
3p14
2.51
1608
0.015
2.1
0.005
7.1
0.211
0.259
21
5
3p1a
1.70
281
0.023
2.0
0.009
5.8
0.162
0.212
2
0
3p1m
2.54
1017
0.013
2.1
0.002
6.9
0.231
0.273
8
0
3p2e
1.68
402
0.021
2.2
0.008
6.6
0.186
0.234
3
0
3p32
1.90
306
0.024
2.3
0.005
7.3
0.204
0.252
3
0
3p4i
2.35
760
0.019
2.1
0.004
7.0
0.195
0.241
2
0
3p4l
1.80
198
0.018
2.0
0.008
7.2
0.200
0.248
1
0
3p5o
1.60
127
0.023
2.1
0.008
6.1
0.156
0.209
1
0
3p5t
2.70
1653
0.016
2.0
0.004
7.4
0.230
0.285
4
0
3p77
1.60
371
0.021
2.3
0.008
6.8
0.173
0.221
2
0
3p7h
2.30
520
0.016
2.0
0.005
8.1
0.200
0.244
2
0
3p8s
2.00
302
0.018
2.2
0.006
7.7
0.165
0.221
2
0
3paj
2.00
599
0.018
2.2
0.006
6.2
0.179
0.251
6
0
3pde
1.75
1149
0.020
2.1
0.006
6.1
0.169
0.212
8
0
3pdt
1.80
251
0.018
2.0
0.005
7.6
0.182
0.237
2
0
3peh
2.75
523
0.019
2.6
0.006
8.4
0.234
0.288
10
0
3pgj
2.49
1075
0.014
1.9
0.003
7.0
0.249
0.287
3
0
3pgy
1.92
1609
0.017
2.0
0.005
6.7
0.173
0.225
8
0
3ph7
2.50
1376
0.016
2.0
0.006
6.4
0.248
0.304
8
0
3phe
2.20
2232
0.016
2.0
0.004
7.7
0.213
0.255
11
1
3pj9
2.10
549
0.021
2.2
0.004
7.3
0.185
0.220
10
0
3pjp
1.60
389
0.022
2.4
0.008
7.7
0.203
0.253
2
0
3pk0
1.74
1043
0.023
2.3
0.007
6.2
0.160
0.207
7
0
Total
66891
573
23
Mean
1.99
0.019
2.2
0.006
7.1
0.193
0.242
No. per 100 residues
0.9
0.03 of the PrimeX-refined structures. Individual bond-angle r.m.s. Z scores are available in the Supplementary Material1. proteins was 0.019 A˚ , compared with 0.015 A˚ for the original
data set. The r.m.s. Z scores (Spronk et al., 2004) for bond
lengths changed from an average value of 0.56 (0.15–1.27) as
deposited to an average value of 0.89 (0.65–1.24) after PrimeX
refinement. The ultrahigh-resolution set had a bond-length
r.m.s.d. of 0.021 A˚ and a mean bond-length r.m.s. 1 Supplementary material has been deposited in the IUCr electronic archive
(Reference: RR5017). Services for accessing this material are described at the
back of the journal. Table 4 (A˚ )
!-Angle standard
deviation ()
R factor Rfree
Clashes Severe
2x3k
2.50
1145
0.019
2.3
0.005
8.0
0.212
0.267
19
0
2xda
1.85
150
0.025
2.5
0.007
6.9
0.175
0.215
3
0
2xn8
1.64
409
0.022
2.2
0.005
6.6
0.170
0.221
2
0
2xpp
1.74
161
0.016
1.9
0.005
5.2
0.197
0.251
3
0
2xs6
2.09
166
0.019
2.0
0.004
6.8
0.195
0.263
1
0
2xsn
2.68
1341
0.017
2.0
0.005
6.6
0.229
0.275
16
0
2xsq
1.72
179
0.022
2.1
0.009
6.4
0.158
0.195
0
0
2xsw
1.90
666
0.021
2.3
0.007
7.0
0.166
0.211
5
0
2xsx
1.70
869
0.023
2.1
0.007
6.4
0.162
0.203
4
0
2xu7
1.90
752
0.020
2.2
0.006
8.0
0.186
0.235
1
0
2xul
2.20
645
0.016
2.2
0.003
7.2
0.189
0.231
1
0
2xvs
1.80
166
0.022
2.3
0.008
6.9
0.177
0.252
1
0
2xvv
2.40
582
0.019
2.3
0.005
6.8
0.182
0.245
3
0
2xxj
1.96
1239
0.017
1.9
0.007
6.5
0.179
0.241
8
0
3acw
1.63
284
0.019
2.0
0.009
5.8
0.201
0.242
1
0
3aey
1.92
698
0.021
2.2
0.008
6.3
0.171
0.204
12
0
3ajx
1.60
828
0.022
2.2
0.006
5.7
0.178
0.211
1
0
3ale
2.50
1460
0.015
2.1
0.004
7.5
0.233
0.272
16
0
3am9
2.17
2584
0.017
2.1
0.005
7.1
0.175
0.241
23
0
3l9w
1.75
695
0.023
2.4
0.009
6.5
0.199
0.233
3
0
3lb4
1.56
274
0.018
2.3
0.012
5.1
0.244
0.277
1
0
3lfl
2.10
686
0.017
1.9
0.003
8.5
0.202
0.269
8
0
3lje
1.75
121
0.025
2.4
0.006
6.5
0.175
0.211
1
0
3ljq
1.90
570
0.020
2.2
0.006
7.2
0.144
0.200
5
0
3lju
1.70
373
0.021
2.2
0.007
6.9
0.192
0.237
1
0
3lpf
2.26
1180
0.017
2.3
0.005
10.2
0.280
0.325
30
1
3lre
2.20
589
0.016
2.1
0.005
7.0
0.218
0.273
2
0
3lrp
2.50
181
0.017
2.2
0.004
7.1
0.184
0.252
3
0
3lt3
2.10
404
0.019
2.4
0.005
7.5
0.188
0.282
2
0
3m0e
2.63
1729
0.015
1.9
0.004
6.8
0.236
0.273
18
0
3m0h
1.58
1685
0.022
2.2
0.008
5.8
0.144
0.178
9
0
3m4z
1.94
309
0.020
2.1
0.007
6.1
0.160
0.203
0
0
3m5o
1.60
410
0.015
2.0
0.007
7.0
0.206
0.247
2
2
3m67
1.80
257
0.026
2.6
0.005
7.9
0.168
0.226
4
0
3mbv
2.00
222
0.017
1.8
0.006
5.9
0.248
0.292
1
0
3mfa
1.63
194
0.021
2.3
0.012
8.6
0.181
0.225
1
0
3mif
2.00
310
0.021
2.4
0.008
9.3
0.194
0.242
3
0
3mk9
2.08
173
0.017
1.9
0.006
6.8
0.186
0.246
2
0
3mke
1.75
265
0.020
2.1
0.010
6.8
0.148
0.191
0
0
3mvi
1.60
698
0.023
2.2
0.008
6.3
0.162
0.205
3
0
3mxe
1.85
198
0.019
2.3
0.005
7.7
0.183
0.251
0
0
3n2v
1.55
158
0.023
2.4
0.008
7.2
0.178
0.227
0
0
3nfy
1.94
498
0.017
2.1
0.004
6.9
0.181
0.259
6
0
3ni0
1.60
182
0.017
1.9
0.003
5.2
0.213
0.254
0
0
3nk4
2.00
581
0.021
2.5
0.006
7.8
0.217
0.247
7
0
3nl6
2.61
1545
0.016
2.4
0.005
7.8
0.225
0.273
23
2
3nm8
2.00
570
0.018
2.2
0.007
7.4
0.191
0.262
2
0
3nmi
2.01
636
0.023
2.4
0.005
6.8
0.198
0.244
9
1
3nof
1.60
213
0.021
2.4
0.010
6.5
0.186
0.216
2
0
3nok
1.65
466
0.016
2.0
0.005
7.4
0.198
0.243
4
0
3nv6
2.20
404
0.015
2.0
0.004
6.6
0.196
0.260
0
0
3nxg
1.95
1291
0.018
2.2
0.006
7.5
0.166
0.213
8
0
3nxp
2.20
363
0.015
2.1
0.005
8.1
0.196
0.238
9
0
3o0a
1.77
425
0.019
2.2
0.007
7.2
0.187
0.248
2
2
3o3p
1.70
635
0.020
2.5
0.006
8.2
0.216
0.251
14
0
3o4h
1.82
2302
0.015
2.2
0.005
6.5
0.216
0.270
16
2
3o79
1.60
202
0.024
2.5
0.012
11.1
0.192
0.239
2
1
3o86
1.60
709
0.021
2.1
0.007
6.8
0.167
0.196
4
0
3oae
2.80
2105
0.017
2.5
0.005
9.4
0.237
0.294
39
0
3oag
2.30
669
0.015
2.1
0.005
8.3
0.210
0.261
1
0
3oc2
1.97
495
0.019
2.3
0.007
7.5
0.171
0.226
5
1
3occ
1.70
1424
0.021
2.1
0.007
7.1
0.156
0.192
15
5
3oep
1.75
485
0.020
2.2
0.008
6.5
0.168
0.210
4
0
3oi7
2.40
1028
0.015
1.9
0.005
6.9
0.241
0.280
7
0
3oia
1.65
405
0.022
2.3
0.008
7.0
0.171
0.237
5
0
3olz
2.75
743
0.014
2.0
0.003
6.8
0.238
0.264
10
0
3om1
1.68
740
0.022
2.4
0.007
6.9
0.183
0.223
6
0
3onw
2.38
702
0.018
2.2
0.005
7.4
0.225
0.263
6
0
3orv
1.91
1707
0.017
2.0
0.006
7.2
0.155
0.207
10
0
3oux
2.40
550
0.017
2.2
0.004
6.4
0.220
0.267
8
0 Table 4
Statistics for structures in the moderate-resolution data set as refined in PrimeX. Table 5 † Corrected for obvious errors in deposited structures, as shown in Tables 1 and 2. ‡ Corrected for clashes owing to errors in the st
clashes and severe clashes per 100 residues are 0.9 and 0.03, respectively. At the same time, some users of REFMAC did very tightly
restrain peptide bonds. The lowest standard deviation for ! was not from among the CNS/CNX-refined structures, but
instead was produced by REFMAC (3pgj; 0.9). group relieved all close contacts and was confirmed by a small
pair of negative and positive difference features in an Fo Fc
map (result not shown). The refinement program CNX did not
correct this situation. A reasonable hypothesis for why it did
not do so is that the interactions between the methionine
methyl group and the other two residues, as represented
through a united-atom model in CNX, were not unfavorable
enough to cause a change in the positions of these atoms. Changes in overall structure quality owing to PrimeX
refinement, as judged by the Ramachandran Z scores (Spronk
et al., 2004), were generally small. Only one change was noted
as significant by the program WHAT_CHECK. This change
was from an original value of 3.16 for the protein 3nl6 as
originally deposited to a value of 2.11 after PrimeX refine-
ment. The mean Ramachandran Z score changed from 0.37
(range 3.16 to +3.69) as originally deposited to 0.51 (range
2.56 to +3.05) after PrimeX refinement. Individual Rama-
chandran Z scores before and after PrimeX refinement are
shown in the Supplementary Material. 3.4.2. Backbone change to relieve clash leads to additional
ligand hydrogen bonds. In PDB entry 3nl6, atoms in the side
chain of ValC209 clash with the side chain of ValC15 (Fig. 2). In producing the all-atom model derived from this structure
through energy minimization, these interactions were suffi-
ciently repulsive that the bond angles around C of ValC15
were distorted rather than allowing atoms to overlap to such
an extreme extent. The close contact was relieved during
PrimeX refinement using the ‘polish’ workflow by motion of
residues C209 and C210 away from residue C15 and towards
the bound thiamine phosphate (TPS), as shown in Fig. 2. This
side-chain motion occurred with a change in the conformation
of the main chain for residue C209. Table 5 This change in the back-
bone position and a few other more subtle atomic shifts
provided multiple additional hydrogen-bond interactions
between the protein and the TPS molecule, a difference that
has potentially major implications for the understanding of
TPS binding. This large structural change during refinement
was probably related to the resolution of the strain of close
contacts in the model, but electrostatic gradients or other
influences during refinement could also play a role. Assuming
that the program was used as intended and in the absence of
any indication to the contrary (Paul et al., 2010), phenix.refine
seems to have tolerated these severe implied all-atom clashes
during refinement. The average Rfree value over the moderate-resolution set
was the same with or without the additional PrimeX all-atom
refinement (0.243 versus 0.242; Tables 2 and 3). The average
for all working R values was somewhat lower for the PrimeX-
refined structures (0.193) versus the average from the original
structures (0.207). research papers in the ultrahigh-resolution set
was 2.5, which is greater than that produced by any of the
other refinement programs, but closest to the value for
PrimeX. The r.m.s. Z score for bond angles in the ultrahigh-
resolution set was 1.12 (0.79–1.46), which is also similar to that 945 Acta Cryst. (2012). D68, 935–952 Bell et al. PrimeX Acta Cryst. (2012). D68, 935–952 research papers Table 5
Summary of geometry and clash statistics. Structure set
No. of
structures
Bond-length
r.m.s.d. (A˚ )
Bond-angle
r.m.s.d. ()
Side-chain planarity
r.m.s.d. (A˚ )
!-Angle standard
deviation ()
Clashes per
100 residues
Severe clashes
per 100 residues
Ultrahigh-resolution set
18
0.021
2.5
0.011
6.4
0.6†
0.03†
Moderate-resolution set + additional
PrimeX refinement
94
0.019
2.2
0.006
7.1
0.7‡
0.03‡
Moderate-resolution set as deposited
94
0.015
1.4
0.005
5.1
4.0
0.5
BUSTER-refined subset
2
0.014
1.7
0.008
2.6
0.9
0.07
CNS/CNX-refined subset
12
0.009
1.3
0.005
2.3
5.9
0.8
PHENIX-refined subset
14
0.008
1.1
0.003
5.4
4.7
0.5
REFMAC-refined subset
66
0.016
1.5
0.005
5.7
3.4
0.5
† Corrected for obvious errors in deposited structures, as shown in Tables 1 and 2. ‡ Corrected for clashes owing to errors in the structures (see text); the numbers of uncorrected
clashes and severe clashes per 100 residues are 0.9 and 0.03, respectively. research papers field and electron-density gradients, which also required the
motion of the glutamine to avoid the formation of a clash with
the lysine. Whatever the causes, the result was a large co-
ordinated movement of the lysine and glutamine side chains
which was dramatic both in terms of the extent of the motion
of the glutamine side chain and in the difference in the key
interactions observed for the binding of ADP to this protein. is less easy to define. MacArthur & Thornton (1996) suggested
from their study of proteins and small polypeptides that a
standard deviation of 6 is appropriate. Bell et al. PrimeX
947
Figure 1
MetC187 in PDB entry 3phe is shown in (a) as originally refined in CNX,
with only a selection of H atoms added for clarity. Clashes between the
terminal methyl group of the methionine and two other residues are
shown as orange dashed lines. These close interactions were tolerated
during the original refinement as a united-atom model. In (b) the location
of the methyl group after PrimeX refinement is shown, where no clashes
involving the methyl group were observed. The electron-density grid for
this region is contoured at 1.0 from a 2Fo Fc composite OMIT map. The molecular model as originally deposited contains
several side chains, including GlnA758, that are misfitted and
thus this structure might be considered by some to be a poor
candidate for automated refinement. In this alternate view of
the situation shown in Fig. 3, residue GlnA758 is positioned
outside of the anticipated radius of convergence for refine-
ment. However, one conclusion is clear: REFMAC was
tolerant of the implied clash as described above either because
it was designed to behave so or because a decision by the users
(Crawley et al., 2011) caused REFMAC to behave in this way. PrimeX all-atom refinement is not tolerant of such inter-
actions because of the highly unfavorable energetics calcu-
lated for such an interaction and it does not allow users to
modify its behavior to tolerate such interactions without
extraordinary efforts. Even when considered in this context,
the ability of the automated PrimeX polish workflow to
improve the model in the manner described in Fig. 3 is
encouraging. 4.1. Summary geometry statistics for PrimeX and other
refinement programs While this study was primarily focused on close nonbonded
contacts and refinement using an all-atom model, other issues
regarding molecular geometry were also of interest and might
best be discussed first. Only moderate-resolution structures
deposited and released in 2010 were used in this study to
ensure that the results reflected current practices in protein
crystallography, especially with respect to geometric restraints. Use of the OPLS all-atom force field in PrimeX produced
reasonable results with respect to summary geometry that
were in line with other programs in terms of bond-length
deviation and side-chain group planarity (Table 5). The results
from the two other summary geometry descriptors monitored
here deserve additional comment. The average of the bond-angle r.m.s.d.s for PrimeX (2.2) is
greater than for any of the other programs that created the
original moderate-resolution structure set (range 1.1–1.7;
Table 5). However, the observation from the ultrahigh-
resolution data set of an average bond-angle r.m.s.d. of 2.5
(range 1.4–3.1; Table 1) clearly suggests that this r.m.s.d. is
reasonable. 3.4. Additional benefits from all-atom refinement The advantages of all-atom refinement of structures at
moderate resolution extend well beyond the prevention and
remediation of clashes. A few examples from the PrimeX
refinements in this study illustrate how a detailed description
of nonbonded contacts influenced and improved the results of
refinement. 3.4.1. Repositioning of a methionine methyl group. Fig. 1
provides an example in which all-atom refinement used in
PrimeX led to a significant improvement in the structural
model. In PDB entry 3phe, clashes of the C" and associated H
atoms of MetC187 with atoms from LeuC293 and TyrC296
suggest that at least one of these residues is in the wrong
position. PrimeX refinement using the ‘polish’ workflow
moved the methyl group as shown in Fig. 1 without manual
intervention. The 2Fo Fc electron-density map as shown did
not give any clear indication of the correct position for this
methyl group. However, the position as deposited was un-
favourable and unlikely to be correct as judged from the
observed clashes. The new position for the methionine methyl 3.4.3. Refinement of two side-chain positions provides a
new view of ADP binding. In its original position in PDB
entry 3pdt, as refined in REFMAC, a clash occurred between
GlnA758 and PheA720 in the all-atom structure (Fig. 3). The
change in structure after PrimeX refinement using the ‘polish’
workflow is hypothesized to have occurred through the
following chain of events. The movement of the GlnA758 side
chain was first driven by relief of this clash. Concurrently, the
LysA722 side chain was moved towards the phosphate group
of the ADP molecule under the influence of both the force 946 Bell et al. PrimeX Acta Cryst. (2012). D68, 935–952 Acta Cryst. (2012). D68, 935–952 Figure 1 MetC187 in PDB entry 3phe is shown in (a) as originally refined in CNX,
with only a selection of H atoms added for clarity. Clashes between the
terminal methyl group of the methionine and two other residues are
shown as orange dashed lines. These close interactions were tolerated
during the original refinement as a united-atom model. In (b) the location
of the methyl group after PrimeX refinement is shown, where no clashes
involving the methyl group were observed. The electron-density grid for
this region is contoured at 1.0 from a 2Fo Fc composite OMIT map. Over-restraint of ! angles in CNS/CNX has been recog-
nized for several years (Priestle, 2003). Considering the time
that has passed since this publication, the number of structures
from BUSTER, CNS/CNX and REFMAC observed with very
low deviation of ! angles is hard to understand. While over-
restraint is easy to recognize, the correct degree of variability 947 Bell et al. PrimeX Acta Cryst. (2012). D68, 935–952 Figure 3 The region near ADP A811 in PDB entry 3pdt is shown in (a) as
originally refined in REFMAC. All H atoms were added to this model and
their positions were minimized while holding non-H atoms fixed, but only
some of these H atoms are shown for clarity. Multiple clashes between the
misplaced residue GlnA758 and PheA720 were apparently tolerated in
the REFMAC refinement. The close contacts (orange dashed lines) were
so severe that the energy minimization distorted the planarity of the
aromatic system rather than allowing the higher energy interpenetration
of atoms. The position of these two residues after refinement with PrimeX
is shown in (b). In order to relieve the strain of the clash, the LysA722
side chain moved toward the ligand with a coordinated motion of the
glutamine side chain into its correct position in strong electron density
below the Lys residue. Hydrogen bonds are shown as purple dashed lines. The electron-density grid for this region is contoured at 1.0 from a
2Fo Fc composite OMIT map. research papers values could be an increase in the number or severity of
clashes. Forcing bond angles or torsion angles toward idealized
values does carry a risk. If an interaction such as a nonbonded
repulsion has driven a particular torsion or bond angle away
from the idealized value, restraining it to be closer to the
idealized value must make that other interaction more un-
favorable. Thus, the result of strictly enforcing these ideal That the same Rfree was obtained with our force-field-based
restraints as with Engh and Huber restraints suggests that
these restraints are reasonably consistent with protein crystal
structures. However, taken together, the decrease in the
average R factor (Rwork), the relatively large r.m.s.d. for bond
angles compared with the deposited structures and the Figure 2
The region near ValC209 in PDB entry 3nl6 is shown in (a) as originally
produced with phenix.refine. All H atoms were added to this model and
their positions were minimized while holding non-H atoms in fixed
positions, but only some of these H atoms are shown for clarity. The
extremely close contacts between the H atom attached to C of ValC15
and atoms of the ValC209 side chain (orange dashed lines) distorted the
bond angles around C. The coordinates of residues C209 and C210
changed after PrimeX refinement as shown in (b), with a shift in the
backbone conformation, relieving the close contacts and resulting in
multiple additional hydrogen bonds to the ligand (purple dashed lines). The electron-density grid for this region is contoured at 1.0 from a g
p
p
Figure 2
The region near ValC209 in PDB entry 3nl6 is shown in (a) as originally
produced with phenix.refine. All H atoms were added to this model and
their positions were minimized while holding non-H atoms in fixed
positions, but only some of these H atoms are shown for clarity. The
extremely close contacts between the H atom attached to C of ValC15
and atoms of the ValC209 side chain (orange dashed lines) distorted the
bond angles around C. The coordinates of residues C209 and C210
changed after PrimeX refinement as shown in (b), with a shift in the
backbone conformation, relieving the close contacts and resulting in
multiple additional hydrogen bonds to the ligand (purple dashed lines). The electron-density grid for this region is contoured at 1.0 from a
2Fo Fc composite OMIT map. research papers Figure 3
The region near ADP A811 in PDB entry 3pdt is shown in (a) as
originally refined in REFMAC. All H atoms were added to this model and
their positions were minimized while holding non-H atoms fixed, but only
some of these H atoms are shown for clarity. Multiple clashes between the
misplaced residue GlnA758 and PheA720 were apparently tolerated in
the REFMAC refinement. The close contacts (orange dashed lines) were
so severe that the energy minimization distorted the planarity of the
aromatic system rather than allowing the higher energy interpenetration
of atoms. The position of these two residues after refinement with PrimeX
is shown in (b). In order to relieve the strain of the clash, the LysA722
side chain moved toward the ligand with a coordinated motion of the
glutamine side chain into its correct position in strong electron density
below the Lys residue. Hydrogen bonds are shown as purple dashed lines. The electron-density grid for this region is contoured at 1.0 from a
2Fo Fc composite OMIT map. Figure 3
The region near ADP A811 in PDB entry 3pdt is shown in (a) as
originally refined in REFMAC. All H atoms were added to this model and
their positions were minimized while holding non-H atoms fixed, but only
some of these H atoms are shown for clarity. Multiple clashes between the
misplaced residue GlnA758 and PheA720 were apparently tolerated in
the REFMAC refinement. The close contacts (orange dashed lines) were
so severe that the energy minimization distorted the planarity of the
aromatic system rather than allowing the higher energy interpenetration
of atoms. The position of these two residues after refinement with PrimeX
is shown in (b). In order to relieve the strain of the clash, the LysA722
side chain moved toward the ligand with a coordinated motion of the
glutamine side chain into its correct position in strong electron density
below the Lys residue. Hydrogen bonds are shown as purple dashed lines. The electron-density grid for this region is contoured at 1.0 from a
2Fo Fc composite OMIT map. Fi
3 Fi
2 4.2. Advantages of all-atom refinement with PrimeX All-atom refinement of moderate-resolution protein crystal
structures with PrimeX resulted in a more than fourfold
decrease in the number of clashes and a 17-fold decrease in
the number of severe clashes. This improvement in model
quality was achieved without sacrificing the goodness of fit to
the X-ray data as judged by the average Rfree values. Impor-
tantly, these models also display good summary statistics, so
that the protein models comply with reasonable molecular-
geometry expectations. What else can be done to reduce the number of clashes in
deposited structures? Perhaps the answer to this question
resides in the standards for structure deposition in the PDB. A committee of the PDB is currently working on structure-
validation tools for use associated with the deposition of
coordinates (Read et al., 2011). A reason for optimism is that
the work of the Richardson group was included in the report
of the committee. From the point of view of many users of
protein structures, the deposition of all-atom models derived
from protein crystal structures should be required. Clashes
determined from an all-atom model should be, at the very
least, measured and documented for all protein models that
are deposited, just as other outliers to molecular-geometry
standards are now listed in the entry header. All-atom refinement with a force field allowed PrimeX
refinement to fix errors that other refinement programs missed
and provided a more accurate picture of critical protein
features such as protein–ligand interactions, as illustrated in
Figs. 1, 2 and 3. Resolution of clashes during refinement can
help to ‘push’ the structure into the correct conformation,
producing potentially remarkably large changes in confor-
mation. They may also serve the role of preventing the
structure from entering nonproductive conformations that are
otherwise allowed in a less restrictive all-atom model. To achieve a higher standard for deposited protein struc-
tures, additional tools that are sensitive to close contacts could
help. PrimeX can contribute to this goal, and the automated
polish workflow presented here was designed to achieve this
goal with the minimum of human intervention. However, the
workflow was designed with the assumption that the crystal
structure coordinates on which it would operate would be
essentially free of errors in the main-chain tracing or side-
chain rotamer selection. 4.2. Advantages of all-atom refinement with PrimeX The prevalence of such errors in the
data set examined here established the need for additional
automated structure tools with the capability of making large
changes in side-chain torsion angles or chain trace. Design of
these workflows is in progress based on the tool set in the
PrimeX refinement package (Bell et al., 2012). Although
similar automated workflows exist for phenix.refine (Afonine
et al., 2005) and indirectly for REFMAC (Murshudov et al.,
2011) through the program SideAide in the PDB_REDO
pipeline (Joosten et al., 2011), the frequency of clashes in
structures refined by phenix.refine and REFMAC (Tables 3
and 5) raises the question whether these automated workflows
can address the issue of all-atom clashes, no matter how
capable and thorough these workflows are intended to be. While the program MolProbity (Davis et al., 2007; Chen,
Arendall et al., 2010) is aimed at solving the right problem,
these same results show that it is not being adequately
employed to deal with the problem at hand. These results also contain an indication of the limits of
usefulness of the polish workflow. Fully 85% of the structures
that entered the workflow with 50 or more total clashes
(Table 3) resulted in an increase in Rfree (Tables 3 and 4). A
large number of clashes is a warning sign that the structure
may contain errors that could have negative consequences
after the application of this refinement process. Figure 2 The region near ValC209 in PDB entry 3nl6 is shown in (a) as originally
produced with phenix.refine. All H atoms were added to this model and
their positions were minimized while holding non-H atoms in fixed
positions, but only some of these H atoms are shown for clarity. The
extremely close contacts between the H atom attached to C of ValC15
and atoms of the ValC209 side chain (orange dashed lines) distorted the
bond angles around C. The coordinates of residues C209 and C210
changed after PrimeX refinement as shown in (b), with a shift in the
backbone conformation, relieving the close contacts and resulting in
multiple additional hydrogen bonds to the ligand (purple dashed lines). The electron-density grid for this region is contoured at 1.0 from a
2Fo Fc composite OMIT map. Bell et al. PrimeX 948 Acta Cryst. (2012). D68, 935–952 research papers programs can be considered to be part of the solution to this
problem. somewhat larger standard deviation for the ! angle above the
optimal value conceived by MacArthur & Thornton (1996)
could be interpreted as evidence that the restraints employed
may require further tuning to decrease the risk of overfitting. This consideration will be examined in future publications. A role for CNS in curbing clashes is also currently available. CNS can be employed with a more complex energy model
than is routinely used by crystallographers. As well as
deploying an Engh and Huber-based restraint system, CNS is
distributed with a force field that includes Lennard–Jones
and electrostatic terms and that is regularly used for the
determination of NMR structures (Linge et al., 2003). This
force field has been employed to produce some very high
quality NMR structures (see, for example, Nozinovic et al.,
2010). Acta Cryst. (2012). D68, 935–952 5. Conclusion Ali, A., Reddy, G. S., Nalam, M. N., Anjum, S. G., Cao, H., Schiffer,
C. A. & Rana, T. M. (2010). J. Med. Chem. 53, 7699–7708. This study documents the existence of numerous unnecessary
close contacts, including many severe ones, implicit in united-
atom models deposited in the PDB. Many of these close
contacts can be readily removed, and doing so need not
damage the agreement of the model with the observed X-ray
diffraction data. Furthermore, attention to close contacts can
bring to light errors in the placement of non-H atoms in
protein crystal structure models. This latter point has also
been made abundantly clear by over a decade of work by Jane
Richardson, David Richardson and coworkers (Word et al.,
1999, Davis et al., 2007; Chen, Arendall et al., 2010). (
)
Allen, F. H. (2002). Acta Cryst. B58, 380–388. Allen, F. H. (2002). Acta Cryst. B58, 380–388. Artz, J. D., Wernimont, A. K., Dunford, J. E., Schapira, M., Dong, A.,
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(
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of science that depend on protein crystal structure models,
such as protein design and drug discovery. Normally, scientists
working in these areas are not in a position to evaluate the
reliability of each protein crystal structure, nor are they able to
judge whether the effects of remediation of crystal structures
might result in different sorts of errors. 5. Conclusion If crystallographers,
who are of course in the best position to do so, do not address
these issues, then eventually other scientists will. The result
will be that protein crystallographers will have less control
over the form in which their experimental results are archived
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Weissig, H., Shindyalov, I. N. & Bourne, P. E. (2000). Nucleic Acids
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Gordeliy, V. (2010). J. Cryst. Growth, 312, 3326–3330. Bosch, D. E., Kimple, A. J., Sammond, D. W., Muller, R. E., Miley,
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statistics are very strictly applied, while the latter receive much
less attention. An understandable explanation for this contrast
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have a positive impact on the issue of clashes in all-atom
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Uncovering spin-orbit coupling-independent hidden spin polarization of energy bands in antiferromagnets
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Nature communications
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cc-by
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Uncovering spin-orbit coupling-independent
hidden spin polarization of energy bands in
antiferromagnets Lin-Ding Yuan1, Xiuwen Zhang
1, Carlos Mera Acosta2 & Alex Zunger1 Received: 17 November 2022
Accepted: 15 August 2023
Check for updates Received: 17 November 2022
Accepted: 15 August 2023
Check for updates Many textbook physical effects in crystals are enabled by some specific sym-
metries. In contrast to such ‘apparent effects’, ‘hidden effect X’ refers to the
general condition where the nominal global system symmetry would disallow
the effect X, whereas the symmetry of local sectors within the crystal would
enable effect X. Known examples include the hidden Rashba and/or hidden
Dresselhaus spin polarization that require spin-orbit coupling, but unlike their
apparent counterparts are demonstrated to exist in non-magnetic systems
even in inversion-symmetric crystals. Here, we discuss hidden spin polariza-
tion effect in collinear antiferromagnets without the requirement for spin-
orbit coupling (SOC). Symmetry analysis suggests that antiferromagnets
hosting such effect can be classified into six types depending on the global vs
local symmetry. We identify which of the possible collinear antiferromagnetic
compounds will harbor such hidden polarization and validate these symmetry
enabling predictions with first-principles density functional calculations for
several representative compounds. This will boost the theoretical and
experimental efforts in finding new spin-polarized materials. Many traditional textbook physical effects in crystals are enabled by
some specific symmetries, encoded in the crystal space group. Such are
the symmetry conditions for the apparent electric polarization which
defines various order parameters such as in ferroelectricity1, circular
dichroism2, and pyroelectricity3. Another example of effects enabled by
the recognized global system symmetry is the removal of spin degen-
eracy of energy bands due to spin-orbit coupling (SOC) in non-magnetic
crystals having broken inversion symmetry (such as the Rashba (R-1)4
and Dresselhaus (D-1)5 effects). When an effect is observed despite the
needed enabling symmetry being absent, it is often assumed that the
system contains some symmetry-altering imperfections. phenomena that are unexpected to exist based on the global sym-
metry of the system. Examples of “Hidden Effect X” that is SOC-induced include (i)
Rashba or Dresselhaus spin polarization, expected exclusively to occur
in non-centrosymmetric crystals, but predicted6,7 and observed8–18 in
centrosymmetric nonmagnetic crystals (denoted R-2 and D-2, respec-
tively). Similar form of Hidden effect X are (ii) X = “anisotropic optical
circular polarized luminescence” expected only in odd-layered transi-
tion-metal dichalcogenides but observed19 also in even-layered crys-
tals. Article https://doi.org/10.1038/s41467-023-40877-8 1Renewable and Sustainable Energy Institute, University of Colorado, Boulder, CO 80309, USA. 2Center for Natural and Human Sciences, Federal University of
ABC, Santo Andre, São Paulo, Brazil.
e-mail: Alex.Zunger@colorado.edu Uncovering spin-orbit coupling-independent
hidden spin polarization of energy bands in
antiferromagnets a SOC-independent hidden spin polarization schematically illustrated as two copies
of spin split energy bands localized on sector-α and sector-β but globally mutually
compensate; (b) three prototypes of spin degenerate bulk; (c) two prototypes of
spin split sector. Sectors in (a) are represented by color-shaded planes, the red and
blue lines in the plane represent the spin-up and spin-down bands. The spin-
splitting prototypes in (b) defined for bulk39 is generalized for sectors in (c). Checkmark and cross in parentheses in (b) and (c) are used to indicate the presence
or absence of the symmetry. a
Hidden spin polarization in AFM polarization was pointed out recently in ref. 38. This involved utilizing
first a few individual symmetry operations: U being a spin rotation of
the SU(2) group acting on the spin 1/2 space that reverses the spin; T
being spatial translation; Θ being time reversal, and I being the spatial
inversion. These individual operations are then used for constructing
two symmetry products: a SOC-free magnetic symmetry ΘIT, and a
spin symmetry UT (where the former product can be simplified to ΘI
by proper choice of inversion center). SOC-independent spin
splitting38,39 would occur only when both symmetry products are
simultaneously violated. Antiferromagnets with ΘIT symmetry28,41,42
will not show such spin splitting. Such symmetry conditions disen-
tangle the SOC-independent splitting from the SOC-induced splitting
by considering the symmetry at the zero SOC limit43–45, where spin and
space are fully decoupled. Hidden spin polarization in AFM a Symmetry
SS
w/o SOC
Magnetism
Prototype
Θ
(✓) and
(X)
No
AFM
SST-1
Θ
(✓) and
(✓)
No
AFM
SST-2
Θ
(X) and
(✓)
No
AFM
SST-3
b
Symmetry classification of the spin degenerate bulk b Given the symmetry conditions, it is thus possible to classify all
different spin splitting prototypes38,39 for magnetic materials. There
are three prototypes with no apparent spin splitting effect: (1) AFM
compounds that violate UT but preserve ΘIT symmetry referred to as
spin splitting prototype 1 (SST-1) antiferromagnets; (2) AFM com-
pounds that preserve both UT and ΘIT symmetry referred to as SST-2
antiferromagnets; (3) AFM compounds that preserve UT but violate
ΘIT symmetry referred to as SST-3 antiferromagnets. Enabling symmetry conditions for hidden SOC-independent
spin polarization in antiferromagnets sp
po a
at o
a t e
o
ag ets
“Hidden spin polarization” is expected in collinear antiferromagnets
when the bulk has zero net spin polarization, but its constituent sec-
tors allow locally a spin splitting and spin polarization effect. Consider
the combination of two possible prototypes constituting sector that
gives hidden spin polarization locally but lead to three possible pro-
totypes of the bulk symmetry (preserving either ΘIT or UT or both)
that disallows apparent spin polarization, one can then classify six
hidden spin polarization cases. Following the previous classification of
spin splitting prototypes for apparent spin degeneracy and apparent
spin splitting38,39, collinear antiferromagnetic materials with “hidden
spin polarization” are those antiferromagnets whose bulk prototype
being SST-I (I = 1, 2, 3) and constitute sector prototype being SST-J
(J = 4, 5). Detailed discussions of the symmetry conditions for hidden
spin polarization in collinear AFM are given in Supplementary Infor-
mation Section A. non-magnetic state has an inversion. Prominently, the hidden spin
polarization in these compounds facilitates the electrical reversal of
their antiferromagnetic ordering25,29. (iv) X = “anomalous Hall effect”
induced by SOC expected only in odd-layered ferromagnetic MnBi2Te4
systems but observed in even-layered antiferromagnetic MnBi2Te4
30
systems via a perturbative applied electric field. Here, we discuss a different form of hidden spin polarization
effect (see Fig. 1a) whose corresponding apparent effect is indepen-
dent of SOC31–40; And the hidden effect exists in antiferromagnetic
materials where spin-up and spin-down bands are paired. This repre-
sents a step further beyond the already known hidden Rashba and
hidden Dresselhaus spin-polarization that unavoidably require a siz-
able contribution from SOC. A careful analysis of the “global (bulk) vs
local (sector)” symmetries suggests that antiferromagnets hosting the
SOC-independent “hidden” spin polarization effect can be delineated
into six types. We scrutinize a vast database of known collinear AFM
materials and performed first-principles calculations on several selec-
ted candidate compounds assuming zero SOC. We show that such
hidden, SOC-independent effects reflect the intrinsic properties of the
perfect crystal rather than an effect due to imperfections. The interest
in this SOC-independent hidden spin polarization effect stems both
from the evolving of the fundamental understanding of general hidden
effects in solids, and from the ability to extend the pool of useful
materials for potential spintronic applications. Enabling symmetry conditions for hidden SOC-independent
spin polarization in antiferromagnets Figure 2 summarizes the six possible types of hidden spin polar-
ization without SOC in antiferromagnets that are spin degenerate but
contain spin split sectors (represented by color-shaped plane). Figure 2a–c illustrates the three cases where the spin degenerate
antiferromagnets of SST-I (I = 1,2,3) can be decomposed into alter-
nating ferromagnetic local sectors that locally violate both UT and ΘIT,
thus allows spin splitting without SOC. FM materials that satisfy the
conditions of violating both UT and ΘIT (always true) are denoted as
SST-5 in Fig. 1. The three magnetic-induced hidden spin polarization
cases can then be denoted as (a) bulk SST-1 sector SST-5; (b) bulk SST-2
sector SST-5, and (c) bulk SST-3 sector SST-5. Figure 2d–f illustrates the
three cases where the spin degenerate AFM of SST-I (I = 1, 2, 3) can be
decomposed into alternating antiferromagnetic local sectors that
locally violate both UT and ΘIT, thus allows spin splitting without SOC. AFM materials that satisfy the condition are denoted as SST-4 in Fig. 1. Uncovering spin-orbit coupling-independent
hidden spin polarization of energy bands in
antiferromagnets In addition, there
are two prototypes with apparent spin splitting effects: (4) AFM
compounds that violate both UT and ΘIT symmetry referred to as SST-
4 antiferromagnets; (5) Ferromagnetic (FM) compounds that violate
both UT and ΘIT symmetry referred to as SST-5 ferromagnets. The
classification defined in bulk crystals38,39 can be generalized to sectors
of a bulk based on the local sector symmetry. Figure 1b,c. summarizes
the classification of “spin degenerate bulk” vs “spin-split sector”. This
will later be applied to describe the symmetry conditions and to define
the different prototypes for the hidden spin polarization effect in
antiferromagnets. Symmetry
SS
w/o SOC
Magnetism
Prototype
(X) and
(X)
Yes
AFM
SST-4
(X) and
(X)
Yes
FM
SST-5
c
Symmetry classification of spin split sector Symmetry classification of spin split sector Fig. 1 | Hidden spin polarization in collinear antiferromagnets without SOC. Fig. 1 | Hidden spin polarization in collinear antiferromagnets without SOC. a SOC-independent hidden spin polarization schematically illustrated as two copies
of spin split energy bands localized on sector-α and sector-β but globally mutually
compensate; (b) three prototypes of spin degenerate bulk; (c) two prototypes of
spin split sector. Sectors in (a) are represented by color-shaded planes, the red and
blue lines in the plane represent the spin-up and spin-down bands. The spin-
splitting prototypes in (b) defined for bulk39 is generalized for sectors in (c). Checkmark and cross in parentheses in (b) and (c) are used to indicate the presence
or absence of the symmetry. Fig. 1 | Hidden spin polarization in collinear antiferromagnets without SOC. a SOC-independent hidden spin polarization schematically illustrated as two copies
of spin split energy bands localized on sector-α and sector-β but globally mutually
compensate; (b) three prototypes of spin degenerate bulk; (c) two prototypes of
spin split sector. Sectors in (a) are represented by color-shaded planes, the red and
blue lines in the plane represent the spin-up and spin-down bands. The spin-
splitting prototypes in (b) defined for bulk39 is generalized for sectors in (c). Checkmark and cross in parentheses in (b) and (c) are used to indicate the presence
or absence of the symmetry. Uncovering spin-orbit coupling-independent
hidden spin polarization of energy bands in
antiferromagnets Such effects were originally dismissed as being due to some
extrinsic sample imperfection20–22 but later on were shown to be an
intrinsic property pertained to the individual layer23. (iii) X = “spin
polarization” induced by SOC in antiferromagnetic systems. The effect
is again expected only in non-centrosymmetric crystals (such as
BiCoO3
24) but shown in centrosymmetric crystals (such as CuMnAs and
Mn2Au25–28) where combined symmetry of inversion and time reversal
disallows splitting. Here, “centrosymmetric” means the crystal in the In contrast, the “Hidden Effect X” in materials that are not sup-
ported by the nominal enabling symmetry, yet effect X exists locally. The “Hidden effect X” reflects the intrinsic properties of the perfect
crystal rather than imperfections that would disappear when the
crystal becomes perfect. The understanding of such hidden intrinsic
effects is important as it can demystify peculiar observations of Nature Communications| (2023) 14:5301 1 https://doi.org/10.1038/s41467-023-40877-8 Article Symmetry
SS
w/o SOC
Magnetism
Prototype
Θ
(✓) and
(X)
No
AFM
SST-1
Θ
(✓) and
(✓)
No
AFM
SST-2
Θ
(X) and
(✓)
No
AFM
SST-3
Symmetry
SS
w/o SOC
Magnetism
Prototype
(X) and
(X)
Yes
AFM
SST-4
(X) and
(X)
Yes
FM
SST-5
c
b
a
Symmetry classification of the spin degenerate bulk
Symmetry classification of spin split sector
Hidden spin polarization in AFM
Fig. 1 | Hidden spin polarization in collinear antiferromagnets without SOC. a SOC-independent hidden spin polarization schematically illustrated as two copie
of spin split energy bands localized on sector-α and sector-β but globally mutuall
compensate; (b) three prototypes of spin degenerate bulk; (c) two prototypes of
spin split sector. Sectors in (a) are represented by color-shaded planes, the red an
blue lines in the plane represent the spin-up and spin-down bands. The spin-
splitting prototypes in (b) defined for bulk39 is generalized for sectors in (c). Checkmark and cross in parentheses in (b) and (c) are used to indicate the presenc
or absence of the symmetry. Symmetry
SS
w/o SOC
Magnetism
Prototype
Θ
(✓) and
(X)
No
AFM
SST-1
Θ
(✓) and
(✓)
No
AFM
SST-2
Θ
(X) and
(✓)
No
AFM
SST-3
Symmetry
SS
w/o SOC
Magnetism
Prototype
(X) and
(X)
Yes
AFM
SST-4
(X) and
(X)
Yes
FM
SST-5
c
b
a
Symmetry classification of the spin degenerate bulk
Symmetry classification of spin split sector
Hidden spin polarization in AFM
Fig. 1 | Hidden spin polarization in collinear antiferromagnets without SOC. Compounds that have SOC-independent hidden spin
polarization We now turn to discuss how the enabling symmetries are applied to
individual sectors to give magnetic hidden spin polarization effects in
real antiferromagnetic materials. Hidden spin polarization from individual ferromagnetic sectors. Figure 3 illustrates the hidden spin polarization effect in tetragonal
CuMnAs54 (bulk belonging to SST-1 class with sectors belonging to SST-
5 class). The crystal is antiferromagnetically ordered with its magnetic
moments collinearly aligned in the (010) direction. The magnetic
space group (MSG) of the crystal is Pm’mn (MSG type III). The unit cell
consists of two MnAs layers (α-sector and β-sector) that are ferro-
magnetically ordered (Fig. 3a, red and blue color shaped polyhedral
are used to indicate oppositely magnetized motifs centered on the
magnetic sites). By considering the bulk antiferromagnets as a com-
bination of two alternating non-centrosymmetric sectors (α-sector and
β-sector), the material has been demonstrated as a useful platform for
electrically switching25,29 the antiferromagnetic magnetization using
the hidden spin polarization from the SOC segregated on each sector. Here, we point out a different SOC-independent scenario that might
also be contributing to the observed electric switching in this material,
i.e., the Zeeman effect within each ferromagnetic MnAs layer creates a
local spin split state anchored on the layer. The two MnAs layers are
connected by the ΘIT symmetry which restores the spin degeneracy of
the bulk and results in a compensated net spin polarization (Fig. 3b). As
shown by the reversed blue and red pattern which are used to map the
relative magnitude of the spin up and spin down polarization, the
hidden spin polarization is non-zero and is compensated by each
other. Examples of hidden spin polarization in spin degenerate bulk As a first step, we will try to find real materials that falls into the six
categories we defined. This can be done straightforwardly by applying
the symmetry conditions to filter out candidate materials in existing
antiferromagnetic databases. We conducted such filtering for MAGN-
DATA database46 and identified a few antiferromagnetic materials of
potential candidates for magnetic hidden spin polarization. Enabling symmetry conditions for SOC-independent apparent
spin polarization in antiferromagnets Enabling symmetry conditions for SOC-independent apparent
spin polarization in antiferromagnets Symmetry is essential to understand the energy bands’ degeneracy of a
material. The symmetry conditions for apparent spin splitting or spin Nature Communications| (2023) 14:5301 2 Article https://doi.org/10.1038/s41467-023-40877-8 Bulk
Sector
SST-1
SST-2
SST-3
SST-5
Hidden spin
polarization
from individual
FM sectors
a
b
c
SST-4
Hidden spin
polarization
from individual
AFM sectors
d
e
f
Θ
(✓) and
(✓)
Θ
(X) and
(✓)
Θ
(X) and
(X)
Θ
(X) and
(X)
Θ
(✓) and
(X)
Fig. 2 | Six types of SOC-independent magnetic hidden spin polarization in
collinear antiferromagnets. These antiferromagnets have global symmetry that
disallows spin splitting without SOC, but have lower local sector symmetry that
allows spin splitting without SOC. Cases (a, b, c) is where hidden spin polarization
arise from local ferromagnetic sectors and cases (d, e, f) is where the hidden spin
polarization arise from local antiferromagnetic sectors. Shaded planes are used to
indicate the individual sectors that have neither ΘIT nor UT symmetry and allow
spin splitting in the absence of SOC; Parallel and antiparallel arrows of red and blue
within the sector plane are used to indicate the ferromagnetic and anti-
ferromagnetic ordering of the sector. Sector symmetry is indicated on top of each
plane, and bulk symmetry is indicated by the arrow connecting the two sectors. c b b a c a d f e f d e Fig. 2 | Six types of SOC-independent magnetic hidden spin polarization in Fig. 2 | Six types of SOC-independent magnetic hidden spin polarization in
collinear antiferromagnets. These antiferromagnets have global symmetry that
disallows spin splitting without SOC, but have lower local sector symmetry that
allows spin splitting without SOC. Cases (a, b, c) is where hidden spin polarization
arise from local ferromagnetic sectors and cases (d, e, f) is where the hidden spin
polarization arise from local antiferromagnetic sectors. Shaded planes are used to indicate the individual sectors that have neither ΘIT nor UT symmetry and allow
spin splitting in the absence of SOC; Parallel and antiparallel arrows of red and blue
within the sector plane are used to indicate the ferromagnetic and anti-
ferromagnetic ordering of the sector. Sector symmetry is indicated on top of each
plane, and bulk symmetry is indicated by the arrow connecting the two sectors. collinear antiferromagnets. Enabling symmetry conditions for SOC-independent apparent
spin polarization in antiferromagnets These antiferromagnets have global symmetry that
disallows spin splitting without SOC, but have lower local sector symmetry that
allows spin splitting without SOC. Cases (a, b, c) is where hidden spin polarization
arise from local ferromagnetic sectors and cases (d, e, f) is where the hidden spin
polarization arise from local antiferromagnetic sectors. Shaded planes are used to The three AFM-induced hidden spin polarization cases can then be
denoted as (d) bulk SST-1 sector SST-4; (e) bulk SST-2 sector SST-4, and
(f) bulk SST-3 sector SST-4. We note that there are multiple ways to
decompose the bulk system into sectors, e.g., the bulk SST-I (I = 1,2,3)
might also be decomposed into sector SST-I (I = 1,2,3) (or equivalently
SST-I (I = 1,2,3) sectors can be used to build the bulk SST-I (I = 1,2,3)
materials), where the local spin polarization of each individual sector is
still zero, therefore, are not the focus of this work. hypothetical or contradictory to enabling symmetry conditions for
AFM spin splitting. Van der Waals compounds with spin splitting not
only allow the potential practical controllability through external
electric fields but also a platform to explore the coexistence of Van der
Waals materials properties and AFM-induced spin splitting. The next step is to validate the predicted hidden spin polarization
effect in some of these identified real materials. We studied the sector-
projected spin textures on certain wavevector planes for three actual
antiferromagnetic materials, CuMnAs54, Ca2MnO4
47 and FeBr2
56 using
PBE + U method59 in the zero SOC limit. The results are presented
below. Additional examples with DFT results are presented in Sup-
plementary Information Section C. These examples proof the exis-
tence of the hidden spin polarization effect. Nature Communications| (2023) 14:5301 https://doi.org/10.1038/s41467-023-40877-8 5a, show that such
splitting is linearly proportional to the applied external electric field,
but in opposite spin polarization ordering for the bottom conduction
bands and the top valence bands. The linear field-dependent splitting
suggests the split states are segregated on either layer (sector). Indeed,
spatial distribution of the spin polarized states, Fig. 5b, c, shows the
spin-up (red) state ΓCB1 is dominantly segregated on the α-sector, while
the spin-down (blue) state ΓCB2 is dominantly segregated on the
β-sector. Therefore, the hidden effect of two-fold degenerate energy
states subspace (when Ez = 0) can be traced back to the individual
FeBr2 layers. Because the applied electric field is small, the main
characteristic of the observed spin polarization is inherited from the
system without electric field. The layer-segregated states shown in
Fig. 5b, c is thus a compelling evidence of the relationship between the
global property of spin splitting induced by a global electric field and
the local spin polarization. We note the hidden spin polarization effect
from local “spin-split” sectors has also been recently exemplified and
revealed via an electric field in some antiferromagnets41,63 where
external electric field lifts the spin degeneracy. We also note that the
layer Hall effect in the even-layered MnBi2Te4—in which electrons from
the top and bottom layers spontaneously deflect in opposite direc-
tions but globally compensate—has been observed with the help of an
applied electric field30. These examples not only verify our under-
standing of the hidden effect being intrinsic to the bulk but also sug-
gest an external electric field as an effective knob for modulating the
hidden effect. antiferromagnets made of spin split ferromagnetic sectors are also
illustrated for CoBr2
56 (bulk belonging to SST-2 with sector belonging
to SST-5) and Ca3Ru2O7
60 (bulk belonging to SST-3 with sector
belonging to SST-5) in Supplementary Information Section C. We note that the corresponding hidden spin polarization pro-
jected onto α-sector and β-sector, shown in Fig. 3c, are all aligned in the
same direction with the magnetization. Thus, the spin remains a good
quantum number. However, the magnitude of the projected spin
polarization (mapped by color changing continuously from blue to
red) may vary depending on the distribution of the degenerate states
on the two sectors. For a pair of degenerate states, the sector projected
spin polarization is the summed contribution from both states. https://doi.org/10.1038/s41467-023-40877-8 The linear field-dependent splitting
suggests the split states are segregated on either layer (sector). Indeed,
spatial distribution of the spin polarized states, Fig. 5b, c, shows the
spin-up (red) state ΓCB1 is dominantly segregated on the α-sector, while
the spin-down (blue) state ΓCB2 is dominantly segregated on the
β-sector. Therefore, the hidden effect of two-fold degenerate energy
states subspace (when Ez = 0) can be traced back to the individual
FeBr2 layers. Because the applied electric field is small, the main
characteristic of the observed spin polarization is inherited from the
system without electric field. The layer-segregated states shown in
Fig. 5b, c is thus a compelling evidence of the relationship between the
global property of spin splitting induced by a global electric field and
the local spin polarization. We note the hidden spin polarization effect
from local “spin-split” sectors has also been recently exemplified and
revealed via an electric field in some antiferromagnets41,63 where
external electric field lifts the spin degeneracy. We also note that the
layer Hall effect in the even-layered MnBi2Te4—in which electrons from
the top and bottom layers spontaneously deflect in opposite direc-
tions but globally compensate—has been observed with the help of an
applied electric field30. These examples not only verify our under-
standing of the hidden effect being intrinsic to the bulk but also sug-
gest an external electric field as an effective knob for modulating the
hidden effect. crystal is the ferromagnetically ordered FeBr2 layer (sector belonging
to SST-5 class). The bilayer slab is built by stacking identical FeBr2 layer
with alternating magnetic ordering. The two layers are connected by
both ΘIT and UT symmetry, the bilayer hexagonal FeBr2 (MSG: PC-3c1)
thus belongs to a bulk SST-2 class, featuring a spin degenerate energy
band. However, the spin degenerate band structure of the SST-2 class
FeBr2 (Fig. 5a, b) is lifted upon the application of an external electric
field perpendicular to the layers (Ez) – a transition from SST-2 to SST-4. The spin splitting arises because of the external electric field Ez creates
a non-equivalent potential on the sectors and breaks the ΘIT and UT
symmetry of the bulk that connects the two layers. DFT calculations for
different values of the applied field, inserted in Fig. https://doi.org/10.1038/s41467-023-40877-8 For
example, the hidden spin polarization of the two spin degenerate
states evenly distributed on both sector-α and sector- β(1/√2(|α↑+ |β↑
and 1/√2(|α↓|β↓〉) is (+0.5) +(−0.5) = 0 when projected onto sector-α
or sector-β; while the hidden spin polarization of the two spin-
degenerate states segregated on one of the sector (|α↑〉and |β↓〉) is
1 when projected onto sector−α and is −1 when projected onto sector. Hidden spin polarization from individual antiferromagnetic sectors. Fig. 4 illustrates the “hidden spin polarization” effect in anti-
ferromagnetic tetragonal Ca2MnO4
47 (bulk belonging to SST-1 class
with
sector
belonging
to
SST-4
class). The
crystal
is
anti-
ferromagnetically ordered with its magnetic moments collinearly
aligned in the (001) direction. The MSG of the crystal is I41’/a’cd’ (MSG
type III). The unit cell consists of two layers of MnO6 octahedral
(α-sector and β-sector) that are antiferromagnetically ordered (Fig. 4a,
red and blue color polyhedral are used to indicate oppositely mag-
netized motifs centered on the magnetic sites). The “magnetic
mechanism” [6] within each AFM-ordered sector then creates a local
spin split state anchored on the layer. The two MnO4 layers are con-
nected by the ΘIT symmetry which restores the spin degeneracy of the
bulk and results in zero net spin polarization (Fig. 4b). However, the
corresponding spin texture projected onto the α-sector and β-sector,
shown in Fig. 4c, are persistently aligned in the same direction as its
magnetization and are compensated to each other (as indicated by the
reversed blue and red pattern which are used to map the relative
magnitude of the spin up and spin down polarization). Examples of
hidden spin polarization in spin degenerate bulk antiferromagnets
made of spin split antiferromagnetic sectors are also illustrated for
MnS2
61 (bulk belonging to SST-2 with sector belonging to SST-4) and
La2NiO4
62 (bulk belonging to SST-3 with sector belonging to SST-4) in
Supplementary Information Section C. https://doi.org/10.1038/s41467-023-40877-8 https://doi.org/10.1038/s41467-023-40877-8 Article Table 1 | Easily exfoliable 2D magnetic compounds with AFM configuration belonging to SST-4 class
Formula
SG
Structure Prototype
Eg (eV)
Eb (meV/Å2)
3D SG
SDB of 3D
ID SDB
FeSe
P4/nmm
FeSe
0.0
22.6
Cmme
ICSD
290411
LaBr
P3m1
ZrCl
0.6
11.7
R3m
ICSD
23354
FeO2
Pmmm
FeO2
0.0
16.3
Cmcm
COD
9015156
PrOI
P4mm
PbClF
0.0
14.9
P4/nmm
COD
1530611
FeOCl
Pmmn
PeOCl
0.0
14.2
Pmmn
COD
1010645
VOBr
Pmmn
FeOCl
0.0
14.7
Pmmn
ICSD
27010
The formula, space group, 2D structure prototype, DFT-PBE calculated bandgap (Eg), and binding energy (Eb) are indicated. The last three columns describe for the experimental parent structure: the
3D space group (3D SG), source database (SDB)58, and the ID in the source database (ID SDB). 1 | Easily exfoliable 2D magnetic compounds with AFM configuration belonging to SST-4 class Table 1 | Easily exfoliable 2D magnetic compounds with AFM configuration bel The formula, space group, 2D structure prototype, DFT-PBE calculated bandgap (Eg), and binding energy (Eb) are indicated. The last three columns des
3D space group (3D SG), source database (SDB)58, and the ID in the source database (ID SDB). type, DFT-PBE calculated bandgap (Eg), and binding energy (Eb) are indicated. The last three columns describe for the experimental parent structure: th
DB)58, and the ID in the source database (ID SDB). crystal is the ferromagnetically ordered FeBr2 layer (sector belonging
to SST-5 class). The bilayer slab is built by stacking identical FeBr2 layer
with alternating magnetic ordering. The two layers are connected by
both ΘIT and UT symmetry, the bilayer hexagonal FeBr2 (MSG: PC-3c1)
thus belongs to a bulk SST-2 class, featuring a spin degenerate energy
band. However, the spin degenerate band structure of the SST-2 class
FeBr2 (Fig. 5a, b) is lifted upon the application of an external electric
field perpendicular to the layers (Ez) – a transition from SST-2 to SST-4. The spin splitting arises because of the external electric field Ez creates
a non-equivalent potential on the sectors and breaks the ΘIT and UT
symmetry of the bulk that connects the two layers. DFT calculations for
different values of the applied field, inserted in Fig. 5a, show that such
splitting is linearly proportional to the applied external electric field,
but in opposite spin polarization ordering for the bottom conduction
bands and the top valence bands. Compounds that have SOC-independent hidden spin
polarization The
identified candidates are: Ca2MnO4
47, CoSe2O5
48 and Fe2TeO6
49,
K2CoP2O7
50 and LiFePO4
51 whose bulk prototype is SST-1 with sector
prototype of SST-4; Sr2IrO4
52 whose bulk prototype is SST-2 with sector
prototype of SST-4; SrCo2V2O8
53 whose bulk prototype is SST-3 with
sector prototype of SST-4; CuMnAs54 and Mn2Au55 whole bulk proto-
type is SST-1 with sector prototype of SST-5; FeCl2 and CoCl2
56 whose
bulk prototype is SST-2 with sector prototype of SST-5; ErAuGe57
whose bulk prototype is SST-3 with sector prototype of SST-5. These
materials formthe platform for the exploration of the magnetic hidden
spin polarization effects. The opposite design philosophy (the bottom-to-top approach) is
to construct layered bulk antiferromagnets with the hidden effect
based on two-dimensional (2D) compounds that belong to SST-4 and
SST-5 prototypes. By searching through the database of predicted
naturally exfoliate 3D Van der Waals materials58, we find a list of 37
ferromagnetic 2D materials and 6 antiferromagnetic 2D monolayers
that can be used as such building blocks (see Tables 1 and 2 for the list). Other predicted and synthesized layered 2D materials are either Nature Communications| (2023) 14:5301 3 The effect of SOC on the predicted hidden spin polarization The effect of SOC on the predicted hidden spin polarization
The SOC-independent hidden spin polarization effect persists in the
presence of SOC. This is because the effect being inherited from the
unusual antiferromagnetic order rather than SOC38. Still, it is important
to note the inclusion of SOC would modify the energy bands in both
non-magnetic materials and magnetic materials43,64,65: (1) it reduces the
degeneracy of certain bands which may cause additional spin splitting. (2) it mixes the spin polarized states of up and down (so spin is no
longer a good quantum number), which results in momentum-
dependent spin polarization that are not unidirectionally aligned; (3)
it opens a gap for the crossing energy bands with opposite spin
polarization. In compounds consist of low-Z elements the SOC-
induced effect can be neglected. Revealing and tailoring the hidden spin polarization by external
electric field. To demonstrate the symmetry connection between
local sectors and the subsequent transition from hidden effect to
apparent effect mediated by the breaking of the symmetryconnection,
we apply in our calculations a perturbative symmetry-breaking exter-
nal electric field on an antiferromagnetic compound with hidden spin
polarization, hexagonal FeBr2 (DFT settings for applying the electric
field is provided in Methods section). The basic building block of the Nature Communications| (2023) 14:5301 4 https://doi.org/10.1038/s41467-023-40877-8 Article Table 2 | Easily exfoliable 2D magnetic compounds with FM
configuration belonging to SST-5 class invariant. By definition, antiferromagnets with primitive lattice trans-
lations that reverse the microscopic magnetic moments are known as
having black and white Bravais lattice that is classified as MSG type IV;
Antiferromagnets without such translation T belongs to MSG type I
and type III66. This suggests there is a one-to-one correspondence
between the existence or absence of the UT in the spin space group
and the MSG being type IV or type I/III. The effect of SOC on the predicted hidden spin polarization Use magnetic symmetry with SOC to describe the spin-splitting
of energy bands without SOC
In collinear antiferromagnetic compounds, the existence of UT in the
Table 2 | Easily exfoliable 2D magnetic compounds with FM
configuration belonging to SST-5 class
Formula
SG
Structure
Prototype
Eg
(eV)
Eb
(meV/Å2)
3D SG
SDB
of 3D
ID SDB
CoBr2
P3m1
CdI2
0.2
16.8
P3m1
COD
9016149
CoCl2
P3m1
CdI2
0.2
10.7
P3m1
COD
9014719
CoO2
P3m1
CdI2
0.0
22.6
P3m1
COD
1522027
FeBr2
P3m1
CdI2
0.0
15.5
P3m1
COD
9009102
FeI2
P3m1
CdI2
0.0
16.9
P3m1
COD
9009103
NiBr2
P3m1
CdI2
0.8
18.1
R3m
COD
9008013
NiCl2
P3m1
CdI2
1.1
16.3
R3m
COD
9009132
NiI2
P3m1
CdI2
0.3
21.5
R3m
COD
9009133
VS2
P3m1
CdI2
0.0
27.7
P3m1
ICSD
651361
VSe2
P3m1
CdI2
0.0
25.4
P3m1
ICSD
86520
VTe2
P3m1
CdI2
0.0
27.1
P3m1
ICSD
603582
TmI2
P3m1
CdI2
0.0
10.5
P3m1
ICSD
43731
LaBr2
P6m2
MoS2
0.6
11.2
P63/
mmc
ICSD
65481
FeTe
P4/
nmm
FeSe
0.0
26.6
P4/
nmm
ICSD
169974
LaCl
P3m1
ZrCl
0.0
11.0
R3m
ICSD
24410
ScCl
P3m1
ZrCl
0.0
13.8
R3m
COD
4343683
TbBr
P3m1
ZrCl
0.0
12.2
R3m
ICSD
23353
YCl
P3m1
ZrCl
0.0
17.6
R3m
ICSD
30708
CuCl2
C2/m
NbTe2
0.2
13.4
C2/m
COD
9001506
EuOBr
P4/
nmm
PbClF
0.0
17.4
P4/
nmm
ICSD
28531
EuOI
P4/
nmm
PbClF
0.0
14.8
P4/
nmm
ICSD
27666
PrOBr
P4/
nmm
PbClF
0.0
24.1
P4/
nmm
COD
2232654
NdOBr
C2/m
PbClF
0.2
21.8
P4/
nmm
COD
9009172
SmOBr
C2/m
PbClF
0.2
18.4
P4/
nmm
COD
1530050
TmOI
C2/m
PbClF
0.2
15.0
P4/
nmm
COD
2310429
TbOBr
Cmme
PbClF
0.0
15.2
P4/
nmm
ICSD
28532
CrOBr
Pmmn
FeOCl
0.5
14.8
Pmmn
ICSD
27092
CrOCl
Pmmn
FeOCl
0.6
13.8
Pmmn
ICSD
4086
CrSBr
Pmmn
FeOCl
0.4
19.5
Pmmn
ICSD
69659
ErSCl
Pmmn
FeOCl
0.3
11.9
Pmmn
ICSD
21009
ErSeI
Pmmn
FeOCl
0.0
11.6
Pmmn
ICSD
50194
HoSI
Pmmn
FeOCl
0.5
10.9
Pmmn
ICSD
425295
ErHCl
P3m1
SmSI
0.0
10.9
R3m
COD
1530725
SmSI
P3m1
SmSI
0.0
11.4
R3m
COD
1008317
YbOCl
P3m1
SmSI
0.0
11.7
R3m
ICSD
6077
CdOCl
P3m1
BiTeI
0.3
25.6
P63mc
COD
9016472
Co(OH)2
C2/m
Mg(OH)2
0.0
18.3
P3m1
ICSD
88940
The formula, space group, 2D structure prototype, DFT-PBE calculated bandgap (Eg), and
binding energy (Eb) are indicated. The effect of SOC on the predicted hidden spin polarization The last three columns describe for the experimental parent
structure: the 3D space group (3D SG), source database (SDB)58, and the ID in the source data-
base (ID SDB). The correspondence relation can be formally established by
introducing an auxiliary MSG—a subgroup of the spin space group
containing only elements of spatial and time reversal symmetries. This
is referred to as “MSG without SOC” in the Appendix of ref. 38 or
equivalently as “magnetic groups with pseudoscalar electron spin” in
ref. 67. Following that, we can prove a chain relation as depicted in
Eq. (1). ð1Þ ðcÞ ΘT in Standard MSG $ ðdÞ Standard MSG being type IV
ð1Þ (a) The existence or not of a UT symmetry in the SSG corresponds
to (b) the existence or not of a ΘT symmetry in the auxiliarygroup. This
is because the ΘU symmetry preserves any collinear magnetic ordering
and is a symmetry of any collinear antiferromagnets43–45. Meanwhile,
(b) the existence or not of a ΘT symmetry in the auxiliary MSG (without
SOC) corresponds to (c) the existence or not of a ΘT symmetry in the
standard MSG (with SOC). Antiferromagnetic materials whose (c) MSG
preserve (or violate) ΘT symmetry is classified as (d) MSG type IV (or
MSG type I/III)66. The established correspondence relation thus justifies the use of
MSG (with SOC)—avoiding the use of the “less familiar” spin
symmetry64—to predict whether the spin splitting effect without SOC
will occur. This also allows the use of the tabulated magnetic structure
symmetry information provided in material database46 to sort out
candidate materials39. For the prediction of the degeneracy of the full
bands without SOC, a comprehensive analyze of the spin symmetry
group and its irreducible representation is necessary43,64,65. Experimental detectability Experimental detectability
Analogous to the detection of SOC-induced hidden spin polarization in
nonmagnetic compounds (also known as R-2 and D-2 effects) [12], a
hidden property can be observed when a probe can resolve the local
sectors where the property is not compensated. Specific to hidden
spin polarization, the spatial segregation of the spin polarization states
allows
in
principle
the
detection
of
the
hidden
effect
in Hidden versus apparent spin polarization in noncollinear
antiferromagnets While the current paper focuses on the hidden spin polarization in
collinear antiferromagnetic compounds, we note that the hidden
effect can also exist in noncollinear antiferromagnetic compounds. When a bulk noncollinear antiferromagnetic compound has ΘIT sym-
metry, the energy bands are spin degenerate. If the system can be
further divided into separate sectors thatlocally violate ΘIT, then there
could exist hidden spin polarization pertaining to the individual sec-
tors. However, one should note that the symmetry condition of having
UT for preserving spin degeneracy in noncollinear antiferromagnetic
compounds39 is not valid anymore, this is because (1) when the spin
arrangement is non-coplanar, the MSG type IV does not guarantee the
existence of UT; Moreover, (2) when the spin arrangement is coplanar,
MSG type IV guarantees the existence of UT, but the existence of such
UT does not always guarantee spin degeneracy. Specifically, when the
spin states are not aligned in the same plane of the coplanar plane, the
UT symmetry will not reverse the spin states as it works in the collinear
magnetic systems. These properties of noncollinear antiferromagnets
offer new knobs to tune the hidden versus apparent spin polarization
via tilting the local magnetic motifs. The formula, space group, 2D structure prototype, DFT-PBE calculated bandgap (Eg), and
binding energy (Eb) are indicated. The last three columns describe for the experimental parent
structure: the 3D space group (3D SG), source database (SDB)58, and the ID in the source data-
base (ID SDB). Use magnetic symmetry with SOC to describe the spin-splitting
of energy bands without SOC The up and down spins are mapped to the color from blue to red. The crystal and
magnetic structure for tetragonal Ca2MnO4 used in our DFT calculations are taken
from ref. 47. degenerate band structure of Ca2MnO4; (c) Hidden spin polarization from each
individual sector of the lowest two conduction bands (C1 and C2) on ΓXR k-plane. The up and down spins are mapped to the color from blue to red. The crystal and
magnetic structure for tetragonal Ca2MnO4 used in our DFT calculations are taken
from ref. 47. Fig. 4 | Hidden spin polarization from the individual antiferromagnetic sector
in bulk tetragonal Ca2MnO4 (bulk belonging to SST-1 class with sector
belonging to SST-4 class). a Crystal structure of antiferromagnetic tetragonal
Ca2MnO4 composed of two antiferromagnetic sectors with opposite magnetic
ordering (the magnetic ordering is indicated by red and blue polyhedra) in the unit
cell. The two layers are referred to as sector-α and sector-β, respectively; (b) Spin degenerate band structure of Ca2MnO4; (c) Hidden spin polarization from each
individual sector of the lowest two conduction bands (C1 and C2) on ΓXR k-plane. The up and down spins are mapped to the color from blue to red. The crystal and
magnetic structure for tetragonal Ca2MnO4 used in our DFT calculations are taken
from ref. 47. spin polarization in AFM, since the unit cell can always be built in
terms of two or more sectors, electric field is a practically direct way
of inducing and controlling the existence of spin splitting (as well as
its magnitude) via modulating the symmetry relationship between
the sectors. For example, in the spin degenerate bulk antiferro-
magnets made of a pair of spin-split antiferromagnetic sectors (e.g.,
FeSe discussed in Supplementary Information Section D) or ferro-
magnetic sectors (e.g., FeBr2 discussed in the Results Section),
external electric field would break the ΘIT and UT symmetry between
the spin split sectors, which then implies a transition from hidden
effect to apparent effect. In fact, the electric field applied couples
with the electron spin through the magnetoelectric effect68, which is
only allowed under specific symmetry conditions41. Additionally,
transport properties that are even functions of the sectors can take
non-vanishing values in a hidden system. For example, non-
reciprocal nonlinear current respond to an applied electric field is
recently demonstrated in antiferromagnetic tetragonal CuMnAs69. This serves as a guide in search for systems exhibiting this particular antiferromagnets. Use magnetic symmetry with SOC to describe the spin-splitting
of energy bands without SOC In collinear antiferromagnetic compounds, the existence of UT in the
spin space group (SSG, symmetry group of the system without SOC)
means there is a spatial translation T that connects the atomic sites
with opposite magnetic moments and keeps the crystal structure Nature Communications| (2023) 14:5301 5 Article https://doi.org/10.1038/s41467-023-40877-8 Fig. 3 | Hidden spin polarization from individual ferromagnetic sectors in bulk
tetragonal CuMnAs (bulk belonging to SST-1 class with sector belonging to
SST-5 class). a Crystal structure of antiferromagnetic CuMnAs composed of two
ferromagnetic layers with opposite magnetization (indicated by red and blue
polyhedra) in the unit cell. The Cu atoms are dismissed. The two layers are referred
to as sector-α and sector-β, respectively; (b) Spin degenerate band structure of
CuMnAs; (c) Hidden spin polarization from each individual sector of the highest
two valence bands (V1 and V2) on ΓXS k-plane. The up and down spins are mapped
to the color from blue to red. The crystal and magnetic structure for tetragonal
CuMnAs used in our DFT calculations are taken from ref. 54. Fig. 3 | Hidden spin polarization from individual ferromagnetic sectors in bulk
tetragonal CuMnAs (bulk belonging to SST-1 class with sector belonging to
SST-5 class). a Crystal structure of antiferromagnetic CuMnAs composed of two
ferromagnetic layers with opposite magnetization (indicated by red and blue
polyhedra) in the unit cell. The Cu atoms are dismissed. The two layers are referred to as sector-α and sector-β, respectively; (b) Spin degenerate band structure of
CuMnAs; (c) Hidden spin polarization from each individual sector of the highest
two valence bands (V1 and V2) on ΓXS k-plane. The up and down spins are mapped
to the color from blue to red. The crystal and magnetic structure for tetragonal
CuMnAs used in our DFT calculations are taken from ref. 54. Fig. 4 | Hidden spin polarization from the individual antiferromagnetic sector
in bulk tetragonal Ca2MnO4 (bulk belonging to SST-1 class with sector
belonging to SST-4 class). a Crystal structure of antiferromagnetic tetragonal
Ca2MnO4 composed of two antiferromagnetic sectors with opposite magnetic
ordering (the magnetic ordering is indicated by red and blue polyhedra) in the unit
cell. The two layers are referred to as sector-α and sector-β, respectively; (b) Spin
degenerate band structure of Ca2MnO4; (c) Hidden spin polarization from each
individual sector of the lowest two conduction bands (C1 and C2) on ΓXR k-plane. Use magnetic symmetry with SOC to describe the spin-splitting
of energy bands without SOC Since this effect is intrinsic to the bulk it can be
distinguished from the surface effect as the latter sensitively depends
on the effective penetration depth of the probing beam [43]. Albeit, for
the hidden spin polarization from individual AFM sectors, to detect the
AFM spin polarization of the individual sectors, one needs to choose
the surface configuration that respects the symmetries of the indivi-
dual sector that ensure the anti-ferromagnetism of the sector, e.g.,
mirror plane symmetries perpendicular to the surface plane that
connect the spin up and spin down magnetic moments of the AFM
sector. Especially, systems with the degenerate states segregated on
the different sectors would result in a minimally compensated hidden
spin polarization, thus contributing to a robust signal when selectively
probing the individual sector, thus being ideal platforms for the
detection of the hidden effect. Electric and magnetic field control of the hidden effect
One of the most desirable features of spin-related phenomena is the
possibility of electric and magnetic control. In the case of the hidden Electric and magnetic field control of the hidden effect Data availability The VASP configuration and output files thatsupport the finding of this
study have been deposited in figshare with the identifier [data
DOI:10.6084/m9.figshare.22693042]. Other
data
related
to
this
research are available from the corresponding author upon reasonable
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layers
with
alternatively
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Energy, Office of Science, Basic Energy Sciences, Materials Sciences
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12229-y (2019). PMID - 31541112. 50. Sale, M., Avdeev, M., Mohamed, Z., Ling, C. D. & Barpanda, P. Magnetic structure and properties of centrosymmetric twisted- Nature Communications| (2023) 14:5301 8 Article https://doi.org/10.1038/s41467-023-40877-8 https://doi.org/10.1038/s41467-023-40877-8 Author contributions A.Z. conceived and supervised the project. L.-D.Y., X.Z. and C.M.A. developed the idea and designed the research. L.-D.Y. and X.Z. con-
ducted the search for candidate antiferromagnetic materials from the
existing database. L.-D.Y. performed the density functional calcula-
tions and plot the Figures. C.M.A. performed the “bottom-to-top”
material design of layered bulk antiferromagnets with the hidden
effect. L.-D.Y., X.Z., C.M.A., and A.Z. analyzed the results and wrote the
manuscript. 65. Liu, P., Li, J., Han, J., Wan, X. & Liu, Q. Spin-group symmetry in
magnetic materials with negligible spin-orbit coupling. Phys. Rev. X
12, 021016, https://doi.org/10.1103/PhysRevX.12.021016 (2022). 66. Zamorzaev, A. Generalization of Fedorov groups. Kristallografiya 2,
15–20 (1957). 67. Turek, I. Altermagnetism and magnetic groups with pseudoscalar
electron spin. Phys. Rev. B 106, 094432, https://doi.org/10.1103/
PhysRevB.106.094432 (2022). Competing interests Competing interests
The authors declare no competing interests. 68. Fiebig, M. Revival of the magnetoelectric effect. J. Phys. D: Appl. Phys. 38, R123, https://doi.org/10.1088/0022-3727/38/8/R01
(2005). 9 Nature Communications| (2023) 14:5301 9 Article Additional information Additional information Open Access This article is licensed under a Creative Commons
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PLACE OF THE THEORY OF ECONOMIC LAW IN THE SYSTEM OF LEGAL REGULATION OF ECONOMY
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Vestnik Universiteta imeni O.E. Kutafina (MGÛA)/Vestnik Universiteta imeni O. E. Kutafina
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V. S. ELISEEV, V. S. ELISEEV,
Professor at the Department of Environmental Law
of the Kutafin Moscow State Law University (MSAL),
Dr. Sci. (Law), Associate Professor
v.s.eliseev@mail.ru
125993, Russia, Moscow, ul. Sadovaya-Kudrinskaya, 9 V. S. ELISEEV,
Professor at the Department of Environmental Law
of the Kutafin Moscow State Law University (MSAL),
Dr. Sci. (Law), Associate Professor
v.s.eliseev@mail.ru
125993, Russia, Moscow, ul. Sadovaya-Kudrinskaya, 9 Елисеев В. С.
Место теории экономического права
в системе правового регулирования экономики Елисеев В. С. Место теории экономического права
в системе правового регулирования экономики Елисеев В. С. 175 175 МЕСТО ТЕОРИИ ЭКОНОМИЧЕСКОГО ПРАВА
В СИСТЕМЕ ПРАВОВОГО РЕГУЛИРОВАНИЯ
ЭКОНОМИКИ Аннотация. Статья анализирует место теории экономического
права в системе правового регулирования экономических отношений,
показывает развитие экономического законодательства в истори-
ческом контексте, сопоставляет теорию экономического права с
хозяйственным, гражданским, предпринимательским, коммерческим
и экономическим правом. Автор обосновывает вывод о том, что
теория экономического права является составной частью теории
права. Обосновывается необходимость использования теории эко-
номического права в ее перспективе, которая позволит согласовы-
вать экономическое законодательство. Теория рассматривает в
качестве основы правового воздействия экономические институты
как первичные области формирования правовых режимов, имеющих
комплексный межотраслевой характер. Вячеслав
Сергеевич ЕЛИСЕЕВ, Вячеслав
Сергеевич ЕЛИСЕЕВ,
профессор кафедры
экологического и
природоресурсного права
Университета
имени О.Е. Кутафина
(МГЮА),
доктор юридических наук,
доцент
v.s.eliseev@mail.ru
125993, Россия, г. Москва,
ул. Садовая-Кудринская, д. 9 Сергеевич ЕЛИСЕЕВ,
профессор кафедры
экологического и
природоресурсного права
Университета
имени О.Е. Кутафина
(МГЮА), Ключевые слова: экономическое право, теория экономического права,
правовое регулирование экономических отношений, концепция (тео-
рия) правового регулирования экономических отношений, развитие
экономического законодательства. доктор юридических наук,
доцент v.s.eliseev@mail.ru
125993, Россия, г. Москва,
ул. Садовая-Кудринская, д. 9 DOI: 10.17803/2311-5998.2020.71.7.175-184 1 См., например: Лаптев В. В. Хозяйственное право — право предпринимательской деятель-
ности // Государство и право. 1993. № 1. С. 33—38 ; Дозорцев В. А. Один кодекс или два?
(Нужен ли хозяйственный кодекс наряду с гражданским?) // Правовые проблемы рыночной
экономики в Российской Федерации : Труды Института законодательства и сравнительного
правоведения при Правительстве Российской Федерации. Вып. 57. М., 1994. С. 115—143.
2 Победоносцев К. П. Курс гражданского права // Сочинения : в 2 т. СПб. : Типография
Мин. пут. сообщения (А. Бенке), 1896. Ч. 1—3. 4 Источники русского торгового права : сборник извлечений из Свода законов, уставов,
полисных условий и договоров. М., 1914. 5 Ершова И. В. Проблемы правового режима государственного имущества в хозяйствен-
ном обороте: теоретические основы и пути совершенствования. М. : Юриспруденция,
2001. С. 10—11. 1 См., например: Лаптев В. В. Хозяйственное право — право предпринимательской деятель-
ности // Государство и право. 1993. № 1. С. 33—38 ; Дозорцев В. А. Один кодекс или два?
(Нужен ли хозяйственный кодекс наряду с гражданским?) // Правовые проблемы рыночной
экономики в Российской Федерации : Труды Института законодательства и сравнительного
правоведения при Правительстве Российской Федерации. Вып. 57. М., 1994. С. 115—143.
2 Победоносцев К. П. Курс гражданского права // Сочинения : в 2 т. СПб. : Типография
Мин. пут. сообщения (А. Бенке), 1896. Ч. 1—3.
3 Шершеневич Г. Ф. Учебник торгового права [по изд. 1914 г.]. М. : Спарк, 1994.
4 Источники русского торгового права : сборник извлечений из Свода законов, уставов,
полисных условий и договоров. М., 1914.
5 Ершова И. В. Проблемы правового режима государственного имущества в хозяйствен-
ном обороте: теоретические основы и пути совершенствования. М. : Юриспруденция,
2001. С. 10—11. 3 Шершеневич Г. Ф. Учебник торгового права [по изд. 1914 г.]. М. : Спарк, 1994. 6 Аскназий С. И. Очерки хозяйственного права в СССР. Л., 1926.
7 Таль Л. Понятие и сущность хозяйственного права // Право и жизнь. 1924. Кн. 9. С. 22.
8 Гинс Г. Поиски нового права // Новая экономика и новое право. Харбин, 1938. С. 26, 38.
9 Либерман С. Последний этап нэпа и хозяйственное право // Проблемы марксизма. 1931.
№ 5—6.
10 Вольф В. Ю. Основы хозяйственного права. М., 1928 ; Стучка П. И. Курс советского
гражданского права. Т. 1. М.-Л., 1931.
11 Стучка П. И. Указ. соч.
12 Стучка П. И., Амфитеатров Г. Н. Основы гражданского законодательства СССР.
М., 1931. С. 5. 12 Стучка П. И., Амфитеатров Г. Н. Основы гражданского законодательства СССР.
М., 1931. С. 5. PLACE OF THE THEORY
OF ECONOMIC LAW IN THE SYSTEM
OF LEGAL REGULATION OF ECONOMY Abstract. The article analyzes the place of the "Theory of economic law"
in the system of legal regulation of economic relations. It shows the histori-
cal development of economic legislation. The "Theory of economic law" is
compared with economic, civil, business, commercial and economic law. The author believes that the " Theory of economic law "is an integral part of
the "Theory of law". The article substantiates the need to use the "Theory
of economic law" in its perspective, which will allow to coordinate economic
legislation. The Theory considers economic institutions as the basis of le- © В. С. Елисеев, 2020 7/2020 176 gal influence. These institutions are the primary areas in the formation of
legal regimes. Legal regimes have a complex cross-sectoral character. Keywords: economic law, theory of economic law, legal regulation of eco-
nomic relations, concept (theory) of legal regulation of economic relations,
development of economic legislation. В
настоящее время вновь возрастает острота дискуссий между представи-
телями различных отраслей права об их месте в системе правового регу-
лирования экономических отношений, актуальность которых приобретает
новый оттенок в силу появления «экономического права», вызвавшего в свой
адрес очередную волну критики, как конструктивной, так и политизированной. В На этом фоне появляются рассуждения о теории экономического права, для
понимания места которой в системе правового регулирования экономики необ-
ходимо обратиться к историческому аспекту развития правовых школ, направ-
ленных на правовое обеспечение экономических отношений с различных пози-
ций, на основе концепций гражданского, хозяйственного, предпринимательского,
коммерческого и экономического права1. В первую очередь следует коснуться дореволюционного периода развития
права в России, основу которого составляли работы таких известных предста-
вителей гражданского права, как К. П. Победоносцев2, Г. Ф. Шершеневич3 и др. Из наиболее показательных документов того времени следует выделить, в
частности, Устав о промышленности, Устав торговый, Устав о векселях и подоб-
ные законы4, которые, по сути, были комплексными нормативными документами,
что объясняется тем, что в дореволюционной России существовал достаточно
разветвленный государственный сектор экономики, место которого в общем рос-
сийском экономическом пространстве было значительно. В научной литературе
отмечается, что государству достаточно часто приходилось выкупать частные
проекты с тем, чтобы «достраивать и перестраивать» начатые стройки, как, в
частности, это имело место в отношении строительства объектов железнодо-
рожной инфраструктуры5. Что касается стратегических объектов, как КВЖД, то
их строительство без участия государства было невозможно в принципе. 7/2020 7/2020 Елисеев В. С. Место теории экономического права
в системе правового регулирования экономики Елисеев В. С. 11 Стучка П. И. Указ. соч. 13 Курс советского хозяйственного права / под ред. Е. Б. Пашуканиса и Л. Я. Гинцбурга. М.,
1935. Т. 1.
14 Мартемьянов В. С. Юридическая трагедия // Юридическая газета. 1992. № 1, 2.
15 Вышинский А. Я. Основные задачи науки советского социалистического права // Совет-
ское государство и право. 1939. № 1. С. 22.
16 Братусь С. Н., Алексеев С. С. О разработке правовых вопросов управления народным
хозяйством // Правоведение. 1963. № 4. С.45.
17 Венедиктов А. В. Государственная социалистическая собственность. М.-Л. : АН СССР, 1948.
18 Лаптев В. В. Предмет и система хозяйственного права. М. : Юрид. лит., 1969.
19 Мамутов В. К. Совершенствование правового регулирования хозяйственной деятель-
ности. Киев, 1982. PLACE OF THE THEORY
OF ECONOMIC LAW IN THE SYSTEM
OF LEGAL REGULATION OF ECONOMY Место теории экономического права
в системе правового регулирования экономи 177 Февральские и особенно октябрьские события 1917 г. изменили ситуацию в
ее основе: произошла национализация и был сформирован государственный и
колхозно-кооперативный сектор экономики; гражданское право уступило место
публичному правовому регулированию. На первое место выходит хозяйственное
право. При этом исследователи отмечают, что термин «хозяйственное право»
частенько подменял термин «гражданское право», иногда мало чем отличаясь
от последнего6. И хотя наука больше шла по пути практического творчества, был накоплен
огромный опыт хозяйствования, планирования, формирования социалистической
(государственной) инфраструктуры. Главной особенностью научных взглядов
того периода была чрезмерная надежда на «революционное правосознание»,
что являлось большим недостатком для формирования права7. Вместе с тем стало очевидно, что хозяйственное право выходит за рамки
частного, а также административного права, являясь самостоятельным комплекс-
ным образованием8, — речь шла в первую очередь о государственном секторе
экономики. В данный период сформировались три основных взгляда на хозяйственное
право: во-первых, оно охватывало все области народного хозяйства (как в тот
период обозначали экономику страны); во-вторых, хозяйственное право регу-
лировало только государственный сектор экономики (социалистические произ-
водственные отношения)9; в-третьих, нельзя не отметить гражданско-правовую
концепцию отрицания роли хозяйственного права как самостоятельной отрасли10. Следует отметить возникновение в 20‑х гг. прошлого столетия «двухсектор-
ной» теории, разработанной П. И. Стучкой11, появившейся вместе с так называ-
емой новой экономической политикой молодого социалистического государства. Суть данного взгляда сводилась к признанию параллельного существования
частного сектора экономики, регламентируемого на основе гражданского права,
и государственного сектора экономики, регулируемого хозяйственным правом. Главной конструктивной мыслью данной теории являлось разделение правового
регулирования для различных секторов экономики. Логическим венцом теории П. И. Стучки были предложения о принятии двух
самостоятельных кодексов12. Однако этот взгляд был принципиально отвергнут
на I Всесоюзном съезде марксистов-государственников в 1931 г. Продолжением марксистской политики государства является появление школы
единого хозяйственного права, связанной с именами Л. Я. Гинцбурга и Е. Б. Па- 7/2020 178 шуканиса13. По сути, это была попытка одним документов в виде Хозяйственного
кодекса объединить государственный и частный сектора экономики на основе
комплексного правового регулирования экономических отношений. Авторы сде-
лали заключение, что административные и гражданско-правовые нормы права
должны гармонично сочетаться, а не противоречить друг другу, на что данная
школа и была направлена. Однако под влиянием представителей цивилистики в сложный период 1937—
1938 гг. авторам была поставлена в вину попытка уничтожения гражданского
права, их объявили врагами народа и репрессировали14. В середине 1930‑х гг. появляется «дуалистическая концепция», сформиро-
ванная под влиянием А. Я. Вышинского: вертикальные экономические отноше-
ния предлагалось регулировать административным правом, а горизонтальные
отношения — гражданским правом. 13 Курс советского хозяйственного права / под ред. Е. Б. Пашуканиса и Л. Я. Гинцбурга. М.,
1935. Т. 1. 19 Мамутов В. К. Совершенствование правового регулирования хозяйственной деятель-
ности. Киев, 1982. PLACE OF THE THEORY
OF ECONOMIC LAW IN THE SYSTEM
OF LEGAL REGULATION OF ECONOMY Сама же концепция единого хозяйственного
права была подвергнута острой критике15. После Великой Отечественной войны появляется концепция хозяйственно-
административного права, сформированная в начале 1960‑х гг. С. Н. Братусем
и С. С. Алексеевым. Согласно данному подходу при административно-правовом
регулировании после «погружения вглубь» хозяйственных отношений образуется
«мертвая зона», которая должна регулироваться хозяйственно-административ-
ным правом — относительно самостоятельной подотраслью юридической науки16. В дальнейшем С. С. Алексеев от этой позиции отказался и перешел на позиции
гражданско-правового регулирования экономических отношений. Для послевоенного периода большое значение имела разработка А. В. Вене-
диктовым конструкции права оперативного управления17, которая и в настоящее
время не потеряла своей актуальности для правового регулирования экономи-
ческих отношений с участием государственных организаций. Наиболее значимым событием является разработка в конце 1950‑х гг. про-
шлого века В. В. Лаптевым18 и В. К. Мамутовым19 третьей школы хозяйственного
права. Согласно данной концепции, правовое регулирование экономики имеет
монистический характер — здесь в основе принцип единства хозяйственно-право-
вого регулирования. Вместе с тем имеет место двойственность частно-публично-
го характера: правовое регулирование вертикальных экономических отношений
предполагает административный характер, а правовое регулирование горизон- 7/2020 7/2020 Елисеев В. С. Место теории экономического права
в системе правового регулирования экономики 179 179 тальных отношений — гражданско-правовой. В целом авторы придерживались
мнения о единстве хозяйственных отношений (их однородности)20. тальных отношений — гражданско-правовой. В целом авторы придерживались
мнения о единстве хозяйственных отношений (их однородности)20. Как отмечал В. К. Мамутов, школа последовательно рассматривала хозяй-
ственное право в качестве самостоятельной отрасли права, но эта отрасль не
является «рафинированной» — она имеет «взаимопроникающие начала с дру-
гими отраслями права»21. Авторы полагали, что необходимо принять Хозяйственный кодекс, проект ко-
торого в СССР был разработан, но не был принят. Что касается населения, то
отношения с его участием относятся к предмету гражданского права. Следует отметить, что после распада Советского Союза В. К. Мамутовым,
возглавившим украинскую школу хозяйственного права, эта цель была достиг-
нута — принят Хозяйственный кодекс Украины22. Что касается России, то уси-
лия В. В. Лаптева и его сподвижников со стороны органов власти поддержки
не нашли. В науке выделяется четвертая школы хозяйственного (предпринимательского)
права, которая связывается с именем В. С. Мартемьянова23. 26 Предпринимательское (хозяйственное) право / под ред. В. В. Лаптева, С. С. Занковского.
М. : Волтерс Клувер, 2006. 560 с. 25 Предпринимательское право России: итоги, тенденции и пути развития : монография /
Е. П. Губин, В. А. Вайпан, И. С. Шиткина [и др. ] ; отв. ред. Е. П. Губин. М. : Юстицинформ,
2019. 664 с. 20 Теоретические проблемы хозяйственного права. М. : Наука, 1975. С. 28.
21 Отграничение хозяйственного права от других отраслей права и их взаимодействие //
Хозяйственное право : учебник / В. К. Мамутов, Г. Л. Знаменский, К. С.Халиулин [и др.] ;
под ред. В. К. Мамутова. Киев : Юринком Интер, 2002. С. 34—38.
22 Господарський кодекс України // Відомості Верховної Ради. 2003. № 18. Ст. 144.
23 Мартемьянов В. С. Хозяйственное право : курс лекций : в 2 т. М. : Бек, 1994.
24 См.: Ершова И. В., Енькова Е. Е., Тарасенко О. А., Трофимова Е. В. Государственное
регулирование предпринимательской деятельности : учебник / отв. ред. И. В. Ершова.
М. : Проспект, 2020. 224 с.
25 Предпринимательское право России: итоги, тенденции и пути развития : монография /
Е. П. Губин, В. А. Вайпан, И. С. Шиткина [и др. ] ; отв. ред. Е. П. Губин. М. : Юстицинформ,
2019. 664 с.
26 Предпринимательское (хозяйственное) право / под ред. В. В. Лаптева, С. С. Занковского.
М. : Волтерс Клувер, 2006. 560 с. 20 Теоретические проблемы хозяйственного права. М. : Наука, 1975. С. 28. 24 См.: Ершова И. В., Енькова Е. Е., Тарасенко О. А., Трофимова Е. В. Государственное
регулирование предпринимательской деятельности : учебник / отв. ред. И. В. Ершова.
М. : Проспект, 2020. 224 с. PLACE OF THE THEORY
OF ECONOMIC LAW IN THE SYSTEM
OF LEGAL REGULATION OF ECONOMY На наш взгляд, кор-
ректнее именовать ее школой предпринимательского права, поскольку, во-первых,
термин «хозяйственное право» фактически выбыл из оборота и используется
лишь как «дань традиции», но не более того; во-вторых, все-таки принадлеж-
ность хозяйственных отношений нужно связывать с государственным сектором
экономики, а не со всей экономикой в целом, а представители данной школы при-
держиваются именно общего охвата экономических отношений; в-третьих, ряд
представителей данной школы придерживаются теории предпринимательского
права как составной части гражданского права. В настоящее время школа предпринимательского права получила развитие
под влиянием кафедры предпринимательского и корпоративного права Универ-
ситета имени О.Е. Кутафина (МГЮА)24 (заведующий кафедрой — И. В. Ершова),
кафедры предпринимательского права МГУ имени М. В. Ломоносова25 (заведу-
ющий кафедрой — Е. П. Губин), сектора предпринимательского и корпоративного
права Института государства и права Российской академии наук26 (руководитель 7/2020 180 сектора — С. С. Занковский). В этом ряду нельзя также не отметить А. Г. Быкова27,
В. К. Андреева28, Н. М. Коршунова29, И. В. Дойникова, В. С. Белых30. На самостоятельном существовании коммерческого права настаивают
Б. И. Пугинский и его последователи, прежде всего с кафедры коммерческого
права МГУ имени М. В. Ломоносова. Они разделяют идеи дуализма гражданского
права и отстаивают идею самостоятельности коммерческого права, выступаю-
щего подотраслью гражданского права31. Вместе с тем все многообразие концепций хозяйственного, предприниматель-
ского и коммерческого права существует при подавляющем господстве граждан-
ско-правовой концепции предпринимательского права. Согласно такому подходу
предпринимательское право, равно как и коммерческое, теряет свою самосто-
ятельность и рассматривается как часть гражданского права. Что касается «хо-
зяйственного права», то такое наименование рассматривается как «пережиток
прошлого»32. Появление экономического права тоже не имеет полной определенности: в
первую очередь необходимо отметить предельно широкий подход, когда эконо-
мическое право рассматривается как обобщенная учебная дисциплина, собрав-
шая различные институты права, в той или иной степени непосредственно отно-
сящиеся к регулированию экономических отношений33, подобно использованию
экономистами термина «хозяйственное право»34. При таком подходе вести речь 29 Предпринимательское право : учебник для студентов вузов, обучающихся по специаль-
ности 030501 «Юриспруденция» / Н. Д. Эриашвили [и др.] ; под ред. Н. М. Коршунова,
Н. Д. Эриашвили, П. В. Алексия. 6-е изд., перераб. и доп. М. : Юнити-Дана ; Закон и
право, 2017. 535 с. 31 Коммерческое право: актуальные проблемы и перспективы развития : сб. статей к юби-
лею доктора юрид. наук, проф. Б. И. Пугинского / сост. Е. А. Абросимова, С. Ю. Филип-
пова. М. : Статут, 2011. 286 с. 32 См., например: Братусь С. Н. Предмет и система советского гражданского права. М.,
1963. С. 30 Предпринимательское право: современный взгляд : монография / МГУ имени М. В. Ло-
моносова ; Е. А. Абросимова, В. К. Андреев, Е. Г. Афанасьев [и др.] ; отв. ред. С. А. Ка-
релина, П. Г. Лахно. М. : Юстицинформ, 2019. 600 с. 27 Быков А. Г. Предпринимательское право: проблемы формирования и развития // Вестник
Моск. ун-та. Серия «Право». 1993. № 6. 31 Коммерческое право: актуальные проблемы и перспективы развития : сб. статей к юби-
лею доктора юрид. наук, проф. Б. И. Пугинского / сост. Е. А. Абросимова, С. Ю. Филип-
пова. М. : Статут, 2011. 286 с. 27 Быков А. Г. Предпринимательское право: проблемы формирования и развития // Вестник
Моск. ун-та. Серия «Право». 1993. № 6.
28 Право и экономическое развитие: проблемы государственного регулирования экономики :
монография / В. К. Андреев, Л. В. Андреева, К. М. Арсланов [и д.] ; под ред. В. А. Вай-
пана, М. А. Егоровой. М. : Юстицинформ, 2017. 432 с.
29 Предпринимательское право : учебник для студентов вузов, обучающихся по специаль-
ности 030501 «Юриспруденция» / Н. Д. Эриашвили [и др.] ; под ред. Н. М. Коршунова,
Н. Д. Эриашвили, П. В. Алексия. 6-е изд., перераб. и доп. М. : Юнити-Дана ; Закон и
право, 2017. 535 с.
30 Предпринимательское право: современный взгляд : монография / МГУ имени М В Ло- 28 Право и экономическое развитие: проблемы государственного регулирования экономики :
монография / В. К. Андреев, Л. В. Андреева, К. М. Арсланов [и д.] ; под ред. В. А. Вай-
пана, М. А. Егоровой. М. : Юстицинформ, 2017. 432 с. 35 Ершов В. В., Ашмарина Е. М., Корнев В. Н. Экономическое право Российской Федера-
ции : монография. М. : РГУП, 2017. 284 с.
36 Альбов А. П., Ручкина Г. Ф. Экономическое право как новое направление развития правовой
науки // Ученые труды Российской академии адвокатуры и нотариата. 2015. № 3 (38). С. 97.
37 Альбов А. П., Ручкина Г. Ф. Указ. соч. С. 99.
38 Ашмарина Е. М. Экономическое право как микромодель системы российского права //
Вопросы экономики и права. 2015. № 84. С. 7—13.
39 Мирошник С. В. Экономическое право в системе российского права // Вопросы эконо-
мики и права. 2016. № 1. С. 12—14.
40 Лаптев В. А. «Мегаотрасли» — экономическое, корпоративное и энергетическое право
или юридические фикции? // Экономика. Право. Общество. 2017. № 3. С. 28—33.
41 Веленто И. И., Елисеев В. С. Теория экономического права : курс лекций. Гродно : ГрГУ,
2004. 417 с. PLACE OF THE THEORY
OF ECONOMIC LAW IN THE SYSTEM
OF LEGAL REGULATION OF ECONOMY 11 ; Толстой Ю. К. Понятие права собственности // Проблемы гражданского
и административного права / под ред. Б. Б. Черепахина, О. С. Иоффе, Ю. К. Толстого. Л., 1962. С. 143 ; Дозорцев В. А. Один кодекс или два? (Нужен ли хозяйственный кодекс
наряду с гражданским?) // Правовые проблемы рыночной экономики в Российской Фе-
дерации : Труды Института законодательства и сравнительного правоведения при Пра-
вительстве Российской Федерации. М., 1994. Вып. 57. С. 115—143. 33 Экономическое право : хрестоматия : в 3 т. СПб. : Питер, 2004. 34 Круглова Н. Ю. Хозяйственное право : учебное пособие для бакалавров. 6-е изд., пере-
раб. и доп. М. : Юрайт, 2015. 883 с. 7/2020 7/2020 Елисеев В. С. Место теории экономического права
в системе правового регулирования экономики 181 181 о новой отрасли права не приходится, поскольку в указанном «сборнике» сосре-
доточены разделы различных отраслей права, в том числе административного,
гражданского, предпринимательского, финансового, аграрного, налогового и т.д. о новой отрасли права не приходится, поскольку в указанном «сборнике» сосре-
доточены разделы различных отраслей права, в том числе административного,
гражданского, предпринимательского, финансового, аграрного, налогового и т.д. Глобальный подход к экономическому праву предлагают ученые Российско-
го государственного университета правосудия (Е. М. Ашмарина, В. В. Ершов,
В. Н. Корнев и др.)35, рассматривая его в качестве мегаотрасли, которая «пред-
ставляет собой рецепцию и аккумуляцию основ всех отраслей первого порядка
по признаку всеобъемлющего характера своего предмета» в «единый правовой
комплекс — экономическое право Российской Федерации». Оно должно «устано-
вить связь между различными отраслями права, регулирующими экономические
отношения, государственной политикой и экономикой»36 и «призвано объединить
в себя все правовые нормы, регулирующие общественные отношения в сфере
экономики, которые будут встроены в мировоззрение и культуру народа, на ко-
тором и будет происходить поступательное развитие экономики»37. Е. М. Ашмариной предлагается примерная структура экономического права:
Общая часть: «экономические основы экономического права», «экономическое
право РФ»; Особенная часть: «правовое регулирование отношений в сфере фи-
нансов», «правовое обеспечение денежного обращения, кредита и банковской
деятельности», «учетные системы и их правовое регулирование»38 и т.п. Наиболее часто встречающиеся аргументы критиков экономического права:
«Экономическое право не отвечает классическим критериям и не может быть
признано самостоятельной отраслью права», поскольку оно «регулирует раз-
нообразные экономические отношения, сочетает в себе как публичные, так и
частные начала»39. В. А. Лаптев отмечает, что по факту «мы опять возвращаемся к давно суще-
ствующему понятию “хозяйственное” либо “предпринимательское право”, что
ставит под сомнение открытие новой отрасли — экономического права»40. 41 Веленто И. И., Елисеев В. С. Теория экономического права : курс лекций. Гродно : ГрГУ,
2004. 417 с. PLACE OF THE THEORY
OF ECONOMIC LAW IN THE SYSTEM
OF LEGAL REGULATION OF ECONOMY Устранению приведенных недостатков может способствовать позиция, постро-
енная на обращении к теории экономического права. Данная позиция разработана
автором настоящей статьи совместно с И. И. Веленто в 1994 г., если считать за
основу дату выпуска первой книги с указанным названием на базе Гродненского
государственного университета имени Я. Купалы (Республика Беларусь)41. 7/2020 182 182 Первоначально авторы определяли место экономического права как главной
правовой отрасли, регулирующей экономические отношения, в основу построения
которой предлагался так называемый Главный кодекс42. Однако впоследствии
от данной идеи они отказались, сделав вывод, что такое обобщение — предмет
исключительно правовой теории, именно на этом (теоретическом) уровне не-
обходимо вести речь о гармонизации различных направлений экономического
законодательства. Вместе с тем, чтобы обобщить все отрасли права, в той или иной степени
регулирующие экономические отношения, актуальным является применение
категории «экономическое право». Собственно, категория «экономические отношения» ранее не приветство-
валась в праве: вместо нее применялся термин «хозяйственные отношения»,
который впоследствии был заменен на «имущественные отношения». С разви-
тием рыночных отношений наступило понимание того, что определенные отрас-
ли права регулируют экономические отношения и табу с данного термина было
снято. С этих позиций категория «экономические отношения» («экономическое
поведение»), по сути, заимствованная из экономической теории, наиболее точно
отражает связь экономики и ее правового регулирования (правового обеспечения). В настоящее время вышла в свет кардинально обновленная книга: «Теория
экономического права: теория отраслей права, обеспечивающих экономические
отношения»43. Теория не ставит своей целью формирование новой мегаотрасли права —
экономического права, как это предлагают представители Российского государ-
ственного университета правосудия. Главная ее идея заключается в том, что в ней
отражены объективные закономерности правового регулирования (правового
обеспечения) экономических отношений, которые едины для всех отраслей права,
в той или иной степени регулирующих экономические отношения. Причем за ос-
нову правового регулирования берутся функционально обособленные институты
(сформированные области) экономических отношений. По указанной причине те-
ория экономического права рассматривается всего лишь как раздел теории права. В свою очередь, экономический институт выступает с позиции права комплекс-
ным образованием, в состав которого входят, во-первых, отношения, подлежащие
правовой регламентации регулятивными нормами права; во-вторых, юридически
нейтральные отношения, в которых не возникают юридические обязательства,
хотя экономические притязания присутствуют (как, например, в игорном бизнесе);
в-третьих, негативное экономическое поведение, которое подлежит сдержива-
нию и подавлению охранительными нормами права, — все указанное входит в
логически оформленную единую область (институт) экономических отношений
и соответствующий институт правового регулирования. Теория не ставит своей целью формирование новой мегаотрасли права —
экономического права, как это предлагают представители Российского государ-
ственного университета правосудия. 42 Елисеев В. С. Проблемы кодификации в регулировании экономических отношений в
Российской Федерации и Республике Беларусь // Российско-белорусская наука XXI века
и проблемы законодательства : материалы Междунар. науч.-практ. конф. Калуга, 2002 /
В. С. Елисеев (отв. ред.). Калуга : Гриф, 2002. С. 51—63. 43 Елисеев В. С., Веленто И. И. Теория экономического права: теория отраслей права, обе-
спечивающих экономические отношения : учебное пособие. М. : Проспект, 2019. 416 с. 42 Елисеев В. С. Проблемы кодификации в регулировании экономических отношений в
Российской Федерации и Республике Беларусь // Российско-белорусская наука XXI века
и проблемы законодательства : материалы Междунар. науч.-практ. конф. Калуга, 2002 /
В. С. Елисеев (отв. ред.). Калуга : Гриф, 2002. С. 51—63.
43 Елисеев В. С., Веленто И. И. Теория экономического права: теория отраслей права, обе-
спечивающих экономические отношения : учебное пособие. М. : Проспект, 2019. 416 с. PLACE OF THE THEORY
OF ECONOMIC LAW IN THE SYSTEM
OF LEGAL REGULATION OF ECONOMY Главная ее идея заключается в том, что в ней
отражены объективные закономерности правового регулирования (правового
обеспечения) экономических отношений, которые едины для всех отраслей права,
в той или иной степени регулирующих экономические отношения. Причем за ос-
нову правового регулирования берутся функционально обособленные институты
(сформированные области) экономических отношений. По указанной причине те-
ория экономического права рассматривается всего лишь как раздел теории права. В свою очередь, экономический институт выступает с позиции права комплекс-
ным образованием, в состав которого входят, во-первых, отношения, подлежащие
правовой регламентации регулятивными нормами права; во-вторых, юридически
нейтральные отношения, в которых не возникают юридические обязательства,
хотя экономические притязания присутствуют (как, например, в игорном бизнесе);
в-третьих, негативное экономическое поведение, которое подлежит сдержива-
нию и подавлению охранительными нормами права, — все указанное входит в
логически оформленную единую область (институт) экономических отношений
и соответствующий институт правового регулирования. 7/2020 7/2020 7/2020 Елисеев В. С. Место теории экономического права
в системе правового регулирования экономики Елисеев В. С. Место теории экономического права
в системе правового регулирования экономики 183 183 Окончательным оформленным правовым образованием, регулирующим эко-
номический институт, с позиции теории экономического права выступает правовой
режим — окончательно сформированная комплексная совокупность норм права
(правовой институт), охватывающая определенную область экономических отно-
шений. Правовой режим состоит из норм права различной природы: императив-
ных и диспозитивных, регулятивных и охранительных. Деление права на отрасли
рассматривается исключительно с позиции юридической необходимости, кото-
рая позволяет правовому регулированию наиболее полно охватить различные
стороны экономического института. С этих позиций отрасль права, построенная
на основе метода правового регулирования, не должна становиться кантовской
«вещью в себе», тем более не должна стремиться занять главенствующее по-
ложение, а должна учитывать свое воздействие наравне с другими правовыми
нормами, принадлежащими различным отраслям права, которые выступают со-
ставной частью правового режима. Теория экономического права имеет следующую структуру: Общая часть «Теоретические основы правового обеспечения экономических
отношений» включает следующие разделы (параграфы): «О предмете право-
вого обеспечения экономических отношений», «О методах правового регулиро-
вания экономических отношений», «Правовой режим как фундамент правового
регулирования экономических отношений», «Особенности формирования охра-
нительного законодательства», «Эффективность правового обеспечения эконо-
мических отношений», «О проблемах развития правовых наук, обеспечивающих
экономические отношения». Особенная часть «Теории правового обеспечения отдельных областей эко-
номических отношений» включает параграфы: «Теория правового обеспечения
рынков», «Теория правового обеспечения экономического саморегулирования
и государственного регулирования экономики», «Теория права собственности»,
«Теория субъекта хозяйствования», «Теория правового обеспечения государ-
ственного сектора экономики», «Общая теория обязательств», «Теория договор-
ных обязательств», «Теория налоговых обязательств» и «Теория обязательств
государственной поддержки», «Теории составных обязательств». PLACE OF THE THEORY
OF ECONOMIC LAW IN THE SYSTEM
OF LEGAL REGULATION OF ECONOMY Наконец, Специальная (аналитическая) часть включает на данном этапе два
параграфа: «Реформирование экономического законодательства СССР» и «Ре-
формирование экономического законодательства Российской Федерации». Очевидно, что в перспективе предполагается развитие структуры теории эко-
номического права. БИБЛИОГРАФИЯ 1. Альбов А. П., Ручкина Г. Ф. Экономическое право как новое направление
развития правовой науки // Ученые труды Российской академии адвокатуры
и нотариата. — 2015. — № 3 (38). — С. 93—99. 2. Братусь С. Н., Алексеев С. С. О разработке правовых вопросов управления
народным хозяйством // Правоведение. — 1963. — № 4. 3. Веленто И. И., Елисеев В. С. Теория экономического права : курс лекций. —
Гродно : ГрГУ, 2004. — 417 с. 7/2020 184 4. Вышинский А. Я. Основные задачи науки советского социалистического пра-
ва // Советское государство и право. — 1939. — № 1. 5. Дозорцев В. А. Один кодекс или два? (Нужен ли хозяйственный кодекс наря-
ду с гражданским?) // Правовые проблемы рыночной экономики в Российской
Федерации : Труды Института законодательства и сравнительного правове-
дения при Правительстве Российской Федерации. — Вып. 57. — М., 1994. —
С. 115—143. 6. Елисеев В. С., Веленто И. И. Теория экономического права: теория отраслей
права, обеспечивающих экономические отношения : учебное пособие. — М. :
Проспект, 2019. — 416 с. 7. Ершов В. В., Ашмарина Е. М., Корнев В. Н. Экономическое право Российской
Федерации : монография. — М. : РГУП, 2017. — 284 с. 8. Ершова И. В. Проблемы правового режима государственного имущества в
хозяйственном обороте: теоретические основы и пути совершенствования. —
М. : Юриспруденция, 2001. 9. Курс советского хозяйственного права / под ред. Е. Б. Пашуканиса и Л. Я. Гинц-
бурга. — М., 1935. — Т. 1. 10. Лаптев В. А. «Мегаотрасли» — экономическое, корпоративное и энергети-
ческое право или юридические фикции? // Экономика. Право. Общество. —
2017. — № 3. — С. 28—33. 11. Лаптев В. В. Предмет и система хозяйственного права. — М. : Юрид. лит.,
1969. 12. Мартемьянов В. С. Хозяйственное право : курс лекций : в 2 т. — М. : Бек,
1994. 13. Мартемьянов В. С. Юридическая трагедия // Юридическая газета. — 1992. —
№ 1, 2. 14. Мирошник С. В. Экономическое право в системе российского права // Вопро-
сы экономики и права. — 2016. — № 1. — С. 12—14. 15. Отграничение хозяйственного права от других отраслей права и их взаимо-
действие // Хозяйственное право : учебник / В. К. Мамутов, Г. Л. Знаменский,
К. С.Халиулин [и др.] ; под ред. В. К. Мамутова. — Киев : Юринком Интер,
2002. 16. Стучка П. И., Амфитеатров Г. Н. Основы гражданского законодательства
СССР. — М., 1931. 17. Экономическое право : хрестоматия : в 3 т. БИБЛИОГРАФИЯ — СПб. : Питер, 2004. 7/2020 7/2020
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https://hal.archives-ouvertes.fr/hal-00930013/file/240%20Justine%20et%20al%20-%20The%20monogenean%20which%20lost%20its%20clamps%20-%20PLoSONE%202013.pdf
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The Monogenean Which Lost Its Clamps
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To cite this version: Jean-Lou Justine, Chahrazed Rahmouni, Delphine Gey, Charlotte Schoelinck, Eric P. Hoberg. The Monogenean Which Lost Its Clamps. PLoS ONE, 2013, 8 (11), pp.e79155. 10.1371/jour-
nal.pone.0079155. hal-00930013 The Monogenean Which Lost Its Clamps
Jean-Lou Justine, Chahrazed Rahmouni, Delphine Gey, Charlotte Schoelinck,
Eric P. Hoberg
To cite this version:
Jean-Lou Justine, Chahrazed Rahmouni, Delphine Gey, Charlotte Schoelinck, Eric P. Hoberg.
The Monogenean Which Lost Its Clamps.
PLoS ONE, 2013, 8 (11), pp.e79155.
10.1371/jour-
nal.pone.0079155. hal-00930013 The Monogenean Which Lost Its Clamps
Jean-Lou Justine, Chahrazed Rahmouni, Delphine Gey, Charlotte Schoelinck,
Eric P. Hoberg Abstract PLoS ONE 8(11)
rnal.pone.0079155 Editor: Gordon Langsley, Institut national de la sante´ et de la recherche me´dicale - Institut Cochin, France itor: Gordon Langsley, Institut national de la sante´ et de la recherche me´dicale - Institut Cochin, France Received June 21, 2013; Accepted September 25, 2013; Published November 22, 2013 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This project was supported by the ‘‘Service de Syste´matique Mole´culaire’’ of MNHN (CNRS UMS 2700), the network ‘‘Bibliothe`que du Vivant’’ funded by
the CNRS, the MNHN, the INRA and the CEA (Genoscope), and a grant (Action The´matique du Muse´um (www.mnhn.fr) ‘‘Formes possibles, Formes re´alise´es’’)
awarded by MNHN to JLJ and CR. (These grants have no numbers). 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: justine@mnhn.fr The Monogenean Which Lost Its Clamps Jean-Lou Justine1*, Chahrazed Rahmouni1, Delphine Gey2, Charlotte Schoelinck ne1*, Chahrazed Rahmouni1, Delphine Gey2, Charlotte Schoelinck1,3, Eric P. Hober Jean-Lou Justine1*, Chahrazed Rahmouni1, Delphine Gey2, Charlotte Schoelinck1,3, Eric P. Hoberg4 1 UMR 7138 ‘‘Syste´matique, Adaptation, E´volution’’, Muse´um National d’Histoire Naturelle, CP 51, Paris, France, 2 UMS 2700 Service de Syste´matique mole´culaire, Muse´um
National d’Histoire Naturelle, Paris, France, 3 Molecular Biology, Aquatic Animal Health, Fisheries and Oceans Canada, Moncton, Canada, 4 United States National Parasite
Collection, United States Department of Agriculture, Agricultural Research Service, Beltsville, Maryland, United States of America Abstract Ectoparasites face a daily challenge: to remain attached to their hosts. Polyopisthocotylean monogeneans usually attach to
the surface of fish gills using highly specialized structures, the sclerotized clamps. In the original description of the
protomicrocotylid species Lethacotyle fijiensis, described 60 years ago, the clamps were considered to be absent but few
specimens were available and this observation was later questioned. In addition, genera within the family
Protomicrocotylidae have either clamps of the ‘‘gastrocotylid’’ or the ‘‘microcotylid’’ types; this puzzled systematists
because these clamp types are characteristic of distinct, major groups. Discovery of another, new, species of the genus
Lethacotyle, has allowed us to explore the nature of the attachment structures in protomicrocotylids. Lethacotyle vera n. sp. is described from the gills of the carangid Caranx papuensis off New Caledonia. It is distinguished from Lethacotyle fijiensis,
the only other species of the genus, by the length of the male copulatory spines. Sequences of 28S rDNA were used to build
a tree, in which Lethacotyle vera grouped with other protomicrocotylids. The identity of the host fish was confirmed with
COI barcodes. We observed that protomicrocotylids have specialized structures associated with their attachment organ,
such as lateral flaps and transverse striations, which are not known in other monogeneans. We thus hypothesized that the
clamps in protomicrocotylids were sequentially lost during evolution, coinciding with the development of other attachment
structures. To test the hypothesis, we calculated the surfaces of clamps and body in 120 species of gastrocotylinean
monogeneans, based on published descriptions. The ratio of clamp surface: body surface was the lowest in
protomicrocotylids. We conclude that clamps in protomicrocotylids are vestigial organs, and that occurrence of
‘‘gastrocotylid’’ and simpler ‘‘microcotylid’’ clamps within the same family are steps in an evolutionary sequence, leading to
the absence of these attributes in species of Lethacotyle. hmouni C, Gey D, Schoelinck C, Hoberg EP (2013) The Monogenean Which Lost Its Clamps. PLoS ONE 8(11): e79155. doi:10.1371/ ation: Justine J-L, Rahmouni C, Gey D, Schoelinck C, Hoberg EP (2013) The Monogenean Which Lost Its Clamps. HAL Id: hal-00930013
https://hal.science/hal-00930013v1
Submitted on 16 Aug 2020 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. PLOS ONE | www.plosone.org Hosts Five specimens of Caranx papuensis Alleyne & MacLeay, 1877
were obtained in Noume´a City, New Caledonia, from amateur
fishermen fishing from the piers of the harbour, or were bought at
the fishmarket, from commercial fishmongers. The latter host
specimens came from professional fisherman who specialize on
mackerels, fish close to Noume´a City and bring back their catch
within hours from the nearby fishing-grounds. Fish specimens are
detailed in Table 1 with registration number, date, locality, length,
weight and availability of photographs. Accurate identification of
marine fish is often a problem in the South Pacific [20–24], and Figure 1. The single specimen of Lethacotyle available for study
before this paper. The slide containing the single specimen of
Lethacotyle available for study before this paper: holotype of Lethacotyle
fijiensis Manter & Prince, 1953 (urn:lsid:zoobank.org:act:DA367684-
AAC2-44D0-A8E8-64894AFA647A), slide USNPC 48718. Our study is
another example of the importance of Museum collections for modern
research [86,87]. doi:10.1371/journal.pone.0079155.g001 Parasites Monogeneans were collected alive or recently dead, flattened in
cold ethanol, and routinely processed, including staining with
carmine and mounting on a microscopic slide in Canada balsam
[25]. Drawings were made using an Olympus BH2 microscope
equipped with a drawing tube and differential interference
contrast (DIC) optics. Measurements were made from pencil
drawings with the help of a custom-made transparent rule,
previously calibrated with a stage micrometer. Drawings were
scanned and redrawn on a computer with Adobe Illustrator. All
measurements are given in micrometres unless otherwise indicat-
ed. In the text and Tables, ‘‘juvenile’’ designates specimens with
incomplete development of genital organs, especially of charac-
teristic sclerotised organs. Further, during our current studies of Lethacotyle including
comparisons among related monogeneans, we noted that clamps
in species of protomicrocotylids were relatively small in compar-
ison to the body. Thus, although individual clamps were not
especially small, all clamps together occupied a small surface area
of the body in comparison to other species of polyopisthocotylean
monogeneans. Our observations suggest that clamps are reduced,
or vestigial, in this family, an assertion based on the ubiquitous
distribution of these attributes among basal polyopisthocotyleans
and the putative phylogenetic relationships for the Protomicroco-
tylidae Johnston & Tiegs, 1922 [4,6]. To test this hypothesis, we
explored the phylogenetic placement of the Protomicrocotylidae
and we compared the ratio for surface of clamps: surface of body
in 120 monogenean species belonging to the Gastrocotylinea
Lebedev, 1972. We found that the protomicrocotylids had the
lowest ratio. Finally, we discuss the evolutionary significance of the
absence of clamps in Lethacotyle spp., a unique feature among
polyopisthocotylean monogeneans. The Monogenean Which Lost Its Clamps The Monogenean Which Lost Its Clamps Thus, in all, our current knowledge of Lethacotyle, in spite of its
uniqueness and interest, is based on the observation of four
specimens, three adults and one juvenile, in which only one has
been kept in a museum and is available to study (Figure 1). No
work has been published on Lethacotyle during the past 40 years and
the doubts concerning the absence of clamps, expressed in the
original description [15] and subsequent comments [16,19], have
remained problematic. photographs of the fish were used to determine species identity by
several ichthyologists. In addition, fish tissues were collected,
stored in 95% or 100% ethanol, and processed for molecular
identification. Specimens of Caranx sexfasciatus Quoy & Gaimard,
1825 from the same locality were examined and provided
specimens of the monogenean Neomicrocotyle sp. used for compar-
ison of morphology and molecules. We collected off New Caledonia, another South Pacific island, a
series of specimens of a previously unrecognized species of
Lethacotyle. Specimens were collected in perfect condition for
morphological study and were submitted to modern molecular
analysis; the new species is described herein. Museum specimens The following museum slides were examined: Bilaterocotyle
novaeguineae Rohde, 1977, paratype, USNPC 74800 (1 slide)
(current status: Bilaterocotyloides novaeguineae (Rohde, 1977) Lebedev,
1986); Neomicrocotyle sp. from Caranx sexfasciatus off New Caledonia,
MNHN JNC3242; Protomicrocotyle celebesensis Yamaguti, 1953,
MNHN HEL80, HEL81; Protomicrocotyle mannarensis Ramalingam,
1960, USNPC 74798, BMNH 1978.6.15.6; Protomicrocotyle manteri
Bravo-Hollis, 1966, paratype, USNPC 75514; Protomicrocotyle
mirabilis (MacCallum, 1918) Johnston & Tiegs, 1922, BMNH
2002.8.12.3-4, BMNH 2007.7.25.34, 2007.7.25.30-33 (2 slides);
Protomicrocotyle pacifica Meserve, 1938, USNPC 100122 (3 slides)
(current status: Neomicrocotyle pacifica (Meserve, 1938) Yamaguti,
1968 [26]); Protomicrocotyle sp., BMNH 1985.11.8.48-47, BMNH
1985.11.8.48-52 (2 slides). The following slides could not be
shipped but photographs were taken by curators: Protomicrocotyle
celebesensis, MPM 22909 (SY6739); Neomicrocotyle carangis Yama-
guti, 1968, holotype, USNPC 63672, and MPM 15660 (B2421-
2423); Lethacotyle fijiensis, holotype, USNPC 48718 (Figure 1);
Protomicrocotyle pacifica, holotype, USNPC 9166. Names in the
above list are those from the original labels, sometimes updated
with correct taxonomy and current usage. Patricia Pilitt
(USNPC) and Eileen Harris (BMNH) are thanked for arranging
specimen loans. Introduction body of the monogenean is deformable and allows it to feed from
blood sucked from the gill [9]. Monogeneans are Platyhelminthes, mostly ectoparasites on fish. Although the monophyly of the Monogenea is dubious [1,2], there
is no doubt that each of the two components of the monogeneans,
namely the Polyopisthocotylea and the Monopisthocotylea, are
each monophyletic and members of the Neodermata, the parasitic
and terminal group of Platyhelminthes, together with the Cestoda
and the Trematoda [3–5]. Members of both monogenean groups
deal with a major issue of parasitic life, attachment to the host, by
a posterior organ named the haptor (or opisthaptor) which
possesses specialized attachment structures [6–8]. Although all known polyopisthocotyleans have suckers or
clamps, a single exception is represented by the species
Lethacotyle fijiensis Manter & Prince, 1953 [15]. This worm is a
parasite on the gills of an unnamed carangid fish off Fiji, a
South Pacific island. The species was described, however, from
only two specimens (among which only one is still in a museum
collection) and the authors mentioned that there was a
possibility that the clamps could have been lost - this is not an
unusual phenomenon when specimens are not collected in
optimal conditions. Hargis (1957) [16] also expressed doubt over
the accuracy of the original description and considered that the
complete absence of clamps was ‘‘unique and puzzling.’’ Later
Ramalingam (1966, 1968) [17,18] found other specimens of
Lethacotyle Manter & Prince, 1953 on a carangid off the
Andaman Islands, and confirmed the absence of clamps in
adult and juvenile worms. However, Ramalingam’s papers
[17,18] were largely ignored, i.e. by Llewellyn (1971) [19] who
commented that ‘‘such extraordinary occurrences deserve re-
examination.’’ In the Polyopisthocotylea (the name means ‘‘many sucker-cups
at the rear’’ [9]) the posterior haptor includes suckers or clamps
[6], and the latter are considered one of the major morphological
synapomorphies of the group [10]. These clamps, ranging in
number from a few to hundreds, are highly specialized structures,
often armed with sclerotised elements [6,11–13]. Clamps attach to
the host’s surface (generally the gill of a marine fish) and thus allow
the worm to resist the flow of water running through the gill
chamber and to maintain position on its host [14]. The anterior November 2013 | Volume 8 | Issue 11 | e79155 1 PLOS ONE | www.plosone.org Molecular sequences Fish DNA was extracted from tissue samples of three specimens
(Table 1) using NucleoSpin 96 tissue kit (Macherey-Nagel)
following the manufacturer’s instructions. The 59 region of the
cytochrome oxidase I (COI) mitochondrial gene was amplified
using
the
primers
FishF1
(59-TCAACCAACCACAAAGA-
CATTGGCAC-39) and FishR1 (59-TAGACTTCTGGGTGGC-
CAAAGAATCA-39) [27]. Species identification was confirmed
using the BOLD identification engine [28]. One monogenean was cut in two parts: the anterior part,
including the key sclerotised reproductive organs, was mounted,
using routine methods, on a microscopic slide [25] as for whole
worms, and the posterior part was used for DNA extraction. Thanks to this method, perfect traceability was insured between
morphological and molecular methods (i.e. both were performed
on the same monogenean individual); in addition, for host-parasite
traceability, the individual host fish of the same individual
monogenean was used for sequencing (Table 1). DNA was also
extracted from another, whole individual monogenean and
provided the same sequence. p
q
For monogeneans, as little tissue was available, DNA was
extracted using NucleoSpin 96 tissue kit with a modified protocol:
the NucleoSpin 96 Tissue Binding Plate was replaced by the
Plasmid Binding Plate (Macherey-Nagel) and elution was per-
formed in 60 mL. A 28S rDNA fragment of 700 bp was amplified
using the universals primers C19 (59-ACCCGCTGAATTTAAG-
CAT-39) and D2 (39-TCCGTGTTTCAAGACGG-59) [29]. PCR
reactions were performed in final volume of 20 ml, containing:
1 ng of DNA, 16 CoralLoad PCR buffer, 3 mM MgCl2, 66 mM
of each dNTP, 0.15 mM of each primer, and 0.5 units of Taq
DNA polymerase (Qiagen). Thermocycles consisted in an initial
denaturation step at 94uC for 49, followed by 38 cycles of
denaturation at 94uC for 300, annealing at 60uC, for 300, and
extension at 72uC for 19. The final extension was conducted at
72uC for 79. PCR products were visualized on a 1.5% agarose gel,
purified and directly sequenced in both directions on 3730xl DNA
Analyzer 96-capillary sequencers (Applied Biosystems) at Geno-
scope (E´vry, France). Sequences were edited and assembled using
CodonCode Aligner software (CodonCode Corporation, Dedham,
MA, USA). Sequences were deposited in GenBank under the
accession numbers KF378583–KF378585 (fish) and KF378588–
KF378589 (monogeneans). The Monogenean Which Lost Its Clamps The Monogenean Which Lost Its Clamps of Caranx papuensis examined, specimens of Lethacotyle vera n. sp., and results. Locality
Fork Length
(mm)
Weight
(g)
Photo
Fish Sequences
Parasites
Parasite Sequences
Noume´a harbour
372
843
Yes
-
3 specimens on slides; 2 adults, 1 juvenile
-
Noume´a harbour
413
1,250
No
-
4 adult specimens on slides
-
Noume´a fish market
275
350
Yes
COI: KF378585
1 adult specimen on slide
-
Noume´a fish market
345
749
Yes
COI: KF378583
3 specimens: 1 juvenile on slide JNC3188A1; 1 adult, cut, anterior
on slide JNC3188A2c, sequenced; 1 adult JNC3188A3c, sequenced
Specimen JNC3188A2c: 28S:
KF378588
Noume´a fish market
.500
unknown
Yes
COI: KF378584
1 adult specimen on slide
- Table 1. Specimens of Caranx papuensis examined, specimens of Lethacotyle vera n. sp., and results. JNC
Date
Locality
Fork Length
(mm)
Weight
(g)
Photo
Fish Sequences
Parasites
Parasite Sequences
JNC1185
05-07-2004
Noume´a harbour
372
843
Yes
-
3 specimens on slides; 2 adults, 1 juvenile
-
JNC1189
06-07-2004
Noume´a harbour
413
1,250
No
-
4 adult specimens on slides
-
JNC1988
04-10-2006
Noume´a fish market
275
350
Yes
COI: KF378585
1 adult specimen on slide
-
JNC3188
17-06-2010
Noume´a fish market
345
749
Yes
COI: KF378583
3 specimens: 1 juvenile on slide JNC3188A1; 1 adult, cut, anterior
on slide JNC3188A2c, sequenced; 1 adult JNC3188A3c, sequenced
Specimen JNC3188A2c: 28S:
KF378588
JNC3209
16-07-2010
Noume´a fish market
.500
unknown
Yes
COI: KF378584
1 adult specimen on slide
-
www.plosone.org Nomenclatural acts The electronic edition of this article conforms to the require-
ments of the amended International Code of Zoological Nomen-
clature, and hence the new names contained herein are available
under that Code from the electronic edition of this article. This
published work and the nomenclatural acts it contains have been
registered in ZooBank, the online registration system for the
ICZN. The ZooBank LSIDs (Life Science Identifiers) can be
resolved and the associated information viewed through any
standard web browser by appending the LSID to the prefix
‘‘http://zoobank.org/’’. The LSID for this publication is: urn:lsid:
zoobank.org:pub:596C3FF5-CD24-4733-95FD-CC060A7FF0EE. The electronic edition of this work was published in a journal
with an ISSN, and has been archived and is available from the
following digital repositories: PubMed Central, LOCKSS. Figure 1. The single specimen of Lethacotyle available for study
before this paper. The slide containing the single specimen of
Lethacotyle available for study before this paper: holotype of Lethacotyle
fijiensis Manter & Prince, 1953 (urn:lsid:zoobank.org:act:DA367684-
AAC2-44D0-A8E8-64894AFA647A), slide USNPC 48718. Our study is
another example of the importance of Museum collections for modern
research [86,87]. d i 10 1371/j
l
0079155 001 doi:10.1371/journal.pone.0079155.g001 November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 2 Phylogenetic analysis of polyopisthocotylean
monogeneans g
The data matrix was built from the published alignment of
Olson & Littlewood [30] (available from http://ebi.edu.au/ftp/
databases/embl/align/ALIGN_000150.dat),
restricted
to
the
Polyopisthocotylea excluding Polystomatidae and Sphyranuridae
(this corresponds to the group designated as Oligonchoinea in
[30]; for equivalences of monogenean terminology, see Table 1 in
[4]) to which were added two newly obtained sequences of 28S:
one from Lethacotyle vera n. sp. (KF378588), and one from an
unidentified
species
of
Neomicrocotyle
Ramalingam,
1960
(KF378589) from Caranx sexfasciatus. There is general agreement
that the Oligonchoinea are monophyletic and that the Hexabo-
thriidae are basal among them [10,30–32], so the two hexabo-
thriids of the original alignment were chosen as an outgroup. Phylogenetic reconstruction was computed using the GTR+I+C
model, selected as the best-fitting model of nucleotide evolution for
28S marker with ModelTest [33], in conjunction with PAUP
4.0b10 [34], following the AIC criterion. Trees were inferred using
two probabilistic approaches: maximum likelihood with a non-
parametric bootstrap (BP) using RaxML [35] and Bayesian PLOS ONE | www.plosone.org November 2013 | Volume 8 | Issue 11 | e79155 3 The Monogenean Which Lost Its Clamps Figure 2. Photograph of the holotype of Lethacotyle fijiensis
Manter & Prince, 1953. Lethacotyle fijiensis Manter & Prince, 1953
(urn:lsid:zoobank.org:act:DA367684-AAC2-44D0-A8E8-64894AFA647A). Holotype, slide USNPC 48718. A, body. B, posterior part of body,
different focus. C, D, spines of male copulatory organ, two different
focuses. E, sclerotised vagina. Original photographs taken by Patricia
Pilitt, USNPC. doi:10.1371/journal.pone.0079155.g002 Inference (BI) using MrBayes version 3.1.2 [36]. Maximum
likelihood (ML) analyses were carried out online on the CIPRES
Science Gateway (The CIPRES Portals. URL: http://www.phylo. org/sub_sections/portal) with RAxML-HPC BlackBox (7.2.7)
[35]. BI analyses were performed using 1,000,000 generations
with sampling every 100 generations and four Metropolis-coupled
Markov chains Monte Carlo (MCMCMC) and other parameters
by default. Two independent analyses were conducted to check for
convergence
of
the
results. The
parameter
estimates
and
convergence were checked using Tracer version 1.4 [37]. The
first 25% of sampled trees were discarded prior to constructing a
50% majority rule consensus tree. Posterior probabilities (PP -
Bayesian analysis) and Bootstrap values (BP - Maximum likelihood
analysis) were used as indicators of node credibility and we used
PP$0.95 and BP$75% as significant values. Morphology of available specimens Museum specimens (or sometimes photographs of specimens) of
species of protomicrocotylids, belonging to the genera Lethacotyle,
Protomicrocotyle, Neomicrocotyle, and Bilaterocotyle were examined for
the presence of clamps and other structures on the haptor. The
number of clamps was found to be consistent with the published
descriptions of species; particularly, we found no specimen with an
incomplete number of clamps (i.e. only 5 clamps when 6 were Ethics statement Fish used for collection of parasites were dead at the time we
acquired them for study, having been commercially caught, and
available for purchase at the Noume´a fish market; no permits were
required for the described study, which complied with all relevant
regulations. described for the species). The single specimen of Lethacotyle fijiensis
has no clamp (Figure 2), as emphasized in its original description
[15]; the same is true for all specimens of our new species (formally
described below). Analysis of relative importance of clamps in
gastrocotylinean monogeneans We compared the structure and the taxonomic distribution of
clamps across the major group, the Gastrocotylinea Lebedev, 1972
[38], which contains the protomicrocotylids [39]. This is one of the
largest groups of polyopisthocotylean monogeneans, which is
characterized by a common, complex clamp structure known as
‘‘gastrocotylid’’ [19,40] (but see below for changes of this structure
in some protomicrocotylids). p
y
Figures in the global literature were extracted from published
PDF files or scanned from printed papers with a table top scanner
with a 600 dpi resolution. The outlines of the body and of
individual clamps were drawn with Adobe Illustrator and then
filled in black. Drawings were exported in JPG format and area
measurements (whole body including clamps and total of all
clamps) were taken with ImageJ [41] on digital files. We checked
against WoRMS [42] (date: 14 May 2013), the list of species of
Gastrocotylinea for which we could obtain illustrations of sufficient
quality. Our database includes 120 species; 9 of these species were
not in WoRMS; the remaining 111 species represented 78% of the
142 species included in WoRMS. The 120 figures are available in
a Supplementary File. The statistical significance of differences
between families was tested with Mann & Whitney U test. Figure 2. Photograph of the holotype of Lethacotyle fijiensis
Manter & Prince, 1953. Lethacotyle fijiensis Manter & Prince, 1953
(urn:lsid:zoobank.org:act:DA367684-AAC2-44D0-A8E8-64894AFA647A). Holotype, slide USNPC 48718. A, body. B, posterior part of body,
different focus. C, D, spines of male copulatory organ, two different
focuses. E, sclerotised vagina. Original photographs taken by Patricia
Pilitt, USNPC. doi:10 1371/journal pone 0079155 g002 Abbreviations Two types of clamps were found in specimens of protomicro-
cotylids (Figure 3), i.e. ‘‘gastrocotylid’’ type (with additional
sclerite) and ‘‘microcotylid’’ type (without the sclerite). Parasitological collections: BMNH, NHMUK: British Museum
(Natural History), London, UK; MNHN, Muse´um National
d’Histoire Naturelle, Paris, France; MPM, Meguro Parasitological
Museum, Tokyo, Japan; USNPC, United States National Parasite
Collection, Beltsville, USA. In addition, we looked for striations on the haptor in specimens,
or sought for the mention of striations in the descriptions. Table 2
shows that haptoral striations were often, but not always,
mentioned
in
the
descriptions
of
protomicrocotylids. Such
striations are apparently not recorded (or observed) in other
members of the Gastrocotylinea (and in polyopisthocotylean
monogeneans
as
well),
with
the
possible
exception
of
a
pseudodiclidophorid [43]. Relative importance of clamps in gastrocotylinean
monogeneans Examples of line drawings are shown in Figure 4; all 120
drawings are in the supplementary file. Data are in Table 3. November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 4 The Monogenean Which Lost Its Clamps Figure 3. Clamps in various genera of Protomicrocotylidae. Examples of clamps in various genera of Protomicrocotylidae. A, Bilaterocotyloides
novaeguineae (Rohde, 1977) Lebedev, 1986 (USNPC 74800). B, Protomicrocotyle sp. (MNHN JNC1163A5). C, Neomicrocotyle sp. (MNHN JNC3242A4). Black: additional sclerite, characteristic of the ‘‘gastrocotylid’’ clamp. Bilaterocotyloides and Protomicrocotyle have clamps of the ‘‘gastrocotylid’’ type,
Neomicrocotyle has clamps of the ‘‘microcotylid’’ type, and Lethacotyle has no clamp. doi:10.1371/journal.pone.0079155.g003 Figure 3. Clamps in various genera of Protomicrocotylidae. Examples of clamps in various genera of Protomicrocotylidae. A, Bilaterocotyloides
novaeguineae (Rohde, 1977) Lebedev, 1986 (USNPC 74800). B, Protomicrocotyle sp. (MNHN JNC1163A5). C, Neomicrocotyle sp. (MNHN JNC3242A4). Black: additional sclerite, characteristic of the ‘‘gastrocotylid’’ clamp. Bilaterocotyloides and Protomicrocotyle have clamps of the ‘‘gastrocotylid’’ type,
Neomicrocotyle has clamps of the ‘‘microcotylid’’ type, and Lethacotyle has no clamp. doi:10.1371/journal.pone.0079155.g003 as C. papuensis based on comparisons to the 12 available specimens
in the database. Results of the comparison are presented in Figure 5 (data shown
for all 120 species) and Figure 6 (data grouped by families). Among
the 25 species with the smallest clamp: body ratios, 21 (84%) are
protomicrocotylids (Figure 5). The clamp: body ratio in proto-
microcotylids is the smallest of all families (Figure 6); ratios are
smaller in protomicrocotylids than in each of the other families,
and the differences are significant, except for the pseudodiclido-
phorids (Table 4). Results of the comparison are presented in Figure 5 (data shown
for all 120 species) and Figure 6 (data grouped by families). Among
the 25 species with the smallest clamp: body ratios, 21 (84%) are
protomicrocotylids (Figure 5). The clamp: body ratio in proto-
microcotylids is the smallest of all families (Figure 6); ratios are
smaller in protomicrocotylids than in each of the other families,
and the differences are significant, except for the pseudodiclido-
phorids (Table 4). Type-locality: Off Noume´a, New Caledonia. Type-locality: Off Noume´a, New Caledonia. Type-material: Holotype MNHH JNC3209A1, collected 16-07-
2010, Noume´a fish market. Paratypes: MNHN, JNC1185,
JNC1189, JNC1988, JNC3188 (whole specimens); NHMUK, 1
slide, 2013.10.8.1; USNPC, 1 slide, 107263. One paratype cut in
two parts, anterior part on slide MNHN JNC3188A2c, posterior
part used for sequencing. doi:10.1371/journal.pone.0079155.t002 Description of the new species Body and clamp surfaces: examples of line
data extraction in each of the 8 families of the Gastrocotylinea. All species drawn to same body length. A, Gotocotylidae, Goto
Bychowskicotylidae, Tonkinopsis transfretanus. C, Gastrocotylidae, Allopseudaxinoides euthynni. D, Neothoracocotylidae, Pr
Allodiscocotylidae, Metacamopia indica. F, Pseudodiclidophoridae, Allopseudodiclidophora opelu. G, Chauhaneidae, Cotyloatlantic
Protomicrocotylidae, Lethacotyle vera n. sp (no clamps). Details in Table 3. doi:10.1371/journal.pone.0079155.g004
The Monogenean Whi Figure 4. Body and clamp surfaces: examples of line drawings in 8 families. Body and clamp surfaces: examples of line drawings used
data extraction in each of the 8 families of the Gastrocotylinea. All species drawn to same body length. A, Gotocotylidae, Gotocotyla niphoni
Bychowskicotylidae, Tonkinopsis transfretanus. C, Gastrocotylidae, Allopseudaxinoides euthynni. D, Neothoracocotylidae, Pricea minimae
Allodiscocotylidae, Metacamopia indica. F, Pseudodiclidophoridae, Allopseudodiclidophora opelu. G, Chauhaneidae, Cotyloatlantica mediterranea
Protomicrocotylidae, Lethacotyle vera n. sp (no clamps). Details in Table 3. doi:10.1371/journal.pone.0079155.g004
The Monogenean Which Lost Its Clam Figure 4. Body and clamp surfaces: examples of line drawings in 8 families. Body and clamp surfaces: examples of line drawings used for
data extraction in each of the 8 families of the Gastrocotylinea. All species drawn to same body length. A, Gotocotylidae, Gotocotyla niphonii. B,
Bychowskicotylidae, Tonkinopsis transfretanus. C, Gastrocotylidae, Allopseudaxinoides euthynni. D, Neothoracocotylidae, Pricea minimae. E,
Allodiscocotylidae, Metacamopia indica. F, Pseudodiclidophoridae, Allopseudodiclidophora opelu. G, Chauhaneidae, Cotyloatlantica mediterranea. H,
Protomicrocotylidae, Lethacotyle vera n. sp (no clamps). Details in Table 3. doi:10.1371/journal.pone.0079155.g004 are herein shown in Figure 2. Other protomicrocotylids: see
Materials and Methods. are herein shown in Figure 2. Other protomicrocotylids: see
Materials and Methods. at two thirds from centre of lappet; median anchors on posterior
edge of lappet; hooks just external to corresponding median
anchors. Lateral anchor with inner root partly divided medially
(Figure 7G), outer root simple, strongly recurved point; median
anchor with flattened triangular root and strongly recurved point
(Figure 7H); hook with elongate, straight shaft (Figure 7I). Transverse striations on posterior part of haptor, including whole
surface of terminal lappet and most terminal part of haptor, but
not lateral flaps (Figure 7F). Pattern of striation similar on ventral
and dorsal sides, 20–25 striations on terminal lappet, regularly
parallel, 15–20 striations on body, less regular. November 2013 | Volume 8 | Issue 11 | e79155 Description of the new species p
q
g
Prevalence: 5/5 (100%); intensity 1–4 (Table 1). Lethacotyle vera Justine, Rahmouni, Gey, Schoelinck &
Hoberg
n. sp. urn:lsid:zoobank.org:act:0B7ABE99-07AF-
4088-97F3-1A154DBA614D Etymology: vera, Latin for true, meaning that Lethacotyle, a genus
differentiated by absence of clamps, was based on true observa-
tions. Type-host: Caranx papuensis Alleyne & MacLeay. Comparative material examined. Lethacotyle fijiensis Manter &
Prince, 1953, holotype, USNPC 48718; the holotype slide
(Figure 1) could not be shipped but photographs were taken and Molecular identification of hosts: The blast search processed on
BOLD engine [28] for the fish specimens MNHN JNC1988,
JNC3188, JNC3209 (Table 1), confirmed the species identification Table 2. Striations and other structures mentioned in protomicrocotylids. Species
Observation
Reference
Subfamily Protomicrocotylinae
Lethacotyle vera n. sp. Figure 2
This paper
Lethacotyle fijiensis
‘‘Dorsal surface of haptor with fine transverse striations’’ p. 105
[15]
Lethacotyle sp. from Andaman I. (as L. fijiensis)
Description of flaps pp. 108–109 (see discussion of present article);
[18]
Protomicrocotyle mirabilis
«Les faces ventrales et dorsales du hapteur et de la languette poste´rieure
posse`dent des stries transversales» (p. 320);
[55]
Protomicrocotyle mirabilis
(as Acanthodiscus mirabile)
‘‘body towards posterior disc transversally striated and spiny along dorsal
surface’’ (p. 93); Figure 49
[64]
Protocotyle celebensis
‘‘The caudal lobe is distinctly striated transversely like the posterior end of the
body proper, giving a serrate appearance in profile’’; Fig. 45
[65]
Bilaterocotyle chirocentrosus
Transversal striations not described, but well visible on Figs. 14, 15
[66]
Neomicrocotyle indicus
‘‘The posterior portion of the body and the dumb-bell shaped haptor show transverse
striations which give a spiny appearance to the surface of the worm’’; Fig. 1
[67]
Bilaterocotyle lucknowensis
Fig. 7.52 (left Fig. and Fig. G)
[68]
Bilaterocotyle mamaevi
‘‘Lappet two discs, each lappet lamellated’’. Fig. 7.53 (left Fig. and Fig. G)
[68]
Subfamily Vallisiopsiinae
Youngiopsis australis
Fig. 42D
[39]
Vallisiopsis contorta
‘‘La partie e´largie raye´e du corps’’ ; Fig. 1
[40]
doi:10.1371/journal.pone.0079155.t002
PLOS ONE | www.plosone.org
5
November 2013 | Volume 8 | Issue 11 | e79155 Table 2. Striations and other structures mentioned in protomicrocotylids. PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 5 The Monogenean Which Lost Its Clamps are herein shown in Figure 2. Other protomicrocotylids: see
at two thirds from centre of lappet; median anch
Figure 4. Body and clamp surfaces: examples of line drawings in 8 families. November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org
7
November 2013 | Volume 8 | Issue 11 | e79155 Taxonomic discussion of the new species Vaginal pore ventral, at midlength of MCO bulb level, on either
side of body midline but opposite to that of haptoral pads. Vagina
comprising anterior smooth part and posterior sclerotised part. Sclerotised part (Figure 7D) cone-shaped, with pointed extremity
posterior;
internal
longitudinal
crests
with
irregular
spines;
posterior end a small sclerotised conical canal. Smooth canal
between sclerotised part and rest of female organs not seen. Taxonomic discussion of the new species
Classification of Lethacotyle. A diversity of taxonomic
opinions illustrates the problematic nature and difficulty for
classification of Lethacotyle and more generally for the Proto-
microcotylidae. For example, Lethacotyle was classified within the
family Discocotylidae Price, 1936, subfamily Vallisiinae Price,
1943 in the original description [15]; in Protomicrocotylidae
Poche, 1926, Lethacotylinae Unnithan, 1962 by Unnithan (1962)
[44]
and
in
Protomicrocotylidae
Johnson
&
Tiegs,
1922,
Lethacotylinae Yamaguti, 1963, independently by Unnithan
(1962) [44] and with a different definition of the subfamily, by
Yamaguti (1963) [8]; in Gastrocotylidae Price, 1943, Valisiinae
Price, 1943 by Hargis, 1957 [16]; and in Protomicrocotylidae
(Johnston & Tiegs, 1922), Protomicrocotylinae Johnston & Tiegs,
1922 in the monograph by Lebedev (1986) [39]. The taxonomic
confusion about the authority for the family Protomicrocotylidae
in this list will not be commented upon here, but the challenge for
classification clearly originates in the structure of the clamps (or
their absence). The classification of polyopisthocotylean monoge-
neans is mainly based on clamp structure, but protomicrocotylids
are unique in that this structure changes relative to each genus
within the family: Protomicrocotyle has clamps of the gastrocotylid
type, but Neomicrocotyle has clamps of the microcotylid type. In
addition, the asymmetrical morphology of the haptor in proto-
microcotylids has been considered as ‘‘extraordinary’’ [19]. Unfortunately, the genus Lethacotyle was not included in
discussions of modern phylogenies of monogeneans [10,45]. Vitellarium in two lateral fields, never dense; anterior extremity
at level of seminal vesicle; posterior extremity at level of haptoral
anterior pad, i.e. anteriormost and posteriormost part of intestine
not coextensive with vitellarium. Vitellarium in two lateral fields, never dense; anterior extremity
at level of seminal vesicle; posterior extremity at level of haptoral
anterior pad, i.e. anteriormost and posteriormost part of intestine
not coextensive with vitellarium. Egg elongate, with long anterior and posterior filaments
(Figure 7E). In utero, egg length 220–225, width 82–95, filament
length 412–467 (n = 2). Egg elongate, with long anterior and posterior filaments
(Figure 7E). Description (Figures 7–8) Body elongate, fusiform (Figure 7A). Tegument of body proper
smooth; tegument of posterior part of haptor with parallel
transverse striations. Haptor devoid of clamps, slightly asymmetrical, comprising
lateral pads and terminal lappet. Lateral pads two, anterior short,
posterior long. Terminal lappet transversally elongate ovate,
symmetrical, armed with 3 pairs of ventral sclerites (1 pair of
hooks, 2 pairs of anchors). Lateral anchors located approximately Mouth subterminal, ventral. Prohaptoral suckers ovoid, asep-
tate, lying diagonally in posterolateral wall of buccal cavity. PLOS ONE | www.plosone.org November 2013 | Volume 8 | Issue 11 | e79155 November 2013 | Volume 8 | Issue 11 | e79155 6 The Monogenean Which Lost Its Clamps The Monogenean Which Lost Its Cla November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 7 The Monogenean Which Lost Its Clamps Figure 5. Ratio between clamp surface and body surface in species of gastrocotylinean monogeneans. Ratio between clamp surface
and body surface in species of gastrocotylinean monogeneans. Ratios are ordered in decreasing sequence. Red: protomicrocotylids; blue: species of
other families. doi:10.1371/journal.pone.0079155.g005 Figure 5. Ratio between clamp surface and body surface in species of gastrocotylinean monogeneans. Ratio between clamp surface
and body surface in species of gastrocotylinean monogeneans. Ratios are ordered in decreasing sequence. Red: protomicrocotylids; blue: species of
other families. doi:10.1371/journal.pone.0079155.g005 Pharynx subovate, muscular, median and immediately posterior to
prohaptoral suckers. Oesophagus long, devoid of diverticula,
bifurcating to 2 intestinal caeca at level of genital atrium. Intestinal
caecum in each lateral field of body proper, extending into haptor
to anteriormost part of lappet; lateral intestinal diverticula
numerous, branched, often indistinct; short diverticula in anterior
haptor, no diverticula in lappet. Phylogenetic position of the new species A phylogenetic analysis of 28S sequences (Figure 9) show that
the new species forms a clade (PP = 0.99, BPML = 95) with
Neomicrocotyle pacifica (from Caranx hippos (Linnaeus, 1766) off
Mexico [30]) and Neomicrocotyle sp. (our specimens from Caranx
sexfasciatus off New Caledonia), the two other protomicrocotylids of
the dataset. Germarium intercaecal, with posterior immature mass, anteri-
orly directed branch, posteriorly directed looped mature branch
(Figure 8E). Small coiled canal with visible wall from extremity of
germarium to posterior part of ootype. Ootype elongate, median,
with basal Mehlis’ glands, continued anteriorly as uterus. Uterus
linear, thin walled, extending up to genital atrium; superposed to
seminal vesicle along part of its path. Median vitelline duct
ventrally superposed to ootype (in holotype), anterior paired
vitelline branches visible only on short distance. Differential diagnosis Lethacotyle vera n. sp. is similar to the single species described in
the genus, L. fijiensis, based on the following characters (Table 5):
body shape, and especially haptoral shape; total absence of clamps;
body dimensions (mean 4340, 2300–5720) vs 3156–3759 in L. fijiensis [15]; presence of a circle of spines in MCO and shape of
individual spines; number of spines 23 (17–27) vs 24–25 in L. fijiensis; shape of cone-like sclerotised vagina; shape and position of
sclerotised haptoral parts. Genital atrium unarmed, median. Testes ovoid, pregermarial,
intercaecal, in 2 bilateral rows along body midline. Vas deferens
expanding just anterior to anteriormost testis into seminal vesicle
filled with sperm; seminal vesicle continued anteriorly by wide
canal to male copulatory organ (MCO); vas efferentia and prostate
not visible. MCO an elongate bulb, with muscular wall and
internal coiled canal, armed with anterior spines; mass of bulb
sometimes protruding anterior to spines. Spines arranged in a tight
circle (‘‘genital corona’’), with tips directed outward and extending
into genital atrium. Spines elongate, with blunt ends, elongate
root, and thumb located at anterior third. General arrangement of
spines
of
genital
corona
slightly
variable
with
specimens
(Figures 7B, 7C, 8C, 8D), but morphology of spines similar in
all adult specimens. It differs in MCO spine length (mean 5065.7, 35–66) vs 24 in L. fijiensis and shape of sclerotised vagina longitudinal crests, with
irregular spines along length vs with minute terminal spines in L. fijiensis. The length of MCO spines in the holotype of L. fijiensis was
ascertained by scaled photographs. Note that in specimens of ‘‘L. fijiensis’’ described by Ramalingam [17,18] the length of the MCO
spines was reported as 15 (vs 24 in original description [15]) and
thus this might represent another species (see below); L. vera n. sp. is distinct from this putative species by the length of MCO spines. November 2013 | Volume 8 | Issue 11 | e79155 Taxonomic discussion of the new species In utero, egg length 220–225, width 82–95, filament
length 412–467 (n = 2). Juvenile specimens (Figures 8A, 8B). Two juvenile specimens
briefly described for differential maturation of organs and
sclerotised parts. One specimen (slide MNHN JNC3188A1,
Figure 8A), 1300 in length, 320 in width: body almost
symmetrical, haptoral pads barely visible; MCO spines incom-
plete, 22–24 in length, no thumbs on spines (Figure 8B); vagina
a denser zone, no sclerotisation; germarium visible, testis zone
an indistinct mass; haptoral hooks not well oriented. One
specimen (slide MNHN JNC1185A3, not figured), 1700 in
length, 350 in width, body symmetrical, MCO a dense mass
without
sclerotisation,
all
other
genital
organs
indistinct;
haptoral sclerotised parts well visible, morphology of lateral
and median anchors similar to adult. Note that the longest
juvenile specimen is apparently the less mature according to less
differentiated sclerotised parts. Our molecular phylogenetic analysis shows that Lethacotyle vera n. sp. groups with two species of Neomicrocotyle and thus confirms that November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 8 The Monogenean Which Lost Its Clamps Table 3. Surface of clamps and body in species of gastrocotylinean monogeneans. Species
Family
Body surface
Clamp surface
Ratio
Reference, page
(mm2)
(mm2)
%
Allodiscocotyla chorinemi Yamaguti, 1953
Allodiscocotylidae
221,079
23,301
10.54
[8] p. 547
Allodiscocotyla diacanthi Unnithan, 1962
Allodiscocotylidae
891,326
53,724
6.03
[8] p. 547
Allodiscocotyla lae Yamaguti, 1968
Allodiscocotylidae
525,572
13,728
2.61
[69] p. 251
Camopia rachycentri Lebedev, 1970
Allodiscocotylidae
12,492,318
150,559
1.21
[39] p. 152
Hargicola oligoplites (Hargis, 1957) Lebedev, 1970
as Vallisia oligoplites Hargis, 1957
Allodiscocotylidae
4,944,569
248,805
5.03
[16] p. 7
Metacamopia chorinemi (Yamaguti, 1953) Lebedev, 1984
as Vallisia chorinemi Yamaguti, 1953
Allodiscocotylidae
1,278,926
16,737
1.31
[65] p. 65
Metacamopia indica (Unnithan, 1962) Lebedev, 1972
Allodiscocotylidae
1,488,158
13,866
0.93
[39] p. 154
Metacamopia oligoplites Takemoto, Amato & Luque, 1996
Allodiscocotylidae
3,273,506
62,243
1.90
[70] p. 167
Vallisia riojai Caballero & Bravo-Hollis, 1963
Allodiscocotylidae
971,416
10,768
1.11
[71] p. 175
Vallisia striata Parona & Perugia, 1890
Allodiscocotylidae
18,448,597
373,475
2.02
[72] p. 19
Bychowskicotyle plectorhynchi Lebedev, 1969
Bychowskicotylidae
857,260
65,536
7.64
[39] p. 100
Gaterina talaensis Lebedev, 1969
Bychowskicotylidae
619,337
33,586
5.42
[39] p. 101
Tonkinopsis transfretanus Lebedev, 1972
Bychowskicotylidae
1,236,435
59,827
4.84
[39] p. 104
Yamaguticotyla jucunda (Lebedev, 1969) Lebedev, 1984
Bychowskicotylidae
1,189,653
50,151
4.22
[39] p. 103
Yamaguticotyla truncata (Goto, 1894)
Bychowskicotylidae
4,924,003
87,494
1.78
[39] p. November 2013 | Volume 8 | Issue 11 | e79155 Taxonomic discussion of the new species Cont. Species
Family
Body surface
Clamp surface
Ratio
Reference, page
(mm2)
(mm2)
%
Engraulicola thrissocles (Tripathi, 1959) Lebedev, 1971
Gastrocotylidae
1,014,407
69,075
6.81
[39] p. 70
Engrauliphila grex Unnithan, 1967
Gastrocotylidae
208,190
24,488
11.76
[77] p. 218
Engrauliscobina triaptella Unnithan, 1967
Gastrocotylidae
1,465,577
91,211
6.22
[77] p. 221
Engraulixenus malabaricus Unnithan, 1967
Gastrocotylidae
556,699
56,562
10.16
[77] p. 215
Eyelavera typica Unnithan, 1968
Gastrocotylidae
9,500,350
844,481
8.89
[39] p. 74
Gastrocotyle indica Subhapradha, 1951
Gastrocotylidae
281,048
27,274
9.70
[68] p. 361
Gastrocotyle kurra Unnithan, 1968
Gastrocotylidae
2,864,859
266,456
9.30
[68] p. 362
Gastrocotyloides dillonhargisi Lebedev, 1980
Gastrocotylidae
1,273,059
233,778
18.36
[39] p. 72
Irinaxine miniata Ghichenok, 1980
Gastrocotylidae
741,990
59,441
8.01
[39] p. 60
Pellonicola arabiana Khan & Karyakarte, 1977
Gastrocotylidae
1,059,163
66,909
6.32
[68] p. 367
Pellonicola elongatus Unnithan, 1967
Gastrocotylidae
353,696
27,079
7.66
[77] p. 225
Pellonicola lanceolatus Kritsky & Bilqees, 1973
Gastrocotylidae
1,785,588
102,492
5.74
[78] p. 198
Pseudaxine bivaginalis Dillon & Hargis, 1965
Gastrocotylidae
1,137,648
44,201
3.89
[79] p. 276
Pseudaxine kurra Unnithan, 1968
Gastrocotylidae
1,909,954
37,999
1.99
[75] p. 268
Pseudaxinoides caballeroi Lebedev, 1977
Gastrocotylidae
2,096,832
93,128
4.44
[39] p. 57
Quadrivalvula asymmetrica Ghichenok, 1980
Gastrocotylidae
3,430,344
994,784
29.00
[39] p. 77
Sibitrema poonui Yamaguti, 1966
Gastrocotylidae
15,402,407
235,741
1.53
[74] p. 430
Cathucotyle cathuaui Lebedev, 1968
Gotocotylidae
1,303,159
261,873
20.10
[80] p. 450
Cathucotyle filipinensis Hayward & Rohde, 1999
Gotocotylidae
2,971,409
413,969
13.93
[80] p. 453
Cathucotyle sinensis Hayward & Rohde, 1999
Gotocotylidae
22,961,031
1,562,400
6.80
[80] p. 455
Gotocotyla acanthura (Parona & Perugia, 1896) Meserve, 1938
Gotocotylidae
3,209,235
466,446
14.53
[80] p. 431
Gotocotyla africanensis Hayward & Rohde, 1999
Gotocotylidae
2,445,683
250,315
10.23
[80] p. 438
Gotocotyla bivaginalis (Ramalingam, 1961) Rohde, 1976
Gotocotylidae
7,414,296
274,996
3.71
[80] p. 440
Gotocotyla elagatis Meserve, 1938
as Gotocotyla meservei Yamaguti, 1953
Gotocotylidae
1,314,267
179,701
13.67
[65] p. 56
Gotocotyla heapae Hayward & Rohde, 1999
Gotocotylidae
1,291,877
69,182
5.36
[80] p. 443
Gotocotyla niphonii Hayward & Rohde, 1999
Gotocotylidae
1,932,305
171,386
8.87
[80] p. 445
Gotocotyla queenslandici Hayward & Rohde, 1999
Gotocotylidae
1,321,566
258,123
19.53
[80] p. 447
Neogotocotyla rohdii Hadi & Bilqees, 2010
Gotocotylidae
2,991,506
347,296
11.61
[81] p. 22
Mexicotyle mexicana (Meserve, 1938) Lebedev, 1984
Neothoracocotylidae
2,566,693
133,237
5.19
[39] p. 90
Neothoracocotyle acanthocybii (Meserve, 1938) Hargis, 1956
Neothoracocotylidae
126,796
12,940
10.21
[39] p. 88
Pricea fotedari Gupta & Sharma, 1979
Neothoracocotylidae
2,219,395
250,628
11.29
[68] p. Taxonomic discussion of the new species 60
Ahpua piscicola Caballero & Bravo-Hollis, 1973
Chauhaneidae
10,832
404
3.73
[73] p. 39
Allopseudopisthogyne constricta Yamaguti, 1965
Chauhaneidae
4,293,278
170,812
3.98
[74] p. 75
Caniongiella australis (Young, 1968) Lebedev, 1976
Chauhaneidae
624,748
7,033
1.1
[39] p. 127
Caniongiella bychowskyi Lebedev, 1976
Chauhaneidae
1,204,269
26,531
2.20
[39] p. 126
Chauhanea madrasensis Ramalingam, 1953
Chauhaneidae
2,631,446
172,925
6.57
[39] p. 120
Cotyloatlantica mediterranea (Euzet & Trilles, 1960)
as Chauhanea mediterranea Euzet & Trilles, 1960
Chauhaneidae
3,309,353
305,081
9.22
[40] p. 190
Gemmaecaputia corrugata Tripathi, 1959
Chauhaneidae
404,195
13,015
3.22
[8] p. 487
Metopisthogyne sphyraenae Yamaguti, 1966
Chauhaneidae
3,415,824
337,042
9.87
[74] p. 426
Oaxacotyle oaxacensis (Caballero & Bravo, 1964) Lebedev, 1984
Chauhaneidae
397,303
56,968
14.34
[39] p. 138
Opisthogyne keralae Unnithan, 1962
Chauhaneidae
262,328
19,843
7.56
[44] p. 318
Paracaniongiella brinkmanni (Unnithan, 1962) Lebedev, 1976
Chauhaneidae
236,647
8,027
3.39
[68] p. 359
Paragemmaecaputia crassicauda Ramalingam, 1960
Chauhaneidae
189,399
9,836
5.19
[68] p. 357
Pentatres sphyraenae Euzet & Razarihelisoa, 1959
Chauhaneidae
497,849
29,309
5.89
[39] p. 132
Pseudochauhanea elongata Kritsky, Bilqees & Leiby, 1972
Chauhaneidae
593,000
16,000
2.70
[39] p. 122
Pseudochauhanea macrorchis Lin, Liu & Zhang in Zhang,
Yang & Liu, 2001
Chauhaneidae
1,037,805
41,361
3.99
[75] p. 261
Pseudochauhanea mexicana Lamothe, 1967
Chauhaneidae
2,354,237
123,241
5.23
[39] p. 120
Pseudochauhanea sphyraenae Yamaguti, 1965
Chauhaneidae
2,795,654
110,955
3.97
[69] p. 251
Pseudomazocraes monsivaisae Caballero & Bravo Hollis, 1955
Chauhaneidae
810,544,311
37,813,692
4.67
[76] p. 108
Pseudomazocraes selene Hargis, 1957
Chauhaneidae
771,077
49,995
6.48
[16] p. 7
Pseudopisthogyne lepidocybii Yamaguti, 1965
Chauhaneidae
2,106,993
189,587
9.00
[57] p. 75
Pseudopisthogynopsis lepidocybii Yamaguti, 1965
Chauhaneidae
8,160,745
821,312
10.06
[39] p. 117
Salinacotyle mexicana (Caballero & Bravo-Hollis, 1963) Lebedev, 1984
Chauhaneidae
1,425,544
103,400
7.25
[39] p. 138
Allopseudaxine macrova (Unnithan, 1957) Yamaguti, 1963
Gastrocotylidae
4,858,980
388,611
8.00
[8] p. 265
Allopseudaxine yaito Yamaguti, 1968
Gastrocotylidae
14,764,474
485,975
3.29
[69] p. 251
Allopseudaxinoides euthynni Yamaguti, 1965
Gastrocotylidae
11,587,179
720,066
6.21
[57] p. 84
Amphipolycotyle chloroscombrus Hargis, 1957
Gastrocotylidae
209,204
22,656
10.83
[16] p. 5
Areotestis sibi Yamaguti, 1965
Gastrocotylidae
33,887,590
629,933
1.86
[57] p. 79
Churavera macrova Unnithan, 1968
Gastrocotylidae
2,844,619
67,889
2.39
[68] p. 368
Cypselurobranchitrema spilonotopteri Yamaguti, 1966
Gastrocotylidae
204,576
13,906
6.80
[74] p. 432
Engraulicola forcepopensis George, 1960
Gastrocotylidae
303,387
13,117
4.32
[68] p. 366
Engraulicola micropharyngella Unnithan, 1967
Gastrocotylidae
293,283
12,295
4.19
[77] p. 212 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 9 The Monogenean Which Lost Its Clamps Table 3. November 2013 | Volume 8 | Issue 11 | e79155 Taxonomic discussion of the new species 383
Pricea microcotylae Chauhan, 1945
Neothoracocotylidae
31,523
3,995
12.67
[66] p. 148
Pricea minimae Chauhan, 1945
Neothoracocotylidae
796,183
170,955
21.47
[66] p. 146
Pricea solandri Gupta & Channa, 1977
Neothoracocotylidae
29,180
4,158
14.25
[68] p. 382
Pseudothoracocotyla ovalis (Tripathi, 1956) Yamaguti, 1963
Neothoracocotylidae
1,104,406
122,155
11.06
[82] p. 164
Pseudothoracocotyla whittingtoni Hayward & Rohde, 1999
Neothoracocotylidae
6,151,442
2,086,478
33.92
[82] p. 167
Scomberocotyle scomberomori (Koratha, 1955) Hargis, 1956
Neothoracocotylidae
2,837,224
293,771
10.35
[39] p. 89
Thoracocotyle crocea MacCallum, 1913
as Paradawesia bychowskyi Bravo & Lamothe, 1976
Neothoracocotylidae
3,604,259
390,338
10.83
[39] p. 94
Pricea multae Chauhan, 1945
Neothoracocotylidae
3,561,945
371,996
10.44
[83] p. 173
Scomberomorocotyle munroi Rohde & Hayward, 1999
Neothoracocotylidae
640,210
74,389
11.62
[84] p. 5
Chauhanocotyle rottleri Khoche & Dad, 1975
Protomicrocotylidae
912,981
13,257
1.45
[68] p. 356
Bilaterocotyle chirocentrosus Chauhan, 1945
Protomicrocotylidae
670,131
15,726
2.35
[66] p. 138
Bilaterocotyle lucknowensis (Agrawal & Sharma, 1986) Pandey &
Agrawal, 2008
Protomicrocotylidae
92,963
2,413
2.60
[68] p. 350
Bilaterocotyle multitesticularis Khan & Karyakarte, 1982
Protomicrocotylidae
1,180,417
9,192
0.78
[68] p. 349
Bilaterocotyle polynemusi Gupta & Krishna, 1980
Protomicrocotylidae
1,358,610
15,508
1.14
[68] p. 347
Bilaterocotyle spindalis Deo & Karyakarte, 1980
Protomicrocotylidae
1,659,994
45,651
2.75
[68] p. 348 November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 10 The Monogenean Which Lost Its Clamps Table 3. Cont. Species
Family
Body surface
Clamp surface
Ratio
Reference, page
(mm2)
(mm2)
%
Bilaterocotyloides carangis Ramalingam, 1961
Protomicrocotylidae
1,135,204
13,434
1.18
[39] p. 114
Bilaterocotyloides madrasensis Radha, 1966
Protomicrocotylidae
441,228
4,984
1.13
[39] p. 116
Bilaterocotyle mamaevi Agrawal, 1988
Protomicrocotylidae
27,004
485
1.80
[68] p. 352
Bilaterocotyloides novaeguineae (Rohde, 1977) Lebedev, 1986
Protomicrocotylidae
442,980
11,411
2.58
[39] p. 114
Bilaterocotyloides spinulosus Liu in Zhang, Yang & Liu, 2001
Protomicrocotylidae
2,197,940
11,976
0.54
[75] p. 247
Lethacotyle fijiensis Manter & Price, 1953
Protomicrocotylidae
2,788,607
0
0
[39] p. 117
Lethacotyle vera n. sp. Protomicrocotylidae
2,562,639
0
0
This paper
Neomicrocotyle carangis Yamaguti, 1968
Protomicrocotylidae
4,287,184
15,571
0.36
[39] p. 110
Neomicrocotyle indica Ramalingam, 1960
Protomicrocotylidae
49,651
232
0.47
[67] p. 375
Neomicrocotyle sp. JNC 3242A7
Protomicrocotylidae
2,663,686
15,327
0.58
This paper
Neomicrocotyle unnithani Yamaguti, 1968
Protomicrocotylidae
2,019,641
13,440
0.67
[44] p. 344
Protomicrocotyle celebesensis Yamaguti, 1953
Protomicrocotylidae
1,791,383
11,869
0.66
[65] p. 56
Protomicrocotyle ivoriensis Wahl, 1972
Protomicrocotylidae
2,939,959
7,682
0.26
[55] p. 324
Protomicrocotyle madrasensis Ramalingam, 1960
Protomicrocotylidae
736,440
5,113
0.69
[67] p. Taxonomic discussion of the new species 375
Protomicrocotyle mannarensis Ramalingam, 1960
Protomicrocotylidae
1,934,754
10,205
0.53
[67] p. 377
Protomicrocotyle manteri Bravo-Hollis, 1966
Protomicrocotylidae
1,608,092
10,245
0.64
[39] p. 106
Protomicrocotyle minuta Ramalingam, 1960
Protomicrocotylidae
334,808
6,589
1.97
[67] p. 377
Protomicrocotyle mirabilis (MacCallum, 1918) Johnston & Tiegs, 1922
Protomicrocotylidae
231,559
7,854
3.39
[55] p. 321
Protomicrocotyle nayaritensis Bravo-Hollis, 1979
Protomicrocotylidae
7,317,320
13,238
0.18
[85] p. 190
Vallisiopsis contorta Subhapradha, 1951
Protomicrocotylidae
29,848
281
0.94
[39] p. 17
Vallisiopsis sphyraenae Yamaguti, 1968
Protomicrocotylidae
6,746,717
48,813
0.72
[69] p. 251
Youngiopsis australis (Young, 1968) Lebedev, 1972
Protomicrocotylidae
1,561,104
16,863
1.08
[39] p. 117
Allopseudodiclidophora opelu Yamaguti, 1965
Pseudodiclidophoridae
5,244,324
20,471
0.39
[57] p. 73
Gephyrocotyle ixoracorona Unnithan, 1966
Pseudodiclidophoridae
482,140
14,380
2.98
[68] p. 340
Pseudodiclidophora decapteri Yamaguti, 1965
Pseudodiclidophoridae
1,163,641
16,908
1.45
[57] p. 70
Quilonella ventrosa Lebedev & Parukhin, 1970
Pseudodiclidophoridae
954,583
13,250
1.39
[39] p. 81
Sawquirahcotyle indica Lebedev, 1976
Pseudodiclidophoridae
2,316,402
45,956
1.98
[39] p. 85
Winkenthughesia australis Robinson, 1961
Pseudodiclidophoridae
8,558,914
244,842
2.86
[43] p. 261
The outlines of body and clamps were redrawn on computer from original publications or from our own drawings, and the surface was calculated using ImageJ. Names
of species follow WoRMS [42]; if different, name used in publication also indicated. All computerized line drawings available as Supplementary Material. Data ordered in
alphabetical order of families and species. doi:10 1371/journal pone 0079155 t003 Table 3. Cont. The outlines of body and clamps were redrawn on computer from original publications or from our own drawings, and the surface was calculated using ImageJ. Names
of species follow WoRMS [42]; if different, name used in publication also indicated. All computerized line drawings available as Supplementary Material. Data ordered in
alphabetical order of families and species. doi:10.1371/journal.pone.0079155.t003 the genus Lethacotyle belongs to the Protomicrocotylidae, in spite of
the absence of clamps. The host of the Lethacotyle species described by Ramalingam is ‘‘C. sexfasciatus’’ but the author mentioned that the carangids were
15 cm [18] and 5.2–26.5 cm [17] in length. Maturity of C. sexfasciatus is attained at 40 cm, common length is 60 cm, and
maximum published weight is 18 kg [48]. Species identification of
carangids, when they are adult, is often difficult, but the validity of
species identification of the small specimens examined by
Ramalingam is certainly dubious. Taxonomic discussion of the new species An hypothesis could be proposed, in which Lethacotyle would be
a primitive species without clamps, with more derived species
having clamps; our analyses clearly falsify this hypothesis, and
demonstrate that the Protomicrocotylidae is not among the basal
groups among the polyopisthocotyleans and the Gastrocotylinea. Species in Lethacotyle Therefore, we consider that: (a) the host of L. fijiensis in Fiji is an
unknown carangid (due to insufficient host identification by
Manter & Prince [15]); (b) the host of the Lethacotyle species
described by Ramalingam is an unknown carangid, due to
identification from immature fish specimens [17,18], and we see
no reason why it should be the same species as Manter & Prince’s
host fish. It might be C. sexfasciatus, as claimed by the author;
however, we examined several C. sexfasciatus from off Australia and
New Caledonia, and found no species of Lethacotyle [49]; (c) it is
likely, based on collections from widely separated areas (Andaman
Islands vs Fiji, which are separated by 9,000 km), the probability of
different host species, and differences in measurements of the Manter & Prince (1953) described L. fijiensis from two specimens
from ‘‘yellow jack’’ [15]; the identification of the host fish is vague,
as often with Manter’s work (other cases: [46,47]), and almost
useless (many carangids are partly yellow). Only one monogenean
specimen, the holotype of L. fijiensis, is kept in the USNPC
collections (Figure 1). Ramalingam [17,18] described a species of Lethacotyle from
‘‘Caranx sexfasciatus’’ from off the Andaman Islands, and claimed it
was the same species as L. fijiensis. No deposition of specimens in a
curated collection or museum is mentioned in the papers. The
MCO spines as described by Ramalingam are 15 mm in length. November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 11 The Monogenean Which Lost Its Clamps Figure 6. Ratio between clamp surface and body surface in families of gastrocotylinean monogeneans. Ratio between clamp surface
and body surface in families of gastrocotylinean monogeneans. Ratios are ordered in decreasing order of mean. Protomicrocotylids have the lowest
mean and lowest minimum. For significance see Table 4. doi:10.1371/journal.pone.0079155.g006 Figure 6. Ratio between clamp surface and body surface in families of gastrocotylinean monogeneans. Ratio between clamp surface
and body surface in families of gastrocotylinean monogeneans. Ratios are ordered in decreasing order of mean. Protomicrocotylids have the lowest
mean and lowest minimum. For significance see Table 4. doi:10.1371/journal.pone.0079155.g006 MCO spines (Table 5), that the species described by Ramalingam
is distinct from both L. fijiensis and L. vera n. sp.; (d) and thus,
Lethacotyle probably comprises, at least, three species. Families are in decreasing order of ratio. P values correspond to Mann & Whitney U tests between each family and the Protomicrocotylidae; all families have a
significantly greater ratio than the Protomicrocotylidae, except the Pseudodiclidophoridae.
doi:10 1371/journal pone 0079155 t004 079155.t004 PLOS ONE | www.plosone.org
12
November 2013 | Volume 8 | Issue 11 | e79155 Species in Lethacotyle Clamps in protomicrocotylids vs other monogeneans
Our results (Figures 5, 6) show that the clamp surface is
significantly smaller in species of the protomicrocotylids in
comparison to other gastrocotylinean monogeneans. In addition,
our description of L. vera n. sp. confirms that clamps are completely
absent in members of the genus Lethacotyle. Clamps are an
important and characteristic part of the anatomy of polyopistho-
cotylean monogeneans, and are clearly the main organ used for
attachment to the host [6,8,11–13]. Protomicrocotylids, no less
than other monogeneans, need to maintain attachment to their
host. In a fluid environment maintenance of position on the Our species is the first referred to Lethacotyle with a precise host
identification. We have examined a number of other carangids
from several genera off New Caledonia [47,49–53] and found L. vera n. sp. only on C. papuensis, suggesting that species of Lethacotyle
are specific to Caranx species. It is likely that the ‘‘yellow jack’’ of
Manter & Prince (1953) [15] and the carangid of Ramalingam
[17,18], both identified with suboptimal precision, were species of
Caranx, but, as explained above, not necessarily conspecific. Table 4. Significant differences of clamp surface: body surface ratios in families of gastrocotylinean monogeneans. Families
n
Minimum
Maximum
Mean
P value
(Total = 118)
(%)
(%)
(%)
Neothoracocotylidae
12
5.19
33.92
13.61
0.000483
Gotocotylidae
9
3.71
20.10
11.47
0.000483
Gastrocotylidae
26
1.53
29
7.60
0.001699
Chauhaneidae
22
1.13
14.34
5.89
0.007222
Bychowskicotylidae
5
1.78
7.64
4.78
0.001699
Allodiscocotylidae
10
0.93
10.54
3.27
0.004136
Pseudodiclidophoridae
6
0.39
2.98
1.84
0.209316
Protomicrocotylidae
28
0
3.39
1.12
-
Families are in decreasing order of ratio. P values correspond to Mann & Whitney U tests between each family and the Protomicrocotylidae; all families have a
significantly greater ratio than the Protomicrocotylidae, except the Pseudodiclidophoridae. doi:10.1371/journal.pone.0079155.t004 Table 4. Significant differences of clamp surface: body surface ratios in families of gastrocotylinean monogeneans. ferences of clamp surface: body surface ratios in families of gastrocotylinean monogeneans. Table 4. Significant differences of clamp surface: body surface ratios in families of gastrocotylinean Families are in decreasing order of ratio. P values correspond to Mann & Whitney U tests between each family and the Protomicrocotylidae; all families have a
significantly greater ratio than the Protomicrocotylidae, except the Pseudodiclidophoridae. doi:10.1371/journal.pone.0079155.t004 November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 12 The Monogenean Which Lost Its Clamps Figure 7. November 2013 | Volume 8 | Issue 11 | e79155 Species in Lethacotyle E, outline of ovary (paratype JNC1189A1). doi:10.1371/journal.pone.0079155.g008 Figure 8. Lethacotyle vera n. sp. Juvenile and other details. Lethacotyle vera n. sp (urn:lsid:zoobank.org:act:0B7ABE99-07AF-4088-97F3-
1A154DBA614D). A, juvenile (specimen MNHN JNC3188A1). B, spines of MCO in juvenile. C, spines of MCO in paratype MNHN JNC3188A2c (posterior
part of body processed for molecular study); D, spines of MCO in paratype MNHN JNC1189A2. E, outline of ovary (paratype JNC1189A1). doi:10.1371/journal.pone.0079155.g008 posterior lappet is perpendicular to the longitudinal axis of the
worm, and firmly applied against the gill surface. coming into contact with the filaments may either press against
them thus helping to hold on to them or after getting a hold
around the filaments may adpress them against the body and thus
effect a hold on to the gills’’. He concluded ‘‘this mode of effecting
attachment to the gills by means of outgrowths of body surface is
unique in Monogenea. An adventious growth of the body surface
as seen in this case is rather unique and possibly nothing parallel is
known among the animal kingdom’’. It is apparent that development of a complex of organs
associated with the haptor, and a reduction in the size and
complexity of the clamps is associated with evolution of the
Protomicrocotylidae. In this group, development of organs for the
attachment on the host, including lateral flaps and posterior
tegumental striations, or a combination of these two structures,
apparently renders clamps of little significance for attachment. It is
not clear which came first (reduction of the clamps or development
of
a
complex
of
tegumental
organs
for
attachment),
and
comprehensive phylogenetic analysis of all members of the family
would be needed to resolve this question [56]. Given the overall
phylogenetic placement of the family, and relative to other
Gastrocotylinea, clamps must be considered vestigial organs in
most
protomicrocotylids
(genera
Protomicrocotyle,
Neomicrocotyle,
Bilaterocotyle and Bilaterocotyloides) and are absent in species of
Lethacotyle. The existence of two major types of clamp structures
(gastrocotylid type in Protomicrocotyle, microcotylid type in Neomi-
crocotyle) which puzzled systematists [8,16,19], is consistent with a
secondary loss of the accessory sclerites in Neomicrocotyle, trans- Unfortunately, we cannot confirm Ramalingam’s observations
and hypotheses, having not observed living worms. Striations are
visible on the posterior lappet of L. vera n. sp., and also on other
protomicrocotylids (Table 5). Such transverse striations are rather
unique among monogeneans. Species in Lethacotyle Lethacotyle vera n. sp. Adult and details. Lethacotyle vera n. sp (urn:lsid:zoobank.org:act:0B7ABE99-07AF-4088-97F3-1A154DBA614
A, whole body; B, spines of male copulatory organ (MCO). C, spines of MCO in other specimen (paratype MNHN JNC1189A3). D, sclerotized vagina
egg, in utero. F, striations on posterior part of body; G, H, I, hooks (paratype MNHN JNC1185A3). A, B, D, F: holotype, MNHN JNC3209A1. doi:10.1371/journal.pone.0079155.g007 Figure 7. Lethacotyle vera n. sp. Adult and details. Lethacotyle vera n. sp (urn:lsid:zoobank.org:act:0B7ABE99-07AF-4088-97F3-1A154DBA614D). A, whole body; B, spines of male copulatory organ (MCO). C, spines of MCO in other specimen (paratype MNHN JNC1189A3). D, sclerotized vagina. E,
egg, in utero. F, striations on posterior part of body; G, H, I, hooks (paratype MNHN JNC1185A3). A, B, D, F: holotype, MNHN JNC3209A1. doi:10.1371/journal.pone.0079155.g007 the haptor, and the striations on the posterior haptoral lappet. Hooks are relatively small in protomicrocotylids and are thus not
considered of importance in attachment. external surfaces of the host represents a challenge, and one
potentially heightened for protomicrocotylids that possess minis-
cule clamps, and for species of Lethacotyle, in which clamps are
completely absent. Ramalingam [18], apparently from a study of living specimens
(although this is not clearly stated in his paper) described the flaps
of the haptor and reported that ‘‘the gap between the flaps in the
anteroposterior axis can be narrowed by the contraction of the
body in this region as well as by the extensile power of the flaps
thus bringing their free ends in contact with each other or may
lead to overlapping condition’’. He explained that the flaps ‘‘on We hypothesize that other structures play a role in host
attachment in protomicrocotylids, as habitat selected by these
monogeneans (the fish gill) does not differ substantially from that
characteristic of other gastrocotylineans which have fully devel-
oped clamps. Among protomicrocotylids, fixation may be attained
by the combined action of the haptoral hooks, the lateral flaps of November 2013 | Volume 8 | Issue 11 | e79155 November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 13 PLOS ONE | www.plosone.org The Monogenean Which Lost Its Clamps Figure 8. Lethacotyle vera n. sp. Juvenile and other details. Lethacotyle vera n. sp (urn:lsid:zoobank.org:act:0B7ABE99-07AF-4088-97F3-
1A154DBA614D). A, juvenile (specimen MNHN JNC3188A1). B, spines of MCO in juvenile. C, spines of MCO in paratype MNHN JNC3188A2c (posterior
part of body processed for molecular study); D, spines of MCO in paratype MNHN JNC1189A2. Species in Lethacotyle Some information about the precise
habitat of protomicrocotylids are available; Rohde [54] stated that
Protomicrocotyle sp. was only found on the posterior surface of the
internal filaments of the first gill of Caranx melampygus Cuvier, 1833. Wahl described the position of specimens of Protomicrocotyle ivoriensis
Wahl, 1972 and P. mirabilis according to their asymmetry and
noted that the posterior lappet was intercalated between two gill
lamellae ([55], p. 329). Indeed, transverse striations are probably
efficient for attachment, by increasing friction, only when the November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 14 The Monogenean Which Lost Its Clamps Table 5. Measurements of Lethacotyle species. Lethacotyle
L. vera n. sp. L. vera n. sp. L. vera n. sp. L. fijiensis
‘‘L. fijiensis’’
‘‘L. Species in Lethacotyle doi:10.1371/journal.pone.0079155.t005 form: mean (minimum–maximum), except for a few measurements with sample .30, for which measurements are in the form: mean
maximum). 005 forming the more complex gastrocotylid clamp into a simpler
microcotylid-like clamp. phylogenetically disparate polyopisthocotyleans. Among some
pseudodiclidophorids, evolution towards a reduced role of clamps
has occurred without attaining the secondarily simplified micro-
cotylid-like structure nor the total absence observed within the
protomicrocotylids. Establishing phylogenetic context is a primary
foundation necessary to differentiate between secondary loss (as
proposed for these lineages of monogeneans) in contrast to
plesiomorphic
absence
[56]. Additionally,
the
phylogenetic
framework is critical for establishing the temporal association
and sequence of evolutionary modification in complex attributes. The Pseudodiclidophoridae also have a small clamp: body ratio,
slightly higher than but not significantly different from the
protomicrocotylids (Figure 6, Table 4). As our study concerns
mainly the protomicrocotylids, we provide here only limited
comments about pseudodiclidophorids. Only 5 pseudodiclido-
phorids were studied, and none has completely lost the clamps;
one has transverse striations [43], and one, Allopseudodiclidophora
opelu Yamaguti, 1965 (Figure 4F) has a ‘‘long anchor-bearing
appendage’’ [57]; several have outstandingly wide posterior bodies
that evoke the possibility of this part working as a sucker, as
suggested for the microcotylid Aspinatrium gallieni Euzet & Ktari,
1971 [58]. These observations suggest that reduction in clamps,
coincidental with development of secondary organs of attachment
is a rare event, but has occurred in multiple lineages of Species in Lethacotyle fijiensis’’
Holotype
Adults
Juveniles
Adult
Adult
Juvenile
Reference
This paper
This paper
This paper
Manter &
Price, 1953
[15]
Ramalingam,
1968 [18]
Ramalingam,
1968 [18]
n
1
8
2
2
1
1
Body Length
5130
4340 (2300–5720, n = 8)
1300, 1700
3156–3759
1540
950
Body Width
750
973 (500–1270, n = 9)
320, 350
663–770
380
130
Pharynx Length
53
66 (53–75, n = 9)
38, 45
64
50
37
Pharynx Width
45
59 (45–70, n = 9)
38,43
50
33
25
Buccal Sucker Length
70
71 (50–83, n = 18)
60, 60
49–52
(diameter)
37
27
Buccal Sucker Width
42–57
59 (42–75, n = 18)
38, 50
25
25
Anterior-Genital Pore Distance
800
648 (360–803, n = 9)
Number of Genital Corona Spines
24
24 (21–27, n = 9)
24–25
24
Length of Genital Corona Spines
52 (43–66,
n = 10)
5065.7 (35–66, n = 138)
24
15
Number of Testes
34
29 (21–34, n = 7)
30
31
Testis Length
4367.9
(25–55, n = 34)
52 (25–82, n = 57)
Testis Width
4267.9
(27–52, n = 34)
145 (63–262, n = 57)
Testicular Mass Length
975
913 (588–1163, n = 6)
370
Testicular Mass Width
125
352 (125–489, n = 6)
Sclerotized Vagina Length
150
157 (125–175, n = 9)
Sclerotized Vagina Width
85
81 (38–100, n = 9)
Unsclerotised Vagina Length
175
170 (60–250, n = 9)
Anterior-Vagina Pore Distance
850
739 (407–938, n = 9)
Ovary Length
542
591 (114–935, n = 8)
Ovary Width
192
340 (192–550, n = 8)
Haptor Total Length
282
325 (245–400, n = 7)
207, 275
130
90
Haptor Total Width
850
764 (588–850, n = 6)
452, 525
260
280
Hamulus Length
24, 30
28 (24–33, n = 15)
27, 32
24
33
30
Posterior Hook Length
16, 16
18 (10–24, n = 13)
15, 16
16
18
22
Small Hook Length
14 (11–16, n = 8)
14
12
12
All measurements are in mm, in the form: mean (minimum–maximum), except for a few measurements with sample .30, for which measurements are in the form: mean
6 standard deviation (minimum–maximum). doi:10.1371/journal.pone.0079155.t005 All measurements are in mm, in the form: mean (minimum–maximum), except for a few measurements with sample .30, for which measurements are in the form: mean
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molecules. Biological Journal of the Linnean Society 66: 75–114. 25. Justine J-L (2005) Species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea:
Diplectanidae) from Epinephelus fasciatus and E. merra (Perciformes: Serranidae) off
New Caledonia and other parts of the Indo-Pacific Ocean, with a comparison of
measurements of specimens prepared using different methods, and a description
of P. caledonicus n. sp. Systematic Parasitology 62: 1–37. 6. Hayward C (2005) Monogenea Polyopisthocotylea (ectoparasitic flukes). In:
Rohde K, editor. Marine Parasitology: CSIRO, Collingwood, Australia &
CABI, Oxon, UK. pp. 55–63. 26. Kritsky DC, McAleese WJ, Bakenhaster MD (2011) Heteronchoineans
(Monogenoidea) from the gills of crevalle jack, Caranx hippos (Perciformes,
Carangidae), from Everglades National Park, Florida, with a redescription of
Protomicrocotyle mirabilis (Gastrocotylinea, Protomicrocotylidae). Comparative
Parasitology 78: 265–274. 7. Whittington ID (2005) Monogenea Monopisthocotylea (ectoparasitic flukes). In:
Rohde K, editor. Author Contributions Conceived and designed the experiments: Conceived and designed the
experiments: JLJ CR. Performed parasite collection and preparation of
specimens: JLJ. Performed microscopical observations on new specimens:
CR JLJ. Performed microscopical observations on Museum specimens: CR
JLJ EPH. Drawn specimens: CR JLJ. Performed analysis of clamp: body
ratios, including statistical tests: CR. Performed molecular analyses: CS
DG. Performed molecular phylogenetic analysis: CS. Discussed manu-
script: JLJ CR DG CS EPH. Performed the experiments: JLJ CR DG CS
EPH. Analyzed the data: JLJ CR DG CS EPH. Wrote the paper: JLJ CR
DG CS EPH. Acknowledgments Various fish specimens were identified (from photographs) by Ronald
Fricke, Jack Randall, Michel Kulbicki, Samuel Igle´sias and Bernard Se´ret. Eric Bureau, student in training, collected some specimens. David Gibson
(BMNH) helped with linguistic discussions, and in the acquisition of rare
literature and comments about species in WoRMS. Patricia Pilitt (USNPC)
provided photomicrographs of the holotype of L. fijiensis, and Takashi Iwaki
(MPM) kindly took photographs of additional museum specimens. Patricia Clamps of protomicrocotylids as vestigial organs Vestigial organs are structures that have apparently lost their
ancestral function in a species, and for which homologous and
functional organs are known in related species. Typical examples
are the loss or reduction of flight organs in some island-dwelling November 2013 | Volume 8 | Issue 11 | e79155 November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 15 The Monogenean Which Lost Its Clamps Figure 9. Tree of gastrocotylinean monogeneans. Tree of gastrocotylinean monogeneans, based on a phylogenetic analysis of 28S sequences. doi:10.1371/journal.pone.0079155.g009 Figure 9. Tree of gastrocotylinean monogeneans. Tree of gastrocotylinean monogeneans, based on a phylogenetic analysis of 28S sequences. doi:10.1371/journal.pone.0079155.g009 various sensory attributes in larvae, used to seek hosts [56,63]. In
Lethacotyle and protomicrocotylids, the loss and modification of organs
concerns the haptor and clamps, body parts of the monogeneans
which are clearly an adaptation to ectoparasitism. The occurrence of
vestigial clamps or the complete absence of clamps, however, does not
demonstrate that these parasites are ‘‘simplified’’. In contrast,
reduction has occurred in the evolutionary context for development
of novel structures for attachment (flaps and striations) which are
unique among any of the lineages of the monogeneans. species (in insects or birds, independently), limbs bones in cetaceans,
or the loss of eyes and pigmentation in cavern-dwelling species which
have occurred under changing regimes for selection [59–62]. Parasites, in old anthropogenic interpretations, were considered
‘‘simpler’’ than free-living animals because they had lost certain
organs (such as the intestine in cestodes)(e.g. [56]). More nuanced
observations have demonstrated the considerable specialization and
structural and biochemical complexity of helminths which often have
complexes of novel organ systems in relation to parasitism, such as November 2013 | Volume 8 | Issue 11 | e79155 November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 16 The Monogenean Which Lost Its Clamps Pilitt (USNPC) and Eileen Harris (BMNH) are thanked for arranging
specimen loans. Pilitt (USNPC) and Eileen Harris (BMNH) are thanked for arranging
specimen loans. Supporting Information File S1
PDF of all figures and measurements of clamp
and body surfaces. Total number of figures: 120. (PDF) The Monogenean Which Lost Its Clamps 41. Rasband WS (1997–2013) ImageJ U. S. National Institutes of Health, Bethesda,
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p
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et P. ivoriensis n. spec., Monoge`nes parasites de Caranx hippos dans la lagune Ebrie´
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59. Darwin C (1872) The origin of species. London: John Murray. 458 p. Darwin C (1872) The origin of species. London: John Murray. 458 p 60. Berta A, Sumich JL (1999) Marine mammals: Evolutionary biology. New York:
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61. Muller GB (2002) Vestigial organs and structures. In: Pagel M, editor. Encylopedia of Evolution. Oxford: Oxford University Press. pp. 1131–1133. 84. Rohde K, Hayward CJ (1999) Scomberomorocotyle munroi n. g., n. sp. (Scomberomorocotylinae n. subf.), a thoracocotylid monogenean from Scomber-
omorus munroi (Scombridae) off Australia and Papua New Guinea. Systematic
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62. Helfman G, Collette B, Facey D, Bowen BW (2009) The diversity of fishes:
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protomicrocotylido nuevo de Caranx hippos caninus Gu¨nther del Pacı´fico
Mexicano. Anales del Centro de Ciencias del Mar y Limnologı´a 6: 189–196. 64. References Manter HW, Prince DF (1953) Some Monogenetic Trematodes of marine fishes
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O. collaris) in the American West. Journal of Parasitology 95: 490–501. November 2013 | Volume 8 | Issue 11 | e79155 PLOS ONE | www.plosone.org 18
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en
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Author Correction: Challenging the sustainability of urban beekeeping using evidence from Swiss cities
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npj urban sustainability
| 2,022
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cc-by
| 311
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www.nature.com/npjUrbanSustain
AUTHOR CORRECTION
OPEN
Author Correction: Challenging the sustainability of urban
beekeeping using evidence from Swiss cities
Joan Casanelles-Abella
and Marco Moretti
npj Urban Sustainability (2022)2:14 ; https://doi.org/10.1038/s42949-022-00059-9
Correction to: npj Urban Sustainability https://doi.org/10.1038/
s42949-021-00046-6, published online 12 January 2022
1234567890():,;
The original version of this Article contained a typographic
error in the Abstract: “an average 10.14 hives per km2 (9370 in
total)” should have read “an average 8.1 hives per km2 (6370
in total)”.
The authors also became aware of an inaccurate assumption in
the original version of the paper, which incorrectly read “we
additionally considered different carrying capacity scenarios,
ranging from 0.5 to an unrealistic value of 75 hives per km2” in
the second last sentence of the first paragraph in the section
“Assessing the sustainability of urban beekeeping” under Results.
In the corrected version, “ranging from 0.5 to” is replaced by
“ranging from 1 to” to avoid confusion.
Published online: 09 June 2022
Published in partnership with RMIT University
This has now been corrected in both the PDF and HTML versions
of the Article.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
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English
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Investigation of reactions between trace gases and functional CuO nanospheres and octahedrons using NEXAFS-TXM imaging
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Scientific reports
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cc-by
| 10,299
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Katja Henzler1,†, Axel Heilemann2,3,4, Janosch Kneer4, Peter Guttmann1, He Jia1,
Eckhard Bartsch2,3, Yan Lu1 & Stefan Palzer4 In order to take full advantage of novel functional materials in the next generation of sensorial
devices scalable processes for their fabrication and utilization are of great importance. Also
understanding the processes lending the properties to those materials is essential. Among the most
sought-after sensor applications are low-cost, highly sensitive and selective metal oxide based gas
sensors. Yet, the surface reactions responsible for provoking a change in the electrical behavior of
gas sensitive layers are insufficiently comprehended. Here, we have used near-edge x-ray absorption
fine structure spectroscopy in combination with x-ray microscopy (NEXAFS-TXM) for ex-situ
measurements, in order to reveal the hydrogen sulfide induced processes at the surface of copper
oxide nanoparticles, which are ultimately responsible for triggering a percolation phase transition. For the first time these measurements allow the imaging of trace gas induced reactions and the
effect they have on the chemical composition of the metal oxide surface and bulk. This makes
the new technique suitable for elucidating adsorption processes in-situ and under real operating
conditions. In recent years micro- and nano-scaled particles of various morphologies and sizes have been investi-
gated in order to utilize their unique properties for a multitude of technological applications, including
energy conversion and storage1, plasmonics2, drug delivery3, catalysis4,5 and gas sensing6. Among the
impediments preventing a quick commercial adaptation of groundbreaking research results is the lack
of scalable interfaces between the nano-regime and macroscopic equipment. One appealing technology
in that regard is the inkjet printing process, which provides a precise and scalable method to deposit
functional nanoparticles onto arbitrary structures. Using this technique colloidal suspensions of metal
oxide inks may be deposited and used as a gas sensitive layer on low-power consuming, micro-machined
silicon-based structures. This enables the investigation of the gas sensitive behavior of metal oxide par-
ticles as a function of size, shape and temperature. In light of the need of highly sensitive and selective
gas sensor solutions for advanced analytical applications the understanding of the underlying processes
as well as the possibility to scale the production processes are of primary concern. p
y
p
p
p
y
Until now, most works are focusing on using n-type metal oxide semiconductors, such as SnO2, WO3
or ZnO for gas sensors. Only limited work has been reported on p-type semiconducting gas sensors 1Institute for Soft Matter and Functional Materials, Helmholtz-Zentrum Berlin für Materialien und Energie GmbH,
Hahn-Meitner-Platz 1, 14109 Berlin, Germany. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Investigation of reactions between
trace gases and functional CuO
nanospheres and octahedrons
using NEXAFS-TXM imaging received: 28 June 2015
accepted: 05 November 2015
Published: 03 December 2015 Katja Henzler1,†, Axel Heilemann2,3,4, Janosch Kneer4, Peter Guttmann1, He Jia1,
Eckhard Bartsch2,3, Yan Lu1 & Stefan Palzer4 Katja Henzler1,†, Axel Heilemann2,3,4, Janosch Kneer4, Peter Guttmann1, He Jia1,
Eckhard Bartsch2,3, Yan Lu1 & Stefan Palzer4 Results and Discussion
Large-scale synthesis route for well-defined Cu2O nanoparticles.
The synthesis of Cu2O nano-
particles relies on a precipitation reaction in alkaline media: Results and Discussion
Large-scale synthesis route for well-defined Cu2O nanoparticles. The synthesis of Cu2O nano-
particles relies on a precipitation reaction in alkaline media: +
+
+
→
+
+
. ( )
+
−
+
N H
H O
Cu
OH
N
H O
Cu O
2
4
4
4
2
1
2
4
2
2
2
3
2 +
+
+
→
+
+
. +
−
+
N H
H O
Cu
OH
N
H O
Cu O
2
4
4
4
2
2
4
2
2
2
3
2 ( )
1 In order to increase the yield and to control the shape and the size of the particles, NaOH has been
added to the reaction12,13. Besides the positive effect on the yield, NaOH may be used to control the
morphology and the size of the resulting Cu2O particles. To this end, the NaOH concentration has been
varied from 0.002–0.5 M NaOH. Up to a NaOH concentration of 0.04 M, the particle size shows a slight
decrease of the hydrodynamic radius from about 240 nm to about 160 nm observed by dynamic light
scattering14–16 shown in Fig. 1a. For these samples a spherical shape and a reasonably narrow size dis-
tribution can be inferred from scanning electron microscopy (SEM) pictures as exemplarily depicted in
Fig. 1b. A sharp transition occurs for NaOH concentrations exceeding 0.04 M (Fig. 1a), when the shape
of Cu2O particles changes from spherical to octahedral. The addition of NaOH into the reaction solution
has been proved to provide the necessary alkalinity for the use of N2H4 as reducing agent17,18. Two fac-
tors are mainly influencing the morphology evolution of Cu2O nanoparticles: One is the capping agent,
Polyvinylpyrrolidone (PVP). As a surfactant, PVP is preferentially adsorbed onto the {111} planes of the
Cu2O crystals, which can decrease the surface energy of the {111} surface and reduce its growth rate. This
favors the Cu2O crystal growth into octahedral structures19,20. Another factor is the amount of NaOH
in the system. During the reaction, Cu(OH)2 is produced by NaOH reacting with Cu2+ ions, which is
the key intermediate. The production of Cu(OH)2 can decrease the reduction rate of Cu2O, which slows
down the growth rate of Cu2O crystals indirectly. Without addition of NaOH, the crystal growth rate is
too fast to be controlled by PVP. Katja Henzler1,†, Axel Heilemann2,3,4, Janosch Kneer4, Peter Guttmann1, He Jia1,
Eckhard Bartsch2,3, Yan Lu1 & Stefan Palzer4 2Institut für Makromolekulare Chemie, University of Freiburg,
79104 Freiburg, Germany. 3Institut für Physikalische Chemie, University of Freiburg, 79104 Freiburg, Germany. 4Laboratory for Gas Sensors, Department of Microsystems Engineering, University of Freiburg, Georges-Köhler
Allee 102, 79110 Freiburg. †Present address: Laboratory for Synchrotron Radiation – Catalysis and Sustainable
Chemistry, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland. Correspondence and requests for materials
should be addressed to S.P. (email: Stefan.palzer@imtek.uni-freiburg.de) Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 1 www.nature.com/scientificreports/ based on materials such as Cu2O and CuO. However, p-type gas sensitive materials show significant
surface reactivity with reducing and oxidizing gases albeit at lower operating temperatures as compared
to n-type materials and consequently they might play a pivotal role in future, low-energy consuming
gas sensing devices. In the past Cu2O and CuO nanostructures have been fabricated using numerous
approaches, few of which are compatible with large-scale production. And while gas-surface interactions
are proposed to explain the gas sensitive behavior of metal oxide layers, analytical tools for directly
observing adsorption processes are scarce7. In this regard, techniques of x-ray absorption spectroscopy
offer unique possibilities, but have so far not been used to directly monitor the chemical structure of
the gas sensitive material itself. In previous works the oxidation state of platinum upon gas exposure has
been investigated8,9 in order to study its effect on the gas sensitive material. To monitor the gas sensitive
material directly Cu2O as well as CuO offer ideal conditions to demonstrate a novel approach com-
bining near-edge x-ray absorption fine structure spectroscopy with x-ray microscopy NEXAFS-TXM10
and gas sensitive characterizations. This technique is able to probe both, the O-K-edge as well as the
Cu-L2,3-edge11 to reveal specific surface processes upon exposure to a trace gas.i ,
g
pi
p
p
p
g
Here, we focus on the reactions between the highly toxic trace gas hydrogen sulfide (H2S) and CuO
to highlight the capabilities of the technique. We employ the novel approach to track the fundamental
changes in CuO caused by the interaction with H2S using the NEXAFS-TXM method to shed light onto
fundamental surface processes. To address both applied as well as fundamental issues of next generation
metal oxide based sensors we present a new route for synthesizing Cu2O nanoparticles with high yield,
and demonstrate how to interface and to use them as sensor material. Using on-chip annealing the Cu2O
nanoparticles may be converted to CuO. Katja Henzler1,†, Axel Heilemann2,3,4, Janosch Kneer4, Peter Guttmann1, He Jia1,
Eckhard Bartsch2,3, Yan Lu1 & Stefan Palzer4 Interestingly, the shape of the nanoparticles remains the same,
even though the crystal structure changes from cubic to monoclinic and the oxygen content increases to
about 50%. Hence, the here presented route allows for producing either CuO or Cu2O nanoparticles, con-
trolling their shape and size and using their specific gas sensitive characteristics. The Cu2O nanoparticles
are dispersed in a solution that is subsequently used as ink for depositing the nanoparticles employing an
inkjet printing system. The solvent of the ink is then evaporated without leaving contaminations in the
sensing layer, i.e. without affecting the gas sensing properties after deposition. The presented approach
is able to pave the way towards a new generation of gas sensitive materials with tailor-made properties
and reproducible, stable base-line resistances. The gas sensing properties of the CuO particles are inves-
tigated and in addition the ex-situ NEXAFS-TXM measurements reveal the changes of the chemical
composition due to the annealing process as well as those caused by exposure of Cu2O and CuO to H2S. The induced chemical changes of the Cu2O/CuO particles by H2S are irreversible at room temperature. Therefore, it was possible to map all stages of the surface reactions between H2S and Cu2O/CuO ex-situ
in the high vacuum setup. This demonstrates the suitability of the NEXAFS-TXM method to provide a
spatially resolved analysis of the chemical bonds formed during gas-surface interaction, which are at the
heart of gas detection using semiconducting metal-oxides as functional material. Results and Discussion
Large-scale synthesis route for well-defined Cu2O nanoparticles. The synthesis of Cu2O nano-
particles relies on a precipitation reaction in alkaline media: Results and Discussion
Large-scale synthesis route for well-defined Cu2O nanoparticles.
The synthesis of Cu2O nano-
particles relies on a precipitation reaction in alkaline media: Adding NaOH to the reaction, the crystal growth rate will be slowed
down. In this case, the previously mentioned formation of Cu2O octahedrons due to the presence of PVP
occurs. Moreover, continuously increasing the concentration of NaOH, [Cu(OH)4]2− can be formed via
the complexation of Cu2+ with OH−, which will increase the edge lengths of the octahedrons as shown
in Fig. 1c,d, a similar result also has been found by W. Zhu and his co-workers13. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 2 www.nature.com/scientificreports/ Figure 1. Both the shape as well as the particle size of the Cu2O particles crucially depend on the NaOH
concentration. (a) The particle radius has been determined using dynamic light scattering. (b) Concentrations
up to 0.03 M NaOH result in spherical particles with a diameter around 200 nm and a reasonably narrow
particle size distribution. (c) Above 0.04 M NaOH the shape changes to octahedrons and (d) further increasing
the NaOH concentration results in larger particle sizes. (b–d) show scanning electron microscopy (SEM)
images for particles produced using 0.03, 0.04 and 0.3 M NaOH, respectively. Figure 1. Both the shape as well as the particle size of the Cu2O particles crucially depend on the NaOH
concentration. (a) The particle radius has been determined using dynamic light scattering. (b) Concentrations
up to 0.03 M NaOH result in spherical particles with a diameter around 200 nm and a reasonably narrow
particle size distribution. (c) Above 0.04 M NaOH the shape changes to octahedrons and (d) further increasing
the NaOH concentration results in larger particle sizes. (b–d) show scanning electron microscopy (SEM)
images for particles produced using 0.03, 0.04 and 0.3 M NaOH, respectively. Characterization of the as-synthesized particles and solid-state transformation to CuO
nanoparticles. Using the scalable wet chemistry process it is possible to control shape and size of
the Cu2O particles. The deposition of the particles is achieved by employing a DIMATIX DMP-283121
system. Tuning the rheological properties by adding polyethylene glycol (PEG) 400 is necessary to match
the requirements imposed by the inkjet printing system. Due to a solid state phase transformation from
Cu2O to CuO at temperatures above 250 °C and ambient pressure it is furthermore possible to convert
the so-produced particles using an on-chip annealing technique. Here we used elevated temperatures
for one hour, which results in pure CuO particles of the same shape as the Cu2O particles. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 Results and Discussion
Large-scale synthesis route for well-defined Cu2O nanoparticles.
The synthesis of Cu2O nano-
particles relies on a precipitation reaction in alkaline media: In order to
examine the long-range as well as the short-range structure of the nanoparticles NEXAFS spectra and
x-ray diffraction (XRD) pattern have been recorded for both spherical and octahedral samples before
and after annealing. The results are presented in Fig. 2.h t
gh
p
g
Figure 2a–d shows the TXM-micrographs of the respective samples. The O-K-edge of the particles
directly after synthesis cannot be investigated due to the remaining PEG, because they have been inkjet
printed in order to ensure complete correspondence between NEXAFS analysis and gas sensitive char-
acterization. However, the Cu-L2,3 edge shown in Fig. 2e reveals the differences in the near range order
when comparing spherical and octahedron particles after synthesis. The Cu-L2,3-edge reveals that the
spherical particles consist of a mixture of Cu+ and Cu2+ in an environment with a highly electronega-
tive partner, like oxygen. The signals at ~931 and 950.9 eV are typical for Cu2+ and the features at 933.8
and 953.6 eV can be assigned to Cu+, respectively11,22–29. The XRD pattern of the spherical particles
after synthesis shown in Fig. 2g does not show any evidence on the coexistence of a copper-(I)- and
copper-(II)-oxide lattice structure within this sample. Without heat treatment, five obvious diffraction
peaks can be found in the pattern, which are indexed to the {110}, {111}, {200}, {220} and {311} planes
of cubic Cu2O (Fig. 2g,h blue lines, JCPDS card no. 65–3288). This means that the detected Cu2+ exists
only in amorphous CuO phases or at defect sites within the spherical particles. On the contrary, as
shown in Fig. 2e, the as-synthesized octahedral particles show features in the NEXAFS spectrum which
can be fully assigned to the chemical nature of copper-(I)-oxide. These differences between the different
particle morphologies can only be detected by NEXAFS spectroscopy. Beam damage as a reason for Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 3 www.nature.com/scientificreports/ Figure 2. (a) TXM-micrograph of the Cu2O spheres after synthesis: overlay of 930.8 eV (yellow) and
933.8 eV (blue) represents the chemical inhomogeneity of the sample. (b) TXM-micrograph of the Cu2O
octahedron after synthesis (933.8 eV). (c) TXM-micrograph of the CuO spheres after oxidation (532.7 eV). (d) TXM-micrograph of the CuO octahedron after oxidation (532.7 eV). (e) NEXAFS spectra at the Cu-
L2,3-edge of the Cu2O nanoparticles after synthesis. (f) NEXAFS spectra at the O-K-edge and Cu-L2,3-edge
of the CuO nanoparticles after oxidation. Results and Discussion
Large-scale synthesis route for well-defined Cu2O nanoparticles.
The synthesis of Cu2O nano-
particles relies on a precipitation reaction in alkaline media: In the XRD pattern, the measured intensity is plotted versus
the scattering angle 2Θ . (g) XRD pattern of Cu2O nanoparticles after synthesis. (h) XRD pattern of CuO
nanoparticles after oxidation. The reference values for the expected Bragg peaks for Cu2O and CuO are
depicted via the δ –function at the corresponding angles. Figure 2. (a) TXM-micrograph of the Cu2O spheres after synthesis: overlay of 930.8 eV (yellow) and
933.8 eV (blue) represents the chemical inhomogeneity of the sample. (b) TXM-micrograph of the Cu2O
octahedron after synthesis (933.8 eV). (c) TXM-micrograph of the CuO spheres after oxidation (532.7 eV). (d) TXM-micrograph of the CuO octahedron after oxidation (532.7 eV). (e) NEXAFS spectra at the Cu-
L2,3-edge of the Cu2O nanoparticles after synthesis. (f) NEXAFS spectra at the O-K-edge and Cu-L2,3-edge
of the CuO nanoparticles after oxidation. In the XRD pattern, the measured intensity is plotted versus
the scattering angle 2Θ . (g) XRD pattern of Cu2O nanoparticles after synthesis. (h) XRD pattern of CuO
nanoparticles after oxidation. The reference values for the expected Bragg peaks for Cu2O and CuO are
depicted via the δ –function at the corresponding angles. this can be excluded because possible beam induced reactions play a minor role in the investigation of
hard-condensed matter like inorganic colloidal particles. Additionally, the vacuum environment during
investigation prevents the sample from oxidation and the soft x-rays (< 1 keV) do not have the energy
needed for crystal rearrangement. Therefore, the found differences between both morphologies can be
related to the nature of the immediate neighbors and the chemical state of the investigated element. Furthermore, this may be a factor contributing to the different behavior of the spherical particles in
comparison to the octahedrons, which is further investigated in the ESI.t p
g
After oxidation both types of particles reveal a complete solid-state transformation from Cu2O to CuO,
which is demonstrated by the NEXAFS spectra shown in Fig. 2f and with the XRD patterns in Fig. 2h. During the oxidation, the oxygen containing PEG in the samples is removed, which can be confirmed by
the TXM images in Fig. 2c,d. Additionally, the spectrum of the O-K-edge in Fig. 2f shows no evidence
of any other oxygen containing substance. All the recorded features in the O-K- and Cu-L2,3-spectra for
the spherical as well as the octahedral particles can be assigned to copper-(II)-oxide. Results and Discussion
Large-scale synthesis route for well-defined Cu2O nanoparticles.
The synthesis of Cu2O nano-
particles relies on a precipitation reaction in alkaline media: Analyzing the peak widths using the Scherrer equation yields a crystallite size
of 21.8 ± 0.8 nm for the octahedral particles (for 0.3 M NaOH), and 9.5 ± 1.6 nm for the spherical Cu2O
particles (0.02 M NaOH), whereas the respective particle sizes are ~1000 nm and ~305 nm (analyzed by
TXM consistent with the results from dynamic light scattering investigation). Spatially resolved surface reactions. The currently used NEXAFS-TXM setup at the electron stor-
age ring BESSY II does not allow in-situ measurements because of the vacuum setup. Therefore, we
have opted for an exemplary gas-surface reaction that is non-reversible at room temperature in normal
atmosphere to demonstrate the potential of our approach. Namely, we have used the known, highly spe-
cific reaction of CuO towards H2S exposure. Even in oxygen depleted atmospheres30,31 the exothermic
reaction32: ( )
2 +
→
+
,
( )
CuO
H S
CuS
H O
2
2
2 causes fundamental changes in the electrical behavior of gas sensitive layers. At temperatures below
200 °C this conversion is irreversible which means that the reaction product is frozen and samples may
be transferred to the NEXAFS-TXM setup without altering the composition of the surface or bulk. Especially since the CuO crystal is transformed to CuS the presence of surface adsorbed species on
either CuO or CuS surfaces do not limit the validity of the NEXAFS-TXM results in the low tempera-
ture regime. In this operational mode the CuO layer may be used to determine the H2S concentration
by using a H2S induced percolation phase transition and measuring the time tPercol necessary to establish
a conducting path via emerging CuS clusters. A second, competing reaction governs the CuO layer’s
behavior for high temperatures33,34, +
→
+
+
,
( )
(
)
−
−
H S
O
SO
H O
e
2
3
2
2
3
3
ads
2
2
2
2 ( )
3 i.e. the layer shows a typical reaction towards reducing gases. In order to confirm the proposed reac-
tions at the employed gas-sensitive materials using the Cu-L2,3 edge and the O-K edge we have taken
the respective spectra of the reference materials Cu, CuS, CuO, and Cu2O. This way we can demonstrate
the formation of CuS even without accessing the S-K edge, which is unavailable at the currently used
beam line. The reference spectra are depicted in Fig. Results and Discussion
Large-scale synthesis route for well-defined Cu2O nanoparticles.
The synthesis of Cu2O nano-
particles relies on a precipitation reaction in alkaline media: The XRD patterns
of the spherical (purple) and octahedral (dark cyan) nanoparticles after annealing are shown in Fig. 2h. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 4 www.nature.com/scientificreports/ Figure 3. NEXAFS-spectra at the O-K- and Cu-L2,3 edge of reference materials. Black: copper-(II)-
sulfide; red: copper; blue: copper-(II)-oxide; green: copper-(I)-oxide. The signal at 932.2 eV in the CuS
sample is attributed to the transition of Cu 2p3/2 to 3d states and shows a distinctly different behavior when
compared to CuO. The O-K edge demonstrates the fundamental differences even more clearly since it does
not show a clear absorption edge, which means that there is no oxygen present in the bulk of the reference
material. Figure 3. NEXAFS-spectra at the O-K- and Cu-L2,3 edge of reference materials. Black: copper-(II)-
sulfide; red: copper; blue: copper-(II)-oxide; green: copper-(I)-oxide. The signal at 932.2 eV in the CuS
sample is attributed to the transition of Cu 2p3/2 to 3d states and shows a distinctly different behavior when
compared to CuO. The O-K edge demonstrates the fundamental differences even more clearly since it does
not show a clear absorption edge, which means that there is no oxygen present in the bulk of the reference
material. After the annealing step, the original Cu2O signals disappear completely, while new peaks appear, which
correspond to the {110}, {002}, and {111} planes of CuO (Fig. 2g and h beige-colored lines, JCPDS card
no. 45–0937). This is further evidence that the Cu2O nanoparticles have completely transformed to CuO
nanoparticles via heating. Analyzing the peak widths using the Scherrer equation yields a crystallite size
of 21.8 ± 0.8 nm for the octahedral particles (for 0.3 M NaOH), and 9.5 ± 1.6 nm for the spherical Cu2O
particles (0.02 M NaOH), whereas the respective particle sizes are ~1000 nm and ~305 nm (analyzed by
TXM consistent with the results from dynamic light scattering investigation). After the annealing step, the original Cu2O signals disappear completely, while new peaks appear, which
correspond to the {110}, {002}, and {111} planes of CuO (Fig. 2g and h beige-colored lines, JCPDS card
no. 45–0937). This is further evidence that the Cu2O nanoparticles have completely transformed to CuO
nanoparticles via heating. Results and Discussion
Large-scale synthesis route for well-defined Cu2O nanoparticles.
The synthesis of Cu2O nano-
particles relies on a precipitation reaction in alkaline media: 4a,b shows the electrical response of the gas sensitive layer for both shapes and at tempera-
tures of 450 °C and 250 °C for 1 ppm of H2S, respectively. At the high temperature of 450 °C, both par-
ticle morphologies react in accordance with equation (3), i.e. the resistivity of the layer increases upon
exposure to H2S. In the low temperature regime both shapes exhibit a steep decline in resistivity because
H2S exposure causes the emergence of a continuous path of highly conducting CuS. Since the reaction
towards H2S is unique and stable CuS structures are formed for temperatures at or below 250 °C the
system used in this work offers an ideal test case to demonstrate the feasibility of directly monitoring
chemical reactions of gas sensitive materials. The corresponding NEXAFS-TXM analysis is depicted in
Fig. 4e,h. In the high temperature regime the morphology of the nanoparticles remains stable which is
verified by NEXAFS-TXM analysis of the layer after exposure to H2S at 450 °C shown in Fig. 4e. The
NEXAFS spectra in Fig. 4d reveal that the chemical composition of the bulk phase is not changed due
to the H2S atmosphere in this temperature regime. All detected signals of the NEXAFS spectra at the
O-K- and Cu-L2,3-edge can be assigned to the signals of copper-(II)-oxide and are identical to the spectra
prior to gas exposure (compare Figs 2 and 3). This means that only the surface of the nanoparticles reacts
during the gas sensing in the high temperature regime. g
g
g
g
p
g
In stark contrast to this, the morphology and the chemical composition of the particles change if
they are exposed to H2S in the low temperature regime which is demonstrated by the NEXAFS-TXM
in Fig. 4f,g. The detected signals at the O-K-edge of both kinds of particles are shown in Fig. 4h, which
can be no longer assigned to CuO bonds. This means that the oxygen lattice of the particles is disturbed
and differences have to be assigned to the different reaction kinetics of spherical and octahedral particle
morphology. The reaction described in equation (2) is not limited to the surface but rather takes place in
the bulk phase as well. Thus, the CuO is completely converted into copper sulfide in the low temperature
regime. Results and Discussion
Large-scale synthesis route for well-defined Cu2O nanoparticles.
The synthesis of Cu2O nano-
particles relies on a precipitation reaction in alkaline media: 3 and a detailed discussion thereof can be found
in the ESI. In our experiments both CuO shapes have been exposed to low levels of hydrogen sulfide in the
same temperature interval. To evaluate the gas sensitive behavior of the layers the electrical conductivity
is determined using interdigitated electrode structures, which form part of a micro-machined hotplate
device35. The combination of inkjet printing technology and the hotplate device allows for quickly testing Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 5 www.nature.com/scientificreports/ Figure 4. Sensing responses towards hydrogen sulfide in two temperature regimes. (a) At 450 °C the sensors
show a chemisorption induced increase in electrical resistance. (b) At 250 °C stable CuS structures form
resulting in a sharp resistance decline without recovery of the sensing signal. Using NEXAFS in combination
with x-ray microscopy all surface processes during gas sensing have been investigated spatially resolved. For
high temperature operation upon H2S exposure the CuO short-range order remains unaltered (c) and (d) and
the NEXAFS spectra for both kind of particles does not change (e), while in the low temperature regime the
structure of the nanoparticles changes dramatically (f,g). (h) In this temperature regime the CuS structures
appear which is confirmed via spectroscopy of the Cu-L2,3 and O-K edge, respectively. Figure 4. Sensing responses towards hydrogen sulfide in two temperature regimes. (a) At 450 °C the sensors
show a chemisorption induced increase in electrical resistance. (b) At 250 °C stable CuS structures form
resulting in a sharp resistance decline without recovery of the sensing signal. Using NEXAFS in combination
with x-ray microscopy all surface processes during gas sensing have been investigated spatially resolved. For
high temperature operation upon H2S exposure the CuO short-range order remains unaltered (c) and (d) and
the NEXAFS spectra for both kind of particles does not change (e), while in the low temperature regime the
structure of the nanoparticles changes dramatically (f,g). (h) In this temperature regime the CuS structures
appear which is confirmed via spectroscopy of the Cu-L2,3 and O-K edge, respectively. novel nano-sized gas sensitive materials. Using NEXAFS-TXM compatible mounts, the experimental
conditions have been duplicated in order to monitor all steps of the gas-surface reaction. Figure 4 shows
both, the gas sensitive response of the CuO layer and the corresponding NEXAFS-TXM analysis, relating
the electrical response of the CuO nanoparticles to the chemical processes at the surface and the bulk
phase. Fig. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 Results and Discussion
Large-scale synthesis route for well-defined Cu2O nanoparticles.
The synthesis of Cu2O nano-
particles relies on a precipitation reaction in alkaline media: At 450 °C the sensors show a chemisorption induced increase in electrical resistance as a function of the
H2S concentration. At 350 °C the behavior differs for spherical and octahedral particles at 1 ppm. For
higher concentrations the formation of CuS structures becomes dominant, yet unstable, as the sensing
resistance recovers after exposure. At 250 °C the CuS structures are stable on the surface and no recovery
of the sensing signal is observed. (b) However, applying a heating protocol the sensor characteristics can be
reproducibly recovered. The temperature is indicated in the middle panel by depicting the corresponding
heater resistivity RH. (c) Measuring tPercol enables selectively determining the H2S concentration using the
trace gas induced phase transition from semiconducting to conducting overall behavior. g
( )
g
p
y
gif
p
At 450 °C the sensors show a chemisorption induced increase in electrical resistance as a function of the
H2S concentration. At 350 °C the behavior differs for spherical and octahedral particles at 1 ppm. For
higher concentrations the formation of CuS structures becomes dominant, yet unstable, as the sensing
resistance recovers after exposure. At 250 °C the CuS structures are stable on the surface and no recovery
of the sensing signal is observed. (b) However, applying a heating protocol the sensor characteristics can be
reproducibly recovered. The temperature is indicated in the middle panel by depicting the corresponding
heater resistivity RH. (c) Measuring tPercol enables selectively determining the H2S concentration using the
trace gas induced phase transition from semiconducting to conducting overall behavior. Because temperature apparently plays a pivotal role in the behavior of the CuO layers we have
expanded the gas sensitive measurements to intermediate temperatures and further concentrations. The
experimental results are depicted in Fig. 5. Lowering the temperature to 350 °C both reaction strengths
are on the same order of magnitude which is highlighted by the shape dependent response to 1 ppm H2S. In order to further investigate the shape and size dependent gas reaction of CuO further experiments
have been performed. The results are presented in the ESI. While sphere-based layers still react according
to equation (2), i.e. by increasing the electrical resistivity upon gas exposure, octahedron-based layers
undergo the percolation phase transition. Increasing the applied H2S concentration above 1 ppm causes
the process described by equation (2) to become dominant for both shapes, i.e. the percolation phase
transition always takes place. Results and Discussion
Large-scale synthesis route for well-defined Cu2O nanoparticles.
The synthesis of Cu2O nano-
particles relies on a precipitation reaction in alkaline media: This is underlined by the detected signals at the Cu-L2,3-edge at 932.3 eV and 952.1 eV which can
be assigned to CuS (compare Ref. 29 and Fig. 3). However, the detected signals in the region between 934
and 941 eV of the Cu-L3-edge are not in complete accordance with the copper-(II)-sulfide structure. It
cannot be ruled out that a distinct amount of Cu2S is also formed under these experimental conditions. By comparing the spectra in Fig. 4h to the reference spectra of CuS in Fig. 3 and to the literature29 this is
further emphasized. Therefore, measurements at the S-K-edge would have to be carried out which is not
possible at the used TXM at the moment. Additionally, measurements at other x-ray microscopes at the
sulfur-K-edge with similar spatial resolution allowing single particle analysis are currently not available. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 6 www.nature.com/scientificreports/ Figure 5. (a) Sensing response towards hydrogen sulfide for different temperatures and concentrations. At 450 °C the sensors show a chemisorption induced increase in electrical resistance as a function of the
H2S concentration. At 350 °C the behavior differs for spherical and octahedral particles at 1 ppm. For
higher concentrations the formation of CuS structures becomes dominant, yet unstable, as the sensing
resistance recovers after exposure. At 250 °C the CuS structures are stable on the surface and no recovery
of the sensing signal is observed. (b) However, applying a heating protocol the sensor characteristics can be
reproducibly recovered. The temperature is indicated in the middle panel by depicting the corresponding
heater resistivity RH. (c) Measuring tPercol enables selectively determining the H2S concentration using the
trace gas induced phase transition from semiconducting to conducting overall behavior. Figure 5. (a) Sensing response towards hydrogen sulfide for different temperatures and concentrations. At 450°C the sensors show a chemisorption induced increase in electrical resistance as a function of the Figure 5. (a) Sensing response towards hydrogen sulfide for different temperatures and concentrations. At 450 °C the sensors show a chemisorption induced increase in electrical resistance as a function of the
H2S concentration. At 350 °C the behavior differs for spherical and octahedral particles at 1 ppm. For Figure 5. (a) Sensing response towards hydrogen sulfide for different temperatures and concentrations. Figure 5. (a) Sensing response towards hydrogen sulfide for different temperatures and concentrations. Results and Discussion
Large-scale synthesis route for well-defined Cu2O nanoparticles.
The synthesis of Cu2O nano-
particles relies on a precipitation reaction in alkaline media: The arrows in (b) indicate the location of
peaks and photon energies used to characterize the different chemical species in (a). Figure 6. (a) TXM-micrographs of the spherical particles after printing and direct exposure to H2S at 200 °C
(overlay 930.8 eV (red), 931.4 eV (green), 932.3 eV (blue)). The false color representation is used as a tool here
in order to visualize the chemical inhomogeneity of the sample. Each color corresponds to a particular type
of chemical bond. This information is extracted from the spectrum of each pixel of the image. (b) NEXAFS
spectra of the O-K-edge and the Cu-L2,3-edge of the marked particles in (a) are represented in green, red
and blue and are labeled 1, 2, and 3, respectively. For comparison, the spectrum of an exemplary octahedron
particle under similar experimental conditions is depicted as well. The arrows in (b) indicate the location of
peaks and photon energies used to characterize the different chemical species in (a). Imaging local surface reactions. Because the gas surface reactions crucially depend on the metal
oxide material we also tested the as-synthesized Cu2O nanoparticles with respect to their gas sensitivity. Unfortunately, due to remaining PEG the sensing layer resistances composed of Cu2O are in excess of
108 Ω making the particles unsuitable for use as gas sensing material. In order to assess the influence
of H2S exposure on Cu2O particles a NEXAFS sample was produced without using the inkjet printing
system, i.e. without using PEG. Future work will entail tuning the rheological properties without using
PEG thus allowing for an electrical read-out of inkjet printed Cu2O layers. To investigate the gas-surface
reactions now, the as-synthesized particles are exposed to 5 ppm H2S in dry synthetic air for 2 hours at
200 °C. Afterwards, the chemical composition of the particles is examined by NEXAFS-TXM. Fig. 6a
shows a resulting TXM-micrograph of the spherical particles, which is given in false color representa-
tion to directly map the chemical inhomogeneity of this sample after the H2S exposure experiment. The
spatially resolved spectroscopy approach allows for extracting information about the chemical bonds
and oxidation state prevailing in each pixel of the image. The false colors are assigned subject to the
marked peak position in Fig. 6b at the Cu-L2,3-edge. The red color in Fig. Results and Discussion
Large-scale synthesis route for well-defined Cu2O nanoparticles.
The synthesis of Cu2O nano-
particles relies on a precipitation reaction in alkaline media: Nevertheless, when removing H2S at 350 °C the CuS structures recede and
both the spherical as well as the octahedral layer return to p-type semiconductivity. Lowering the tem-
perature to 250 °C, the conversion to conductive behavior becomes permanent, i.e. even after removing
H2S from the test chamber the CuS structures remain stable as indicated by the stable, low resistivity
which can be seen in Fig. 4b. Consequently, a temperature treatment might be used to reset the func-
tional layers. This approach is demonstrated in Fig. 5b where the temperature of the gas sensitive layer
is increased to 450 °C after the end of the gas exposure. This is indicated by the temperature dependent
resistivity of the microheater RH in the center panel of the graph. By applying 450 °C to the layer for
30 min it returns to p-type semiconductivity thus resetting the original gas sensing conditions. Fig. 5c
shows the behavior of the layers after the onset of the exposure to a concentration of 1 ppm, 5 ppm, and
10 ppm H2S, respectively, at 250 °C in greater detail. It demonstrates how the percolation time tPercol may
be used to infer the H2S concentration. To characterize the layer’s behavior the time tPercol is defined as
the time from the onset of gas exposure until the resistivity of the layer drops below the threshold value
of 300 Ω. This novel approach may be used to determine the H2S concentration in a fundamentally new,
highly specific way because only H2S causes the percolation phase transition. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 7 7 www.nature.com/scientificreports/ Figure 6. (a) TXM-micrographs of the spherical particles after printing and direct exposure to H2S at 200 °C
(overlay 930.8 eV (red), 931.4 eV (green), 932.3 eV (blue)). The false color representation is used as a tool here
in order to visualize the chemical inhomogeneity of the sample. Each color corresponds to a particular type
of chemical bond. This information is extracted from the spectrum of each pixel of the image. (b) NEXAFS
spectra of the O-K-edge and the Cu-L2,3-edge of the marked particles in (a) are represented in green, red
and blue and are labeled 1, 2, and 3, respectively. For comparison, the spectrum of an exemplary octahedron
particle under similar experimental conditions is depicted as well. Results and Discussion
Large-scale synthesis route for well-defined Cu2O nanoparticles.
The synthesis of Cu2O nano-
particles relies on a precipitation reaction in alkaline media: 6a is assigned to photon energy
930.8 eV, which means that at this energy a chemical bond of Cu2+ in an oxygen environment will show
a maximum of contrast in x-ray absorption whereas the other copper components show a much smaller
absorption. The blue color in Fig. 6a is assigned to photon energy 932.3 eV, which is the characteristic
absorption energy of Cu2+ in a sulfide environment. The green color presents a transition state between
the oxygen and sulfide environment of Cu2+. This cannot be detected in a conventional x-ray absorp-
tion spectroscopy experiment with a spot size of a few microns. The NEXAFS-TXM method employed
here features a spatial resolution on the order of 20 nm and demonstrates how NEXAFS-TXM helps
uncovering the underlying surface processes. The respective NEXAFS spectra of the marked particles in
Fig. 6a are shown in Fig. 6b. Additionally, a representative NEXAFS spectrum of an octahedron particle
prepared under similar experimental conditions as the spherical particles is shown in Fig. 6b. The cor-
responding TXM-micrograph is presented in ESI Figure S1.h p
g
g p
p
g
The analysis of the NEXAFS spectra has utilized the O-K-edge in the energy range 525 eV–555 eV as
well as at the Cu-L2,3-edge in the energy range 925 eV–970 eV. In this way information about the chemical
state of both atomic species constituting the gas sensitive material can be obtained.h p
g
g
The comparison of the spectra of the octahedral particles with literature data and the measured ref-
erences are in good accordance for copper-(I)-oxide22,23,24,27. In detail: The recorded NEXAFS spectra at
the O-K-edge show the typical pronounced absorption peak at 532.7 eV and minor features at higher
photon energy for copper-(I)-oxide. The main absorption feature can be explained by the transition of
an electron from the 1 s core level to the final states of p symmetry at the oxygen site. The minor features
at higher photon energy are caused by the hybridization of the O 2p states with the Cu d and s states23. The recorded main signals of the octahedron particles at the Cu-L3-edge (2p3/2; around 933.8 eV) and
the Cu-L2-edge (2p1/2; around 953.6 eV) are ascribed to the electron transition from the Cu p states to
the unoccupied 3d level due to the unusual crystal structure of Cu2O22. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 Results and Discussion
Large-scale synthesis route for well-defined Cu2O nanoparticles.
The synthesis of Cu2O nano-
particles relies on a precipitation reaction in alkaline media: p
y
2
In contrast, the NEXAFS spectra at the O-K-edge of all marked spherical particles in Fig. 6a sho
significant difference in comparison to the spectrum of the octahedral particles and to the Cu2O refere Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 8 www.nature.com/scientificreports/ For the marked particle 3, no distinct absorption signal is recorded, which leads to the assumption that
the oxygen lattice structure of the blue area in Fig. 6a is more or less disturbed. The Cu-L2,3-edge spec-
trum of particle 3 clearly shows that a copper sulfide structure is formed. The peak at 932.3 eV indicates
the existence for Cu2+ in a sulfide environment, whereas the region between 934 to 940 eV does not
precisely show the minima and maxima of the pure CuS structure (compare Fig. 3 and the Ref. 29). This
means that there is a coexistence of CuS and Cu2S. Additionally, the shoulder around 930.8 eV marks the
existence of Cu2+ in an environment with an electronegative anion, like oxygen27. Similar spectra can be
obtained for all the blue areas of Fig. 6a for both absorption edges. The recorded spectra of particle 1 and
2 presents in our view transitions states in this solid-state transformation process, because the recorded
signals for both particles cannot be fully assigned to the Cu2O, CuO or to the CuS structure.h g
p
y
g
These results clearly indicate that the as-synthesized spherical particles undergo a solid-state trans-
formation into copper sulfide upon H2S exposure while octahedral particles do show a fundamentally
different behavior. This is a direct observation of the profound influence of particle shape on gas-surface
reactions. Possibly, the exposed crystallographic plane and the associated surface energies play a central
role in this process36 and consequently influence on the gas sensitive behavior is expected. Additionally,
this example illustrates the high potential for combining the spatial resolution of x-ray microscopy with
the chemical information obtained from NEXAFS spectroscopy for functional materials development. Conclusion Because of the highly complex nature of gas-solid interactions and the current lack of possibilities to
directly monitor surface reactions the behavior of solid-state gas sensors based on functional metal
oxides is inadequately understood. Within this work the use of NEXAFS-TXM as a means to shed light
onto chemisorption processes at metal oxide surface has been demonstrated using nanoparticles syn-
thesized using a wet chemistry approach. Here, the combination of a bottom-up approach to produce
nanoparticles with well-defined shape and form with the inkjet technology proved to be a promising
method to directly apply basic research results in sensorial devices. The NEXAFS-TXM samples have
been prepared copying all steps of the gas detection, i.e. after synthesis of Cu2O, after transformation to
CuO, after exposure to H2S at low temperatures and after resetting the functional layer, i.e. after convert-
ing CuS back to CuO. Using narrow bandwidth x-ray absorption spectroscopy it was possible to reveal
the underlying changes to the material system, morphology and chemical composition. The reaction
between CuO/Cu2O surfaces and H2S has provided a suitable sample system since the surface states of
the different reactions may be retained for examination in the HZB NEXAFS-TXM. Consequently, the
existing explanations for changes in electrical conductivity observed during the gas characterization could
be substantiated using spectral analysis of the Cu-L2,3 and O-K edges. In particular, the NEXAFS-TXM
analysis demonstrated that for temperatures below 250 °C CuO is converted to CuS whenever H2S is
present. The emergence of highly conducting CuS structures as well as the chemical structure of CuO
have been imaged spatially resolved revealing the sensing mechanism responsible for the selective detec-
tion of H2S. This process is not limited to the particle surface but encompasses the whole particle thus
completely destroying the CuO crystalline structure. Furthermore, the transformation is reversible in the
absence of H2S and applying temperatures of 450 °C to the layer. This makes the reaction mechanism a
suitable starting point for a new type of gas sensor based on the percolation effect induced by H2S. Future
setups based on this approach should offer the possibility for in-situ measurements under real-world
conditions by combining a gas measurement apparatus with a NEXAFS setup, thus enabling a spatially
resolved imaging of the chemisorption processes at metal oxide surfaces under gas exposure. Methods
D
i l The glass ampule was opened directly before the
measurement and some crystals of the reference materials have been spread over the prepared grids and
directly transferred into the oxidation protective vacuum of the TXM measurement.i y
p
A chemical vapor deposition (CVD) process on a TEM grid with holey carbon support film was used
to prepare the copper metal reference (film thickness ~30 nm). The sample was stored and placed on
the sample holder under argon atmosphere. Only during the time for the transfer from one apparatus to
another the sample to the TXM microscope was not protected by inert gas. Near edge X-ray absorption fine structure-transmission X-ray microscopy (NEXAFS-TXM). The
NEXAFS-TXM spectra were recorded on the O-K-edge and the Cu-L2,3-edge with the HZB-TXM
which is installed at the undulator beamline U41-FSGM at the electron storage ring BESSY II, Berlin,
Germany. It provides a high spatial resolution close to 10 nm (half-pitch) and a spectral resolution up
to E/Δ E ≈ 104. Typical spectra are presented for each set of measurements. The TXM allows measure-
ments to be taken at room or liquid nitrogen temperature in a vacuum of 1.3 × 10−9 bar. The spectra
were recorded at room temperature in transmission mode by taking a sequence of images over a range
of photon energies covering the investigated absorption edges with a calculated E/Δ E > 5800 for the
Cu-L2,3-edge and E/Δ E > 12000 for the O-K-edge. Note that the exit slit of the monochromator was set
to 9 μ m for the Cu-L2,3-edge and 7 μ m for the O-K-edge resulting in the given calculated monochroma-
ticity values. The exposure time for one image with 1340 × 1300 pixels was 24 s for the Cu-L2,3-edge and
4 s for the O-K-edge to achieve a sufficient signal to noise ratio in the images. Taking an image stack
with up to 226 images at different energies needs inherently about 45 to 120 min because of all necessary
movements, exposure time, and camera read out time and image storage. The NEXAFS spectra were
normalized since the photon flux varies as a function of photon energy (hν ) and time in the object
field (x, y). Methods
D
i l The remaining protocol was the same as in the standard synthesis without
NaOH. Inkjet printing. After washing the Cu2O particles are dispersed in pure ethanol and 60 s of ultrasound
are applied prior to printing in order to obtain a homogeneous dispersion. 2 ml of the dispersion are
transferred into the printer cartridges. The dispersions containing spherical particles were modified with
0.5 ml PEG 400 to improve printability.h The inkjet deposition is performed using a DIMATIX DMP 2831 printer system with 10 pl print heads. The nozzle is kept at room temperature and the substrate at 30 °C. The nozzle droplet firing voltage was
set to 27 ± 3 V. The drop-to-drop distance (pitch) was set to 40 μ m. For gas sensor devices 6 stacked lay-
ers were deposited. For NEXAFS measurement particles were deposited on a silicon nitride membrane. Sample preparation printer parameters have been identical for gas sensor devices and NEXAFS-TXM
samples. Conversion of Cu2O to CuO. For the gas sensing experiments Cu2O particles were converted on-chip
by the integrated heaters at a temperature of 350 °C for several hours prior to exposing the sensors to
trace gases. This is well above the limit for stable Cu2O. gh
2
For the NEXAFS-TXM characterization the Cu2O particles were oxidized in dry ambient air in a
quartz tube furnace (ATV PEO 603) by linearly ramping the temperature for 1 h to the destination tem-
perature of 400 °C and holding it for 1 h before cooling down. XRD. X-ray diffraction measurements were operated in the 2θ range from 20 to 80° on a Bruker
D8-Advance X-ray diffractometer with Cu Kα 1 radiation (λ = 1.5406 Å). The radiation source was oper-
ated at a voltage of 40 kV and a current of 40 mA. The step size of the measurements is 0.02° with an
integration time of 3.5 seconds per step. Sample preparation of reference-samples for NEXAFS-TXM. The copper grids with carbon
support film (200 meshes, Science Services, Munich, Germany) have been pretreated by 10 s of glow
discharge. Reference materials for Cu2O (Sigma-Aldrich; product no. 566284; purity > 99.99%), CuO
(Sigma-Aldrich, product no.203130; purity > 99.999%) and CuS (Sigma Aldrich, product no. 450820,
purity > 99.99%) are commercially ordered. These high purity materials are delivered in a glass ampule,
which is completely sealed under inert gas atmosphere. Methods
D
i l Dynamic light scattering. Particle sizes were determined by dynamic light scattering (DLS), using
a He:Ne gas laser (λ = 632.8 nm, 22.5 mW) and a goniometer (ALV/SP-86; from ALV Company,
Langen, Germany) equipped with an index match bath. As detector two Perkin Elmer photomultipli-
ers (SPCM-CD2696 Rev.G) were used and cross correlated by an ALV 5000/E correlator. The intensity
auto-correlation function was transformed into the intermediate scattering function by using the Siegert
relation. Diffusion coefficients were calculated by cumulant analysis and used to determine the hydro-
dynamic radii via the Stokes–Einstein equation. The measurements were performed at an angle of 100°. Synthesis of Cu2O nanoparticles. Cu2O nanospheres with radii ranging from 150 nm to 1600 nm
were prepared with high yield needed for inkjet printing. In the synthesis protocol Cu(NO3) 2•3H2O was
dissolved in a 1000 ml three-necked flask in 1/3 of the total water (500 ml) and then kept stirring for
about 30 min at 250 rpm. Meanwhile 5 g Polyvinylpyrrolidone (PVP, average MW= 55000) were dissolved
in the remaining water. After complete dissolution the PVP solution was added dropwise to the Cu(NO3)
solution and the reaction mixture was then stirred for another 2 hours. Then 453 μ l of hydrazine solution
(35% N2H4 in water) was added to the reaction mixture drop-wisely. The color of the solution changed
into orange immediately after the introduction of N2H4, which indicates the production of Cu2O nano-
particles. The resulting solution was kept stirring for 30 min at room temperature and was then washed
with H2O and EtOH several times by centrifugation and redispersion (Multifuge 3 SR, Heraeus: 900 g,
45 min, 21 °C). In a final step the Cu2O nanoparticles were redispersed in a small amount of EtOH and
stowed away in the dark until further use. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 9 www.nature.com/scientificreports/ To further optimize the synthesis for higher yield this standard protocol was modified by the addition
of NaOH. To achieve the wanted NaOH concentration in the reaction mixture water in the Cu(NO3)
solution was reduced by a certain amount to be later on replaced by the same volume of a 2 mol/l NaOH
stock solution. After drop-wise addition of the PVP solution the reaction mixture was stirred for 2 hours
and then the NaOH stock solution volume added under stirring (250 rpm). The N2H4 was then added
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i l The normalization was performed by dividing the intensity I(x, y, hν ) recorded on a single
nanostructure by the intensity I0 (x + Δ x, y + Δ y, hν ) recorded in its sample free proximity at position
(x + Δ x, y + Δ y). Both I(x, y, hν ) and I0(x, y, hν ) were recorded within the same image stack since bare
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0
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MOX layers as well as the composition of the gas matrix is described in detail in38. Here, the read-out
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Mater. Chem. 19, 5220–5225 (2009). Mater. Chem. 19, 5220 5225 (2009). 20. Huang, W.-C., Lyu, L.-M., Yang, Y.-C. & Huang, M. H. Synthesis of Cu2O nanocrystals from cubic to rhombic dodecahedral
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35. Author Contributions K.H. performed the NEXAFS-TXM measurements and the related analysis. P.G. supported K.H. during
the NEXAFS-TXM measurement. J.H. and A.H. performed the particle synthesis and characterization. A.H. performed the dynamic light scattering experiments. J.K. produced the gas sensitive MEMS devices
and performed the gas sensitive characterisation. E.B. and Y.L. supervised the particle synthesis and
characterization. K.H., J.K. and S.P. designed the experiments and analysed the data. P.G., E.B., Y.L. edited the manuscript. K.H. and S.P. wrote the manuscript. Acknowledgementst A.H. thanks “Stiftung Industrieforschung” for financial support. This work has been partially funded
by the German Federal ministry for Education and Science (BMBF) under grant number 16SV5943
(SensOdor). We thank HZB for the allocation of neutron/synchrotron radiation beamtime. The article Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 11 www.nature.com/scientificreports/ processing charge was funded by the German Research Foundation (DFG) and the Albert Ludwigs
University Freiburg in the funding programme Open Access Publishing. processing charge was funded by the German Research Foundation (DFG) and the Albert Ludwigs
University Freiburg in the funding programme Open Access Publishing. Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Henzler, K. et al. Investigation of reactions between trace gases and functional
CuO nanospheres and octahedrons using NEXAFS - TXM imaging. Sci. Rep. 5, 17729; doi: 10.1038/
srep17729 (2015). How to cite this article: Henzler, K. et al. Investigation of reactions between trace gases and functional
CuO nanospheres and octahedrons using NEXAFS - TXM imaging. Sci. Rep. 5, 17729; doi: 10.1038/
srep17729 (2015). This work is licensed under a Creative Commons Attribution 4.0 International License. The
images or other third party material in this article are included in the article’s Creative Com-
mons license, unless indicated otherwise in the credit line; if the material is not included under the
Creative Commons license, users will need to obtain permission from the license holder to reproduce
the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports | 5:17729 | DOI: 10.1038/srep17729 12
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Measuring ice- and liquid-water properties in mixed-phase cloud layers at the Leipzig Cloudnet station
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Atmospheric chemistry and physics
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cc-by
| 10,567
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Measuring ice- and liquid-water properties in mixed-phase clou
layers at the Leipzig Cloudnet station
Johannes Bühl, Patric Seifert, Alexander Myagkov, and Albert Ansmann
Leibniz Institute for Tropospheric Research (TROPOS), Permoserstr. 15, 04318 Leipzig, Germany Correspondence to: Johannes Bühl (buehl@tropos.de) Received: 12 January 2016 – Published in Atmos. Chem. Phys. Discuss.: 27 January 2016
Revised: 23 June 2016 – Accepted: 18 July 2016 – Published: 26 August 2016 Received: 12 January 2016 – Published in Atmos. Chem. Phys. Discuss.: 27 January 2016
Revised: 23 June 2016 – Accepted: 18 July 2016 – Published: 26 August 2016 Abstract. An analysis of the Cloudnet data set collected at
Leipzig, Germany, with special focus on mixed-phase lay-
ered clouds is presented. We derive liquid- and ice-water
content together with vertical motions of ice particles falling
through cloud base. The ice mass flux is calculated by
combining measurements of ice-water content and particle
Doppler velocity. The efficiency of heterogeneous ice forma-
tion and its impact on cloud lifetime is estimated for different
cloud-top temperatures by relating the ice mass flux and the
liquid-water content at cloud top. Cloud radar measurements
of polarization and Doppler velocity indicate that ice crys-
tals formed in mixed-phase cloud layers with a geometrical
thickness of less than 350 m are mostly pristine when they
fall out of the cloud. spectrum of vertical air motions in detail. Laboratory mea-
surements have already delivered a lot of useful information,
e.g., about the ice nucleation efficiency of aerosol particles
with temperature (Murray et al., 2012; DeMott et al., 2015). Observations of the process of ice nucleation in nature, how-
ever, are limited. By means of active remote sensing, quan-
tities that are directly connected with ice nucleation events,
e.g., the ice-water content (IWC) of ice crystals from cloud
layers, can be measured (D. Zhang et al., 2010; Bühl et al.,
2013). In the European Union research project BACCHUS
(Impact of Biogenic vs. Anthropogenic emissions on Clouds
and Climate: towards a Holistic UnderStanding) the ice nu-
cleating properties of aerosols are investigated. It is one ma-
jor task of this project to study the life cycle of aerosols
from its source through the clouds by means of aircraft,
in situ, and remote-sensing observations. Combined remote-
sensing observations in the framework of Cloudnet (Illing-
worth et al., 2007) constitute one main pillar of the BAC-
CHUS project. Beyond other things, Cloudnet provides a tar-
get classification scheme for identifying the physical phase
of hydrometeors. Measuring ice- and liquid-water properties in mixed-phase clou
layers at the Leipzig Cloudnet station
Johannes Bühl, Patric Seifert, Alexander Myagkov, and Albert Ansmann
Leibniz Institute for Tropospheric Research (TROPOS), Permoserstr. 15, 04318 Leipzig, Germany A similar multi-sensor approach is used by
the ARM (Atmospheric Radiation Measurement) program
(Shupe, 2007), which recently performed several measure-
ment campaigns in the Arctic in order to study the interaction
between aerosols and clouds (Zhang et al., 2014). 1
Introduction Understanding the process of heterogeneous ice formation is
currently one of the major topics in weather and climate re-
search (Cantrell and Heymsfield, 2005; Hoose et al., 2008). Heterogeneous ice formation drives the generation of rain
(Mülmenstädt et al., 2015) and impacts cloud stability (Mor-
rison et al., 2005) and atmospheric radiative transfer (Sun
and Shine, 1994). It is therefore a crucial component in the
hydrological cycle in the Earth’s atmosphere. The interaction
between aerosol and clouds in general involves very complex
processes. Vertical motions keep mixed-phase clouds alive
by activating aerosol particles to cloud droplets, while at the
same time ice crystals nucleate and remove water from the
cloud. To understand these complex interactions it is neces-
sary to know all influences, process aspects, involved aerosol
particles, cloud droplets, ice crystal ensembles, as well as the Since 2011, the Leipzig Aerosol and Cloud Remote Obser-
vations System (LACROS) (Wandinger, 2012) has belonged
to the Cloudnet consortium. In this article, remote measure-
ments of LACROS analyzed with Cloudnet algorithms are
used to describe ice formation processes under ambient con-
ditions. Such remote-sensing measurements fill a critical gap
in the study of mixed-phase processes because they deliver
the information about the entire cloud column from the base Atmos. Chem. Phys., 16, 10609–10620, 2016
www.atmos-chem-phys.net/16/10609/2016/
doi:10.5194/acp-16-10609-2016
© Author(s) 2016. CC Attribution 3.0 License. Atmos. Chem. Phys., 16, 10609–10620, 2016
www.atmos-chem-phys.net/16/10609/2016/
doi:10.5194/acp-16-10609-2016
© Author(s) 2016. CC Attribution 3.0 License. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds For a residence time of 20 min
within a mixed-phase cloud layer, particles could still be
considered pristine. Additionally, Yano and Phillips (2010)
found that within this time, secondary processes like riming
do not influence heterogeneous ice formation significantly. According to Fukuta and Takahashi (1999), a residence time
of 20 min corresponds to a geometrical thickness of a mixed-
phase cloud-top layer of 350 m. Hence, for the present study
only clouds with a geometrical thickness of below 350 m are
selected to avoid altering of the ice crystals by riming, splin-
tering, or aggregation processes. g
g
y
y
Shallow mixed-phase cloud layers like altocumulus, al-
tostratus, or stratocumulus have been used before by dif-
ferent groups as atmospheric laboratories in order to study
aerosol–cloud-dynamics interaction under ambient condi-
tions (Fleishauer et al., 2002; Y. Zhang et al., 2010; D. Zhang
et al., 2010; Bühl et al., 2013; Schmidt et al., 2015; Seifert
et al., 2015). These cloud types are especially well suited
for process studies purposes because they show narrow con-
straints on basic environmental variables like temperature,
pressure, humidity, and the number of potentially involved
microphysical processes (Tao and Moncrieff, 2009). The
well-defined base and top of shallow cloud layers are opti-
mal to study aerosol effects on ice nucleation as well as the
impact of up- and downdraft on cloud ice production. As an
additional benefit, these shallow cloud layers can easily be
penetrated by lidar and cloud radar systems, which is not pos-
sible for deep convective clouds due to massive signal atten-
uation and strong turbulence within their cores. For climate
research these shallow cloud layers are important due to their
hard-to-predict impact on Earth’s radiative budget. From the
meteorological point of view, the understanding of ice forma-
tion processes in deep convective mixed-phase clouds may
be more important. However, such clouds are difficult to ob-
serve and may not allow to resolve the basic ice processes
and aerosol- and dynamics-related aspects of ice formation. Both questions can be answered only by studying the process
of ice formation itself in the atmosphere. p
All of the statistical analysis of ice formation in our for-
mer studies (Kanitz et al., 2011; Bühl et al., 2013; Schmidt
et al., 2015; Seifert et al., 2015) has been done manually. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 10610 (2013), in which properties of ice and cloud water have been
analyzed separately and independently. Secondly, statistics
about Doppler velocity (terminal fall velocity of the ice crys-
tals plus vertical velocity of air) and radar depolarization of
the ice crystals are compiled in order to directly assess ice
crystal sedimentation rates and to derive basic information
about the shape of particles at the same time (not only quan-
titative knowledge about the particles themselves is gathered,
but also the usability of cloud layers as atmospheric “lab-
oratories” is characterized). Only if ice crystals are pristine
(i.e., do not show signs of riming growth, aggregation, or
secondary ice formation) is there a direct link between the
properties of the ice (e.g., size, shape, and mass) and their
formation process within the mixed-phase cloud-top layer. These measurements of ice particle properties are compared
with laboratory studies of Fukuta and Takahashi (1999) in
order to assess the quality of the Cloudnet measurements. Based on our data set, the IWC produced by particles falling
from cloud layers is derived and compared with the avail-
able liquid water within the cloud-top layer. Together with
the quality-assured measurements of fall velocity (Doppler
velocity averaged over a complete cloud case), a direct con-
nection between the liquid water in the cloud-top layer and
the resulting ice mass flux is established, which can be re-
garded as a quantitative measure of heterogeneous ice forma-
tion in the atmosphere. With this approach, the impact of ice
formation on cloud lifetime is also estimated for the temper-
ature regime between −35 and 0 ◦C. Fukuta and Takahashi
(1999) also provide comprehensive laboratory measurements
of the growth of ice crystals. They found different distinct
features in the resulting shape of ice crystals for different
growth times and calculated corresponding residence times
within a cloud layer, taking into account increasing fall speed
with increasing particle size. For a residence time of 20 min
within a mixed-phase cloud layer, particles could still be
considered pristine. Additionally, Yano and Phillips (2010)
found that within this time, secondary processes like riming
do not influence heterogeneous ice formation significantly. According to Fukuta and Takahashi (1999), a residence time
of 20 min corresponds to a geometrical thickness of a mixed-
phase cloud-top layer of 350 m. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds Hence, for the present study
only clouds with a geometrical thickness of below 350 m are
selected to avoid altering of the ice crystals by riming, splin-
tering, or aggregation processes. h
i
d
f ll
i
i
h to the top, which is not possible with aircraft measurements
alone. In this way, the temperature level at which ice nucle-
ation takes place can be derived and at the same time the
resulting ice water falling from the layer can be analyzed. (2013), in which properties of ice and cloud water have been
analyzed separately and independently. Secondly, statistics
about Doppler velocity (terminal fall velocity of the ice crys-
tals plus vertical velocity of air) and radar depolarization of
the ice crystals are compiled in order to directly assess ice
crystal sedimentation rates and to derive basic information
about the shape of particles at the same time (not only quan-
titative knowledge about the particles themselves is gathered,
but also the usability of cloud layers as atmospheric “lab-
oratories” is characterized). Only if ice crystals are pristine
(i.e., do not show signs of riming growth, aggregation, or
secondary ice formation) is there a direct link between the
properties of the ice (e.g., size, shape, and mass) and their
formation process within the mixed-phase cloud-top layer. These measurements of ice particle properties are compared
with laboratory studies of Fukuta and Takahashi (1999) in
order to assess the quality of the Cloudnet measurements. Based on our data set, the IWC produced by particles falling
from cloud layers is derived and compared with the avail-
able liquid water within the cloud-top layer. Together with
the quality-assured measurements of fall velocity (Doppler
velocity averaged over a complete cloud case), a direct con-
nection between the liquid water in the cloud-top layer and
the resulting ice mass flux is established, which can be re-
garded as a quantitative measure of heterogeneous ice forma-
tion in the atmosphere. With this approach, the impact of ice
formation on cloud lifetime is also estimated for the temper-
ature regime between −35 and 0 ◦C. Fukuta and Takahashi
(1999) also provide comprehensive laboratory measurements
of the growth of ice crystals. They found different distinct
features in the resulting shape of ice crystals for different
growth times and calculated corresponding residence times
within a cloud layer, taking into account increasing fall speed
with increasing particle size. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 3
Automated selection and classification of cloud layers
in a Cloudnet data set The goal of this study is to obtain a data set of mixed-phase
cloud layers that fulfill certain quality criteria such as tem-
poral and spatial homogeneity. As stated above, the contin-
uous, homogenized Cloudnet-processed data set is used as
a basis for the approach. The automated Cloudnet algorithm
reduces data from a set of remote-sensing instruments on a
common grid that has a temporal resolution of 30 s and a
height resolution of 30.2 m (similar to the one of the cloud
radar). In a further step, the physical state of the atmosphere
in all height bins is classified into different categories, e.g.,
containing cloud droplets, ice particles, or both. Other defi-
nitions concerning aerosol are also present but do not play a
role in the context of this work. A detailed description of the
target categorization scheme of Cloudnet is given in Illing-
worth et al. (2007). Basically, liquid-water droplets are de-
tected by a threshold in lidar signal followed by a characteris-
tic decrease of the latter above liquid cloud base. Ice particles
are in general defined to be present if the radar-observed ver-
tical velocity of the targets indicates falling particles and the
dew-point temperature within a range gate is below 0 ◦C. If,
in addition, the analysis of the lidar signal of the considered
pixel meets the criteria for the presence of liquid droplets, the
pixel is categorized as mixed phase. The height of the melt-
ing layer is derived either from the meteorological data (dew- For the statistical analysis, a cloud must pass certain qual-
ity criteria: a coherent cloud structure must be found for more
than 15 min, no seeding of particles from higher-level clouds
must be present, and for at least 85 % of the cloud’s occur-
rence time a liquid- or mixed-phase cloud top must be de-
tected (height range where water vapor saturation over liquid
water is close to 1; see Fig. 1). The properties of the detected
clouds (e.g., CTH, geometrical cloud thickness δh, standard
deviation of cloud-top height σCTH, cloud-top temperature
(CTT), radar reflectivity factor (Z), IWC, liquid-water con-
tent (LWC), LDR, lidar attenuated backscatter coefficient
(β), and lidar volume linear depolarization ratio) are stored
for further analysis. See Fig. 1 for an overview where the
different properties are derived for one cloud case. 2
Data set point temperature is 0 ◦C) or from measurements of radar lin-
ear depolarization ratio (LDR) larger than −15 dB. Thus, the
decision between liquid-only, mixed-phase, or ice-only cloud
layers is made primarily based on the modeled temperature
and changes in the vertical-velocity profile. However, on the
basis of temperature only, there is no way to unambiguously
decide between drizzle and/or falling ice crystals below 0 ◦C. The data analyzed within the frame of this work have
been collected with LACROS (Wandinger, 2012) at TRO-
POS Leipzig, Germany (51.3◦N, 12.4◦E), between 2011
and 2015. The time coverage of Cloudnet observations at
Leipzig is about 85 %. Instruments relevant for the present
work are the PollyXT Raman/depolarization lidar (Althausen
et al., 2009; Engelmann et al., 2016), the Jenoptik ceilometer
CHM15kx, the MIRA-35 cloud radar (Görsdorf et al., 2015),
and the HATPRO (Humidity and Temperature Profiler) mi-
crowave radiometer (Rose et al., 2005). The measurements
of these instruments are analyzed by the Cloudnet algorithms
(Illingworth et al., 2007) to derive microphysical properties
of hydrometeors on a continuous basis. Additionally, model
input of environmental variables like temperature and hu-
midity is used. For the Cloudnet data set of Leipzig, fore-
cast data of COSMO-EU (Consortium for Small-scale Mod-
eling – Europe) were used from 2011 to May 2014. Since
June 2014, forecast data of the integrated forecast system of
the ECMWF (European Centre for Medium-Range Weather
Forecasts) have been used. In the rare cases when these data
are not available, COSMO-EU is used as a fall-back option. The resulting Cloudnet data set is the basis for the following
analysis of cloud layers over Leipzig presented in the follow-
ing. g
y
The target classification of Cloudnet only takes into ac-
count single range gates. Taking into account measurements
of a complete cloud case facilitates the disambiguation be-
tween a mixed-phase and a liquid-only case. Hence, for this
work, an automated algorithm has been developed that runs
on this basic target classification product of Cloudnet. Sin-
gle 30 s profiles are analyzed to search for liquid water at
T < 0 ◦C. If liquid water is found, the base and top height
of the liquid layer is stored and the height range below this
liquid-water bin is searched for ice. If ice is found below,
the height of transition between liquid and ice is also stored. 2
Data set This procedure is done for all profiles of the data set. Af-
terwards neighboring cloud profiles are merged to coherent
cloud layers if they lie within 300 s of temporal and 350 m of
vertical distance. The 300 s horizontal separation is derived
from experience. Increasing the value increases the homo-
geneity of the cloud cases but reduces the total number of
cases at the same time. The 350 m cloud thickness is mo-
tivated by Fukuta and Takahashi (1999), as it probably ex-
cludes secondary ice formation processes and particle rim-
ing. Cloud-top height (CTH) of the cloud layers is specified
to be larger than 1500 m in order to exclude clouds influenced
by the boundary layer. D. Zhang et al. (2010) went with a
similar approach. A set of connected profiles constitutes a
cloud layer for which we assume that the cloud properties
are similar. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 10611 J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds Such an approach is time consuming and cloud selection
criteria can not be applied on a fully objective basis. Until
now, some Cloudnet stations have been running continuously
for more than 10 years (e.g., Chilbolton and Lindenberg),
providing a wealth of measurement values each day. There-
fore, the analysis of clouds within such a data set can only
be effective with an automated algorithm. For the present
work, a method has been developed to automatically evaluate
measurements from the Cloudnet data set collected between
2011 and 2015 at TROPOS. A modified cloud-classification
scheme from Bühl et al. (2013) is used to automatically dis-
criminate liquid- and mixed-phase cloud layers. The method
is generally applicable to any Cloudnet data set of arbitrary
size. Hence, the method can be used to quickly analyze any
data set with the same objective criteria, thus harmonizing
Cloudnet measurements from all over the world. The paper is structured as follows. Section 2 gives a short
overview about the data set used in the context of this work. In Sect. 3 the methodology to analyze the data set is pre-
sented. At the beginning of Sect. 4 the ice-detection capabil-
ity of different cloud radar systems is analyzed. After that,
quantitative statistics of ice and liquid water within mixed-
phase cloud layers are derived. The focus of the present work is twofold. Firstly, quanti-
tative statistics about ice and water mass in shallow mixed-
phase cloud layers are derived from the Cloudnet data set,
taking into account values of each Cloudnet profile individu-
ally. This constitutes a step forward compared to Bühl et al. www.atmos-chem-phys.net/16/10609/2016/ Atmos. Chem. Phys., 16, 10609–10620, 2016 3
Automated selection and classification of cloud layers
in a Cloudnet data set The pic-
ture also shows that some measurement values are taken only
from a height level 60 m below the mixed-phase cloud base. At this point, cloud droplets should be absent and ice parti-
cles should still be largely unaltered by evaporation or aggre-
gation processes. Hence their size and shape should only be
related to processes that take place within the mixed-phase
cloud-top layer. In the context of this work, all measurement
values derived in this way are marked with the index CB Atmos. Chem. Phys., 16, 10609–10620, 2016 www.atmos-chem-phys.net/16/10609/2016/ (2014), state of water saturation is indicated for the
different parts of the clouds. Figure 1. Schematic representation of the different measurement Figure 1. Schematic representation of the different measurement
and averaging schemes in a mixed-phase cloud layer. Water droplets
within the mixed-phase top layer are detected by lidar. The ice pre-
cipitation below is mainly detected by the cloud radar. IWC and
LWC are provided by Cloudnet and are a function of height (h)
and time (t). IWP and LWP are the column integrated values of
LWC and IWC over the liquid cloud top and the ice precipitation
respectively. IWCCB represents the mean of all IWC values mea-
sured about 60 m below current cloud-base height (CBH). Follow-
ing Zhang et al. (2014), state of water saturation is indicated for the
different parts of the clouds. mixed Figure 2. Flowchart of the mixed-phase cloud discrimination
method from Bühl et al. (2013) as it is applied in the current work. Most clouds are successfully analyzed with combined lidar/radar. (for “cloud base”). In addition, by this definition of coher-
ent cloud layers the average vertical velocities can be used
as an estimate of the particle fall velocity. In cloud layers,
the size of turbulent eddies is restricted to the layer depth. The maximum scale length of free turbulence within the
layer is hence approximately 2 times its geometrical thick-
ness (Moin, 2009). For cloud layers of 350 m of vertical geo-
metrical extent, such small-scale fluctuations cancel out over
the course of 15 min or longer. The average Doppler velocity
of falling particles measured over the timespan of cloud layer
occurrence is therefore free of influences of small-scale tur-
bulence. However, large-scale vertical air motions equal to or
longer than 15 min still influence the measurements. used (Engelmann et al., 2016), if available. In Fig. 3 three
example cases with different CTTs from different dates are
shown together. Cloud radar measurements of Z, LDR, and
v are shown together with the attenuated backscatter coeffi-
cient from the lidar. The CTTs of the three cases are chosen
in such a way that distinct differences in LDR measurements
are visible between the cases. As an example for cloud detec-
tion/selection, all clouds with δh < 350 m and σCTH < 150 m
detected on 2 October 2012 at Leipzig are marked in Fig. 4. www.atmos-chem-phys.net/16/10609/2016/ The CTT statistics of all selected and classified cloud layers
with these selection criteria (δh < 350 m and σCTH < 150 m)
are shown in Fig. 5a and b. It is visible that no mixed-phase
clouds are detected below −40 ◦C. The result of this auto-
mated analysis is within the statistical accuracy of 15 % of
the results of the study of Bühl et al. (2013), which was done
on the basis on manual cloud selection. After cloud identification, the cloud-classification scheme
from Bühl et al. (2013) is used to discriminate between
liquid- and mixed-phase cloud layers (see Fig. 2). This clas-
sification method reduces the dependence on model temper-
ature by taking into account information from all cloud pro-
files to make a decision between the microphysical states
“liquid” or “mixed phase”. Depolarization measurements
from lidar and radar are used to directly identify ice crys-
tals falling from a cloud layer. Mixed-phase clouds close to
0 ◦C also often show a melting layer, which is the most un-
ambiguous sign of the presence of ice particles (Di Giro-
lamo et al., 2012). High LDR values are also produced by
the needle-like ice crystals prevailing for clouds with a CTT
between −8 and −2 ◦C (Fukuta and Takahashi, 1999). Such
clear LDR signals make the decision between ice and liq-
uid water fortunately very easy close to the 0 ◦C level, where
model temperature in most cases is not accurate enough and
the increase in particle fall speed due to melting is not signif-
icant. For low values of Z (typically below −30 dBZ) and no
detection of a melting layer, the depolarized signal is usually
too weak to be detected by the cross-polarized channel of the
MIRA-35 cloud radar. In this case, measurements of volume
linear depolarization ratio from a collocated PollyXT lidar is www.atmos-chem-phys.net/16/10609/2016/ Atmos. Chem. Phys., 16, 10609–10620, 2016 J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 10612 ing ice- and liquid-water properties in mixed-phase clouds
MIRA-35
Cloud Radar
PollyXT
Lidar
Cloud layer
detection
Melting layer
present? LDR > -30 dB
at T < 0 °C
Terminal velocity
< 0.8 m s ¹
⁻
Lidar volume
depol. > 0.2
liquid
mixed
NO
YES
YES
YES
YES
YES
NO
NO
NO / N.A. Virga present? NO
Cloudnet
Data Synthesis
Figure 2. Flowchart of the mixed-phase cloud discrimination
method from Bühl et al. (2013) as it is applied in the current work. Most clouds are successfully analyzed with combined lidar/radar. 10612
J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds
Figure 1. Schematic representation of the different measurement
and averaging schemes in a mixed-phase cloud layer. Water droplets
within the mixed-phase top layer are detected by lidar. The ice pre-
cipitation below is mainly detected by the cloud radar. IWC and
LWC are provided by Cloudnet and are a function of height (h)
and time (t). IWP and LWP are the column integrated values of
LWC and IWC over the liquid cloud top and the ice precipitation
respectively. IWCCB represents the mean of all IWC values mea-
sured about 60 m below current cloud-base height (CBH). Follow-
ing Zhang et al. (2014), state of water saturation is indicated for the
different parts of the clouds. MIRA-35
Cloud Radar
PollyXT
Lidar
Cloud layer
detection
Melting layer
present? LDR > -30 dB
at T < 0 °C
Terminal velocity
< 0.8 m s ¹
⁻
Lidar volume
depol. > 0.2
liquid
mixed
NO
YES
YES
YES
YES
YES
NO
NO
NO / N.A. Virga present? NO
Cloudnet
Data Synthesis
Figure 2. Flowchart of the mixed-phase cloud discrimination Figure 1. Schematic representation of the different measurement
and averaging schemes in a mixed-phase cloud layer. Water droplets
within the mixed-phase top layer are detected by lidar. The ice pre-
cipitation below is mainly detected by the cloud radar. IWC and
LWC are provided by Cloudnet and are a function of height (h)
and time (t). IWP and LWP are the column integrated values of
LWC and IWC over the liquid cloud top and the ice precipitation
respectively. IWCCB represents the mean of all IWC values mea-
sured about 60 m below current cloud-base height (CBH). Follow-
ing Zhang et al. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 10613 Figure 3. Three example case studies of mixed-phase clouds identified with the automated algorithm described in Sect. 3. Figure 3. Three example case studies of mixed-phase clouds identified with the automated algorithm ree example case studies of mixed-phase clouds identified with the automated algorithm described in Sect. 3. Figure 4. Example of automated detection of mixed-phase cloud
layers on the basis of the Cloudnet target classification scheme for
2 October 2012. Clouds are marked due to the selection criteria ex-
plained in the text. Blue squares mark liquid-only layers and red
squares mark mixed-phase layers. The colors are only for a very
basic visualization of the layer detection. The decision between
mixed-phase and liquid clouds in the following analysis is more
complex and described in the text. restrictions to certain scenarios or they do not take into ac-
count any temperature dependence of the ice properties. Uncertainties in the measurements of Z add to these er-
rors. Amongst these, for the quantitative understanding of
ice formation in the atmosphere, knowledge about the ac-
curacy and – especially – about the signal detection thresh-
old of the cloud radar is critical. In the case of ground-based
radar, different factors can affect the measured values of Z,
e.g., unknown attenuation in rain and uncertainties in radar
calibration. Attenuation induced by water vapor and liquid
cloud layers is corrected in Cloudnet. Additionally, attenu-
ation is avoided by excluding clouds from the analysis that
are measured above other clouds or rain. The LACROS cloud
radar is calibrated by the manufacturer with the method de-
scribed in Görsdorf et al. (2015). The calibration is estimated
to be accurate to 3 dB, resulting in an additional bias in the
IWC retrieval of about 35 % (for the range between −60 and
0 dBZ and −40 to 0 ◦C), making them an estimation within
the order of magnitude. Figure 4. Example of automated detection of mixed-phase cloud
layers on the basis of the Cloudnet target classification scheme for
2 October 2012. Clouds are marked due to the selection criteria ex-
plained in the text. Blue squares mark liquid-only layers and red
squares mark mixed-phase layers. The colors are only for a very
basic visualization of the layer detection. The decision between
mixed-phase and liquid clouds in the following analysis is more
complex and described in the text. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds The starting point for the characterization of the IWC data
set is Fig. 6. In this figure, the signal-to-noise ratio (SNR)
detected within cloud virgae (streams of ice particles falling
from cloud top in which water is close to saturation over
ice; see Fig. 1) is depicted together with the detected aver-
age LDR (color scale). The LACROS cloud radar can detect between the methods is about a factor of 2 for the temper-
ature interval −30 to −15 ◦C and about a factor of 5 in the
interval −15 to 0 ◦C. Other methods to derive the IWC from
continuous remote-sensing observations, as they are for in-
stance summarized in Shupe et al. (2008), suffer from their Atmos. Chem. Phys., 16, 10609–10620, 2016 4.1
Ice mass retrieval and detection thresholds A quantitative retrieval of ice mass is done by Cloudnet
via the method of Hogan et al. (2006). IWC values are ob-
tained for each range bin with a simple empirical function
depending on Z and the ambient temperature. The uncer-
tainty of the method is estimated by Hogan et al. (2006)
to be (+50/−30) % below a temperature of −10 ◦C and
(+100/−50) % above. A possible bias of (+15/−10) % is
estimated by Hogan et al. (2006). Comparison between the
retrieval of Hogan et al. (2006) and three other Z−T param-
eterizations from Protat et al. (2007) shows that the spread Atmos. Chem. Phys., 16, 10609–10620, 2016 www.atmos-chem-phys.net/16/10609/2016/ Measuring ice- and liquid-water properties in mixed-phase clouds 4.2
Particle Doppler velocity and radar depolarization
of pristine ice crystals In contrast to the extensive properties ZCB and IWCCB, the
measurements of the cloud radar can also be used to de-
rive the intensive properties of the ice crystals (e.g., vCB and
LDR). The latter are connected to size, shape, and orienta-
tion of the ice particles. Values of LDR and vCB averaged for
each cloud case are shown in Fig. 8c and d. Note that LDR is
dependent on both particle shape and particle orientation, so
this information is not unambiguous (Reinking et al., 1997). However, if particles are oriented, high LDR values indicate
prolate (column-shaped) particles and low values point to-
wards more oblate particles like dendrites. For randomly ori-
ented aspherical particles, LDR is always elevated. In this
way, LDR gives only basic information about particle shape,
but LDR has the advantage that it can be derived easily to-
gether with vCB values with a vertical-pointing radar. Zthr = −10 ×
2log(50002/r2)
−45dBZ
(1) (1) due to the quadratic decrease of received radiation with
range. The corresponding thresholds of IWC (IWCthr) for
different radar systems are drawn within the plots. Please
note that the ice detection threshold is dependent on not only
the radar signal threshold but also temperature, according
to the retrieval of Hogan et al. (2006). For spaceborne sys-
tems Zthr is nearly constant for the complete troposphere. The measurement distance of about 400–800 km leads to a
range-induced signal variation of maximum 5 % between 0
and 12 km height. For ground-based systems, however, the
detection threshold varies significantly for different heights. This phenomenon is depicted in Fig. 7c, where mean ZCB is
plotted against CTH instead of CTT. The height-dependent
detection threshold of the LACROS cloud radar is shown. The single values (30 s integration time and 30 m height
resolution) of LDR and vCB from all cases are shown in
Fig. 8a and b. The values are taken from the virgae where
the target classification of Cloudnet states “ice only” (red-
zone in Fig. 1). These representations already show inter-
esting features. In Fig. 7a, for example, it is shown that at
temperatures above −10 ◦C the average value of ZCB is of-
ten below −30 dBZ. The depolarization measurements show
a clear feature of elevated LDR values in this temperature
range, pointing towards the presence of highly prolate and
oriented ice particles. The vertical-velocity measurements in
Fig. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 10614 Figure 6. The 90 % percentile of cloud radar SNR is shown for each
cloud case together with mean detected LDR. For ±2.5 ◦C intervals
mean values (white squares) and standard deviation (black bars) are
given. Figure 5. Distribution of cloud-top temperature for all pure liq-
uid (a) and mixed-phase (b) cloud layers detected between 2011
and 2015 over Leipzig. Figure 5. Distribution of cloud-top temperature for all pure liq-
uid (a) and mixed-phase (b) cloud layers detected between 2011
and 2015 over Leipzig. Figure 6. The 90 % percentile of cloud radar SNR is shown for each
cloud case together with mean detected LDR. For ±2.5 ◦C intervals
mean values (white squares) and standard deviation (black bars) are
given. a signal down to a SNR of −23 dB. From Fig. 6 it becomes
obvious that particle detection at higher temperatures above
−10 ◦C are often close to the detection limit. In this temper-
ature regime, the detection of some ice below cloud bases
might be missed and clouds could be erroneously be classi-
fied as liquid clouds. In contrast, ice detection seems to be
quite reliable below −10 ◦C, where all cases have a mean
SNR well above the detection threshold. It is also visible
from the figure that LDR values can only be detected if a
certain SNR threshold is reached. olds within the troposphere of −27 dBZ and −33 dBZ re-
spectively. Hence, the CloudSat and EarthCare satellites are
both able to detect most of the ice formation in clouds with
CTT < −10 ◦C. At temperatures warmer than this, probably
90 % of the ice signals below the cloud layers will be missed
(see Fig. 7a). Figure 7a depicts all measurements of ZCB sorted by CTT. In Fig. 7b the values of ZCB are shown averaged for individ-
ual cloud cases. The equivalent values of IWCCB are shown
in Fig. 7c. The LACROS MIRA-35 cloud radar has a de-
tection threshold of Zthr = −45 dBZ at a range of 5000.0 m
(Görsdorf et al., 2015). For other ranges r we hence find a
threshold of J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 10615 Figure 7. (a) All values of column-normalized ZCB. Maximum values in each column are marked with white bars. (b) ZCB averaged for
each cloud case together with averaged LDR values. (c) IWCCB averaged for each cloud case. (d) Values of ZCB depicted depending on CTH
instead of CTT; the cutoff at lower heights appears due to the selection criterion CTH > 1500 m. Thresholds for Z and IWC are illustrated
within the graphs as solid lines with labels. Figure 7. (a) All values of column-normalized ZCB. Maximum values in each column are marked with white bars. (b) ZCB averaged for
each cloud case together with averaged LDR values. (c) IWCCB averaged for each cloud case. (d) Values of ZCB depicted depending on CTH
instead of CTT; the cutoff at lower heights appears due to the selection criterion CTH > 1500 m. Thresholds for Z and IWC are illustrated
within the graphs as solid lines with labels. val is too large for a definite identification of this phe-
nomenon. indicating the different prevailing particle habits over the
temperature range of heterogeneous ice formation. Fukuta and Takahashi (1999) also found several distinct
features in the distribution of ice particle size, shape, and
mass with temperature. Some of these features can be seen
within the measurements of LDR and vCB. We note that by our definition of “pristine particles” we
follow the laboratory experiments of Fukuta and Takahashi
(1999). We consider all particles pristine that have not un-
dergone riming growth, aggregation, or splintering. As ex-
plained above, these processes should be excluded by the
cloud selection criteria. Non-pristine crystals that would re-
sult, for example, from ice particle break-up, aggregation,
or graupel formation would be asymmetric and would there-
fore increase the LDR values. However, the LDR values we
find are very close to the literature values of Reinking et al. (1997), which yield −28 dB for plate-like crystals and about
−20 dB for columnar-shaped particles. These calculations,
however, depend strongly on the orientation of the ice crys-
tals (i.e., at what angle they “wobble”). Larger angles of ori-
entation would increase the measured LDR values. J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds Finding
these very low values of down to −30 dB is therefore an in-
dication that pristine particles dominate and secondary ice
formation only plays a minor role within the selected cloud
layer type. – An enhanced growth of ice crystal mass around −14 ◦C
was found by Fukuta and Takahashi (1999). The effect
can also be seen in Fig. 7a and b as a strong increase of
ZCB at this temperature. – The high values of LDR measured at a CTT of −5 ◦C
correspond to a needle- or column-like particle shape
(see Fig. 8a and c). In the temperature range around
−14 ◦C LDR values can be found to be around −28 dB,
corresponding to plate-like crystal shapes. Please note
that these features are also displayed in Fig. 3. In Reink-
ing et al. (1997) the LDR values of −15 to −20 dB are
computed for these ice crystals shapes. g
p
g
−14 ◦C LDR values can be found to be around −28 dB,
corresponding to plate-like crystal shapes. Please note
that these features are also displayed in Fig. 3. In Reink-
ing et al. (1997) the LDR values of −15 to −20 dB are
computed for these ice crystals shapes. – Hints about the presence of these isometric ice crystals
are found in the increase of Doppler velocity in Fig. 8d. Measured Doppler velocities peak at around −10 and
−22 ◦C, while minima of LDR can be found at −12
and −22 ◦C. This connection also points towards more
isometric, compact ice crystals around these tempera-
tures. Actually, the increase of Doppler velocities in the
temperature interval between −5 and −0 ◦C bin is also
found in Fukuta and Takahashi (1999). However, the un-
certainty of the measurements in this temperature inter- 4.2
Particle Doppler velocity and radar depolarization
of pristine ice crystals 8b also show features of enhanced Doppler velocities, The LACROS cloud radar has a depolarization decoupling
of −33 dB, which stands out from all radars currently op-
erated within the framework of Cloudnet. Only this techni-
cal prerequisite makes high-quality measurements of LDR
possible. Also, the detection threshold of −47 dBZ at a
range of 5000 m is outstanding. Satellite missions equipped
with cloud radars like CloudSat (Stephens et al., 2002) and
EarthCare (Illingworth et al., 2014) have detection thresh- Atmos. Chem. Phys., 16, 10609–10620, 2016 www.atmos-chem-phys.net/16/10609/2016/ 4.3
IWCCB and LWC at cloud top In the previous sections the properties of the ice particles pro-
duced within mixed-phase clouds were investigated. For the
estimation of cloud stability by approaches like the one pre-
sented by Korolev and Field (2008), however, the ratio of
IWCCB/LWC = ILCR (ice- to liquid-water content mass ra- www.atmos-chem-phys.net/16/10609/2016/ www.atmos-chem-phys.net/16/10609/2016/ Atmos. Chem. Phys., 16, 10609–10620, 2016 J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds 10616 J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds Figure 8. All values of (a) LDR and (b) vCB measured with cloud radar MIRA-35 in the virgae below cloud layers over Leipzig. The visible
spread in vCB is due to vertical air motion (see velocity plots in Fig. 3). Averaged values for the individual cloud cases are depicted in (c, d)
respectively. Maximum values in each column are marked with white bars. MIRA 35 i
h
i
b l
l
d l
L i
i
Th
i ibl Figure 8. All values of (a) LDR and (b) vCB measured with cloud radar MIRA-35 in the virgae below cloud layers over Leipzig. The visible
spread in vCB is due to vertical air motion (see velocity plots in Fig. 3). Averaged values for the individual cloud cases are depicted in (c, d)
respectively. Maximum values in each column are marked with white bars. tio) at cloud top is important. For that estimation, the LWC
has to be retrieved in addition to the IWC. phase layer have the same properties as within the layer, this
estimate of ILCR is representative for the average ratio be-
tween ice- and liquid-water content within the mixed-phase
cloud layer. The uncertainty of ILCR is still quite large. As
mentioned above, the absolute accuracy of the measurements
of LWC and IWCCB is 1 order of magnitude due to unknown
biases of the retrieval itself and the radar calibration. Nev-
ertheless, the standard deviation within a temperature inter-
val of about −5 ◦C is only a factor of 2. That comparably
low value might be partially due to the reason that both the
IWCCB and the LWC retrieval method rely on the same tem-
perature field, reducing this part of the variability. Systematic
uncertainties of both the IWCCB and LWC, however, remain. In this work, the LWC of a cloud layer is calculated for
each cloud profile adiabatically between cloud bases and
cloud tops, assuming an adiabaticity of 1. Cloudnet also pro-
vides operationally adiabatic profiles scaled with the liquid-
water path (LWP) measured with the microwave radiome-
ter (Merk et al., 2016). However, the LWP measurements
of the microwave radiometer have an uncertainty of about
±20 g m−2. www.atmos-chem-phys.net/16/10609/2016/ Since the average LWP of the cloud under study
is actually around 20 g m−2, the adiabatically calculated pro-
files are used in the context of this work. An overview about
the LWP of all cloud layers under study is given in Fig. 9a. Zhang et al. (2014) found a similar relationship between
LWP and T for Arctic supercooled midlevel clouds. For the
current work, the retrieved adiabatic LWP can be considered
as a maximum guess. The actual LWP may be lower, which
is described by the adiabaticity factor f . Merk et al. (2016)
report f to be within 0.6 to 1.0. Airborne studies of mixed-
phase clouds found rather good agreement between observed
and adiabatic LWC profiles for shallow cloud layers (Larson
et al., 2006; Noh et al., 2013). Hence, the adiabatic LWC
profiles serve as an estimation until better calibration meth-
ods for the microwave radiometers are available. Such meth-
ods are currently under investigation by different groups,
e.g., Maschwitz et al. (2013). An alternative approach may
be LWP measurements with depolarization lidar (Hu et al.,
2010; Donovan et al., 2015). temperature range of heterogeneous ice formation (−40 to
0 ◦C). temperature range of heterogeneous ice formation (−40 to
0 ◦C). temperature range of heterogeneous ice formation (−40 to
0 ◦C). at temperatures below −20 ◦C it can be seen that the flux of
ice mass is only weakly dependent on temperature. In this
temperature range IWCCB (Fig. 7c) is decreasing with tem-
perature while vCB (Fig. 8d) is increasing. Also, the peak at
−15 ◦C is less pronounced compared to Fig. 7b and c as it
coincides with a minimum in particle fall velocity. LWP (2) Defined in this way, Tl is time measured in seconds. Assum-
ing static conditions, Tl is the time the liquid cloud-top layer
would have depleted all its liquid water by ice sedimentation
alone. It is a theoretical quantity, but it gives an impression of
the relative impact of ice formation on different cloud layers. An overview of Tl for all cloud cases under study is shown
in Fig. 10a, indicating that Tl varies over 4 orders over the Defined in this way, Tl is time measured in seconds. Assum-
ing static conditions, Tl is the time the liquid cloud-top layer
would have depleted all its liquid water by ice sedimentation
alone. It is a theoretical quantity, but it gives an impression of
the relative impact of ice formation on different cloud layers. An overview of Tl for all cloud cases under study is shown
in Fig. 10a, indicating that Tl varies over 4 orders over the It is demonstrated in this work that a detailed insight into
the microphysics of mixed-phase cloud layers is possible
with a combination of the LACROS instrumentation and
Cloudnet. Vertical-velocity measurements show the dynami-
cal state of the turbulent layer and cloud radar measurements 4.4
Estimating the ice mass flux from a cloud layer The ILCR connects measurements of ice- and liquid-water
mass. However, ice crystals formed inside the mixed-
phase cloud-top layer are falling with vCB > 0.2 m s−1 (see
Fig. 8d), while the majority of cloud droplets have negligible
fall velocities. The same number of particles creates a differ-
ent IWC when falling at different terminal velocities, because
the stream of particles is “stretched” differently. Hence, the
ice flux F = IWCCB × vCB at cloud base gives the most ac-
curate description of ice formation per time interval inside
the cloud-top layer. In this very simple picture, F describes
the flux quite coarsely. However, since both vCB and IWCCB
are calculated from the same radar signal, a direct multipli-
cation can be applied. The resulting parameter is an estima-
tion within the order of magnitude, but it can safely be com-
pared to the other flux values presented here. Figure 10a dis-
plays averaged F for all cloud cases under study. Especially In Fig. 9b, IWCCB is divided by the mean LWC in the
mixed-phase cloud top in order to derive an estimate of
ILCR. Assuming that particles directly below the mixed- Atmos. Chem. Phys., 16, 10609–10620, 2016 www.atmos-chem-phys.net/16/10609/2016/ www.atmos-chem-phys.net/16/10609/2016/ J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds
10617 J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds Measuring ice- and liquid-water properties in mixed-phase clouds 10617 Figure 9. (a) LWP of all clouds under study is shown in dependence of temperature and mean cloud-top thickness. (b) The ratio between
IWCCB and mean LWC is calculated for each cloud profile and averaged for each cloud case. Figure 9. (a) LWP of all clouds under study is shown in dependence of temperature and mean cloud-top thickness. (b) The ratio between
IWCCB and mean LWC is calculated for each cloud profile and averaged for each cloud case. Figure 10. (a) The ice mass flux at cloud base. (b) The estimated static lifetime index Tl = LWP/F of each cloud. Figure 10. (a) The ice mass flux at cloud base. (b) The estimated static lifetime index Tl = LWP/F of each cloud. ce mass flux at cloud base. (b) The estimated static lifetime index Tl = LWP/F of each cloud. 5
Summary and conclusions The concept of ice mass flux also opens the possibility
to derive basic information about the impact of ice forma-
tion on static cloud lifetime. Water particles most probably
glaciate at cloud top and fall through the mixed-phase layer. Having connected vCB with IWCCB to the ice flux, it is also
possible to relate this quantity to the available LWP within
the ice-generating liquid cloud layer. Since ice particles grow
through the Wegener–Bergeron–Findeisen process (Korolev
and Field, 2008), there is an indirect connection between
the amount of available water vapor and ice crystal growth. Hence, a dynamic view of ice formation in the cloud layers
can be established by dividing F and LWP profile-wise. Quantitative retrievals of ice crystal properties like basic in-
formation about particle shape and Doppler velocity have
been found to be quantitatively in line with theoretical com-
putations of Reinking et al. (1997) and laboratory studies of
Fukuta and Takahashi (1999). The dominating part of the ice
particles falling from mixed-phase cloud layers with a geo-
metrical thickness of the mixed-phase top layer < 350 m are
apparently mostly pristine. Hence, these particles are prob-
ably the result of primary ice formation, and secondary ice
formation is only a minor process in these cloud layers. Ad-
ditionally, a profile-based connection between the measured
LWP and the retrieved IWCCB has been established. The flux
of ice mass at cloud-base height is found to increase within 2
orders of magnitude within the CTT range from −40 to 0 ◦C. The relative influence of the loss of ice on static lifetime in-
dex is found to increase even by 4 orders of magnitude within
the same range of CTT. LWP
F
=
LWP
IWCCB × vCB
!= Tl
(2) J. Bühl et al.: Measuring ice- and liquid-water properties in mixed-phase clouds show the ice flux from that layer. Together with the retrieval
of ice nuclei properties with Raman lidar (Mamouri and Ans-
mann, 2015) the life cycle of an ice nucleus in mixed-phase
clouds from entrainment over activation to ice nucleation and
sedimentation can be closed. quests, please contact Johannes Bühl (buehl@tropos.de) at
TROPOS. We will gladly provide the requested data used in
this study. Please note that the full data set contains about
600 GB of data. quests, please contact Johannes Bühl (buehl@tropos.de) at
TROPOS. We will gladly provide the requested data used in
this study. Please note that the full data set contains about
600 GB of data. It is an important finding that the dominating number of
ice crystals in mixed-phase cloud layers with δh < 350 m is
pristine. This means that the flux of ice crystals measured at
cloud base is directly connected to the rate of ice nucleation
within the mixed-phase layer. The direct measurement of the
complete process of ice nucleation seems therefore feasible
with remote sensing. However, in future, more advanced par-
ticle typing methods such as presented in Myagkov et al. (2016a, b) should be applied to further characterize shape
and size of the particles on an operational basis. Acknowledgements. The research leading to these results has re-
ceived funding from the European Union’s Horizon 2020 research
and innovation programme under grant agreement no. 654109
(ACTRIS-2) and previously from the European Union Seventh
Framework Programme (FP7/2007–2013) under grant agreement
no. 262254 (ACTRIS) and 603445 (BACCHUS) as well as from
the Cloudnet project (European Union contract EVK2-2000-
00611). Funding was also received from the HD(CP)2 project
(FKZ 01LK1209C and 01LK1212C) of the German Ministry for
Education and Research. The relative impact of the loss of ice water on a mixed-
phase cloud layer has been measured. However, it has to be
noted again that the cloud static lifetime index presented here
might not directly be connected to the absolute lifetime of
a cloud. Even the definition of a cloud lifetime is difficult
because particles are mixed between cloud parcels and the
apparent motion of clouds can be independent from horizon-
tal wind speed. However, the static lifetime value presented
here can be used to study the impact of ice on predominantly
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Data availability The Cloudnet data set used in this study is available in the
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Launching a new English language program
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Abstract In the rapidly globalizing world, English language proficiency is becoming increasingly pivotal for academic and
professional success. This research article presents a comprehensive study surrounding the launch of a novel English
language program, termed the IELTS 4.0, by the Mentor Language School. The article delves into the objectives,
development, and promotion processes of the program, aiming to elucidate its distinctive features and potential
benefits. Drawing from both qualitative and quantitative data, the research evaluates the program's curriculum
design, pedagogical strategies, and its early impact on students' language proficiency outcomes. Initial feedback
reveals a positive reception, with particular emphasis on its modern teaching methodologies, expert-led instruction,
and cultural immersion components. Additionally, the program's structured approach, segmented into Beginner,
Intermediate, Advanced, and IELTS Preparation levels, facilitates tailored learning experiences, catering to diverse
student needs. The article concludes by underscoring the potential of the IELTS 4.0 program in shaping the future
landscape of English language education, with recommendations for further enhancements based on early
observations and feedback. Keywords: Pedagogical Strategies, Language Proficiency, Cultural Immersion Article Information:
Article history: Received: 15/07/2023 Accepted: 05/08/2023 Online: 06/10/2023 Publish
DOI: https://doi.org/10.60087/jklst.vol2.n3.p ̣̣̣̣45
i Correspondence author: Thuan, Phan Kim
Email: thuanphanvus84@gmail.com Article Information:
Article history: Received: 15/07/2023 Accepted: 05/08/2023 Online: 06/10/2023 Published: 20/12/2023
DOI: https://doi.org/10.60087/jklst.vol2.n3.p ̣̣̣̣45
i Correspondence author: Thuan, Phan Kim
Email: thuanphanvus84@gmail.com Launching a new English language program Thuan, Phan Kim MAE, Hoa Sen University 1. Introduction Mentor Language School is a reputable language teaching organization that has been providing high-quality
language programs to students from various backgrounds for many years. Our organization has a strong
commitment to meeting the needs of our students, and we are continually striving to provide innovative and
effective language education programs. This report introduces our new English language program, the IELTS 4.0 program, and outlines the project's
development and promotion process. It includes an overview of the curriculum, objectives, and marketing
strategies used to promote the program, as well as feedback received since its launch. The report aims to
showcase the program's unique features and benefits while providing insight into its development and
marketing process. ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 2023
Journal of Knowledge Learning and Science Technology
journal homepage: https://jklst.org/index.php/home ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 2023
Journal of Knowledge Learning and Science Technology
journal homepage: https://jklst.org/index.php/home ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 2023
Journal of Knowledge Learning and Science Technology
journal homepage: https://jklst.org/index.php/home ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 2023
Journal of Knowledge Learning and Science Technology
journal homepage: https://jklst.org/index.php/home 2.1. Explanation of the program's goals The IELTS 4.0 program is designed to help high school students graduate from high school and assist other
students in improving their English language proficiency to increase their chances of securing better job
opportunities. The program aims to achieve the following goals: (Nick Kenny and Bruce Rogers, 2015). 35 Journal of Knowledge Learning and Science Technology ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 2023 Enhance students' language skills: The IELTS 4.0 program is designed to help students develop their
English language skills, including reading, writing, listening, and speaking. By improving their language
proficiency, students can communicate more effectively and confidently, which can increase their chances
of academic and career success. Prepare students for academic success: The program aims to help high school students improve their
English language proficiency to meet the requirements for graduation. It provides a structured curriculum
and expert instructors to ensure that students have the necessary language skills to excel in their academic
studies. Increase employability: The IELTS 4.0 program also targets students who are looking to improve their
English language proficiency to increase their chances of finding better job opportunities. By enhancing
their language skills, students can communicate effectively with colleagues, clients, and customers in the
workplace, making them more desirable to potential employers. Provide a high-quality English language program: The program is designed to provide a high-quality
English language learning experience for students. It includes a comprehensive curriculum, expert
instructors, and modern teaching methods to ensure that students receive the best possible education. Overall, the IELTS 4.0 program aims to provide students with the language skills and knowledge necessary
to achieve academic success and improve their employability, ultimately leading to better opportunities for
their future. Provide a high-quality English language program: The program is designed to provide a high-quality
English language learning experience for students. It includes a comprehensive curriculum, expert
instructors, and modern teaching methods to ensure that students receive the best possible education. Overall, the IELTS 4.0 program aims to provide students with the language skills and knowledge necessary
to achieve academic success and improve their employability, ultimately leading to better opportunities for
their future. Level 2: Intermediate The intermediate level is designed for students who have some experience with the English language but
still need to improve their language proficiency. In this level, students will learn more advanced vocabulary,
sentence structure, and grammar rules. They will also learn how to read and write more complex texts and
hold more advanced conversations. By the end of this level, students should be able to communicate more
effectively in English and understand more complex information. Level 1: Beginner The beginner level is designed for students who have little or no experience with the English language. In
this level, students will learn basic vocabulary, sentence structure, and grammar rules. They will also learn
how to read and write simple texts and hold basic conversations. By the end of this level, students should
be able to understand and communicate basic information in English. 2.2. Detailed program description and curriculum
2.2.1. Detailed program description The IELTS 4.0 program is a comprehensive English language program designed to help high school students
graduate from high school and assist other students in improving their English language proficiency to
increase their chances of securing better job opportunities. The program is divided into four levels, each
designed to help students develop their language skills in reading, writing, listening, and speaking. (Nick
Kenny and Bruce Rogers, 2015) Level 3: Advanced The advanced level is designed for students who have a good understanding of the English language but
still need to improve their language proficiency. At this level, students will learn advanced vocabulary,
sentence structure, and grammar rules. They will also learn how to read and write academic texts and hold Thuan Phan Kim 36 advanced conversations. By the end of this level, students should be able to communicate fluently in English
and understand complex academic information. The IELTS preparation level is designed for students who need to prepare for the IELTS exam. In this level,
students will learn advanced vocabulary, sentence structure, and grammar rules specifically for the IELTS
exam. They will also learn test-taking strategies and practice taking mock exams to prepare for the actual
exam. By the end of this level, students should be prepared to take the IELTS exam and achieve their desired
score. 2.3. Our strengths and features that distinguish our program from others. These include:
Potential
Weakness
Descriptions
Cost
Our program may be more expensive than other language programs, due to the personalized
nature of the instruction and the use of specialized materials and technology. The IELTS 4.0 program offered by our language teaching organization is designed to provide students with
a high-quality English language learning experience that sets it apart from other programs. Here are some
strengths and features that distinguish our program: Strengths
and
Features
Description
Tailored
curriculum
The program's curriculum is designed to be flexible and customizable, depending on each
student's specific needs and goals. This approach ensures that students receive personalized
instruction that meets their individual learning needs. Experienced
instructors
The program has experienced and highly qualified instructors who are trained to provide expert
guidance and support to students. Instructors are native English speakers who have extensive
experience teaching English to students from diverse backgrounds. Advanced
technology
The program uses advanced technology to enhance the learning experience. Multimedia
resources, online tools, and interactive activities are incorporated to make learning engaging
and effective. Students can access the online learning platform, which includes video lectures,
quizzes, and practice exercises. Small class sizes The program emphasizes small class sizes to ensure that students receive personalized attention
and guidance from their instructors. This approach allows instructors to provide individualized
feedback and support to each student, which can lead to better learning outcomes. IELTS
test
preparation
The program includes a dedicated IELTS preparation level that is designed to help students
prepare for the IELTS exam. This level provides students with the skills and knowledge
necessary to succeed on the exam, including test-taking strategies and mock exams. Academic
and
career support
The program provides academic and career support to students, including academic counseling,
college application assistance, and career counseling. The organization is committed to helping
students achieve their academic and career goals. Overall, the IELTS 4.0 program offered by our language teaching organization stands out from other
programs due to its tailored curriculum, experienced instructors, advanced technology, small class sizes,
dedicated IELTS test preparation level, and academic and career support. These strengths and features
ensure that students receive a high-quality English language learning experience that can help them achieve
their goals. 2.3. Our strengths and features that distinguish our program from others. 2.3. Our strengths and features that distinguish our program from others. The IELTS 4.0 program offered by our language teaching organization is designed to provide students with
a high-quality English language learning experience that sets it apart from other programs. Here are some
strengths and features that distinguish our program:
Strengths
and
Features
Description
Tailored
curriculum
The program's curriculum is designed to be flexible and customizable, depending on each
student's specific needs and goals. This approach ensures that students receive personalized
instruction that meets their individual learning needs. Experienced
instructors
The program has experienced and highly qualified instructors who are trained to provide expert
guidance and support to students. Instructors are native English speakers who have extensive
experience teaching English to students from diverse backgrounds. Advanced
technology
The program uses advanced technology to enhance the learning experience. Multimedia
resources, online tools, and interactive activities are incorporated to make learning engaging
and effective. Students can access the online learning platform, which includes video lectures,
quizzes, and practice exercises. Small class sizes The program emphasizes small class sizes to ensure that students receive personalized attention
and guidance from their instructors. This approach allows instructors to provide individualized
feedback and support to each student, which can lead to better learning outcomes. IELTS
test
preparation
The program includes a dedicated IELTS preparation level that is designed to help students
prepare for the IELTS exam. This level provides students with the skills and knowledge
necessary to succeed on the exam, including test-taking strategies and mock exams. Academic
and
career support
The program provides academic and career support to students, including academic counseling,
college application assistance, and career counseling. The organization is committed to helping
students achieve their academic and career goals. Overall, the IELTS 4.0 program offered by our language teaching organization stands out from other
programs due to its tailored curriculum, experienced instructors, advanced technology, small class sizes,
dedicated IELTS test preparation level, and academic and career support. These strengths and features
ensure that students receive a high-quality English language learning experience that can help them achieve
their goals. 2.4. Some potential weaknesses
While we believe our new English language program has many strengths, it is important to acknowledge
that there are also some potential weaknesses. 2.2.2. Curriculum
The curriculum for each level includes the following components:
Fig. 1 Detailed program description and curriculum
Reading
•
Developing reading comprehension skills
•
Building vocabulary through reading
•
Identifying and analyzing the main ideas and supporting details of texts
Writing
•
Developing writing skills for different purposes (e.g., essays, emails, reports)
•
Building vocabulary for writing
•
Developing organization and structure in writing
Listening
•
Developing listening comprehension skills
•
Building vocabulary through listening
•
Identifying and analyzing the main ideas and supporting details of spoken language
Speaking
•
Developing speaking skills for different purposes (e.g., interviews, presentations, group
discussions)
•
Building vocabulary for speaking
•
Developing fluency and accuracy in speaking
CURRICULUM
Speaking
Writing
Reading
Listening The curriculum for each level includes the following components: Reading Speaking Writing CURRICULUM Speaking Listening Reading g
•
Developing reading comprehension skills
•
Building vocabulary through reading
•
Identifying and analyzing the main ideas and supporting details of texts
Writing •
Developing writing skills for different purposes (e.g., essays, emails, reports) •
Building vocabulary for writing •
Developing organization and structure in writing
Listening •
Developing listening comprehension skills •
Building vocabulary through listening •
Identifying and analyzing the main ideas and supporting details of spoken language
Speaking •
Developing speaking skills for different purposes (e.g., interviews, presentations, group
discussions) •
Building vocabulary for speaking •
Developing fluency and accuracy in speaking Journal of Knowledge Learning and Science Technology ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 2023 37 Overall, the IELTS 4.0 program provides a comprehensive and structured curriculum that is designed to
help students develop their language skills and achieve their goals in academic and professional contexts. 2.5. Some threats While we see many opportunities in the current situation for the English program, we should also pay
attention to some threats. While we see many opportunities in the current situation for the English program, we should also pay
attention to some threats. 1. Competition from other language teaching organizations offering similar English language
programs, potentially limiting enrollment and revenue. 2. Economic downturns or recessions affect the ability of individuals to afford language education,
decreasing the program's revenue. 3. Changes in government policies or regulations impacting the availability of student visas or funding
for language education, potentially limiting the program's reach and appeal. 4. Political instability or social unrest affecting the safety and security of students, potentially
impacting enrollment and the program's reputation. 3. Project development process 3.1. Market research to identify student preferences and needs. 2.4. Some potential weaknesses reased online education platform use enables the program to reach a wider audience. Increased online education platform use enables the program to reach a wider audience. Access to a diverse pool of experienced and qualified English language teachers, allowing for the
development of a high-quality program. Access to a diverse pool of experienced and qualified English language teachers, allowing for the
development of a high-quality program. The ability to incorporate new technologies and teaching methods to improve the program's effectiveness. The ability to incorporate new technologies and teaching methods to improve the program' The ability to incorporate new technologies and teaching methods to improve the program's effectiveness. The ability to incorporate new technologies and teaching methods to improve the program's effectiveness. 2 5 Some threats 2.4. Some potential weaknesses While we believe our new English language program has many strengths, it is important to acknowledge
that there are also some potential weaknesses. These include: Potential
Weakness
Descriptions
Cost
Our program may be more expensive than other language programs, due to the personalized
nature of the instruction and the use of specialized materials and technology. Potential
Weakness
Descriptions
Cost
Our program may be more expensive than other language programs, due to the personalized
nature of the instruction and the use of specialized materials and technology. 38 Thuan Phan Kim Availability
Our program may not be available in all locations, as we may not have the resources to expand
to all areas. Time
commitment
Our program requires a significant time commitment from students, as it is designed to be
comprehensive and thorough. Students will need to dedicate themselves to regular study and
practice in order to see the full benefits of the program. Teaching style
Our program may not be suitable for students who prefer a more traditional teaching style, as it
is designed to be interactive and engaging, using a range of multimedia materials and real-life
scenarios. rogram may not be available in all locations, as we may not have the resources to expand
areas Overall, while our program has many strengths, it is important to acknowledge that there may be some
limitations or weaknesses that could impact its suitability for certain students or situations. Overall, while our program has many strengths, it is important to acknowledge that there may be some
limitations or weaknesses that could impact its suitability for certain students or situations. In addition to the strengths and weaknesses outlined above, the IELTS 4.0 program offers students a range
of opportunities to enhance their language skills and gain valuable experiences. These are some of the
opportunities of the current situation of the program:
Growing demand for English language proficiency globally, creating opportunities for the program to attract
new students. In addition to the strengths and weaknesses outlined above, the IELTS 4.0 program offers students a range
of opportunities to enhance their language skills and gain valuable experiences. These are some of the
opportunities of the current situation of the program: Growing demand for English language proficiency globally, creating opportunities for the program to attract
new students. Increased online education platform use enables the program to reach a wider audience. 3.1. Market research to identify student preferences and needs. To ensure that our IELTS 4.0 program meets the preferences and needs of our students, we conducted
extensive market research. Our research involved surveys, focus groups, and interviews with current and
potential students, as well as analysis of industry trends and best practices. The findings from our research
helped us to tailor the program's curriculum and features to meet the specific needs and preferences of our
target audience. We are confident that our program will provide a high-quality English language learning
experience that meets the needs and expectations of our students. Based on the findings of the market research, the IELTS 4.0 program should be designed to meet the
preferences and needs of potential students. A blended learning approach that includes both online and in-
person classes would be ideal, with a curriculum that is relevant, up-to-date, and includes multimedia nal of Knowledge Learning and Science Technology ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 202 39 content. The class size should be kept small, with no more than 15 students per class, and teachers should
be highly qualified and experienced. By meeting these preferences and needs, the new English program will
be well-positioned to attract and retain students. 3.2. Development of program objectives and curriculum To develop an effective program, several steps must be taken to ensure that it meets the needs of the target
audience and achieves its intended learning outcomes: Needs Assessment: Identify the target audience's English proficiency level, learning needs, and preferences. Program Objectives: Establish specific, measurable, achievable, relevant, and time-bound objectives. Needs Assessment: Identify the target audience's English proficiency level, learning needs, and preferences. Program Objectives: Establish specific, measurable, achievable, relevant, and time-bound objectives. Curriculum Design: Develop a curriculum that aligns with the program objectives and includes clear
learning outcomes, lesson plans, and assessments. Needs Assessment: Identify the target audience s English proficiency level, learning needs, and preferences. Program Objectives: Establish specific, measurable, achievable, relevant, and time-bound objectives. Curriculum Design: Develop a curriculum that aligns with the program objectives and includes clear
learning outcomes, lesson plans, and assessments. Curriculum Design: Develop a curriculum that aligns with the program objectives and includes clear
learning outcomes, lesson plans, and assessments. Teaching Methodology: Design an interactive, learner-centred methodology that caters to different learning
styles. Teaching Methodology: Design an interactive, learner-centred methodology that caters to different learning
styles. Course Materials and Resources: Select and design relevant, up-to-date materials and resources that support
the curriculum and teaching methodology. Course Materials and Resources: Select and design relevant, up-to-date materials and resources that support
the curriculum and teaching methodology. Program Evaluation: Conduct regular evaluation and feedback to measure program effectiveness and make
necessary improvements. Continuous Improvement: Continuously update and improve the program based on feedback received. Collaboration among program designers, language experts, and potential participants is crucial to ensure
program success. 3.3. Timeline and milestones Developing a timeline and milestones for the new English program is an essential step in ensuring that the
program is implemented effectively and efficiently. The following is an example of a timeline and
milestones for a 12-week program (Peter Sommerville, 2023). Week
Activities
Weeks 1-2
Conduct a needs assessment, establish program objectives, design the curriculum, teaching
methodology, and course materials. Weeks 3-4
Recruit and train English language teachers who meet the program's requirements and
qualifications. Train teachers on the program's objectives, curriculum, teaching methodology, and
assessment methods. Weeks 5-6
Finalize the program schedule, lesson plans, and course materials. Establish communication
channels with participants and provide orientation on the program's objectives, schedule, and
expectations. Weeks 7-11
Implement the program. Participants attend classes and engage in learning activities. Conduct
regular assessments to measure progress and adjust teaching strategies and materials if necessary. Week 12
Conduct program evaluations to measure the effectiveness of the program in achieving its
objectives. Participants who have successfully completed the program will receive a certificate of
completion. Overall, developing a timeline and milestones for the new English program will help ensure that the program
is delivered effectively and efficiently and that its objectives are achieved within the desired timeframe. 3.4. Budget and resources 40 Thuan Phan Kim Developing a budget and identifying the necessary resources for the new English program is an important
step in ensuring the program's success. The following are some of the main expenses and resources that
may be needed for the program: (Mike McKeever, 2011) Developing a budget and identifying the necessary resources for the new English program is an important
step in ensuring the program's success. The following are some of the main expenses and resources that
may be needed for the program: (Mike McKeever, 2011) Personnel Costs: The largest expense for the program is likely to be personnel costs, including salaries and
benefits for the English language teachers and program staff. Course Materials and Resources: Course materials and resources such as textbooks, workbooks, audio-
visual aids, and technology equipment will be needed to support the curriculum and teaching methodology. Facility Costs: The program may require a dedicated facility or classrooms equipped with appropriate
technology and learning resources. Marketing and Promotion: A budget for marketing and promotion may be required to promote the program
and attract potential participants. This may include website development, online advertising, and brochures. 3.3. Timeline and milestones Administrative Costs: Administrative costs such as licensing fees, insurance, and legal fees should also be
included in the budget. Program Evaluation: A budget for program evaluation should be included to measure the effectiveness of
the program and identify areas for improvement. Program Evaluation: A budget for program evaluation should be included to measure the effectiveness of
the program and identify areas for improvement. Contingency: It is also important to include a contingency budget to account for unexpected costs or
emergencies. Contingency: It is also important to include a contingency budget to account for unexpected costs or
emergencies. In addition to budgeting for expenses, resources such as expert advisors, consultants, and partners may also
be needed to support the program's development and implementation. Other resources, such as research
materials, training materials, and software may also be required. Overall, developing a budget and identifying the necessary resources for the new English program is crucial
in ensuring that the program is adequately funded and that the resources needed to deliver high-quality
instruction are available. The following table displays the break-even analysis of the new product. The following table displays the break-even analysis of the new product. Table 1. The break-even analysis of the new product. Table 1. The break-even analysis of the new product. Table 1. The break-even analysis of the new product. Account
Rate in thousand VND
Revenue
Tuition (on each course)
20,00
Total per student (on each course)
20,000
Cost
Variable costs per student
Material
500
Copying
500
Total variable
1,000
Gross Profit
19,000
Fixed cost Account
Rate in thousand VND
Revenue
Tuition (on each course)
20,00
Total per student (on each course)
20,000
Cost
Variable costs per student
Material
500
Copying
500
Total variable
1,000 Account
Rate in thousand VND
Revenue
Tuition (on each course)
20,00
Total per student (on each course)
20,000 500
500
1,000
19,000 500
500
1,000
19,000 Journal of Knowledge Learning and Science Technology ISSN: 2959-6386 (Online), Vol. 3.5. Recruitment and training of teachers for the new program Recruiting and training teachers for the new English program is an essential step in ensuring that the
program is delivered effectively and efficiently. The following are some considerations for teacher
recruitment and training: (Peter Sommerville, 2023) Requirements and qualifications: The program development team should identify the requirements and
qualifications for English language teachers. These may include minimum educational requirements,
teaching experience, language proficiency, and teaching certifications. Recruitment: The program development team should develop a recruitment plan that identifies potential
candidates and outlines the recruitment process. Recruitment channels may include job boards, social
media, personal networks, and professional associations. Selection: Once applications are received, a selection process should be established to identify the most
suitable candidates. This may include an initial screening, interviews, and reference checks. Selection: Once applications are received, a selection process should be established to identify the most
suitable candidates. This may include an initial screening, interviews, and reference checks. Orientation: Newly hired teachers should receive an orientation that includes an overview of the program's
objectives, curriculum, teaching methodology, and assessment methods. They should also be provided with
relevant teaching resources and technology equipment. Training: Teachers should receive ongoing training and professional development to support their growth
and development. This may include workshops, mentoring, coaching, and online training. Training: Teachers should receive ongoing training and professional development to support their growth
and development. This may include workshops, mentoring, coaching, and online training. Assessment and Feedback: Teachers should receive regular assessment and feedback on their performance,
including observations, evaluations, and feedback from participants. This can help identify areas for
improvement and support ongoing development. Assessment and Feedback: Teachers should receive regular assessment and feedback on their performance,
including observations, evaluations, and feedback from participants. This can help identify areas for
improvement and support ongoing development. Overall, recruiting and training teachers for the new English program is a critical step in ensuring the
program's success. By identifying the requirements and qualifications for teachers, developing a recruitment
plan, providing orientation and training, and assessing performance, the program can attract and retain
qualified and effective teachers to deliver high-quality instruction to participants. 3.6. Establishment of program pricing When establishing program pricing, it's crucial to consider multiple factors to ensure accessibility,
affordability, and profitability. The team should evaluate program costs, conduct market research, assess
program value, consider the target market, and develop a pricing strategy that aligns with program
objectives. Promotions and discounts can also attract and retain participants. Ultimately, the pricing strategy
should generate enough revenue to cover costs and generate profit to support the program's growth and
sustainability. 3.3. Timeline and milestones 2, Issue 3, December 2023 41 And each class with maximum number of students is 15, the profit will be:
Account
Rate in thousand VND
Revenue
Tuition (on each course) (with 15 students)
300,000
Total of each class (15 students)
300,000
Cost
Variable costs per student
Material
7,500
Copying
7,500
Total variable
15,000
Gross Profit of 15 students class
285,000
Fixed cost
Personnel Costs (Teaching salaries/ benefits)
57,600
Facility Costs
30,000
Marketing and Promotion
35,000
Administrative Costs
10,000
Program Evaluation
5,000
Contingency Budget
19,000
Total fixed cost
156,600
FC
Fixed costs
156,600
CR
Class revenue
300,000
Personnel Costs (Teaching salaries/ benefits)
57,600
Facility Costs
30,000
Marketing and Promotion
35,000
Administrative Costs
10,000
Program Evaluation
5,000
Contingency Budget
19,000
Total fixed cost
156,600
FC
Fixed costs
156,600
Unit Revenue
Unit revenue or price/unit
20,000
Unit VC
Unit variable cost
1,000
GP
Gross Profit
19,000
BE
Break-even point in units
8.2 And each class with maximum number of students is 15, the profit will be:
Account
Rate in thousand VND
Revenue
Tuition (on each course) (with 15 students)
300,000
Total of each class (15 students)
300,000
Cost
Variable costs per student
Material
7,500
Copying
7,500
Total variable
15,000
Gross Profit of 15 students class
285,000
Fixed cost
Personnel Costs (Teaching salaries/ benefits)
57,600
Facility Costs
30,000
Marketing and Promotion
35,000
Administrative Costs
10,000
Program Evaluation
5,000
Contingency Budget
19,000
Total fixed cost
156,600
FC
Fixed costs
156,600
CR
Class revenue
300,000
Personnel Costs (Teaching salaries/ benefits)
57,600
Facility Costs
30,000
Marketing and Promotion
35,000
Administrative Costs
10,000
Program Evaluation
5,000
Contingency Budget
19,000
Total fixed cost
156,600
FC
Fixed costs
156,600
Unit Revenue
Unit revenue or price/unit
20,000
Unit VC
Unit variable cost
1,000
GP
Gross Profit
19,000
BE
Break-even point in units
8.2 42 Thuan Phan Kim CVC
Class variable cost
15,000
GP
Gross Profit
285,000
NP
Net profit
128,400 Class variable cost
Gross Profit
Net profit 4.3. Measuring the effectiveness of marketing strategies To measure the marketing strategies' effectiveness for the new English program, the program development
team can track website traffic, monitor social media engagement, analyze SEM results, track referral
marketing, and monitor enrollment numbers. By analyzing data regularly, the team can evaluate the
strategies and make adjustments as needed to improve the program's visibility and enrollment numbers. The target audience for the new English program: IELTS 4.0 course would be: 4.1. The target audience for the new English program: IELTS 4.0 course would be 4.1. The target audience for the new English program: IELTS 4.0 course would be: 1. High school students who are seeking to graduate from high school without attending the final
English exam. This may include students who struggle with the English language or who have failed
previous English exams. 1. High school students who are seeking to graduate from high school without attending the final
English exam. This may include students who struggle with the English language or who have failed
previous English exams. Journal of Knowledge Learning and Science Technology ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 2023 43 2. Individuals who want to improve their English language proficiency to get a better job or advance
their careers. This may include individuals who work in industries that require strong English
language skills, such as international business, hospitality, and customer service. 2. Individuals who want to improve their English language proficiency to get a better job or advance
their careers. This may include individuals who work in industries that require strong English
language skills, such as international business, hospitality, and customer service. 4.2. Marketing channels used to reach the target audience. B
i
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k ti
h
l
th
d
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t t
i
th
' Events and workshops: The team may host events or workshops to showcase the program's curriculum,
teaching methodology, and outcomes, and to engage with potential participants directly. Events and workshops: The team may host events or workshops to showcase the program's curriculum,
teaching methodology, and outcomes, and to engage with potential participants directly. By using a combination of marketing channels, the program development team can increase the program's
visibility and attract potential participants who are most likely to benefit from the program. By using a combination of marketing channels, the program development team can increase the program's
visibility and attract potential participants who are most likely to benefit from the program. 4.2. Marketing channels used to reach the target audience. To reach the target audience for the new English program: IELTS 4.0 course, the program development
team may consider using a variety of marketing channels to promote the program and attract potential
participants. Here are some examples of marketing channels that may be effective for reaching the target
audience: (Simon Kingsnorth, 2019) Social media advertising: The program development team may use social media advertising on platforms
such as Facebook, TikTok, and YouTube to target potential participants based on their demographics,
interests, and behavior. Search engine marketing (SEM): The team may use search engine marketing to target potential participants
who are actively searching for English language courses or IELTS exam preparation. Referral marketing: The team may encourage current and former program participants to refer friends and
family members to the program in exchange for discounts or other incentives. Public relations (PR): The team may use PR strategies to generate media coverage and build awareness of
the program, such as issuing press releases or pitching stories to local media outlets. Public relations (PR): The team may use PR strategies to generate media coverage and build awareness of
the program, such as issuing press releases or pitching stories to local media outlets. the program, such as issuing press releases or pitching stories to local media outlets. Community partnerships: The team may establish partnerships with local schools, community
organizations, or businesses to promote the program to their networks and members. Events and workshops: The team may host events or workshops to showcase the program's curriculum,
teaching methodology, and outcomes, and to engage with potential participants directly. By using a combination of marketing channels, the program development team can increase the program's
visibility and attract potential participants who are most likely to benefit from the program. Community partnerships: The team may establish partnerships with local schools, community
organizations, or businesses to promote the program to their networks and members. Community partnerships: The team may establish partnerships with local schools, community
organizations, or businesses to promote the program to their networks and members. Events and workshops: The team may host events or workshops to showcase the program's curriculum,
teaching methodology, and outcomes, and to engage with potential participants directly. 5. Accountability Here are some of the important positions at a language center and their duties: (Peter Sommerville, 2023)
Director of the Language Center: The Director of the Language Center oversees the overall management
of the center. He is responsible for developing and implementing the center's policies and procedures,
overseeing budget management, and ensuring that the center is meeting its objectives and goals. Academic Director: The Academic Director is responsible for developing and overseeing the center's
academic programs, including curriculum development, teacher training, and evaluation. They ensure that
the center's academic programs meet industry standards and that the teaching methodology is effective. Operations Manager: The Operations Manager oversees the daily operations of the center, including
managing administrative staff, scheduling classes, and managing the facility. They ensure that the center
runs smoothly and efficiently. Here are some of the important positions at a language center and their duties: (Peter Sommerville, 2023)
Director of the Language Center: The Director of the Language Center oversees the overall management
of the center. He is responsible for developing and implementing the center's policies and procedures,
overseeing budget management, and ensuring that the center is meeting its objectives and goals. Academic Director: The Academic Director is responsible for developing and overseeing the center's
academic programs, including curriculum development, teacher training, and evaluation. They ensure that
the center's academic programs meet industry standards and that the teaching methodology is effective. Academic Director: The Academic Director is responsible for developing and overseeing the center's
academic programs, including curriculum development, teacher training, and evaluation. They ensure that
the center's academic programs meet industry standards and that the teaching methodology is effective. Operations Manager: The Operations Manager oversees the daily operations of the center, including
managing administrative staff, scheduling classes, and managing the facility. They ensure that the center
runs smoothly and efficiently. Thuan Phan Kim 44 Course Coordinator: The Course Coordinator is responsible for coordinating the center's courses and
ensuring that they are running effectively. They work closely with teachers to ensure that course content
and delivery meet the center's objectives and standards. Language Teachers: Language Teachers are responsible for delivering high-quality language instruction to
students. They develop lesson plans, deliver lectures, and provide feedback to students. They also evaluate
students' progress and adjust lesson plans as needed. 6. Results and Feedback Enrollment numbers and revenue generated are two key indicators of the success of a language program. However, success should not be measured solely by these numbers, as they can be influenced by various
factors such as program pricing, competition, and marketing efforts. Enrollment numbers and revenue generated are two key indicators of the success of a language program. However, success should not be measured solely by these numbers, as they can be influenced by various
factors such as program pricing, competition, and marketing efforts. To determine the success of the program, it is important to consider other factors such as student satisfaction,
teacher satisfaction, student outcomes, and program reputation. To determine the success of the program, it is important to consider other factors such as student satisfaction,
teacher satisfaction, student outcomes, and program reputation. Student satisfaction can be measured through surveys and feedback forms, which can provide information
about how students perceive the program, their overall experience, and areas for improvement. Student satisfaction can be measured through surveys and feedback forms, which can provide information
about how students perceive the program, their overall experience, and areas for improvement. Teacher satisfaction can be measured through teacher evaluations, which can provide information about
how teachers perceive the program, their level of engagement, and their satisfaction with their roles and
responsibilities. Teacher satisfaction can be measured through teacher evaluations, which can provide information about
how teachers perceive the program, their level of engagement, and their satisfaction with their roles and
responsibilities. Student outcomes can be measured through assessments, evaluations, and graduation rates. These can
provide information about how well students are learning and achieving their goals. Student outcomes can be measured through assessments, evaluations, and graduation rates. These can
provide information about how well students are learning and achieving their goals. Program reputation can be measured through word-of-mouth recommendations, online reviews, and other
forms of feedback from students and stakeholders. This can provide insight into how the program is
perceived in the community and its overall reputation. By considering these factors in addition to enrollment numbers and revenue generated, the language centre
can determine the overall success of the program and make informed decisions about how to improve and
grow the program over time. 5. Accountability Administrative Staff: Administrative staff members provide support to the center, including managing
registrations and payments, answering phone calls, messages, comments and emails, and managing the
center's database. Each of these positions plays an important role in the success of a language center, and they work together
to ensure that the center is providing high-quality language instruction and meeting the needs of its students. [1] Mike McKeever (2011). How to Write a Business Plan 10th Edition. Delta Printing Solutions, Inc.
[2] Nick Kenny and Bruce Rogers (2015). The Complete Guide To IELTS Student's Book. Cengage
Learning.
[3] Peter Sommerville (2023). Managing Teacher Quality and Training: A Guide for Language School
Managers (e-book). Oxford, UK.
[4] Simon Kingsnorth (2019). Digital Marketing Strategy. Kogan Page Limited. 8. Conclusion The report on the new English program, IELTS 4.0, for students who want to graduate from high school
without attending the final English exam and other students who want to improve their English to get a
better job, found that there is a significant demand for such a program in the market. Through market
research, the target audience's preferences and needs were identified, which informed the program's
objectives and curriculum. The report outlined the timeline and milestones for the program's development, as well as the budget and
resources required for its implementation. The recruitment and training of teachers for the program were
also discussed, as were the program's pricing strategies. In terms of marketing, the report recommended various channels for reaching the target audience, including
social media, email campaigns, and advertising. The importance of monitoring and evaluating the program's
effectiveness was emphasized, and several plans for future improvements and adjustments were proposed,
including curriculum revisions, teacher training and development, and enhanced student support. The report also highlighted the importance of establishing monitoring and evaluation systems to assess the
program's effectiveness and identify areas for improvement. Overall, the report found that the IELTS 4.0 In terms of marketing, the report recommended various channels for reaching the target audience, including
social media, email campaigns, and advertising. The importance of monitoring and evaluating the program's
effectiveness was emphasized, and several plans for future improvements and adjustments were proposed,
including curriculum revisions, teacher training and development, and enhanced student support. The report also highlighted the importance of establishing monitoring and evaluation systems to assess the
program's effectiveness and identify areas for improvement. Overall, the report found that the IELTS 4.0
program has the potential to be a successful and profitable venture for the language center, given the high
demand for such a program in the market. 7. Monitoring & evaluation systems Monitoring and evaluation systems are essential for assessing the effectiveness of the English program and
identifying areas for improvement. Some key elements of a monitoring and evaluation system for the
program above include: Student and teacher feedback: Collecting feedback from students and teachers through surveys, evaluations,
and other feedback mechanisms can provide valuable insights into the program's effectiveness, areas for
improvement, and overall satisfaction. Assessment data: Monitoring and analyzing student assessment data, such as test scores and graduation
rates, can provide insight into student outcomes and identify areas where the program may need
improvement. Journal of Knowledge Learning and Science Technology ISSN: 2959-6386 (Online), Vol. 2, Issue 3, December 2023 45 Program metrics: Tracking program metrics, such as enrollment numbers, revenue generated, and
marketing effectiveness, can provide data-driven insights into the program's performance and areas for
improvement. Program metrics: Tracking program metrics, such as enrollment numbers, revenue generated, and
marketing effectiveness, can provide data-driven insights into the program's performance and areas for
improvement. Performance indicators: Establishing performance indicators, such as student satisfaction rates, teacher
engagement levels, and student outcomes, can help to measure the program's success over time and provide
a basis for ongoing improvement efforts. Regular program reviews: Conducting regular program reviews, including external evaluations and internal
assessments, can provide a comprehensive overview of the program's performance and help to identify areas
for improvement. By establishing a robust monitoring and evaluation system that incorporates feedback from students,
teachers, and other stakeholders, the language center can ensure the program remains relevant and effective
over time and make data-driven decisions about program improvements and adjustments.
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Development and Pilot Evaluation of an Online Retrofit Decision-Making Tool for Homeowners
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SEDDIKI, M., BENNADJI, A., ALABID, J., GRAY, D. and DEVECI, G. 2022. Development and pilot evaluation of an online
retrofit decision-making tool for homeowners. Buildings [online], 12(10), article 1513. Available from:
https://doi.org/10.3390/buildings12101513 SEDDIKI, M., BENNADJI, A., ALABID, J., GRAY, D. and DEVECI, G. 2022. Development and pilot evaluation of an online
retrofit decision-making tool for homeowners. Buildings [online], 12(10), article 1513. Available from:
https://doi.org/10.3390/buildings12101513 SEDDIKI, M., BENNADJI, A., ALABID, J., GRAY, D. and DEVECI, G. 2022. Development and pilot evaluation of an online
retrofit decision-making tool for homeowners. Buildings [online], 12(10), article 1513. Available from:
https://doi.org/10.3390/buildings12101513 Citation: Seddiki, M.; Bennadji, A.;
Alabid, J.; Gray, D.; Deveci, G. Development and Pilot Evaluation of
an Online Retrofit Decision-Making
Tool for Homeowners. Buildings 2022,
12, 1513. https://doi.org/10.3390/
buildings12101513 Keywords: decision-making; online tool; retrofit; pilot evaluation; homeowners; moisture-safe Academic Editor: Antonio Caggiano Article Mohammed Seddiki 1,*, Amar Bennadji 1, Jamal Alabid 2, David Gray 3 and Gokay Deveci 1 1
Scott Sutherland School of Architecture and Built Environment, Robert Gordon University, Garthdee House,
Garthdee Road, Aberdeen AB10 7QB, UK 1
Scott Sutherland School of Architecture and Built Environment, Robert Gordon University, Garthdee House
Garthdee Road, Aberdeen AB10 7QB, UK 2
Faculty of Engineering & Informatics, University of Bradford, Bradfor BD7 1DP, UK
3
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AB10 7QB UK 2
Faculty of Engineering & Informatics, University of Bradford, Bradfor BD7 1DP, UK
3
Cultural and Creative Business School, Robert Gordon University, Aberdeen AB10 7QB, UK
*
Correspondence: m.seddiki1@rgu.ac.uk Abstract: Many retrofit projects went wrong in the UK principally because of the application of
inappropriate retrofit solutions, which resulted in damp issues, with some leaving houses in worse
conditions than pre-retrofit. Various online tools were developed to inform homeowners about
the benefits of retrofitting. Prior to this study, little was known about users’ evaluation of these
tools and the effects of calculator use. Furthermore, no retrofit tool aims to raise the awareness of
homeowners about moisture risks in a retrofit project. The originality of this study is to develop
and evaluate an online moisture-safe retrofit decision-making tool for homeowners. The adopted
methodology consisted of two phases. Phase one aimed to develop the tool. In phase two, semi-
structured interviews were conducted to evaluate the tool. The results indicate that the tool has
been well received by homeowners. The tool significantly increased participants’ awareness of
moisture risks related to a retrofit project. Most participants considered the tool an eye-opener,
while few of them found it scary. However, the tool did not result in an increased willingness to
invest in energy efficiency measures. The discouragement was related to high investment costs
and long payback periods of some retrofit measures. Based on our findings, we formulate a set
of design recommendations to improve the proposed tool and help retrofit calculators, in general,
overcome challenges. Development and pilot evaluation of an online
retrofit decision-making tool for homeowners. 2022 © 2022 by the authors. Licensee MDPI, Basel, Switzerland. © 2022 by the authors. Licensee MDPI, Basel, Switzerland. © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This document was downloaded from
https://openair.rgu.ac.uk buildings buildings buildings 1. Introduction Several retrofit tools are available in the UK to help homeowners understand how
their houses and flats use energy and how their home’s energy is used. The identified tools
in the UK are “Home Energy Saving” tool developed by Halifax (part of Lloyds Banking
Group) [9], “Energy Efficiency calculator” developed by the UK government [10], “Home
Energy Check tool” developed by home energy Scotland [11], “AECB Home Energy Check”
developed by AECB—the sustainable building association [12], “Quick Scan tool” devel-
oped by the city of Aberdeen (Scotland) [13], and “Reducing home heat loss” developed by
energy saving trust [14]. Another tool available for England that is worth mentioning even
though it is not destinated for homeowners and targets professionals is the TABULA tool
which was developed as part of the European project EPISCOPE [15]. Most available tools for homeowners evaluate retrofit options by taking into consid-
eration energetic, financial, and environmental criteria, while other key characteristics
that need to be considered when selecting an appropriate package of improvements are
neglected, such as the disruptions caused by the renovation work or more importantly the
risks related to moisture after a retrofitting work [6]. In fact, the perceived hassle of installation has been found to be one of the factors influ-
encing house owners’ preferences on energy retrofits [16]. As indicated in Glew et al. [17],
disruption is an important indicator because it can be a serious barrier to the implementa-
tion of retrofit measures, especially if the property cannot be used while the retrofit work is
carried out. Another crucial aspect is the moisture risk in retrofitting projects. Many homeowners
are reluctant to undertake renovation works, particularly in traditional houses, because
they are afraid that this may cause damp and mould growth issues in the building [18]
which would negatively affect the value of the building, the energy performance, and
the health of occupants. These worries are sustained by the fact that during the last
decade, a large number of retrofit projects went wrong in the UK mainly because of the
application of inappropriate retrofit solutions which resulted in damp and mould growth
issues leaving homeowners with houses in a worse condition than pre-retrofit [19]. 1. Introduction In 2020, the UK residential sector emitted 67.7 MtCO2, accounting for 20.8% of all
carbon dioxide emissions [1]. UK homes’ greenhouse gas emissions need to fall by at least
24% by 2030 from 1990 levels to meet the UK’s ambitious goal, which is reaching zero
emissions from existing buildings in 2050 [2]. The current rate of retrofitting progress is
around 9000 measures installed per week across the whole UK housing stock and needs to
rise by around seven times to meet its climate targets. The UK has the ambition to retrofit
almost all homes to a minimum of Energy Performance Certificate (EPC) band C standard
by 2035 [3]. Published: 23 September 2022 Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. y
Researchers have found that insufficient support measures hinder homeowners from
investing in energy renovations and recommended states to identify a set of support
measures that is appropriate to homeowners by involving them in the preparation and
implementation of their strategies [4]. It has been found that information on the capital
cost of energy efficiency measures and the amount of energy savings is one of the strongest
incentives to encourage homeowners to invest in energy renovation [5]. 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
gy
There have been several projects that aimed to develop simple energy tools to assist
homeowners in their renovation projects by providing them with information on energy
improvement solutions suitable for their homes. Seddiki et al. [6] provided a comprehensive https://www.mdpi.com/journal/buildings Buildings 2022, 12, 1513. https://doi.org/10.3390/buildings12101513 Buildings 2022, 12, 1513 2 of 18 literature review on existing energy retrofit web decision tools available for homeowners. For instance, Request2Action, which is a European Union Intelligent Energy programme
funded project, developed a web-based tool named Home Energy Check (HEC) that aims
to familiarise the user with the different available energy improvement solutions and to
highlight the implementation of EPCs recommendations and energy savings achieved [7]. Elsewhere, the city of Ghent in Belgium developed a user-friendly web application,
“Check Je Huis”, that guides homeowners/tenants in their renovation projects. The appli-
cation helps users to identify the energy efficiency of their home and the possibilities of
improving them [8]. 1. Introduction Not
only does this undermine the UK target to meet net zero by 2050, but it also costs a fortune
to remove the applied measures and redo the work for already vulnerable householders
having generally to rectify problems themselves, not to mention the disturbance, stress,
and often health issues caused to homeowners. In fact, damp and mould development due
to poorly designed and installed retrofit measures are likely to cause respiratory problems,
respiratory infections, allergies, or asthma [20]. A report produced by the UK Office of Gas
and Electricity Markets (Ofgem’s) indicated that 6.9% of the almost 1.5 million measures
installed in the UK between January 2013 and March 2015 were inspected, and 9.9% of
the examined measures haven’t been installed correctly in the first instance and required
additional work to be undertaken [21]. After the observation that many retrofit projects failed, the Secretaries of State of the
Department for Energy and Climate Change (DECC-now BEIS) and the Department for
Communities and Local Government (DCLG-now MHCLG) commissioned an industry-led
report “Each Home Counts review” [21]. The aims of the review were to restore the confi-
dence of consumers and of Government in the retrofit industry in order to facilitate new
central and local government policies to promote and support a national programme of
domestic retrofit. The recommendation made in the report “Each Home Counts review” led Buildings 2022, 12, 1513 3 of 18 3 of 18 the UK government to introduce the PAS2035 standard, which provides a comprehensive
methodology on how to perform a retrofit project. The standard introduced a new actor,
namely the retrofit coordinator, which is responsible for overseeing the assessment of
dwellings, elaborating a risk assessment and management plan, as well as the subsequent
specification, monitoring, and evaluation of energy efficiency measures, in accordance with
PAS 2035. The retrofit coordinator should have an adequate training to assess moisture risks
related to the implementation of retrofit measures which is anticipated to prevent catas-
trophic situations. However, PAS 2035 is currently compulsory only for public buildings,
while for private buildings, landlords and homeowners could install retrofitting measures
simply on the basis of an assessment of installers or domestic energy assessors which are
not qualified to assess moisture risks and related issues in retrofit measures [22]. q
Hence, including moisture risks assessment in an energy retrofit online tool for home-
owners presents various advantages. 1. Introduction On one hand, this could reassure reluctant homeown-
ers that are afraid of moisture issues on the feasibility of managing the risks and would
likely encourage them to invest in retrofit measures. On the other hand, for homeowners
that are not aware of moisture risks, the online tool would prevent them from implementing
risky solutions recommended by a retrofit assessor or an installer and would raise their
awareness on moisture risks. This paper extends the current knowledge by including moisture risks in an online
energy retrofit tool for homeowners. In contrast, existing tools evaluate retrofit options by
considering only energy, financial, and environmental criteria. Furthermore, little is known
about the (positive and negative, intended and unintended) effects retrofit calculators
may have on the awareness and the willingness to pursue renovations of their users. The
proposed paper extends existing findings by shedding light on the experiences of retrofit
calculator users. The paper focuses on the (1) users’ acceptance of and satisfaction with the
developed retrofit calculator, (2) effects of the tool on moisture awareness and behaviour,
and (3) effects of the tool on the willingness to invest in energy efficiency. 2.2. Creation and Validation of Energy Models of Building Typologies Energy models were developed using the Standard Assessment Procedure (SAP),
which is the methodology used by the UK government to assess and compare the energy
and environmental performance of dwellings. SAP quantifies a dwelling’s performance
in terms of energy use per unit floor area, a fuel-cost-based energy efficiency rating (the
SAP Rating) and emissions of CO2 (the Environmental Impact Rating). Other SAP outputs
include space heating demand, total delivered energy, cooling load, and so on. PAS 2035
recommends using Full SAP for retrofit assessments [22]. The assumptions regarding
building typologies can be found in Bennadji et al. [23]. g yp
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j
[
]
To validate the energy models, the calculated energy efficiency ratings (the SAP Rating)
of the building typologies were compared with the median energy efficiency rating from
statistics as it is the only indicator available for building typologies in the four UK countries. Moreover, it was not possible to compare the SAP rating of the 20 building types
separately as the data available in the statistics indicates median SAP ratings for different
UK countries either per dwelling type or per dwelling age, and there are no data that
combines these two factors in a matrix of typologies. Median SAP ratings were retrieved
from the office of national statistics for England and Wales [24], the Scottish house condition
survey 2019 [25], and the Northern Ireland house condition survey 2016 [26]. g yp
g
j
To validate the energy models, the calculated energy efficiency ratings (the SAP Rating)
of the building typologies were compared with the median energy efficiency rating from
statistics as it is the only indicator available for building typologies in the four UK countries. Moreover, it was not possible to compare the SAP rating of the 20 building types
l
h d
l bl
h
d
d
f
d ff To validate the energy models, the calculated energy efficiency ratings (the SAP Rating)
of the building typologies were compared with the median energy efficiency rating from
statistics as it is the only indicator available for building typologies in the four UK countries. Moreover, it was not possible to compare the SAP rating of the 20 building types
separately as the data available in the statistics indicates median SAP ratings for different
UK countries either per dwelling type or per dwelling age, and there are no data that
combines these two factors in a matrix of typologies. 2. Materials and Methods As indicated in Seddiki et al. [6], four major methodologies have been adopted in
developing energy retrofit tools for homeowners; empirical data-driven methods, pre-
simulated databases, simplified normative calculation methods, and advanced calculation
methods. In this paper, the tool development was based on a database of multicriteria
assessments carried out for 20 building typologies representative of the UK housing stock. In a pre-simulated database, the energy performance of several combinations of building
envelope, HVAC systems, and renewable energy integration is predicted using detailed
numerical models of representative buildings. The choice to base the developed moisture-
safe tool on a pre-simulated database is motivated by the fact that this approach allows the
creation of extremely easy to use tools as they require few inputs and are highly accurate as
simulations are performed in advanced simulation engines. The methodology used to create the tool consists of different steps (Figure 1): First,
building typologies representative of the UK housing stock were defined, then the energy
model of each building typology was created and validated against statistical data. Then,
individual retrofit measures as well as packages of solutions were generated. Afterward,
the retrofitting measures were evaluated according to four criteria: energy saving, capital
cost, disruption level, and moisture risks. Then, the results of the multicriteria assessment
were compiled into a user-friendly online tool. Finally, a pilot evaluation was performed
to collect feedback about the tool and requirements for user interface design. A detailed
explanation of the different steps is presented below:
VIEW
4 of 18 Fi
1 Th
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d t
t th
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l
Definition of
building
typologies
Reference
building
models and
validation
Generation of
retrofit
measures
Multicriteria
evaluation
Prototype
website
development
Pilot
evaluation
Figure 1. The methodology used to create the online tool. Figure 1. The methodology used to create the online tool. Buildings 2022, 12, 1513 4 of 18 2.1. Definition of Building Typologies 2.1. Definition of Building Typologies The first step in developing the moisture-safe energy calculator, which is based on a pre-
simulated database, is to define building typologies representative of the UK housing stock. The number of reference buildings is directly related to the accuracy of the data generated
by the online tool for homeowners; the greater the number of reference buildings is, the
more accurate the results are. However, increasing the number of building typologies makes
the development of the tool extremely time-consuming, which is an inherent limitation of
the pre-simulated database approach. Hence, a compromise should be reached between
the number of simulations and the accuracy of the data provided to homeowners. The
reference buildings developed by Bennadji et al. [23] as part of the European project
Stronghouse and based on the European project EPISCOPE [15] were adopted in this
paper to represent the housing stock of the UK for their geometric data, construction, and
energy-related properties. gy
p
p
This study considered 20 building typologies split into five construction periods
(pre–1919, 1919–1944, 1945–1964, 1965–1980, post–1980) and four building sizes, including
single-family house (SFH), terraced house (TH), multi-family house (MFH), and apartment
block (AB). The assumptions regarding building typologies in Bennadji et al. [23] took
into consideration the current condition of the housing stock as opposed to the EPISCOPE
project, where the energy efficiency characteristics of building typologies have been defined
for un-modernised conditions. 2.4. Multicriteria Assessment of Renovation Alternatives To provide users with sufficient information to help them proceed in their renovation
journey, individual retrofit measures and packages of solutions were evaluated according
to the various criteria indicated in PAS 2035. A brief description of the evaluation criteria
recommended by PAS 2035 is presented as follows. 2.3. Generation of Retrofit Measures The generated retrofit measures took into consideration three areas of improvement,
which were the building envelope, the building services, and renewable energies. The
measures targeted a level close to nearly zero building energy requirements, following the
concept that if a retrofit measure is implemented, it must be highly energy efficient to avoid
missed opportunities, especially in a step-by-step retrofit which represents about 80% of
retrofits in reality [28]. In fact, a building that starts with a shallow retrofit will likely never
achieve a high level of energy performance in the future, which is called “the locked in
effect” [29]. Retrofit measures were evaluated individually and in packages to take into
consideration the integrated effect. The saving attributable to a package of improvement
measures will be less than the sum of the savings attributable to individual measures. For each housing type, one pre-retrofit simulation was performed, between eleven
(for single-family and terraced houses) and five (multifamily houses and apartment blocks)
single Energy Efficiency Measures (EEMs), and one application of all EEMs for a total of
about 157 simulations. The list of the selected energy improvement measures included
exclusively measures that are commonly implemented in UK retrofitting projects. The retrofitting measures were customised to each building typology. For example, an
ASHP was simulated for single-family and terraced houses, while a new efficient boiler
was simulated for multifamily houses and apartment blocks. The general assumptions of
the retrofit measures and the energy simulations are detailed in Supplementary Material S1
(General assumptions of the retrofit measures). 2.2. Creation and Validation of Energy Models of Building Typologies Median SAP ratings were retrieved
from the office of national statistics for England and Wales [24], the Scottish house condition
survey 2019 [25], and the Northern Ireland house condition survey 2016 [26]. Therefore, to allow the comparison of the calculated data and the statistics, median
energy efficiency scores were calculated per dwelling type and per dwelling age. So, for
example, the median calculated SAP score for SFH represents the average calculated SAP
scores of 5 building types (SFH Pre–1919, SFH 1919–1944, SFH 1945–1964, SFH 1965–1980,
and SFH Post–1980). Similarly, the median calculated SAP score for buildings built pre–1919
represents the average calculated SAP scores of 4 building types (SFH Pre–1919, TH
Pre–1919, MFH Pre–1919, and AB Pre–1919). The comparison of median SAP scores from statistics and calculated median SAP
scores is presented in Table 1. The result of the comparison reveals an overestimation of
about ±10 EPC points, which is considered acceptable as indicated in Crawley et al. [27]
which compared values from the UK national database of all registered EPCs for properties
that have had more than one EPC. Buildings 2022, 12, 1513 5 of 18 Table 1. Comparison of median SAP rating from statistics and calculated median SAP rating. Table 1. Comparison of median SAP rating from statistics and calculated median SAP rating. Median Energy Efficiency Scores (MEES) from Statistics
Calculated MEES
Dwelling Type
England
Scotland
Wales
Northern Ireland
UK
UK
Difference MEES
SFH
63
61
62
65
62.75
68
5.25
TH
65
65
62
68
65
69
4
MFH
72
67
58
74
67.75
73
5.25
AB
72
68
70
77
71.75
74
2.25
Dwelling Age
Pre–1919
57
56
55
52
55
66
11
1919–1944
57
62
59
61
59.75
70
10.25
1945–1964
64
64
62
64
63.5
70
6.5
1965–1980
63
66
64
67
65
71
6
Post–1980
71
73
73
72
72.25
77
4.75 2.4.3. The Simple Payback Period A simple payback period (in years) is the capital cost of the EEM or a package of EEMs
divided by the estimated annual fuel cost savings [22]. 2.4.2. Capital Cost The capital cost took into consideration only the cash flow required to execute the
renovation work (Gross Capital Cost), while concepts such as the ongoing need for capital
upgrades not related to energy efficiency, the avoided capital costs, the cost of savings
through design, and the cost of subsidies and incentives were not considered. Assumptions
for the capital costs of retrofit solutions were retrieved from the latest updated assumptions
published by the UK Department of Energy & Climate Change about the costs of different
energy efficiency interventions in existing homes [30], from the cost analysis report of the
Retrofit for the Future programme [31] and from other sources [32,33]. 2.4.1. Annual Energy Cost Saving The annual energy cost savings of the retrofit measures were evaluated using the
SAP method. PAS 2035 recommends using Full SAP for retrofit assessments [22]. Retrofit
measures were evaluated individually and in packages to take into consideration the
integrated effect. The fuel costs embedded in the SAP energy rating were used and the
results for each building typology were expressed in GBP/year. Buildings 2022, 12, 1513 6 of 18 2.4.4. Disruption Level The level of disruption to occupants has been recently introduced by the UK standard
PAS 2035 as well as the retrofit academy in the retrofit coordinator course as a key element in
the evaluation of retrofit solutions [34]. Other scientific studies highlighted the importance
of inconvenience and disruption during retrofit activities on the decision of households to
invest in energy renovation [35]. Issues such as the duration of the work and the incapacity
to occupy the building for a prolonged period are important elements of households’
decision process along with other aspects such as cost and energy savings. Hence, it is
important that occupants have a clear idea about how the retrofit work will affect their use
of the building. Assumptions regarding the level of disruption of each renovation measures
were expressed in a qualitative manner. The score of renovations measures were taken
from the work carried out by the experts of the retrofit academy (see Table 2). Table 2. Disruption levels [34]. Disruption
Actions
Minimal
Low-energy lamps, energy-efficient appliances
Low
Heating control, cavity wall insulation, draught stripping, loft insulation
Moderate
Replacement boiler, solar water heating, solar PV panels
High
Replacement windows, whole house ventilation, external wall insulation
Significant
Ground floor insulation, internal wall insulation, new heating installation Table 2. Disruption levels [34]. Disruption
Actions
Minimal
Low-energy lamps, energy-efficient appliances
Low
Heating control, cavity wall insulation, draught stripping, loft insulation
Moderate
Replacement boiler, solar water heating, solar PV panels
High
Replacement windows, whole house ventilation, external wall insulation
Significant
Ground floor insulation, internal wall insulation, new heating installation Table 2. Disruption levels [34]. 1.
Green icon: means that the solution does not present a risk of moisture. 2.5. Prototype Website Development
2.5. Prototype Website Development The aim of this step is to deploy the pre-simulated retrofit data on a prototype dy-
namic website. The content management system Wix was used to developing the Portal. Content management systems such as Wix and WordPress have been widely used in the
literature to develop prototype websites. For example, Ali et al. [37] used WordPress for
the development of a custom app radiator control system for retrofitting legacy heating
systems in existing buildings. Similarly, Carvalho et al. [38] used Wordpress to create a
web portal for COVID-19 information in Brazil, which helps users publish, organise, and
manage content posted on the web. Merritt & Zhao [39] used WIX in order to develop a
website for a prototype supermarket. Authors argued that this method offers an intuitive
drag and drop site building which can outline the visual aspects of the site. The aim of this step is to deploy the pre-simulated retrofit data on a prototype dy-
namic website. The content management system Wix was used to developing the Portal. Content management systems such as Wix and WordPress have been widely used in the
literature to develop prototype websites. For example, Ali et al., [37] used WordPress for
the development of a custom app radiator control system for retrofitting legacy heating
systems in existing buildings. Similarly, Carvalho et al., [38] used Wordpress to create a
web portal for COVID-19 information in Brazil, which helps users publish, organise, and
manage content posted on the web. Merritt & Zhao [39] used WIX in order to develop a
website for a prototype supermarket. Authors argued that this method offers an intuitive
drag and drop site building which can outline the visual aspects of the site. The website front end of the proposed prototype in the paper was developed using
the free and code-free website builder Wix. The purpose of selecting this method was that
it is an easy-to-use editor, the system is intuitive, accessibility is built into the system, and
the drag and drop functions make editing pages or updating content fairly straightforward
(Figure 2). The website front end of the proposed prototype in the paper was developed using
the free and code-free website builder Wix. 2.4.5. Moisture Risks As mentioned previously, one of the main contributions of this paper is to include an
assessment of each suggested retrofit solution in terms of moisture risks. Moisture safe design does not mean that the risks of moisture are all eliminated; it only
means that the causes and consequences of moisture are understood, and that appropriate
actions are taken to reduce these risks. Often there will be conflicts between the different
objectives (energy saving, moisture, etc.). Therefore, a compromise should be reached,
and measures to manage the accepted risks should be installed. In some extreme cases,
it is even recommended to not insulate because of the high risk of moisture. Moisture
risks can be evaluated using dynamic hygrothermal simulation (WUFI), condensation risk
analysis (Glaser method), calculation of the properties of thermal bridges and prescriptive
guidance [34]. In this paper moisture risks of each solution were evaluated using the
UK perspective guidance “Management of moisture in buildings–Code of practice” BS
5020 [36]. For each retrofit solution, the tool presents the results of moisture risks using
coloured icons: 1. Green icon: means that the solution does not present a risk of moisture. 1. Green icon: means that the solution does not present a risk of moisture. Buildings 2022, 12, 1513 7 of 18
e risks 7 of 18
e risks 2. Red Icon: means there is a risk, but the situation may not be serious. It is advisable to
further investigate the solution by numerical simulations. 2. Red Icon: means there is a risk, but the situation may not be serious. It is advisable to
further investigate the solution by numerical simulations. h
l
f
h
f
l
f
k
d h
f The evaluation of each retrofit solution in terms of moisture risks and the information
displayed for the user are detailed in Supplementary Material S2 (moisture risks). The evaluation of each retrofit solution in terms of moisture risks and the information
displayed for the user are detailed in Supplementary Material S2 (moisture risks). 2.5. Prototype Website Development
2.5. Prototype Website Development The purpose of selecting this method was that
it is an easy-to-use editor, the system is intuitive, accessibility is built into the system, and
the drag and drop functions make editing pages or updating content fairly straightfor-
ward (Figure 2). Figure 2. Wix drag-and-drop functionality. Figure 2. Wix drag-and-drop functionality. The website back end was developed using the content manager Wix functionality. Content collections were created to store information about retrofit measures. The content
is stored in a grid layout made up of items (rows) and fields (columns) (Figure 3). 2.6. Pilot Evaluation
2.6. Pilot Evaluation The pilot study involved semi-structured interviews with homeowners that were
asked to test the online tool before the interviews. The objectives of the pilot evaluation
were to investigate (1) users’ acceptance of and satisfaction with the developed retrofit
calculator, (2) effects of the tool on moisture awareness and behaviour, and (3) effects of
the tool on the willingness to invest in energy efficiency. The pilot also aimed to collect
feedback about the tool and requirements for user interface design. The pilot study involved semi-structured interviews with homeowners that were
asked to test the online tool before the interviews. The objectives of the pilot evaluation
were to investigate (1) users’ acceptance of and satisfaction with the developed retrofit
calculator, (2) effects of the tool on moisture awareness and behaviour, and (3) effects of
the tool on the willingness to invest in energy efficiency. The pilot also aimed to collect
feedback about the tool and requirements for user interface design. q
g
Semi-structured interviews have been wildly used in the literature to test pilot new
web-based tools [40–42]. The main advantage of conducting interviews is that they pro-
vide in-depth information, as they allow the interviewer to expose information regarding
the online tool that are unlikely to be captured through questionnaires [43]. Semi-structured interviews have been wildly used in the literature to test pilot new
web-based tools [40–42]. The main advantage of conducting interviews is that they provide
in-depth information, as they allow the interviewer to expose information regarding the
online tool that are unlikely to be captured through questionnaires [43]. Figure 2. Wix drag-and-drop functionality. Figure 2. Wix drag-and-drop functionality. The website back end was developed using the content manager Wix functionality. Content collections were created to store information about retrofit measures. The content
is stored in a grid layout made up of items (rows) and fields (columns) (Figure 3). Buildings 2022, 12, 1513 8 of 18
nality. ontent Figure 3. Data entered into Wix Content Manager. Figure 3. Data entered into Wix Content Manager. Figure 3. Data entered into Wix Content Manager. Figure 3. Data entered into Wix Content Manager. 2.6.1. Participants
2.6.1. Participants p
The potential testers were recruited through a snowball sampling method, which
means the author identified a few testers through convenience sampling who then indi-
cated the contact information of other potential testers and so forth. Eligible participants
(i) were homeowners; (ii) were located in the UK; (ⅲ) were able to read English profi-
ciently, and (ⅳ) were able to give informed consent. A number of studies in the literature
have successfully used snowball sampling in order to pilot online tools [43–45]. From May
2022 to July 2022, a sum of 15 homeowners were interviewed. The participants were aged
between 30 to 65 years and had a broad range of educational backgrounds. Their proper-
ties were built from pre 1919 to post 1980
The potential testers were recruited through a snowball sampling method, which
means the author identified a few testers through convenience sampling who then indi-
cated the contact information of other potential testers and so forth. Eligible participants
(i) were homeowners; (ii) were located in the UK; (iii) were able to read English proficiently,
and (iv) were able to give informed consent. A number of studies in the literature have
successfully used snowball sampling in order to pilot online tools [43–45]. From May 2022
to July 2022, a sum of 15 homeowners were interviewed. The participants were aged
between 30 to 65 years and had a broad range of educational backgrounds. Their properties
were built from pre–1919 to post–1980. 2 6 2 Data Collectio
a d A alysis
2.6.2. Data Collection and Analysis 2.6.2. Data Collection and Analysis
Participants were contacted via email and provided with the following information:
(1) an information letter outlining study details and requirements of participants; (2) a link
to the web-based tool (https://mseddiki1.wixsite.com/my-site); and (3) a consent form. The
interview questions included: “Was the tool easy to use?” and “What did you think of the
Participants were contacted via email and provided with the following information:
(1) an information letter outlining study details and requirements of participants; (2) a link
to the web-based tool (https://mseddiki1.wixsite.com/my-site); and (3) a consent form. The interview questions included: “Was the tool easy to use?” and “What did you think
of the amount of information in the tool?” Participants were also asked what they liked
or disliked about the website, how it could be improved, and how the tool affected their
understanding of energy improvement options and moisture risks in a retrofit project. Buildings 2022, 12, 1513 9 of 18
ners w 9 of 18
ners w Supplementary Material S3 contains the interview guide used in this research. To
verify the guideline for the interview, pilot interviews of two homeowners were carried out
before the formal semi-structured interview (the pilot interviews were not included in the
sum of 15 respondents). The results of the pilot interviews helped to improve the guideline
for the interview. i
e u
o
e po
e
)
e e u
o
e pi o i
e
ie
e pe
o i
guideline for the interview. Interviews were conducted and recorded using Zoom and transcribed into
documents. Interviews lasted between 30 and 45 minutes. Content analysis usi
ware NVivo has been adopted to analyse the qualitative data from interview Interviews were conducted and recorded using Zoom and transcribed into electronic
documents. Interviews lasted between 30 and 45 min. Content analysis using the software
NVivo has been adopted to analyse the qualitative data from interview transcripts. Content
analysis has been commonly used in piloting of tool studies [43,46,47]. Content analysis was
chosen as it allows a descriptive approach in both coding of the data and its interpretation
of quantitative counts of the codes [48]. Content analysis has been commonly used in piloting of tool studies [43,46,47
analysis was chosen as it allows a descriptive approach in both coding of the d
interpretation of quantitative counts of the codes [48]. 3. Prototype Demonstration 3. Prototype Demonstration
The user interface is
As homeowners are unlik The user interface is crucial, as it is the connection between a person and the system. As homeowners are unlikely to be knowledgeable about moisture risks inherent in retrofit
projects, a simple web-based moisture-safe retrofit decision-making tool has been developed
for them. Some of the interface forms are shown and explained below. Additional details
are available on the tool’s website [49]. The tool interface and procedure for its operation
involve three basic steps:
y
g
projects, a simple web-based moisture-safe retrofit decision-making tool has b
oped for them. Some of the interface forms are shown and explained below. details are available on the tool’s website [49]. The tool interface and proced
operation involve three basic steps:
Step 1: Aims to present the tool and educate homeowners on moisture ma Step 1: Aims to present the tool and educate homeowners on moisture management
in buildings. p
p
in buildings. Figure 4 shows “My home page” which is the very first phase of the decisio Figure 4 shows “My home page”, which is the very first phase of the decision process. In this phase, the tool provides information concerning the moisture-safe retrofit calculator,
moisture in buildings, the importance of moisture management in a retrofit project, the
consequences of moisture unbalance retrofit, and the user interface of the tool. Figure 4 shows My home page , which is the very first phase of the decisio
In this phase, the tool provides information concerning the moisture-safe retro
tor, moisture in buildings, the importance of moisture management in a retro
the consequences of moisture unbalance retrofit, and the user interface of the t Figure 4. My home page. Figure 4. My home page. Step 2: Input the building type.
Step 2: Input the building type.
In this step users are asked to se Step 2: Input the building type. Step 2: Input the building type. In this step users are asked to se In this step, users are asked to select their dwelling type (Figure 5). The tool auto-
matically provides information regarding existing specifications and energy improvement
solutions according to the selected building type. The tool considers 20 building typologies
split into five construction periods (pre–1919, 1919–1944, 1945–1964, 1965–1980, post–1980)
and four building sizes, including single-family houses, terraced houses, multifamily
houses, and apartment blocks. Single-family houses encompass detached houses and
bungalows. Terraced house includes end and mid terraced houses as well as semi-detached
houses. Multifamily house includes converted and low-rise flats. Apartment block includes
purpose built and high-rise flats. The reference buildings are based on the European project
EPISCOPE [15]. In this step, users are asked to select their dwelling type (Figure 5). The tool automat-
ically provides information regarding existing specifications and energy improvement so-
lutions according to the selected building type. The tool considers 20 building typologies
split into five construction periods (pre–1919, 1919–1944, 1945–1964, 1965–1980, post–
1980) and four building sizes, including single-family houses, terraced houses, multifam-
ily houses, and apartment blocks. Single-family houses encompass detached houses and
bungalows. Terraced house includes end and mid terraced houses as well as semi-de-
tached houses. Multifamily house includes converted and low-rise flats. Apartment block
includes purpose built and high-rise flats. The reference buildings are based on the Euro-
pean project EPISCOPE [15]. Figure 5. Select your dwelling type page. Figure 5. Select your dwelling type page. Figure 5. Select your dwelling type page. Figure 5. Select your dwelling type page. Step 3: Display the results. Step 3: Display the results. p
p y
This step shows the output results of the system. The first section, “Existing specifi-
cations”, of the results page provides information on the existing specifications of the se-
lected building type, which are based on assumptions in Bennadji et al., [23] and based on
Loga et al., [15]. The displayed data (Figure 6) includes property size, type of windows,
walls, roof, floor, heating system, ventilation, airtightness, final energy consumption, and
Energy Performance Certificate (EPC). p
p y
This step shows the output results of the system. The first section, “Existing speci-
fications”, of the results page provides information on the existing specifications of the
selected building type, which are based on assumptions in Bennadji et al. Figure 4. My home pag
Figure 4. My home page. Buildings 2022, 12, 1513
Buildings 2022, 12, x FOR 10 of 18
10 of 18 10 of 18
10 of 18 Step 2: Input the building type.
Step 2: Input the building type.
In this step users are asked to se [23] and based
on Loga et al. [15]. The displayed data (Figure 6) includes property size, type of windows,
walls, roof, floor, heating system, ventilation, airtightness, final energy consumption, and
Energy Performance Certificate (EPC). gy
(
)
gy
The second section, “Your suggested energy efficiency home improvements”, provides
a list of suggested energy efficiency home improvements (Figure 7). 11 of 18
11 of 1 Buildings 2022, 12, 1513
ildings 2022, 12, x FOR PE Figure 6. Existing specifications section. The second section, “Your suggested energy efficiency home imp
vides a list of suggested energy efficiency home improvements (Figure
Figure 6. Existing specifications section. Figure 6. Existing specifications section. The second section, “Your suggested energy efficiency home improv
vides a list of suggested energy efficiency home improvements (Figure 7). Figure 7. Your suggested energy efficiency home improvements section. Figure 6. Existing specifications section. The second section, “Your suggested energy efficiency home imp Figure 6. Existing specifications section. Figure 6. Existing specifications section. vides a list of suggested energy efficie Figure 6. Existing specifications section. Figure 6. Existing specifications section. vides a list of suggested energy efficie Figure 6. Existing specifications section. Figure 6. Existing specifications section. vides a list of suggested energy efficien g
g p
The second section, “Your suggested energy efficiency home imp
vides a list of suggested energy efficiency home improvements (Figure
Figure 7. Your suggested energy efficiency home improvements section. The second section, “Your suggested energy efficien Figure 7. Your suggested energy efficiency home improvements section. Buildings 2022, 12, 1513 12 of 18 12 of 18 Each solution is evaluated individually in terms of estimated cost, yearly savings,
payback period, disruption, and risk of moisture. In addition, users can click the “i” icon to
learn more about moisture risks related to each solution (Figure 8). Information includes
simplified explanations of moisture risks as well as mitigation strategies. Each solution is evaluated individually in terms of estimated cost, yearly savings,
payback period, disruption, and risk of moisture. In addition, users can click the “i” icon
to learn more about moisture risks related to each solution (Figure 8). Information in-
cludes simplified explanations of moisture risks as well as mitigation strategies. Each solution is evaluated individually in terms of estimated cost, yearly savings,
payback period, disruption, and risk of moisture. Figure 8. “i” icon to learn more about moisture risks related to each solution. Figure 8. “i” icon to learn more about moisture risks related to each solution. Figure 8. “i” icon to learn more about moisture risks related to each solution. In the third section, “Upgrade recommendation summary”, the upgrade recommen-
dation summary (Figure 9) shows the effect of undertaking all of the improvement
measures listed in your suggested energy efficiency home improvements table. Again, the
package of solutions is evaluated in terms of estimated cost, yearly savings, payback pe-
riod, disruption, and risk of moisture. In the third section, “Upgrade recommendation summary”, the upgrade recommenda-
tion summary (Figure 9) shows the effect of undertaking all of the improvement measures
listed in your suggested energy efficiency home improvements table. Again, the package of
solutions is evaluated in terms of estimated cost, yearly savings, payback period, disruption,
and risk of moisture. In the third section, Upgrade recommendation summary , the upgrade recommen-
dation summary (Figure 9) shows the effect of undertaking all of the improvement
measures listed in your suggested energy efficiency home improvements table. Again, the
package of solutions is evaluated in terms of estimated cost, yearly savings, payback pe-
riod, disruption, and risk of moisture. Step 2: Input the building type.
Step 2: Input the building type.
In this step users are asked to se In addition, users can click the “i” icon
to learn more about moisture risks related to each solution (Figure 8). Information in-
cludes simplified explanations of moisture risks as well as mitigation strategies. Figure 8. “i” icon to learn more about moisture risks related to each solution. Figure 8. “i” icon to learn more about moisture risks related to each solution. Figure 8. “i” icon to learn more about moisture risks related to each solution. 4.1. User Acceptance and Satisfaction The pilot-testing phase revealed that the tool was positively received, with all partic-
ipants reporting that the website was easy to use and easy to navigate. The majority of
participants found that they could understand the information. Participants highlighted the importance of a retrofit tool to combine text explanations
with visual illustrations (P1: “you can better understand with images”). “These findings
are in line with those by Kok and Barendregt [50]. Participants agreed that data entry was
easy and not too time-consuming. y
g
Preferably, online retrofit tools should not require users to enter technical details (P3: “I
was thinking maybe I need to measure space in my home [ . . . ] what kind of windows
do I have, or what sort of walls, do I have, so the website did that, for me, by just saying
just give us what type of property and we’ll do the rest which is a relief because you don’t
want obviously to overload the user with the burden of finding out all these technical
details”). In fact, it is argued that many people prefer a quick-test calculator over a more
comprehensive version [51]. Participants indicated that the tool would have been more accessible if it had been
adapted to phones’ displays (P7: “I have tried to open the website on my mobile phone,
but it didn’t work”). Indeed, scholars have argued that mobile-like advice tool should be
used in energy retrofit communication [44]. Moreover, most participants felt the amount of information was sufficient and that no
parts needed to be left out or removed. Participants liked to see the multicriteria evaluation
of the retrofit solutions (P11: “the breakdown and the detail provided in terms of the
estimated cost and yearly savings and the payback period that was really kind of very
helpful in kind of giving me kind of understanding of options going forward”). Participants
also appreciated the possibility to click “i” icon to learn more about moisture risks related
to each solution (P3: “very much appreciated also to have the additional information next
to each of the recommendations, so if I wanted to learn more about each one of the types
of improvement, I can go to the to the relevant sites”). They liked that the tool indicates
how to find accredited professionals (P9: “The feedback at the end and how to contact a
contractor make things easy”). Figure 9. Upgrade recommendation summary. Figure 9. Upgrade recommendation summary. Buildings 2022, 12, 1513 13 of 18 13 of 18 The fourth section, “Your step-by-step retrofit plan”, provides advice regarding the
sequencing of the renovation work in the case where the package of solutions cannot be
implemented at once (lack of time, money, too much inconvenience, or any other reason). This section introduces the concept of a “medium-term improvement plan” according to
the British standard for domestic retrofitting PAS2035. The tool only aims to raise homeowners’ awareness on energy improvement options
and moisture risks related to a retrofit project. The results presented by the tool are only
estimates and not valid as an actual EPC. To continue their renovation journey, homeowners would have to contact an accredited
retrofit coordinator. Hence, the fifth section, “What’s Next?” provides useful links to help
homeowners find an accredited retrofit coordinator, determine how they can pay for
improvements, and explore hints and tips on dealing with damp and condensation. 4. Discussion This article describes the development and pilot testing of a web-based moisture-safe
retrofit decision-making tool for homeowners. The discussion section is organised into four
sections, including the three focal points of this study: user acceptance and satisfaction,
increased awareness about moisture, willingness to pursue a renovation project, and the
study’s limitations. We illustrate our findings with representative quotes from participants. 4.1. User Acceptance and Satisfaction All participants reported that they would recommend the
website to other homeowners. This is in line with the study by Collins et al. [52] in which
more than 66% of respondents recommended a footprint calculator to others. Buildings 2022, 12, 1513 14 of 18 4.3. Willingness to Pursue a Renovation Project Most participants reported that using the tool did not increase their willingness to
pursue a renovation project. This disagrees with another study in which homeowners
evaluated a green home decision-making tool, and it has been found that the tool increased
homeowners’ willingness to pursue a green renovation [44]. Many participants reported
feeling discouraged from investing in energy efficiency measures because of high invest-
ment costs and long payback periods of some measures. The discouragement was not
related to an increased awareness of moisture issues. P8 said, “holy crap thirty-two grands
for windows wow”. P3 said, “discouraged is maybe the word, because obviously of the esti-
mated cost and the payback period kind of It makes you think whether it is worth it or not”). For instance, installing solid floor with insulation beneath the floor might show poor return
of investment with long payback periods for up to 113 years which exceeds the estimated
lifespan of most construction products and buildings themselves. This agrees with a study
conducted by Alabid et al. [54] that argued that long payback periods represent one of the
main barriers to the implementation of a deep retrofitting approach. The Association for
Environment Conscious Building (AECB) argued that to encourage homeowners to invest
in a deep retrofit, economic decision-making should not focus only on payback periods and
should consider co-benefits associated with improving the energy performance of existing
buildings including upgraded health, comfort and perceived wellbeing combined with
grants or financial incentives, lower maintenance and running costs, and higher energy
prices [55]. Therefore, it may be helpful for online retrofit tools to include co-benefits
associated with a retrofit in the financial evaluation in order to reassure homeowners about
their investment. Finally, online retrofit tools should not present the users only with the
best energy efficiency solutions, which are generally expensive for homeowners. Tools
should instead provide users the opportunity to select between a range of retrofit measures
(e.g., various thicknesses of insulation, different window types, etc.) from the most efficient
to the least efficient solutions in order not to scare homeowners with high investment costs
of measure targeting high energy standards (P8: “I think it probably would put me off
doing anything, right now, because it it’s giving you the most expensive scenario”). 4.2. Awareness about Moisture Risks 4.2. Awareness about Moisture Risks Most participants indicated that the tool improved their awareness of moisture risks
in a retrofit project. This is in line with the findings of Dreijerink and Paradies [53] that
online calculators increased participants’ awareness of environmental issues. Participants
were made aware of different consequences of moisture-imbalanced retrofits (P13: “I was
really surprised to know that renovation solutions can lead to dampness”). In that sense,
the tool was considered an eye-opener. The new awareness of moisture issues was scary for
a few participants (P12: “It was kind of scary for me”). Only one participant said the tool
had not increased his awareness (P3: “I would say not by much because obviously here my
situation is a little bit [ . . . ] I had kind of some pre knowledge of some of the options that
were available to me”). By educating homeowners on moisture risk issues such as damp
and mould, the developed tool is expected to protect homeowners’ health. 5. Conclusions While there are several online tools available to inform homeowners about the benefits
of retrofitting, to the best of our knowledge, this is the first study that aims to develop and
evaluate an online tool that aspires to raise homeowners’ awareness about moisture risks in
a retrofit project. Furthermore, the proposed paper extends existing findings by shedding
light on the experiences of retrofit calculator users. The paper focuses on the (1) users’
acceptance of and satisfaction with the developed retrofit calculator, (2) effects of the tool on
moisture awareness and behaviour, and (3) effects of the tool on the willingness to invest in
energy efficiency. The results of the pilot study demonstrate that the developed web-based
tool has been well received by homeowners. The tool significantly increased participants’
awareness of moisture risks related to a retrofit project. Most participants considered the
tool an eye-opener, while few of them found it scary. However, another important finding
is that the tool did not result in an increased willingness to invest in energy efficiency
measures. The discouragement was not related to an increased awareness of moisture
issues and was linked to high investment costs and long payback periods of some retrofit
measures. The pilot study also generated valuable insights on how to improve the design
and development of online retrofit calculators to stimulate the adoption of energy efficiency
measures. Future improvements would be to: 1. Include co-benefits associated with a retrofit in the financial evaluation, which will
enable retrofit tools to make a stronger case for investment in retrofitting. The co-
benefits that may be quantified are the Net Present Value (NPV) calculation, including
available grants, operational and maintenance cost savings, carbon savings, thermal
comfort, and reduced incidence of asthma. 2. Provide users with the opportunity to select between a range of retrofit measures (e.g.,
various thicknesses of insulation, different window types, etc.) from the most efficient
to the least efficient solutions in order not to scare homeowners with high investment
costs of measures targeting high energy standards. g
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3. Use simple language, a clear design, and use graphics to clarify information. 4. Facilitate easy and quick data entry without asking users for technical details. This
can be done by using automated data collection. 5. Use a responsive layout to adapt to various screen sizes. 4.4. Limitations Although the developed online tool aims to increase homeowners’ awareness regard-
ing moisture issues related to a retrofit project, homeowners cannot rely on decision aids
to replace a retrofit coordinator whose job is to oversee the management and design of all
retrofit measures [56]. Risks associated with domestic retrofit comprises a much broader
range of issues than those addressed by the developed tool, including technical risk such as
putting the correct package of improvements in place, managing the interaction between
measures, and process risks such as assessing existing dwelling(s) adequately and ensuring
that appropriate retrofit designs are developed [34]. Therefore, while the developed tool
provides useful and relevant information for homeowners, it is not designed to replace
consultation with a retrofit coordinator. Buildings 2022, 12, 1513 15 of 18 15 of 18 The proposed tool in its actual form has several limitations. It currently considers
only a limited number of retrofit measures. A future improvement could be to allow
users to select between a range of retrofit measures (e.g., various thicknesses of insulation,
different window types, etc.) from the most efficient to the least efficient solutions. Another
improvement could be to filter different retrofit options according to budget limitations. Furthermore, to ensure quick and easy data entry, the developed tool is based on a pre-
simulated database that includes the most common building typologies in the UK. The
user can get an idea of energy-saving options only by indicating the building typology. However, the shortcoming of using a pre-simulated database is that the accuracy of the
result is limited. In fact, every house in the UK is unique (occupation pattern, history of
modifications, location, etc.). Therefore, it is impossible to generate accurate results for
every homeowner based on a pre-simulated database and the displayed information for the
users could be significantly different from their actual building data. An alternative way to
reach a balance between ease of use and precision of the results is to use automated data
collection, as suggested by Bekaroo et al. [57] and Guzman et al. [58]. The future version of
the tool could be connected to national EPC databases to collect users’ data automatically. Moreover, the evaluation provided valuable feedback, which will lead to the future
development of the proposed tool. Finally, given the small pilot nature of the study, the
generalisability of study findings is limited. Further research with larger sample size
is necessary. 5. Conclusions For future works, the recommendations proposed in this study will be implemented
in the future version of the developed tool. Furthermore, large-scale deployment of the Buildings 2022, 12, 1513 16 of 18 proposed tool is envisaged while also planning for feedback from the larger audience. This will also enable a comparison of usability, awareness, and willingness to pursue a
renovation project based on the demographic details of the participants. proposed tool is envisaged while also planning for feedback from the larger audience. This will also enable a comparison of usability, awareness, and willingness to pursue a
renovation project based on the demographic details of the participants. Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/buildings12101513/s1, Supplementary Material S1: General
assumptions of the retrofit measures; supplementary Material S2: Moisture risks evaluation; Supple-
mentary Material S3: Interview guide. Author Contributions: Conceptualization, M.S., A.B., J.A., D.G. and G.D.; methodology, M.S., A.B. and J.A.; software, M.S., A.B. and J.A.; validation, M.S., A.B. and J.A.; writing—original draft
preparation, M.S., A.B. and J.A.; writing—review and editing, M.S., A.B. and J.A.; supervision, project
administration, and funding acquisition, A.B., D.G. and G.D. All authors have read and agreed to the
published version of the manuscript. Funding: This research was carried out within Stronghouse project and was funded by Interreg
North Sea region programme grant number [J-NO: 38-2-15-19]. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Not applicable. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We would like to thank all partners from the Stronghouse project for their
continuous and rich discussions throughout the development of this research. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. National-Statistics 2020 UK Greenhouse Gas Emissions, Provisional Figures. 2020. Available online: https://assets.publishing. service.gov.uk/government/uploads/system/uploads/attachment_data/file/972583/2020_Provisional_emissions_statistics_
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training-course/ (accessed on 5 January 2022). 56. Edwards, J. Retrofitting traditional buildings for improved energy performance in the context of BSI PAS 2035. J. Build. Surv. Apprais. Valuat. 2021, 9, 294–304. 57. Bekaroo, G.; Roopowa, D.; Bokhoree, C. Mobile-based Carbon footprint calculation: Insights from a usability study. In Proceedings
of the 2019 Conference on Next Generation Computing Applications (NextComp), Mauritius, Madagascar, 19–21 September 2019;
IEEE: Piscataway, NJ, USA, 2019; pp. 1–6. y
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International Workshop on Social Sensing, Montreal, QC, Canada, 15 April 2019; pp. 24–29.
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COLLOQUIA THEOLOGICA OTTONIANA 38 (2022)
s. 9–30
DOI: 10.18276/cto.2022.38-01
ISSN (print) 1731-0555 | ISSN (online) 2353-2998 ZOLTÁN JÓZSEF BARA
Universitatea UBB Cluj Napoca, Romania | Facultatea de teologie romano catolică
ORCID: 0000-0002-1254-8458 Introduzione La Chiesa Ortodossa Romena, come pure la sua teologia, rappresentano una sintesi
tra Oriente e Occidente, grazie al carattere specifico del popolo romeno, che ha
un’origine latina, ma vive una spiritualità bizantina. “Dumitru Stăniloae (1903–1993)
è ritenuto insieme con Romano Guardini, Paul Tillich, Karl Barth, Karl Rahner,
Yves Congar, Nikos Nissiotis ed altri, tra i più rinomati teologi del ventesimo secolo”1. L’opera di riflessione teologica e filosofica di Dumitru Stăniloae, elaborata durante
i sei decenni di vita accademica e pubblicistica svolta nei due centri dell’Orto
dossia romena, Sibiu e Bucarest, ammonta a circa 20 libri originali, 1300 arti-
coli e 30 traduzioni dai Padri della Chiesa e dai grandi teologi contemporanei2. Nell’opera di Dumitru Stăniloae esiste una grande varietà di temi e problemi ecu-
menici, in cui egli ha assunto delle posizioni originali, a volte radicali, ma sempre
favorevoli al dialogo3. In questo brano vorrei presentare due temi che, in un certo
senso, sintetizzano la visione ecumenico originale di Stăniloae sull’unione delle
Chiese. Si tratta del tema della cattolicità o sinodalità aperta4 e quello dell’interco-
munione eucaristica come tipologia ecumenica5. Summary In Dumitru Stăniloae’s work, he takes original, albeit sometimes radical, positions on a great
variety of ecumenical themes and problems; however, Stăniloae is always in favor of dialogue. Following Stăniloae’s arguments, I will present two themes that summarize Stăniloae’s original
ecumenical vision on the union of the Churches: the theme of catholicity or open synodality, and
Eucharistic intercommunion. Stăniloae suggests an open synodality or catholicity as a dogmatic
and spiritual opening of the Christian Churches to rediscover their unity. At the basis of this open
catholicity is the Sacred Scripture where there are diverse traditions and meanings – differences
that come from the diversity of God’s actions in the history of salvation. The Catholic or synodal
approach can become, in all the senses of the Scripture, the meeting point for all Christians. On the other hand, in Stăniloae’s vision, Eucharistic intercommunion must be a full Eucharistic
communion. Orthodoxy considers the unity of the Church as deeply and ontologically anchored
in Christ, and it considers the Eucharist as the mean that sustains this unity. This unity can-
not bring the Eucharist down to the level of being psychologically and physically lax among
Christians. Indeed, without the unity of faith, and without communion with the body and
blood of Christ, the Church could not exist in the full sense of the word; just as without unity
in faith and without the Church, Eucharistic communion cannot be achieved. Eucharistic
intercommunion makes a separation between the unity of faith and the Church, inconceivable
because it leads to the dissolution of the Church, of the unity of faith, and of the Eucharist
itself. Communion with the body and blood of Christ cannot be separated from unity in faith
or from the conviction that the Church represents a profound ontological unity of the faithful
in Christ and among them. Keywords: Dumitru Stăniloae, ecumenism, open catholicity (sinodality), union of
Churches, eucharistic inter-communion 10 Zoltán József Bara 1
Cf. Theologen des 20. Jahrhunderts. Eine Einführung, a cura di P. Neuner, G. Wenz, Darm
stadt 2002, p. 145–156. 2
Z. Bara, Un approccio ortodosso ecclesiologico alla dimensione sacramentale della Chiesa:
la Chiesa, Sacramento di Cristo, secondo Dumitru Stăniloae, in: Sakramenty nowym wyzwa-
niem dla Kościoła Katolickiego, a cura di J. Radkiewicz, Szczecin 2019, p. 155–156. 4
sobornicitatea deschisă 1. La sinodalità aperta Il tema della sinodalità aperta è stato descritto da Stăniloae in un articolo
pubblicato nel 19716, in cui voleva sottolineare la necessità dell’apertura dogma-
tica e spirituale delle Chiese cristiane, per ritrovare la loro unità. Il progresso del
mondo – secondo Stăniloae – è anche un progresso della Chiesa e un progresso 2
Z. Bara, Un approccio ortodosso ecclesiologico alla dimensione sacramentale della Chiesa:
la Chiesa, Sacramento di Cristo, secondo Dumitru Stăniloae, in: Sakramenty nowym wyzwa-
niem dla Kościoła Katolickiego, a cura di J. Radkiewicz, Szczecin 2019, p. 155–156. 3
M. Stavrou, Teologia cristiana ortodossa, in: Dizionario del sapere storico-religioso del Nove-
cento, Bologna 2010, p. 1514; G. R. Roberson, Ecumenism in the Thought of Dumitru Stăniloae,
a cura di A. Melloni, New York 1999, p. 51. 4
sobornicitatea deschisă 5
N. Moşoiu, “Sobornicitatea deschisă” ca tipologie ecumenică sau De la teologia confesională la teo-
logia ecumenică în perspectiva viziunii despre “sobornicitatea deschisă”, “Revista Teologică” 4
(1997), p. 87–117. 5
N. Moşoiu, “Sobornicitatea deschisă” ca tipologie ecumenică sau De la teologia confesională la teo-
logia ecumenică în perspectiva viziunii despre “sobornicitatea deschisă”, “Revista Teologică” 4
(1997), p. 87–117. 6
D. Stăniloae, Sobornicitate deschisă, “Ortodoxia” 23 (1971) 2, p. 165–180. Cf. V. Coman, “Open
Sobornicity” and “Receptive Ecumenism”: Fruitful Models of Ecumenical Interaction, in: Just Do It? Recognition and Reception in Ecumenical Relations: Proceedings of the 19th Academic Consul-
tation of the Societas Oecumenica, a cura di D. Heller, M. Hietamaki, Leipzig 2018, p. 241–251;
V. Coman, Le Saint Esprit comme liaison de l’amour éternel entre le Père et le Fils: un cas de ‘sobor-
nicité ouverte’ dans la théologie orthodoxe, “Irénikon” 89 (2016) 1, p. 25–51; R. Bordeianu, La sinodalità e l’intercomunione in Dumitru Stăniloae 11 del cristianesimo verso la Chiesa Una e Cattolica. L’arricchimento spirituale
e l’ascensione del mondo verso i veri valori porta anche ad un arricchimento spi-
rituale, ad un’amplificazione e ad una scoperta del contenuto spirituale che è più
propriamente della Chiesa. E se la Chiesa deve comprendere tutta la verità, tutta
la realtà di Dio, essa non si può chiudere ai valori che, nel suo continuo sforzo,
l’umanità svela, aiutata anche dall’opera di Dio7. Punto di partenza quindi per l’approfondimento di questo tema lo rappresenta
il documento Scrittura e Tradizione emanato dalla Comissione Fede e Costituzione
del Consiglio Ecumenico delle Chiese8. (In) Voluntary Ecumenism: Dumitru Stăniloae’s Interaction with the West as Open Soborni-
city, in: Orthodox Constructions of the West, a cura di E.G. Demacopoulos, A. Papanikolaou,
New York 2013, p. 240–253; L. Turcescu, Eucharistic Ecclesiology or Open Sobornicity?, in: Dumi-
tru Stăniloae: Tradition and Modernity in Theology, a cura di L. Turescu, Palm Beach 2002,
p. 83–103. 9
Nouveauté dans l’oecumenisme, Taizé 1968, p. 58.
10 Ibidem. 7
“Progresul lumii este astfel și un progres al Bisericii și un progres al creștinismului spre Bise-
rica cea una și sobornicească. Îmbogățirea spirituală și înălțarea lumii spre valorile adevărate,
prilejuiește și o îmbogățire spirituală, o amplificare și o descoperire a conținutului spiritual cel
mai propriu al Bisericii. Și dacă Biserica trebuie să cuprindă tot adevărul, să înțeleagă toată rea-
litatea lui Dumnezeu, ea nu se poate închide valorilor pe care le dezvăluie omenirea în efortul
ei continuu, ajutată și ea prin lucrarea lui Dumnezeu”. Cf. D. Stăniloae, Coordonatele ecumeni-
smului din punct de vedere ortodox, “Ortodoxia” 19 (1967) 4, p. 540. Cf. C. Sonea, Teologia dia-
logului intercreștin. O introducere, Cluj Napoca 2017, p. 273–278. 8
Questo documento era difatti il risultato delle discussioni ecumeniche tenute tra diversi gruppi
di teologi, in seguito alla decisione presa all’incontro di Aarhus nel 1964, risultato che è stato
presentato all’incontro di Bristol nel 1967. Oltre al tema ricordato, il documento include anche
altri tre temi: Dio nella natura e nella storia, l’Eucaristia e la Chiesa e il popolo ebreo. (In) Voluntary Ecumenism: Dumitru Stăniloae’s Interaction with the West as Open Soborni-
city, in: Orthodox Constructions of the West, a cura di E.G. Demacopoulos, A. Papanikolaou, y
f
p
p
New York 2013, p. 240–253; L. Turcescu, Eucharistic Ecclesiology or Open Sobornicity?, in: Dumi-
tru Stăniloae: Tradition and Modernity in Theology, a cura di L. Turescu, Palm Beach 2002,
p. 83–103. 11 “Sobornicitatea deschisă are o perspectivă profetică, deoarece insistă asupra însemnătăţii uni-
tăţii creştinilor pentru reconcilierea lumii, fapt realizabil în măsura trecerii lui Hristos pascal
în viaţa umană sub “forma Bisericii”. Cf. D. Stăniloae, Dogmatica I, București 2003, p. 61;
ibidem, Dogmatica II, p. 206. 12 “E un mister această ținere la un loc a unor aspecte contradictorii. Iar unitatea părților are un
sens cu mult mai bogat decât suma sensurilor părților, un sens inepuizabil rațional. Rațiunea
are totuși un rol în sesizarea aspectelor care sunt împletite în această unitate, făcând ca să nu
fie redus în mod simplist la unul singur, cu negarea celorlalte”. Cf. D. Stăniloae, Sobornicitate
deschisă..., p. 167. 1. La sinodalità aperta Questo documento, dopo aver notato che
nella Bibbia c’è una unità nella diversità, afferma che la diversità delle tradizioni
delle Chiese attuali può essere ridotta alla diversità delle tradizioni esistenti nella
Sacra Scrittura, per cui la ricerca dell’unità tra le diverse tradizioni della Chiesa dovrà
appropriarsi dell’unità del Vangelo, così come la riflette la pluralità delle diverse
tradizioni bibliche9. Il documento sostiene inoltre che la diversità delle tradizioni
nella Bibbia riflette difatti la diversità delle azioni di Dio nella storia umana, come
pure la diversità delle risposte umane a queste azioni. In questo senso il documento
raccomanda agli esegeti di non attacarsi ad una sola riflessione biblica, anche se
questa sembra centrale, perché tale scelta li porterebbe a non comprendere più
la diversità di questa ricchezza10. Secondo Stăniloae, questa raccomandazione vale non solo per gli esegeti,
ma anche per le Chiese cristiane, perché molte delle divisioni apparse in seno al
cristianesimo sono dovute ad un attaccamento unilaterale a certe affermazioni 12 Zoltán József Bara bibliche, ignorando le altre, che non sono meno importanti. Perciò, accettare
la diversità dei sensi presenti nella Scrittura, può diventare una via che porta alla
vera cattolicità (sobornicitate) della Chiesa e quindi all’avvicinamento ecumenico,
tanto desiderato dal mondo cristiano11. L’approccio unilaterale alla diversità dei sensi presenti nella Sacra Scrittura,
osserva il teologo romeno, si è manifestato fin dall’inizio nel cristianesimo, e questo
ha portato a delle dolorose divisioni. Di questo peccato si sono fatte colpevoli, per
esempio, le eresie antitrinitarie, le quali riducevano sia la divinità all’unità di una
sola persona, sia scioglievano l’unità in tre dei. Di fronte a queste eresie, la Chiesa,
tenendo conto delle espressioni bibliche, ha affermato sia l’unità d’essere della
divinità, che la trinità delle persone. Quindi, mentre le eresie si lasciavano portare
da un giudizio semplicistico, la Chiesa era animata dalla realtà complessa, in cui
sono uniti aspetti diversi, complementari e a volte anche contraddittori. La realtà
che guida la Chiesa, ha perciò un carattere misterioso, in quanto è un mistero questo
tenere insieme aspetti contraddittori, e l’unità delle parti ha un senso molto più ricco
che la somma dei sensi delle parti, un senso inesauribile. La ragione ha comunque
un ruolo nella percezione degli aspetti che sono impliciti in questa unità, facendo sì
che essi non siano ridotti in modo semplicistico ad un solo, negando tutti gli altri12. 13 “Nu s-a plasat pe o altă poziție unilaterală și contradictorie față de poziția unilaterală a unei
erezii sau alteia, ci s-a menținut pe o poziție de largă îmbrățișare a tuturor aspectelor credinței,
inclusiv a celor afirmate de erezii. Erezia își vedea în învățătura Bisericii și aspectul susținut de
ea, deși încadrat în toate celelalte aspecte ale învățăturii creștine. Încât, îndată ce se renăștea
în ea tendința spre plenitudine, ajungea vrând-nevrând la întâlnirea cu Biserica în învățătura
pe care o păstrase aceasta. Iar tendința spre plenitudine se naște aproape firesc în erezie,
după perioada de afirmare inițială a unei teze unilaterale din opoziția pasionată față de Bise-
rică și față de plenitudinea susținută de ea”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 167–168. 1. La sinodalità aperta In questo senso, è interessante notare che, nell’Oriente cristiano, tutte le eresie,
a eccezione di quelle cristologiche, sono state riassorbite nella grande Chiesa, e questo
è dovuto, secondo Stăniloae, non solo all’aiuto dato dall’imperatore bizantino nel
riportare i cristiani separati all’interno della Chiesa, ma anche al fatto che questa
non si è situata su un’altra posizione unilaterale e contraddittoria nei confronti
della posizione unilaterale delle eresie, ma si è mantenuta su una posizione che
abbracciava tutti gli aspetti della fede, inclusivamente quelli affermati dalle eresie. L’eresia vedeva nella dottrina della Chiesa anche l’aspetto sostenuto da essa, anche
se inquadrato in tutti gli altri aspetti della dottrina cristiana. Tanto che, una
volta che nasceva dentro di essa la tendenza verso la pienezza, arrivava, volendo
o no, all’incontro con la dottrina che la Chiesa aveva conservato. E la tendenza
verso la pienezza nasce quasi naturalmente nell’eresia, dopo il periodo iniziale La sinodalità e l’intercomunione in Dumitru Stăniloae 13 d’affermazione di una tesi unilaterale, a causa di un’opposizione passionale verso
la Chiesa e verso la pienezza sostenuta da essa13. Un caso particolare lo rappresenta lo scisma tra la Chiesa d’Oriente e quella
dell’Occidente, consumatasi nel 1054, perché allora la divisione non è stata più
provocata, afferma Stăniloae, dalla negazione di un aspetto dottrinale, ma dall’accen
tuazione esagerata da parte della Chiesa occidentale di un punto dottrinale relativo
all’unità della Chiesa, accentuazione che ha portato alla subordinazione e all’inter-
pretazione dell’intera dottrina alla luce di questo punto. Perciò, l’unità della Chiesa
non era più vista essenzialmente come un’unità d’equilibrio, come un’unità dei
contrari, ma come un’unità in cui una parte viene innalzata alla dignità di centro
onnipotente, che soffoca sempre di più tutte le altri parti componenti, che sempli-
fica la complessità, uniformizza la diversità, portando ad un impoverimento della
vita della Chiesa in Dio, impoverimento uguale a quello provocato dalle eresie,
che negavano parti essenziali della dottrina cristiana14. Pur ammettendo che il canone 28 del Concilio di Calcedonia aveva stabilito una
gradazione delle prime cinque sedi episcopali, in base alla loro origine apostolica
e all’importanza politica delle loro città e che, secondo questa gradazione, il vescovo
di Roma era il primo, il teologo romeno sottolinea che questa gradazione era soltanto
presidenziale, rappresentando una certa autorità morale dei loro vescovi. 14 “Nu ca o unitate de echilibru, ca o unitate a contrariilor, ci ca o unitate în care o parte e ridi-
cată la rang de centru atotputernic care sufocă tot mai mult celelalte părți componente, simpli-
fică complexitatea, uniformizează varietatea, ducând la o sărăcire a vieții Bisericii în Dumne-
zeu, egală cu cea pe care o provocau ereziile care negau părți esențiale ale învățăturii creștine”.
Cf. D. Stăniloae, Sobornicitate deschisă..., p. 68. 15 “Despărțirea Apusului creștin de Biserica răsăriteană, care a rămas în complexitatea echili-
brată a învățăturii creștine. Scaunul Romei a suprimat în Apus prin aceasta desfășurarea liberă
a vieții în Dumnezeu, sau posibilitatea ei nestingherită de îmbogățire spirituală, implicit posi-
bilitatea de creștere neîmpiedicată a umanului”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 169.
16 Infatti, i sacramenti sono “gli atti parziali per mezzo dei quali si offre a ciascuno, personalmente,
la vita di Cristo dalla Chiesa”. In questo senso, è giusto affermare che “per mezzo dei sacramenti
cresce e si nutre la Chiesa, cioè il Corpo mistico del Signore, che non è altro che l’allargamento
e il prolungamento dell’Incarnazione di Cristo”. D. Stăniloae, Sinteză eclesiologică, “Studii Teo-
logice” 7 (1955), 5–6, p. 267–284, 268. D. Stăniloae, Transparenţa Bisericii în viaţa sacramen-
tală, “Ortodoxia” 22 (1970) 4, p. 501–516. Cf. Z. Bara, La realtà mistica della chiesa nella rifles-
sione ortodossa di Dumitru Stăniloae, “Studia Theologia Catholica Latina” 66 (2021) 2, p. 83–84. 1. La sinodalità aperta I vescovi di
queste sedi primaziali, grazie alla loro autorità morale, svolgevano quindi un servizio
a favore dell’unità della Chiesa, ma senza restringere la sua libertà, in quanto l’unità
si manteneva prima di tutto, mediante la forza dell’amore e della stessa fede, per
l’opera dello Spirito Santo. Purtroppo, aggiunge lui, la sede romana, accentuando
la sua autorità esclusiva e dittatoriale, dopo aver soppresso la libertà della Chiesa
Occidentale, ha cercato di sopprimere anche la libertà della Chiesa Orientale, fatto
che ha avuto come conseguenza la separazione dell’Occidente cristiano dalla Chiesa
orientale, che è rimasta nella complessità equilibrata della dottrina cristiana. La Sede 14 Zoltán József Bara romana ha soppresso in Occidente lo sviluppo libero della vita in Dio, ossia la sua
possibilità di arricchimento spirituale e, implicitamente, la possibilità di crescita
senza restrizioni dell’umanità15. L’opzione della sede romana a favore di un’autorità contraria alla libertà ha
portato poi, continua Stăniloae, ad una nuova divisione nel seno del cristiane-
simo, divisione provocata dalla Riforma protestante del XVI secolo. Se nel 1054
si aveva scelto l’unità della Chiesa contro la sua libertà, adesso veniva contestata
la Chiesa stessa, in quanto contraria alla libertà, affermandosi così una fede cri-
stiana individuale. Dal complesso bipolare Chiesa – individuo, il protestantesimo
aveva optato per l’individuo, in quanto il cattolicesimo aveva accentuato l’unità
della Chiesa contro la libertà dell’individuo. Insieme a questo però, sono state
contestate tutte le strutture che sostengono la comunione della Chiesa con Dio:
i sacramenti, la partecipazione alla grazia divina, la santità, la comunione nella fede
e nell’amore.16 La Riforma ha contestato queste strutture ecclesiali, perché prima
di tutto contestava la struttura gerarchica, per mezzo di cui si mantenevano le altre
strutture. Per legittimare la sua autorità quindi, scrive Stăniloae, la Sede romana
aveva scelto dalla complessità contraddittoria dei testi biblici, quelli che sembra-
vano confermare l’idea di un primato di Pietro; la Riforma invece, per legittimare
il rifiuto di ogni struttura ecclesiale, quelli che affermavano il sacerdozio generale
dei fedeli, la dimensione fraterna della comunità cristiana e la diversità dei ministeri. La situazione era diventata tanto più senza via d’uscita, quanto ambedue le parti,
il cattolicesimo e il protestantesimo, rifiutando la complessità contraddittoria dei
testi scritturistici, definivano ciascuna se stessa in contrapposizione con l’altra. Lo spirito occidentale era diventato uno spirito di dure alternative, taglienti,
abituato con le semplificazioni di un razionalismo semplicistico, inferiore. 17 “Spiritul occidental devenise un spirit de alternative dure, tranșante, obișnuit cu simplificările
unui raționalism simplist, inferior. Pierduse suplețea unei rațiuni superioare care se orientează
după complexitatea suplă, echilibrată a realității, exprimată de Evanghelie, și a unei vieți spirituale
bogate și autentice... A înflorit teologia produsă de rațiune ca o valoare in sine, independentă de
viața complexă a Bisericii; a înflorit o organizație juridică clădită... din câteva principii rațion-
ale, organizație care și-a aservit ei și teologia”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 170. 18 A questo proposito, Stăniloae non è sicuro sulla provenienza di questa tendenza del cristia-
nesimo occidentale, cioè se è il risultato del suo incontro reale con l’Ortodossia, oppure se
è nato da sé. Comunque, egli è certo che essa porta in modo naturale verso l’Ortodossia. Inol-
tre, egli è contento di notare che oggi sempre più cristiani occidentali guardano con più inte-
resse la Chiesa Orientale, interesse che non è una semplice curiosità intellettuale, ma l’espres-
sione della convinzione che l’Ortodossia potrebbe aiutare al rinnovamento del cristianesimo
in genere. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 171. 19 “Fără Ortodoxie creștinismul occidental nu se poate mișca decât de la o extremitate la alta...
Refacerea unității e pentru creștinismul occidental o chestiune de ieșire din planul alternati-
velor exclusiviste. El trebuie să regăsească spiritul Ortodoxiei care nu se opune uneia sau alteia
din alternative, ci îmbrățișează în învățătura și în echilibrul ei și punctele afirmate de ambele
forme al creștinismului apusean”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 171. 1. La sinodalità aperta Aveva
perso l’agilità di una ragione superiore che si orienta secondo la complessità equi-
librata della realtà, espressa dal Vangelo, come pure di una vita spirituale ricca ed
autentica [...] Ha fatto fiorire una teologia prodotta dalla ragione come valore in sé, La sinodalità e l’intercomunione in Dumitru Stăniloae 15 indipendente dalla vita complessa della Chiesa; ha fatto fiorire un’organizzazione
giuridica, costruita anche essa... su alcuni principi razionali, su una organizzazione
che ha asservito anche la teologia17. Oggi però, osserva lui, in Occidente si nota il risveglio del desiderio verso
la complessità della dottrina e della vita cristiana, e questa tendenza va incontro
all’Ortodossia, che ha conservato la pienezza e l’equilibrio della vera spiritualità18. Senza l’Ortodossia, il cristianesimo occidentale non si può muovere che da un’e-
stremità all’altra [...] Il rifacimento dell’unità è per il cristianesimo occidentale
il problema d’uscire dal piano delle alternative esclusivistiche. Esso deve ritrovare
lo spirito dell’Ortodossia, che non si oppone ad una o all’altra di queste alternative,
ma abbraccia, nella sua dottrina e nel suo equilibrio, anche i punti affermati da
ambedue le forme del cristianesimo occidentale19. L’affermazione che l’unità delle Chiese cristiane si deve realizzare sotto il segno
dell’Ortodossia, non significa certo, riconosce Stăniloae, che la Chiesa Ortodossa
ha attualizzato finora, in modo soddisfacente, nel piano della spiritualità e della
vita dei fedeli, tutti i valori potenziali compresi nel tesoro dell’Ortodossia. Perciò
anch’essa ha bisogno di aprirsi ai valori spirituali attualizzati dai cristiani occi-
dentali, come, per esempio, il valore delle forme esterne dell’unità della Chiesa
e il valore della Sacra Scrittura. Forse in Oriente, dopo un certo tempo, l’unità
cristiana non è stata più vissuta, con tutta la sua intensità, come all’inizio della
Chiesa, sotto la forma superiore della comunità universale tra le Chiese locali,
e allora all’Occidente è sembrato necessario, forse a causa di speciali circostanze,
di vivere questa unità in un modo più pieno, senza rendersi conto che con il tempo 16 Zoltán József Bara Zoltán József Bara questa unità sarebbe stata pagata con un sempre più grande sacrificio. D’altra parte,
l’apparizione del cattolicesimo, quale affermazione giuridico-centrale dell’unità
del cristianesimo, ha portato praticamente gli ortodossi ad accentuare sempre più
l’indipendenza delle Chiese locali quali forme per mantenere la loro unità. 20 “Poate în Răsărit nu s-a mai trăit, începând de la o vreme, cu toată intensitatea unitatea creștină
ca la începutul Bisericii, sub forma superioară a comunității universale între Bisericile locale,
și atunci Occidentului i-a apărut necesar, poate din cauza unor împrejurări speciale, să trăiască
această unitate într-un mod mai deplin, fără să-și dea seama că această unitate avea să fie plătită
cu vremea cu un tot mai mare sacrificiu. Pe de altă parte, apariția catolicismului ca afirmare
juridic-centralistă a unității creștinismului, a făcut și pe ortodocși ca în practică să accen-
tueze mai mult independența Bisericilor lor locale decât formele de menținere a unității lor.
Iar când a apărut protestantismul, a preluat în practică de la catolicism un fel de antireformă,
accentuând Tradiția în dauna Sfintei Scripturi și importanța actelor obiective ale Tainelor
și a ierarhiei în dauna trăirii personale și în comuniunea legăturii cu Dumnezeu”. Cf. D. Stăni
loae, Sobornicitate deschisă..., p. 171. 21 “Ea trebuie să fie soborul a toată lumea, în care toți creștinii își aduc înțelegerea întregii realități
divine revelate și a întregii realități umane văzute în lumina revelației integrale, pentru a o împ-
ărtăși tuturor și pentru ca fiecare să se împărtășească de înțelegerea tuturor”. Cf. D. Stăniloae,
Sobornicitate deschisă..., p. 172. 1. La sinodalità aperta Quando
è apparso il protestantesimo, [la Chiesa Ortodossa] ha preso praticamente dal cat-
tolicesimo un certo tipo di controriforma, accentuando la Tradizione a scapito della
Sacra Scrittura e l’importanza degli atti oggettivi dei Sacramenti e della gerarchia
a scapito del vivere personale la comunione con Dio20. La cattolicità-sinodale della Chiesa rappresenta l’universalità della fede cristiana
vissuta sotto la forma di comunione. In questo senso, scrive Stăniloae, essa significa
non solo l’unità universale dei cristiani in forma di comunione, ma anche l’unità
onnicomprensiva della dottrina cristiana, vissuta dalla comunità universale e libera
dei cristiani. Perciò essa equivale all’universalità degli aspetti della Rivelazione,
percepiti dalla totalità delle prospettive umane e diventati il bene comune della
comunità umana universale. Essa dev’essere l’assemblea (soborul) di tutto il mondo,
in cui tutti i cristiani portano la loro comprensione della realtà divina rivelata
e dell’intera realtà umana vista alla luce della Rivelazione integrale, per comunicarla
a tutti e perché ciascuno partecipi alla comprensione di tutti21. Le Chiese cristiane sono chiamate quindi ad attualizzare la comprensione
dell’intera realtà divina e umana, riflessa e presente in una forma concentrata nel
Vangelo. Ma come si potrebbe inquadrare l’esperienza cattolica e protestante in
quella ortodossa? Secondo Stăniloae, per dare una risposta soddisfacente a questa
domanda, bisogna trovare anzitutto la spiegazione della diversità spesso contrad-
ditoria delle affermazioni bibliche. Il documento sopra ricordato della Comissione
Fede e Costituzione aveva detto che questa diversità proviene dalla diversità delle 21 “Ea trebuie să fie soborul a toată lumea, în care toți creștinii își aduc înțelegerea întregii realități
divine revelate și a întregii realități umane văzute în lumina revelației integrale, pentru a o împ-
ărtăși tuturor și pentru ca fiecare să se împărtășească de înțelegerea tuturor”. Cf. D. Stăniloae,
Sobornicitate deschisă..., p. 172. 21 “Ea trebuie să fie soborul a toată lumea, în care toți creștinii își aduc înțelegerea întregii realități
divine revelate și a întregii realități umane văzute în lumina revelației integrale, pentru a o împ-
ărtăși tuturor și pentru ca fiecare să se împărtășească de înțelegerea tuturor”. Cf. D. Stăniloae,
Sobornicitate deschisă..., p. 172. La sinodalità e l’intercomunione in Dumitru Stăniloae 17 azioni di Dio e dalla diversità delle risposte umane ad esse. 23 “Dumnezeu se face cunoscut și lucrează prin acte, cuvinte și imagini mereu diferite, pentru că
nu-și epuizează ființa în nici una din ele, dar prin toate se revelează. Pe nici unul din aceste
moduri nu trebuie să-l disprețuim, o dată ce Dumnezeu a găsit potrivit să-l folosească, dar nu
trebuie să socotim pe nici unul și nici pe toate cele din Revelație și din istoria evlaviei de până
acum ca exprimând integral pe Dumnezeu, ca excluzând pentru viitor alte moduri posibile
de a exprima pe Dumnezeu, chiar dacă cadrele adevărului divin au fost descoperite în totalita-
tea Revelației”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 173. 22 “Care ne oferă revelația sa prin Biblie ca ceva unitar cu toată diversitatea prezentă în ea, care ni
se prezintă general obligatorie de crezut în tot ansamblul ei și nu se vrea obligatorie pentru unii
în unele părți, pentru alții în altele”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 172. 22 “Care ne oferă revelația sa prin Biblie ca ceva unitar cu toată diversitatea prezentă în ea, care ni
se prezintă general obligatorie de crezut în tot ansamblul ei și nu se vrea obligatorie pentru unii
în unele părți, pentru alții în altele”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 172.
23 “Dumnezeu se face cunoscut și lucrează prin acte, cuvinte și imagini mereu diferite, pentru că
nu-și epuizează ființa în nici una din ele, dar prin toate se revelează. Pe nici unul din aceste
moduri nu trebuie să-l disprețuim, o dată ce Dumnezeu a găsit potrivit să-l folosească, dar nu
trebuie să socotim pe nici unul și nici pe toate cele din Revelație și din istoria evlaviei de până
acum ca exprimând integral pe Dumnezeu, ca excluzând pentru viitor alte moduri posibile
de a exprima pe Dumnezeu, chiar dacă cadrele adevărului divin au fost descoperite în totalita-
tea Revelației”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 173. 1. La sinodalità aperta Ma se questo fosse
vero, allora si potrebbe dire, scrive Stăniloae, che la diversità delle situazioni in cui
vivono oggi i cristiani, giustificherebbe la persistenza nella loro diversità, ogni
gruppo attaccandosi a quelle azioni divine che corrispondono alla loro situazione
storica specifica. Questa conclusione non corrisponde però alla volontà di Dio,
che ci offre la sua Rivelazione mediante la Bibbia come qualcosa di unitario, nono-
stante la diversità presente in essa, che ci si presenta generalmente obbligatoria da
credere nel suo insieme e che non si vuole obbligatoria per alcuni in alcune delle
sue parti, mentre per gli altri in altri parti22. Nella diversità delle sue azioni e delle sue parole, Dio persegue un unico piano
con l’intera umanità, e questo piano consiste nella ricapitolazione degli uomini nella
ricchezza infinita della sua vita e della sua verità, rivelate nella persona di Gesù
Cristo. Perciò, tutti i cristiani devono superare la diversità dei modi in cui Dio si
rivela e in cui essi lo comprendono, per arrivare alla sua unità integrale, che è al
di là di questi modi diversi di Rivelazione e di comprensione. Dio si fa conoscere
e agisce attraverso atti, parole e immagini sempre diversi, perché il suo essere non
si esaurisce in nessuno di questi, ma si rivela in tutti. Non dobbiamo disprezzare
nessuno di questi modi, se Dio ha trovato conveniente di utilizzarli. Così pure
non dobbiamo considerare che uno o tutti i modi della Rivelazione e della storia
della spiritualità sviluppatasi finora, esprimono integralmente Dio, o escludono
per il futuro altri modi possibili per esprimere Dio, anche se il quadro della verità
divina è stato svelato nella totalità dalla Rivelazione23. Di conseguenza, le Chiese cristiane non si devono attaccare ai modi della Rive-
lazione divina come alla realtà ultima, perché questi rappresentano solo la realtà
penultima; oltre a questo, bisogna ammettere anche la possibilità di nuovi modi
per esprimere Dio. Questo doppio atteggiamento della coscienza cristiana, cioè,
da una lato, riconoscere il valore dei modi utilizzati finora per esprimere Dio e,
dall’altra parte, il loro valore relativo o incompiuto, può diventare, secondo Stăniloae,
un importante aiuto per ritrovare la strada dell’unità tra i cristiani. 23 “Dumnezeu se face cunoscut și lucrează prin acte, cuvinte și imagini mereu diferite, pentru că
nu-și epuizează ființa în nici una din ele, dar prin toate se revelează. 24 “A rămâne numai la unele moduri ale exprimării lui Dumnezeu ca la ultima și exclusiva reali-
tate înseamnă a nu trăi prezența lui decât într-un mod mărginit, sau poate deloc, și a absolutiza
diversitatea noastră, nevăzând prin această diversitate pe Același Unic Dumnezeu, cu atât mai
bogat cu cât îl trăim prin mai multe moduri de revelare și exprimare. Iar aceasta ne menține în
dezbinare, socotind fiecare că prin modul său de a trăi și exprima pe Dumnezeu îl trăiește și-l
exprimă pe Dumnezeu în întregime, și acest mod al său e unicul mod de a trăi și exprima pe
Dumnezeu”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 173. 1. La sinodalità aperta Pe nici unul din aceste
moduri nu trebuie să-l disprețuim, o dată ce Dumnezeu a găsit potrivit să-l folosească, dar nu
trebuie să socotim pe nici unul și nici pe toate cele din Revelație și din istoria evlaviei de până
acum ca exprimând integral pe Dumnezeu, ca excluzând pentru viitor alte moduri posibile
de a exprima pe Dumnezeu, chiar dacă cadrele adevărului divin au fost descoperite în totalita-
tea Revelației”. Cf. D. Stăniloae, Sobornicitate deschisă..., p. 173. 18 Zoltán József Bara Zoltán József Bara Rimanere attaccati solo ad alcuni modi che esprimono Dio, come alla realtà
ultima ed esclusiva, significa vivere la sua presenza in un modo limitato, o forse
affatto, e assolutizzare la nostra diversità, non vedendo più per questa diversità lo
stesso Unico Dio, tanto più ricco, quanto lo viviamo in più modi di rivelazione e di
espressione. E questo ci mantiene in disunione perché ognuno ritiene che il suo
modo sia l’unico per vivere ed esprimere integralmente Dio24. Grazie al suo approccio cattolico-sinodale alla pienezza della Rivelazione, l’Orto
dossia, afferma Stăniloae, è tutelata dall’attaccamento rigido ad un certo mezzo della
Rivelazione. Ciò non significa però, che i suoi fedeli vivano praticamente questo
movimento di trascendere la realtà penultima, per arrivare alla realtà ultima. Oltre
ad essere tutelata da questo attaccamento rigido, l’Ortodossia ha la possibilità di
inquadrare nel suo modo di vivere anche gli aspetti accentuati dalla Chiesa Cattolica
e da quella Protestante. L’Ortodossia può dare un’importanza molto più grande
alla Scrittura e alla fede, che nel passato, senza darle un valore assoluto. Essa può
dare una più grande attenzione che nel passato, all’unità ecclesiale e alla sua orga-
nizzazione, riattivando l’autorità morale e il ruolo presidenziale delle antiche sedi
patriarcali, senza accordare un valore supremo e assolutista alla sede romana25. Per evitare l’attacamento rigido ad alcuni mezzi della Rivelazione come alla
realtà ultima, di grande aiuto sono anche le critiche reciproche che si fanno
le Chiese cristiane. 25 “Ortodoxia poate să dea o importanță foarte mare Scripturii și credinței - mai mare decât s-a dat
în trecut - fără să le dea o valoare exclusivă. Ea poate acorda o mai mare atenție unității biseri-
cești și organizării decât i-a dat în trecut, reactivând autoritatea morală și rolul prezidențial al
vechilor scaune patriarhale, fără să acorde o valoare suprempă și absolutistă scaunului roman”.
Cf. D. Stăniloae, Sobornicitate deschisă..., p. 175. 26 “Critica romano-cattolicismului ne poate fi de folos pentru ca să întărim mai mult unitatea noa-
stră, dar nu prin dependența de un centru exterior, ci printr-o ancorare mai profundă a tuturor
în Dumnezeu, Cel ma presus de toate modurile în care ni se revelează și în care îl adorăm. Cri-
tica protestantismului ne poate îndemna ca, afirmând valoarea tuturor modurilor de revelare
și de închinare a lui Dumnezeu..., să le socotim numai mijloace transparente pentru Dumnezeu
Cel mai presus de ele, trepte de pe care să contemplăm pe Dumnezeu în bogăția lui nesfârșită”.
Cf. D. Stăniloae, Sobornicitate deschisă..., p. 176. 1. La sinodalità aperta L’approccio cattolico-sinodale alla totalità dei sensi della Rivelazione implica,
sottolinea infine Stăniloae, anche un certo pluralismo teologico. Il pluralismo
teologico si basa sulla convinzione che ogni teologia rappresenta la contempla-
zione del mistero apofatico di Dio, da un certo punto di vista. In questo modo
si realizza una comprensione sinfonica di tutte queste prospettive teologiche e,
quindi, una comprensione comune, che è più corrispondente alla realtà divina. 1. La sinodalità aperta Per esempio, alla critica fatta dal cattolicesimo che l’Ortodos-
sia ha indebolito l’unità al suo interno, oppure a quella del protestantesimo, che
essa è troppo attaccata alle forme esterne, egli risponde: La critica del romano-
-cattolicesimo ci può essere utile per confermare di più la nostra unità, però non
per una dipendenza da un centro esteriore, ma per un ancoraggio più profondo di
tutte le cose in Dio, il quale è al di là di tutti i modi in cui ci si rivela e in cui noi
lo adoriamo. La critica del protestantesimo, oltre all’affermazione del valore di
tutti i modi di rivelazione e di adorazione di Dio, ci può suggerire di considerarli La sinodalità e l’intercomunione in Dumitru Stăniloae 19 soltanto come mezzi trasparenti per il Dio, che è al di là di essi, come gradini dai
quali possiamo contemplare Dio nella sua ricchezza infinita26. L’approccio cattolico-sinodale alla totalità dei sensi della Rivelazione implica,
sottolinea infine Stăniloae, anche un certo pluralismo teologico. Il pluralismo
teologico si basa sulla convinzione che ogni teologia rappresenta la contempla-
zione del mistero apofatico di Dio, da un certo punto di vista. In questo modo
si realizza una comprensione sinfonica di tutte queste prospettive teologiche e,
quindi, una comprensione comune, che è più corrispondente alla realtà divina. Il mistero infinito di Dio infatti, non può essere esaurito dai concetti e dalle com-
prensioni umane cristiane, ma viene percepito, sotto diversi aspetti, non solo dalle
singole persone, ma anche dalle diverse epoche storiche, come pure dalle diverse
comunità cristiane, nazionali e geografiche. Tutte queste diverse percezioni, che si
condizionano reciprocamente, ma senza uniformarsi, producono poi un adegua-
mento del linguaggio cristiano ai modi di percezione del mistero divino rivelato. Così si realizza un arricchimento della comprensione umana, pur rimanendo in
continuità e all’interno della stessa fede apostolica. Secondo Stăniloae, dunque,
nella prospettiva della cattolicità aperta, vengono valorizzate non solo le immagini
e le forme ecclesiali, ma anche quelle terrene. Le parole, le azioni e le immagini
utilizzate dalla Rivelazione sottolineano l’importanza che hanno le immagini,
le forme, i concetti e le parole umane nel conoscere Dio e la sua opera nel mondo,
come pure nel comprendere il contenuto profondo della Scrittura. Alla luce della
Rivelazione inoltre, anche l’uomo appare in tutta la sua ampiezza, in un continuo
movimento di attualizzazione e di superamento, quale immagine espressiva di Dio. 27 D. Stăniloae, Iubire și adevăr: pentru o depășire a dilemei ecumenismului contemporan pe mar-
ginea întâlnirii ecumeniste de la Taizé, “Ortodoxia” 19 (1967) 2, p. 288–290. Il tema di questo
incontro, a cui avevano preso parte, oltre ai circa 1200 giovani, anche rappresentanti della Chiesa
Cattolica, Ortodossa e Protestante, è stato dedicato alla riconciliazione tra le Chiese cristiane.
Alla fine dell’incontro, i giovani chiesero alle autorità ecclesiali e ai teologi delle rispettive Chiese
di trovare una soluzione positiva alle divergenze dogmatiche che impediscono la comune par-
tecipazione al banchetto eucaristico. Nella sua risposta, il cardinale Bea, allora presidente del
Secretariato per l’unità dei cristiani, ha affermato che la communicatio in sacris è strettamente
collegata all’unità nella fede e alla Chiesa, in quanto i sacramenti sono dentro la Chiesa e non
fuori di essa. Per questo motivo, la communicatio in sacris si può fare soltanto in casi eccezionali.
Prendendo spunto da questa dichiarazione, Stăniloae espone di seguito la posizione ortodossa
relativa al tema del’intercomunione, sottolineando soprattutto il legame tra l’amore e la verità. 29 “Este produsul celui mai patent spirit relativist și sincretist, al unei diplomații și porniri senti-
mentale tranzacționiste pe tema învățăturii despre Euharistie. Ea reflectă importanța margi-
nală, secundară acordată acestei învățături și practici în formațiile creștine de unde a apărut” –
D. Stăniloae, În problema intercomuniunii..., p. 561. 28 D. Stăniloae, În problema intercomuniunii, “Ortodoxia” 4 (1971), p. 561–584. 30 “Poți oare să fii în Hristos pe de o parte unit, pe de alta neunit cu alții? Poți să cugeți în pun-
cte importante altfel decât alții, aflându-te cu ei în același Hristos? Sau, poți să te afli deplin
în Hristos cu cei ce cugetă în multe privințe altfel decât tine despre El? În ce mai constă comu-
niunea cu ei în Hristos, o dată ce nu ai aceeași cugetare despre El cu cei cu care afirmi că ești în
El?”. Cf. D. Stăniloae, În problema intercomuniunii..., p. 562. “Noi non possiamo avere accesso
al Padre se non nello stato di sacrificio puro; ma non perveniamo a tale stato se non mediante
l’imprimersi in noi del sacrificio puro del Cristo, che ci permette di darci totalmente al Padre…
Così la comunione eucaristica spesso ripetuta nella vita terrena e l’ascesa spirituale realizzata
dalla nostra offerta al Cristo sempre più profonda e ininterrotta, nutrita dalla comunione eucari-
stica, ci conducono verso la comunione perfetta e pienamente svelata del Cristo nella vita futura,
dove la distinzione fra la comunione eucaristica con il Cristo e l’apice della nostra vita in lui ces-
serà interamente”. Cf. D. Stăniloae, La Liturgie de la communauté el la Liturgie intèrieure dans
la vision philocalique (Gestes et paroles dans les diverses familles liturgiques. Confèrences Saint
Serge XXV-c semaine d’étude, juillet 1977), Roma 1978, p. 270. 2. L’intercomunione eucaristica Il secondo tema che sintetizza la visione ecumenica del nostro autore riguarda
l’intercomunione eucaristica. Pur riconoscendo che il movimento ecumenico ha
il grande merito di aver riproposto il problema dell’unità tra le Chiese cristiane
e di aver stimolato il dialogo tra esse, Stăniloae sottolinea che, a volte, questo
desiderio verso l’unità ha generato in molti cristiani sia un entusiasmo facile,
secondo cui le differenze dottrinali tra le Chiese possono essere superate mediante 20 Zoltán József Bara il calore sentimentale dell’amore, sia uno spirito diplomatico, secondo cui l’unità tra
le Chiese può essere realizzata mediante un compromesso tra gli aspetti divergenti. Un caso significativo in questo senso è rappresentato appunto dall’intercomunione
eucaristica. Su questo tema, Stăniloae si è espresso inizialmente in un’articolo pubblicato
nel 196727, in cui commenta il tema dell’incontro internazionale della gioventù
(2–5 ottobre 1966) promosso dalla Comunità di Taizé. Uno studio più approfondito
su questo argomento è apparso nel 197128, in cui espone i motivi per cui la Chiesa
Ortodossa non può accettare, nello stadio attuale, la comunione eucaristica con
le altre Chiese cristiane. La tendenza verso l’intercomunione eucaristica rappresenta per Stăniloae il pro-
dotto del più chiaro spirito relativistico e sincretista, di una diplomazia e di una
mossa sentimentale transazionista sul tema della dottrina eucaristica. Essa riflette
l’importanza marginale, secondaria accordata a questa dottrina e pratica nelle
formazioni cristiane dov’è apparsa29. Prodotto di uno spirito di transazione, il concetto di intercomunione eucaristica
esprime, anzitutto, una grave contraddizione logica. Infatti, quale comunione
eucaristica, l’intercomunione dovrebbe essere una comunione piena; quale interco-
munione, però, essa è qualcosa di meno della comunione eucaristica piena, per cui
non può essere considerata come comunione eucaristica, perché i gruppi che sono
o vogliono essere in intercomunione eucaristica, non sono e non saranno in comu-
nione totale, così come viene concepita la comunione eucaristica. Puoi forse essere,
da una parte, unito in Cristo e, dall’altra parte, disunito con gli altri? Puoi pensare
in punti importanti diversamente dagli altri, essendo insieme con loro nello stesso La sinodalità e l’intercomunione in Dumitru Stăniloae 21 Cristo? Oppure, puoi essere pienamente in Cristo con quelli che pensano di lui
in molti aspetti diversamente da te? 31 “Prin Euharistie suntem adunați în mod real în El, că El ni se face prin Euharistie fundamen-
tul comun în care viețuim și factorul care crează în toți creștinii această simțire”. Cf. D. Stăni
loae, În problema intercomuniunii..., p. 562. 2. L’intercomunione eucaristica In che cosa consisterebbe la comunione con
essi in Cristo, dato che non hai la stessa concezione su di lui con quelli che affermi
che sei in lui?30 Una unione con Cristo, nonostante la differenza di concezione su di lui, si potrebbe
ammettere, afferma Stăniloae, soltanto nel caso di una comunione metanoetica,
vissuta nel sentimento. Ma anche in questo caso si presuppone la fede comune
in qualcosa di essenziale relativo a Cristo, la fede cioè che mediante l’Eucaristia
siamo radunati realmente in lui, che, per l’Eucaristia, egli diventa il fondamento
comune in cui viviamo e il fattore che crea in tutti i cristiani questo sentimento31. Questo tipo d’unità però, non si trova in quelli che tendono verso l’intercomunione,
in quanto manca il fondamento di una comunione nella fede. L’intercomunione che
si desidera realizzare in questo senso è piuttosto una comunione formale, che vuole
far convivere la comunione eucaristica con la non comunione cosciente e volontaria
in molti punti essenziali della fede. Perciò, la mossa sentimentale e diplomatica,
che ha lanciato l’idea e la pratica dell’intercomunione, ha creato una formula ibrida,
contradditoria in se stessa, che può essere accettata soltanto da una coscienza
addormentata, che non pensa fino in fondo al senso dell’atto che compie. Per una coscienza sveglia, invece, una comunione eucaristica reale e durabile non
è pensabile senza la comunione nella fede. Privi di questa coscienza, i sostenitori
dell’idea di intercomunione esprimono, quindi, un certo disprezzo, sia nei confronti
del tesoro della fede, sia nei confronti dell’Eucaristia. Infatti, se considerano che
l’intercomunione eucaristica è una comunione piena, essi mettono l’Eucaristia
al di sopra di ogni cosa e disprezzano la comunione nei punti importanti della 22 Zoltán József Bara fede. Ma siccome tra i punti di fede diversi fa parte anche l’Eucaristia, sulla quale
le Chiese hanno delle concezioni molto divergenti, si può dire piuttosto che questa
alternativa rappresenta un atteggiamento relativistico non solo riguardo al tesoro
della fede, ma anche nei confronti della dottrina eucaristica della propria Chiesa32. Se invece, ammettono che per l’intercomunione non si vuole realizzare una
comunione ontologica in Cristo, ma soltanto una comunione psicologica parziale,
quale mezzo per l’avvicinamento dottrinale tra le Chiese, essi disprezzano l’Euca-
ristia, mentre rispettano il tesoro della fede proprio di ogni Chiesa, con tutte le sue
differenze, dato che non possono rinunciare a queste differenze. 32 “Pun Euharistia mai presus de orice și disprețuiesc comuniunea în puncte importante ale cre-
dinței... Dar întrucât dintre punctele de credință deosebite face parte și Eucaristia, despre care
Bisericile au concepții foarte deosebite, se poate spune mai degrabă că această alternativă repre-
zintă o atitudine relativistă nu numai față de tezaurul de credință, ci și față de învățătura despre
Euharistie a propriei Biserici”. D. Stăniloae, În problema intercomuniunii..., p. 563. 33 “Ortodoxia, considerând Biserica drept o unitate profund și ontologic ancorată în Hristos, iar
Euharistia ca mijloc culminant de susținere continuă a acestei unități, nu poate coborî Euha-
ristia la nivelul unui mijloc de unitate laxă, psihologică și superficială între creștini. Aceasta
ar avea repercusiuni pentru însăși sensul ei despre Biserică... Căci ar fi greu să conceapă și să
practice Euharistia în două sensuri, ca având două roluri: într-unul când e primită împreună
cu membrii Bisericii și într-altul când e primită cu cei ce nu sunt membrii Bisericii Ortodoxe”.
Cf. D. Stăniloae, În problema intercomuniunii..., p. 564. 32 “Pun Euharistia mai presus de orice și disprețuiesc comuniunea în puncte importante ale cre-
dinței... Dar întrucât dintre punctele de credință deosebite face parte și Eucaristia, despre care
Bisericile au concepții foarte deosebite, se poate spune mai degrabă că această alternativă repre-
zintă o atitudine relativistă nu numai față de tezaurul de credință, ci și față de învățătura despre
Euharistie a propriei Biserici”. D. Stăniloae, În problema intercomuniunii..., p. 563.
33 “Ortodoxia, considerând Biserica drept o unitate profund și ontologic ancorată în Hristos, iar
Euharistia ca mijloc culminant de susținere continuă a acestei unități, nu poate coborî Euha-
ristia la nivelul unui mijloc de unitate laxă, psihologică și superficială între creștini. Aceasta
ar avea repercusiuni pentru însăși sensul ei despre Biserică... Căci ar fi greu să conceapă și să
practice Euharistia în două sensuri, ca având două roluri: într-unul când e primită împreună
cu membrii Bisericii și într-altul când e primită cu cei ce nu sunt membrii Bisericii Ortodoxe”.
Cf. D. Stăniloae, În problema intercomuniunii..., p. 564. 34 Stăniloae si è espresso in termini chiari contro l’intercomunione in assenza di unità dottrinale:
“Non posso capire come la comunione alla santa eucaristia possa compensare in qualche modo
la non comunione nella fede”. Ha sostenuto che l’ortodossia non è risultato dell’eucaristia, ma
che la vera eucaristia è risultato dell’ortodossia, cosicché la comunione eucaristica può aver luogo
soltanto in un contesto in cui si condivide la stessa fede. Cf. R. Bordeianu, Ospitalità eucari-
stica: un’analisi fenomenologica della recente teologia ortodossa, in: Il dono dell’ospitalità. Atti del
XXV Convegno ecumenico internazionale di spiritualità ortodossa (Bose, 6–9 settembre 2017),
a cura di L. Chremaschi, L.D’Ayala Valva, A. Mainardi, Magnano (BI) 2018, p. qui 271–272.
Le parole di Stăniloae in: T. Ware, Church and Eucharist, Communion and Intercommunion,
“Sobornost” 7 (1978), p. 558. 2. L’intercomunione eucaristica Alla base di questo
atteggiamento si trova difatti una diversa concezione sull’unità della Chiesa, come
pure sulla natura stessa della Chiesa, concezione che l’Ortodossia non può accettare. L’Ortodossia, considerando la Chiesa come un’unità profondamente e ontologi-
camente ancorata in Cristo, e l’Eucaristia come il mezzo culminante che sostiene
quest’unità, non può far scendere l’Eucaristia al livello di un mezzo di unità lassa,
psicologica e superficiale tra i cristiani. Questo avrebbe delle ripercussioni sulla
sua stessa concezione sulla Chiesa... Perché sarebbe difficile concepire e praticare
l’Eucaristia in due sensi, o come avente due ruoli: il primo quando è ricevuta insieme
con i membri della Chiesa, il secondo quando è ricevuta insieme con quelli che non
sono membri della Chiesa Ortodossa33. Nella concezione della Chiesa Ortodossa, sottolinea lui, l’unità della fede
e la convinzione che nell’Eucaristia ci si comunica al Corpo e al Sangue di Cristo
sono strettamente collegate con la convinzione che la Chiesa rappresenta un’unità
ontologica profonda dei fedeli con Cristo e tra di loro. Infatti, senza l’unità della
fede e senza la comunione con il Corpo e il Sangue di Cristo, non potrebbe esi-
stere la Chiesa nel senso pieno della parola, così come senza l’unità nella fede
e senza la Chiesa, non si può realizzare la comunione eucaristica. Nello stesso modo,
senza la Chiesa e senza la comunione eucaristica, l’unità della fede non è durabile. Perciò, per la Chiesa Ortodossa è inconcepibile un’intercomunione eucaristica, La sinodalità e l’intercomunione in Dumitru Stăniloae 23 che separa l’unità della fede dalla Chiesa, perché porta alla dissoluzione della Chiesa,
dell’unità della fede e dell’Eucaristia34. Tenendo conto di queste tre coordinate essenziali della vera e piena comunione tra
i cristiani in Cristo, Stăniloae esprime il suo disaccordo nei confronti di quei teologi
ortodossi, che, sotto l’influsso del movimento ecumenico, inclinano ad ammettere
che la Chiesa Ortodossa può dare, in certe circostanze, l’Eucaristia ai membri della
Chiesa Romano-Cattolica, della Chiesa Vecchio-Cattolica e delle Chiese Antiche
Orientali, in virtù del principio dell’economia, perché, secondo lui, ciò significa
un’interpretazione errata della nozione e della pratica dell’economia. Nella con-
cezione ortodossa, difatti, l’economia significa il riconoscimento dei sacramenti
(Battesimo, Confermazione, Matrimonio e Ordine) ricevuti da qualcuno fuori della
Chiesa Ortodossa, nel momento in cui diventa membro di questa Chiesa, e non
l’amministrazione di un sacramento a qualcuno che continua a rimanere membro
di un’altra Chiesa. 35 “În fața unei atitudini favorabile față de intercomunione e cazul să ne întrebăm: membrii unei
Biserici care se împărtășesc de Tainele altei Biserici, cărei Biserici aparțin?... Ei nu mai aparțin
deplin nici Bisericii lor, o dată ce primesc Euharistia de la altă Biserică, nici celei de la care pri-
mesc uneori Euharistia, pentru că nu au credința ei. Ei plutesc oarecum în spațiul spiritual 2. L’intercomunione eucaristica Perciò, la comunione sacramentale della rispettiva persona
con i membri della Chiesa Ortodossa ha come fondamento l’avvenuta comunione
nella fede con questi. In questo modo non si rompe il legame tra i sacramenti,
la Chiesa e l’unità di fede, come avviene invece nel caso dell’intercomunione. Di fronte ad un atteggiamento favorevole verso l’intercomunione, è il caso
di chiederci: i membri di una Chiesa che partecipano ai sacramenti di un’altra
Chiesa, a quale Chiesa appartengono? [...] Essi non appartengono pienamente
né alla loro Chiesa, dato che ricevono l’Eucaristia in un’altra Chiesa, né a quella
dove ricevono a volte l’Eucaristia, perché non hanno la sua fede. Essi galleggiano
in un certo senso nello spazio spirituale tra le due Chiese. Non avendo una fede
comune con i membri della Chiesa dove si comunicano, e non conservando più
una piena comunione nei sacramenti neanche con i membri della loro Chiesa, essi
hanno soltanto una comunione imperfetta con ciascuna delle due Chiese35. 35 “În fața unei atitudini favorabile față de intercomunione e cazul să ne întrebăm: membrii unei
Biserici care se împărtășesc de Tainele altei Biserici, cărei Biserici aparțin?... Ei nu mai aparțin
deplin nici Bisericii lor, o dată ce primesc Euharistia de la altă Biserică, nici celei de la care pri-
mesc uneori Euharistia, pentru că nu au credința ei. Ei plutesc oarecum în spațiul spiritual 24 Zoltán József Bara Per quanto riguarda l’intercomunione tra la Chiesa Ortodossa e la Chiesa Roma-
no-Cattolica, Stăniloae sostiene che questa non può essere proposta, finché non si
arriva all’unità della fede ortodossa, fondamento della piena comunione eucaristica. In questo senso, egli esprime la sua disapprovazione nei confronti degli esponenti
della Chiesa di Costantinopoli, che inclinano ad accettare la piena comunione
eucaristica con la Chiesa Romano-Cattolica, argomentando che, dopo la sospensione
reciproca della scomunica pronunciata nel 1054, le due Chiese si trovano, dal punto
di vista dogmatico, esattamente nella situazione anteriore al 1054 e quindi non
esiste più alcun ostacolo per il ristabilimento della comunione eucaristica. Di fronte
a quest’affermazione, dobbiamo ricordare che allora gli anatemi sono stati indetti
sulla base delle differenze apparse riguardo alla processione dello Spirito Santo
e all’inizio dell’affermazione del diritto di giurisdizione del Papa sulla gerarchia
orientale. E dopo questo, la divisione nella fede ha continuato con la formulazione
dogmatica del primato e dell’infallibilità papale, dell’immacolata concezione,
del purgatorio, ecc. 2. L’intercomunione eucaristica [...] Avendo solo pochi fedeli, gli esponenenti di quella Chiesa
autocefala si affrettano a rispondere positivamente all’offerta del Papa in questo
senso, in quanto non conoscono più la responsabilità verso le masse dei fedeli e non
si rendono conto che per una simile intercomunione eucaristica, rendono le masse
indifferenti nei confronti del rifiuto del primato papale e le portano sotto il giogo
del primato papale, oppure nell’uniatismo e nella divisione36. dintre cele două Biserici. Neavând o credință comună cu membrii Bisericii celelalte cu care se
împărtășesc, dar nemaipăstrând o comuniune deplină în Taine nici cu membrii Bisericii sale,
ei n-au decât o comuniune nedeplină cu fiecare Biserică”. Cf. D. Stăniloae, În problema inter-
comuniunii..., p. 565–566. 36 “Față de această afirmație, trebuie să amintim că anatemele s-au rostit atunci pe baza deosebirii
apărute în privința purcederii Duhului Sfânt și a începutului de afirmare a dreptului de jurisdi-
cție al papei asupra ierarhiei orientale. Iar de atunci încoace dezbinarea în credință a progresat,
prin formularea dogmatică a primatului și a infailibilității papale, a imaculatei concepții, a pur-
gatorului etc... Lipsită aproape total de credincioși, conducerea acelei Biserici Ortodoxe auto-
cefale, se grăbește să răspundă pozitiv unei oferte a papei în acest sens, întrucât nu mai cuno-
aște experiența răspunderii față de masele de credincioși și nu-și dă seama că printr-o astfel de
intercomuniune euharistică, făcându-le indiferente față de respingerea primatului papal, le-ar
arunca sub jugul primatului papal, sau în uniatism și dezbinare”. Cf. D. Stăniloae, În problema
intercomuniunii..., p. 568. Infatti, Dumitru Stăniloae si è espresso contro l’intercomunione in
assenza di unità dottrinale: “Non posso capire come la comunione alla santa eucaristia possa
compensare in qualche modo la non comunione nella fede”. Ha sostenuto che l’ortodossia non
è risultato dell’eucaristia, ma che la vera eucaristia è risultato dell’ortodossia, cosicché la comu-
nione eucaristica può aver luogo soltanto in un contesto in cui si condivide la stessa fede. Tale
conclusione deriva dall’ordo della divina liturgia in cui la comunità dapprima confessa la mede-
sima fede, e poi ha luogo l’epiclesi. Ma Stăniloae non vuole insinuare che la chiesa cattolica ha
perduto la vera fede a tal punto che la sua eucaristia non sia valida. dintre cele două Biserici. Neavând o credință comună cu membrii Bisericii celelalte cu care se
împărtășesc, dar nemaipăstrând o comuniune deplină în Taine nici cu membrii Bisericii sale,
ei n-au decât o comuniune nedeplină cu fiecare Biserică”. Cf. D. Stăniloae, În problema inter-
comuniunii..., p. 565–566. 2. L’intercomunione eucaristica Riconosce che la chiesa cat-
tolica professa la presenza del corpo e del sangue di Cristo nell’eucaristia e, ancor più, afferma 36 “Față de această afirmație, trebuie să amintim că anatemele s-au rostit atunci pe baza deosebirii
apărute în privința purcederii Duhului Sfânt și a începutului de afirmare a dreptului de jurisdi-
cție al papei asupra ierarhiei orientale. Iar de atunci încoace dezbinarea în credință a progresat,
prin formularea dogmatică a primatului și a infailibilității papale, a imaculatei concepții, a pur-
gatorului etc... Lipsită aproape total de credincioși, conducerea acelei Biserici Ortodoxe auto-
cefale, se grăbește să răspundă pozitiv unei oferte a papei în acest sens, întrucât nu mai cuno-
aște experiența răspunderii față de masele de credincioși și nu-și dă seama că printr-o astfel de
intercomuniune euharistică, făcându-le indiferente față de respingerea primatului papal, le-ar
arunca sub jugul primatului papal, sau în uniatism și dezbinare”. Cf. D. Stăniloae, În problema
intercomuniunii..., p. 568. Infatti, Dumitru Stăniloae si è espresso contro l’intercomunione in
assenza di unità dottrinale: “Non posso capire come la comunione alla santa eucaristia possa
compensare in qualche modo la non comunione nella fede”. Ha sostenuto che l’ortodossia non
è risultato dell’eucaristia, ma che la vera eucaristia è risultato dell’ortodossia, cosicché la comu-
nione eucaristica può aver luogo soltanto in un contesto in cui si condivide la stessa fede. Tale
conclusione deriva dall’ordo della divina liturgia in cui la comunità dapprima confessa la mede-
sima fede, e poi ha luogo l’epiclesi. Ma Stăniloae non vuole insinuare che la chiesa cattolica ha
perduto la vera fede a tal punto che la sua eucaristia non sia valida. Riconosce che la chiesa cat-
tolica professa la presenza del corpo e del sangue di Cristo nell’eucaristia e, ancor più, afferma La sinodalità e l’intercomunione in Dumitru Stăniloae 25 Con la Chiesa Vecchio-Cattolica invece, pur essendo più vicina, egli considera
che, anche in questo caso, la comunione eucaristica non si può realizzare finché
non viene firmata una dichiarazione comune sulla piena unità nella fede. Per quanto riguarda, infine, le Chiese Antiche-Orientali, secondo lui, l’inter-
comunione è ancora più facile, dato che tra queste Chiese e la Chiesa Ortodossa,
come hanno mostrato le comissioni teologiche miste, non sussiste più alcuna
differenza dottrinale essenziale, anzi tra esse c’è una profonda identità relativa alla
spiritualità e alla concezione sacramentale ed ecclesiologica. 37 “Tocmai pentru a păstra neslăbită conștiința supremei iubiri a lui Hristos față de lume nși putința
de a trăi această iubire pentru toți creștinii și pentru toți oamenii, tocmai pentru a nu goli Euha-
ristia de eficacitatea acestei supreme iubiri, Ortodoxia nu se învoiește cu tratarea Euharistiei
ca o ceremonie mai mult simbolică, ca o prezență echivocă și insuficient de eficace a trupului
lui Hristos, ca o ceremonie cu o semnificație mai mult teoretică și sentimentală, despărțită mai
mult sau mai puțin de prezența reală a trupului lui Hristos și a energiilor lui dumnezeiești; toc-
mai de aceea ea nu se învoiește să considere împărtășania comună ca pe un act lipsit de supor-
tul unei credințe comune, lipsit de calitatea de bază a unei înaintări moralascetice în sfințenie, che la chiesa cattolica, così come la chiesa ortodossa, ha conservato la fede nella Trinità e in Cri-
sto, anche se ha aggiunto all’antica fede della chiesa il dogma del primato e dell’infallibilità del
papa, il Filioque e il purgatorio. Tra tutti questi dogmi, quello del primato del papa e della sua
infallibilità sono ostacoli alla comunione, e dividono la chiesa. Cf. T. Ware, Church and Eucha-
rist..., p. 558; D. Stăniloae, Spiritualitate şi comunione în Liturghia Orthodoxă, Craiova 1986,
p. 389–399 e 401–402. che la chiesa cattolica, così come la chiesa ortodossa, ha conservato la fede nella Trinità e in Cri-
sto, anche se ha aggiunto all’antica fede della chiesa il dogma del primato e dell’infallibilità del
papa, il Filioque e il purgatorio. Tra tutti questi dogmi, quello del primato del papa e della sua
infallibilità sono ostacoli alla comunione, e dividono la chiesa. Cf. T. Ware, Church and Eucha-
rist..., p. 558; D. Stăniloae, Spiritualitate şi comunione în Liturghia Orthodoxă, Craiova 1986,
p. 389–399 e 401–402. ca pe un act lipsit de puterea care unește într-o Biserică pe cei ce îl practică”. Cf. D. Stăniloae,
În problema intercomuniunii..., p. 582.
38 “Membrii Bisericii Ortodoxe sunt chemați să se comporte ca mâinile lucrătoare ale lui Hristos,
ca ochii înțelegători ai lui Hristos, ca urechile atente ale lui Hristos, ca inima iubitoare a lui
Hristos față de toți, față de toate trebuințele și chemările altora, dovedind cu fapta că sunt măd-
ularele lui Hristos”. Cf. D. Stăniloae, În problema intercomuniunii..., p. 582. 2. L’intercomunione eucaristica Nonostante questo,
egli afferma che, prima di una dichiarazione ufficiale, firmata dai sinodi locali delle
due Chiese riguardo all’unità della fede, non si può ammettere che una comunione
sacramentale parziale. Il rifiuto netto dell’intercomunione con le altre Chiese cristiane, non significa
però una mancanza d’amore della Chiesa Ortodossa nei confronti degli altri cristiani? Secondo Stăniloae, questo rifiuto della Chiesa Ortodossa non dev’essere inteso
come mancanza d’amore, ma come testimonianza a favore del vero amore di Cristo: Proprio per conservare forte la coscienza dell’amore supremo di Cristo per
l’umanità, come pure la possibilità di vivere questo amore per tutti i cristiani e per
tutti gli uomini, proprio per non svuotare l’Eucaristia dall’efficacia di quest’amore,
l’Ortodossia non accetta di trattare l’Eucaristia come una cerimonia simbolica, come
una presenza equivoca ed insufficientemente efficace del Corpo di Cristo, come una
cerimonia con un significato teoretico e sentimentale, separata più o meno dalla
presenza reale del Corpo di Cristo e dalle sue energie divine; proprio per questo
essa non accetta di considerare la comunione come un atto privo del supporto delle
fede comune, privo della qualità fondamentale per un progresso morale e ascetico
nella santità, come un atto privo della forza che unisce in una Chiesa tutti coloro
che lo praticano37. 26 Zoltán József Bara Secondo Stăniloae, dunque, la Chiesa Ortodossa respinge l’intercomunione
eucaristica con le altre Chiese cristiane, perché vuole conservare l’Eucaristia come
il nucleo supremo e inesauribile, da dove irradia l’amore di Cristo. Ciò non signi-
fica una chiusura in se stessa, oppure una diminuzione della sua tensione verso
l’universalità; ma l’amore che unisce i suoi membri nel Corpo mistico di Cristo,
essendo alimentato dall’Eucaristia, si irradia da questi verso tutti i cristiani. Diventati
membra del Corpo di Cristo, i cristiani ortodossi possono perciò esprimere in tante
altre forme il loro amore verso gli altri cristiani: I membri della Chiesa Ortodossa sono chiamati a comportarsi come le mani
operose di Cristo, come gli occhi comprensivi di Cristo, come gli orecchi attenti
di Cristo, come il cuore amorevole di Cristo verso tutti e verso tutte le necessità
e le vocazioni degli altri, manifestando praticamente che sono membra di Cristo38. ca pe un act lipsit de puterea care unește într-o Biserică pe cei ce îl practică”. Cf. D. Stăniloae,
În problema intercomuniunii..., p. 582. Conclusione Stăniloae ha definito le coordinate dell’ecumenismo dal punto di vista ortodosso. La prima coordinata, quale non abbiamo discusso in questo brano si riferisce
all’unione di tutte le Chiese nell’integrità della fede apostolica, fede vissuta dalla
Chiesa indivisa del primo millennio cristiano. La fede apostolica include gli scritti,
la tipologia e la tradizione apostolica. Custode della fede apostolica è la Chiesa, per cui
alla fede apostolica appartiene anche la dimensione istituzionale della Chiesa, cioè
la vita di comunione con Dio mediante i sacramenti celebrati dai ministri ordinati. La piena valorizzazione della fede apostolica si realizza però mediante l’unità
cattolico-sinodale della Chiesa. L’unità cattolico-sinodale della Chiesa è una unità
dogmatica, che concilia in una misteriosa e indissolubile tensione l’unità con la diver-
sità. Perciò, la seconda coordinata dell’ecumenismo riguarda l’unità dogmatica nella
diversità e nell’indipendenza delle Chiese locali. In questo senso, l’unità delle Chiese
si realizza attraverso la loro unione nella verità rivelata, e non attraverso una unione
di tipo politico o amministrativo. Infine, la terza coordinata consiste nell’aper-
tura delle Chiese al mondo. Il motivo di quest’apertura deriva dal fatto che Dio
agisce anche al di là delle frontiere visibili della Chiesa. Riconoscendo che Dio agisce
non solo per mezzo della Chiesa sul mondo, ma anche per mezzo del mondo sulla
Chiesa, le Chiese cristiane comprendono in una nuova luce non solo la Rivelazione, La sinodalità e l’intercomunione in Dumitru Stăniloae 27 ma anche la creazione, l’Incarnazione del Figlio di Dio, la dimensione cosmica
della salvezza, la comunità umana e la cattolicità-sinodale della Chiesa. In questo
modo si realizza anche un’avvicinamento dottrinale tra esse. Alla luce di questi
principi ecumenici, Stăniloae ha pronosticato una sinodalità o cattolicità aperta,
quale apertura dogmatica e spirituale delle Chiese cristiane per ritrovare la loro
unità. Alla base di questa cattolicità aperta si trova il fatto che nella Sacra Scrittura
c’è una diversità di tradizioni e significati, diversità che proviene dalla diversità delle
azioni di Dio nella storia di salvezza. Se l’approccio unilaterale ai sensi della Scrittura
è stato nel passato causa di divisione tra i cristiani, l’approccio cattolico-sinodale
a tutti i sensi della Scrittura può diventare il punto d’incontro tra tutti i cristiani. Per arrivare a questo punto d’incontro, le Chiese cristiane devono quindi evitare
di attaccarsi ad uno o più di questi sensi, perché essi rappresentano solo la realtà
penultima. 39 R. Bordeianu, Dumitru Staniloae: An Ecumenical Ecclesiology, Edinburgh 2013, p. 29.
40 D. Stăniloae, Coordonatele ecumenismului din punct de vedere ortodox, in “Ortodoxia” 19
(1967) 4, p. 494–540. D. Stăniloae, Coordonatele ecumenismului din punct de vedere ortodox, in “Ortodoxia” 19
(1967) 4, p. 494–540. 39 R. Bordeianu, Dumitru Staniloae: An Ecumenical Ecclesiology, Edinburgh 2013, p. 29. Conclusione Quello che le Chiese cristiane devono cercare, è invece la realtà ultima,
cioè Dio infinitamente ricco, che si manifesta in diversi modi, ma che prosegue
un unico scopo: la ricapitolazione di tutte le cose in Cristo. Di grande aiuto per
la realizzazione dell’approccio cattolico-sinodale sono le critiche reciproche delle
Chiese, come anche la promozione di un sano pluralismo teologico.39 Nel desiderio di superare le differenze dogmatiche che separano le Chiese e per
arrivare alla piena comunione eucaristica, molti – secondo Stăniloae – sono caduti
in un entusiasmo facile, pensando che le differenze possano essere superate con
il calore dell’amore, oppure in uno spirito diplomatico, realizzando un compromesso
tra le posizioni divergenti. Il risultato di queste due tendenze, che hanno alla base
un diversa concezione ecclesiologica, è conosciuto con il termine di intercomunione
eucaristica. Secondo Stăniloae, l’intercomunione eucaristica, oltre ad essere l’espres-
sione di una mentalità relativista e transazionista, comporta una contraddizione
logica, perché l’intercomunione non è comunione eucaristica piena e, quindi,
i gruppi che vogliono essere in intercomunione non possono essere in comunione
totale, così come invece viene concepita la comunione eucaristica. La comunione con
il Corpo e il Sangue di Cristo non può essere separata dall’unità nella fede e dalla
convinzione che la Chiesa rappresenta una profonda unità ontologica dei fedeli
in Cristo e tra essi. Questi tre elementi sono inseparabili, e non si può avere uno
senza l’altro. Per questo motivo, egli non è d’accordo con quelli che propongono
l’intercomunione eucaristica tra la Chiesa Ortodossa e le altre Chiese cristiane,
senza arrivare prima ad un accordo dogmatico40. Il rifiuto dell’intercomunione 28 Zoltán József Bara Zoltán József Bara non dev’essere però visto come una mancanza d’amore nei confronti di queste
Chiese, ma come una testimonianza a favore dell’Eucaristia, quale nucleo supremo
e inesauribile da dove irradia l’amore di Cristo. In 50 anni il dialogo tra le Chiese
si è approfondito e arricchito di molti argomenti. Comunque le tesi di Stăniloae
sono ancora stimolanti, specialmente quella sulla sinodalità aperta. Però, se biso-
gna accettare le contraddizioni, come sostiene nella sinodalità aperta, perché non
accettarle anche nella intercomunione? Bibliografia Stăniloae, D., În problema intercomuniunii, “Ortodoxia” 4 (1971), p. 561–584. Stăniloae D., Sobornicitate deschisă, “Ortodoxia” 23 (1971) 2, p. 165–180. Stăniloae D., La Liturgie de la communauté el la Liturgie intèrieure dans la vision philoca-
lique (Gestes et paroles dans les diverses familles liturgiques. Confèrences Saint Serge
XXV-c semaine d’étude, juillet 1977), Roma 1978. Stăniloae, D., Dogmatica I, București 2003. Stăniloae, D., Dogmatica I, București 2003. Stăniloae D., Dogmatica II, București 2003. Stăniloae D., Spiritualitate şi comunione în Liturghia Orthodoxă, Craiova 1986. Theologen des 20. Jahrhunderts. Eine Einführung, a cura di P. Neuner, G. Wenz, Darmstadt
2002, p. 145–156. Turcescu L., Eucharistic Ecclesiology or Open Sobornicity?, in: Dumitru Stăniloae: Tradi-
tion and Modernity in Theology, a cura di L. Turcescu, Palm Beach 2002, p. 83–103. Ware T., Church and Eucharist, Communion and Intercommunion, “Sobornost” 7 (1978),
p. 550–567. Bibliografia Bara Z., Un approccio ortodosso ecclesiologico alla dimensione sacramentale della Chiesa:
la Chiesa, Sacramento di Cristo, secondo Dumitru Stăniloae, in: Sakramenty nowym
wyzwaniem dla Kościoła Katolickiego, a cura di J. Radkiewicz, Szczecin 2019, p. 155–180. Bara Z., La realtà mistica della Chiesa nella riflessione ortodossa di Dumitru Stăniloae,
“Studia Theologia Catholica Latina” 66 (2021) 2, p. 77–107. Bordeianu R., Dumitru Staniloae: An Ecumenical Ecclesiology, Edinburgh 2013. Bordeianu R., (In)Voluntary Ecumenism: Dumitru Stăniloae’s Interaction with the West
as Open Sobornicity, in: Orthodox Constructions of the West, a cura di E.G. Demaco-
poulos, A. Papanikolaou, New York 2013, p. 240–253. Bordeianu R., Ospitalità eucaristica: un’analisi fenomenologica della recente teologia
ortodossa, in: Il dono dell’ospitalità. Atti del XXV Convegno ecumenico internazionale
di spiritualità ortodossa (Bose, 6–9 settembre 2017), a cura di L. Chremaschi, L.D’Ayala
Valva, A. Mainardi, Magnano (BI) 2018, p. 263–285. Coman V., “Open Sobornicity” and “Receptive Ecumenism”: Fruitful Models of Ecumenical
Interaction, in: Just Do It? Recognition and Reception in Ecumenical Relations: Procee-
dings of the 19th Academic Consultation of the Societas Oecumenica, a cura di D. Heller,
M. Hietamaki, Leipzig 2018, p. 241–251. Coman V., Le Saint Esprit comme liaison de l’amour éternel entre le Père et le Fils: un cas
de ‘sobornicité ouverte’ dans la théologie orthodoxe, “Irénikon” 89 (2016) 1, p. 25–51. Moşoiu N., “Sobornicitatea deschisă” ca tipologie ecumenică sau De la teologia confesională
la teologia ecumenică în perspectiva viziunii despre “sobornicitatea deschisă”, “Revista
Teologică” 4 (1997), p. 87–117. Nouveauté dans l’oecumenisme, Taizé 1968. Nouveauté dans l’oecumenisme, Taizé 1968. Roberson G.R., Ecumenism in the Thought of Dumitru Stăniloae, in: T. Damian, G. Alexe,
E. Pentiuc, R. Roberson, J. Jonita, L. Turcescu, The Theological Legacy of Fr. Dumitru
Stăniloae and its Ecumenical Actuality, Symposium VI/1, New York 1999, p. 43–54. Stavrou M., Teologia cristiana ortodossa, in: Dizionario del sapere storico-religioso del
Novecento, a cura di A. Melloni, Bologna 2010. Stăniloae D., Sinteză eclesiologică, “Studii Teologice” 7 (1955) 5–6, p. 267–284. La sinodalità e l’intercomunione in Dumitru Stăniloae 29 Stăniloae D., Coordonatele ecumenismului din punct de vedere ortodox, „Ortodoxia” 19
(1967) 4, p. 494–540. Stăniloae D., Coordonatele ecumenismului din punct de vedere ortodox, „Ortodoxia”
(1967) 4, p. 494–540. Stăniloae D., Iubire și adevăr: pentru o depășire a dilemei ecumenismului contemporan
pe marginea întâlnirii ecumeniste de la Taizé, “Ortodoxia” 19 (1967) 2, p. 288–290. Stăniloae D., Transparenţa Bisericii în viaţa sacramentală, “Ortodoxia” 22 (1970) 4,
p. 501–516. Sommario Nell’opera di Dumitru Stăniloae esiste una grande varietà di temi e problemi ecumenici, in cui
egli ha assunto delle posizioni originali, a volte radicali, ma sempre favorevoli al dialogo. In questo brano vorrei presentare specialmente due temi che, in un certo senso, sintetizzano
la visione ecumenico originale di Stăniloae sull’unione delle Chiese: il tema della cattolicità
o sinodalità aperta, e quella dell’intercomunione eucaristica, seguendo i suoi argomenti. Stăniloae
ha pronosticato una sinodalità o cattolicità aperta, quale apertura dogmatica e spirituale delle
Chiese cristiane per ritrovare la loro unità. Alla base di questa cattolicità aperta si trova il fatto
che nella Sacra Scrittura c’è una diversità di tradizioni e significati, diversità che provengono
dalla diversità delle azioni di Dio nella storia di salvezza. L’approccio cattolico-sinodale a tutti
i sensi della Scrittura può diventare il punto d’incontro tra tutti i cristiani. Dall’altra parte, nella visione di Stăniloae, l’intercomunione eucaristica deve essere una comu-
nione eucaristica piena. L’Ortodossia, considerando la Chiesa come un’unità profondamente 30 Zoltán József Bara e ontologicamente ancorata in Cristo, e l’Eucaristia come il mezzo culminante che sostiene
quest’unità, non può far scendere l’Eucaristia al livello di un mezzo di unità lassa, psicologica
e superficiale tra i cristiani. Infatti, senza l’unità della fede e senza la comunione con il Corpo
e il Sangue di Cristo, non potrebbe esistere la Chiesa nel senso pieno della parola, così come
senza l’unità nella fede e senza la Chiesa non si può realizzare la comunione eucaristica. È inconcepibile un’intercomunione eucaristica che separa l’unità della fede dalla Chiesa, perché
porta alla dissoluzione della Chiesa, dell’unità della fede e dell’Eucaristia. La comunione con
il Corpo e il Sangue di Cristo non può essere separata dall’unità nella fede e dalla convin-
zione che la Chiesa rappresenta una profonda unità ontologica dei fedeli in Cristo e tra essi. Parole Chiave: Dumitru Stăniloae, ecumenismo, cattolicità aperta (sinodalità), unione delle
Chiese, intercomunione eucaristica Nota autorska Zoltán József Bara – duchowny rzymskokatolicki, adiunkt na Wydziale Teologii Rzym-
skokatolickiej Uniwersytetu Babeșa i Bolyaia w Cluj-Napoca w Rumunii oraz na Wydziale
Rzymskokatolickiej Teologii Pastoralnej w Alba Iulia (Seminarium Incarnatae Sapientiae);
e-mail: barazoli@gmail.com; zoltan.bara@ubbcluj.ro. Bara Z.J., La sinodalità e l’intercomunione in Dumitru Stăniloae, „Colloquia Theologica Otto-
niana” 38 (2022), s. 9–30. DOI: 10.18276/cto.2022.38-01. Cytowanie Bara Z.J., La sinodalità e l’intercomunione in Dumitru Stăniloae, „Colloquia Theologica Otto-
niana” 38 (2022), s. 9–30. DOI: 10.18276/cto.2022.38-01.
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Transduction and BRICS
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CDD: 337.1 CDD: 337.1 DOI: http://dx.doi.org/10.1590/0100-6045.2015.V38N3.DP DOI: http://dx.doi.org/10.1590/0100-6045.2015.V38N3.DP Abstract: BRICS has philosophical significance. It creates new pressure on
cross-cultural skill. This is analysed here as requiring transduction: a variety of
defeasible practical reasoning. This replaces a simplistic model of the relation between
knowledge and action with a more realistic and contemporary model. The transduction
format has utility in cross-cultural training. Keywords: BRICS; cross-cultural skill; context; executive function; schema theory. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. Don Peterson Shantou University
Department of Computer Science
Da Xue Lu, 243, Guangdong, 515063, China. 中国广东省汕头市大学路243号汕头大学计算机系 | 邮编:515063
peterson@stu.edu.cn Shantou University
Department of Computer Science
Da Xue Lu, 243, Guangdong, 515063, China. 中国广东省汕头市大学路243号汕头大学计算机系 | 邮编:515063
peterson@stu.edu.cn Shantou University
Department of Computer Science
Da Xue Lu, 243, Guangdong, 515063, China. 中国广东省汕头市大学路243号汕头大学计算机系 | 邮编:515063
peterson@stu.edu.cn 中国广东省汕头市大学路243号汕头大学计算机系 | 邮编:515063
peterson@stu.edu.cn Received: 17.01.2015; Revised: 22.10.2015; Accepted: 30.12.2015 BRICS BRICS is a collaboration between Brazil, Russia, India, China, and
South Africa: it was identified as a possibility by O'Neill (2001), official
discussion began in New York in 2006, and the bloc was formally initiated in
Yekaterinburg in 2009. Since 2010, the BRICS nations have met annually at
formal summits. Bilateral relations among BRICS nations emphasise mutual
'win-win' benefit, non-interference, and equality. There are other blocs: ASEAN (Association of Southeast Asian
Nations), initiated in 1967, now includes Indonesia, Malaysia, the Philippines,
Singapore, Thailand, Brunei, Cambodia, Laos, Myanmar, and Vietnam. O'Neill,
who first identified BRICS, has identified: MIKT (Mexico, Indonesia, South
Korea, and Turkey), MINT (Mexico, Indonesia, Nigeria, and Turkey), and the Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. 16 Don Peterson Next Eleven (Bangladesh, Egypt, Indonesia, Iran, Mexico, Nigeria, Pakistan,
the Philippines, Turkey, South Korea, and Vietnam). Next Eleven (Bangladesh, Egypt, Indonesia, Iran, Mexico, Nigeria, Pakistan,
the Philippines, Turkey, South Korea, and Vietnam). These blocs are, of course, an issue for economics and politics
(Gutemberg, 2015). They are also an issue and direction for contemporary
philosophy. The world has changed, life has changed, and the challenge to our
cognitive apparatus has changed. There may be 'nothing new under the sun',
but the need to interact and collaborate with 'the other' is greater now than
before. In the past, of course, some people needed to interact internationally,
but the obligation is greater now. The modern condition in the 21st Century is
one of cooperating heterogeneity, in which we keep our differences, but we
have to collaborate. In the past, we had our differences (in culture, language,
religion, cuisine, legal systems, business practice, art, etc.) but we could ignore
each other, or not take each other seriously, even under colonialism. In the
present, we have our differences but we cannot ignore each other. There has
occurred, in fact, a sea change in the demands on human cognition. This may
be a change in degree rather than principle, but it is a sea change. The emergence of blocs such as BRICS makes two things clear in the
problematic of the philosophy of globalisation. The first is that we will not
homogenise. We will not acquire one unified world system of politics, religion,
culture, law, ethics, cuisine, language, art, etc. These blocs are collaborations,
they are not melting pots. The second is that we have to collaborate. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. TRANSDUCTION and BRICS17 A key point here is that what we need to achieve is win-win
collaboration. We do not have to become the other, be like the other, agree
with the other, nor like everything about the other. In fact, collaboration may
be best if we retain our difference. Retaining our difference, we may have more
to offer each other. This casts a sceptical philosophical light on some 'dead'
metaphors. We talk of 'seeing' the other's point of view, 'knowing' their culture,
putting ourselves 'in their shoes', and so on. Is this helpful? Is this accurate? The actual objective is synergy. The Greek etymology of the word 'syn-ergy' is
'together-work', and this is the point. Under BRICS, under the other blocs,
under cooperating heterogeneity, we need to do this. In short, the main issue is
action. We need to do the right thing, and avoid doing the wrong thing, in
complex situations. Of course, knowledge matters. We need to know etiquette. We need to
know that the other culture likes this and does not like that. Even better, we
know their history and some reasons for their preferences. Even better, we
grow to appreciate some of their gems, such as Brazilian dance, Russian bass,
Indian yoga, Chinese tea, or South African music. But knowledge is not
enough. We need adaptive action. At home, we might do one thing. In our own
culture, we might do things a certain way. But in a trans-national working
situation we may not have this simple option. We may need to adjust to the
additional factor of the other culture. This is inherently more complex than
operating within our own culture, but it is a feature of modern life under
BRICS and other blocs, where it is now clear that we will not homogenise, and
we must collaborate. The issue of adaptive action is therefore prioritised in the modern age. The basic task is to retain our integrity while working with the other. As is well
known, expatriate maturity involves a conjunction of these two factors. Progress in this is illustrated as a U-curve moving through initial elation, culture
shock, and then differentiated adjustment (Adler and Gundersen, 2007). Of
course, we acquire knowledge of the other culture as we progress. Of course
this may be enlightening, and we may change as a result. But in the end
intercultural business relationships are collaborative rather than synthetic. BRICS Ignoring
other cultures is no longer an option. Disrespecting other cultures is no longer
an option. International business and multinational companies have become a
norm rather than an exception. Accordingly, a service industry in cross-cultural skill has arisen, and is
growing fast. In international business, management, and human resources
management, cross-cultural skill is now seen as crucial. The field is new, and
several names are currently in use: 'cross-cultural' may become 'inter-cultural',
'skill' may become 'communication', 'competence', or 'capability', etc. Countries
such as those in BRICS need to do business with each other on a routine basis. Collaboration is essential. Multinational teams are normal (Halverson and
Tirmizi, 2008). Expatriate failure is expensive (Chew, 2004). As a live area of
applied psychology, support for cross-cultural skill is now supplied worldwide
in the forms of training, consultancy, and psychometric testing (Livermore,
2015; Hofstede and Minkov, 2010; Ang and Van Dyne, 2008). Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. TRANSDUCTION and BRICS17 Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. TRANSDUCTION and BRICS17 We
do not really become a member of the other culture, but we create productive
synergy with members of the other culture. We now introduce the concept of transduction as an analysis of
adaptive action. This does not pretend to solve all the problems of the modern 18 Don Peterson TRANSDUCTION and BRICS19 Transduction is a form of adjusted action. In neuropsychology, this is
the second function identified by Norman and Shallice. In logic, it is a form of
defeasible practical reasoning (reasoning to a conclusion on action, which is
influenced by context which can change our decision). We start with an example. [E1] You have a standard route to work. This includes walking down a
particular pavement. Today, however, you see a big puddle of water covering
the width of the pavement. So on this occasion, you stop, walk round on the
road, and continue after the puddle. Your feet stay dry, and nothing is
disturbed. (Tomorrow, you find no puddle, and you return to your usual
enactment.) We see here a pattern of four components: [S] A basic schema, which generates standard or routine action. [S] A basic schema, which generates standard or routine action. [D] A default action, which would normally be performed, in the
absence of anything to the contrary. [S] A basic schema, which generates standard or routine action. [D] A default action, which would normally be performed, in the
absence of anything to the contrary. [D] A default action, which would normally be performed, in the
absence of anything to the contrary. [D] A default action, which would normally be performed, in the
absence of anything to the contrary. [C] A context, comprising circumstances relevant to the consequences
of our performed action. [V] A variant action, this being a version of [D] whose details of
enactment are adjusted to [C]. [V] A variant action, this being a version of [D] whose details of
enactment are adjusted to [C]. This is a basic case, but it reveals a reasoning format which is often
required. We now consider a cross-cultural example. [E2] A group of long-term resident expatriates in Dubai planned to
visit a museum at 2.00 pm on a Wednesday. The sign on the museum wall says
that it is open at this time. On arrival, they find that the door is shut, and a man
comes out and tells them to go away and come back later. The expatriates
become angry. They do in fact have the necessary knowledge. They know that
this is the month of Ramadan, which involves extra and obligatory prayer times
for Muslims, which take priority over work. Transduction We turn first to the human brain. We turn to the basic equipment
which we have. Work in neuropsychology on 'executive function' has
established that our performance (behaviour, action) results from two factors,
not one. Our performance results from a combination of knowledge (in the
form of schemas) and executive function (control over the enactment of
schemas) (Elliott, 2003). Piaget (1952) defined a schema as 'a cohesive,
repeatable action sequence possessing component actions that are tightly
interconnected and governed by a core meaning'. Schemas are rule-of-thumb
formulae for action in standard situations. They tell us what to do normally, all
things being equal. The first challenge here is that we need to select which schema to
activate at any given time. We cannot do everything at once, and a juggling act
is needed. We need to control when we start, when we stop, and when we
temporarily suspend our schemas. Norman and Shallice (1986) attribute this
function to 'contention scheduling'. This capability is associated with the
prefrontal cortex. This area is at the top, and at the front of the human brain. This is thought to be only an association: executive function relies also on other
areas of the brain, and this area of the brain does other things too (Alverez et
al, 2006). Nevertheless, it is a strong association. The second challenge is that, having selected a schema, we may need to
modulate its enactment. In standard, routine situations, we may not need to do
this, but in novel situations, we may need to produce adjusted action. We may
need to adjust how, where, when, and with whom we do things and say things,
which may be crucial to success. Norman and Shallice recognise this as a
function of the 'Supervisory Attention System' (SAS). The point for BRICS and
the age of globalisation, is that 'novel' situations have become normal in
international business. This is not a challenge which happens once a year. It is a
challenge which happens every day. We rely on standard schemas, formulae,
principles, and routines: but we also need to steer their enactment. We need to
steer the detail of how we do what we do, in response to the other party, the
other culture. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. Situation Table for example [E2]. [S] The signs say that the museum will be open at 2.00 pm on
nesday but it is not open [S] The signs say that the museum will be open at 2.00 pm on
ednesday, but it is not open. Wednesday, but it is not open. TRANSDUCTION and BRICS19 It takes them some time to engage
this knowledge of context [C] with their default response [D] of getting angry,
so as to produce a practical variant [V] in which they retain their plan to visit Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. 20 Don Peterson the museum [S] but adjust its details of enactment, so as to go away and return
at 3.00 pm as suggested. (In future, when it is not Ramadan, they rely on the
sign as before.) This example introduces several new factors: mental states, culture,
planning, etc. Our claim, though, is that these are details, and that the basic
pattern of transduction remains the same. Both of our examples can be
expressed in 'situation tables', which use the pattern of four components
identified above, as follows. Situation Table for example [E1]. Situation Table for example [E1]. Situation Table for example [E1]. [S] I have a standard route to work. [D] I walk straight down the pavement. [C] Today there is a big puddle across the pavement. [V] Stop, walk round the puddle, then continue. Situation Table for example [E2]. Situation Table for example [E2]. TRANSDUCTION and BRICS21 TRANSDUCTION and BRICS21 conclusion is an action). So we might speak here of 'contrapragma' ('against
circumstances'), rather than 'contradiction' ('against speaking'). The affinity with
Da Costa's thinking is that an opposition (whether 'contrapragma' or
'contradiction') between [C] and [D] may be a constructive synergy. We attempt here to explicate constructive synergy. What is articulated
is a process of working with the other. This is not a process of identifying with
the other, agreeing with the other, or becoming the other. If these things come,
then well and good: but they will be additions. The basic function is
collaboration. In structural terms, [D] is changed to [V] under the influence of
[C]. We see a reasoning format here which is under new pressure in the 21st
Century. It is normal, it is common sense, it is not new. It is basic, it does not
always work, and its articulation in situation tables is useful in cross-cultural
training. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. Wednesday, but it is not open. Wednesday, but it is not open. [D] Get angry. [C] This is the month of Ramadan, when Muslims have obligatory
extra prayers. [C] This is the month of Ramadan, when Muslims have obligatory
extra prayers. [V] Go away, and come back later. [V] Go away, and come back later. [V] Go away, and come back later. This pattern of reasoning is an addition to that identified in classical
deductive logic. The pattern there is a one-step, monotonic (non-defeasible)
process. That is: however complex the derivation, we deal with one set of
premises and one conclusion, and introduction of new premises cannot cause
retraction of a conclusion. Here we deal with two steps, [S/D] and [C/V], and
we derive [V] as a version of [D]: not as a simple rejection of [D] or revision of
[S], as identified in classical non-monotonic logic. Da Costa (1958, 1982),
makes a related and illuminating point in paraconsistent logic. Da Costa's
point is that inconsistency is not always a disaster. A database, for example, can
be contradiction-tolerant, as is often needed in business systems. Da Costa is
concerned with propositional reasoning (in which our conclusion is a
statement), and we are concerned here with practical reasoning (in which our Discussion For the cross-cultural skill industry, then, we suggest that situation
tables, as above, backed up by the analytical framework of transduction, with its
four basic components, are useful in training. These provide a simple, graphical
way of explaining cross-cultural situations, and the ways in which we succeed or
fail to adjust to their circumstances. For philosophy, in the age of BRICS, we need to abandon a model
introduced by Socrates and expressed by Plato. On this model, right action
(arete) is produced by right knowledge (episteme). In the early Dialogues,
unsuccessful attempts are made to produce super-schemas (logoi), and in the
later Dialogues, these are sent to heaven as 'Platonic Forms' (eidoi) (Plato,
429-347 BCE/1997). On this model, action is determined by a single unit of
knowledge. The better this unit is, the better the probability that we produce
right action. In terms of schema theory, we seek super-schemas, which
guarantee right action. For cross-cultural action, we might seek super-schemas
which encompass all cross-cultural situations. We might develop perfect units
of knowledge, which tell us what to do internationally. We could file these in a
Library of Super-Schemas, thus assisting education and international
understanding. Just as the Ten Commandments tell us how to act, and the 22 Don Peterson Highway Code (DVSA, 2015) tells us how to drive, so this Library would tell us
what to do in international business. Of course this is naive. We need the Ten
Commandments, we need the Highway Code, we need principles and formulae:
but these are subject to transduction. j
Can we ever escape from transduction? Let us take a simple case: the
game of 'noughts and crosses' (or 'tic-tac-toe'). As Zaslavsky (1982) explains,
the game is determinate. That is: there is an algorithm for perfect play,
producing a draw or win. There are 765 different positions, and 26,830 possible
games. If we know this algorithm, we cannot lose. We have a super-schema. Consider, though, the situation where we are in the middle of a business deal
with a person of vanity who does not like to lose any game. It might then be
expedient to lose deliberately. We have a super-schema, but we modify its
enactment so as to satisfy a larger purpose: getting the deal done. What this
illustrates is that we cannot escape transduction, and that Socrates was wrong. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. TRANSDUCTION and BRICS23 TRANSDUCTION and BRICS23 Conclusion This article is intended as a contribution to the theory and practice of
cross-cultural skill. In theory, it replaces an idealist model of knowledge and
action with a realistic and contemporary schema-based model. In practice, it
provides an analytical framework, and a format of situation tables, for use in
training. Discussion However much we optimise and expand a schema, it will never be
autonomously adequate to all circumstances and surprises, since these are
infinite. A related point is made by Wittgenstein (1953/2009) in his
'rule-following paradox', where he recognises that, having learned a rule, the
ability to 'go on' with it to new situations is something additional. We call the
idealist fantasy the 'Fallacy of Completeness'. The burden of interaction entailed
by globalisation may bring a welcome release from this fallacy. We propose here a contemporary model of the relation between
knowledge and action. Ideally, we would employ vast lookup tables mapping
situations to facts and actions, or vast algorithms which did the same. We
would employ huge but perfect formulae, there would be no need for
situation-checking, and we would have the security of being right all the time. Socrates, then, would be right. Realistically, though, we are simple creatures
facing a complex world, which under BRICS and other blocs has become more
complex. Our cognitive resources are limited, and such an ideal strategy is not
actually tenable. The point about limitation is well made by Simon (1957, 1982)
in his treatment of bounded rationality. We therefore use a cognitive 'hack', and
this hack is transduction. The old idealist model of knowledge and action is
unrealistic and out-of-date. In the contemporary condition of BRICS,
transduction is a necessity. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. References ADLER, N.J., GUNDERSEN, A. International Dimensions of Organizational Behavior. 5th Edition. South-Western College Publications, 2007. ANG, S., VAN DYNE, L. (eds). Handbook of Cultural Intelligence: Theory, Measurement
and Application. Routledge, 2008. CHEW, J. "Managing MNC Expatriates through Crises: A Challenge for
International Human Resource Management, Research and Practice". Human Resource Management, 12 (2), pp. 1-30, 2004. DA COSTA, N.C.A. "Nota sobre o conceito de contradição". Anuário da
Sociadade Paranaense de Matemática, 1, pp. 6-8, 1958. ––––––. "The philosophical import of paraconsistent logic". The Journal of
Non-Classical Logic, 1 (1), pp. 1-19, 1982. DRIVER AND VEHICLE STANDARD AGENCY (DVSA). "The Official Highway
Code". UK Stationery Office, 2015. GUTEMBERG, P.L. "The Sino-Brazilian Principles in a Latin American and
BRICS Context: The Case for Comparative Public Budgeting Legal
Research". Wisconsin International Law Journal, Volume 33, No. 1, 2015. HALVERSON, C. B., AQEEL TIRMINIZ, S. (eds). Effective Multicultural Teams:
Theory and Practice. Advances in Group Decision and Negotiation (Book
3). Springer, 2008. HOFSTEDE, G.J., MINKOV, M. Cultures and Organizations: Software of the Mind,
Third Edition. McGraw-Hill Education, 2010. Manuscrito – Rev. Int. Fil., Campinas, v. 38, n.3, pp. 15-24, set.-dez. 2015. 24 Don Peterson LIVERMORE, D. Leading with Cultural Intelligence: The Real Secret to Success, 2nd
Edition. AMACOM, 2015. NORMAN, D. A., SHALLICE, T. "Attention to action: Willed and automatic
control of behaviour". In Davidson, R. J., G. E. Schwartz, and D. Shapiro, (eds). Consciousness and Self-Regulation: Advances in Research and
Theory. Plenum Press, 1986. O'NEILL, J. "Building Better Global Economic BRICs". Global Economics
Paper No: 66. Goldman Sachs, 2001. PIAGET, J. "Autobiography". In E. Boring (ed), History of Psychology in
Autobiography. Clark University Press, 1952. PLATO, A. Complete Works. c. 429-347 BCE. J. M. Cooper (ed). Hackett, 1997. SIMON, H.A. Models of Man. Wiley & Sons, 1957. SIMON, H.A. Models of Man. Wiley & Sons, 1957. ––––––. Models of Bounded Rationality, Volumes 1 and 2. MIT Press, 1982. WITTGENSTEIN, L. "Philosophische Untersuchungen". Translated by G.E.M. Anscombe as Philosophical Investigations. Blackwell, 1953. Fourth edition, P. M. S. Hacker, J. Schulte (eds), Blackwell, 2009. ZASLAVSKY, C. Tic Tac Toe: And Other Three-In-A Row Games from Ancient Egypt to
the Modern Computer. Crowell, 1982.
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English
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Search for supersymmetry in pp collisions at s = 13 $$ \sqrt{s}=13 $$ TeV in the single-lepton final state using the sum of masses of large-radius jets
|
The Journal of high energy physics/The journal of high energy physics
| 2,016
|
cc-by
| 27,082
|
Published for SISSA by
Springer
Received: May 15, 2016
Accepted: July 20, 2016
Published: August 22, 2016
Search for supersymmetry in pp collisions at
√s = 13 TeV in the single-lepton final state using the
sum of masses of large-radius jets
The CMS collaboration
E-mail: cms-publication-committee-chair@cern.ch Published for SISSA by
Springer
Received: May 15, 2016
Accepted: July 20, 2016
Published: August 22, 2016
Search for supersymmetry in pp collisions at
√s = 13 TeV in the single-lepton final state using the
sum of masses of large-radius jets
The CMS collaboration
E-mail: cms-publication-committee-chair@cern.ch Published for SISSA by
Springer Received: May 15, 2016
Accepted: July 20, 2016
Published: August 22, 2016 Received: May 15, 2016
Accepted: July 20, 2016
Published: August 22, 2016 Received: May 15, 2016
Accepted: July 20, 2016
Published: August 22, 2016 Search for supersymmetry in pp collisions at
√s = 13 TeV in the single-lepton final state using the
sum of masses of large-radius jets JHEP08(2016)122 The CMS collaboration ,
py g
,
for the benefit of the CMS Collaboration. The CMS collaboration Contents
1
Introduction
1
2
Detector
4
3
Simulated event samples
4
4
Event reconstruction
5
5
Trigger and event selection
8
6
Background estimation
9
6.1
Method
9
6.2
Implementation
13
6.3
Systematic uncertainties
15
7
Results and interpretation
17
8
Summary
21
The CMS collaboration
30 Contents
1
Introduction
1
2
Detector
4
3
Simulated event samples
4
4
Event reconstruction
5
5
Trigger and event selection
8
6
Background estimation
9
6.1
Method
9
6.2
Implementation
13
6.3
Systematic uncertainties
15
7
Results and interpretation
17
8
Summary
21
The CMS collaboration
30 Contents
1
Introduction
1
2
Detector
4
3
Simulated event samples
4
4
Event reconstruction
5
5
Trigger and event selection
8
6
Background estimation
9
6.1
Method
9
6.2
Implementation
13
6.3
Systematic uncertainties
15
7
Results and interpretation
17
8
Summary
21
The CMS collaboration
30 JHEP08(2016)122 The CMS collaboration The CMS collaboration E-mail: cms-publication-committee-chair@cern.ch Abstract: Results are reported from a search for supersymmetric particles in proton-
proton collisions in the final state with a single, high transverse momentum lepton; multiple
jets, including at least one b-tagged jet; and large missing transverse momentum. The data
sample corresponds to an integrated luminosity of 2.3 fb−1 at √s = 13 TeV, recorded by
the CMS experiment at the LHC. The search focuses on processes leading to high jet
multiplicities, such as gluino pair production with eg →t¯t eχ0
1. The quantity MJ, defined as
the sum of the masses of the large-radius jets in the event, is used in conjunction with other
kinematic variables to provide discrimination between signal and background and as a key
part of the background estimation method. The observed event yields in the signal regions
in data are consistent with those expected for standard model backgrounds, estimated from
control regions in data. Exclusion limits are obtained for a simplified model corresponding
to gluino pair production with three-body decays into top quarks and neutralinos. Gluinos
with a mass below 1600 GeV are excluded at a 95% confidence level for scenarios with low
eχ0
1 mass, and neutralinos with a mass below 800 GeV are excluded for a gluino mass of
about 1300 GeV. For models with two-body gluino decays producing on-shell top squarks,
the excluded region is only weakly sensitive to the top squark mass. Keywords: Hadron-Hadron scattering (experiments), Supersymmetry ArXiv ePrint: 1605.04608 doi:10.1007/JHEP08(2016)122 doi:10.1007/JHEP08(2016)122 Open Access, Copyright CERN,
for the benefit of the CMS Collaboration. Article funded by SCOAP3. 1
Introduction Supersymmetry (SUSY) [1–8] is an extension of the standard model (SM) of particle physics
that is motivated by several considerations, including the gauge hierarchy problem [9–14],
the existence of astrophysical dark matter [15–17], and the possibility of gauge coupling
constant unification at high energy [18–22]. In SUSY models, each SM particle has a
corresponding supersymmetric partner (or partners) whose spin differs by one-half, such
that fermions are mapped to bosons and vice versa. Gauge quantum numbers are preserved
by this symmetry, and to preserve degrees of freedom, a SM spin-1/2 Dirac particle, such
as the top quark, has two spin-0 partners, the top squarks. The SUSY partner of the
(spin-1) gluon, the massless mediator of the strong interactions in the SM, is the spin-1/2
gluino. In R-parity–conserving models [23, 24], SUSY particles are produced in pairs, and
the lightest supersymmetric particle (LSP) is stable. If the LSP is the lightest neutralino
(eχ0
1), an electrically neutral mixture of the SUSY partners of the neutral electroweak gauge
and Higgs bosons, then it has weak interactions only and can in principle account for some
or all of the dark matter. The gauge hierarchy problem has become more urgent with the discovery of the Higgs
boson [25–30]. Although the SM is conceptually complete, the Higgs boson mass, together – 1 – with the electroweak scale, is unstable against enormous corrections from loop processes,
which pull the Higgs mass to the cutoffscale of the theory, for example, the Planck scale. This outcome can be avoided within the framework of the SM only with extreme fine tuning
of the bare Higgs mass parameter, a situation that is regarded as unnatural, although not
excluded. This problem suggests that additional symmetries and associated degrees of
freedom may be present that ameliorate these effects. So-called natural SUSY models [31–
34], in which sufficiently light SUSY partners are present, are a major focus of current new
physics searches at the CERN LHC. In natural models, several of the SUSY partners are
constrained to be light [33]: both top squarks, etL and etR, which have the same electroweak
couplings as the left- (L) and right- (R) handed top quarks, respectively; the bottom squark
with L-handed couplings (ebL); the gluino (eg); and the Higgsinos (eh). 1
Introduction While the gluino mass
is not constrained by naturalness considerations as strongly as that of the lighter top squark
mass eigenstate, et1, the cross section for gluino pair production is substantially larger than
that for top squark pair production, for a given mass. As a consequence, the two types
of searches can have comparable sensitivity to these models. Both types of searches are
currently of intense interest, and CMS and ATLAS data taken at √s = 8 TeV have provided
significant constraints [35] on natural SUSY scenarios. JHEP08(2016)122 This study uses the first LHC proton-proton collision data taken by the CMS experi-
ment at √s = 13 TeV to search for gluino pair production. Searches targeting this process in
the single-lepton final state using 8 TeV data have been performed by both ATLAS [36, 37]
and CMS [38]. For meg = 1.5 TeV, somewhat above the highest gluino masses excluded
at √s = 8 TeV, the cross section for gluino pair production increases dramatically with
center-of-mass energy, from about 0.4 fb at √s = 8 TeV to about 14 fb at √s = 13 TeV [39]. In contrast, the cross section for the dominant background, tt production, increases much
more slowly, from about 248 pb at √s = 8 TeV to 816 pb at √s = 13 TeV [40]. As a
consequence, the sensitivity of this search can be significantly extended with respect to
searches performed at √s = 8 TeV, even though the 13 TeV data sample has an integrated
luminosity of only 2.3 fb−1, roughly one-tenth of that acquired at 8 TeV. The search targets gluino pair production with eg →tteχ0
1, which arises from eg →
et1t, where the top squark is produced either on or offmass shell. The off-mass-shell
scenario is shown in figure 1 (left) and is often designated T1tttt [41] in simplified model
scenarios [42–44]. Results are also obtained for scenarios with on-shell top squark masses. This scenario is shown in figure 1 (right) and will be denoted by T5tttt. (For this scenario,
the small contribution from the direct production of top squark pairs is also taken into
account.) Regardless of whether the top squark is produced on or offmass shell, the final
state is characterized by a large number of jets, four of which are b jets from top quark
decays. 1
Introduction Depending on the decay modes of the accompanying W bosons, a range of lepton
multiplicities is possible; we focus here on the single-lepton final state, where the lepton
is either an electron or a muon. Because the two neutralinos (eχ0
1) are undetected, their
production in SUSY events typically gives rise to a large amount of missing (unobserved)
momentum, whose value in the direction transverse to the beam axis can be inferred from
the momenta of the observed particles. The missing transverse momentum, ⃗p miss
T
, is a key
element of searches for R-parity-conserving SUSY, and its magnitude is denoted by Emiss
T
. – 2 – P1
P2
˜g
˜g
¯t
t
eχ0
1
eχ0
1
¯t
t
P1
P2
eg
eg
et1
et1
¯t
t
eχ0
1
eχ0
1
¯t
t
Figure 1. Gluino pair production and decay for the simplified models T1tttt (left) and T5tttt
(right). In T1tttt, the gluino undergoes three-body decay eg →tteχ0
1 via a virtual intermediate
top squark. In T5tttt, the gluino decays via the sequential two-body process eg →et1t, et1 →teχ0
1. Because gluinos are Majorana particles, each one can decay to et1t and to the charge conjugate final
state et1t. P1
P2
eg
eg
et1
et1
¯t
t
eχ0
1
eχ0
1
¯t
t P1
P2
˜g
˜g
¯t
t
eχ0
1
eχ0
1
¯t
t ¯t JHEP08(2016)122 Figure 1. Gluino pair production and decay for the simplified models T1tttt (left) and T5tttt
(right). In T1tttt, the gluino undergoes three-body decay eg →tteχ0
1 via a virtual intermediate
top squark. In T5tttt, the gluino decays via the sequential two-body process eg →et1t, et1 →teχ0
1. Because gluinos are Majorana particles, each one can decay to et1t and to the charge conjugate final
state et1t. A challenge in performing searches for SUSY particles is obtaining sufficient sensitivity
to the signal, while at the same time understanding the background contribution from SM
processes in a robust manner. This analysis is designed such that the background in the
signal regions arises largely from a single process, dilepton tt production, in which both
W bosons from t →bW+ decay leptonically, but only one lepton satisfies the criteria
associated with identification, the minimum transverse momentum (pT) requirement, and
isolation from other energy in the event. 2
Detector The central feature of the CMS detector is a superconducting solenoid of 6 m internal di-
ameter, providing a magnetic field of 3.8 T. Within the solenoid volume are the tracking
and calorimeter systems. The tracking system, composed of silicon-pixel and silicon-strip
detectors, measures charged particle trajectories within the pseudorapidity range |η| < 2.5,
where η ≡−ln[tan(θ/2)] and θ is the polar angle of the trajectory of the particle with
respect to the counterclockwise proton beam direction. A lead tungstate crystal electro-
magnetic calorimeter (ECAL), and a brass and scintillator hadron calorimeter (HCAL),
each composed of a barrel and two endcap sections, provide energy measurements up to
|η| = 3. Forward calorimeters extend the pseudorapidity coverage provided by the barrel
and endcap detectors up to |η| = 5. Muons are identified and measured within the range
|η| < 2.4 by gas-ionization detectors embedded in the steel magnetic flux-return yoke out-
side the solenoid. The detector is nearly hermetic, permitting the accurate measurement
of ⃗p miss
T
. A more detailed description of the CMS detector, together with a definition of
the coordinate system used and the relevant kinematic variables, is given in ref. [51]. JHEP08(2016)122 1
Introduction The search signature is characterized not only by
the presence of high-pT jets and b-tagged jets, an isolated high-pT lepton, and large Emiss
T
,
but also by additional kinematic variables. Apart from resolution effects, the transverse
mass of the lepton + ⃗p miss
T
system, mT, is bounded above by mW for events with a single
leptonically decaying W, and this variable is very effective in suppressing the otherwise
dominant single-lepton tt background. The quantity MJ, the scalar sum of the masses of
large-radius jets, is used both to characterize the mass and energy scale of the event, pro-
viding discrimination between signal and background, and as a key part of the background
estimation. A property of MJ exploited in this analysis is that, for the dominant back-
ground, this variable is nearly uncorrelated with mT. Because of the absence of correlation
between MJ and mT, the background shape at high mT, including the signal region, can
be measured to a very good approximation using a low-mT control sample. The quantity
MJ was first discussed in phenomenological studies, for example, in refs. [45–47]. Similar
variables have been used by ATLAS for SUSY searches in all-hadronic final states using
8 TeV data [48, 49]. We have presented studies of basic MJ properties and performance
using early 13 TeV data [50]. This paper is organized as follows. Section 2 gives a brief overview of the CMS de-
tector. Section 3 discusses the simulated event samples used in the analysis. The event
reconstruction is discussed in section 4, while section 5 describes the trigger and event
selection. Section 6 presents the methodology used to predict the SM background from the
event yields in control regions in data. The associated systematic uncertainties are also
discussed. The event yields observed in the signal regions are presented in section 7. These – 3 – yields are compared with background predictions and used to obtain exclusion regions for
the gluino pair production models shown in figure 1. Finally, section 8 presents a summary
of the methodology and the results. 3
Simulated event samples The analysis makes use of several simulated event samples for modeling the SM background
and signal processes. While the background estimation in the analysis is performed largely
from control samples in the data, simulated event samples provide correction factors, typ-
ically near unity. The equivalent integrated luminosity of the simulated event samples is
at least six times that of the data, and at least 100 times that of the data in the case of tt
and signal processes. The production of tt+jets, W+jets, Z+jets, and QCD multijet events is simulated
with the Monte Carlo (MC) generator MadGraph5 aMC@NLO 2.2.2 [52] in leading-
order (LO) mode. Single top quark events are modeled at next-to-leading order (NLO)
with MadGraph5 aMC@NLO for the s-channel and powheg v2 [53, 54] for the t-channel
and W-associated production. Additional small backgrounds, such as tt production in asso-
ciation with bosons, diboson processes, and t¯tt¯t are similarly produced at NLO with either
MadGraph5 aMC@NLO or powheg. All events are generated using the NNPDF 3.0 [55]
set of parton distribution functions (PDF). Parton showering and fragmentation are per-
formed with the pythia 8.205 [56] generator with the underlying event model based on
the CUETP8M1 tune detailed in ref. [57]. The detector simulation is performed with
Geant4 [58]. The cross sections used to scale simulated event yields are based on the
highest order calculation available. For tt, in addition to using the next-to-next-to-leading
order + next-to-next-to-leading logarithmic cross section calculation [40], the modeling of – 4 – the event kinematics is improved by reweighting the top quark pT spectrum to match the
data [59], keeping the overall normalization fixed. Signal events for the T1tttt and T5tttt simplified models are generated in a man-
ner similar to that for the SM backgrounds, with the MadGraph5 aMC@NLO 2.2.2
generator in LO mode using the NNPDF 3.0 PDF set and followed with pythia 8.205
for showering and fragmentation. The detector simulation is performed with the CMS
fast simulation package [60] with scale factors applied to account for any differences with
respect to the full simulation used for backgrounds. Event samples are generated for a
representative set of model scenarios by scanning over the relevant mass ranges for the
eg and eχ0
1, and the yields are normalized to the NLO + next-to-leading-logarithmic cross
section [39, 61–64]. JHEP08(2016)122 Throughout this paper, two T1tttt benchmark models are used to illustrate typ-
ical signal behavior. 3
Simulated event samples The T1tttt(1500,100) model, with masses meg = 1500 GeV and
meχ0
1 = 100 GeV, corresponds to a scenario with a large mass splitting (referred to as
non-compressed, or NC) between the gluino and the neutralino. This mass combination
probes the sensitivity of the analysis to a low cross section (14 fb) process that has a hard
Emiss
T
spectrum, which results in a relatively high signal efficiency. The T1tttt(1200,800)
model, with masses meg = 1200 GeV and meχ0
1 = 800 GeV, corresponds to a scenario with
a small mass splitting (referred to as compressed, or C) between the gluino and the neu-
tralino. Here the cross section is much higher (86 fb) because the gluino mass is lower than
for the T1tttt(1500,100) model, but the sensitivity suffers from a low signal efficiency due
to the soft Emiss
T
spectrum. Finally, to model the presence of additional proton-proton collisions from the same or
adjacent beam crossing as the primary hard-scattering process (“pileup” interactions), the
simulated events are overlaid with multiple minimum bias events, which are also generated
with the pythia 8.205 generator with the underlying event model based on the CUETP8M1
tune. The distribution of the number of overlaid minimum bias events is broad and peaks
in the range 10–15. 4
Event reconstruction The reconstruction of physics objects in an event proceeds from the candidate particles
identified by the particle-flow (PF) algorithm [65, 66], which uses information from the
tracker, calorimeters, and muon systems to identify the candidates as charged or neutral
hadrons, photons, electrons, or muons. Charged particle tracks are required to originate
from the event primary vertex (PV), defined as the reconstructed vertex, located within
24 cm (2 cm) of the center of the detector in the direction along (perpendicular to) the
beam axis, that has the highest value of p2
T summed over the associated charged particle
tracks. The charged PF candidates associated with the PV and the neutral PF candidates are
clustered into jets using the anti-kT algorithm [67] with distance parameter R = 0.4, as
implemented in the fastjet package [68]. The estimated pileup contribution to the jet pT
from neutral PF candidates is removed with a correction based on the area of the jet and – 5 – the average energy density of the event [69]. The jet energy is calibrated using pT- and
η-dependent corrections; the resulting calibrated jet is required to satisfy pT > 30 GeV and
|η| ≤2.4. Each jet must also meet loose identification requirements [70] to suppress, for
example, calorimeter noise. Finally, jets that have PF constituents matched to an isolated
lepton, as defined below, are removed from the jet collection. A subset of the jets are “tagged” as originating from b quarks using the combined
secondary vertex (CSV) algorithm [71, 72]. For the CSV medium working point chosen
for this analysis, the signal efficiency for b jets in the range pT = 30 to 50 GeV is 60–67%
(51–57%) in the barrel (endcap), increasing with pT. Above pT ≈150 GeV the b tagging
efficiency decreases. The probability to misidentify jets arising from c quarks is 13–15%
(11–13%) in the barrel (endcap), while the misidentification probability for light-flavor
quarks or gluons is 1–2%. JHEP08(2016)122 Throughout this paper, quantities related to the number of jets (Njets) or to the number
of b-tagged jets (Nb) are based only on small-R jets, not on the large-R jets discussed below. Electrons are reconstructed by associating a charged particle track with an ECAL
supercluster [73]. 4
Event reconstruction The resulting candidate electrons are required to have pT > 20 GeV and
|η| < 2.5, and to satisfy identification criteria designed to remove light-parton jets, photon
conversions, and electrons from heavy flavor hadron decays. Muons are reconstructed by
associating tracks in the muon system with those found in the silicon tracker [74]. Muon
candidates are required to satisfy pT > 20 GeV and |η| < 2.4. To preferentially select leptons that originate in the decay of W bosons, leptons are
required to be isolated from other PF candidates. Isolation is quantified using an optimized
version of the “mini-isolation” variable originally suggested in ref. [75], in which the trans-
verse energy of the particles within a cone in η-φ space surrounding the lepton momentum
vector is computed using a cone size that scales as 1/pℓ
T, where pℓ
T is the transverse momen-
tum of the lepton. In this analysis, mini-isolation, Irel
mini, is defined as the transverse energy
of particles in a cone of radius Rmini-iso around the lepton, divided by pℓ
T. The transverse
energy is computed as the scalar sum of the pT values of the charged hadrons from the PV,
neutral hadrons, and photons. The neutral hadron and photon contributions to this sum
are corrected for pileup. The cone radius Rmini-iso varies with the pℓ
T according to Rmini-iso =
0.2,
pℓ
T ≤50 GeV
(10 GeV)/pℓ
T,
pℓ
T ∈(50 GeV, 200 GeV)
0.05,
pℓ
T ≥200 GeV. (4.1) Rmini-iso =
0.2,
pℓ
T ≤50 GeV
(10 GeV)/pℓ
T,
pℓ
T ∈(50 GeV, 200 GeV)
0.05,
pℓ
T ≥200 GeV. (4.1) (4.1) The 1/pℓ
T dependence is motivated by considering a two-body decay of a massive parent
particle with mass M and large pT, for which the angular separation of the daughter
particles is roughly ∆Rdaughters ≈2M/pT. The pT-dependent cone size reduces the rate
of accidental overlaps between the lepton and jets in high-multiplicity or highly Lorentz-
boosted events, particularly overlaps between b jets and leptons originating from a boosted
top quark. The cone remains large enough to contain b-hadron decay products for non-
prompt leptons across a range of pℓ
T values. Muons (electrons) must satisfy Irel
mini < 0.2 (0.1). 4
Event reconstruction The combined efficiency for the electron reconstruction and isolation requirements is about – 6 – [GeV]
J
M
0
200
400
600
800
1000
% events/(40 GeV)
1
−
10
1
10
2
10
Simulation
CMS
13 TeV
Simulation
CMS
13 TeV
(1500,100)
1
0
χ∼
tt
→
g~
,
g~
g~
, 1 true lepton
tt
, 2 true leptons
tt
< 10 GeV
T
ISR p
[GeV]
J
M
0
200
400
600
800
1000
% events/(40 GeV)
1
−
10
1
10
2
10
Simulation
CMS
13 TeV
Simulation
CMS
13 TeV
(1500,100)
1
0
χ∼
tt
→
g~
,
g~
g~
, 1 true lepton
tt
, 2 true leptons
tt
> 100 GeV
T
ISR p
Figure 2. Distributions of MJ, normalized to the same area, from simulated event samples with
a small ISR contribution (left) and a significant ISR contribution (right). These components are
defined according to whether the pT of the tt system (or, in the case of signal events, that of
the egeg system) is <10 GeV or >100 GeV, respectively. The T1tttt(NC) signal model (dashed red
line), is described in section 3; the first model parameter in parentheses corresponds to meg and the
second to meχ0
1, both in units of GeV. The events satisfy the requirements Emiss
T
> 200 GeV and
HT > 500 GeV and have at least one reconstructed lepton. [GeV]
J
M
0
200
400
600
800
1000
% events/(40 GeV)
1
−
10
1
10
2
10
Simulation
CMS
13 TeV
Simulation
CMS
13 TeV
(1500,100)
1
0
χ∼
tt
→
g~
,
g~
g~
, 1 true lepton
tt
, 2 true leptons
tt
< 10 GeV
T
ISR p [GeV]
J
M
0
200
400
600
800
1000
% events/(40 GeV)
1
−
10
1
10
2
10
Simulation
CMS
13 TeV
Simulation
CMS
13 TeV
(1500,100)
1
0
χ∼
tt
→
g~
,
g~
g~
, 1 true lepton
tt
, 2 true leptons
tt
> 100 GeV
T
ISR p % events/(40 GeV) JHEP08(2016)122 Figure 2. Distributions of MJ, normalized to the same area, from simulated event samples wi Figure 2. Distributions of MJ, normalized to the same area, from simulated event samples with
a small ISR contribution (left) and a significant ISR contribution (right). 4
Event reconstruction The variable MJ is defined as the sum of all large-R jet
masses: 50% at a pℓ
T of 20 GeV, increasing to 65% at 50 GeV and reaching a plateau of 80% above
200 GeV. The combined reconstruction and isolation efficiencies for muons are about 70%
at a pℓ
T of 20 GeV, increasing to 80% at 50 GeV and reaching a plateau of 95% at 200 GeV. We cluster R = 0.4 (“small-R”) jets and the isolated leptons into R = 1.2 (“large-R”)
jets using the anti-kT algorithm. The mass of the large-R jets retains angular information
about the clustered objects, as well as their pT and multiplicity. Clustering small-R jets
instead of PF candidates incorporates the jet pileup corrections, thereby reducing the
dependence of the mass on pileup. The variable MJ is defined as the sum of all large-R jet
masses: We cluster R = 0.4 (“small-R”) jets and the isolated leptons into R = 1.2 (“large-R”)
jets using the anti-kT algorithm. The mass of the large-R jets retains angular information
about the clustered objects, as well as their pT and multiplicity. Clustering small-R jets
instead of PF candidates incorporates the jet pileup corrections, thereby reducing the
dependence of the mass on pileup. The variable MJ is defined as the sum of all large-R jet
masses: MJ =
X
Ji=large-R jets
m(Ji). (4.2) (4.2) The technique of clustering small-R jets into large-R jets has been used previously by
ATLAS in, for example, ref. [76]. Leptons are included in the large-R jets to include the
full kinematics of the event, and the choice R = 1.2 optimizes the background rejection
power of MJ while retaining signal efficiency. Larger distance parameters were found to
offer no significant additional discriminating power, while smaller parameters decrease the
background rejection up to a factor of two for models with small mass splittings between
the gluino and neutralino. For tt events with a small contribution from initial-state radiation (ISR), the MJ
distribution has an approximate cutoffat twice the mass of the top quark, as shown in
figure 2 (left). In contrast, the MJ distribution for signal events extends to larger values. The presence of a significant amount of ISR generates a high-MJ tail in the tt background,
as shown in figure 2 (right). 4
Event reconstruction These components are
defined according to whether the pT of the tt system (or, in the case of signal events, that of
the egeg system) is <10 GeV or >100 GeV, respectively. The T1tttt(NC) signal model (dashed red
line), is described in section 3; the first model parameter in parentheses corresponds to meg and the
second to meχ0
1, both in units of GeV. The events satisfy the requirements Emiss
T
> 200 GeV and
HT > 500 GeV and have at least one reconstructed lepton. Figure 2. Distributions of MJ, normalized to the same area, from simulated event samples with
a small ISR contribution (left) and a significant ISR contribution (right). These components are
defined according to whether the pT of the tt system (or, in the case of signal events, that of
the egeg system) is <10 GeV or >100 GeV, respectively. The T1tttt(NC) signal model (dashed red
line), is described in section 3; the first model parameter in parentheses corresponds to meg and the
second to meχ0
1, both in units of GeV. The events satisfy the requirements Emiss
T
> 200 GeV and
HT > 500 GeV and have at least one reconstructed lepton. 50% at a pℓ
T of 20 GeV, increasing to 65% at 50 GeV and reaching a plateau of 80% above
200 GeV. The combined reconstruction and isolation efficiencies for muons are about 70%
at a pℓ
T of 20 GeV, increasing to 80% at 50 GeV and reaching a plateau of 95% at 200 GeV. 50% at a pℓ
T of 20 GeV, increasing to 65% at 50 GeV and reaching a plateau of 80% above
200 GeV. The combined reconstruction and isolation efficiencies for muons are about 70%
at a pℓ
T of 20 GeV, increasing to 80% at 50 GeV and reaching a plateau of 95% at 200 GeV. We cluster R = 0.4 (“small-R”) jets and the isolated leptons into R = 1.2 (“large-R”)
jets using the anti-kT algorithm. The mass of the large-R jets retains angular information
about the clustered objects, as well as their pT and multiplicity. Clustering small-R jets
instead of PF candidates incorporates the jet pileup corrections, thereby reducing the
dependence of the mass on pileup. 5
Trigger and event selection The data sample used in this analysis was obtained with triggers that require HT > 350 GeV
and at least one electron or muon with pT > 15 GeV, where these variables are computed
with online (trigger-level) quantities and typically have somewhat poorer resolution than
the corresponding offline variables. To ensure high trigger efficiency with respect to the
offline definition of lepton isolation described in the previous section (mini-isolation), we
designed these triggers with very loose lepton isolation requirements and fixed the isolation
cone size to R = 0.2. For events passing the offline selection, the total trigger efficiencies,
measured in data control samples that are independently triggered, are found to be (95.1±
1.1)% for the muon channel and (94.1±1.2)% for the electron channel and are independent
of the analysis variables within the uncertainties. These efficiencies are applied to the
simulation as a correction. The offline event selection is summarized in table 1, which lists the event yields ex-
pected from simulation for both SM background processes and for the two benchmark
T1tttt signal models. We select events with exactly one isolated charged lepton (an elec-
tron or a muon), HT > 500 GeV, Emiss
T
> 200 GeV, and at least six jets, at least one of
which is b-tagged. After this set of requirements, referred in the following as the baseline
selection, more than 80% of the remaining SM background arises from tt production. The
contributions from events with a single top quark or a W boson in association with jets are
each about 6–7%. The background from QCD multijet events after the baseline selection
is negligible due to the combination of leptonic, Emiss
T
, and Njets requirements. After the baseline selection requirements are applied, events are binned in several other
kinematic variables, both to increase the signal sensitivity and to define control regions,
as described in section 6.1. To illustrate the effect of additional requirements, table 1 lists
the expected yields for examples of event selection requirements on MJ, mT, Njets, and
Nb. The events satisfying the baseline selection are divided in the MJ–mT plane into a
signal region, defined by the additional requirements MJ > 400 GeV and mT > 140 GeV,
and three control samples, bounded by MJ > 250 GeV, that are used in the background
estimation. Approximately 37% of signal T1tttt events are selected with the single-lepton
requirement only. 4
Event reconstruction The missing transverse energy, Emiss
T
, is given by the magnitude of ⃗p miss
T
, the negative
vector sum of the transverse momenta of all PF candidates [65, 66]. Correspondence to the – 7 – true undetectable energy in the event is improved by replacing the contribution of the PF
candidates associated with a jet by the calibrated four-momentum of that jet. To separate
backgrounds characterized by the presence of a single W boson decaying leptonically but
without any other source of missing energy, the lepton and the Emiss
T
are combined to
obtain the transverse mass, mT, defined as: mT =
q
2pℓ
TEmiss
T
[1 −cos(∆φℓ,⃗p miss
T
)],
(4.3) (4.3) where ∆φℓ,⃗p miss
T
is the difference between the azimuthal angles of the lepton momentum
vector and the missing momentum vector, ⃗p miss
T
. Finally, we define the quantity HT as the
scalar sum of the transverse momenta of all the small-R jets passing the selection. JHEP08(2016)122 5
Trigger and event selection The benchmark signal models, T1tttt(NC) and T1tttt(C), are
described in section 3. The event selection requirements listed above the horizontal line in the
middle of the table are defined as the baseline selection. The background estimates before the HT
requirement are not specified because some of the simulated event samples do not extend to the
low HT region. Given the size of the MC samples described in section 3, rows with zero yield have
statistical uncertainties of at most 0.16 events, and below 0.05 events in most cases. than meχ0
1, more than half of the events passing the lepton requirement lie in the signal
region. For compressed spectrum models, where meχ0
1 ≈meg −2mt, the MJ, HT, and Emiss
T
spectra become much softer and, as a result, only 5–10% of the single-lepton signal events
are selected. As shown in figure 3, backgrounds with a single W boson decaying leptonically are
strongly suppressed after the mT > 140 GeV requirement, so the total SM background in
the signal region is dominated by dilepton tt events. This dilepton background falls into
two categories, which make roughly equal contributions. The first involves an identified
electron or muon and a hadronically decaying τ from W decay. The second source involves
two leptons, each of which is an electron or a muon. One of the leptons fails to satisfy
the lepton selection criteria, which include the pT and isolation requirements. This missed
lepton can be produced either directly or indirectly in W decay, where in the indirect case
the lepton is the daughter of a τ. 5
Trigger and event selection In non-compressed spectrum models, for which meg is significantly larger – 8 – L = 2.3 fb−1
Other
QCD
ttV
Single t
W+jets
tt (1ℓ)
tt (2ℓ)
SM bkg. T1tttt(NC)
T1tttt(C)
No selection
—
—
—
—
—
—
—
—
31.3
190.0
1ℓ, pT > 20 GeV
—
—
—
—
—
—
—
—
11.9
68.7
HT > 500 GeV
4131.9
31831.5
721.9
2926.6
31885.1
27628.7
3357.8
102483.4
11.9
44.9
Emiss
T
> 200 GeV
310.6
154.7
89.1
457.2
4343.1
2183.6
584.0
8122.3
10.5
21.5
Njets ≥6, pT > 30 GeV
27.3
8.0
36.8
82.8
278.7
792.3
171.4
1397.4
9.6
20.4
Nb ≥1
9.4
2.7
29.6
63.9
66.3
632.2
137.4
941.4
9.1
19.1
MJ > 250 GeV
6.7
2.6
22.6
43.8
46.1
455.2
87.2
664.2
9.0
16.5
mT > 140 GeV
0.7
1.4
3.0
3.5
1.2
5.5
32.5
47.9
7.0
9.2
MJ > 400 GeV
0.4
0.8
1.1
1.4
0.6
2.8
9.7
16.7
6.4
4.5
Nb ≥2
0.16
0.04
0.55
0.68
0.00
1.29
4.52
7.24
4.87
3.47
Emiss
T
> 400 GeV
0.02
0.00
0.12
0.31
0.00
0.07
0.72
1.24
3.60
1.48
Njets ≥9, pT > 30 GeV
0.01
0.00
0.03
0.00
0.00
0.01
0.11
0.16
1.64
1.00
Table 1. Event yields obtained from simulated event samples, as the event selection criteria are
applied. The category Other includes Drell-Yan, ttH(→bb), tttt, WZ, and WW. The yields for
tt events in fully hadronic final states are included in the QCD multijet category. The category
ttV includes ttW, ttZ, and ttγ. The benchmark signal models, T1tttt(NC) and T1tttt(C), are
described in section 3. The event selection requirements listed above the horizontal line in the
middle of the table are defined as the baseline selection. The background estimates before the HT
requirement are not specified because some of the simulated event samples do not extend to the
low HT region. Given the size of the MC samples described in section 3, rows with zero yield have
statistical uncertainties of at most 0.16 events, and below 0.05 events in most cases. JHEP08(2016)122 Table 1. Event yields obtained from simulated event samples, as the event selection criteria are
applied. The category Other includes Drell-Yan, ttH(→bb), tttt, WZ, and WW. The yields for
tt events in fully hadronic final states are included in the QCD multijet category. The category
ttV includes ttW, ttZ, and ttγ. 6.1
Method The prediction of the background yields in each of the signal bins takes advantage of the
fact that the MJ and mT distributions of events with a significant amount of ISR are largely
uncorrelated. The correlation coefficients for the single-lepton and dilepton tt events in the
MJ–mT plane after the baseline selection (as shown in figure 4) are small, in the range 0.03
to 0.05. The absence of a substantial correlation allows us to measure the MJ distribution – 9 – Events/(35 GeV)
1
−
10
1
10
2
10
3
10
4
10
5
10
6
10
CMS
(13 TeV)
-1
2.3 fb
Data
(1500,100)
1
0
χ∼
tt
→
g~
,
g~
g~
(1200,800)
1
0
χ∼
tt
→
g~
,
g~
g~
, 1 true lepton
tt
, 2 true leptons
tt
W+jets
Single t
ttV
Other
[GeV]
T
m
0
100
200
300
400
Data / MC
0.5
1
1.5
Figure 3. Distribution of mT in data and simulated event samples after the baseline selection is
applied. The background contributions shown here are from simulation, and their total yield is
normalized to the number of events observed in data. The signal distributions are normalized to
the expected cross sections. The dashed vertical line indicates the mT > 140 GeV threshold that
separates the signal regions from the control samples. Events/(35 GeV)
1
−
10
1
10
2
10
3
10
4
10
5
10
6
10
CMS
(13 TeV)
-1
2.3 fb
Data
(1500,100)
1
0
χ∼
tt
→
g~
,
g~
g~
(1200,800)
1
0
χ∼
tt
→
g~
,
g~
g~
, 1 true lepton
tt
, 2 true leptons
tt
W+jets
Single t
ttV
Other
[GeV]
T
m
0
100
200
300
400
Data / MC
0.5
1
1.5 JHEP08(2016)122 Figure 3. Distribution of mT in data and simulated event samples after the baseline selection is
applied. The background contributions shown here are from simulation, and their total yield is
normalized to the number of events observed in data. The signal distributions are normalized to
the expected cross sections. The dashed vertical line indicates the mT > 140 GeV threshold that
separates the signal regions from the control samples. 6.1
Method [GeV]
J
M
0
200
400
600
800
1000
[GeV]
T
m
0
50
100
150
200
250
300
350
400
=0.05
ρ
(1l),
tt
=0.03
ρ
(2l),
tt
(1500,100)
0
1
χ∼t
t
→
g~
,
g~
g~
R1
R2
R3
R4
CMS
Simulation
13 TeV
Figure 4. Distribution of simulated single-lepton tt events (dark-blue triangles), dilepton tt events
(light-blue inverted triangles), and T1tttt(1500,100) events (red squares) in the MJ–mT plane after
the baseline selection. Each marker represents one expected event at 2.3 fb−1. Overflow events are
placed on the edge of the plot. The values of the correlation coefficients ρ for each background
process are given in the legend. Region R4 is the nominal signal region, while R1, R2, and R3 serve
as control regions. The small signal contributions in the control regions are taken into account in
one of the global fits, as discussed in the text. [GeV]
J
M
0
200
400
600
800
1000
[GeV]
T
m
0
50
100
150
200
250
300
350
400
=0.05
ρ
(1l),
tt
=0.03
ρ
(2l),
tt
(1500,100)
0
1
χ∼t
t
→
g~
,
g~
g~
R1
R2
R3
R4
CMS
Simulation
13 TeV Figure 4. Distribution of simulated single-lepton tt events (dark-blue triangles), dilepton tt events
(light-blue inverted triangles), and T1tttt(1500,100) events (red squares) in the MJ–mT plane after
the baseline selection. Each marker represents one expected event at 2.3 fb−1. Overflow events are
placed on the edge of the plot. The values of the correlation coefficients ρ for each background
process are given in the legend. Region R4 is the nominal signal region, while R1, R2, and R3 serve
as control regions. The small signal contributions in the control regions are taken into account in
one of the global fits, as discussed in the text. – 10 – of the background at low mT with good statistical precision, and extrapolate it to high
mT. The underlying explanation for this behavior is not immediately obvious, given that
low-mT events originate mainly from tt events where only one of the top quarks decays
leptonically (1ℓtt), while the high-mT regions are dominated by dilepton tt events (2ℓtt). 6.1
Method In particular, as shown in figure 2 (left), in the absence of significant ISR, the dileptonic
tt events have a softer MJ spectrum than single-lepton tt events, simply because the
reconstructed mass of a leptonically decaying top quark does not include the undetected
neutrino. In events with substantial ISR, however, the contributions to MJ from the accidental
overlap of jets can dominate the contributions due to the intrinsic mass of the top quarks. This effect is illustrated in figure 5, which compares the Njets and MJ distributions of
single-lepton and dilepton tt events at high and low mT after the baseline selection is
applied. Since we require at least 6 jets, single-lepton tt events must have at least 2 ISR
jets and dilepton tt events must have at least 4. In this regime, the probability of additional
ISR jets is similar for events with a given number of partons of similar momenta, and, as
a result, the number of objects contributing to MJ (jets plus the reconstructed lepton) is
comparable in 1ℓand 2ℓtt events. When these ISR jets overlap with the top quark decay
products, the masses of the resulting large-R jets are dominated by the accidental overlap
and, thus, the shapes of the MJ distribution of 1ℓand 2ℓtt events become more similar. This is the case for MJ > 250 GeV, where figure 5 (right) shows that the distributions of
the 1ℓand 2ℓtt backgrounds have nearly the same shape, and the low-mT to high-mT
extrapolation is warranted. JHEP08(2016)122 We thus divide the MJ–mT plane into four regions, three control regions (CR) and
one signal region (SR): • Region R1 (CR): mT ≤140 GeV, 250 ≤MJ ≤400 GeV
• Region R2 (CR): mT ≤140 GeV, MJ > 400 GeV
• Region R3 (CR): mT > 140 GeV, 250 ≤MJ ≤400 GeV
• Region R4 (SR): mT > 140 GeV, MJ > 400 GeV. • Region R1 (CR): mT ≤140 GeV, 250 ≤MJ ≤400 GeV
• Region R2 (CR): mT ≤140 GeV, MJ > 400 GeV
• Region R3 (CR): mT > 140 GeV, 250 ≤MJ ≤400 GeV • Region R3 (CR): mT > 140 GeV, 250 ≤MJ ≤400 GeV • Region R4 (SR): mT > 140 GeV, MJ > 400 GeV. 6.1
Method These regions are further subdivided into 10 bins of Emiss
T
, Njets, and Nb to increase signal
sensitivity: • Six bins with 200 < Emiss
T
≤400 GeV: (6 ≤Njets ≤8, Njets ≥9) × (Nb = 1, Nb =
2, Nb ≥3) • Six bins with 200 < Emiss
T
≤400 GeV: (6 ≤Njets ≤8, Njets ≥9) × (Nb = 1, Nb =
2, Nb ≥3) • Four bins with Emiss
T
> 400 GeV: (6 ≤Njets ≤8, Njets ≥9) × (Nb = 1, Nb ≥2), • Four bins with ET
> 400 GeV: (6 ≤Njets ≤8, Njets ≥9) × (Nb = 1, Nb ≥2),
where the multiplication indicates that the binning is two dimensional in Njets and Nb. Given that the main background processes have two or fewer b quarks, the total SM
contribution to the Nb ≥3 bins is very small and is driven by the b-tag fake rate. Signal
events in the T1tttt and T5tttt models are expected to populate primarily the bins with
Nb ≥2, while bins with Nb = 1 mainly serve to test the method in a background dominated
region. here the multiplication indicates that the binning is two dimensional in Njets and Nb. – 11 – % events
0
20
40
60
Simulation
CMS
13 TeV
140 GeV
≤
T
, 1 true lepton: m
tt
> 140 GeV
T
, 2 true leptons: m
tt
jets
N
6
8
10
12
T
/ Low m
T
High m
0
0.05
0.1
0.15
% events/(50 GeV)
2
−
10
1
−
10
1
10
2
10
Simulation
CMS
13 TeV
140 GeV
≤
T
, 1 true lepton: m
tt
> 140 GeV
T
, 2 true leptons: m
tt
[GeV]
J
M
0
500
1000
1500
T
/ Low m
T
High m
0
0.05
0.1
0.15
Figure 5. Comparison of Njets and MJ distributions, normalized to the same area, in simulated tt
events with two true leptons at high mT and one true lepton at low mT, after the baseline selection
is applied. The shapes of these distributions are similar. These two contributions are the dominant
backgrounds in their respective mT regions. The dashed vertical line on the right-hand plot indicates
the MJ > 400 GeV threshold that separates the signal regions from the control samples. 6.1
Method The shaded
region corresponding to MJ < 250 GeV is not used in the background estimation. % events/(50 GeV)
2
−
10
1
−
10
1
10
2
10
Simulation
CMS
13 TeV
140 GeV
≤
T
, 1 true lepton: m
tt
> 140 GeV
T
, 2 true leptons: m
tt
[GeV]
J
M
0
500
1000
1500
T
/ Low m
T
High m
0
0.05
0.1
0.15 % events
0
20
40
60
Simulation
CMS
13 TeV
140 GeV
≤
T
, 1 true lepton: m
tt
> 140 GeV
T
, 2 true leptons: m
tt
jets
N
6
8
10
12
T
/ Low m
T
High m
0
0.05
0.1
0.15 % events/(50 GeV) % events JHEP08(2016)122 Figure 5. Comparison of Njets and MJ distributions, normalized to the same area, in simulated tt
events with two true leptons at high mT and one true lepton at low mT, after the baseline selection
is applied. The shapes of these distributions are similar. These two contributions are the dominant
backgrounds in their respective mT regions. The dashed vertical line on the right-hand plot indicates
the MJ > 400 GeV threshold that separates the signal regions from the control samples. The shaded
region corresponding to MJ < 250 GeV is not used in the background estimation. To obtain an estimate of the background rate in each of the signal bins, a modified
version of an “ABCD” method is used. Here, the symbols A, B, C, and D refer to four
regions in a two-dimensional space in the data, where one of the regions is dominated by
signal and the other three by backgrounds. In a standard ABCD method, the background
rate in the signal region is estimated from the yields in three control regions with the
expression µbkg
R4 = NR2 NR3/NR1,
(6.1) (6.1) where the labels on the regions correspond to those shown in figure 4. The background
prediction is unbiased in the limit that the two variables that define the plane (in this
case, MJ and mT) are uncorrelated. The effect of any residual correlation is corrected with
factors κ that can be obtained from simulated event samples: κ = NMC,bkg
R4
/N MC,bkg
R3
NMC,bkg
R2
/N MC,bkg
R1
. (6.2) (6.2) When the two ABCD variables are uncorrelated or nearly so, the κ factors are close to
unity. 6.1
Method This procedure ignores potential signal contamination in the control regions, which
is accounted for by incorporating the constraints in eqs. (6.1) and (6.2) into a fit that
includes both signal and background components, as described in section 6.2. In principle, the background in the 10 signal bins could be estimated by applying this
procedure in 10 independent planes. However, this procedure would incur large statistical
uncertainties in some bins due to low numbers of events in R3. This problem is especially – 12 – 4
5
6
7
8
9
≥
4
5
6
7
8
9
≥
)
T
R(m
0
0.05
0.1
0.15
0.2
0.25
Simulation
CMS
13 TeV
Baseline selection
Baseline selection
= 1
b
N
= 2
b
N
3
≥
b
N
400 GeV
≤
J
M
> 400 GeV
J
M
jets
N
Figure 6. The ratio R(mT) of high-mT (R3 and R4) to low-mT (R1 and R2) event yields for the
simulated SM background, as a function of Njets and Nb. The baseline selection requires Njets ≥6. The uncertainties shown are statistical only. 4
5
6
7
8
9
≥
4
5
6
7
8
9
≥
)
T
R(m
0
0.05
0.1
0.15
0.2
0.25
Simulation
CMS
13 TeV
Baseline selection
Baseline selection
= 1
b
N
= 2
b
N
3
≥
b
N
400 GeV
≤
J
M
> 400 GeV
J
M
jets
N
Figure 6. The ratio R(mT) of high-mT (R3 and R4) to low-mT (R1 and R2) event yields for the
simulated SM background as a function of Njets and Nb The baseline selection requires Njets ≥6 )
T
R(m JHEP08(2016)122 Figure 6. The ratio R(mT) of high-mT (R3 and R4) to low-mT (R1 and R2) event yields for the
simulated SM background, as a function of Njets and Nb. The baseline selection requires Njets ≥6. The uncertainties shown are statistical only. important in bins with a high number of jets, where the MJ spectrum shifts to higher
values and the number of background events expected in R4 can exceed the background
in R3. To alleviate this problem, we exploit the fact that, after the baseline selection, the
background is dominated by just one source (tt events), and the shapes of the Njets dis-
tributions are nearly identical for the single-lepton and dilepton components (due to the
large amounts of ISR). 6.1
Method As a result, the mT distribution is approximately independent of
Njets and Nb. We study this behavior with the ratio of the number of events at high to
low mT: R(mT) ≡N(mT > 140 GeV)
N(mT ≤140 GeV). (6.3) (6.3) Because, as seen in figure 6, the values of R(mT) do not vary substantially across Njets and
Nb bins, the predicted value of R(mT) is not sensitive to the modeling of the distributions
of those quantities. We exploit this result by integrating the yields of the low-MJ regions
(R1 and R3) over the Njets and Nb bins for each Emiss
T
bin. This procedure increases
the statistical power of the ABCD method but also introduces a correlation among the
predictions (eq. (6.1)) for the Njets and Nb bins associated with a given Emiss
T
bin. Figure 7
shows the κ factors for the 10 signal bins after summing over Njets and Nb in R1 and R3. In all cases, their values are close to unity. 6.2
Implementation The method outlined in section 6.1 is implemented with a likelihood function that incor-
porates the statistical and systematic uncertainties in κ, accounts for correlations arising
from the common R1 and R3 yields, and corrects for signal contamination in the control
regions. – 13 – 400
≤
miss
T
E
> 400
miss
T
E
400
≤
miss
T
E
> 400
miss
T
E
κ
0
0.5
1
1.5
2
Simulation
CMS
13 TeV
= 1
b
N
= 2
b
N
3
≥
b
N
2
≥
b
N
8
≤
jets
N
≤
6
9
≥
jets
N 400
≤
miss
T
E
> 400
miss
T
E
400
≤
miss
T
E
> 400
miss
T
E
κ
0
0.5
1
1.5
2
Simulation
CMS
13 TeV
= 1
b
N
= 2
b
N
3
≥
b
N
2
≥
b
N
8
≤
jets
N
≤
6
9
≥
jets
N
Figure 7. Values of the double-ratio κ in each of the 10 signal bins, calculated using the simulated
SM background. The κ factors are close to unity, indicating the small correlation between MJ and
mT. The uncertainties shown are statistical only. JHEP08(2016)122 Figure 7. Values of the double-ratio κ in each of the 10 signal bins, calculated using the simulated
SM background. The κ factors are close to unity, indicating the small correlation between MJ and
mT. The uncertainties shown are statistical only. The SM background contribution for each region is described as follows. We define
µbkg
Ri
as the estimated (Poisson) mean background in each region Ri, with i = 1, 2, 3, 4. Then, in an ABCD background calculation, these four rates can be expressed in terms of
three floating fit parameters µ, R(mT), and R(MJ), and the correlation correction factor
κ, as µbkg
R1 = µ,
µbkg
R2 = µ R(MJ),
µbkg
R3 = µ R(mT),
µbkg
R4 = κ µ R(MJ) R(mT). (6.4) µbkg
R2 = µ R(MJ),
µbkg
R4 = κ µ R(MJ) R(mT). (6.4) µbkg
R1 = µ,
µbkg
R2 = µ R(MJ),
µbkg
R3 = µ R(mT),
µbkg
R4 = κ µ R(MJ) R(mT). (6.4) (6.4) Here, µ is the background rate fit parameter for R1, R(MJ) is the ratio of the R2 to R1
rates, and R(mT) is the ratio of the R3 to R1 rates. The quantity κ is given by eq. 6.2
Implementation (6.2)
after replacing the yields NMC,bkg
Ri
by the background rate fit parameters µMC,bkg
Ri
. Here, µ is the background rate fit parameter for R1, R(MJ) is the ratio of the R2 to R1
rates, and R(mT) is the ratio of the R3 to R1 rates. The quantity κ is given by eq. (6.2)
after replacing the yields NMC,bkg
Ri
by the background rate fit parameters µMC,bkg
Ri
. Similarly, we define Ndata
Ri
as the observed data yield in each region, µMC,sig
Ri
as the
expected signal rate in each region, and r as the parameter quantifying the signal strength
relative to the expected yield across all analysis regions. We can then write the likelihood
function as L = Ldata
ABCD LMC
κ
LMC
sig ,
(6.5) L = Ldata
ABCD LMC
κ
LMC
sig ,
(6.5)
Ldata
ABCD =
4
Y
i=1
Nbins(Ri)
Y
k=1
Poisson(Ndata
Ri,k |µbkg
Ri,k + r µMC,sig
Ri,k
),
(6.6)
LMC
κ
=
4
Y
i=1
Nbins(Ri)
Y
k=1
Poisson(NMC,bkg
Ri,k
|µMC,bkg
Ri,k
),
(6.7)
LMC
sig =
4
Y
i=1
Nbins(Ri)
Y
k=1
Poisson(NMC,sig
Ri,k
|µMC,sig
Ri,k
). (6.8) (6.5) Ldata
ABCD =
4
Y
i=1
Nbins(Ri)
Y
k=1
Poisson(Ndata
Ri,k |µbkg
Ri,k + r µMC,sig
Ri,k
),
(6.6) (6.6) LMC
κ
=
4
Y
i=1
Nbins(Ri)
Y
k=1
Poisson(NMC,bkg
Ri,k
|µMC,bkg
Ri,k
),
(6.7)
4
Nbins(Ri) (6.7) (6.8) – 14 – The indices k run over each of the Emiss
T
, Njets, and Nb bins defined in the previous section;
these indices were suppressed in eq. (6.4) for simplicity. Given the integration over Njets
and Nb at low MJ, Nbins(R1) = Nbins(R3) = 2, while Nbins(R2) = Nbins(R4) = 10. In eq. (6.5), Ldata
ABCD accounts for the statistical uncertainty in the observed data yield in
the four ABCD regions, and LMC
κ
and LMC
sig account for the uncertainty in the computation
of the κ correction factor and signal shape, respectively, due to the finite size of the MC
samples. The systematic uncertainties in κ and the signal efficiency are described in the following
sections. These effects are incorporated in the likelihood function as log-normal constraints
with a nuisance parameter for each uncorrelated source of uncertainty. These terms are
not explicitly shown in the likelihood function above for simplicity. JHEP08(2016)122 The likelihood function defined in eqs. 6.2
Implementation (6.5)–(6.8) is employed in two separate types
of fits that provide complementary but compatible background estimates based on an
ABCD model. The first type of fit, which we call the predictive fit, allows us to more easily
establish the agreement of the background predictions and the observations in the null (i.e.,
the background-only) hypothesis. We do this by excluding the observations in the signal
regions in the likelihood (that is, by truncating the first product in eq. (6.6) at i = 3) and
fixing the signal strength r to 0. This procedure leaves as many unknowns as constraints:
three data floating parameters (µ, R(MJ), and R(mT)) and three observations (Ndata
Ri,k with
i = 1, 2, 3) for each ABCD plane. In the likelihood function there are additional floating
parameters associated with MC quantities, which have small uncertainties. As a result,
the estimated background rates in regions R1, R2, and R3 converge to the observed values
in those bins, and we obtain predictions for the signal regions that do not depend on the
observed Ndata
R4 . The predictive fit thus converges to the standard ABCD method, and the
likelihood machinery becomes just a convenient way to solve the system of equations and
propagate the various uncertainties. Additionally, we implement a global fit which, by making use of the observations in the
signal regions, can provide an estimate of the signal strength r, while allowing for signal
events to populate the control regions. This is achieved by including all four observations,
Ndata
Ri,k with i = 1, 2, 3, 4, in the likelihood function. Since there are four observations and
three floating background parameters in each ABCD plane, there are enough constraints
for the signal strength also to be determined in the fit. 6.3
Systematic uncertainties This section describes the systematic uncertainties in the background prediction, which are
incorporated into the analysis as an uncertainty in the κ correction. Because the dominant
background arises from 2ℓtt events, we use a control sample with two reconstructed leptons
to validate our background estimation procedure and to quantify the associated uncertainty. The resulting uncertainty is augmented with simulation-based studies of effects that are not
covered by this dilepton test. Table 2 summarizes all of the uncertainties in the background
prediction. The ability of the ABCD method to predict the 2ℓtt background is studied using a
modified ABCD plane, in which the high-mT regions, R3 and R4, are replaced with regions – 15 – Source
Fractional uncertainty [%]
Data sample size
28–118
Dilepton control sample test
37–88
Simulation sample size
5–17
Jet energy resolution
2–10
Jet energy corrections
1–5
ISR pT
1–5
Top pT
1–4
Non-tt background
2–11 JHEP08(2016)122 Table 2. Summary of uncertainties in the background predictions. All entries in the table except
for data sample size correspond to a relative uncertainty on κ. The ranges indicate the spread of
each uncertainty across the signal bins. Uncertainties from a particular source are treated as fully
correlated across bins, while uncertainties from different sources are treated as uncorrelated. D3 and D4, which have two reconstructed leptons. These regions have low and high MJ,
respectively, just as R3 and R4. The events in D3 and D4 pass the same selection as those
in R3 and R4, except for the following changes: Njets bin boundaries are lowered by one to
keep the number of large-R jet constituents the same as in the single-lepton samples; the
mT requirement is not applied; and events with Nb = 0 are included to increase the size of
the event sample, while events with Nb ≥3 are excluded to avoid signal contamination. We
perform this test only for low Emiss
T
to further avoid the potentially large signal contribution
in the high-Emiss
T
region. The low-MJ regions (R1 and D3) are integrated over Njets, while
the high-MJ regions (R2 and D4) are binned in low and high Njets. The predictive fit
is then used to predict the D4 event yields for both Njets bins. We predict 11.0 ± 2.3
(1.5 ± 0.5) events for the low (high) Njets bin, and we observe 12 (2) events. 6.3
Systematic uncertainties Given the
good agreement between prediction and observation, the statistical precision of the test is
taken as a systematic uncertainty in κ. These uncertainties are 37% and 88% for the low-
and high-Njets regions, respectively. Since the event composition of regions D3 and D4 is not fully representative of that in
R3 and R4, we perform studies on potential additional sources of systematic uncertainty
in the simulation. We find that the main source of 1ℓtt events in the high-mT region is jet
energy mismeasurement. We study the impact of mismodeling the size of this contribution
by smearing the jet energies by an additional 50% with respect to the jet energy resolution
measured in data [70] and calculating the corresponding shift in κ. To ensure that there
are no further significant differences between the MJ shapes of events reconstructed with
one or two leptons, we also calculate the shift in κ due to jet energy corrections, potential
ISR pT and top quark pT mismodeling, as well as the amount of non-tt background. Even
though each of these can alone have a significant effect on the MJ shape, the κ factor, as
a double ratio, remains largely unaffected (table 2). Including these uncertainties in the
likelihood fit produces a negligible contribution to the total uncertainty. – 16 – )
2
Simulated events/(800 GeV
2
−
10
1
−
10
1
[GeV]
J
M
0
200
400
600
800
1000
1200
[GeV]
T
m
0
100
200
300
400
500
600
Data
(1500,100)
0
1
χ∼
t
t
→
g~
,
g~
g~
CMS
(13 TeV)
-1
2.3 fb
Figure 8. Two-dimensional distributions for data and simulated event samples in the variables
mT and MJ in the Nb ≥2 region after the baseline selection. The distributions integrate
over the Njets and Emiss
T
bins. The black dots are the data; the colored histogram is the total
simulated background, normalized to the data; and the red dots are a particular signal sam-
ple drawn from the expected distribution for gluino pair production in the T1tttt model with
meg = 1500 GeV and meχ0
1 = 100 GeV for 2.3 fb−1. Overflow events are shown on the edges of the
plot. The definitions of the signal and control regions are the same as those shown in figure 4. 6.3
Systematic uncertainties )
2
Simulated events/(800 GeV
2
−
10
1
−
10
1
[GeV]
J
M
0
200
400
600
800
1000
1200
[GeV]
T
m
0
100
200
300
400
500
600
Data
(1500,100)
0
1
χ∼
t
t
→
g~
,
g~
g~
CMS
(13 TeV)
-1
2.3 fb JHEP08(2016)122 Figure 8. Two-dimensional distributions for data and simulated event samples in the variables
mT and MJ in the Nb ≥2 region after the baseline selection. The distributions integrate
over the Njets and Emiss
T
bins. The black dots are the data; the colored histogram is the total
simulated background, normalized to the data; and the red dots are a particular signal sam-
ple drawn from the expected distribution for gluino pair production in the T1tttt model with
meg = 1500 GeV and meχ0
1 = 100 GeV for 2.3 fb−1. Overflow events are shown on the edges of the
plot. The definitions of the signal and control regions are the same as those shown in figure 4. 7
Results and interpretation Figure 8 shows the two-dimensional distribution of the data in the mT–MJ plane after the
baseline selection, but with the additional requirement Nb ≥2. The baseline requirements
include Emiss
T
> 200 GeV and Njets ≥6, but no further event selection is applied. For
comparison, the plot also shows the expected total SM background based on simulation, as
well as a particular sample of the expected signal distribution. The overall distribution of
events in data is consistent with the background expectation, where the majority of events
are concentrated at low mT and MJ. In R4, the nominal signal region, we observe only
two events in data, while, as shown in table 3, the predicted SM background is about 5
events. The T1tttt(1500,100) (NC) model would be expected to contribute 5 additional
events to R4. The validity of the central assumption of the background estimation method can be
checked in the nearly signal-free Nb = 1 region by comparing the MJ shapes observed
in the high- and low-mT regions in data. Figure 9 (left) shows the MJ shapes in the
Nb = 1 sample, integrating over the Njets and Emiss
T
bins. The low mT data have been
normalized to the overall yields in the corresponding high-mT data. The shapes of the MJ
distributions for the high- and low-mT regions are consistent. Figure 9 (right) shows that
the corresponding distributions in the Nb ≥2 sample are also consistent, as expected in
the absence of signal. – 17 – [GeV]
J
M
200
400
600
Events/(75 GeV)
0
5
10
15
20
CMS
(13 TeV)
-1
2.3 fb
> 140 GeV
T
Data, m
140 GeV
≤
T
Data, m
(1500,100)
1
0
χ∼
tt
→
g~
,
g~
g~
(1200,800)
1
0
χ∼
tt
→
g~
,
g~
g~
= 1
b
N
[GeV]
J
M
200
400
600
Events/(75 GeV)
0
5
10
15
CMS
(13 TeV)
-1
2.3 fb
> 140 GeV
T
Data, m
140 GeV
≤
T
Data, m
(1500,100)
1
0
χ∼
tt
→
g~
,
g~
g~
(1200,800)
1
0
χ∼
tt
→
g~
,
g~
g~
2
≥
b
N
Figure 9. Comparison of the MJ distributions for low- and high-mT in data with Nb = 1 (left) and
Nb ≥2 (right) after the baseline selection. The expected MJ distributions of the two benchmark
T1tttt scenarios for mT > 140 GeV are overlaid. 7
Results and interpretation The distributions integrate over the Njets and
Emiss
T
bins. The low-mT distribution is normalized to the number of events in the high-mT region. The dashed vertical lines indicate the MJ > 400 GeV threshold that separates the signal regions
from the control samples. [GeV]
J
M
200
400
600
Events/(75 GeV)
0
5
10
15
CMS
(13 TeV)
-1
2.3 fb
> 140 GeV
T
Data, m
140 GeV
≤
T
Data, m
(1500,100)
1
0
χ∼
tt
→
g~
,
g~
g~
(1200,800)
1
0
χ∼
tt
→
g~
,
g~
g~
2
≥
b
N [GeV]
J
M
200
400
600
Events/(75 GeV)
0
5
10
15
20
CMS
(13 TeV)
-1
2.3 fb
> 140 GeV
T
Data, m
140 GeV
≤
T
Data, m
(1500,100)
1
0
χ∼
tt
→
g~
,
g~
g~
(1200,800)
1
0
χ∼
tt
→
g~
,
g~
g~
= 1
b
N JHEP08(2016)122 Figure 9. Comparison of the MJ distributions for low- and high-mT in data with Nb = 1 (left) an Figure 9. Comparison of the MJ distributions for low- and high-mT in data with Nb = 1 (left) and
Nb ≥2 (right) after the baseline selection. The expected MJ distributions of the two benchmark
T1tttt scenarios for mT > 140 GeV are overlaid. The distributions integrate over the Njets and
Emiss
T
bins. The low-mT distribution is normalized to the number of events in the high-mT region. The dashed vertical lines indicate the MJ > 400 GeV threshold that separates the signal regions
from the control samples. Figure 9. Comparison of the MJ distributions for low- and high-mT in data with Nb = 1 (left) and
Nb ≥2 (right) after the baseline selection. The expected MJ distributions of the two benchmark
T1tttt scenarios for mT > 140 GeV are overlaid. The distributions integrate over the Njets and
Emiss
T
bins. The low-mT distribution is normalized to the number of events in the high-mT region. The dashed vertical lines indicate the MJ > 400 GeV threshold that separates the signal regions
from the control samples. Table 3 summarizes the observed event yields, the fitted backgrounds, and the expected
signal yields for the two T1tttt benchmark model points. 7
Results and interpretation Two background estimates are
given: the predictive fit (PF), which uses only the yields in regions R1, R2, and R3, and
the global fit (GF), which also incorporates region R4, as described in section 6. In both
versions of the fit, the signal strength r is fixed to zero, giving results that are model
independent. (When setting limits on individual models, we allow r to float, as discussed
below.) The rows labeled R4 give the results for each of the ten signal regions, as well as
the corresponding κ factors. In the absence of signal, the predictive fit and the version of the global fit performed
under the null hypothesis, r = 0, should be consistent with each other. However, because
the global fit incorporates more information, specifically the yields in R4, this fit has
a smaller uncertainty. The regions with Nb = 1 have small expected contributions from
signal. Summing over all four such signal regions (R4), the number of estimated background
events from the PF and GF are 6.1±2.2 and 5.5±1.3, respectively, compared with 8 events
observed in data. The consistency between the two predictions and between the predicted
and observed yields in the R4 regions with Nb = 1, where the signal contribution is expected
to be small, serves as a further check on the background estimation method. Summing the
yields over the six signal bins with Nb ≥2, the number of estimated background events
from PF and GF is 5.6±1.6 and 4.9±1.0, respectively. In data, we observe 2 events, lower
than, but consistent with the background-only hypothesis. Given the absence of any significant excess, the results are interpreted first as exclusion
limits on the production cross section for T1tttt model points as a function of meg and meχ0
1. Table 4 shows the ranges for the systematic uncertainties associated with predictions for
the expected signal yields, including those on the signal efficiency. The largest uncertainties
arise from the jet energy corrections and from the modeling of ISR. These uncertainties
are generally in the range 10–20% but can increase to ∼30% as the mass splitting between – 18 – Region: bin
κ
T1tttt(NC)
T1tttt(C)
Fitted µbkg (PF)
Fitted µbkg (GF)
Obs. 7
Results and interpretation 200 < Emiss
T
≤400 GeV
R1: all Njets, Nb
—
0.1
3.2
336.0 ± 18.3
335.3 ± 18.2
336
R2: 6 ≤Njets ≤8, Nb = 1
—
0.1
0.2
47.1 ± 6.9
49.5 ± 6.9
47
R2: Njets ≥9, Nb = 1
—
0.1
0.3
7.0 ± 2.6
7.5 ± 2.7
7
R2: 6 ≤Njets ≤8, Nb = 2
—
0.1
0.3
42.0 ± 6.5
41.1 ± 6.2
42
R2: Njets ≥9, Nb = 2
—
0.1
0.5
7.0 ± 2.6
6.6 ± 2.5
7
R2: 6 ≤Njets ≤8, Nb ≥3
—
0.1
0.2
12.0 ± 3.5
11.1 ± 3.2
12
R2: Njets ≥9, Nb ≥3
—
0.2
0.6
1.0 ± 1.0
0.9 ± 0.9
1
R3: all Njets, Nb
—
0.2
3.8
21.0 ± 4.6
21.6 ± 4.2
21
R4: 6 ≤Njets ≤8, Nb = 1
1.12 ± 0.09 ± 0.43
0.2
0.2
3.3 ± 1.4
3.6 ± 1.0
6
R4: Njets ≥9, Nb = 1
0.91 ± 0.06 ± 0.81
0.2
0.4
0.4 ± 0.3
0.4 ± 0.2
1
R4: 6 ≤Njets ≤8, Nb = 2
1.11 ± 0.06 ± 0.42
0.3
0.4
2.9 ± 1.2
2.9 ± 0.8
2
R4: Njets ≥9, Nb = 2
1.05 ± 0.11 ± 0.94
0.3
0.6
0.5 ± 0.3
0.4 ± 0.2
0
R4: 6 ≤Njets ≤8, Nb ≥3
1.25 ± 0.11 ± 0.47
0.3
0.3
0.9 ± 0.4
0.9 ± 0.3
0
R4: Njets ≥9, Nb ≥3
1.05 ± 0.10 ± 0.93
0.3
0.7
0.1 ± 0.1
0.1 ± 0.1
0
Emiss
T
> 400 GeV
R1: all Njets, Nb
—
0.1
0.5
16.0 ± 4.0
17.1 ± 4.0
16
R2: 6 ≤Njets ≤8, Nb = 1
—
0.2
0.1
8.0 ± 2.8
6.8 ± 2.5
8
R2: Njets ≥9, Nb = 1
—
0.1
0.2
1.0 ± 1.0
1.7 ± 1.2
1
R2: 6 ≤Njets ≤8, Nb ≥2
—
0.5
0.3
3.0 ± 1.7
2.5 ± 1.4
3
R2: Njets ≥9, Nb ≥2
—
0.4
0.6
1.0 ± 1.0
0.9 ± 0.9
1
R3: all Njets, Nb
—
0.4
0.9
4.0 ± 2.0
2.9 ± 1.4
4
R4: 6 ≤Njets ≤8, Nb = 1
1.09 ± 0.16 ± 0.42
0.7
0.2
2.2 ± 1.7
1.2 ± 0.7
0
R4: Njets ≥9, Nb = 1
0.98 ± 0.16 ± 0.87
0.4
0.3
0.2 ± 0.3
0.3 ± 0.2
1
R4: 6 ≤Njets ≤8, Nb ≥2
1.29 ± 0.22 ± 0.50
1.9
0.5
1.0 ± 0.8
0.5 ± 0.4
0
R4: Njets ≥9, Nb ≥2
0.90 ± 0.14 ± 0.80
1.6
1.0
0.2 ± 0.3
0.1 ± 0.1
0
Table 3. 7
Results and interpretation Observed and predicted event yields for the signal regions (R4) and background regions
(R1–R3) in data (2.3 fb−1). Expected yields for the two SUSY T1tttt benchmark scenarios are also
given. The results from two types of fits are reported: the predictive fit (PF) and the version of
the global fit (GF) performed under the assumption of the null hypothesis (r = 0). The predictive
fit uses the observed yields in regions R1, R2, and R3 only and is effectively just a propagation of
uncertainties. The global fit uses all four regions. The values of κ obtained from the simulation fit
are also listed. The first uncertainty in κ is statistical, while the second corresponds to the total
systematic uncertainty. The benchmark signal models, T1tttt(NC) and T1tttt(C), are described in
section 3. JHEP08(2016)122 Table 3. Observed and predicted event yields for the signal regions (R4) and background regions
(R1–R3) in data (2.3 fb−1). Expected yields for the two SUSY T1tttt benchmark scenarios are also
given. The results from two types of fits are reported: the predictive fit (PF) and the version of
the global fit (GF) performed under the assumption of the null hypothesis (r = 0). The predictive
fit uses the observed yields in regions R1, R2, and R3 only and is effectively just a propagation of
uncertainties. The global fit uses all four regions. The values of κ obtained from the simulation fit
are also listed. The first uncertainty in κ is statistical, while the second corresponds to the total
systematic uncertainty. The benchmark signal models, T1tttt(NC) and T1tttt(C), are described in
section 3. the gluino and LSP decreases [77]. The uncertainty associated with the renormalization
and factorization scales is determined by varying the scales independently up and down by
a factor of two; these are applied only as an uncertainty in the signal shape, i.e., the cross
section is held constant. The uncertainty associated with the b tagging efficiency is in the
range 1–15%. Uncertainties due to pileup, luminosity [78], lepton selection, and trigger
efficiency are found to be ≤5%. Uncertainties for each particular source are treated as
fully correlated across bins. 7
Results and interpretation A 95% confidence level (CL) upper limit on the production cross section is estimated
using the modified frequentist CLS method [79–81], with a one-sided profile likelihood – 19 – Source
Fractional uncertainty [%]
Lepton efficiency
1–5
Trigger efficiency
1
b tagging efficiency
1–15
Jet energy corrections
1–30
Renormalization and factorization scales
1–5
Initial state radiation
1–35
Pileup
5
Integrated luminosity
3
Table 4. Typical values of the signal-related systematic uncertainties. Uncertainties due to a
particular source are treated as fully correlated between bins, while uncertainties due to different
sources are treated as uncorrelated. JHEP08(2016)122 Table 4. Typical values of the signal-related systematic uncertainties. Uncertainties due to a
particular source are treated as fully correlated between bins, while uncertainties due to different
sources are treated as uncorrelated. Table 4. Typical values of the signal-related systematic uncertainties. Uncertainties due to a
particular source are treated as fully correlated between bins, while uncertainties due to different
sources are treated as uncorrelated. ratio test statistic. For this test, we perform the global fit under the background-only and
background-plus-signal (r floating) hypotheses. The statistical uncertainties from data
counts in the control regions are modeled by the Poisson terms in eq. (6.6). All systematic
uncertainties are multiplicative and are treated as log-normal distributions. Exclusion
limits are also estimated for ±1σ variations on the production cross section based on the
NLO+NLL calculation [39]. Figure 10 shows the corresponding excluded region at a 95% CL for the T1tttt model
in the meg–meχ0
1 plane. At low eχ0
1 mass we exclude gluinos with masses of up to 1600 GeV. The highest limit on the eχ0
1 mass is 800 GeV, attained for meg of approximately 1300 GeV. The observed limits are within the 1σ uncertainty in the expected limits. The central
value is slightly higher because the observed event yield is less than the SM background
prediction, as shown in table 3. In the context of natural SUSY models, it is important to extend the interpretation to
scenarios in which the top squark is lighter than the gluino. Rather than considering a large
set of models with independently varying top squark masses, we consider the extreme case in
which the top squark has approximately the smallest mass consistent with two-body decay,
met1 ≈mt + meχ0
1, for a range of gluino and neutralino masses. 7
Results and interpretation The decay kinematics for
such extreme, compressed mass spectrum models correspond to the lowest signal efficiency
for given values of meg and meχ0
1, because the top quark and the eχ0
1 are produced at rest
in the top squark frame. As a consequence, the excluded signal cross section for fixed
values of meg and meχ0
1 and with meg > met1 ≥mt + meχ0
1 is minimized around this extreme
model point. For physical consistency, the signal model used in this study, both in the fit
procedure and in the theoretical cross section used to obtain mass limits, includes not only
gluino-pair production, but also direct et1et1 production. However, the effect of the direct
top squark contribution on the results is small, ≲2% for meχ0
1 > 400 GeV and up to 20%
for low values of meχ0
1. χ1
Figure 11 shows the excluded region in the meg–meχ0
1 plane for this combined model
with both gluino-mediated top squark production and direct top squark pair production. – 20 – [GeV]
g~
m
800
1000
1200
1400
1600
1800
[GeV]
1
0
χ∼
m
0
200
400
600
800
1000
1200
1400
1600
1800
3
−
10
2
−
10
1
−
10
1
(13 TeV)
-1
2.3 fb
CMS
NLO+NLL exclusion
1
0
χ∼
t
t
→
g~
,
g~
g~
→
pp
theory
σ
1
±
Observed
experiment
σ
1
±
Expected
95% CL upper limit on cross section [pb]
Figure 10. Interpretation of results in the T1tttt model. The colored regions show the upper
limits (95% CL) on the production cross section for pp →egeg,eg →tteχ0
1 in the meg–meχ0
1 plane. The
curves show the expected and observed limits on the corresponding SUSY particle masses obtained
by comparing the excluded cross section with theoretical cross sections. [GeV]
g~
m
800
1000
1200
1400
1600
1800
[GeV]
1
0
χ∼
m
0
200
400
600
800
1000
1200
1400
1600
1800
3
−
10
2
−
10
1
−
10
1
(13 TeV)
-1
2.3 fb
CMS
NLO+NLL exclusion
1
0
χ∼
t
t
→
g~
,
g~
g~
→
pp
theory
σ
1
±
Observed
experiment
σ
1
±
Expected
95% CL upper limit on cross section [pb] JHEP08(2016)122 Figure 10. Interpretation of results in the T1tttt model. 7
Results and interpretation In this kinematic region, the sensitivity to the signal is dominated by the events
that have at least two leptonic W boson decays, which produce additional Emiss
T
, as well as
a tail in the mT distribution. Although such dilepton events are nominally excluded in the
analysis, a significant number of these signal events escape the dilepton veto. These events
include both W decays to τ leptons that decay hadronically, and W decays to electrons or
muons that are below kinematic thresholds or are outside of the detector acceptance. 7
Results and interpretation The colored regions show the upper
limits (95% CL) on the production cross section for pp →egeg,eg →tteχ0
1 in the meg–meχ0
1 plane. The
curves show the expected and observed limits on the corresponding SUSY particle masses obtained
by comparing the excluded cross section with theoretical cross sections. The top squark mass is assumed to be 175 GeV above that of the neutralino. For most of
the excluded region, the boundary is close to that obtained for the T1tttt model, showing
that there is only a weak sensitivity to the value of the top squark mass. The uncertainty
on the boundary of the excluded region for the T5tttt model is similar to that shown for
the T1tttt model in figure 10. For meχ0
1 > 150 GeV, the excluded value of meg is typically
within 60 GeV of that excluded for T1tttt. Models that have low values of meχ0
1 show a
reduced sensitivity because the neutralino carries very little momentum, reducing the value
of mT. In this kinematic region, the sensitivity to the signal is dominated by the events
that have at least two leptonic W boson decays, which produce additional Emiss
T
, as well as
a tail in the mT distribution. Although such dilepton events are nominally excluded in the
analysis, a significant number of these signal events escape the dilepton veto. These events
include both W decays to τ leptons that decay hadronically, and W decays to electrons or
muons that are below kinematic thresholds or are outside of the detector acceptance. The top squark mass is assumed to be 175 GeV above that of the neutralino. For most of
the excluded region, the boundary is close to that obtained for the T1tttt model, showing
that there is only a weak sensitivity to the value of the top squark mass. The uncertainty
on the boundary of the excluded region for the T5tttt model is similar to that shown for
the T1tttt model in figure 10. For meχ0
1 > 150 GeV, the excluded value of meg is typically
within 60 GeV of that excluded for T1tttt. Models that have low values of meχ0
1 show a
reduced sensitivity because the neutralino carries very little momentum, reducing the value
of mT. 8
Summary Using a sample of proton-proton collisions at √s = 13 TeV with an integrated luminosity
of 2.3 fb−1, a search for supersymmetry is performed in the final state with a single lepton, – 21 – [GeV]
g~
m
600
800
1000
1200
1400
1600
[GeV]
1
0
χ∼
m
0
200
400
600
800
1000
1200
CMS
(13 TeV)
-1
2.3 fb
1
0
χ∼
t
t
→
g~
,
g~
g~
→
pp
= 175 GeV)
1
0
χ∼
- m
1t~
(m
1
0
χ∼
t
→
1t~
t,
1t~
→
g~
,
1t~
1t~
+
g~
g~
→
pp
Expected
Observed
Figure 11. Excluded region (95% CL), shown in blue, in the meg–meχ0
1 plane for a model combining
T5tttt, gluino pair production, followed by gluino decay to an on-shell top squark, together with
a model for direct top squark pair production. The top squarks decay via the two-body process
et →teχ0
1. The neutralino and top squark masses are related by the constraint met1 = meχ0
1 +175 GeV. For comparison, the excluded region (95% CL) from figure 10 for the T1tttt model, which has three-
body gluino decay, is shown in red. The small difference between the two boundary curves shows
that the limits for the scenarios with two-body gluino decay have only a weak dependence on the
top squark mass. [GeV]
g~
m
600
800
1000
1200
1400
1600
[GeV]
1
0
χ∼
m
0
200
400
600
800
1000
1200
CMS
(13 TeV)
-1
2.3 fb
1
0
χ∼
t
t
→
g~
,
g~
g~
→
pp
= 175 GeV)
1
0
χ∼
- m
1t~
(m
1
0
χ∼
t
→
1t~
t,
1t~
→
g~
,
1t~
1t~
+
g~
g~
→
pp
Expected
Observed JHEP08(2016)122 Figure 11. Excluded region (95% CL), shown in blue, in the meg–meχ0
1 plane for a model combining
T5tttt, gluino pair production, followed by gluino decay to an on-shell top squark, together with
a model for direct top squark pair production. The top squarks decay via the two-body process
et →teχ0
1. The neutralino and top squark masses are related by the constraint met1 = meχ0
1 +175 GeV. For comparison, the excluded region (95% CL) from figure 10 for the T1tttt model, which has three-
body gluino decay, is shown in red. 8
Summary The small difference between the two boundary curves shows
that the limits for the scenarios with two-body gluino decay have only a weak dependence on the
top squark mass. b-tagged jets, and large missing transverse momentum. The search focuses on final states
resulting from the pair production of gluinos, which subsequently decay via eg →tteχ0
1,
leading to high jet multiplicities. A key feature of the analysis is the use of the variable MJ, the sum of the masses
of large-R jets, which are formed by clustering anti-kT R = 0.4 jets and leptons. Used
in conjunction with the variable mT, the transverse mass of the system consisting of the
lepton and the missing transverse momentum vector, MJ provides a powerful background
estimation method that is well suited to this high jet multiplicity search. After the baseline selection is applied, signal (R4) and control regions (R1, R2, and R3)
are defined in the MJ–mT plane, which are further divided into bins of Emiss
T
, Njets, and
Nb to provide additional sensitivity. In regions R3 and R4, the requirement mT > 140 GeV
provides strong suppression of the single-lepton tt background, so that dilepton tt events
dominate over all other background sources. For these dilepton events to enter a signal – 22 – region, however, they must contain a substantial amount of initial-state radiation (ISR). For
this extreme range of ISR jet momentum and multiplicity, the single-lepton and dilepton
tt events have very similar kinematic properties. The variables MJ and mT are nearly
uncorrelated, even though different processes dominate the low- and high-mT regions. As
a consequence, the low-mT regions (R1 and R2) can be used to measure the background
shape for the MJ distribution at high mT. A correction factor, near unity, is taken from
simulation and is used to account for a possible correlation between MJ and mT. The observed event yields in the signal regions are consistent with the predictions for
the SM background contributions, and exclusion limits are set on the gluino pair production
cross sections in the meg–meχ0
1 plane, as described by the simplified models T1tttt and
T5tttt, where the latter is augmented with a model of direct top squark pair production
for consistency. In the T1tttt model, gluinos decay via the three-body process eg →tteχ0
1,
which proceeds via a virtual top squark in the intermediate state. 8
Summary Under the assumption
of a 100% branching fraction to this final state, the cross section limit for each model point
is compared with the theoretical cross section to determine the excluded particle masses. Gluinos with a mass below 1600 GeV are excluded at a 95% CL for scenarios with low eχ0
1
mass, and neutralinos with a mass below 800 GeV are excluded for a gluino mass of about
1300 GeV. In the T5tttt model, the top squark is lighter than the gluino, which therefore
decays via a two-body process. The boundary of the excluded region in the meg–meχ0
1 plane
for T5tttt is found to be only weakly sensitive to the top squark mass. These results
significantly extend the sensitivity of single-lepton searches based on data at √s = 8 TeV. JHEP08(2016)122 Acknowledgments Forschungszentren, Germany; the General Secretariat for Research and Technology, Greece;
the National Scientific Research Foundation, and National Innovation Office, Hungary; the
Department of Atomic Energy and the Department of Science and Technology, India; the
Institute for Studies in Theoretical Physics and Mathematics, Iran; the Science Founda-
tion, Ireland; the Istituto Nazionale di Fisica Nucleare, Italy; the Ministry of Science, ICT
and Future Planning, and National Research Foundation (NRF), Republic of Korea; the
Lithuanian Academy of Sciences; the Ministry of Education, and University of Malaya
(Malaysia); the Mexican Funding Agencies (BUAP, CINVESTAV, CONACYT, LNS, SEP,
and UASLP-FAI); the Ministry of Business, Innovation and Employment, New Zealand;
the Pakistan Atomic Energy Commission; the Ministry of Science and Higher Education
and the National Science Centre, Poland; the Funda¸c˜ao para a Ciˆencia e a Tecnologia,
Portugal; JINR, Dubna; the Ministry of Education and Science of the Russian Federation,
the Federal Agency of Atomic Energy of the Russian Federation, Russian Academy of
Sciences, and the Russian Foundation for Basic Research; the Ministry of Education, Sci-
ence and Technological Development of Serbia; the Secretar´ıa de Estado de Investigaci´on,
Desarrollo e Innovaci´on and Programa Consolider-Ingenio 2010, Spain; the Swiss Funding
Agencies (ETH Board, ETH Zurich, PSI, SNF, UniZH, Canton Zurich, and SER); the
Ministry of Science and Technology, Taipei; the Thailand Center of Excellence in Physics,
the Institute for the Promotion of Teaching Science and Technology of Thailand, Special
Task Force for Activating Research and the National Science and Technology Development
Agency of Thailand; the Scientific and Technical Research Council of Turkey, and Turkish
Atomic Energy Authority; the National Academy of Sciences of Ukraine, and State Fund
for Fundamental Researches, Ukraine; the Science and Technology Facilities Council, U.K.;
the US Department of Energy, and the US National Science Foundation. JHEP08(2016)122 Individuals have received support from the Marie-Curie program and the European Re-
search Council and EPLANET (European Union); the Leventis Foundation; the A.P. Acknowledgments Finally, we acknowledge the enduring support for the construction and operation
of the LHC and the CMS detector provided by the following funding agencies: the Austrian
Federal Ministry of Science, Research and Economy and the Austrian Science Fund; the
Belgian Fonds de la Recherche Scientifique, and Fonds voor Wetenschappelijk Onderzoek;
the Brazilian Funding Agencies (CNPq, CAPES, FAPERJ, and FAPESP); the Bulgarian
Ministry of Education and Science; CERN; the Chinese Academy of Sciences, Ministry of
Science and Technology, and National Natural Science Foundation of China; the Colombian
Funding Agency (COLCIENCIAS); the Croatian Ministry of Science, Education and Sport,
and the Croatian Science Foundation; the Research Promotion Foundation, Cyprus; the
Ministry of Education and Research, Estonian Research Council via IUT23-4 and IUT23-
6 and European Regional Development Fund, Estonia; the Academy of Finland, Finnish
Ministry of Education and Culture, and Helsinki Institute of Physics; the Institut National
de Physique Nucl´eaire et de Physique des Particules/CNRS, and Commissariat `a l’´Energie
Atomique et aux ´Energies Alternatives/CEA, France; the Bundesministerium f¨ur Bildung
und Forschung, Deutsche Forschungsgemeinschaft, and Helmholtz-Gemeinschaft Deutscher – 23 – Forschungszentren, Germany; the General Secretariat for Research and Technology, Greece;
the National Scientific Research Foundation, and National Innovation Office, Hungary; the
Department of Atomic Energy and the Department of Science and Technology, India; the
Institute for Studies in Theoretical Physics and Mathematics, Iran; the Science Founda-
tion, Ireland; the Istituto Nazionale di Fisica Nucleare, Italy; the Ministry of Science, ICT
and Future Planning, and National Research Foundation (NRF), Republic of Korea; the
Lithuanian Academy of Sciences; the Ministry of Education, and University of Malaya
(Malaysia); the Mexican Funding Agencies (BUAP, CINVESTAV, CONACYT, LNS, SEP,
and UASLP-FAI); the Ministry of Business, Innovation and Employment, New Zealand;
the Pakistan Atomic Energy Commission; the Ministry of Science and Higher Education
and the National Science Centre, Poland; the Funda¸c˜ao para a Ciˆencia e a Tecnologia,
Portugal; JINR, Dubna; the Ministry of Education and Science of the Russian Federation,
the Federal Agency of Atomic Energy of the Russian Federation, Russian Academy of
Sciences, and the Russian Foundation for Basic Research; the Ministry of Education, Sci-
ence and Technological Development of Serbia; the Secretar´ıa de Estado de Investigaci´on,
Desarrollo e Innovaci´on and Programa Consolider-Ingenio 2010, Spain; the Swiss Funding
Agencies (ETH Board, ETH Zurich, PSI, SNF, UniZH, Canton Zurich, and SER); the
Ministry of Science and Technology, Taipei; the Thailand Center of Excellence in Physics,
the Institute for the Promotion of Teaching Science and Technology of Thailand, Special
Task Force for Activating Research and the National Science and Technology Development
Agency of Thailand; the Scientific and Technical Research Council of Turkey, and Turkish
Atomic Energy Authority; the National Academy of Sciences of Ukraine, and State Fund
for Fundamental Researches, Ukraine; the Science and Technology Facilities Council, U.K.;
the US Department of Energy, and the US National Science Foundation. Acknowledgments We congratulate our colleagues in the CERN accelerator departments for the excellent per-
formance of the LHC and thank the technical and administrative staffs at CERN and at
other CMS institutes for their contributions to the success of the CMS effort. In addition,
we gratefully acknowledge the computing centers and personnel of the Worldwide LHC
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J. Lingemann, A. Nehrkorn, A. Nowack, I.M. Nugent, C. Pistone, O. Pooth, A. Stahl15 V. Cherepanov, Y. Erdogan, G. Fl¨ugge, F. Hoehle, B. Kargoll, T. Kress, A. K¨unsken,
J. Lingemann, A. Nehrkorn, A. Nowack, I.M. Nugent, C. Pistone, O. Pooth, A. Stahl15 National and Kapodistrian University of Athens, Athens, Greece National and Kapodistrian University of Athens, Athens, Greece
A. Agapitos, S. Kesisoglou, A. Panagiotou, N. Saoulidou, E. Tziaferi A. Agapitos, S. Kesisoglou, A. Panagiotou, N. Saoulidou, E. Tziaferi A. Agapitos, S. Kesisoglou, A. Panagiotou, N. Saoulidou, E. Tziaferi
University of Io´annina, Io´annina, Greece
I. Evangelou, G. Flouris, C. Foudas, P. Kokkas, N. Loukas, N. Manthos, I. Papadopoulos,
E. Paradas
MTA-ELTE Lend¨ulet CMS Particle and Nuclear Physics Group, E¨otv¨os Lor´and
University
N. Filipovic
Wigner Research Centre for Physics, Budapest, Hungary
G. Bencze, C. Hajdu, P. Hidas, D. Horvath20, F. Sikler, V. Veszpremi, G. Vesztergombi21,
A.J. Zsigmond
Institute of Nuclear Research ATOMKI, Debrecen, Hungary
N. Beni, S. Czellar, J. Karancsi22, A. Makovec, J. Molnar, Z. Szillasi
University of Debrecen, Debrecen, Hungary
M. Bart´ok21, P. Raics, Z.L. Trocsanyi, B. Ujvari
National Institute of Science Education and Research, Bhubaneswar, India
S. Bahinipati, S. Choudhury23, P. Mal, K. Mandal, A. Nayak24, D.K. Sahoo, N. Sahoo,
S.K. Swain
Panjab University, Chandigarh, India
S. Bansal, S.B. Beri, V. Bhatnagar, R. Chawla, R. Gupta, U.Bhawandeep, A.K. Kalsi,
A. Kaur, M. Kaur, R. Kumar, A. Mehta, M. Mittal, J.B. Singh, G. Walia
University of Delhi, Delhi, India
Ashok Kumar, A. Bhardwaj, B.C. Choudhary, R.B. Garg, S. Keshri, A. Kumar, S. Mal-
hotra, M. Naimuddin, N. Nishu, K. Ranjan, R. Sharma, V. Sharma University of Io´annina, Io´annina, Greece I. Evangelou, G. Flouris, C. Foudas, P. Kokkas, N. Loukas, N. Manthos, I. Papadopoulos,
E. Paradas Institute of Nuclear Research ATOMKI, Debrecen, Hungary
N. Beni, S. Czellar, J. Karancsi22, A. Makovec, J. Molnar, Z. Szillasi
University of Debrecen, Debrecen, Hungary
M. Bart´ok21, P. Raics, Z.L. Trocsanyi, B. Ujvari Institute of Nuclear Research ATOMKI, Debrecen, Hungary
N. Beni, S. Czellar, J. Karancsi22, A. Makovec, J. Molnar, Z. Szillasi National Institute of Science Education and Research, Bhubaneswar, India
S. Bahinipati, S. Choudhury23, P. Mal, K. Mandal, A. Nayak24, D.K. Sahoo, N. Sahoo,
S.K. Swain S. Bahinipati, S. Choudhury23, P. Mal, K. Mandal, A. Nayak24, D.K. Sahoo, N. Saho
S.K. Swain University of Hamburg, Hamburg, Germany V. Blobel, M. Centis Vignali, A.R. Draeger, T. Dreyer, E. Garutti, K. Goebel, D. Gonzalez,
J. Haller, M. Hoffmann, A. Junkes, R. Klanner, R. Kogler, N. Kovalchuk, T. Lapsien,
T. Lenz, I. Marchesini, D. Marconi, M. Meyer, M. Niedziela, D. Nowatschin, J. Ott,
F. Pantaleo15, T. Peiffer, A. Perieanu, J. Poehlsen, C. Sander, C. Scharf, P. Schleper,
A. Schmidt, S. Schumann, J. Schwandt, H. Stadie, G. Steinbr¨uck, F.M. Stober, M. St¨over,
H. Tholen, D. Troendle, E. Usai, L. Vanelderen, A. Vanhoefer, B. Vormwald – 33 – Panjab University, Chandigarh, India S. Bansal, S.B. Beri, V. Bhatnagar, R. Chawla, R. Gupta, U.Bhawandeep, A.K. Kalsi,
A. Kaur, M. Kaur, R. Kumar, A. Mehta, M. Mittal, J.B. Singh, G. Walia Institut f¨ur Experimentelle Kernphysik, Karlsruhe, Germany C. Barth, C. Baus, J. Berger, E. Butz, T. Chwalek, F. Colombo, W. De Boer, A. D C. Barth, C. Baus, J. Berger, E. Butz, T. Chwalek, F. Colombo, W. De Boer, A. Dierlamm,
S. Fink, R. Friese, M. Giffels, A. Gilbert, D. Haitz, F. Hartmann15, S.M. Heindl,
U. Husemann, I. Katkov16, P. Lobelle Pardo, B. Maier, H. Mildner, M.U. Mozer, T. M¨uller,
Th. M¨uller, M. Plagge, G. Quast, K. Rabbertz, S. R¨ocker, F. Roscher, M. Schr¨oder,
G. Sieber, H.J. Simonis, R. Ulrich, J. Wagner-Kuhr, S. Wayand, M. Weber, T. Weiler,
S. Williamson, C. W¨ohrmann, R. Wolf Institute of Nuclear and Particle Physics (INPP), NCSR Demokritos, Aghia
Paraskevi, Greece JHEP08(2016)122 G. Anagnostou, G. Daskalakis, T. Geralis, V.A. Giakoumopoulou, A. Kyriakis, D. Loukas,
I. Topsis-Giotis Saha Institute of Nuclear Physics, Kolkata, India Saha Institute of Nuclear Physics, Kolkata, India
R. Bhattacharya, S. Bhattacharya, K. Chatterjee, S. Dey, S. Dutt, S. Dutta, S. Ghosh,
N. Majumdar, A. Modak, K. Mondal, S. Mukhopadhyay, S. Nandan, A. Purohit, A. Roy,
D. Roy, S. Roy Chowdhury, S. Sarkar, M. Sharan, S. Thakur Saha Institute of Nuclear Physics, Kolkata, India R. Bhattacharya, S. Bhattacharya, K. Chatterjee, S. Dey, S. Dutt, S. Dutta, S. Ghos R. Bhattacharya, S. Bhattacharya, K. Chatterjee, S. Dey, S. Dutt, S. Dutta, S. Ghosh,
N. Majumdar, A. Modak, K. Mondal, S. Mukhopadhyay, S. Nandan, A. Purohit, A. Roy,
D. Roy, S. Roy Chowdhury, S. Sarkar, M. Sharan, S. Thakur D. Roy, S. Roy Chowdhury, S. Sarkar, M. Sharan, S. Thakur Indian Institute of Technology Madras, Madras, India
P.K. Behera Bhabha Atomic Research Centre, Mumbai, India Bhabha Atomic Research Centre, Mumbai, India
R. Chudasama, D. Dutta, V. Jha, V. Kumar, A.K. Mohanty15, P.K. Netrakanti, L.M. Pant,
P. Shukla, A. Topkar Bhabha Atomic Research Centre, Mumbai, India R. Chudasama, D. Dutta, V. Jha, V. Kumar, A.K. Mohanty15, P.K. Netrakanti, L.M. Pant,
P. Shukla, A. Topkar JHEP08(2016)122 Tata Institute of Fundamental Research-A, Mumbai, India Tata Institute of Fundamental Research-A, Mumbai, India
T. Aziz, S. Dugad, G. Kole, B. Mahakud, S. Mitra, G.B. Mohanty, N. Sur, B. Sutar T. Aziz, S. Dugad, G. Kole, B. Mahakud, S. Mitra, G.B. Mohanty, N. Sur, B. Sutar Tata Institute of Fundamental Research-B, Mumbai, India Tata Institute of Fundamental Research-B, Mumbai, India
S. Banerjee, S. Bhowmik25, R.K. Dewanjee, S. Ganguly, M. Guchait, Sa. Jain, S. Kumar,
M. Maity25, G. Majumder, K. Mazumdar, B. Parida, T. Sarkar25, N. Wickramage26 S. Banerjee, S. Bhowmik25, R.K. Dewanjee, S. Ganguly, M. Guchait, Sa. Jain, S. Kuma M. Maity25, G. Majumder, K. Mazumdar, B. Parida, T. Sarkar25, N. Wickramage26 Indian Institute of Science Education and Research (IISER), Pune, India
S. Chauhan, S. Dube, A. Kapoor, K. Kothekar, A. Rane, S. Sharma Institute for Research in Fundamental Sciences (IPM), Tehran, Iran Institute for Research in Fundamental Sciences (IPM), Tehran, Iran
H. Bakhshiansohi, H. Behnamian, S. Chenarani27, E. Eskandari Tadavani, S.M. Etesami27,
A. Fahim28, M. Khakzad, M. Mohammadi Najafabadi, M. Naseri, S. Paktinat Mehdiabadi,
F. Rezaei Hosseinabadi, B. Safarzadeh29, M. Zeinali University College Dublin, Dublin, Ireland
M. Felcini, M. Grunewald University College Dublin, Dublin, Ireland
M. Felcini, M. Grunewald INFN Sezione di Baria, Universit`a di Barib, Politecnico di Baric, Bari, Italy
M. Abbresciaa,b, C. Calabriaa,b, C. Caputoa,b, A. Colaleoa, D. Creanzaa,c, L. Cristellaa,b,
N. De Filippisa,c, M. De Palmaa,b, L. Fiorea, G. Iasellia,c, G. Maggia,c, M. Maggia,
G. Minielloa,b, S. Mya,b, S. Nuzzoa,b, A. Pompilia,b, G. Pugliesea,c, R. Radognaa,b,
A. Ranieria, G. Selvaggia,b, L. Silvestrisa,15, R. Vendittia,b, P. Verwilligena University of Delhi, Delhi, India Ashok Kumar, A. Bhardwaj, B.C. Choudhary, R.B. Garg, S. Keshri, A. Kumar, S. Mal-
hotra, M. Naimuddin, N. Nishu, K. Ranjan, R. Sharma, V. Sharma – 34 – INFN Sezione di Bolognaa, Universit`a di Bolognab, Bologna, Italy INFN Sezione di Bolognaa, Universit`a di Bolognab, Bologna, Italy INFN Sezione di Bologna , Universita di Bologna , Bologna, Italy
G. Abbiendia, C. Battilana, D. Bonacorsia,b, S. Braibant-Giacomellia,b, L. Brigliadoria,b,
R. Campaninia,b,
P. Capiluppia,b,
A. Castroa,b,
F.R. Cavalloa,
S.S. Chhibraa,b,
G. Codispotia,b, M. Cuffiania,b, G.M. Dallavallea, F. Fabbria, A. Fanfania,b, D. Fasanellaa,b,
P. Giacomellia, C. Grandia, L. Guiduccia,b, S. Marcellinia, G. Masettia, A. Montanaria,
F.L. Navarriaa,b, A. Perrottaa, A.M. Rossia,b, T. Rovellia,b, G.P. Sirolia,b, N. Tosia,b,15 F.L. Navarriaa,b, A. Perrottaa, A.M. Rossia,b, T. Rovellia,b, G.P. Sirolia,b, N. Tosia,b,15 NFN Sezione di Firenzea, Universit`a di Firenzeb, Firenze, Italy INFN Sezione di Firenzea, Universit`a di Firenzeb, Firenze, Italy
G. Barbaglia, V. Ciullia,b, C. Civininia, R. D’Alessandroa,b, E. Focardia,b, V. Goria,b,
P. Lenzia,b, M. Meschinia, S. Paolettia, G. Sguazzonia, L. Viliania,b,15 G. Barbaglia, V. Ciullia,b, C. Civininia, R. D’Alessandroa,b, E. Focardia,b, V. Goria
P. Lenzia,b, M. Meschinia, S. Paolettia, G. Sguazzonia, L. Viliania,b,15 INFN Laboratori Nazionali di Frascati, Frascati, Italy
L. Benussi, S. Bianco, F. Fabbri, D. Piccolo, F. Primavera15 INFN Sezione di Genovaa, Universit`a di Genovab, Genova, Italy
V. Calvellia,b, F. Ferroa, M. Lo Veterea,b, M.R. Mongea,b, E. Robuttia, S. Tosia,b INFN Sezione di Milano-Bicoccaa, Universit`a di Milano-Bicoccab, Milano, Italy
L. Brianza, M.E. Dinardoa,b, S. Fiorendia,b, S. Gennaia, A. Ghezzia,b, P. Govonia,b,
S. Malvezzia,
R.A. Manzonia,b,15,
B. Marzocchia,b,
D. Menascea,
L. Moronia,
M. Paganonia,b, D. Pedrinia, S. Pigazzini, S. Ragazzia,b, T. Tabarelli de Fatisa,b JHEP08(2016)122 INFN Sezione di Napolia, Universit`a di Napoli ’Federico II’b, Napoli, Italy,
Universit`a della Basilicatac, Potenza, Italy, Universit`a G. Marconid, Roma,
Italy S. Buontempoa,
N. Cavalloa,c,
G. De Nardo,
S. Di Guidaa,d,15,
M. Espositoa S. Buontempoa,
N. Cavalloa,c,
G. De Nardo,
S. Di Guidaa,d,15,
M. Espositoa,b,
F
Fabozzia,c
A O M
Iorioa,b
G
Lanzaa
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Listaa
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Meolaa,d,15
M
Merolaa S. Buontempoa,
N. Cavalloa,c,
G. De Nardo,
S. Di Guidaa,d,15,
M. Espositoa,b,
F. Fabozzia,c, A.O.M. Iorioa,b, G. Lanzaa, L. Listaa, S. Meolaa,d,15, M. Merolaa,
P P
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a 15 C S
a b F Th S. Buontempoa,
N. Cavalloa,c,
G. De Nardo,
S. Di Guidaa,d,15,
M. Espositoa,b,
F. Fabozzia,c, A.O.M. Iorioa,b, G. Lanzaa, L. Listaa, S. Meolaa,d,15, M. Merolaa,
P. Paoluccia,15, C. Sciaccaa,b, F. Thyssen F. Fabozzia,c, A.O.M. Iorioa,b, G. Lanzaa, L. Listaa, S. Meolaa,d,15, M. Merolaa,
P. Paoluccia,15, C. Sciaccaa,b, F. Thyssen INFN Sezione di Padovaa, Universit`a di Padovab, Padova, Italy, Universit`a di
Trentoc, Trento, Italy P. Azzia,15,
L. Benatoa,b,
D. Biselloa,b,
A. Bolettia,b,
R. Carlina,b,
A. Carvalho
Antunes De Oliveiraa,b,
P. Checchiaa,
M. Dall’Ossoa,b,
P. De Castro Manzanoa, ,
,
,
,
,
Antunes De Oliveiraa,b,
P. Checchiaa,
M. Dall’Ossoa,b,
P. De Castro Manzanoa,
T. Dorigoa, U. Dossellia, F. Gasparinia,b, F. Gonellaa, S. Lacapraraa, M. Margonia,b,
A T
Meneguzzoa,b
J
Pazzinia,b,15
M
Pegoraroa
N
Pozzobona,b
P
Ronchesea,b T. Dorigoa, U. Dossellia, F. Gasparinia,b, F. Gonellaa, S. Lacapraraa, M. Margonia,b,
A.T. Meneguzzoa,b, J. Pazzinia,b,15, M. Pegoraroa, N. Pozzobona,b, P. Ronchesea,b, M. Sgaravattoa, F. Simonettoa,b, E. Torassaa, M. Zanetti, P. Zottoa,b, A. Zucchettaa,b,
G. Zumerlea,b INFN Sezione di Paviaa, Universit`a di Paviab, Pavia, Italy INFN Sezione di Cataniaa, Universit`a di Cataniab, Catania, Ita INFN Sezione di Cataniaa, Universit`a di Cataniab, Catania, Italy S. Albergoa,b, M. Chiorbolia,b, S. Costaa,b, A. Di Mattiaa, F. Giordanoa,b, R. Potenzaa,b,
A. Tricomia,b, C. Tuvea,b A. Tricomia,b, C. Tuvea,b – 35 – INFN Sezione di Firenzea, Universit`a di Firenzeb, Firenze, Italy INFN Sezione di Paviaa, Universit`a di Paviab, Pavia, Italy A. Braghieria, A. Magnania,b, P. Montagnaa,b, S.P. Rattia,b, V. Rea, C. Riccardia,b,
P. Salvinia, I. Vaia,b, P. Vituloa,b INFN Sezione di Perugiaa, Universit`a di Perugiab, Perugia, Italy L. Alunni Solestizia,b,
G.M. Bileia,
D. Ciangottinia,b,
L. Fan`oa,b,
P. Laricciaa,b,
R. Leonardia,b, G. Mantovania,b, M. Menichellia, A. Sahaa, A. Santocchiaa,b INFN Sezione di Pisaa, Universit`a di Pisab, Scuola Normale Superiore di Pisac,
Pisa, Italy K. Androsova,30, P. Azzurria,15, G. Bagliesia, J. Bernardinia, T. Boccalia, R. Castaldia,
M.A. Cioccia,30, R. Dell’Orsoa, S. Donatoa,c, G. Fedi, A. Giassia, M.T. Grippoa,30,
F. Ligabuea,c, T. Lomtadzea, L. Martinia,b, A. Messineoa,b, F. Pallaa, A. Rizzia,b, A. Savoy-
Navarroa,31, P. Spagnoloa, R. Tenchinia, G. Tonellia,b, A. Venturia, P.G. Verdinia F. Ligabuea,c, T. Lomtadzea, L. Martinia,b, A. Messineoa,b, F. Pallaa, A. Rizzia,b, A. Savoy-
Navarroa,31, P. Spagnoloa, R. Tenchinia, G. Tonellia,b, A. Venturia, P.G. Verdinia – 36 – INFN Sezione di Romaa, Universit`a di Romab, Roma, Italy L. Baronea,b, F. Cavallaria, M. Cipriania,b, G. D’imperioa,b,15, D. Del Rea,b,15, M. Diemoz S. Gellia,b, C. Jordaa, E. Longoa,b, F. Margarolia,b, P. Meridiania, G. Organtinia,b,
R. Paramattia, F. Preiatoa,b, S. Rahatloua,b, C. Rovellia, F. Santanastasioa,b INFN Sezione di Torinoa, Universit`a di Torinob, Torino, Italy, Universit`a del
Piemonte Orientalec, Novara, Italy N. Amapanea,b,
R. Arcidiaconoa,c,15,
S. Argiroa,b,
M. Arneodoa,c,
N. Bartosik N. Amapanea,b,
R. Arcidiaconoa,c,15,
S. Argiroa,b,
M. Arneodoa,c,
N. Bartosika,
R. Bellana,b, C. Biinoa, N. Cartigliaa, F. Cennaa,b, M. Costaa,b, R. Covarellia,b, R. Bellana,b, C. Biinoa, N. Cartigliaa, F. Cennaa,b, M. Costaa,b, R. Covarellia,b, A. Deganoa,b,
N. Demariaa,
L. Fincoa,b,
B. Kiania,b,
C. Mariottia,
S. Maselli A. Deganoa,b,
N. Demariaa,
L. Fincoa,b,
B. Kiania,b,
C. Mariottia,
S. Masellia,
E. Migliorea,b, V. Monacoa,b, E. Monteila,b, M.M. Obertinoa,b, L. Pachera,b, N. Pastronea, JHEP08(2016)122 E. Migliorea,b, V. Monacoa,b, E. Monteila,b, M.M. Obertinoa,b, L. Pachera,b, N. Pastrone M. Pelliccionia, G.L. Pinna Angionia,b, F. Raveraa,b, A. Romeroa,b, M. Ruspaa,c,
R. Sacchia,b, K. Shchelinaa,b, V. Solaa, A. Solanoa,b, A. Staianoa, P. Traczyka,b INFN Sezione di Triestea, Universit`a di Triesteb, Trieste, Italy S. Belfortea, M. Casarsaa, F. Cossuttia, G. Della Riccaa,b, C. La Licataa,b, A. Schizzia,b,
A. Zanettia Kyungpook National University, Daegu, Korea Warsaw, Poland K. Bunkowski, A. Byszuk35, K. Doroba, A. Kalinowski, M. Konecki, J. Krolikowski,
M. Misiura, M. Olszewski, M. Walczak Laborat´orio de Instrumenta¸c˜ao e F´ısica Experimental de Part´ıculas, Lisboa,
Portugal Kyungpook National University, Daegu, Korea D.H. Kim, G.N. Kim, M.S. Kim, S. Lee, S.W. Lee, Y.D. Oh, S. Sekmen, D.C. Son,
Y.C. Yang Chonbuk National University, Jeonju, Korea
H. Kim, A. Lee Chonbuk National University, Jeonju, Korea Chonbuk National University, Jeonju, Korea
H. Kim, A. Lee y,
j ,
H. Kim, A. Lee Hanyang University, Seoul, Korea J.A. Brochero Cifuentes, T.J. Kim Korea University, Seoul, Korea
S. Cho, S. Choi, Y. Go, D. Gyun, S. Ha, B. Hong, Y. Jo, Y. Kim, B. Lee, K. Lee, K.S. Lee,
S. Lee, J. Lim, S.K. Park, Y. Roh Seoul National University, Seoul, Korea
J. Almond, J. Kim, S.B. Oh, S.h. Seo, U.K. Yang, H.D. Yoo, G.B. Yu y,
,
J. Almond, J. Kim, S.B. Oh, S.h. Seo, U.K. Yang, H.D. Yoo, G.B. Yu University of Seoul, Seoul, Korea M. Choi, H. Kim, H. Kim, J.H. Kim, J.S.H. Lee, I.C. Park, G. Ryu, M.S. Ryu M. Choi, H. Kim, H. Kim, J.H. Kim, J.S.H. Lee, I.C. Park, G. Ryu, M.S. Ryu Sungkyunkwan University, Suwon, Korea Sungkyunkwan University, Suwon, Korea Y. Choi, J. Goh, C. Hwang, D. Kim, J. Lee, I. Yu Y. Choi, J. Goh, C. Hwang, D. Kim, J. Lee, I. Yu Vilnius University, Vilnius, Lithuania
V. Dudenas, A. Juodagalvis, J. Vaitkus Vilnius University, Vilnius, Lithuania V. Dudenas, A. Juodagalvis, J. Vaitkus V. Dudenas, A. Juodagalvis, J. Vaitkus National Centre for Particle Physics,
Universiti Malaya,
Kuala Lumpur,
Malaysia I. Ahmed, Z.A. Ibrahim, J.R. Komaragiri, M.A.B. Md Ali32, F. Mohamad Idris33,
W.A.T. Wan Abdullah, M.N. Yusli, Z. Zolkapli – 37 – Centro de Investigacion y de Estudios Avanzados del IPN, Mexico City, Mexico
H. Castilla-Valdez, E. De La Cruz-Burelo, I. Heredia-De La Cruz34, A. Hernandez-Almada,
R. Lopez-Fernandez, J. Mejia Guisao, A. Sanchez-Hernandez g
y
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y,
H. Castilla-Valdez, E. De La Cruz-Burelo, I. Heredia-De La Cruz34, A. Hernandez-Almada,
R. Lopez-Fernandez, J. Mejia Guisao, A. Sanchez-Hernandez Universidad Iberoamericana, Mexico City, Mexico
S. Carrillo Moreno, C. Oropeza Barrera, F. Vazquez Valencia Benemerita Universidad Autonoma de Puebla, Puebla, Mexico
S. Carpinteyro, I. Pedraza, H.A. Salazar Ibarguen, C. Uribe Estrada Universidad Aut´onoma de San Luis Potos´ı, San Luis Potos´ı, Mexico
A. Morelos Pineda JHEP08(2016)122 University of Auckland, Auckland, New Zealand
D. Krofcheck University of Canterbury, Christchurch, New Zealand
P.H. Butler National Centre for Physics, Quaid-I-Azam University, Islamabad, Pakistan
A. Ahmad, M. Ahmad, Q. Hassan, H.R. Hoorani, W.A. Khan, M.A. Shah, M. Shoaib,
M. Waqas National Centre for Nuclear Research, Swierk, Poland National Centre for Nuclear Research, Swierk, Poland National Centre for Nuclear Research, Swierk, Poland
H. Bialkowska, M. Bluj, B. Boimska, T. Frueboes, M. G´orski, M. Kazana, K. Nawrocki,
K. Romanowska-Rybinska, M. Szleper, P. Zalewski H. Bialkowska, M. Bluj, B. Boimska, T. Frueboes, M. G´orski, M. Kazana, K. Nawrock
K. Romanowska-Rybinska, M. Szleper, P. Zalewski Institute of Experimental Physics, Faculty of Physics, University of Warsaw,
Warsaw, Poland Moscow, Russia A. Baskakov, A. Belyaev, E. Boos, M. Dubinin41, L. Dudko, A. Ershov, A. Gribushin,
V. Klyukhin, O. Kodolova, I. Lokhtin, I. Miagkov, S. Obraztsov, S. Petrushanko, V. Savrin,
A. Snigirev Institute for Theoretical and Experimental Physics, Moscow, Russia Institute for Theoretical and Experimental Physics, Moscow, Russia V. Epshteyn, V. Gavrilov, N. Lychkovskaya, V. Popov, I. Pozdnyakov, G. Safronov,
A. Spiridonov, M. Toms, E. Vlasov, A. Zhokin National Research Nuclear University ‘Moscow Engineering Physics Institute’
(MEPhI), Moscow, Russia
M. Chadeeva40, M. Danilov40, O. Markin P.N. Lebedev Physical Institute, Moscow, Russia V. Andreev, M. Azarkin37, I. Dremin37, M. Kirakosyan, A. Leonidov37, S.V. Rusakov,
A. Terkulov JHEP08(2016)122 Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University,
Moscow, Russia Portugal P. Bargassa, C. Beir˜ao Da Cruz E Silva, A. Di Francesco, P. Faccioli, P.G. Ferreira Parracho,
M. Gallinaro, J. Hollar, N. Leonardo, L. Lloret Iglesias, M.V. Nemallapudi, J. Rodrigues
Antunes, J. Seixas, O. Toldaiev, D. Vadruccio, J. Varela, P. Vischia Joint Institute for Nuclear Research, Dubna, Russia
S. Afanasiev, P. Bunin, M. Gavrilenko, I. Golutvin, I. Gorbunov, A. Kamenev, V. Kar-
javin, A. Lanev, A. Malakhov, V. Matveev36,37, P. Moisenz, V. Palichik, V. Perelygin,
S. Shmatov, S. Shulha, N. Skatchkov, V. Smirnov, N. Voytishin, A. Zarubin Petersburg Nuclear Physics Institute, Gatchina (St. Petersburg), Russia
L. Chtchipounov, V. Golovtsov, Y. Ivanov, V. Kim38, E. Kuznetsova39, V. Murzin,
V. Oreshkin, V. Sulimov, A. Vorobyev Institute for Nuclear Research, Moscow, Russia
Yu. Andreev, A. Dermenev, S. Gninenko, N. Golubev, A. Karneyeu, M. Kirsanov,
N. Krasnikov, A. Pashenkov, D. Tlisov, A. Toropin Institute for Nuclear Research, Moscow, Russia Yu. Andreev, A. Dermenev, S. Gninenko, N. Golubev, A. Karneyeu, M. Kirsano
N. Krasnikov, A. Pashenkov, D. Tlisov, A. Toropin – 38 – State Research Center of Russian Federation, Institute for High Energy
Physics, Protvino, Russia State Research Center of Russian Federation, Institute for High Energy
Physics, Protvino, Russia I. Azhgirey, I. Bayshev, S. Bitioukov, D. Elumakhov, V. Kachanov, A. Kalinin, D. Kon-
stantinov, V. Krychkine, V. Petrov, R. Ryutin, A. Sobol, S. Troshin, N. Tyurin, A. Uzunian,
A. Volkov University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear
Sciences, Belgrade, Serbia
P. Adzic42, P. Cirkovic, D. Devetak, J. Milosevic, V. Rekovic Centro
de
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(CIEMAT), Madrid, Spain (CIEMAT), Madrid, Spain ,
,
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A. Delgado Peris, A. Escalante Del Valle, C. Fernandez Bedoya, J.P. Fern´andez Ramos,
J. Flix, M.C. Fouz, P. Garcia-Abia, O. Gonzalez Lopez, S. Goy Lopez, J.M. Hernandez,
M.I. Josa, E. Navarro De Martino, A. P´erez-Calero Yzquierdo, J. Puerta Pelayo, A. Quin-
tario Olmeda, I. Redondo, L. Romero, M.S. Soares Universidad Aut´onoma de Madrid, Madrid, Spain
J.F. de Troc´oniz, M. Missiroli, D. Moran Universidad Aut´onoma de Madrid, Madrid, Spain
J.F. de Troc´oniz, M. Missiroli, D. Moran Universidad de Oviedo, Oviedo, Spain J. Cuevas, J. Fernandez Menendez, I. Gonzalez Caballero, J.R. Gonz´alez Fern´andez,
E. Palencia Cortezon, S. Sanchez Cruz, J.M. Vizan Garcia Instituto de F´ısica de Cantabria (IFCA), CSIC-Universidad de Cantabria,
Santander, Spain I.J. Cabrillo, A. Calderon, J.R. Casti˜neiras De Saa, E. Curras, M. Fernandez, J. Garcia-
Ferrero, G. Gomez, A. Lopez Virto, J. Marco, C. Martinez Rivero, F. Matorras, J. Piedra – 39 – Gomez, T. Rodrigo, A. Ruiz-Jimeno, L. Scodellaro, N. Trevisani, I. Vila, R. Vilar
Cortabitarte Gomez, T. Rodrigo, A. Ruiz-Jimeno, L. Scodellaro, N. Trevisani, I. Vila, R. Vilar
Cortabitarte CERN, European Organization for Nuclear Research, Geneva, Switzerland
D. Abbaneo, E. Auffray, G. Auzinger, M. Bachtis, P. Baillon, A.H. Ball, D. Barney,
P. Bloch, A. Bocci, A. Bonato, C. Botta, T. Camporesi, R. Castello, M. Cepeda, G. Cermi-
nara, M. D’Alfonso, D. d’Enterria, A. Dabrowski, V. Daponte, A. David, M. De Gruttola,
F. De Guio, A. De Roeck, E. Di Marco43, M. Dobson, M. Dordevic, B. Dorney, T. du Pree,
D. Duggan, M. D¨unser, N. Dupont, A. Elliott-Peisert, S. Fartoukh, G. Franzoni, J. Fulcher,
W. Funk, D. Gigi, K. Gill, M. Girone, F. Glege, D. Gulhan, S. Gundacker, M. Guthoff,
J. Hammer, P. Harris, J. Hegeman, V. Innocente, P. Janot, H. Kirschenmann, V. Kn¨unz,
A. Kornmayer15, M.J. Kortelainen, K. Kousouris, M. Krammer1, P. Lecoq, C. Louren¸co,
M.T. Lucchini, L. Malgeri, M. Mannelli, A. Martelli, F. Meijers, S. Mersi, E. Meschi,
F. Moortgat, S. Morovic, M. Mulders, H. Neugebauer, S. Orfanelli44, L. Orsini, L. Pape,
E. Perez, M. Peruzzi, A. Petrilli, G. Petrucciani, A. Pfeiffer, M. Pierini, A. Racz, T. Reis,
G. Rolandi45, M. Rovere, M. Ruan, H. Sakulin, J.B. Sauvan, C. Sch¨afer, C. Schwick,
M. Seidel, A. Sharma, P. Silva, M. Simon, P. Sphicas46, J. Steggemann, M. Stoye,
Y. Takahashi, M. Tosi, D. Treille, A. Triossi, A. Tsirou, V. Veckalns47, G.I. Veres21,
N. Wardle, A. Zagozdzinska35, W.D. (CIEMAT), Madrid, Spain Zeuner CERN, European Organization for Nuclear Research, Geneva, Switzerland
D. Abbaneo, E. Auffray, G. Auzinger, M. Bachtis, P. Baillon, A.H. Ball, D. Barney,
P. Bloch, A. Bocci, A. Bonato, C. Botta, T. Camporesi, R. Castello, M. Cepeda, G. Cermi-
nara, M. D’Alfonso, D. d’Enterria, A. Dabrowski, V. Daponte, A. David, M. De Gruttola,
F. De Guio, A. De Roeck, E. Di Marco43, M. Dobson, M. Dordevic, B. Dorney, T. du Pree,
D. Duggan, M. D¨unser, N. Dupont, A. Elliott-Peisert, S. Fartoukh, G. Franzoni, J. Fulcher,
W. Funk, D. Gigi, K. Gill, M. Girone, F. Glege, D. Gulhan, S. Gundacker, M. Guthoff,
J. Hammer, P. Harris, J. Hegeman, V. Innocente, P. Janot, H. Kirschenmann, V. Kn¨unz,
A. Kornmayer15, M.J. Kortelainen, K. Kousouris, M. Krammer1, P. Lecoq, C. Louren¸co,
M.T. Lucchini, L. Malgeri, M. Mannelli, A. Martelli, F. Meijers, S. Mersi, E. Meschi,
F. Moortgat, S. Morovic, M. Mulders, H. Neugebauer, S. Orfanelli44, L. Orsini, L. Pape,
E. Perez, M. Peruzzi, A. Petrilli, G. Petrucciani, A. Pfeiffer, M. Pierini, A. Racz, T. Reis,
G. Rolandi45, M. Rovere, M. Ruan, H. Sakulin, J.B. Sauvan, C. Sch¨afer, C. Schwick,
M. Seidel, A. Sharma, P. Silva, M. Simon, P. Sphicas46, J. Steggemann, M. Stoye,
Y. Takahashi, M. Tosi, D. Treille, A. Triossi, A. Tsirou, V. Veckalns47, G.I. Veres21,
N. Wardle, A. Zagozdzinska35, W.D. Zeuner JHEP08(2016)122 Universit¨at Z¨urich, Zurich, Switzerland T.K. Aarrestad, C. Amsler49, L. Caminada, M.F. Canelli, V. Chiochia, A. De Cosa,
C. Galloni, A. Hinzmann, T. Hreus, B. Kilminster, C. Lange, J. Ngadiuba, D. Pinna,
G. Rauco, P. Robmann, D. Salerno, Y. Yang National Central University, Chung-Li, Taiwan Institute for Particle Physics, ETH Zurich, Zurich, Switzerland Institute for Particle Physics, ETH Zurich, Zurich, Switzerland Institute for Particle Physics, ETH Zurich, Zurich, Switzerland
F. Bachmair, L. B¨ani, L. Bianchini, B. Casal, G. Dissertori, M. Dittmar, M. Doneg`a,
P. Eller, C. Grab, C. Heidegger, D. Hits, J. Hoss, G. Kasieczka, P. Lecomte†, W. Lus-
termann, B. Mangano, M. Marionneau, P. Martinez Ruiz del Arbol, M. Masciovecchio,
M.T. Meinhard, D. Meister, F. Micheli, P. Musella, F. Nessi-Tedaldi, F. Pandolfi,
J. Pata, F. Pauss, G. Perrin, L. Perrozzi, M. Quittnat, M. Rossini, M. Sch¨onenberger,
A. Starodumov48, M. Takahashi, V.R. Tavolaro, K. Theofilatos, R. Wallny Paul Scherrer Institut, Villigen, Switzerland W. Bertl, K. Deiters, W. Erdmann, R. Horisberger, Q. Ingram, H.C. Kaestli, D. Kotlinski,
U. Langenegger, T. Rohe National Central University, Chung-Li, Taiwan V. Candelise, T.H. Doan, Sh. Jain, R. Khurana, M. Konyushikhin, C.M. Kuo, W. Lin,
Y.J. Lu, A. Pozdnyakov, S.S. Yu National Taiwan University (NTU), Taipei, Taiwan
Arun Kumar, P. Chang, Y.H. Chang, Y.W. Chang, Y. Chao, K.F. Chen, P.H. Chen,
C. Dietz, F. Fiori, W.-S. Hou, Y. Hsiung, Y.F. Liu, R.-S. Lu, M. Mi˜nano Moya, E. Paganis,
A. Psallidas, J.f. Tsai, Y.M. Tzeng A. Psallidas, J.f. Tsai, Y.M. Tzeng – 40 – Chulalongkorn University, Faculty of Science, Department of Physics, Bangkok,
Thailand B. Asavapibhop, G. Singh, N. Srimanobhas, N. Suwonjandee B. Asavapibhop, G. Singh, N. Srimanobhas, N. Suwonjandee Cukurova University, Adana, Turkey
A. Adiguzel, S. Cerci50, S. Damarseckin, Z.S. Demiroglu, C. Dozen, I. Dumanoglu, S. Gir-
gis, G. Gokbulut, Y. Guler, E. Gurpinar, I. Hos, E.E. Kangal51, O. Kara, A. Kayis Topaksu,
U. Kiminsu, M. Oglakci, G. Onengut52, K. Ozdemir53, D. Sunar Cerci50, H. Topakli54,
S. Turkcapar, I.S. Zorbakir, C. Zorbilmez Middle East Technical University, Physics Department, Ankara, Turkey
B. Bilin, S. Bilmis, B. Isildak55, G. Karapinar56, M. Yalvac, M. Zeyrek JHEP08(2016)122 Bogazici University, Istanbul, Turkey
E. G¨ulmez, M. Kaya57, O. Kaya58, E.A. Yetkin59, T. Yetkin60 Bogazici University, Istanbul, Turkey
E. G¨ulmez, M. Kaya57, O. Kaya58, E.A. Yetkin59, T. Yetkin60 Istanbul Technical University, Istanbul, Turkey
A. Cakir, K. Cankocak, S. Sen61 Institute for Scintillation Materials of National Academy of Science of Ukraine,
Kharkov, Ukraine
B. Grynyov National Scientific Center, Kharkov Institute of Physics and Technology,
Kharkov, Ukraine
L. Levchuk, P. Sorokin L. Levchuk, P. Sorokin University of Bristol, Bristol, United Kingdom R. Aggleton, F. Ball, L. Beck, J.J. Brooke, D. Burns, E. Clement, D. Cussans, H. Flacher,
J. Goldstein, M. Grimes, G.P. Heath, H.F. Heath, J. Jacob, L. Kreczko, C. Lucas,
D.M. Newbold62, S. Paramesvaran, A. Poll, T. Sakuma, S. Seif El Nasr-storey, D. Smith,
V.J. Smith Rutherford Appleton Laboratory, Didcot, United Kingdom
K.W. Bell, A. Belyaev63, C. Brew, R.M. Brown, L. Calligaris, D. Cieri, D.J.A. Cockerill,
J.A. Coughlan, K. Harder, S. Harper, E. Olaiya, D. Petyt, C.H. Shepherd-Themistocleous,
A. Thea, I.R. Tomalin, T. Williams Imperial College, London, United Kingdom Imperial College, London, United Kingdom
M. Baber, R. Bainbridge, O. Buchmuller, A. Bundock, D. Burton, S. Casasso, M. Citron,
D. Colling, L. Corpe, P. Dauncey, G. Davies, A. De Wit, M. Della Negra, P. Dunne,
A. Elwood, D. Futyan, Y. Haddad, G. Hall, G. Iles, R. Lane, C. Laner, R. Lucas62,
L. Lyons, A.-M. Magnan, S. Malik, L. Mastrolorenzo, J. Nash, A. Nikitenko48, J. Pela,
B. Penning, M. Pesaresi, D.M. Raymond, A. Richards, A. Rose, C. Seez, A. Tapper,
K. Uchida, M. Vazquez Acosta64, T. Virdee15, S.C. Zenz Brown University, Providence, U.S.A. JHEP08(2016)122 G. Benelli, E. Berry, D. Cutts, A. Garabedian, J. Hakala, U. Heintz, O. Jesus, E. Laird,
G. Landsberg, Z. Mao, M. Narain, S. Piperov, S. Sagir, E. Spencer, R. Syarif University of California, Davis, Davis, U.S.A. R. Breedon, G. Breto, D. Burns, M. Calderon De La Barca Sanchez, S. Chauhan,
M. Chertok, J. Conway, R. Conway, P.T. Cox, R. Erbacher, C. Flores, G. Funk, M. Gardner,
W. Ko, R. Lander, C. Mclean, M. Mulhearn, D. Pellett, J. Pilot, F. Ricci-Tam, S. Shalhout,
J. Smith, M. Squires, D. Stolp, M. Tripathi, S. Wilbur, R. Yohay University of California, Los Angeles, U.S.A. R. Cousins, P. Everaerts, A. Florent, J. Hauser, M. Ignatenko, D. Saltzberg, E. Takasugi,
V. Valuev, M. Weber University of California, Riverside, Riverside, U.S.A. K. Burt, R. Clare, J. Ellison, J.W. Gary, G. Hanson, J. Heilman, P. Jandir, E. Kennedy,
F. Lacroix, O.R. Long, M. Malberti, M. Olmedo Negrete, M.I. Paneva, A. Shrinivas, H. Wei,
S. Wimpenny, B. R. Yates S. Wimpenny, B. R. Yates University of California, San Diego, La Jolla, U.S.A. J.G. Branson, G.B. Cerati, S. Cittolin, M. Derdzinski, R. Gerosa, A. Holzner, D. Klein,
J Letts I Macneill D Olivito S Padhi M Pieri M Sani V Sharma S Simon M Tadel J.G. Branson, G.B. Cerati, S. Cittolin, M. Derdzinski, R. Gerosa, A. Holzner, D. Klein,
J. Letts, I. Macneill, D. Olivito, S. Padhi, M. Pieri, M. Sani, V. Sharma, S. Simon, M. Tadel,
A. Vartak, S. Wasserbaech65, C. Welke, J. Wood, F. W¨urthwein, A. Yagil, G. Zevi Della J.G. Branson, G.B. Cerati, S. Cittolin, M. Derdzinski, R. Gerosa, A. Holzner, D. Klei J.G. Branson, G.B. Cerati, S. Cittolin, M. Derdzinski, R. Gerosa, A. Holzner, D. Klei
J. Letts, I. Macneill, D. Olivito, S. Padhi, M. Pieri, M. Sani, V. Sharma, S. Simon, M. Tade J.G. Branson, G.B. Cerati, S. Cittolin, M. Derdzinski, R. Gerosa, A. Holzner, D. Klein,
J. Letts, I. Macneill, D. Olivito, S. Padhi, M. Pieri, M. Sani, V. Sharma, S. Simon, M. Tadel,
A. Vartak, S. Wasserbaech65, C. Welke, J. Wood, F. W¨urthwein, A. Yagil, G. Zevi Della
Porta A. Vartak, S. Wasserbaech65, C. Welke, J. Wood, F. W¨urthwein, A. Yagil, G. Zevi Della
Porta University of California, Santa Barbara, Santa Barbara, U.S.A. University of California, Santa Barbara, Santa Barbara, U.S.A. R. Bhandari, J. Bradmiller-Feld, C. Campagnari, A. Dishaw, V. Dutta, K. Flowers,
M. Franco Sevilla, P. Geffert, C. George, F. Brunel University, Uxbridge, United Kingdom Brunel University, Uxbridge, United Kingdom J.E. Cole, P.R. Hobson, A. Khan, P. Kyberd, D. Leslie, I.D. Reid, P. Symonds, L. Teodor-
escu, M. Turner – 41 – Baylor University, Waco, U.S.A. Baylor University, Waco, U.S.A. A. Borzou, K. Call, J. Dittmann, K. Hatakeyama, H. Liu, N. Pastika The University of Alabama, Tuscaloosa, U.S.A. O. Charaf, S.I. Cooper, C. Henderson, P. Rumerio The University of Alabama, Tuscaloosa, U.S.A. The University of Alabama, Tuscaloosa, U.S.A
O. Charaf, S.I. Cooper, C. Henderson, P. Rumerio Boston University, Boston, U.S.A. D. Arcaro, A. Avetisyan, T. Bose, D. Gastler, D. Rankin, C. Richardson, J. Rohlf, L. Sulak,
D. Zou Brown University, Providence, U.S.A. Fairfield University, Fairfield, U.S.A. Fairfield University, Fairfield, U.S.A. Fairfield University, Fairfield, U.S.A. D. Winn JHEP08(2016)122 D. Winn Fermi National Accelerator Laboratory, Batavia, U.S.A. Fermi National Accelerator Laboratory, Batavia, U.S.A. S. Abdullin, M. Albrow, G. Apollinari, S. Banerjee, L.A.T. Bauerdick, A. Beretvas,
J. Berryhill, P.C. Bhat, G. Bolla, K. Burkett, J.N. Butler, H.W.K. Cheung, F. Chlebana,
S. Cihangir, M. Cremonesi, V.D. Elvira, I. Fisk, J. Freeman, E. Gottschalk, L. Gray,
D. Green, S. Gr¨unendahl, O. Gutsche, D. Hare, R.M. Harris, S. Hasegawa, J. Hirschauer,
Z. Hu, B. Jayatilaka, S. Jindariani, M. Johnson, U. Joshi, B. Klima, B. Kreis, S. Lammel,
J. Linacre, D. Lincoln, R. Lipton, T. Liu, R. Lopes De S´a, J. Lykken, K. Maeshima,
N. Magini, J.M. Marraffino, S. Maruyama, D. Mason, P. McBride, P. Merkel, S. Mrenna,
S. Nahn, C. Newman-Holmes†, V. O’Dell, K. Pedro, O. Prokofyev, G. Rakness, L. Ristori,
E. Sexton-Kennedy, A. Soha, W.J. Spalding, L. Spiegel, S. Stoynev, N. Strobbe, L. Taylor,
S. Tkaczyk, N.V. Tran, L. Uplegger, E.W. Vaandering, C. Vernieri, M. Verzocchi, R. Vidal,
M. Wang, H.A. Weber, A. Whitbeck S. Nahn, C. Newman-Holmes†, V. O’Dell, K. Pedro, O. Prokofyev, G. Rakness, L. Ristori, Sexton-Kennedy, A. Soha, W.J. Spalding, L. Spiegel, S. Stoynev, N. Strobbe, L. Taylor, S. Tkaczyk, N.V. Tran, L. Uplegger, E.W. Vaandering, C. Vernieri, M. Verzocchi, R. Vidal,
M. Wang, H.A. Weber, A. Whitbeck M. Wang, H.A. Weber, A. Whitbeck Brown University, Providence, U.S.A. Golf, L. Gouskos, J. Gran, R. Heller,
J. Incandela, N. Mccoll, S.D. Mullin, A. Ovcharova, J. Richman, D. Stuart, I. Suarez,
C. West, J. Yoo C. West, J. Yoo California Institute of Technology, Pasadena, U.S.A. California Institute of Technology, Pasadena, U.S.A. D. Anderson, A. Apresyan, J. Bendavid, A. Bornheim, J. Bunn, Y. Chen, J. Duarte,
A. Mott, H.B. Newman, C. Pena, M. Spiropulu, J.R. Vlimant, S. Xie, R.Y. Zhu D. Anderson, A. Apresyan, J. Bendavid, A. Bornheim, J. Bunn, Y. Chen, J. Duart
A. Mott, H.B. Newman, C. Pena, M. Spiropulu, J.R. Vlimant, S. Xie, R.Y. Zhu Carnegie Mellon University, Pittsburgh, U.S.A. Carnegie Mellon University, Pittsburgh, U.S.A. M.B. Andrews, V. Azzolini, B. Carlson, T. Ferguson, M. Paulini, J. Russ, M. Sun, H. Vogel,
I. Vorobiev M.B. Andrews, V. Azzolini, B. Carlson, T. Ferguson, M. Paulini, J. Russ, M. Sun, H. Vogel,
I. Vorobiev – 42 – University of Florida, Gainesville, U.S.A. University of Florida, Gainesville, U.S.A. D. Acosta, P. Avery, P. Bortignon, D. Bourilkov, A. Brinkerhoff, A. Carnes, M. Carver,
D. Curry, S. Das, R.D. Field, I.K. Furic, J. Konigsberg, A. Korytov, P. Ma, K. Matchev, H. Mei, P. Milenovic66, G. Mitselmakher, D. Rank, L. Shchutska, D. Sperka, L. Thomas,
J. Wang, S. Wang, J. Yelton J. Wang, S. Wang, J. Yelton Florida International University, Miami, U.S.A. S. Linn, P. Markowitz, G. Martinez, J.L. Rodriguez University of Colorado Boulder, Boulder, U.S.A. University of Colorado Boulder, Boulder, U.S.A. J.P. Cumalat, W.T. Ford, F. Jensen, A. Johnson, M. Krohn, T. Mulholland, K. Stenson,
S.R. Wagner Cornell University, Ithaca, U.S.A. J. Alexander, J. Chaves, J. Chu, S. Dittmer, K. Mcdermott, N. Mirman, G. Nicolas
Kaufman, J.R. Patterson, A. Rinkevicius, A. Ryd, L. Skinnari, L. Soffi, S.M. Tan, Z. Tao,
J. Thom, J. Tucker, P. Wittich, M. Zientek Florida International University, Miami, U.S.A. Florida International University, Miami, U.S.A. S. Linn, P. Markowitz, G. Martinez, J.L. Rodriguez Florida State University, Tallahassee, U.S.A. A. Ackert, J.R. Adams, T. Adams, A. Askew, S. Bein, B. Diamond, S. Hagopian,
V. Hagopian, K.F. Johnson, A. Khatiwada, H. Prosper, A. Santra, M. Weinberg Florida Institute of Technology, Melbourne, U.S.A. gy,
,
M.M. Baarmand, V. Bhopatkar, S. Colafranceschi67, M. Hohlmann, D. Noonan, T. Roy,
F. Yumiceva University of Illinois at Chicago (UIC), Chicago, U.S.A. M.R. Adams, L. Apanasevich, D. Berry, R.R. Betts, I. Bucinskaite, R. Cavanaugh,
O. Evdokimov, L. Gauthier, C.E. Gerber, D.J. Hofman, P. Kurt, C. O’Brien, I.D. Sandoval
Gonzalez, P. Turner, N. Varelas, Z. Wu, M. Zakaria, J. Zhang – 43 – The University of Iowa, Iowa City, U.S.A. B. Bilki68, W. Clarida, K. Dilsiz, S. Durgut, R.P. Gandrajula, M. Haytmyradov, V. Khris-
tenko, J.-P. Merlo, H. Mermerkaya69, A. Mestvirishvili, A. Moeller, J. Nachtman, H. Ogul,
Y. Onel, F. Ozok70, A. Penzo, C. Snyder, E. Tiras, J. Wetzel, K. Yi Johns Hopkins University, Baltimore, U.S.A. Johns Hopkins University, Baltimore, U.S.A. I. Anderson, B. Blumenfeld, A. Cocoros, N. Eminizer, D. Fehling, L. Feng, A.V. Gritsan,
P. Maksimovic, M. Osherson, J. Roskes, U. Sarica, M. Swartz, M. Xiao, Y. Xin, C. You I. Anderson, B. Blumenfeld, A. Cocoros, N. Eminizer, D. Fehling, L. Feng, A.V. Gritsan,
P. Maksimovic, M. Osherson, J. Roskes, U. Sarica, M. Swartz, M. Xiao, Y. Xin, C. You I. Anderson, B. Blumenfeld, A. Cocoros, N. Eminizer, D. Fehling, L. Feng, A.V. Gritsa P. Maksimovic, M. Osherson, J. Roskes, U. Sarica, M. Swartz, M. Xiao, Y. Xin, C. You The University of Kansas, Lawrence, U.S.A. A. Al-bataineh, P. Baringer, A. Bean, J. Bowen, C. Bruner, J. Castle, R.P. Kenny III,
A. Kropivnitskaya, D. Majumder, W. Mcbrayer, M. Murray, S. Sanders, R. Stringer,
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Haute Alsace Mulhouse, CNRS/IN2P3, Strasbourg, France 3: Also at Institut Pluridisciplinaire Hubert Curien, Universit´e de
Haute Alsace Mulhouse, CNRS/IN2P3, Strasbourg, France 4: Also at Universidade Estadual de Campinas, Campinas, Brazil 4: Also at Universidade Estadual de Campinas, Campinas, Brazil 5: Also at Centre National de la Recherche Scientifique (CNRS) — IN2P3, Paris, France 5: Also at Centre National de la Recherche Scientifique (CNRS) — IN2P3, Paris, France 6: Also at Universit´e Libre de Bruxelles, Bruxelles, Belgium 6: Also at Universit´e Libre de Bruxelles, Bruxelles, Belgium 7: Also at Deutsches Elektronen-Synchrotron, Hamburg, Germany 7: Also at Deutsches Elektronen-Synchrotron, Hamburg, Germany 8: Also at Joint Institute for Nuclear Research, Dubna, Russia 8: Also at Joint Institute for Nuclear Research, Dubna, Russia 8: Also at Joint Institute for Nuclear Researc 9: Also at Helwan University, Cairo, Egypt 9: Also at Helwan University, Cairo, Egypt 10: Now at Zewail City of Science and Technology, Zewail, Egypt 10: Now at Zewail City of Science and Technology, Zewail, Egypt 11: Also at Ain Shams University, Cairo, Egypt 11: Also at Ain Shams University, Cairo, Egypt 12: Also at Fayoum University, El-Fayoum, Egypt 12: Also at Fayoum University, El-Fayoum, Egypt 13: Now at British University in Egypt, Cairo, Egypt 13: Now at British University in Egypt, Cairo, Egypt 14: Also at Universit´e de Haute Alsace, Mulhouse, France 14: Also at Universit´e de Haute Alsace, Mulhouse, France – 46 – 15: Also at CERN, European Organization for Nuclear Research, Geneva, Switzerland 16: Also at Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University,
Moscow, Russia 17: Also at RWTH Aachen University, III. Texas A&M University, College Station, U.S.A. Physikalisches Institut A, Aachen, Germany 18: Also at University of Hamburg, Hamburg, Germany 19: Also at Brandenburg University of Technology, Cottbus, Germany 20: Also at Institute of Nuclear Research ATOMKI, Debrecen, Hungary 21: Also at MTA-ELTE Lend¨ulet CMS Particle and Nuclear Physics Group, E¨otv¨os Lor´and
University, Budapest, Hungary 22: Also at University of Debrecen, Debrecen, Hungary 23: Also at Indian Institute of Science Education and Research, Bhopal, India JHEP08(2016)122 24: Also at Institute of Physics, Bhubaneswar, India 25: Also at University of Visva-Bharati, Santiniketan, India 26: Also at University of Ruhuna, Matara, Sri Lanka 27: Also at Isfahan University of Technology, Isfahan, Iran 28: Also at University of Tehran, Department of Engineering Science, Tehran, Iran 28: Also at University of Tehran, Department of Engineering 29: Also at Plasma Physics Research Center, Science and Research Branch, Islamic Aza
University, Tehran, Iran 30: Also at Universit`a degli Studi di Siena, Siena, Italy 31: Also at Purdue University, West Lafayette, U.S.A. 31: Also at Purdue University, West Lafayette, U.S.A. 32: Also at International Islamic University of Malaysia, Kuala Lumpur, Malaysia 32: Also at International Islamic University of Malaysia, Kuala Lumpur, Malaysia 33: Also at Malaysian Nuclear Agency, MOSTI, Kajang, Malaysia 34: Also at Consejo Nacional de Ciencia y Tecnolog´ıa, Mexico city, Mexico 35: Also at Warsaw University of Technology, Institute of Electronic Systems, Warsaw, Polan 36: Also at Institute for Nuclear Research, Moscow, Russia 36: Also at Institute for Nuclear Research, Moscow, Russia t Institute for Nuclear Research, Moscow, Russia 37: Now at National Research Nuclear University ’Moscow Engineering Physics Institute’
(MEPhI), Moscow, Russia (
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38: Also at St. Petersburg State Polytechnical University, St.˜Petersburg, Russia 38: Also at St. Petersburg State Polytechnical University, St.˜Petersburg, Russia 39: Also at University of Florida, Gainesville, U.S.A. 40: Also at P.N. Lebedev Physical Institute, Moscow, Russia 41: Also at California Institute of Technology, Pasadena, U.S.A. Texas A&M University, College Station, U.S.A. 42: Also at Faculty of Physics, University of Belgrade, Belgrade, Serbia 42: Also at Faculty of Physics, University of Belgrade, Belgrade, Serbia 43: Also at INFN Sezione di Roma, Universit`a di Roma, Roma, Italy at National Technical University of Athens, Athe 44: Also at National Technical University of Athens, Athens, Greece 45: Also at Scuola Normale e Sezione dell’INFN, Pisa, Italy 46: Also at National and Kapodistrian University of Athens, Athens, Greece 47: Also at Riga Technical University, Riga, Latvia 48: Also at Institute for Theoretical and Experimental Physics, Moscow, Russia 49: Also at Albert Einstein Center for Fundamental Physics, Bern, Switzerland 49: Also at Albert Einstein Center for Fundamental Physics, Bern, Switzerland 50: Also at Adiyaman University, Adiyaman, Turkey 51: Also at Mersin University, Mersin, Turkey 52: Also at Cag University, Mersin, Turkey 53: Also at Piri Reis University, Istanbul, Turkey 53: Also at Piri Reis University, Istanbul, Turkey 54: Also at Gaziosmanpasa University, Tokat, Turkey 54: Also at Gaziosmanpasa University, Tokat, Turkey 55: Also at Ozyegin University, Istanbul, Turkey 55: Also at Ozyegin University, Istanbul, Turkey 56: Also at Izmir Institute of Technology, Izmir, Turkey 56: Also at Izmir Institute of Technology, Izmir, Turkey 57: Also at Marmara University, Istanbul, Turkey 57: Also at Marmara University, Istanbul, Turkey 58: Also at Kafkas University, Kars, Turkey 59: Also at Istanbul Bilgi University, Istanbul, Turkey 59: Also at Istanbul Bilgi University, Istanbul, Turkey – 47 – 60: Also at Yildiz Technical University, Istanbul, Turkey 61: Also at Hacettepe University, Ankara, Turkey 62: Also at Rutherford Appleton Laboratory, Didcot, United Kingdom 63: Also at School of Physics and Astronomy, University of Southampton, Southampton, Unit
Kingdom 64: Also at Instituto de Astrof´ısica de Canarias, La Laguna, Spain 65: Also at Utah Valley University, Orem, U.S.A. 66: Also at University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences,
Belgrade, Serbia 67: Also at Facolt`a Ingegneria, Universit`a di Roma, Roma, Ital Also at Argonne National Laboratory, Argonne JHEP08(2016)122 69: Also at Erzincan University, Erzincan, Turkey 70: Also at Mimar Sinan University, Istanbul, Istanbul, Turkey 70: Also at Mimar Sinan University, Istanbul, Istanbul, Turkey 71: Also at Texas A&M University at Qatar, Doha, Qatar 72: Also at Kyungpook National University, Daegu, Korea – 48 –
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Coupling Mechanism and Coupling Degree Measurement Model of Shipbuilding Industry Cluster
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Polish Maritime Research
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POLISH MARITIME RESEARCH Special Issue 2016 S1 (91) 2016 Vol. 23; pp. 78-85
10.1515/pomr-2016-0049 POLISH MARITIME RESEARCH Special Issue 2016 S1 (91) 2016 Vol. 23; pp. 78-85
10.1515/pomr-2016-0049 POLISH MARITIME RESEARCH Special Issue 2016 S1 (91) 2016 Vol. 23; pp. 78-85
10.1515/pomr-2016-0049 COUPLING MECHANISM AND COUPLING DEGREE MEASUREMENT
MODEL OF SHIPBUILDING INDUSTRY CLUSTER AN Xiao li
a) School of Economics and Management, Harbin Engineering University,P.R.China,150001
b) School of Economics and Management, Tsinghua University,P.R.China,10010 ABSTRACT This article was aimed to analyse the working condition of the coupling mechanism in Chinese shipbuilding industry
cluster. Literature review was conducted to unveil the coupling mechanism of shipbuilding industry cluster, providing
analysis of the present development of shipbuilding industry. A theoretical model was proposed by using the efficacy
function and the capacity of the coupling coefficient while measuring the coupling of the shipbuilding industry cluster. Four coupling characteristics were found including relevant, systemic, diversity, and collaborative in the shipbuilding
industry cluster. The shipbuilding industry cluster has two levels of coupling mechanism including the core layer coupling
mechanism and the support layer coupling mechanism respectively. The improvement through coupling the core layer,
support layer, and the coupling between two levels, is an effective way to achieve long-term stable development in
Chinese shipbuilding industry cluster. Keywords: coupling mechanism, coupling degree measurement model, shipbuilding industry cluster INTRODUCTION greater contribution to military and civilian. Shipbuilding
industry cluster is an effective way for the development of
shipbuilding industry as a strategic tool (Huang, 2006)[1]. Scholars focus on the studies on development elements (Cao
and Liu, 2007)[2], development models (Tao and Yang, 2007;
Li, 2008; Hu and Yu, 2013)[3-5], the formation mechanism of
shipbuilding industrial cluster (Tao, 2005; Zhao, 2007; Wang,
2010)[6-8], and the life cycle of South Korea’s shipbuilding
industry cluster (Shin et al., 2011)[9], shipbuilding industry
cluster development theoretical model (Zong, 2013)[10],
development opportunities and challenges of shipbuilding
industry cluster (Zhang and Zeng, 2014)[11], etc. However, the
literatures rarely involve the study of the complex mechanism
of the shipbuilding industry cluster. The coupling theory
provides a new theoretical support for this field of research. Chinese Shipbuilding Industry is the supporting industry
of Chinese maritime transportation, ocean development
and national defense construction, and strategic industry
of combination of military and civilian. It has important
significance to the national defense capability and the national
economic growth. The construction of shipbuilding industrial
cluster is an effective way to enhance the competitiveness
of shipbuilding industry, and the appropriate coupling
mechanism can make positive synergy effect on shipbuilding
industry cluster. The industrial clusters of shipbuilding
industry can take appropriate measures to improve the
coupling degree to make the cluster maintain high coupling
operation in a state of highly efficient operation, and make POLISH MARITIME RESEARCH, No S1/2016 78 The coupling theory is applied to the study of the complex
relationships within the systems, which is mainly used in
manufacturing industry and other related fields. Li and Li
(2005) presented that the equipment manufacturing industry
cluster coupling contains three layers coupling mechanisms,
namely the manufacturing enterprise’s coupling mechanism,
coupling mechanism of industry cluster and regional social
network coupling mechanism, proposed the coupling
degree model of equipment manufacturing industry cluster,
analyzed the influence factors of equipment manufacturing
industry cluster coupling degree and coupling mechanism,
and finally made empirical analysis on Shenyang equipment
manufacturing industry cluster[12]. Ruan and Zuan (2010) put
forward the coupling degree model of manufacturing industry
cluster, and made empirical analysis of the coupling degree
on Qingdao manufacturing industry cluster[13]. Yuan et al. (2012) divided the network structure of the manufacturing
industry into core manufacturing layer, industry matching
layer, service support layer and analyzed coupling mechanism
among layers[14]. Fu et al. INTRODUCTION (2012) made the research on the
coupling of multiple wastewater using network design to
achieve agile manufacturing[16]. Du et al. (2012) established the
coupling coordination model about the industrial technology
innovation ability. The model mainly includes three basic
parts, the efficiency function, the coupling degree model
and the evaluation index system. This coupling coordination
model was used to evaluate whether the coal resource cities
should develop some leading industries, and took Qitaihe as
an example[17].Liu and Sun (2014) established the coupling
degree measurement model of knowledge innovation and
technology innovation in high-end equipment manufacturing
industry, and proved the validity of the coupling degree
measure model from the empirical point of view[15]. Zou et
al. (2014) analyzed the development trend of the equipment
manufacturing industry in Liaoning by using factor analysis
method, and proposed the coupling development “Trilogy” of
technology innovation and technological upgrading of Liaoning
province equipment manufacturing industry combined
with technological innovation and technological upgrading
coupling improvement mechanism, respectively, exploration
and development model, the leading domestic development
model and international development model[18].The research
of the coupling mechanism and coupling degree measurement
model of shipbuilding industry cluster can provide theoretical
guidance and decision-making basis for the industrial cluster
of shipbuilding industry, which has important significance to
both theoretical and practical development. DEVELOPMENT STATUS QUO OF CHINESE
SHIPBUILDING INDUSTRY completion. The market share of China was more than 45%
both in the year of 2013 and 2014, followed by South Korea
and Japanese with the market share of about 30% and 20%
respectively. hi The new received orders in China kept the world’s first in
2013 and 2014. From the perspective of market share, China,
South Korea and Japan are the world shipbuilding countries
led by China. China’s shipbuilding industry development is
quite superior and stability. Fig.1. Market share of major shipbuilding countries in the world in 2013 Fig.1. Market share of major shipbuilding countries in the world in 2013 Fig.1. Market share of major shipbuilding countries in the world in 2013 Fig.2. Market share of major shipbuilding countries in the world in 2014 Fig.2. Market share of major shipbuilding countries in the world in 2014 DEVELOPMENT STATUS QUO OF CHINESE
SHIPBUILDING INDUSTRY From 2001 to 2014, China’s annual shipbuilding output ,
the new received orders, the overall holding orders had showed
a rising trend, and reached the highest value in 2012, 2007
and 2008, respectively. China’s annual shipbuilding output
had been declining in the last three years, but the degree of
declining has slowed. The number of new received orders had
increased significantly, and then fell slightly. China’s annual
shipbuilding output, the new received orders, and the overall
holding orders had been showing a fluctuation states, however
the general trends are still upward. INDUSTRIAL CONCENTRATION HAS BEEN
INCREASED, AND THE COMPREHENSIVE
STRENGTH HAS BEEN GREATLY IMPROVED On one hand, from the ecological point of
view, the shipbuilding industry cluster is a industry species
ecological system; on the other hand, from the innovation
economics point of view, shipbuilding industry cluster is a
special kind of regional innovation system[19].This paper
argues that the shipbuilding industry cluster refers to taking
ship manufacturing enterprises as the center, and united the
related enterprises or organizations of ship industry, such as:
supporting enterprises, universities, research institutions,
vocational training institutions and shipbuilding industry
association, gathering together to form an organic system in
a particular geographic location [8][20]. The industry cluster,
which has complete industrial structure, robust support
system, can be formed through specialization and depth
cooperation of subjects within the cluster. Initially coupling is
a physical concept, which has been widely used in the field of
system science and social sciences. The concept of coupling is
used to explore or describe the relationship, mutual influence
and common cooperation and evolution process of various
systems. In physics, coupling refers to the phenomenon that
two or more than two of the circuit elements or the input
and output of the electrical network are closely matched
with each other, and the transmission of energy from one
side to the other side through the interaction. In this article,
the coupling is defined as a dynamic relationship formed
by positive interaction, matching and coordination among
system elements or subsystems to achieve the coordinated
development. The coupling of Chinese shipbuilding industry
cluster is a kind of dynamic relationship and synergistic action
process among the related enterprises and organizations of
shipbuilding industry. The coupling of shipbuilding industry
cluster is not only coordination among the agents, but is also
on collaborative development, so as to achieve the purposes,
such as complementary advantages, mutual promotion and
coordinated development. In the year of 2014, there were more than 90 percent orders
has been completed in the three major shipbuilding bases,
which were in the place of the Bohai Bay region, Yangtze
River Delta and Pearl River Delta. The concentration degree
of top 10 enterprises, top 20 enterprises and top 30 enterprises
was up to 50.6%, 71.6% , 83.8%, respectively, which increased
3.2%,5.9% and 7.4% respectively comparing to the year of
2013. This means the degree of industrial concentration has
been significantly improved. INDUSTRIAL CONCENTRATION HAS BEEN
INCREASED, AND THE COMPREHENSIVE
STRENGTH HAS BEEN GREATLY IMPROVED There has been formed a group
of key enterprises in China, and four of them are the world’s
top ten enterprises, in terms of new orders. We can obtain that
Chinese shipbuilding enterprises’ market competitiveness
and the international status had been improved. CHINA REMAINED THE WORLD’S LARGEST
SHIPBUILDING COUNTRY The data of Figure 1 and 2 are obtained from China
Association of the National Shipbuilding Industry Website. The new orders of China, South Korea, and Japan already
occupied more than 90% of the world shipbuilding total 79 POLISH MARITIME RESEARCH, No S1/2016 MARKET DEMAND IS NOT BALANCED, AND
THE DEVELOPMENT POTENTIAL IS SLIGHTLY
INADEQUATE In the year of 2014, China’s new receive orders quantity
maintained a high level, which was about 60 million deadweight
tons. The quantity of new receive orders was showing a
quarterly decline trend, which was 25 million deadweight
tons, 14 million deadweight tons, 11 million deadweight tons
and 7 million deadweight tons, from first quarter to the fourth
quarter, respectively. Except the first quarter, the situation
of shipbuilding enterprises had deteriorated significantly
in the year of 2014. The main reason is as following: first
of all, shipping market supply and demand contradictions
problems have not been fundamentally resolved, and the
order quantity is gradually reduced. Secondly, as a significant
reduction in international oil prices, which dampened the
enthusiasm of the enterprise to manufacture energy-saving
ships, and postponed dismantling of old ships, the market
demand for new ships continued to decrease. In recent years,
the Chinese shipbuilding outputhas reached 20 million
tons. Facing the depresseding market, China’s shipbuilding
industry capacity utilization efficiency is not high, and does
not reach a reasonable level of the industry. THE COUPLING CHARACTERISTICS OF
SHIPBUILDING INDUSTRY CLUSTER Shipbuilding industry cluster has the characteristics of
relevance, systematic, diversity, collaborative etc. Firstly,
correlation. The coupling agents including core ship
manufacturing enterprises, the ship supporting enterprises,
the ship parts production enterprises, and the service
organizations are interrelated and interact with each other. Shipbuilding industry cluster is not a closed static system. Obviously, Chinese shipbuilding industry cluster has the
prerequisites for application of coupling theory. Secondly,
systematic. The main subjects or elements in the coupling
system form a new system through cooperating with
each other according to certain rules. The main bodies of
Chinese shipbuilding industry cluster form the new system
of shipbuilding industry cluster through cooperating with
each other according to the rules and regulations, such as
cooperation contract, contract etc. Thirdly, diversity. Each INDUSTRIAL CONCENTRATION HAS BEEN
INCREASED, AND THE COMPREHENSIVE
STRENGTH HAS BEEN GREATLY IMPROVED Unique symbiotic cluster of shipbuilding industry enterprises
was formed through the exchange of knowledge, information,
human resources, equipment and capital of each subject
in the cluster. On one hand, from the ecological point of
view, the shipbuilding industry cluster is a industry species
ecological system; on the other hand, from the innovation
economics point of view, shipbuilding industry cluster is a
special kind of regional innovation system[19].This paper
argues that the shipbuilding industry cluster refers to taking
ship manufacturing enterprises as the center, and united the
related enterprises or organizations of ship industry, such as:
supporting enterprises, universities, research institutions,
vocational training institutions and shipbuilding industry
association, gathering together to form an organic system in
a particular geographic location [8][20]. The industry cluster,
which has complete industrial structure, robust support
system, can be formed through specialization and depth
cooperation of subjects within the cluster. Initially coupling is
a physical concept, which has been widely used in the field of
system science and social sciences. The concept of coupling is
used to explore or describe the relationship, mutual influence
and common cooperation and evolution process of various
systems. In physics, coupling refers to the phenomenon that
two or more than two of the circuit elements or the input
and output of the electrical network are closely matched
with each other, and the transmission of energy from one
side to the other side through the interaction. In this article,
the coupling is defined as a dynamic relationship formed
by positive interaction, matching and coordination among
system elements or subsystems to achieve the coordinated
development. The coupling of Chinese shipbuilding industry
cluster is a kind of dynamic relationship and synergistic action
process among the related enterprises and organizations of
shipbuilding industry. The coupling of shipbuilding industry
cluster is not only coordination among the agents, but is also
on collaborative development, so as to achieve the purposes,
such as complementary advantages, mutual promotion and
coordinated development. Unique symbiotic cluster of shipbuilding industry enterprises
was formed through the exchange of knowledge, information,
human resources, equipment and capital of each subject
in the cluster. THE COUPLING CONNOTATION OF SHIPBUILDING
INDUSTRY CLUSTER As a typical large-scale equipment manufacturing industry,
shipbuilding industry enterpriseshave many characteristics
such as long industrial chain involved and high requirements
for supporting industry [4].A mature shipbuilding industry
cluster should include the shipping core manufacturing
enterprises, the ship supporting enterprises, the ship parts
production enterprises, and the service oriented institutions. POLISH MARITIME RESEARCH, No S1/2016 80 enterprises, the ship matching enterprises, and the ship core
manufacturing enterprises are closely linked to form a vertical
industrial chain. coupled subsystem has its own organizational characteristics. The links between the elements of the subsystems are
complicated, which constitute a variety of coupling modes. Each subject of shipbuilding industry cluster has its own
organization and management mode and characteristics,
so the links of enterprises and support service agencies are
very complex, and the relationship between them is dynamic. Finally, collaborative. The coupled subsystems in shipbuilding
industry cluster interact with each other through the way
of complementary advantages to achieve the collaborative
development of the entire system [21]. Fig.3. The coupling structure of shipbuilding industry cluster core layer THE COUPLING MECHANISM OF
SHIPBUILDING INDUSTRY CLUSTER Large ship manufacturing enterprises are the core of
agglomeration of Chinese shipbuilding industry cluster, and
a series of related businesses(ship matching enterprises, ship
parts production enterprises and shipbuilding supporting
enterprise) and service organizations (such as scientific
research institutions, intermediary institutions, government
agencies, educational institutions, and shipbuilding industry
association) are derived from the large ship manufacturing
enterprise. These service institutions provide the related
services, such as technical, information, policy guidance and
coordination for the cluster. Chinese Shipbuilding Industry
Cluster coupling structure contains two levels of coupling
mechanisms: the core layer coupling mechanism, and the
support layer coupling mechanism[12]. The coupling of the
shipbuilding industry cluster is the complex combination of
the two levels of coupling mechanisms rather than a simple
superposition. The two coupling layers are closely linked
to each other based on knowledge sharing and economic
transactions, but independent of each other. The coupling
dynamics of one level will have an effect on the other
coupling layer, thus affecting the overall function of the
cluster. A positive effect is produced by the good coupling
of two levels, to promote the formation of a high coupling
degree of the entire cluster, so as to maintain sustainable
competitiveness. Fig.3. The coupling structure of shipbuilding industry cluster core layer Fig.3. The coupling structure of shipbuilding industry cluster core layer The main contents of the core layer coupling mechanism
are ship manufacturing enterprises, which are the core of
the shipbuilding industry cluster and play the role of the
components supplied by the supporting enterprises to be
assembled into the ship. The matching enterprises and the
ship spare parts manufacturing enterprises provide the
ship core manufacturing enterprises with spare parts and
necessary components of the ship. Of course, the ship spare
parts production enterprises also provide spare parts for the
ship matching enterprises. Shipbuilding supply enterprises
provide the cores of ship manufacturing enterprises, the
ship matching enterprises and ship spare parts production
enterprises with all kinds of raw materials for shipbuilding. These adaptation stages are the initial stage of coupling, and
the closely and efficiently cooperation and interaction among
the enterprises are the key points. Each enterprise needs
follow-up and support of other links as key link in the vertical
industry chain, and the benign interaction among enterprises
to produce the adhesive effect, so as to achieve the coordinated COUPLING MECHANISM OF SUPPORT LAYER Fig.4 .The support coupling layer structure of shipbuilding industrial cluster CONSTRUCTION OF COUPLING
DEGREE MEASUREMENT MODEL OF
SHIPBUILDING INDUSTRY CLUSTER Previous research illustrates the coupling mechanism
of shipbuilding industry cluster. In reality, it is necessary
to measure the coupling degree of shipbuilding industry
cluster scientifically in order to clear the coupling condition of
shipbuilding industrial cluster. Then relevant institutions can
take pertinent measures to improve cluster coupling degree. Construction process of coupling degree measurement model
of shipbuilding industry cluster is as follows. Fig.4 .The support coupling layer structure of shipbuilding industrial cluster Fig.4 .The support coupling layer structure of shipbuilding industrial cluster Support coupling mechanism is also the outer coupling
mechanism, mainly provides technology information,
resource, policy guidance and other services for shipbuilding
industry cluster. The coupling layer mainly includes the relation
and coupling between some support service institutions and
the inner cluster. The inner cluster includes the shipping core
manufacturing enterprises, the ship matching enterprises, the
ship spare parts production enterprises and the shipbuilding
supply enterprises. This coupling layer is important, but can
easily be neglected. Support service institutions, which are
parts of support coupling layer, include government agencies,
intermediary institutions, scientific research institutions,
education institutions, financial institutions and shipbuilding
industry association, etc. In general, these support service
institutions support cluster inner layer. Support coupling
layer structure is as follows.h COUPLING MECHANISM OF CORE LAYER The core layer coupling mechanism is mainly the coupling
of the core ship manufacturing enterprises, the ship matching
enterprises, the ship spare parts manufacturing enterprises
and the shipbuilding supply enterprises. In this coupling
layer, the cooperation of the enterprises is not only between
supply and demand, but the matching of the upstream
and downstream enterprises in the product, service and
manpower, etc, based on the complete vertical industrial
chain. Industrial connection is a prerequisite for the coupling
of industry cluster, and the information flow in unobstructed
and timely manner are the conditions of realizing the benign
coupling. Shipbuilding industry cluster is a typical vertical
industry chain agglomeration model, which is longer,
involving a wide range of services, with high requirements
for matching services. The ship spare parts manufacturing POLISH MARITIME RESEARCH, No S1/2016 81 services for the cluster, playing the role of middle bridge
and avoiding low coupling degree of cluster caused by the
non-smooth information. Scientific research institutions
include universities and research institutes, which are the
most important innovation institutions of knowledge and
technology. Shipbuilding industry cluster inner layer can
cooperate with scientific research institutions, such as
forming the Research Alliance to continue to learn new
technologies, to absorb shipbuilding talents. Scientific
research institutions become the birthplace of the high-
tech and technical talents of the cluster inner layer in this
way. Education and training institutions train managers,
so cluster coupling degree is improved by the managers by
making the scientific management more effectively in the
cluster inner layer. Financial institutions provide funding
for enterprises. Shipbuilding industry associations offer a
variety of social services for shipbuilding industry cluster,
and maintain the legitimate rights of interests of the member
units and the industry. The overall effect is much greater
than their respective effect, resulting in efficiency overflow
proliferation and positive effect on the whole cluster, thus
forming a virtuous circle, expanding the overall positive effect
of the cluster, so as to achieve benign coupling by support
service institutions coordinating with cluster inner layer, and
producing synergistic effect in dynamic interaction. development and the coupling. Good coupling mechanism
can promote the formation of positive coupling effect bundle. The positive effect of the coupling effect bundle is amplified
to achieve positive cycle. COUPLING MECHANISM OF SUPPORT LAYER EFFICIENCY FUNCTION OF SHIPBUILDING
INDUSTRY CLUSTER Set variable (i=1,2,...,n) is the order parameter of the
shipbuilding industry cluster. The core ship manufacturing
enterprise, the ship matching enterprise, the ship spare parts
manufacturing enterprise, the shipbuilding supply enterprise,
and support service institution are the five order parameters. uij is the j-index of i-parameter, the value is Xij (i=1,2,..., n). Aij, Bij are the upper and lower limits of order parameter of
shipbuilding industry cluster steady state. And efficiency
function of the core ship manufacturing enterprise, the ship
matching enterprise, the ship spare parts manufacturing
enterprise, the shipbuilding supply enterprise, and support
service institution orderly to shipbuilding industrial cluster
system can be expressed by: The main contents of support layer coupling mechanism
are government agencies involving in shipbuilding
industrial cluster coupled by providing policy support,
macro-environment creation, etc. for cluster inner layer. Government agencies play a guiding role on the behavior
of all the subjects in the cluster inner layer by industrial
preferential policies, tax policies to bring the cluster to
benign coupling. Intermediary agencies provide information uij has a negative effect
uij has a positive effect
(1) (1) POLISH MARITIME RESEARCH, No S1/2016 82 The uij is the efficiency contribution size of variable Xij
to the system. uij represents the satisfaction degree of each
index reaching the goal, uij is between 0 and 1, reaching to 0
for the most unsatisfied, closing to 1 for the most satisfactory. The core ship manufacturing enterprise, the ship matching
enterprise, the ship spare parts manufacturing enterprise, the
shipbuilding supply enterprise and support service institution
are mutually connected and influenced subsystems, the
total contribution of each index to subsystems is achieved
by integrated method, and the linear weighted method is
generally used[22]. same. Therefore, it is obviously not a good reflection of the
shipbuilding industry cluster synergy and the overall effect
only using the coupling degree to evaluate the development
of the cluster. In this case, the significance of conclusion by
using the coupling degree to the development of cluster is not
obvious. The modified coupling degree measurement model
of shipbuilding industry cluster is[25-27]: (4) (4) (2) (2) where, C is coupling degree; C’ is modified coupling
degree; T is subsystem comprehensive harmonic index of
shipbuilding industrial cluster, which reflects the overall
synergistic effect of shipbuilding industrial cluster; a, b, c,
etc. are undetermined coefficients, which can determined
by absorbing the experts’ advice. The modified coupling
is divided into four levels. CONCLUSIONS Through the review of the prior literature on the Chinese
shipbuilding industrial cluster and manufacturing, this
paper focuses on the current development of the Chinese
shipbuilding industry and related concepts. The shipbuilding
industrial cluster includes two basic coupling mechanism
layers: the core layer and support layer on top of the
prior papers, this paper developed a new coupling degree
measurement model In order to achieve the long-term
stable development of the shipbuilding industry cluster, the
following aspects were proposed. (3) (3) According to the above equation, the core layer consists
of four sub-systems, namely the core ship manufacturing
enterprise, the ship matching enterprise, the ship spare parts
manufacturing enterprise, the shipbuilding supply enterprise. Support layer consists of five subsystems, namely the core ship
manufacturing enterprise, the ship matching enterprise, the
ship spare parts manufacturing enterprise, the shipbuilding
supply enterprise and support service institution. The core
layer coupling degree measurement model and the support
layer coupling degree measurement model are constructed
with subsystem number instead of n of the formula. The value
of C is between 0 and 1, 1 represents subsystem coupled with
maximum degree and the coupling condition of subsystems
benign. 0 represents subsystems coupled with minimum
degree, and the subsystems are independent. First of all, the ship matching enterprises play an important
role in the shipbuilding industry cluster. Accelerated
development of matching industries is useful for improving
the coupling degree of the core layer, promoting the
development of the shipbuilding industry cluster. When the
matching capacity cannot catch up with the development of
the shipbuilding industry, the ship manufacturing enterprises
will not carry out effective production activities, in other
words, the core layer coupling degree of shipbuilding industry
cluster is delayed by the supporting layer. The development
of matching industries can be accelerated by implementing
professional operation, e.g. establishing the main auxiliary
instrument, machinery and other professional company;
concentrating financial resources, material resources,
manpower to engage in specialized production[6].Specialized
production can be applied to improve the matching capacity
of shipbuilding industry cluster, to promote the establishment
of a good coupling mechanism. Coupling degree C is an important parameter to judge the
coupling state of the subsystems, but it is difficult to reflect the
overall effect of the cluster coupling in some cases, especially
in the cluster contrast study. Due to the development strategy,
the characteristics of each shipbuilding industry cluster
are different. EFFICIENCY FUNCTION OF SHIPBUILDING
INDUSTRY CLUSTER Coupling layer of shipbuilding
industrial cluster is in the low coordination coupling stage, if
0 < C’ ≤ 0.4. Coupling layer of shipbuilding industrial
cluster is in the moderate coordination coupling stage, if
0.4 < C’ ≤ 0.6. Coupling layer of shipbuilding industrial cluster
is in the high coordination coupling stage, if 0.6 < C’ ≤ 0.8. Coupling layer of shipbuilding industrial cluster is in the
extreme coordination coupling stage, if 0.8 < C’ ≤ 1. (2) ui is the contribution of subsystem to total system order
degree. m is the number indicators of subsystem, λij is index
weights. The weights of each index can be determined by
analytic hierarchy process and entropy weight method[22]
[23]. COUPLING DEGREE FUNCTION OF SHIPBUILDING
INDUSTRY CLUSTER The calculation of the coupling degree of shipbuilding
industry cluster draw lessons from the concept of capacity
coupling and capacity coupling coefficient model in
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Direct in situ photolithography of perovskite quantum dots based on photocatalysis of lead bromide complexes
|
Nature communications
| 2,022
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cc-by
| 10,945
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Direct in situ photolithography of perovskite
quantum dots based on photocatalysis of
lead bromide complexes Pingping Zhang1, Gaoling Yang
2
, Fei Li3, Jianbing Shi1 & Haizheng Zhong
1 Photolithography has shown great potential in patterning solution-processed
nanomaterials for integration into advanced optoelectronic devices. However,
photolithography of perovskite quantum dots (PQDs) has so far been hindered
by the incompatibility of perovskite with traditional optical lithography pro-
cesses where lots of solvents and high-energy ultraviolet (UV) light exposure
are required. Herein, we report a direct in situ photolithography technique to
pattern PQDs based on the photopolymerization catalyzed by lead bromide
complexes. By combining direct photolithography with in situ fabrication of
PQDs, this method allows to directly photolithograph perovskite precursors,
avoiding the complicated lift-off processes and the destruction of PQDs by
solvents or high-energy UV light, as PQDs are produced after lithography
exposure. We further demonstrate that the thiol-ene free-radical photo-
polymerization is catalyzed by lead bromide complexes in the perovskite
precursor solution, while no external initiators or catalysts are needed. Using
direct in situ photolithography, PQD patterns with high resolution up to 2450
pixels per inch (PPI), excellent fluorescence uniformity, and good stability, are
successfully demonstrated. This work opens an avenue for non-destructive
direct photolithography of high-efficiency light-emitting PQDs, and potentially
expands their application in various integrated optoelectronic devices. Perovskite quantum dots (PQDs) exhibit narrow photoluminescence
(PL) emission with high quantum yields which is tunable over the entire
visible range, have shown great potential in a range of optoelectronic
applications1–4. In particular, there is intense interest in applying PQDs in
displays5–8. Despite the great progress, PQDs based light-emitting
diodes (LEDs) still suffer from some issues that prevent their incor-
poration into commercial display products such as cell phones or lap-
tops. One of them is patterning, multicolor LEDs have been hindered by
the difficulty in patterning red, green, and blue (RGB) PQD films into
arrays of individual devices rather than bulk films9–12. Therefore, the
capability of patterning PQDs into pixel arrays is urgently needed. Over the past few years, various patterning techniques have been
developed, including inkjet printing13, nanoimprinting14, laser direct
writing15, photolithography16, and so on. Among all these methods,
photolithography has attracted considerable attention owing to its high
resolution, wide availability, and high throughput. There are two cate-
gories of this technique, conventional photoresist-assisted method, and
direct photolithography. The former uses the conventional lift-off
technique where the photoresist is first patterned before QDs are
deposited17–20. Article https://doi.org/10.1038/s41467-022-34453-9 Description of direct in situ photolithography method Cross-section transmis-
sion electron microscopy (TEM) measurement was performed to clearly
demonstrate the QD structure of perovskite, and exhibit their homo-
geneous dispersion in micropatterns, as shown in Fig. 1c. High crystal-
linity and continuous lattice fringes can be seen clearly from further
high-resolution TEM (HRTEM) image in Fig. 1d, the lattice constant of
0.301 nm is consistent with (002) interplanar spacing of MAPbBr3. The
bright and uniform PQD-polymer films can be ascribed to the good
protection of polymer matrix on the exposed region, which was proved
by the calculation of the retention of PQDs defined as the percentage of
PQDs retained in the exposed films after developing24. Inductively
coupled plasma-optical emission spectroscopy (ICP-OES) and induc-
tively coupled plasma-mass spectroscopy (ICP-MS) were used for the
analysis of Pb atom in the developer solvent and polymer film to
determine the retention of PQDs. And the figure was recorded as up to
85%, which accounts for the good protection effect of polymer on
perovskite thus obtaining high luminescent patterns. Exploration of photochemical mechanism
In general, external initiators or catalysts are essential for poly- Most of these photolithography technologies are based on pho-
topolymerization, which is the curing process of monomers triggered
by photosensitizers or photoinitiators exposed to light. Recently,
perovskite has shown great promise as an efficient catalyst31,32, and is
used to directly initiate the photopolymerization process for the fab-
rication of various PQD-polymer compounds, such as PQD-polymethyl
methacrylate33, PQD-polystyrene34, and PQD-polyaniline35. However,
these photopolymerizations can be easily inhibited in the air due to the
quenching effect of oxygen on carbon radicals36. While the thiol-ene
reaction is insensible to oxygen and high-efficiency, which has been
applied in photolithography for microdevice fabrication37. A typical
radical thiol-ene reaction often starts from thiyl radicals, then they
attack double bonds to generate carbon radicals, which in turn react
with thiol monomers to release thiyl radicals, resulting in the chain
growth and transfer38. The insensitivity to oxygen comes from that the
ready hydrogen abstraction of peroxy radicals from thiols to generate
active thiyl radicals, which have the ability to continue the free-radical
chain process39. Thioethers have been photosynthesized with PQDs as
a photocatalyst40. However, most reactions with PQDs as a photo-
catalyst normally involve blue or white light irradiation for a long time
of several hours which may also potentially destroy the PQDs41 and are
hard to incorporate existing photolithography process. Description of direct in situ photolithography method Description of direct in situ photolithography method
Fine PQDs patterns were fabricated from the original perovskite pre-
cursor solution with monomers by direct in situ photolithography
method. As shown in Fig. 1a, prior to photolithography, the glass sub-
strate is cleaned by sonication in various solvents, then silane coupling
agents are used to functionalize the substrate with ethenyl or thiol
groups exposing. Such functionalized substrate creates strong covalent
bonding sites for the resultant polymer film, which is critical to the
successful patterning of perovskite films. The patterning of PQDs can
start from simply casting the photosensitive perovskite precursor resist
(PPR) directly onto the functionalized substrate. The PPR is key to the
direct in situ photolithography, which is prepared by dissolving reagent
salts, multifunctional thiol and ethenyl monomers in polar aprotic sol-
vents. Specifically, for green PPR, MABr (MA = methylammonium) and
PbBr2 are chosen as perovskite precursor salts, which have been proved
to be very effective reagents to generate perovskite. For monomers,
trimethylolpropane tris (3-mercaptopropionate) (TTMP), a multi-thiol
crosslinking reagent, is used as a thiol monomer, while triallyl iso-
cyanurate (TAIC) provides electron-rich vinyl groups. To dissolve per-
ovskite precursor and monomers better, polar aprotic solvents such as
N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) are the
best choice. Unlike previous methods, direct in situ photolithography
approach directly patterns original perovskite precursors instead of
prepared QD inks, which often need extensive cleaning steps and can be
destroyed under high-energy UV light in the presence of oxygen51,52. Second, perovskite precursor film is exposed with UV light through a
photomask. Upon UV irradiation, a photochemically activated reaction
between TTMP and TAIC occurs, leading to the solidification of the PPR
in the exposed area. Third, the unexposed PPR is removed by spin-
washing with chloroform as a developer. Cured products on exposed
areas can adhere to the substrate strongly during spin-washing because
of the covalent interactions formed between functionalized substrates
and TTMP or TAIC in PPRs, making the developing process much easier. Finally, the prepared perovskite precursor patterns are directly
annealed in the ambient air to evaporate residual solvents, when the
concentration reaches its critical value for in situ nucleation, lumines-
cent perovskite patterns are formed. Figure 1b shows the corresponding
fluorescence image of the patterned arrays of square with a size of
30 μm on a substrate, the patterned pixel arrays emit bright and uni-
form green fluorescence under UV irradiation. Direct in situ photolithography of perovskite
quantum dots based on photocatalysis of
lead bromide complexes However, PQDs are easily dissolved in polar solvents and
re-dispersed in nonpolar solvents due to their ionic nature, making
them usually incompatible with traditional photolithography processes 1MIIT Key Laboratory for Low Dimensional Quantum Structure and Devices, School of Materials Science and Engineering, Beijing Institute of Technology,
Beijing 100081, China. 2MIIT Key Laboratory for Low Dimensional Quantum Structure and Devices, School of Optics and Photonics, Beijing Institute of
Technology, Beijing 100081, China. 3QD LAB, Hefei Innovation Research Institute of Beihang University, Hefei, Anhui 230001, China. e-mail: glyang@bit.edu.cn 1MIIT Key Laboratory for Low Dimensional Quantum Structure and Devices, School of Materials Science and Engineering, Beijing Institute of Technology,
Beijing 100081, China. 2MIIT Key Laboratory for Low Dimensional Quantum Structure and Devices, School of Optics and Photonics, Beijing Institute of
Technology, Beijing 100081, China. 3QD LAB, Hefei Innovation Research Institute of Beihang University, Hefei, Anhui 230001, China. e-mail: glyang@bit.edu.cn Nature Communications| (2022) 13:6713 Nature Communications| (2022) 13:6713 Nature Communications| (2022) 13:6713 1 Article https://doi.org/10.1038/s41467-022-34453-9 synthesis strategy, clearly provides an efficient platform for the man-
ufacture of high-resolution patterned optoelectronic devices. where a lot of solvents are needed in photoresists, developers, etchants,
or strippers11. Alternatively, direct photolithography is quite simple and
effective, which patterns photosensitive QDs directly through the light-
induced change in their solubility21. Recently, several research groups
presented high-resolution QD patterns based on direct photo-
lithography with different photochemistry mechanisms, such as unsa-
turated double bond cross-linking22–24, azide cross-linking24–27, polarity
change28–30, and so on. Description of direct in situ photolithography method Hence, PQDs
may not be the best option for photocatalysis during the existing
photolithography process. Additionally, in the existing direct photolithography, almost all
photosensitive QDs are prepared through the hot injection approach
and subsequent ligand exchange method beforehand, which usually
involve high reaction temperature, harsh inert gas environment,
complex synthesis steps, and extensive cleaning steps. In addition,
these photosensitive QDs suffer from preserving their optical prop-
erties in the lithography exposure process, especially for PQDs. In situ
fabrication has been proved to be a powerful method to synthesize
PQDs directly on a substrate or in a matrix42, including polymer
matrix43, glass matrix44, molecular sieves45, or crystals46, due to the low
formation enthalpy and high defect tolerant of ionic perovskite47,48. Recently, in situ fabrication has been utilized to provide patterned
PQDs pixels with tunable luminescence for integrating into optoelec-
tronic devices49,50. Thus motivated, here we propose a direct photolithography
method to pattern in situ fabricated PQDs based on polymerization
catalyzed by lead bromide complexes. This method produces high-
resolution patterns by direct photolithography of perovskite pre-
cursors using UV light to trigger the photopolymerization, PQDs are
in situ fabricated in polymer by final annealing, avoiding high-energy
UV light to destroy PQDs since they were produced after exposure. In
particular, we demonstrate that the photopolymerization under UV
irradiation is based on lead bromide complexes catalyzing the thiol-
ene free-radical reaction, without any external initiators or catalysts
that are harmful to the stability of patterns. Furthermore, uniform and
residual-free luminescent pixel arrays are fabricated via direct photo-
lithography of in situ fabricated PQDs, in which the high uniformity of
emission intensities reflects the homogeneous distribution of PQDs. Lastly, we successfully fabricate red, green and blue PQD patterns, the
minimum feature size of 5 μm corresponding to a resolution of up to
2450 pixels per inch (PPI), the maximal thickness of 10 μm enables to
absorb blue or UV light more efficiently when used as color convention
pixels. The direct photolithography method combined with in situ Exploration of photochemical mechanism p
p
In general, external initiators or catalysts are essential for poly-
merization, but things are different in our case. It seems perovskite
precursors have the ability of photocatalysis, which makes direct Nature Communications| (2022) 13:6713 2 https://doi.org/10.1038/s41467-022-34453-9 Article Casting
UV exposure
Developing
Annealing
In situ crystallization
Photopolymerization
MABr + PbBr2 + monomers
in DMF and (or) DMSO
Silane coupling agent
MA
+
Br
−
Pb
2+
Monomer
Polymer
PQD
PPR
Developer
d (002) = 0.301 nm
2 nm
200 nm
90 μm
a
b
c
d
g. 1 | Schematic illustration of PQD patterning method. a Schematic descrip-
n of the direct in situ photolithography method from the perovskite precursor
ist (PPR), annealing refers to heating the samples at a specific temperature. Fluorescence image of green MAPbBr3 PQD patterns (30 μm size square)
fabricated by the direct in situ photolithography method. c Cross-section TEM
image of MAPbBr3 PQD patterns fabricated by the direct in situ photolithograp
method. d HRTEM image of single MAPbBr3 PQD in the patterns fabricated by t
direct in situ photolithography method. Casting
UV exposure
Developing
Annealing
In situ crystallization
Photopolymerization
MABr + PbBr2 + monomers
in DMF and (or) DMSO
PPR
Developer
a a Developer Casting Annealing Developing MABr + PbBr2 + monomers
in DMF and (or) DMSO Photopolymerization In situ crystallization MABr + PbBr2 + monomers
in DMF and (or) DMSO Silane coupling agent Monomer 90 μm
b b fabricated by the direct in situ photolithography method. c Cross-section TEM
image of MAPbBr3 PQD patterns fabricated by the direct in situ photolithography
method. d HRTEM image of single MAPbBr3 PQD in the patterns fabricated by the
direct in situ photolithography method. Fig. 1 | Schematic illustration of PQD patterning method. a Schematic descrip-
tion of the direct in situ photolithography method from the perovskite precursor
resist (PPR), annealing refers to heating the samples at a specific temperature. b Fluorescence image of green MAPbBr3 PQD patterns (30 μm size square) 100 s irradiation (Fig. 2b), confirming the thiol-ene photopolymeriza-
tion system. The extent of polymerization was also determined by FT-
IR spectra, where 83% and 84% were ascribed to the conversion rate of
the ethenyl and thiol group, suggesting the nearly 1:1 stoichiometry
rection (Supplementary Fig. 2). Furthermore, various monomers with
varying quantities of functional groups (Supplementary Fig. Exploration of photochemical mechanism 3a, b)
were also examined under the same reaction conditions, all the com-
binations were cured within 30 min under a low power UV light
(10 mW cm−2) (Supplementary Fig. 3c and Supplementary Table 2),
demonstrating the universality of thiol-ene reactions photocatalyzed
by perovskite precursors. in situ photolithography of PQDs possible. We seek to understand the
photocatalysis mechanism by investigating the type of polymerization,
photocatalytic species, and reactive site. To verify the photocatalysis
of perovskite precursors, equal volumes of monomer systems with and
without perovskite precursors were irradiated under 365 nm UV light
for 2 min. As can be seen in Fig. 2a, with perovskite precursors, soli-
dified gelatin was formed at the bottom of the bottle, while without
precursors, the ink was still liquid, appreciably illustrating the photo-
catalysis of perovskite precursors. To further explore whether the
photopolymerization was conducted by self-polymerization31,33 or
copolymerization37, monomer systems either with one single mono-
mer (ethenyl or thiol) or with both of them mixing with perovskite
precursor were irradiated under UV light. Evidently, the monomer
system containing both ethenyl and thiol monomers was cured within
30 s (entry 1 in Supplementary Table 1 and Supplementary Fig. 1), while
for a single monomer, no polymerization occurred (entries 2 and 3 in
Supplementary Table 1 and Supplementary Fig. 1). This indicates that
the PPR underwent the thiol-ene reaction. Additionally, this reaction
system was further verified by Fourier-transform infrared (FT-IR)
spectra of the PPR before and after UV irradiation, both peaks of
–CH=CH2 (3080 cm−1) and –SH (2527 cm−1) reduced dramatically after in situ photolithography of PQDs possible. We seek to understand the
photocatalysis mechanism by investigating the type of polymerization,
photocatalytic species, and reactive site. To verify the photocatalysis
of perovskite precursors, equal volumes of monomer systems with and
without perovskite precursors were irradiated under 365 nm UV light
for 2 min. As can be seen in Fig. 2a, with perovskite precursors, soli-
dified gelatin was formed at the bottom of the bottle, while without
precursors, the ink was still liquid, appreciably illustrating the photo-
catalysis of perovskite precursors. To further explore whether the
photopolymerization was conducted by self-polymerization31,33 or
copolymerization37, monomer systems either with one single mono-
mer (ethenyl or thiol) or with both of them mixing with perovskite
precursor were irradiated under UV light. Exploration of photochemical mechanism Therefore, we speculated that the photopolymerization
in this system may be catalyzed by the lead bromide complexes. To
confirm the catalytic capacity of lead bromide complexes, PPRs with
different bromide concentrations were tested. As can be seen in
Fig. 3a, with the increase of [MABr], the curing time dramatically
reduced, with a maximum increase in catalytic effect by a factor of ten. Since high coordinated lead bromide complexes are formed at high
bromide concentration, which can be confirmed by the redshift of
absorption (Supplementary Fig. 10a)56, the enhanced catalytic effect at
high [MABr] can imply the stronger catalytic capacity of high coordi-
nated lead bromide complexes. To further demonstrate that, per-
ovskite
precursors
were
dissolved
in
solvents
with
different
coordination abilities (DMSO > DMF > GBL (γ-butyrolactone))55. As
solvent molecules can compete with halides for coordination with lead
ions, when the coordination ability of a solvent was reduced, the
coordination of halide and Pb2+ would increase to form lead halide
complexes with high coordination numbers57, which can be proved by
the redshift of their absorption spectra (Supplementary Fig. 10b). As
shown in Fig. 3b, when the curing yields after 2 min 30 s UV light
irradiation were plotted against the decreasing coordination ability of
solvents, a clear upward trend emerged (0% for DMSO, 20% for DMF,
70% for complex DMF/GLB), strongly suggesting the key photo-
catalytic role of high coordinated lead bromide complexes. In per-
ovskite precursor solutions, PbBr2, PbBr3
‒, PbBr4
2‒ have been reported
in the literature56, while PbBr2 has been excluded in the previous
experiment, we speculated that high coordinated PbBr4
2‒ is the key
photocatalyst in this reaction. To prove that, UV lights with various
wavelengths were used, since lead bromide complexes with different
coordination numbers show different absorption of light at different
wavelengths56,58. As shown in Fig. 3c, when exposed under 311 nm UV
light close to the absorption peak of PbBr3
‒, an acceptable exposure
dose for completely curing were required, combining with the abso-
lutely predominated concentration of PbBr3
‒ in the PPR, indicating a
relatively weak catalytic effect of PbBr3
‒. When replacing with 365 nm
UV light close to the absorption peak of PbBr4
2‒, only half exposure
dose was needed even under a relatively low concentration, implying
the strong photocatalytic capacity of PbBr4
2‒. Exploration of photochemical mechanism c EPR spectra of the PPR collected after 0 min and
2 min UV irradiation. Source data are provided as a Source Data file. 3100
2600
2500
2400
Absorbance (arb. u.)
Wavenumbers (cm−1)
PPR
Cured PPR
b With
precursors
Without
precursors
1 cm
a
Gelatin
Liquid 3460
3480
3500
3520
3540
3560
Intensity (arb. u.)
Magnetic field (G)
0 min
2 min
c b c a spectra of the perovskite precursor resist (PPR) and cured PPR after 365 nm UV
light (5 W) irradiation for 100 s. c EPR spectra of the PPR collected after 0 min and
2 min UV irradiation. Source data are provided as a Source Data file. Fig. 2 | Investigation of the photopolymerization type. a Photograph of the
resultant inks without (left side) and with (right side) perovskite precursors in the
monomer system after irradiating with 365 nm UV light (20 W) for 2 min. b FT-IR The ink with HBr had a comparable curing rate to that with MABr
(Supplementary Fig. 9), along with the photocatalysis capacity of PbBr2
demonstrated before, which absolutely excluded the photocatalysis
effect of PQDs in our case. These results suggest that the primary
catalyst of this reaction is neither from any single reagent salts and
their constituent ions in the perovskite precursor solution nor PQDs. system is not the thiol-Michael addition mechanism. When a negligible
amount of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used as a free-
radical inhibitor, no polymerization happened under the same UV light
environment (entries 6 vs 4 in Supplementary Table 3 and Supple-
mentary Fig. 4), confirming the inhibiting effect of the free-radical
inhibitor for this reaction system. To further confirm the presence of
free radicals, electron paramagnetic resonance (EPR) spectroscopy
measurement was carried out. A clear EPR signal assigned to carbon
radicals was detected after 2 min irradiation of PPR using a UV light
source (Fig. 2c), unambiguously proving the growth and transfer of
polymer chain was initiated by free radicals. Together, both the inhi-
bition reaction and EPR results strongly confirm the free-radical reac-
tion pathway. Perovskite precursor solutions are actually colloid54, where
halides and solvent molecules coordinate with lead ions to obtain a
variety of lead halide complexes PbXn
2−n (X = Cl, Br or I; n = the coor-
dination number of halide), all the various complexes are in dynamic
equilibrium55. Exploration of photochemical mechanism Evidently, the monomer
system containing both ethenyl and thiol monomers was cured within
30 s (entry 1 in Supplementary Table 1 and Supplementary Fig. 1), while
for a single monomer, no polymerization occurred (entries 2 and 3 in
Supplementary Table 1 and Supplementary Fig. 1). This indicates that
the PPR underwent the thiol-ene reaction. Additionally, this reaction
system was further verified by Fourier-transform infrared (FT-IR)
spectra of the PPR before and after UV irradiation, both peaks of
–CH=CH2 (3080 cm−1) and –SH (2527 cm−1) reduced dramatically after 100 s irradiation (Fig. 2b), confirming the thiol-ene photopolymeriza-
tion system. The extent of polymerization was also determined by FT-
IR spectra, where 83% and 84% were ascribed to the conversion rate of
the ethenyl and thiol group, suggesting the nearly 1:1 stoichiometry
rection (Supplementary Fig. 2). Furthermore, various monomers with
varying quantities of functional groups (Supplementary Fig. 3a, b)
were also examined under the same reaction conditions, all the com-
binations were cured within 30 min under a low power UV light
(10 mW cm−2) (Supplementary Fig. 3c and Supplementary Table 2),
demonstrating the universality of thiol-ene reactions photocatalyzed
by perovskite precursors. Generally,
the
thiol-ene
reaction
has
two
polymerization
mechanisms: thiol-Michael addition which is catalyzed mainly by bases
or nucleophiles53, and thiol-ene free-radical addition which is induced
by photoinitiators or high-energy UV light irradiation37. To unravel the
polymerization mechanism of the PPRs, proton source and free-radical
inhibitors were added, which can effectively inhibit the thiol-Michael
addition and free-radical addition, respectively. When a proton source
such as HCl was added in the PPR, the cured polymer was obtained
after 19 s irradiation using a 365 nm UV light source (entries 5 vs 4 in
Supplementary Table 3 and Supplementary Fig. 4), implying this Nature Communications| (2022) 13:6713 3 https://doi.org/10.1038/s41467-022-34453-9 Article 3100
2600
2500
2400
Absorbance (arb. u.)
Wavenumbers (cm−1)
PPR
Cured PPR
With
precursors
Without
precursors
1 cm
3460
3480
3500
3520
3540
3560
Intensity (arb. u.)
Magnetic field (G)
0 min
2 min
b
c
a
Gelatin
Liquid
Fig. 2 | Investigation of the photopolymerization type. a Photograph of the
resultant inks without (left side) and with (right side) perovskite precursors in the
monomer system after irradiating with 365 nm UV light (20 W) for 2 min. b FT-IR
spectra of the perovskite precursor resist (PPR) and cured PPR after 365 nm UV
light (5 W) irradiation for 100 s. Exploration of photochemical mechanism At 405 nm, the y
We were intrigued by the unexpected photocatalytic ability of
perovskite precursors and curious about which part plays a major role. Firstly, to explore whether the reagent salts MABr and PbBr2 were the
primary catalyst, a series of control experiments were performed with
various constituents. We found that nearly all the liquid was cured in
6 min when MABr and PbBr2 were used together (entry 7 in Supple-
mentary Table 4 and Supplementary Fig. 5a), reaction conducted using
PbBr2 also gave cured products but at a low yield after 20 min irra-
diation (entry 8 in Supplementary Table 4 and Supplementary Fig. 5b),
no cured product appeared when MABr was used (entry 9 in Supple-
mentary Table 4 and Supplementary Fig. 5b). This result implies the
fast catalytic effect of the mixture of MABr and PbBr2, the slow cata-
lytic effect of PbBr2, and excludes the catalytic possibility of MABr, MA+
and Br– in this system. Obviously, PbBr2 does not play a major catalytic
role in this photocatalytic reaction, but it does show some catalytic
effect, so we wondered if Pb2+ might be the catalyst. Therefore, we
examined its catalytic ability by changing the halides from Br to I and
Cl. However, no cured product was obtained under UV light (311, 365,
and 405 nm) irradiation (entries 11 and 12 vs 10 in Supplementary
Table 5, Supplementary Fig. 6), excluding the catalytic effect of Pb2+
either. Since the PQDs have been reported to be used as photocatalyst
for thiol-ene reactions under blue light irradiation40, it is easy to relate
the photocatalytic effect to the PQDs. However, both the PL and
ultraviolet-visible (UV–Vis) spectra (Supplementary Fig. 7) of the PPR
exhibit distinctly different emission and large Stokes shift compared
with the typical PQDs, demonstrating that there was no PQD generated
from PPR no matter how long the solution was stirred. All the PPRs with
different stirring time can be cured within 2 min (the left five in Sup-
plementary Fig. 8), while no cured product was found in the ink using
PQDs as photoinitiator (the rightest in Supplementary Fig. 8), which at
least indicates that PQDs cannot photocatalyze the polymerization as
efficiently as the perovskite precursors. Furthermore, MABr was sub-
stituted by HBr to eliminate A site ion that is necessary for the fabri-
cation of perovskite, avoiding any possibility to produce perovskite. Exploration of photochemical mechanism f PLE (solid line) and PL (dot-dash line) spectra of the perovskite precurso
solution (PPS) and PPR. g Proposed photochemical mechanism of lead bromide
complexes catalyzed initiation process of thiol-ene free-radical addition, L stands
for a solvent molecule. Source data are provided with this paper. Electron ho
Pb
Br
Br
Br
B
LUMO
HOMO
UV light
O
Pb
Br
Br
Br
Br
L
H
S
Pb
Br
Br
Br
H
Br
S δ+
δ−
Coordination
g Electron hole transfer
H
Pb
Br
Br
Br
Br
S δ+
δ−
h+
e−
LUMO
HOMO
e−
UV light
O2
O2·−
r
H
Br
S δ+
δ−
ation
Pb
Br
Br
Br
Br
L
H+
S·
Initiation Electron hole transfer
H
Pb
Br
Br
Br
Br
S δ+
δ−
h+
e−
LUMO
HOMO
e−
UV light
O2
O2·−
Pb
Br
Br
Br
Br
L
H
S
Pb
Br
Br
Br
H
Br
S δ+
δ−
Coordination
Pb
Br
Br
Br
Br
L
H+
S·
Initiation
g Electron hole transfer wavelengths (line graph corresponding to the right axis). EPR spectra of the inks
respectively mixed by d TAIC and e TTMP with the same perovskite precursor
solution. f PLE (solid line) and PL (dot-dash line) spectra of the perovskite precursor
solution (PPS) and PPR. g Proposed photochemical mechanism of lead bromide
complexes catalyzed initiation process of thiol-ene free-radical addition, L stands
for a solvent molecule. Source data are provided with this paper. Fig. 3 | Exploration of the photochemical mechanism. a Curing time of per-
ovskite precursor resists (PPRs) with an increasing ratio of MABr to PbBr2 at a
constant concentration of PbBr2. b Curing yield of PPRs with different solvents at
constant concentrations of all reagent salts and monomers. c Exposure dose of UV
lights with different wavelengths to cure the PPR completely (histogram corre-
sponding to the left axis) and absorbance of the PPR at different UV light the perovskite precursor solution (Fig. 3e), while no carbon radical was
detected when TAIC was used (Fig. 3d). The EPR measurements sug-
gest that the monomers catalyzed by lead bromide complexes are
thiols. Together, all these results confirm that the sulfur–lead coordi-
nation bond is the reactive site. absorbance is nearly 0, indicating the low concentration of higher
coordinated lead bromide complexes. Exploration of photochemical mechanism [MABr]:[PbBr2] molar radio
0:1
0.5:1
1:1
2:1
4:1
0
2
4
6
8
10
Curing time (min)
DMSO
DMF
DMF&GBL
0.0
0.2
0.4
0.6
0.8
Solvent
Curing ratio
a
b [MABr]:[PbBr2] molar radio
0:1
0.5:1
1:1
2:1
4:1
0
2
4
6
8
10
Curing time (min)
DMSO
DMF
DMF&GBL
0.0
0.2
0.4
0.6
0.8
Solvent
Curing ratio
Ab
b
( b
)
a
b
c
Exposure dose (J cm‒2)
311 nm
365 nm
405 nm
0
3
6
9
12
15
18
UV light
0
2
4
6
8
10 [MABr]:[PbBr2] molar radio
0:1
0.5:1
1:1
2:1
4:1
0
2
4
6
8
10
Curing time (min)
a DMSO
DMF
DMF&GBL
0.0
0.2
0.4
0.6
0.8
Solvent
Curing ratio
b Absorbance (abs. u.)
c
Exposure dose (J cm‒2)
311 nm
365 nm
405 nm
0
3
6
9
12
15
18
UV light
0
2
4
6
8
10 b c a 3460
3480
3500
3520
3540
Intensity (arb. u.)
Magnetic field (G)
0 min
8 min f
PL intensity (arb. u.)
320
380
440
500
560
620
0
50
100
150
200
250
0
50
100
150
200
250
PPS
PPS
PPR
PPR
PLE intensity (arb. u.)
Wavelength (nm) d
3460
3480
3500
3520
3540
3560
Intensity (arb. u.)
Magnetic field (G)
0 min
8 min f d Intensity (arb. u.)
e e PLE intensity (arb. u.) Intensity (arb. u.) Wavelength (nm) Magnetic field (G) Wavelength (nm) g Electron hole transfer
H
Pb
Br
Br
Br
Br
S δ+
δ−
h+
e−
LUMO
HOMO
e−
UV light
O2
O2·−
Pb
Br
Br
Br
Br
L
H
S
Pb
Br
Br
Br
H
Br
S δ+
δ−
Coordination
Pb
Br
Br
Br
Br
L
H+
S·
Initiation
g
Fig. 3 | Exploration of the photochemical mechanism. a Curing time of per-
ovskite precursor resists (PPRs) with an increasing ratio of MABr to PbBr2 at a
constant concentration of PbBr2. b Curing yield of PPRs with different solvents at
constant concentrations of all reagent salts and monomers. c Exposure dose of UV
lights with different wavelengths to cure the PPR completely (histogram corre-
sponding to the left axis) and absorbance of the PPR at different UV light
wavelengths (line graph corresponding to the right axis). EPR spectra of the inks
respectively mixed by d TAIC and e TTMP with the same perovskite precursor
solution. Exploration of photochemical mechanism Nature Communications| (2022) 13:6713 4 https://doi.org/10.1038/s41467-022-34453-9 Article Electron hole transfer
H
Pb
Br
Br
Br
Br
S δ+
δ−
h+
e−
LUMO
HOMO
e−
UV light
O2
O2·−
Pb
Br
Br
Br
Br
L
H
S
Pb
Br
Br
Br
H
Br
S δ+
δ−
Coordination
Pb
Br
Br
Br
Br
L
H+
S·
Initiation
g
[MABr]:[PbBr2] molar radio
3460
3480
3500
3520
3540
Intensity (arb. u.)
Magnetic field (G)
0 min
8 min
0:1
0.5:1
1:1
2:1
4:1
0
2
4
6
8
10
Curing time (min)
DMSO
DMF
DMF&GBL
0.0
0.2
0.4
0.6
0.8
Solvent
Curing ratio
Absorbance (abs. u.)
e
f
d
a
b
c
3460
3480
3500
3520
3540
3560
Intensity (arb. u.)
Magnetic field (G)
0 min
8 min
PL intensity (arb. u.)
320
380
440
500
560
620
0
50
100
150
200
250
0
50
100
150
200
250
PPS
PPS
PPR
PPR
PLE intensity (arb. u.)
Wavelength (nm)
Exposure dose (J cm‒2)
311 nm
365 nm
405 nm
0
3
6
9
12
15
18
UV light
0
2
4
6
8
10
ig. 3 | Exploration of the photochemical mechanism. a Curing time of per-
vskite precursor resists (PPRs) with an increasing ratio of MABr to PbBr2 at a
onstant concentration of PbBr2. b Curing yield of PPRs with different solvents at
onstant concentrations of all reagent salts and monomers. c Exposure dose of UV
ghts with different wavelengths to cure the PPR completely (histogram corre-
ponding to the left axis) and absorbance of the PPR at different UV light
wavelengths (line graph corresponding to the right axis). EPR spectra of the inks
respectively mixed by d TAIC and e TTMP with the same perovskite precursor
solution. f PLE (solid line) and PL (dot-dash line) spectra of the perovskite precursor
solution (PPS) and PPR. g Proposed photochemical mechanism of lead bromide
complexes catalyzed initiation process of thiol-ene free-radical addition, L stands
for a solvent molecule. Source data are provided with this paper. Demonstration of PQD patterns via direct in situ
photolithography p
g
p y
Through the direct in situ lithography method, we fabricated a series
of micropatterns from mesoscale to microscale. Figure 4a shows the
fluorescence microscope image of 20 μm blue stripes with high con-
trast between the bright blue and dark states, exhibiting clearly
defined and sharp edges. The scanning electron microscope (SEM)
image further demonstrates the smooth surface and high contrast
between the exposed and unexposed regions in these pattern arrays
(Fig. 4b). Figure 4c and Supplementary Fig. 13a show the corre-
sponding cross-section SME image, the sharp and clean edges indicate
that all the exposed line patterns were cured and the unexposed parts
were removed completely. The thickness of patterns was adjustable by
altering UV exposure time (Supplementary Fig. 13a), and a propor-
tional relationship was recorded (Supplementary Fig. 13b). The inset
image of the enlarged view of a single stripe (Fig. 4c) shows the
thickness of patterns was more than 10 μm, which is much larger than
most of the patterns within 1 μm obtained by conventional patterning
processes. The absorbance spectra of the PQD-polymer films were
shown in Supplementary Fig. 14, the large absorption suggests these
thick films enable UV or blue light to be fully absorbed and blocked to
improve energy efficiency and avoid the leakage of light60. We further
demonstrated the luminescence uniformity of PQD patterns using a
fluorescence microscope. Figure 4d depicts the emission intensity of a
PQD array composed of 196 circle pixels with a diameter of 20 μm,
desirable fluorescence homogeneity across all pixels can be seen. The
corresponding statistical histogram of their fluorescence intensity is
shown in Fig. 4e, about 70% of pixels were distributed in the range of
2000–2200, indicating the good luminescence uniformity of the PQD
pixel arrays. Moreover, the radial and surface fluorescence intensity
distribution of an individual 20 μm circle pixel were shown in Fig. 4f,
illustrating the great fluorescence homogeneity of each pixel in PQD
patterns. The 3D fluorescence image of 50 μm blue stripe patterns
further confirmed the uniformity of each stripe while no residue can be
seen at the unexposed regions (Supplementary Fig. 15), verifying the
high efficiency and feasibility of the direct in situ photolithography. The higher-resolution images were created, including circle and
square patterns with a size of 10 μm in Supplementary Fig. 21a and
Supplementary Fig. 21b, circle patterns with a diameter of 5 μm in
Fig. Demonstration of PQD patterns via direct in situ
photolithography 5f, corresponding to the resolution of up to 2540 PPI, sufficiently
enabling for the resolution of augmented reality (AR) and virtual reality
(VR) display. This resolution was limited by the apparatus we used,
whose minimum feature size is 5 μm, the method can be extended to
fabricate higher resolution patterns by using advanced apparatus. We
further evaluated the applicability of this method to various substrates
which were functioned with VTMS or MPTS to enable ethenyl or thiol
groups exposing, the PPR can react with these active groups to form
covalent bonding under UV exposure. Clear patterns were formed not
only on rigid substrates, such as glasses and wafers (Fig. 5h), but also
on flexible substrates like polyethylene terephthalate (PET) (Fig. 5g,
Supplementary Fig. 21c), implying broader integration of the direct
in situ photolithography method. What’s more, owing to no external
initiators involved and effective polymer encapsulation, the fabricated
PQD patterns exhibited good stability, which is highly important for
further applications. The films with and without 2 w% initiators (1-
hydroxycyclohexyl phenyl ketone) were evaluated with respect to UV
and heat. Supplementary Fig. 22a shows the sample with initiators
dropped to 7% of the original PL intensity after continuous six-day
irradiation, while the one without initiators remained basically
unchanged despite fluctuations. Under the heat of 60 °C, the PL
intensity of the sample with initiators decreased by 82% in the first 16 h
and continued dropping to 6% of the origin, while the figure for that
without initiators fluctuated and remained the origin intensity until the
end of test for 64 h (Supplementary Fig. 22b). In addition, at ambient
temperature and normal atmosphere with an average humidity of 54%,
the PL intensity of the sample without initiators can maintain 85% of
the origin after 30 days (Supplementary Fig. 22c). The good stability of
the PQDs might benefit from the effective encapsulation of polymer to
discourage PQDs from ion migration, crystal aggregation, as well as
permeation of O2 and moisture65. By contrast, the films with external
initiators may generate radicals upon irradiation, which quenched
PQDs easily. Furthermore, harsher solvent tests were performed to
confirm the good protection of the polymer. As can be seen in Fig. 5h
and Supplementary Fig. Exploration of photochemical mechanism Colorful PQDs (Fig. 5e and Supplementary Fig. 19) were also fabricated
on a substrate by using SiO2 as an intermediate layer between different
layers to efficiently protect the preceding layer from being destroyed
by DMF and DMSO. The UV-Vis absorbance, PL emission and time-
resolved PL spectra of the patterned film with stripes of 100 μm period
(Fig. 5d) are shown in Supplementary Fig. 20. The peak wavelengths, PL
quantum yields (QYs) and average PL lifetimes are 464 nm, 17% and
36.4 ns for blue patterns, 521 nm, 87% and 40.3 ns for green patterns,
638 nm, 58% and 61.6 ns for red patterns, respectively. A mechanism of
in situ PQD fabrication can account for the good performance: a
polymer matrix forms before the perovskite nucleation; the developer
acts as an antisolvent to increase the supersaturation instantaneously
and produce plenty of smaller nuclei62; further annealing of the film
facilitates the growth of nuclei into larger PQDs due to the removal of
solvent63 and heat-induced diffusion of the precursor ions in the
polymer matrix. The polymer matrix formed beforehand enables the
controlled nucleation and growth of perovskite to facilitate a uniform
PQD distribution due to the spatial confinement and the local deple-
tion of precursors43,64. thiol positive radicals, which can remove the hydrogen ions immedi-
ately to generate sulfur radicals with the assistance of bromides. Finally, the lead bromide complexes that have lost holes return to the
ground state under the oxidation of oxygen in the solution to com-
plete the catalytic regeneration cycle in the aerobic atmospheric
conditions. The role of oxygen as the electron scavenger was con-
firmed by the increasing amount of thiyl radicals in the mixture of the
perovskite precursor solution and TTMP from N2, to air, to O2 atmo-
sphere (Supplementary Fig. 11). In addition, the universe photo-
catalytic effect of lead bromide complexes was also demonstrated in
other radical reactions by transforming aniline into polyaniline (Sup-
plementary Fig. 12). Exploration of photochemical mechanism Based on these results, it can be
concluded that the highly coordinated lead bromide complex has a
strong catalytic capacity, while in our case, it is PbBr4
2‒. To gain further insight into the photopolymerization mechanism,
severalexperiments wereconducted to determine the reactive site. We
first added different kinds of monomers into the perovskite precursor
solution separately (entries 13 and 14 in Supplementary Table 6), where
pH went down evidently only after TTMP was added. Meanwhile, a
significant decrease in pH can be seen when PbBr2 was added to the
monomer system (entry 15 in Supplementary Table 6). The increased
acidity
of
the
solutions
may
come
from
the
weakening
of
sulfur–hydrogen
bonding, indicating
the effective
coordination
between sulfur in thiol groups and lead ions. To further verify this
statement, oleic acid (OA), which can coordinate with lead ions
effectively was added into the PPR59. Almost double curing time can be
explained by the reduction of coordination between sulfur and lead
ions, verifying the reactive site is on the lead ion (entries 16 and 17 in
Supplementary Table 7). Moreover, strong sulfur radical signals were
detected after 8 min irradiation when TTMP was added separately into Furthermore, fluorescence quenching studies were performed to
prove the electron–hole transfer between PbBr4
2‒ and organic mono-
mers. As shown in Fig. 3f, both the intensity of photoluminescence
excitation (PLE) and PL spectra of the perovskite precursor solution
drop more than 35% with limited blue shift after adding organic
monomers, indicating that the organic monomers may act as
quenchers to extract the electrons and/or holes from the excited
PbBr4
2‒ 58. Based on our photopolymerization reaction results and
mechanism studies, we proposed a mechanism of thiol-ene free-radi-
cal addition photocatalyzed by lead bromide complexes as shown in
Fig. 3g. Firstly, the sulfur atoms in thiol monomers coordinate with the
lead ions, while the hydrogen atoms coordinate with bromides. Upon
UV light irradiation, lead bromide complexes transition from the
ground state to the excited state accompanied by the generation of
electrons and holes. The holes transfer to the thiol groups to generate Nature Communications| (2022) 13:6713 5 https://doi.org/10.1038/s41467-022-34453-9 Article red perovskite due to the ascending sequence lying in the solubility of
precursors61 and formation enthalpy48. Examples of the cartoon, let-
ters, and the school logo composed of circle red, green, and blue pixels
with a diameter and spacing of 20 μm were demonstrated in Fig. 5a–c. Demonstration of PQD patterns via direct in situ
photolithography a
b
c
40 μm
40 μm
40 μm
10.4 μm a
b
40 μm
40 μm c
40 μm
10.4 μm b c f 0
5
10
15
20
25
30
0.0
0.2
0.4
0.6
0.8
1.0
Norm. PL intensity
Distance (μm)
f PL intensity (abr. u.)
d d e
1800 2000 2200 2400 2600
0
35
70
105
140
Average count
PL intensity (arb. u.) e Average count 196 circle pixels with a diameter of 20 μm. Error bars represent standard deviation
(the statistical data and errors were presented in Supplementary Table 8). f Radial
emission intensity distribution of a single 20 μm circle pixel, inlet image is the
surface-emission intensity of the pixel. Source data are provided with this paper. Fig. 4 | Characterization of PQD patterns prepared via direct in situ photo-
lithography. a Fluorescence image, b SEM image, and c cross-section SEM image
(inset image is enlarged view of a single stripe) of blue stripe patterns. d Emission
intensity distribution image, e average emission intensity statistical distribution of Patterned PQDs
Ethanol
50 μm
0.2 cm
100 μm
100 μm
100 μm
0.5 cm
50 μm
0.2 cm
0.2 cm
50 μm
500 μm
50 μm
0.5 cm
a
b
c
d
e
f
g
h
Fig. 5 | Multicolored PQDs patterns via direct in situ photolithography. Fluor-
escence images of a red cartoon, b green letters, c blue logo and corresponding
pixels; d red, green and blue stripe patterns; e red and green double color pat-
terned film with squares of 250 μm; f blue circle patterns with a diameter of 5 μm,
the scale bar in the inset image is 10 μm. g Photograph of green cartoon on the
flexible PET under UV light. h Photograph of green logo dipping into ethanol under
UV light. 100 μm
100 μm
100 μm
500 μm
50 μm
d
e
f a b Patterned PQDs
Ethanol
0.5 cm
h 0.5 cm
g h 50 μm
0.2 cm
c g c the scale bar in the inset image is 10 μm. g Photograph of green cartoon on the
flexible PET under UV light. h Photograph of green logo dipping into ethanol under
UV light. Fig. 5 | Multicolored PQDs patterns via direct in situ photolithography. Demonstration of PQD patterns via direct in situ
photolithography 23a, the luminescent patterns still kept com-
plete morphology and bright fluorescence when immersing into water
and polar ethanol which can destroy PQDs easily, about 66 and 60% of
the origin PL QY were retained even after 10 h (Supplementary
Fig. 23b). The migration of PQDs in ethanol was determined by The direct in situ photolithography can also be extended to other
color PQD patterns. To make sure blue and red PPRs can be patterned,
it is critical to determine the proportion of Br‒ substituted by Cl‒ or I‒ to
ensure enough lead bromide complexes exist in the PPR. That is
because both the amounts of PbBr4
2– and the curing rates of corre-
sponding PPRs decreased with the increasing ratio of [Cl‒] and [I‒]
(Supplementary Fig. 16a), and a proportional linear relationship was
recorded between the curing rate and the content of PbBr4
2‒ (Sup-
plementary Fig. 16b). To balance the ratio involving Cl‒ and I‒ to satisfy
the formation of blue and red PQDs and the quantity of PbBr4
2‒
required to photocatalyze the polymerization, suitable ratios were
chosen for blue and red PQDs and their catalysis capacities were
confirmed by the variation of PL and PLE spectra (Supplementary
Fig. 17). To obtain PQDs with good performance, annealing process
was optimized for different PQDs (Supplementary Fig. 18), the climb-
ing annealing temperature is respectively applied from blue, green and Nature Communications| (2022) 13:6713 6 Article https://doi.org/10.1038/s41467-022-34453-9 0
5
10
15
20
25
30
0.0
0.2
0.4
0.6
0.8
1.0
Norm. PL intensity
Distance (μm)
a
b
c
40 μm
40 μm
40 μm
PL intensity (abr. u.)
d
e
f
10.4 μm
1800 2000 2200 2400 2600
0
35
70
105
140
Average count
PL intensity (arb. u.)
Fig. 4 | Characterization of PQD patterns prepared via direct in situ photo-
lithography. a Fluorescence image, b SEM image, and c cross-section SEM image
(inset image is enlarged view of a single stripe) of blue stripe patterns. d Emission
intensity distribution image, e average emission intensity statistical distribution of
196 circle pixels with a diameter of 20 μm. Error bars represent standard deviation
(the statistical data and errors were presented in Supplementary Table 8). f Radial
emission intensity distribution of a single 20 μm circle pixel, inlet image is the
surface-emission intensity of the pixel. Source data are provided with this paper. Article Article an attenuated total reflection (ATR) accessory, with zinc-selenide dia-
mond coated plate. EPR spectra were measured by Bruker EMXplus-6/
1, no chemicals were needed to detect carbon radicals, while 5,5-
dimethyl-1-pyrroline-N-oxide (DMPO) was used as a radical trapping
agent to detect thiyl radical. Steady-state UV–Vis absorption spectra
were measured by PerkinElmer Lambda1050+, and the texting solu-
tions were loaded in a standard quartz cuvette with an optical path of
1 mm. PLE and PL spectra were measured using an F-380 fluorescence
spectrometer (Tianjin Gangdong Sci. & Tech. Development Co., Ltd.),
the standard quartz cuvette with an optical path of 1 mm loaded with
texting solutions was placed at a 45° angle to the light source. an attenuated total reflection (ATR) accessory, with zinc-selenide dia-
mond coated plate. EPR spectra were measured by Bruker EMXplus-6/
1, no chemicals were needed to detect carbon radicals, while 5,5-
dimethyl-1-pyrroline-N-oxide (DMPO) was used as a radical trapping
agent to detect thiyl radical. Steady-state UV–Vis absorption spectra
were measured by PerkinElmer Lambda1050+, and the texting solu-
tions were loaded in a standard quartz cuvette with an optical path of
1 mm. PLE and PL spectra were measured using an F-380 fluorescence
spectrometer (Tianjin Gangdong Sci. & Tech. Development Co., Ltd.),
the standard quartz cuvette with an optical path of 1 mm loaded with
texting solutions was placed at a 45° angle to the light source. analyzing the content of Pb atoms in the film from ICP-MS and the
immersed solution from ICP-OES, the proportion of 14% was assigned
to the PQDs leaching from the film after 10 h immersion, illustrating
the robustness of the direct in situ photolithography method. Collec-
tively, these results demonstrate that the direct in situ photo-
lithography technology allows for high-resolution, full-color and
patterned applications. In conclusion, by combining direct optical lithography and in situ
fabrication of PQDs, we propose a non-destructive lithography
method that can directly lithograph perovskite precursor solutions
and then grow PQDs in situ. We demonstrate the photocatalytic role of
lead bromide complexes in polymerization, thus enabling fast photo-
lithography without the need for additional initiators or catalysts. Different from the traditional photolithography method, PQDs are
generated after the lithography process, which eliminates the complex
process required to prepare the PQDs in advance, at the same time
effectively avoids the degradation of PQDs by solvents and high-
energy UV light in the lithography process. Reporting summary Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article. Preparation and characterization of PPRs The data generated in this study are provided in the Source Data
file. Source data are provided with this paper. All operations were performed under atmospheric conditions. A green
PPR was prepared by dissolving 0.3 mmol MABr, 0.15 mmol PbBr2,
1 mmol TAIC, and 1 mmol TTMP into 0.8 mL DMF and 0.2 mL DMSO. A
blue PPR was prepared by dissolving 0.4 mmol MACl, 0.2 mmol PbBr2,
0.1 mmol PEABr, 1 mmol TAIC, and 1 mmol TTMP into 0.8 mL DMF and
0.2 mL DMSO. A red PPR was prepared by dissolving 0.09 mmol CsI,
0.21 mmol MAI, 0.05 mmol PbI2, 0.15 mmol PbBr2, 0.12 mmol PEABr
1 mmol TAIC and 1 mmol TTMP into 0.8 mL DMF and 0.2 mL DMSO. All
PPRs need to be stirred for 4 h at room temperature. References Materials All reagents were used as received without further purification: lead
bromide (PbBr2, 99%), lead iodide (PbI2, 98%), cesium bromide (CsBr,
99.9%), cesium iodide (CsI, 99.9%), methylamine hydrobromide
(MABr, 98%), methylamine hydroiodide (MAI, 98%), methylamine
hydrochloride (MACl, 98%), isocyanuric acid tris(2-acryloyloxyethyl)
ester (IATE, >80%) were purchased from Aladdin. 2-Phenylethanamine
bromide, (PEABr, >99.5%) was purchased from Xi’an Polymer Light
Technology Corp. Triallyl isocyanurate (TAIC, 98%), trimethylolpro-
pane trimethacrylate (TMPTMA, 90%), trimethylolpropane tris(3-
mercaptopropionate) (TTMP, 85%), pentaerythritol tetra(3-mercapto-
proionate)
(PTMP,
95%),
vinyltrimethoxysilane
(VTMS,
97%),
3-mercaptopropyltriethoxysilane (MPTS, 98%) were purchased from
Meryer. 1,4-Butanediol bis(thioglycolate) (BBT, 98%) was purchased
from Macklin. N,N-dimethylformamide (DMF, 99.5%), dimethyl sulf-
oxide (DMSO, ≥99.8%), trichloromethane (CHCl3, ≥99%) were pur-
chased from Beijing Tong Guang Fine Chemicals Company Co., Ltd. 5,5-Dimethyl-1pyrroline N-oxide (DMPO, 98%) was purchased from
Energy-Chemical Co., Ltd. Article By controlling the poly-
merization and in situ fabrication conditions, non-destructive efficient
PQD pixel patterns can be obtained, with pixel resolution up to 2450
PPI, thicknesses up to 10 μm, and good stability to light, heat, and
solvents. The direct in situ photolithography technology provides a
useful idea for the preparation of non-destructive and high-efficiency
light-emitting PQD pixels, and offers a simple technical route for the
development of high-resolution patterned optoelectronic applica-
tions, such as Micro-LED, laser, and AR/VR devices. In the future, lead-
free perovskite, including Sn-based, Bi-based, Cu-based and double
perovskite, can be explored for direct in situ photolithography to
avoid the use of lead. It is of great importance to develop scale-up film
coating methods with high utilization of materials, such as blade, spray
coating, and roll-to-roll processes. Patterning and characterization of PQDs All operations were performed under atmospheric conditions. All
substrates were cleaned in an ultrasonic bath using deionized water,
ethanol, acetone, ethanol, isopropanol for 15 min each and were blown
dry with a nitrogen gun. VTMS (MPTS) modifying substrates were
carried out with the vapor-deposition method: a small drop of VTMS
(MPTS) was dropped onto the substrates, subsequently, these sub-
strates were heated to 80 (100) °C for 4 h. After casting 10 μL of PPR on
the center of the substrate, the substrate was then bound between a
black PMMA board and a patterned chrome mask. Then, a 20 W
365 nm handheld-LED was put to expose above the mask for 3–10 s for
green patterns, 5–12 s for blue patterns, and 4–8 min for red patterns. After exposure, the substrate was placed on a spin coater. The uncured
zone was rinsed out by spin-washing with clean chloroform, yielding
micropatterns. The substrate was then annealed at 80 °C for 3 min for
green patterns, room temperature for blue patterns, and 130 °C for
5 min for red patterns to remove residual solvents, and PQDs were
in situ fabricated in the polymer matrix. For colorful patterns on the
same substrate, an intermediate layer of SiO2 was deposited between
each PQD layer via vacuum magnetron sputtering. Methods
i l Fluorescent images were obtained using a Nikon N-SIM A1R
microscope. SEM images were taken on a ZEISS GeminiSEM. The
retention and the leaching rate of the PQD-polymer films were deter-
mined by analyzing the content of Pb atom in the solvents and films
based on the Agilent ICP-OES and ICP-MS 7800, and the testing sam-
ples were digested in an acidic condition and diluted by deionized
water. The PL intensity of the optical images was manipulated using
the camera software or ImageJ. Optical absorption spectra of pat-
terned films were collected using a UV-6100 UV–Vis spectro-
photometer
(Shanghai
Mapada
Instruments
Co.,
Ltd.). Photoluminescence spectra were collected with an F-380 fluorescence
spectrometer (Tianjin Gangdong Sci. & Tech. Development Co., Ltd.). The ultrathin section samples were analyzed using a Tecnai G2 F30
TEM machine operating at an acceleration voltage of 300 kV. The
ultrathin section samples were prepared using Leica EM UC7
ultramicrotome. Demonstration of PQD patterns via direct in situ
photolithography Fluor-
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pixels; d red, green and blue stripe patterns; e red and green double color pat-
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The authors declare no competing interests. Acknowledgements © The Author(s) 2022 The authors would like to acknowledge the Experimental Center of
Advanced Materials of Beijing Institute of Technology for the support in The authors would like to acknowledge the Experimental Center of
Advanced Materials of Beijing Institute of Technology for the support in 10 Nature Communications| (2022) 13:6713
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Bacterial Toxins Fuel Disease Progression in Cutaneous T-Cell Lymphoma
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Toxins
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Toxins 2013, 5, 1402-1421; doi:10.3390/toxins5081402
toxins
ISSN 2072-6651
www.mdpi.com/journal/toxins
Review
Bacterial Toxins Fuel Disease Progression in Cutaneous
T-Cell Lymphoma
Andreas Willerslev-Olsen 1, Thorbjørn Krejsgaard 1, Lise M. Lindahl 2,
Charlotte Menne Bonefeld 1, Mariusz A. Wasik 3, Sergei B. Koralov 4, Carsten Geisler 1,
Mogens Kilian 5, Lars Iversen 2, Anders Woetmann 1 and Niels Odum 1,*
1 Department of International Health, Immunology and Microbiology, University of Copenhagen,
Copenhagen 2200, Denmark; E-Mails: awo@sund.ku.dk (A.W.-O.); thorkr@sund.ku.dk (T.K.);
cmenne@sund.ku.dk (C.M.B.); cge@sund.ku.dk (C.G.); awoetmann@sund.ku.dk (A.W.)
2 Department of Dermatology, Aarhus University Hospital, Aarhus 8000, Denmark;
E-Mails: lise.lindahl@ki.au.dk (L.M.L.); lars.iversen@ki.au.dk (L.I.)
3 Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia,
PA 19104, USA; E-Mail: wasik@mail.med.upenn.edu
4 Department of Pathology, NYU Langone Medical Center, New York, NY 10016, USA;
E-Mail: sergei.koralov@nyumc.org
5 Department of Biomedicine, Aarhus University, Aarhus 8000, Denmark;
E-Mail: kilian@microbiology.au.dk
* Author to whom correspondence should be addressed; E-Mail: ndum@sund.ku.dk;
Tel.: +45-3532-7879.
Received: 4 July 2013; in revised form: 2 August 2013 / Accepted: 6 August 2013 /
Published: 14 August 2013
Abstract: In patients with cutaneous T-cell lymphoma (CTCL) bacterial infections
constitute a major clinical problem caused by compromised skin barrier and a progressive
immunodeficiency. Indeed, the majority of patients with advanced disease die from
infections with bacteria, e.g., Staphylococcus aureus. Bacterial toxins such as
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OPEN ACCESS Toxins 2013, 5, 1402-1421; doi:10.3390/toxins5081402 toxins
ISSN 2072-6651
www.mdpi.com/journal/toxins
OPEN ACCESS toxins
ISSN 2072-6651
www.mdpi.com/journal/toxins
OPEN ACCESS Keywords:
cutaneous
T-cell
lymphoma;
infections;
Staphylococcus
aureus;
enterotoxins; superantigens Keywords:
cutaneous
T-cell
lymphoma;
infections;
Staphylococcus
aureus;
enterotoxins; superantigens 1. Introduction Cutaneous T-cell lymphoma (CTCL) comprises a heterogeneous group of lymphoproliferative
disorders defined by primary expansion of malignant T lymphocytes in the skin. The two most
common forms, mycosis fungoides (MF) and Sézary syndrome (SS), constitute approximately
50%–70% of all de novo cases of CTCL, with MF accounting for the majority of cases. In this review,
CTCL will refer exclusively to mycosis fungoides and Sézary syndrome. Early skin lesions in CTCL
usually present as erythematous patches that notoriously resemble benign inflammatory skin disorders
like psoriasis, chronic eczema or atopic dermatitis—collectively, making an early diagnosis very
difficult [1–6] even though promising new approaches using miRNA expression profiling seem to
discriminate between the benign inflammatory and malignant conditions inflammation with high
accuracy [7,8]. Although patients diagnosed in the early stages of disease often experience an indolent
disease course, a subgroup of patients experience an aggressive clinical course with tumor
development, skin ulceration, involvement of lymph nodes, bone marrow and internal organs and
gradual development of immunodeficiency at later stages of disease. Concomitant with disease
progression there is a decrease in normal lymphocyte count and functionality and, consequently,
advanced disease may be associated with profound immune deregulation [1,2,9,10]. The etiology of
CTCL has long puzzled researchers and a wide range of risk factors has been examined in this regard. Chromosomal instability and abnormal expression of genes involved in cell cycle control and
proliferation has been reported several times in CTCL studies [11–13]. However, in contrast to other
hematological disorders, in CTCL well documented etiological or predisposing genetic factors remain
elusive. Occupational and environmental factors have been proposed in some studies but with limited
reproducibility and a lack of any evident biological causality [14–16]. Yet, a recent finding by Duvic
and colleagues sheds light on a previously suspected link between drugs (thiazide used in the treatment
of hypertension) and CTCL [17] indicating that environmental factors might indeed play a role in a
subset of patients with chemical or biological agents acting as inciting agents in the context of this
T cell lymphoma. Familial aggregation of CTCL incidences has been described [18] and a correlation
between CTCL disease occurrence and certain human leukocyte antigen (HLA) alleles has also been
observed [19]. Andreas Willerslev-Olsen 1, Thorbjørn Krejsgaard 1, Lise M. Lindahl 2,
Charlotte Menne Bonefeld 1, Mariusz A. Wasik 3, Sergei B. Koralov 4, Carsten Geisler 1,
Mogens Kilian 5, Lars Iversen 2, Anders Woetmann 1 and Niels Odum 1,* Andreas Willerslev-Olsen 1, Thorbjørn Krejsgaard 1, Lise M. Lindahl 2,
Charlotte Menne Bonefeld 1, Mariusz A. Wasik 3, Sergei B. Koralov 4, Carsten Geisler 1,
Mogens Kilian 5, Lars Iversen 2, Anders Woetmann 1 and Niels Odum 1,* Andreas Willerslev-Olsen 1, Thorbjørn Krejsgaard 1, Lise M. Lindahl 2,
Charlotte Menne Bonefeld 1, Mariusz A. Wasik 3, Sergei B. Koralov 4, Carsten Geisler 1,
Mogens Kilian 5, Lars Iversen 2, Anders Woetmann 1 and Niels Odum 1,* 1 Department of International Health, Immunology and Microbiology, University of Copenhagen,
Copenhagen 2200, Denmark; E-Mails: awo@sund.ku.dk (A.W.-O.); thorkr@sund.ku.dk (T.K.);
cmenne@sund.ku.dk (C.M.B.); cge@sund.ku.dk (C.G.); awoetmann@sund.ku.dk (A.W.) 1 Department of International Health, Immunology and Microbiology, University of Copenhagen,
Copenhagen 2200, Denmark; E-Mails: awo@sund.ku.dk (A.W.-O.); thorkr@sund.ku.dk (T.K.);
cmenne@sund ku dk (C M B ); cge@sund ku dk (C G ); awoetmann@sund ku dk (A W ) 2 Department of Dermatology, Aarhus University Hospital, Aarhus 8000, Denmark;
E-Mails: lise.lindahl@ki.au.dk (L.M.L.); lars.iversen@ki.au.dk (L.I.) 3 Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia,
PA 19104, USA; E-Mail: wasik@mail.med.upenn.edu 4 Department of Pathology, NYU Langone Medical Center, New York, NY 10016, USA;
E-Mail: sergei.koralov@nyumc.org 5 Department of Biomedicine, Aarhus University, Aarhus 8000, Denmark;
E-Mail: kilian@microbiology.au.dk * Author to whom correspondence should be addressed; E-Mail: ndum@sund.ku.dk;
Tel.: +45-3532-7879. Received: 4 July 2013; in revised form: 2 August 2013 / Accepted: 6 August 2013 /
Published: 14 August 2013 Abstract: In patients with cutaneous T-cell lymphoma (CTCL) bacterial infections
constitute a major clinical problem caused by compromised skin barrier and a progressive
immunodeficiency. Indeed, the majority of patients with advanced disease die from
infections with bacteria, e.g., Staphylococcus aureus. Bacterial toxins such as
staphylococcal enterotoxins (SE) have long been suspected to be involved in the
pathogenesis in CTCL. Here, we review links between bacterial infections and CTCL with
focus on earlier studies addressing a direct role of SE on malignant T cells and recent data
indicating novel indirect mechanisms involving SE- and cytokine-driven cross-talk
between malignant- and non-malignant T cells. 1403 Toxins 2013, 5 Toxins 2013, 5 progressive disease die more frequently from infection rather than from the CTCL per se [21,23]. These important findings prompt the question whether the high incidence of infections in CTCL
patients is a mere consequence of a compromised skin barrier, a suppressed immune system, or a
combination of both. 2. High Prevalence of Infections High incidence of infections is a common clinical experience in CTCL [20–22]. Axelrod et al. examined and quantified different types of infection in 356 CTCL patients [21]. Among the 478
documented infections, 396 were of bacterial origin with the remaining identified as viral, fungal or
parasitic. Their study documented that skin was by far the most prevalent site of infection and that risk
of infection was intimately associated with the disease stage. Thus, these findings supported the
clinical experience that major morbidity and mortality stems from infections and also that patients with 1404 Toxins 2013, 5 In CTCL, the early infiltrating CD4 T cells display a TH1 phenotype and in concert, these immune
cells are seemingly capable of controlling CTCL cell expansion via cytokines and cytotoxicity [25–28]. Accordingly, it has been shown that the presence of cytotoxic CD8 T cells within the CTCL
lesions is a positive prognostic factor, and several case reports have evidenced that use of the
immunosuppressant cyclosporine in treatment of CTCL accelerates disease progression and large cell
transformation [10,29,30]. During the disease progression, the concentration of TH1 cytokines
decreases in contrast to an increased production of TH2 cytokines and angiogenetic and lymphangiogenetic
factors such as VEGF-A and VEGF-C [10,31–35]. This increasing bias towards a TH2 immune
response obstructs an effective cellular immune response and can be framed within the immunoediting
hypothesis as a process in which the malignancy transitions from an equilibrium phase to a tumor
escape phase. Indeed, as the disease progresses, the malignant T cells display an increased expression
of B lymphoid tyrosine kinase (BLK), and cyclooxygenase 2 (COX-2) as well as activation of signal
transducers and activators of transcription-3 (STAT3) and STAT5 [36–39], which in turn drive
expression of TH2 cytokines, oncomiRs (miR-155), and the suppressor of cytokine signaling 3
(SOCS3) [37,40]. The enhanced expression of SOCS3 has been shown to protect malignant T cells
from growth-inhibition by pro-inflammatory cytokines such as interferon-alpha (IFNα) [41]. Because
IFNα is used for treatment of CTCL, the development of IFNα resistance comprises a pressing clinical
problem [41]. Furthermore, direct diversion of anti-tumor immune response been attributed to the malignant
T cells in CTCL. Several studies have demonstrated forkhead box P3 (FOXP3) expression in
malignant T cells in a subset of patients [42–45] and upregulation of cytotoxic T-lymphocyte antigen
4 (CTLA-4) in a stage-dependent manner [46]. Likewise the interaction of programmed death protein 1
(PD-1) and its ligand, PD-L1 has been associated with immune evasion in CTCL [47,48] as these cell
surface molecules are involved in the induction and maintenance of peripheral T cell tolerance. The
increased expression of PD-L1 on neoplastic T cells has been hypothesized to involve the aberrant and
constitutive activation of the janus associated kinase 3 (JAK3)-dependent STAT3 cell signaling
pathway which is also allegedly a key player in sustaining tissue inflammation while antagonizing
tumor immunity [49]. Furthermore, the constitutively active STAT3 induces the secretion of the two
potent immunosuppressants; IL-10 and transforming growth factor-beta (TGFβ) [44,45,50]. 3. Immunopathogenesis CTCL progression is typically associated with immune suppression. The malignant cells
normally exhibit a mature memory CD4 T cell phenotype and express a range of skin-homing
receptors in the initial disease stages, which contribute to the characteristic epidermotropism of
malignant T cells [6,10]. The immunopathogenesis in CTCL is characterized by a gradual shift of cytokine profile in lesional
tissue. Early lesions contain a large proportion of non-malignant cells, which primarily consist of
dendritic cells, macrophages and tumor-infiltrating cytotoxic CD8 and CD4 T cells [6,10,24]. CD4 T
cells may display several different phenotypes depending on their specific activation as illustrated in
Figure 1. While the CD4 T cell helper type 1 (TH1) is crucial in promoting an effective cellular
immune response and as such beneficial in an anti-tumor response, the TH2 phenotype is on the
contrary promoting a humoral immune response. The more recently recognized TH17 cell is believed
to be important in certain microbial infection while the T regulatory phenotype is paramount in
establishing and sustaining peripheral tolerance. Figure 1. Schematic illustration of the antigen presenting cells (APC) antigen presentation
and cytokine release together with the subsequent induction of different lymphocyte helper
subsets. (1) The APC delivers three signals required for successful lymphocyte activation;
antigen presentation, co-stimulation and cytokine release with cytokines being the major
determinant of lymphocyte subset induction; (2) Additionally dendritic cells DC are able to
induce a regulatory phenotype either by the absence of co-stimulation (immature DC’s lack
CD80/86) or by activation of lymphocytes in a regulatory cytokine environment
(tolerogenic DC’s). 1405 Toxins 2013, 5 Collectively,
the expression and secretion of the above mentioned molecules supports the model originally proposed
by Berger and colleagues who suggest that CTCL T cells maintain dendritic cell immaturity by the
release of regulatory cytokines. Further, according to their hypothesis, this results in polarization of the
DC’s towards a tolerogenic phenotype, rather than an activating phenotype. In turn, this subtype of
DC should promote malignant T cell proliferation and the acquisition of immunosuppressive
charactheristics [50,51]. Finally, the immunodeficiency in late stage CTCL could also caused by a gradual displacement of
non-malignant T cells by the expanding malignant T cell clones; in other words, that the malignant
T cells eventually outcompete and substitute the non-malignant T cell population, which results in a
state reminiscent of advanced AIDS with a lack of functional CD4 T helper cells and severe
immunosuppression [1,2,10,52]. Figure 2 shows an illustration of our current view of the dynamic immunological changes during
disease progression. Thus, the interplay between malignant T cells, dendritic cells and infiltrating Toxins 2013, 5 malignant T cells with capacities resembling either regulatory T cells or different T helper subsets. Two members of the IL-17 family of cytokines: IL-17A and IL-17F, have recently been implicated in
CTCL pathogenesis [54–56]. Expression of IL-17A and IL-17F is increased in skin lesions and
comparable to the expression levels in lesional psoriatic skin [55]. Noteworthy, heterogeneity in
IL-17A and IL-17F expression existed among CTCL patients with some patients having normal or
near normal expression whereas others had highly increased levels of IL-17 cytokines. Importantly, an
elevated expression of IL-17F correlated with progressive disease [55]. Given the observations of
increased IL-17 expression in CTCL patients with bacterial infections [54], we propose a link between
bacterial infection, expression of IL-17F and the disease progression. However, it remains to be
determined whether IL-17F and other IL-17 family cytokines are fostering disease progression via
induction of angiogenesis or other as yet unidentified mechanisms or if an increased expression of
these cytokines is a sign of a “frustrated” immune response unable to combat the bacterial infection. Toxins 2013, 5 1406 Toxins 2013, 5 and/or skin-resident, non-malignant T cells change dramatically as the disease progress from an
indolent condition to an aggressive cancer. In early stages and non-progressive disease, dendritic cells
produce interferon-alpha (IFNa), non-malignant T cells produce TH1 cytokines (such as IL-12 and
IFNg), and CD8 cytotoxic T cells produce granzymes and mediate direct killing of malignant T cells. These events generate a hostile environment inhibiting malignant proliferation—yet without
eradicating the malignant T cell clone—i.e., the tumor lesion is kept in a “state of equilibrium” without
expansion and spreading (Figure 2(1)). As malignant T cells change and begin expressing immune
modulatory molecules and cytokines (which inhibit the immune control), a “tumor immunological
privilege” is established. This “immune privilege” shelter malignant T cells from inhibitory signals
allowing for malignant proliferation and induction of immunosuppression and eventually,
immunodeficiency (Figure 2(2)). Figure 2. Schematic illustration of the transition from a state of tumor equilibrium to a
state of tumor immune privilege. The tumor equilibrium state (1) is characterized by T cell-
and cytokine-mediated control of tumor progression. Conversely, the state of tumor
immune privilege (2) is predominated by regulatory signals and cytokines allowing for
immune evasion and tumor progression and metastasis. (Yellow: DC; blue: nonmalignant
T cell; red: malignant T cell). Malignant T cells in CTCL display a considerable degree of phenotypic heterogeneity, which
ongst other things, is believed to impact disease aggressiveness and response to treatment [6,24,53]. ent studies indicate that this heterogeneity is highly dependent upon crosstalk between malignant
ells and the tumor environment, in that malignant T cells have been shown to secrete a wide array
cytokines, which collectively may activate keratinocytes and surrounding stromal cells and thus
ain tissue inflammation. In return, the activated microenvironment impregnates the malleable Malignant T cells in CTCL display a considerable degree of phenotypic heterogeneity, which
amongst other things, is believed to impact disease aggressiveness and response to treatment [6,24,53]. Recent studies indicate that this heterogeneity is highly dependent upon crosstalk between malignant
T cells and the tumor environment, in that malignant T cells have been shown to secrete a wide array
of cytokines, which collectively may activate keratinocytes and surrounding stromal cells and thus
sustain tissue inflammation. In return, the activated microenvironment impregnates the malleable 1407 Toxins 2013, 5 Toxins 2013, 5 1408 chronic expansion of Chlamydia-specific T cells and the combination of SAF and chronic T cell
activation was hypothesized to lead steadily to the development of CTCL [76]. Borrelia burgdorferi
has also been implicated and in 2006 Bonin and colleagues [77] reported on a minor association
between Borrelia burgdorferi and CTCL in a population endemic for Borrelia infection. Later they
suggested that Borrelia in conjunction with HTLV-1 (or other infectious agents) can provide a
persistent antigenic stimulation, which contributes to the transformation and expansion of T
lymphocytes [20]. However, subsequent studies failed to detect a significant presence of
Chlamydia pneumonia and Borrelia burgdorferi in CTCL skin specimens [78,79]. Sporadic case
reports [80–82] describe other infectious agents of various types but generally they may reflect the
findings by Axelrod and colleagues, of a very high degree of diversity in infectious agents present in
CTCL patients [21]. As mentioned above, a multitude of pathogens have been isolated from CTCL
patients and recurrent infections comprise a large clinical challenge in the care of CTCL patients. The
pathogens most frequently associated with CTCL are listed in Tables 1 and 2. Table 1. Prevalence of the most frequent bacterial and viral pathogens associated with
cutaneous T-cell lymphoma (CTCL) disease. Patient cohort included 356 CTCL patients. Modified from Axelrod et al. 1992 [21]. Bacteria
Number of infections
Frequency
Staphylococcus aureus
117
33%–38% *
Enterobacteriaceae
38
10.7%
Beta-hemolytic streptococci
35
9.8%
Pseudomonas aeruginosa
12
3.4%
Viruses
Herpes zoster
34
9.6%
Herpes simplex
30
8.4%
* A general study by Axelrod et al. (1992) [21] reports that 33% of infections in CTCL are Staphylococcus
aureus. Jackow et al. (1997) [83] detects Staphylococcus aureus in 38% of examined CTCL patients. Table 2. Complications associated with infections in CTCL. Table 2. Complications associated with infections in CTCL. Table 2. Complications associated with infections in CTCL. Co-morbidity from infections
Bacterial infections
bacteremia, pneumonia, intra-abdominal infections
Viral infections
ulcerative skin lesions,
dissemination (Kaposi varicelliform eruption) 4. Infectious Etiology It has previously been hypothesized that infectious agents (such as a retrovirus) were responsible
for the outgrowth of neoplastic T cells and as such are a primary etiological factor in CTCL. MacKie
originally launched this hypothesis in 1981 by proposing that CTCL arises from an initial viral
infection of epidermal antigen presenting cells [57]. Mackie was inspired by observations of distinctive
aggregates of epidermal dendritic cells and T cells in MF patients called Pautrier’s abscesses and also
from reports of retrovirus-like particles observed in malignant CTCL T cell cultures [58]. Furthermore,
viral antigens have the potential to induce loss of T cell receptor (TCR) diversity, which is a
characteristic feature of CTCL [59,60]. This may occur when cross-reactivity exists between viral and
auto antigens. According to the hypothesis, autoantigen can sustain proliferation and activation of an
autoreactive T cell population following virus eradication thereby resulting in a narrowing of the TCR
repertoire [61]. The idea of an infectious etiologic agent gained momentum by the earlier discovery of HTLV-1 and
its association with adult T cell lymphoma (ATL) [62]. The distinction between the two diseases was
not initially recognized due to the clinical, pathological, and histological similarities—although ATL
was later established as an unique HTLV-1 induced entity [63]. However, the analogy between
CTCL and ATL and the—at the time newly discovered—T-lymphotropic retrovirus, HIV-1 seemed
conspicuous [64] and motivated researchers to search for a retroviral culprit in CTCL. Concordantly,
retroviral activity and HTLV-like particles in peripheral blood mononuclear cells derived from CTCL
patients [65], and successful polymerase chain reaction (PCR) amplification of HTLV-1 sequences
was also reported in CTCL skin biopsy specimens [66]. However, controversies arose and later,
well-performed, controlled studies failed to associate HTLV-1 with CTCL and the hypothesis of
HTLV-1 as the etiological factor in CTCL was put to rest [67–69]. Other viruses such as Epstein-Barr,
herpes virus 6-8, and cytomegalovirus were later suspected to be involved in CTCL but so far, the
associations have been relatively weak and not (yet) convincingly reproduced [70–74]. In addition, bacterial agents have been assigned a direct role in the etiology of CTCL. One
candidate was Chlamydia pneumonia, which was suggested to foster CTCL through the secretion of a
Sézary T cell activating factor (SAF) [75]. Chronic infection with Chlamydia was believed to facilitate Toxins 2013, 5 malignant CTCL cells responded to SE in a TCR variable β chain (TCRVβ) restricted manner
suggesting a possible involvement in the disease [87]. Later, Duvic and colleagues [83] examined
42 CTCL patients with advanced disease (SS or advanced MF with erythrodermia) for bacterial
colonization in skin and blood and found that 76% of the patients harbored staphylococci, of which
50% were SE-producing strains of S. aureus. Moreover, all patients with toxic shock syndrome toxin-1
(TSST-1)-producing Staphylococcus aureus infections had an expansion of TSST-1 specific T cells
expressing the appropriate Vβ2 T cell receptors [83]. This observation suggests that superantigens such
as SE may be involved in CTCL pathogenesis, as these toxins can facilitate the observed Vβ-restricted
T cell expansion [88]. It was hypothesized that SE provide a persistent antigen stimulus for
T lymphocytes driving malignant T cell expansion. This notion has been propelled by multiple reports
of skewed or diminished T cell receptor repertoire in CTCL patients as discussed below. However, as
these studies examined only patients with advanced disease, they actually do not provide evidence for
a key role of SE in the etiology and early stage of CTCL. 5. Staphylococcus Staphylococcus aureus is a major source of morbidity in CTCL causing chronic or recurrent skin
infections and life-threatening systemic infections such as sepsis, pneumonia and intra-abdominal
infections [21–23,84]. S. aureus is renowned for its ability to produce staphylococcal enterotoxins (SE)
(also known as superantigens) [85,86]. Superantigens are characterized by their ability to activate large
fractions of T lymphocytes by crosslinking MHC class 2 molecules and T-cell receptors (independently of
antigen specificity of the TCR and the antigen-peptide-binding groove in the MHC) thereby
circumventing normal antigen processing and presentation. In 1992, Tokura et al. showed that 1409 6. TCRVβ Restriction By spectratyping the variable regions of the TCR’s β-chain Yawalkar et al. [60] demonstrated that
half of all early-stage patients and all late-stage patients exhibited a highly diminished complexity of
the TCR repertoire compared to the diverse repertoire displayed by normal peripheral T cells [60]. The
shrunken TCR repertoire could not reflect a simple monoclonal expansion, as multiple Vβ-families
were overrepresented while others were underrepresented or completely absent. In short, Vβ-family
distribution failed to follow a normal Gaussian distribution pattern. This skewing of the TCR repertoire
was hypothesized to be the result of superantigens such as SE. Superantigens may skew the TCR Vβ
repertoire by two mechanisms: (1) One involves the previously mentioned direct mechanism by polyclonal activation and
proliferation of Vβ-specific T cells following TCR ligation [83,85,86]. Such Vβ-specific expansion by
superantigens was suggested by Linneman [89], based on an early-stage CTCL patient, who displayed
a dominant Vβ5 T cell population in the skin biopsies. The idea is that superantigen responsive
malignant T cells receive activation-signals and thus obtain a growth advantage allowing them to
out-compete non-transformed cells. (2) The other mechanism involves polyclonal expansion followed by activation-induced cell death
of superantigen reactive T cells, which results in a relative expansion of the remaining Vβ-families and
thus induces a reciprocal superantigen Vβ-signature. This mechanism has been demonstrated by
McCormack and colleagues in a series of mouse studies [90] and subsequently expanded to humans by
Vonderheid and colleagues [91] in a cohort of 49 CTCL patients in which a majority of whom
exhibited increased Vβ5 usage relative to other Vβ families, usually predominant in normal CD4 T
cells. By investigating the TCRα and TCRβ gene rearrangement in 29 CTCL patients Van der Fits [92]
concluded that the absence of an unambiguous similarity in the complementarity-determining regions
argues against the notion of a single ordinary antigen delivering persistent and pathogenic antigen
stimulation in CTCL. However, the skewed Vβ and Jβ gene usage suggested the possibility that also
here superantigens may be responsible for the restricted TCR repertoire. It remains to be definitely
demonstrated by which mechanism superantigens induce polyclonal T cell proliferation in CTCL. 1410 Toxins 2013, 5 However, since Fas receptor expression has been shown to be effectively down-regulated [25,93,94]
and anti-apoptotic pathways such as B-cell lymphoma 2 (Bcl-2) and programmed cell death protein 10
(PDCD10) are enhanced in the CTCL clones [95,96], it is tempting to speculate that malignant T cells
can evade Fas induced apoptosis after superantigen activation whereas non-malignant T cells
expressing the corresponding Vβ TCR families are deleted. Removal by apoptosis of TH1 T cell
subsets producing interferon and other inflammatory cytokines, which keep malignant T cells in check,
might indirectly promote expansion of malignant T cells. Although several studies provide circumstantial evidence of superantigen-induced Vβ
TCR-associated oligo/poly-clonality in CTCL patients, other groups fail to see “Vβ-signatures”
indicative of superantigen involvement. In contrast, these studies observe a monoclonal expansion of
malignant T cells [97–100]. This discrepancy might, amongst other factors, depend in part on the
disease-stage of the examined patients, as studies tend to show increasing monoclonality of T cell
populations with progression [1,2,4,60]. Indeed, it may also simply rely on the inherent heterogeneity
of CTCL; i.e., the disease may in some patients manifest itself as an oligoclonal or skewed polyclonal
expansion of T cells while in others it develops as a monoclonal entity. Collectively the above
mentioned observations fail to clarify whether infections and infectious superantigens such as SE,
function as a primary causative factor—or if they are a secondary event resulting from a weakened
immune system and compromised skin barriers. Therefore, further studies are required in order to
ascertain whether or not superantigens directly facilitate early expansion of pre-malignant T cells in
CTCL, and it is justified to conclude that definitive evidence for an etiological role of superantigens in
CTCL is currently still lacking. Toxins 2013, 5 activation of ZAP70/p72syk, PLCg1, and expression of the IL-2RA subunit [108–113]. Combined,
these findings suggest a novel mechanism of tumor growth promotion by SE involving an indirect
stimulation of malignant cell proliferation involving LFA-3/CD2-mediated cell-cell contact and
soluble growth factors such as IL-2 and other as yet unidentified factors provided by the
toxins-activated non-malignant T cells [104]. Figure 3. Schematic illustration of SE-mediated cross-talk between malignant and
non-malignant T cells. Malignant T cells often display deficient expression and function of
the TCR/CD3 complex and may not respond directly to bacterial superantigens such as
staphylococcal enterotoxins (SE). Instead, malignant T cells often express MHC class II
molecules, which are high-affinity receptors for SE (1). Non-malignant T cells with the
appropriate Vb TCR respond to SE presented by malignant T cells (2, 3) or by antigen
presenting cells (APC) (not shown). SE-mediated cross-talk between malignant and
non-malignant T cells triggers cell-to-cell contact and production of growth factors, which
in turn promote proliferation of malignant T cells (3) [104]. If this mechanism is operating in patients, it indicates that bacterial infections, and especially
infections with superantigen-producing bacteria, indirectly activate malignant T cells through help
from non-malignant T cells. The activation is not restricted by the Vβ-family on the malignant T cells,
but only by their expression of MHC class II molecules or MHC class II expression by other
surrounding cells types and their presentation of superantigens to Vβ-specific non-malignant T cells. The ability of non-malignant T cells to inadvertently promote tumor expansion is not unique to CTCL
and has previously been reported in other malignancies. Specifically, in a squamous cell carcinoma
model, deletion of non-malignant CD4 T cells decreased neoplastic cell progression and tumor
incidence [114] underscoring the intimate relationship between inflammation and cancer. In CTCL, the
indirect mechanism by which bacterial superantigens activate otherwise non-specific or TCR-deficient
malignant T cells predicts that bacterial infections promote expansion of malignant T cells in an
inflammatory setting with non-malignant T cells. In contrast, this model does not imply (but does not
exclude) that bacterial superantigens have an etiological role in CTCL but does suggest a critical role If this mechanism is operating in patients, it indicates that bacterial infections, and especially
infections with superantigen-producing bacteria, indirectly activate malignant T cells through help
from non-malignant T cells. 7. Indirect Mechanism of Action The mechanism of oligoclonal expansion of premalignant T cells, should it occur by superantigen
stimulation, is confounded by the fact that several studies of T cells from CTCL patients (including the
early patch-plaque stage) show that malignant T cells often display deficient function and/or deficient
expression of CD3 TCR complex [101–103]. Recently, our group has proposed a novel role of
bacterial toxins in disease progression [104]. As illustrated in Figure 2, we observed that whereas
malignant T cells did not respond directly to bacterial superantigens, they proliferated vigorously in
response to SE in co-cultures with non-malignant T cells indicating an indirect mechanism for growth
promotion by SE [104]. Noteworthy, malignant T cells often express major histocompatibility complex
(MHC) class II molecules [104], which are high-affinity ligands for bacterial toxins such as SE [105]. Thus, even with defective TCR/CD3 complex, malignant T cells are able to bind SE (Figure 3(2)) and
stimulate non-malignant T cells to produce growth factors such as interleukin-2 (IL-2), which in turn
promotes growth of the malignant T cells (Figure 3(3)). In addition, toxin-induced cell-to-cell contact
between malignant and non-malignant T cells also triggers growth-promoting signals via lymphocyte
function associated antigen 3 (LFA-3)/CD2- dependent mechanism (Figure 3(3)) [104]. Importantly,
both growth factor and cell-to-cell contact dependent growth stimulation of malignant T cells require
MHC class II ligation by the bacterial toxin and expression of a functional TCR/CD3 complex by the
non-malignant T cells [104]. In this regard, it is worth mentioning that MHC class II ligation by SE
enhances cell-to-cell adhesion [106,107] and IL-2-induced T cell proliferation through an increased 1411 Toxins 2013, 5 Toxins 2013, 5 1412 of superantigens in the progression of CTCL. In keeping, it substantiates the general assumption that in
progressive disease, malignant T cells in conjunction with the tumor environment are driven towards
promoting a diverted inflammatory response, which boosts malignant T cell growth, exacerbates
disease and likely increases susceptibility to additional infection. 9. Conclusions In conclusion, bacterial infections are a major clinical problem in CTCL and an important driver of
morbidity and mortality in this disease. Despite much effort, definitive evidence supporting a direct
etiological role of bacterial toxins is still lacking but other evidence suggests that toxins may also
promote malignant T-cell expansion through a mechanism involving cross-talk between the malignant
and non-malignant T cells. Given the proposed model for toxins as drivers of disease progression and
the promising clinical data showing a beneficial effect of antibiotics on both morbidity and disease
progression, we propose that an aggressive strategy for anti-bacterial therapy should be considered in
all patients with clinically relevant and verified infections with S. aureus. 8. Clinical Improvement after Antibiotic Treatment Because many patients are suffering from recurrent bacterial infections, clinicians are often
reluctant to undertake an aggressive treatment of skin infections with antibiotics due to the risk of
increasing resistance to antibiotics. However, important clinical findings lend support to the
hypothesis, that bacterial infections complicate and promote disease progression in CTCL. In several
small series and case studies, elimination of S. aureus infection with antibiotics was associated with a
rapid clinical improvement: in some patients treatment resulted in complete clinical response with no
residual skin involvement by CTCL [83,115,116]. In an early study by Tokura and colleagues, clinical
improvement in skin disease was observed after treating two CTCL patients with antibacterial
agents [116]. In addition, Duvic and co-workers reported that in patients infected with SE-producing
S. aureus, treatment with antibiotics resulted in clear clinical improvement [83]. Another study [115]
reported on a high degree of S. aureus colonization in CTCL patients with an increased incidence in
advanced erythrodermic SS patients when compared to non-erythrodermic MF patients. Eradication of
S. aureus from the nostrils with oral and topical antibiotics was achieved in 85% of cases and similar
treatment of skin lesions was effective in 91%. Consequently, after 4–8 weeks significant clinical
improvement was seen in a majority of the treated patients [115]. Collectively, these observations
speak in favor of an aggressive antibiotic treatment of bacterial infections in CTCL patients. Moreover,
they are in support of the mechanism proposed above that bacterial toxins promote CTCL disease
progression in CTCL. Toxins 2013, 5 The activation is not restricted by the Vβ-family on the malignant T cells,
but only by their expression of MHC class II molecules or MHC class II expression by other
surrounding cells types and their presentation of superantigens to Vβ-specific non-malignant T cells. The ability of non-malignant T cells to inadvertently promote tumor expansion is not unique to CTCL
and has previously been reported in other malignancies. Specifically, in a squamous cell carcinoma
model, deletion of non-malignant CD4 T cells decreased neoplastic cell progression and tumor
incidence [114] underscoring the intimate relationship between inflammation and cancer. In CTCL, the
indirect mechanism by which bacterial superantigens activate otherwise non-specific or TCR-deficient
malignant T cells predicts that bacterial infections promote expansion of malignant T cells in an
inflammatory setting with non-malignant T cells. In contrast, this model does not imply (but does not
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Murinometric measurements and retroperitoneal adipose tissue in young rats exposed to the high-fat diet: Is there correlation?
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Abstract Aim: This study aimed to verify the correlation between murine measurements and retroperitoneal adipose tissue in
rats exposed to the high-fat diet. Material and methods: Wistar male adult rats, descendants of mothers who consumed
a high-fat diet during pregnancy and lactation and fed the same diet after weaning were used. At 60 days of life, body
weight, longitudinal axis and waist circumference (WC) were measured. The Body Mass Index (BMI) and the Lee Index
were calculated for a posterior analysis of the correlation with the amount of retroperitoneal adipose tissue dissected
on the same day. For analysis of the data, the Pearson correlation test was used, considering statistical significance for
p <0.05. Results: Body weight had a weak correlation (r= 0.31; p= 0.38) with retroperitoneal adipose tissue. While
the longitudinal correlated moderately and negative (r= -0.40; p= 0.25). Abdominal circumference (r= 0.62; p= 0.05),
body mass index (r= 0.61; p= 0.03) and Lee (r= 0.69; p= 0.03) correlated moderately and positively with adipose
tissue. Conclusion: Among the measured murine measurements, weight and longitudinal axis were not good indicators
to represent accumulation of retroperitoneal adipose tissue in rats. However, Lee’s index seems to be the best murine
marker to diagnose the accumulation of retroperitoneal fat. BMI, CA and Lee index were murine parameters with
higher correlation. Keywords: high-fat diet, obesity, rats, murinometric index, retroperitoneal adipose tissue. Brazilian Journal of Biology https://doi.org/10.1590/1519-6984.221405
Original Article https://doi.org/10.1590/1519-6984.221405
Original Article https://doi.org/10.1590/1519-6984.221405 A. P. A Macêdoa* , G. S. Cordeirob , L. S. Santosb , D. A. E. Santob , G. S. Perezb ,
R. D. Coutoa,c , M. E. P. C. Machadob,d , J. M. Barreto Medeirosb,d A. P. A Macêdoa* , G. S. Cordeirob , L. S. Santosb , D. A. E. Santob , G. S. Perezb ,
R. D. Coutoa,c , M. E. P. C. Machadob,d , J. M. Barreto Medeirosb,d bPrograma de Pós-Graduação em Alimento, Nutrição e Saúde, Escola de Nutrição, Universidade Federal da Bahia,
Avenida Araújo Pinho, 32, Canela, Código Postal 40110-150, Salvador, BA, Brasil. cDepartamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal da Bahia,
Rua Barão de Jeremoabo, s/n, Ondina, Código Postal 41170-290, Salvador, BA, Brasil. *e-mail: paulamacedo.nut@gmail.com Brazilian Journal of Biology
ISSN 1519-6984 (Print)
ISSN 1678-4375 (Online) Brazilian Journal of Biology
ISSN 1519-6984 (Print)
ISSN 1678-4375 (Online) 2. Material and Methods For analysis of the correlation of the murinometric
indicators with retroperitoneal adipose tissue, we used
the Pearson correlation test, considering the following
classification: weak (0 <r <0.4); moderate (0.4 ≤ r < 0.7)
and strong (r≥ 0.7). Correlations with statistical significance
were considered those with a moderate or strong correlation
degree and p≤0.05 (Siqueira and Tibúrcio, 2011). This study was approved by the Ethics Committee on
Animal Experimentation, Faculty of Veterinary Medicine and
Animal Science, UFBA, according to protocol n° 59/2017. Young rats (Rattus norvegicus), of the Albinus variety,
of the Wistar strain at the Laboratory of Experimental
Nutrition of the Federal University of Bahia (UFBA),
were carried out, in which all animals were kept under
the same conditions, with a temperature of 23 ± 2 °C and
light / dark cycle of 12 hours. 2.2. High-fat diet This diet consisting of a mixture of commercial ration
(Nuvilab® CR1) with roasted peanuts, milk chocolate,
and Maria type biscuit, in a ratio of 3: 2: 2: 1. These
ingredients were milled, mixed and heated in an oven
at 60 °C. Subsequently, they were offered in the form
of pellets, containing the percentage of lipids of 23%
(Oliveira et al., 2011). It is well established in the literature that the ingestion
of hypercaloric and high-fat diets are directly related
to the development of obesity in rodents (Souza et al.,
2018; Lima et al., 2018). Thus, in recent years, health
professionals and researchers in the field have developed
experimental studies to understand this clinical condition
(Mittwede et al., 2016). 1. Introduction used between 90 and 100 days, with a minimum body
weight of 220 g and a maximum of 280 g.i Obesity is considered a public health problem due
to the high prevalence and its comorbidities (Banik and
Rahman, 2018; Luz et al., 2017). The accumulation of fat
in the abdominal region has been reported as a determining
factor for the development of chronic non-transmissible
diseases (Almeida et al., 2016). The onset of gestation was confirmed by the vaginal
smear test (Perez et al., 2015). On the first day of gestation,
it was offered to the fat high diet (n = 4). The females
maintained the consumption of the high-fat diet until the
end of the lactation (21st day of life of the descendants). After weaning, which occurred on the 22nd day of life of
the offspring, the high-fat diet was offered to the offspring
(n = 10) until the sixtieth day of life of the animal. The increasing number of obese individuals is associated
with excessive consumption of high-calorie and high-fat
diets, especially saturated fats (Nasreddine et al., 2018). Animal scientific research has sought to reproduce the
human dietary habit using high-fat diets, inducing the
accumulation of body fat, to similarly reflect the metabolic
alterations found in human obesity (West and York, 1998;
Casper et al., 2008). 3. Results The descriptive analysis of the murine parameters and
the amount of retroperitoneal adipose tissue of the high-fat
offspring on the sixtieth day of life is shown in Table 1. The body weight presented a weak correlation (r = 0.31,
p=0.38), while the longitudinal correlated moderately 2.3. Data collect On the sixtieth day of life of the offspring, measurements
were measured as: weight, longitudinal axis, abdominal
circumference and weight of retroperitoneal adipose tissue. And then calculated the BMI, the Lee Index. In experimental studies with rodents exposed to the
high-fat diet the murinometric parameters such as body
weight, longitudinal axis, and circumference of the abdomen
(CA) have been used extensively; and Lee and body mass
indexes (BMI) to identify adiposity and diagnose obesity
(Licholai et al., 2018; Mcnay and Speakman, 2013). However, it is known that the measurement of body fat
is the most sensitive indicator to diagnose obesity (Hariri
and Thibault, 2010). The animals’ body weight was recorded using a digital
electronic scale -Mart, model S-4000, with a capacity of
4 kg and a sensitivity of 0.001g. Body length was assessed
by measuring the longitudinal axis of the naso-anal. With
the aid of a pen, the points between the muzzle and the base
of the tail were marked, and the distance between them was
evaluated by a digital caliper of 0.01 (BTS digital caliper)
accuracy. The abdominal circumference was determined
by the circumference of the midpoint between the anterior
and posterior legs, a method used by Pini et al. (2016). Despite its accuracy, analyzing adipose tissue is
a difficult and costly method when compared to basic
murine measurements, which makes it usual to measure
these measurements in most of the work in rats. Thus,
it is relevant to evaluate whether there is a correlation
between the murine metric indicators and the amount
of retroperitoneal adipose tissue. Therefore, the purpose
of this study is to verify the correlation between murine
measurements and retroperitoneal adipose tissue in high‑fat
diet exposed rats. BMI was determined by body weight (g) / length2 (cm)
(Novelli et al., 2007) and for the Lee Index, proposed by
Lee in 1928, the formula consists of dividing the cubic
root weight in grams by the naso-anal length in centimeters
(Bernardis and Patterson, 1968). For the collection of retroperitoneal adipose tissue, the
adipose tissue was dissected, sterilized in saline and heavy. Resumo Objetivo: Este estudo teve como objetivo verificar a correlação entre medidas murinométricas e tecido adiposo
retroperitoneal em ratos expostos à dieta hiperlipídica. Material e métodos: Foram utilizados ratos Wistar machos adultos,
descendentes de mães que consumiram dieta hiperlipídica durante a gestação e lactação e alimentados com a mesma
dieta após o desmame. Aos 60 dias de vida, foram medidos o peso corporal, o eixo longitudinal e a circunferência da
cintura (CC). O Índice de Massa Corporal (IMC) e o Índice de Lee foram calculados para posterior análise da correlação
com a quantidade de tecido adiposo retroperitoneal dissecado no mesmo dia. Para análise dos dados, utilizou‑se o teste
de correlação de Pearson, considerando significância estatística para p <0.05. Resultados: O peso corporal apresentou
uma correlação fraca (r= 0,31; p= 0,38) com o tecido adiposo retroperitoneal. Enquanto o longitudinal correlacionou
moderadamente e negativo (r= -0,40; p= 0,25). A circunferência abdominal (r = 0,62; p = 0,05), índice de massa
corporal (r= 0,61; p= 0,03) e Lee (r= 0,69; p= 0,03) correlacionaram-se moderada e positivamente com o tecido
adiposo. Conclusão: Entre as medidas murinométricas, o peso e o eixo longitudinal não foram bons indicadores para
representar o acúmulo de tecido adiposo retroperitoneal em ratos. No entanto, o índice de Lee parece ser o melhor
indicador murinométrico para diagnosticar o acúmulo de gordura retroperitoneal. O IMC, índice de Lee e CA foram
parâmetros murinométricos com maior correlação. lavras-chave: dieta hiperlipídica, obesidade, ratos, índice murinométrico, tecido adiposo retroperitoneal. Braz. J. Biol., 2021 , vol. 81, no. 2 pp.246-250 246 Murinometric measurements and retroperitoneal adipose tissue Braz. J. Biol., 2021 , vol. 81, no. 2 pp.246-250 4. Discussion The studies that investigated, the longitudinal axis,
another murinometric parameter, are few. It is mainly
measured to perform the calculation of BMI and the Lee
index (Sospedra et al., 2015). The results of this analysis
showed a moderate and negative correlation with adipose
tissue, without statistical significance. It may be that the
longitudinal growth of the animal has contributed to a
greater distribution of body fat (Nascimento et al., 2008). In the present study, the high-fat diet-induced accumulation
of retroperitoneal adipose tissue in rats and the correlation
with the commonly used murine measurements was used to
diagnose obesity. Among the measured murine measurements,
BMI, CA and Lee index were murine parameters with a
higher correlation. However, weight and longitudinal axis
were not good indicators to represent the accumulation of
retroperitoneal adipose tissue in rats. The consumption of
foods high in fat and high energy density has increased
worldwide (Kratz et al., 2013; Santos et al., 2019). This
excessive intake causes an increase in body adiposity and
has a strong association with chronic non-transmissible
diseases (Kratz et al., 2013). In the present study, a moderate correlation was found
between the abdominal circumference and adipose tissue,
but no statistical significance. suggesting that this is a good
parameter to evaluate the accumulation of retroperitoneal
fat. Simillary, Angéloco et al. (2012) identified a moderate
and positive correlation for abdominal circumference with
carcass fat in rodents submitted to a high-fat or high-
sucrose diet. Differently, Gerbaix et al. (2010) considered
abdominal circumference as a useful tool after having
correlated strongly and significantly with the amount
of adipose tissue evaluated by radiographic dual-energy
absorptiometry (DEXA) and by dissection of the perirenal
and peri-epididymal tissues. According to the results found in this study, the weak
correlation of body weight with retroperitoneal adipose
tissue, without statistical significance. May be related to
the fact that weight does not evaluate body composition
(Gerbaix et al., 2010). Considering that body weight is
determined by bone, muscle and adipose tissues, it may
be that changes in the amount of bone and muscle tissues
have influenced the results found. Corroborating with
the findings in this study, Lac et al. (2008), rodents fed a
high-fat diet in adulthood showed lower levels of bone
minerals, bone mineral density, and skeletal area. Similarly,
Pomar et al. 2.1. Experimental design The animals were from primiparous females fed with
high-fat during pregnancy and lactation. Wistar rats were 247 Braz. J. Biol., 2021 , vol. 81, no. 2 pp.246-250 Macêdo, A.P.A. et al. Table 1. Murine measurements and amount of retroperitoneal adipose tissue in young rats submitted to the high-fat diet. N
Mean
Standard deviation
Weight (g)
10
199.52
12.54
Longitudinal axis (cm)
10
20.34
0.57
Abdominal Circumference (cm)
10
14.81
0.59
Body Mass Index (g.cm-2)
10
0.48
0.03
Lee Index ( g.cm-1)
10
0.29
0.01
Retroperitoneal adipose tissue (g)
10
4.50
0.95 Table 2. Correlation coefficient between the murinometric indicators and the amount of retroperitoneal adipose tissue in
young rats submitted to the high-fat diet. r
p-value
Weight (g)
0.31
0.38
Longitudinal axis (cm)
-0.40
0.25
Abdominal Circumference (cm)
0.62
0.05
Body Mass Index (g.cm-2)
0.61
0.06
Lee Index ( g.cm-1)
0.69*
0.03
* Statistically significant correlation, p <0.05. and negative (r = -0.40, p=0.25) with the amount of
retroperitoneal adipose tissue. Abdominal circumference
(r = 0.62, p=0.05), body mass index (r = 0.61, p=0.03)
and Lee (r = 0.69, p=0.03) correlated moderately and
positively with adipose tissue. Only Lee’s index showed
a statistically significant correlation (Table 2). and negative (r = -0.40, p=0.25) with the amount of
retroperitoneal adipose tissue. Abdominal circumference
(r = 0.62, p=0.05), body mass index (r = 0.61, p=0.03)
and Lee (r = 0.69, p=0.03) correlated moderately and
positively with adipose tissue. Only Lee’s index showed
a statistically significant correlation (Table 2). Obesity causes chronic inflammation that can affect
the integrity and function of tissues. In muscle tissue, there
is a greater deposition of adipose tissue. This promotes
specific changes in the metabolism of muscle proteins,
impairing protein synthesis in the muscle (Tardif et al.,
2014; Collins et al., 2016). This mechanism justifies the
possible loss of lean mass in the animals studied, and,
consequently, the loss of weight. Braz. J. Biol., 2021 , vol. 81, no. 2 pp.246-250 Murinometric measurements and retroperitoneal adipose tissue Current Obesity Reports, vol. 7, no. 4, pp. 1-7. http://dx.doi. org/10.1007/s13679-018-0323-x. PMid:30349968. Current Obesity Reports, vol. 7, no. 4, pp. 1-7. http://dx.doi. org/10.1007/s13679-018-0323-x. PMid:30349968. a strong and positive correlation. However, in the work of
Novelli et al. (2007), the result of the classification obtained
in his study was a very strong correlation. This difference
in the intensity of classification can be attributed to the fact
that the researchers used a different classification for the
correlation values and because they evaluated the carcass
fat. And BMI accurately comprises calculating body mass,
not retroperitoneal mass. BERNARDIS, L. L. and PATTERSON B. D. 1968, Correlation
between ‘Lee index’ and carcass fat content in weanling and adult
female rats with hypothalamic lesions. Journal of Endocrinology,
vol. 40, no. 4, pp. 527-528. https://doi.org/10.1677/joe.0.0400527 CASPER, R.C., SULLIVAN, E.L. and TECOTT, L., 2008. Relevance of animal models to human eating disorders and obesity. Psychopharmacology, vol. 199, no. 3, pp. 313-329. http://dx.doi. org/10.1007/s00213-008-1102-2. PMid:18317734. Contrary to body mass index, the Lee index was
developed specifically for rats to evaluate obesity in these
animals (Bernardis and Patterson, 1968). As the Lee index is
a specific indicator for rats, this specificity may have resulted
in a moderate correlation with statistically significant found
with the amount of adipose tissue. Angelóco et al. (2012)
showed a positive and strong correlation between the Lee
index and carcass fat, suggesting that the Lee index is a
good indicator to evaluate body fatness. The difference in
the intensity of the classification, as well as the BMI, is
because Angelóco et al. (2012) analyzed the total fat of the
carcass of the animal. COLLINS, K.H., HART, D.A., REIMER, R.A., SEERATTAN,
R.A., WATERS‐BANKER, C., SIBOLE, S.C. and HERZOG, W.,
2016. High‐fat high‐sucrose diet leads to dynamic structural and
inflammatory alterations in the rat vastus lateralis muscle. Journal
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A.S. and MOTA, M.R., 2018. Association between adiposity
indices and blood pressure is stronger in sarcopenic obese women. Current Hypertension Reviews, vol. 14, no. 1. http://dx.doi.org/1
0.2174/1573402114666181031145341. PMid:30381084. DUTRA, M.T., MARTINS, K.G., VIEIRA, D.D.R., OLIVEIRA,
A.S. and MOTA, M.R., 2018. Association between adiposity
indices and blood pressure is stronger in sarcopenic obese women. Current Hypertension Reviews, vol. 14, no. 1. http://dx.doi.org/1
0.2174/1573402114666181031145341. PMid:30381084. GERBAIX, M., METZ, L., RINGOT, E. and COURTEIX, D.,
2010. Murinometric measurements and retroperitoneal adipose tissue Visceral fat mass determination in rodent: validation of
dual-energy X-ray absorptiometry and anthropometric techniques
in fat and lean rats. Lipids in Health and Disease, vol. 9, no. 1, pp. 140. http://dx.doi.org/10.1186/1476-511X-9-140. PMid:21143884. 5. Conclusion The high-fat diet was able to promote the accumulation
of retroperitoneal fat in animals exposed to the diet until
adult life. Considering the results of the present study, it can
be concluded that the weight and the longitudinal axis did
not present a good correlation with retroperitoneal adipose
tissue in young rats, but the BMI, CA and the Lee index
were murine parameters with higher correlation. Lee’s
index seems to be the best murine marker to diagnose
retroperitoneal fat accumulation. Therefore, scientific
research using rodents to simulate human obesity should
investigate the murine measurements more carefully and
associate these with other indicators to ensure the reliability
of the results reported. HARIRI, N. and THIBAULT, L., 2010. High-fat diet-induced
obesity in animal models. Nutrition Research Reviews, vol. 23, no. 2, pp. 270-299. http://dx.doi.org/10.1017/S0954422410000168. PMid:20977819. KRATZ, M., BAARS, T. and GUYENET, S., 2013. The relationship
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and metabolic disease. European Journal of Nutrition, vol. 52,
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evidenced, with no statistical significance. It is important
to consider, during the BMI analysis, that this indicator
was formulated to assess the nutritional status of humans
(Dutra et al. 2018). With this in mind, great care must be
taken in adapting this parameter to animals. In agreement
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080/1028415X.2016.1149294. PMid:28277186. WEST, D. B. and YORK, B., 1998. Dietary fat, genetic predisposition,
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Injeksi Kortikosteroid Intratimpani Sebagai Salvage Therapy pada Pasien Tuli Mendadak
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Laporan Kasus
Injeksi Kortikosteroid Intratimpani Sebagai Salvage Therapy
pada Pasien Tuli Mendadak
Ricko Mariza Putra1, Jacky Munilson2, Yan Edward2, Nirza Warto2, Rossy Rosalinda2
Abstrak
Pendahuluan : Tuli mendadak merupakan salah satu kegawatdaruratan di bagian telinga hidung tenggorok
bedah kepala dan leher (THT-KL). Tuli mendadak atau sudden sensorineural hearing loss (SSNHL) adalah tuli
sensorineural lebih dari 30 dB pada 3 frekuensi berturut turut secara mendadak dalam waktu 3 hari. Etiologi tuli
mendadak hingga saat ini belum diketahui dengan pasti, namun penyebab tersering tuli mendadak, yaitu idiopatik
(71%). Penatalaksanaan tuli mendadak meliputi terapi konservatif, salah satunya dengan pemberian kortikosteroid
secara sistemik dan lokal. Pemberian lokal dapat dilakukan dengan cara injeksi langsung intratimpani. Terapi
kortikosteroid secara lokal dapat diberikan sebagai terapi primer, terapi adjuvan (kombinasi) dan salvage therapy.
Laporan kasus : seorang pasien perempuan berusia 36 tahun dengan diagnosis tuli mendadak pada telinga kanan
yang dilakukan salvage therapy dengan penyuntikan deksametason intratimpani sebanyak empat kali secara selang
3 hari setelah gagal dengan terapi sistemik. Kesimpulan : Injeksi kortikosteroid intratimpani digunakan sebagai salvage
therapy dapat menjadi pilihan untuk pasien yang gagal diterapi dengan kortikosteroid sistemik.
Kata kunci: tuli mendadak; salvage therapy; deksametason; intratimpani
Abstract
Introduction: Sudden deafness is one of the emergency in otorhinolaryngology head and neck surgery (ORLHNS). Sudden deafness or sudden sensorineural hearing loss (SSNHL) is sensorineural deafness of more than 30 dB
at 3 frequencies within 3 days. The etiology of sudden deafness is recently remain unknown, but the most common
cause of deafness is idiopathic (71%). Management of sudden deafness includes conservative therapy, one of which is
corticosteroids systemically and locally. Local delivery is done by intratympanic injection. Local corticosteroid therapy
can be administered as primary therapy, adjuvant therapy (combination) or salvage therapy. Case report : a female
patient aged 36 years old with diagnosis of sudden deafness at right ear which is performed salvage therapy with
dexamethasone intratympanic injection four times with interval of 3 days after systemic therapy was failed. Conclusion
: corticosteroid intratympanic injection as salvage therapy may be an option for patients who failed therapy with
systemic corticosteroids.
Keywords: sudden deafness, salvage therapy, dexamethasone, intratympanic
Affiliasi penulis : 1Bagian THT-KL Fakultas Kedokteran Universitas
Andalas, 2Bagian THT-KL Fakultas Kedokteran Universitas Andalas
Korespondensi : Ricko Mariza Putra, dr.rickomp@yahoo.co.id Telp:
08111891391
PENDAHULUAN
Tuli mendadak merupakan salah satu
kegawatdaruratan yang memerlukan penanganan
segera, efektif dan tepat. Walaupun pada beberapa
kepustakaan menyatakan bahwa tuli mendadak dapat
mengalami pemulihan secara spontan,
sekitar
32-70 %.1 Etiologi tuli mendadak hingga saat ini belum
diketahui dengan pasti, namun terdapat beberapa
kemungkinan penyebab tuli mendadak, yaitu idiopatik
(71%), penyakit infeksi (12,8%), penyakit telinga
(4,7%) seperti penyakit Meniere, otosklerosis, penyakit
autoimun telinga bagian dalam, operasi telinga atau
dasar otak, trauma (4,2%), vaskular dan hematologik
(2,8%), neoplasma (2,3%), serta penyebab lainnya
(2,2%).2,3 Derajat ketulian dapat bervariasi mulai dari
ringan, sedang, sedang berat, berat dan sangat berat.
Telinga yang terkena biasanya unilateral, hanya 1-5%
kasus bilateral.4 Penatalaksanaan tuli mendadak
meliputi terapi konservatif dengan beberapa modalitas.
Penanganan harus dilakukan sedini mungkin karena
penanganan yang terlambat akan menyebabkan tuli
yang permanen. Pemberian kortikostreroid dapat
dilakukan secara sistemik dan lokal. Kortikosteroid
lokal dapat diberikan sebagai terapi primer, adjuvan
terapi dan salvage therapy.5,6
Anatomi vaskularisasi telinga dalam
Vaskularisasi telinga dalam berasal dari a.
labirintin yang merupakan cabang dari a. cerebelaris
anteroinferior. Arteri ini masuk ke meatus akustikus
internus dan bercabang menjadi a. vestibularis
anterior dan a. koklearis communis yang kemudian
akan bercabang menjadi a. kohlearis dan a.
vestibulokohlearis.
Arteri
vestibularis
anterior
memperdarahi n. vestibularis, urtikulus dan sebagian
duktus semisirkularis. Sedangkan a. vestibulokoklearis
sampai di mediolus daerah putaran basal koklea
bercabang menjadi cabang terminal vestibularis dan
cabang kohlear.7
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Cabang vestibular memperdarahi sakulus,
sebagian besar kanalis semisirkularis dan ujung basal
kohlea. Cabang kohlear memperdarahi ganglion
spiralis, lamina spiralis ossea, limbus dan ligamen
spiralis. a. koklearis berjalan mengitari n. akustikus di
kanalis akustikus internus dan di dalam kohlea
mengitari modiolus7 (Gambar 1).
Gambar 1. Vaskularisasi telinga dalam
Definisi dan Epidemiologi
Tuli mendadak atau sudden deafness atau
sudden sensorineural hearing loss (SSNHL) adalah
hilangnya pendengaran sensorineural yang lebih dari
30 dB pada 3 frekuensi berturut turut secara
mendadak dalam waktu 3 hari, sering unilateral dan
idiopatik. Insiden kasus tuli mendadak berkisar 5-20
orang tiap 100.000 orang pertahun.1,4
Penderita tuli mendadak mencapai 1% dari
seluruh penderita ketulian di seluruh dunia, dengan
15.000 kasus baru setiap tahunnya.8 Distribusi
kejadian antara laki-laki dan perempuan hampir sama,
dengan puncak usia 40-54 tahun.4 Sedangkan
menurut Fedriani puncak usia 50-60 tahun.8 Telinga
yang terkena sebagian besar satu telinga (unilateral),
dan hanya 1-5 % kasus yang terjadi pada kedua
telinga (bilateral).4
Etiopatogenesis
Penyebab tuli mendadak hingga saat ini
belum diketahui dengan pasti, namun banyak teori
yang mengemukakan faktor resiko penyebab
terjadinya tuli mendadak. Sekitar 10 – 15 % penyebab
tuli mendadak yang dapat teridentifikasi.4,8 Empat teori
utama yang dapat menjelaskan terjadinya tuli
mendadak diantaranya infeksi virus, gangguan
pembuluh darah, ruptur membran intra koklea dan
penyakit autoimun telinga dalam.4
Diagnosis
Diagnosis
ditegakkan
berdasarkan
anamnesis, pemeriksaan fisik, audiometri dan
pemeriksaan
penunjang
lainnya.
Anamnesis
didapatkan hingga 80% datang dengan keluhan
telinga penuh. Tinitus terjadi pada sekitar 80% pasien
dan vertigo 30%. Pemeriksaan fisik umum, seperti
tekanan darah, nadi, nafas, suhu serta pemeriksaan
lokalis THT-KL. Pasien dengan tuli sensorineural
hampir selalu memiliki pemeriksaan otoskopi yang
normal.9,10 Pemeriksan pendengaran dengan penala
didapatkan Rinne positif, Weber lateralisasi ke telinga
yang sehat, Schwabach memendek. Pada audiometri
nada murni didapatkan tuli sensorineural ringan
hingga sangat berat.9-11 Tuli mendadak juga dapat
dievaluasi dengan speech reception threshold, speech
discrimination score, refleks stapedius, timpanogram
dan tone decay.11 Pemeriksaan penunjang lainnya
dapat juga diperiksa seperti pada (Tabel 1)
Tabel 1. Pemeriksaan laboratorium dan pencitraan4
Test
Tujuan
Darah lengkap
Polisitemia, leukemia,
dengan
trombositosis
differensial
Tingkat
Skrining penyakit auto immun
sedimentasi, LED atau inflamasi, diikuti dengan test
ANA atau 68 kD Ab
Tes koagulasi
Koagulopati (jika ada indikasi,
atau riwayat)
Tes fungsi tiroid
Hypothyroidism (jika ada indikasi,
atau riwayat)
FTA-Abs atau
Tes antibodi, Treponema
MHA-TP
pallidum, kongenital atau didapat
MRI
Lesi Retrocochlear, multiple
sclerosis, perdarahan intra koklea
Tomografi
kelainan kapsul otic pada pasien
komputer
pediatrik
68 KD Ab, 68 kiloDalton antibodi; ANA. antibodi
antinuclear; FTA-Abs,Fluorescent treponemal anibodyabsoption test ; MHA-TP. microhemagglutinationTreponema pallidum
Penatalaksanaan
Pengobatan
tuli
mendadak
idealnya
didasarkan pada penyebabnya tetapi umumnya
bersifat idiopatik, sehingga pengobatan dilakukan
secara empiris. Terapi tuli mendadak umumnya
berupa steroid sistemik, vasodilator (histamin,
papaverin, verapamil, carbogen), hemodilusi (dextran,
pentoxifylline, manitol, dan heparin), atau antiviral
(acyclovir, valacyclovir). Terapi lainnya dapat berupa
oksigen hiperbarik 100% 2 hingga 2,5 ATA selama
90 menit setiap hari selama 10-20 kali terapi. Selain
itu, pasien dianjurkan tirah baring selama 2 minggu,
diet rendah garam dan rendah kolesterol, penggunaan
neurotonik, vitamin C, vitamin E, dan preparat herbal
(gingko biloba).8,9
Pemberian kortikosteroid pada kasus tuli
mendadak bervariasi dari segi jenis, dosis maupun
teknik pemberian (Tabel 2) :
a. Terapi kortikosteroid sistemik
Kortikosteroid sistemik yang paling sering
digunakan antara lain prednison, metilprednisolon dan
deksametason. Prednison diberikan dengan dosis 1
mg/kg berat badan/hari atau maksimal 60 mg/hari
tapering off selama 12-15 hari. Metilprednisolon
diberikan dengan dosis 48 mg tapering off.
Deksametason diberikan dengan dosis 10 mg/hari
tapering off. Efek samping yang ditimbulkan
penggunaan kortikosteroid antara lain insomnia,
dizziness, gastritis, kenaikan berat badan, berkeringat,
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perubahan mood, hiperglikemia dan fotosensitif. Efek
samping yang cukup berat, tetapi jarang ditemukan
yakni pankreatitis, perdarahan, hipertensi, katarak,
miopati, infeksi oportunistik, osteoporosis, dan
osteonekrosis.2 Pasien dengan kondisi medis sistemik,
seperti insulin-dependent diabetes mellitus (IDDM),
diabetes tidak terkontrol, hipertensi labil, tuberkulosis,
dan
ulkus
peptikum
tidak
diajurkan
terapi
kortikosteroid sistemik.2
b. Terapi kortikosteroid intratimpani
Injeksi kortikosteroid intratimpani pertama kali
dilaporkan penggunaannya oleh Itoh (1991) untuk
pengobatan penyakit telinga bagian dalam pada
pasien dengan Penyakit Meniere. Pada kasus tuli
mendadak terapi kortikosteroid intratimpani pertama
kali dilaporkan oleh Silverstein (1996). Meskipun
efektifitasnya belum terbukti secara definitif, terapi
kortikosteroid intratimpani untuk tuli sensorineural
menjadi lebih banyak digunakan.5,6
Protokol pengobatan tuli mendadak dengan
kortikosteroid intratimpani terdiri dari:5,6,8,9
•
Terapi Primer; terapi intratimpani sebagai
pengobatan pertama untuk tuli mendadak, tanpa
kortikosteroid sistemik
•
Terapi Adjuvan; terapi diberikan bersamaan
dengan sistemik kortikosteroid
•
Salvage therapy: terapi dimulai setelah terapi
kortikosteroid sistemik gagal.
Pemberian kortikosteroid intratimpani sebagai
terapi primer biasanya pada kelompok pasien yang
tidak bisa mentolerir efek samping sistemik dosis
tinggi dari terapi kortikosteroid sistemik, seperti pasien
diabetes atau tekanan darah tinggi yang sulit untuk
dikontrol. Sebuah studi mengenai terapi kombinasi
kortikosteroid sistemik dosis tinggi dan kortikosteroid
intratimpani menunjukkan hasil perbaikan fungsi
pendengaran secara signifikan. Namun, studi lainnya
tidak
menghasilkan
perbedaan
pemulihan
pendengaran antara terapi kombinasi kortikosteroid
oral dan intratimpani dengan terapi kortikosteroid oral
saja.12 Kebanyakan penggunaan kortikosteroid
intratimpani dilakukan setelah pengobatan tuli
mendadak menggunakan kortikosteroid sistemik
gagal.
Jumlah yang disuntikkan antara 0,3 – 0,5 ml,
sesuai dengan perkiraan volume ruang telinga tengah.
Normalnya pada dewasa volume telinga tengah 0,6 1,5 ml. Periode waktu dan jumlah penyuntikan juga
berbeda-beda yang bervariasi mulai dari 3 hari sampai
7 hari selama 3 sampai 4 kali pemberian (Tabel 2).8,9
Metoda yang dilakukan juga bervariasi : penyuntikan
melalui membran timpani langsung dengan jarum
suntik (Gambar 2a), melalui miringotomi dengan
tabung (grommet) (Gambar 2b), menggunakan pipa
(wick) yang diletakkan pada miringotomi (Gambar 2c),
atau melalui pompa yang ditanamkan untuk
mengantarkan steroid secara konstan (Gambar 2d).13
Tabel 2. Pedoman umum terapi kortikosteroid pda tuli
mendadak
Oral
Intratimpani
Kortikosteroid
Kortikosteroid
Waktu
Segera, ≤ 14
Segera,
pengobatan
hari pertama.
Salvage (rescue)
Keberhasilan
setelah pengobatan
dilaporkan
sistemik gagal
hingga 6
minggu onset
tuli mendadak
Dosis
Prednison
Dexamethasone
1 mg/kg/hr
24 mg/ml atau 16
(maks 60 mg/hr) mg/ml (ditambah),
atau
atau 10 mg/ml
Methylprednisol (stok)
one 48 mg/hr
Methylprednisolone
atau
40 mg/ml atau 30
Deksametason
mg/ml
10 mg/hr
Durasi/
Dosis penuh
Injeksi 0,4-0,8 ml
frekuensi
selama 7-14
ke telinga tengah
hari, dilanjutkan setiap 3-7 hari
dengan
untuk 3-4 sesi
tappering
Teknik
Jangan
Diberikan obat bius
membagi dosis
topikal, lalu
penyuntikkan ke
kuadran posterior
inferior dengan
jarum suntik untuk
mengisi ruang
telinga tengah.
Kepala posisi
otologic (telinga
yang sakit berada
diatas) selama 1530 menit
Pemantauan Audiogram :
Audiogram :
Setelah selesai
Sebelum setiap
pengobatan dan suntikan, setelah
pada delayed
selesai pengobatan
interval
dan pada delayed
interval
Periksa membran
timpani (TM) untuk
memastikan
penyembuhan
setelah selesai
pengobatan dan
pada delayed
interval
Modifikasi
Efek samping
Dapat dipasang
seperti insomnia tabung bertekanan
Memantau
jika direncanakan
hiperglikemia,
beberapa kali
dan hipertensi
suntikan, tetapi
pada pasien
meningkatkan risiko
yang rentan
perforasi membran
timpani
Dapat juga
dipertimbangkan
menambahkan jalur
transportasi jendela
bundar
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Gambar 2. Metoda penyuntikan (a) membran timpani
langsung dengan jarum suntik (b) melalui tabung
(grommet), (c) menggunakan pipa (wick), (d) melalui
pompa yang ditanamkan.
Prognosis
Prognosis dan keberhasilan penatalaksanaan
tuli mendadak dipengaruhi oleh beberapa faktor
diantaranya usia, onset kehilangan pendengaran,
derajat ketulian, jenis audiogram pertama ada tidaknya
vertigo, ada tidaknya tinnitus, respon 2 minggu
pengobatan
pertama,
dan
adanya
faktor
predisposisi.2,4,11
Derajat ketulian menurut ISO diklasifikasikan
sebagai ringan (>25-40), sedang (>40-55), sedang
berat (>55-70), berat (>70-90), sangat berat (>90),
sesuai rata-rata nada murni pada frekuensi 500Hz,
1000Hz, 2000Hz, 4000Hz.10 Semakin berat derajat
ketulian maka prognosis menjadi lebih rendah.14 Tipe
kehilangan pendengaran diklasifikasikan menjadi 4
kelompok yaitu ascending type (frekuensi 250-500Hz),
descending type (frekuensi 4000-8000), flat type
(<20dB perbedaan antara yg terbaik dan terburuk),
total atau sub total (>85dB).14 Ascending type memiliki
prognosis yang lebih baik bila dibandingkan dengan
descending, flat serta total atau sub total. Sedangkan
menurut
Atay
pembagian
tipe
kehilangan
pendengaran terdiri dari upward-sloping type
(gangguan lebih berat pada frekuensi rendah),
downward-sloping type (gangguanlebih berat pada
frekuensi tinggi), flat type (tidak lebih dari 10dB dari
ambang dengar) dan profound type (lebih dari 70dB
pada semua frekuensi).1 Awal mulai pengobatan oleh
Kasaplogu dkk dibagi menjadi 2 kelompok yaitu early
(5 hari pertama), late (hari ke 5-15).14 Pengobatan
dimulai kurang dari lima hari akan memberikan hasil
yang lebih baik. Vertigo dapat digunakan sebagai
indikator tingkat keparahan lesi dan berhubungan
dengan prognosis pengobatan yang rendah. Menurut
Kasaplogu menyimpulkan hasil penelitian Danino
bahwa
adanya
gejala
tinitus
mempengaruhi
reversibilitas hingga 80%. Usia lanjut, hipertensi,
diabetes dan hiperlipidemia berkaitan dengan
disfungsi mikrovaskuler di koklea yang akan
memperburuk prognosis kesembuhan.1,11,14
Namun pasien tuli mendadak yang tidak
diobati dapat juga mengalami pemulihan spontan.
Menurut Jones seperti yang dikutip oleh Conlin
menyatakan tuli mendadak dapat mengalami
pemulihan spontan sekitar 32-70%.15 Menurut Matox
seperti yang dikutip oleh Kasapoglu menyatakan
sekitar 65% kasus tuli mendadak terjadi pemulihan
spontan, sedangkan Battaglia menyatakan sekitar
32-64% tuli mendadak dapat mengalami pemulihan
spontan.14
Evaluasi fungsi pendengaran dilakukan
setiap minggu selama satu bulan. Kriteria perbaikan
pendengaran menurut Kallinen: Sangat baik, apabila
perbaikan lebih dari 30 dB pada 5 frekuensi. Sembuh,
apabila perbaikan ambang pendengaran kurang dari
30 dB pada frekuensi 250 Hz, 500 Hz, 1000Hz, 2000
Hz dan di bawah 25 dB pada frekuensi 4000 Hz. Baik,
apabila rerata perbaikan 10-30 dB pada 5 frekuensi.
Tidak ada perbaikan, apabila terdapat perbaikan
kurang dari 10 dB pada 5 frekuensi.11
Evaluasi menurut Ho seperti yang dikutip
oleh Filipo menyatakan pembagian evaluasi menjadi 4
kelompok yaitu pulih total dengan ambang dengar
kurang dari 25 dB, pulih bermakna dengan
peningkatan ambang dengar lebih dari 30 dB, pulih
minimal dengan peningkatan ambang dengar sekitar
10-30 dB, dan tidak terjadi pemulihan dengan
peningkatan ambang dengar kurang dari 10 dB.16
Sedangkan Siegel seperti yang dikutip oleh Ferri dkk
membagi kriteria perbaikan pendengaran setelah
3 bulan terapi menjadi : sembuh sempurna bila
perbaikan lebih dari 30 dB dan pendengaran lebih baik
dari 25 dB, penyembuhan parsial bila perbaikan lebih
dari 15 dB dan pendengaran antara 25–45 dB,
penyembuhan ringan (slight) bila perbaikan lebih dari
15 dB tetapi pendengaran lebih buruk dari 45 dB, tidak
ada perbaikan bila perbaikan kurang dari 15 dB dan
pendengaran lebih buruk dari 75 dB.17
LAPORAN KASUS
Seorang pasien perempuan usia 36 tahun
datang ke IGD RSUP dr. M. Djamil Padang dengan
keluhan telinga kanan tiba-tiba tidak dapat mendengar
sejak 2 hari yang lalu. Keluhan ini dirasakan saat
pasien bangun tidur. Telinga kanan dirasakan
berdenging kadang bergemuruh sejak kejadian dan
terus menerus. Pasien sudah berobat ke Spesialis
THT-KL di klinik swasta di kota Padang dan telah
dilakukan pemeriksaan audiometri serta disarankan
untuk dirawat. Riwayat kedua telinga berair
sebelumnya tidak ada, nyeri pada kedua telinga tidak
ada, riwayat trauma tidak ada, riwayat terpapar bising
tidak ada. Riwayat demam, batuk dan pilek tidak ada.
Pusing berputar tidak ada. Riwayat sakit kepala hebat
tidak ada. Riwayat diabetes dan tekanan darah tinggi
tidak ada. Riwayat stroke dan penyakit jantung tidak
ada. Pasien bekerja sebagai staf di kantor notaris.
Satu minggu terakhir pasien bekerja lembur.
Pemeriksaan status generalis didapatkan
keadaan umum sedang, kesadaran komposmentis
kooperatif, frekuensi nafas 18 kali permenit, frekuensi
nadi 80 kali permenit, tekanan darah 120/80 mmHg
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dan suhu 36,9° C, Berat badan 65 Kg. Pemeriksaan
telinga kanan dan kiri didapatkan liang telinga lapang,
membran timpani utuh, refleks cahaya positif. Tes
penala didapatkan Rinne positif pada telinga kanan
dan kiri, Weber lateralisasi ke kiri, Schwabach telinga
kanan memendek, telinga kiri sama dengan
pemeriksa. Kesan tuli sensorineural pada telinga
kanan. Pada pemeriksaan hidung dan tenggorok
dalam batas normal.
Tes
keseimbangan
sederhana
dapat
dilakukan. Pemeriksaan audiometri nada murni
didapatkan kesan telinga kanan tuli sensorineural
derajat sangat berat dengan ambang dengar
103,75 dB dan telinga kiri normal dengan ambang
dengar 11,25 dB (Gambar 3).
spesialis penyakit dalam untuk hiperkoagulasi dan dari
bagian penyakit dalam pasien diterapi dengan heparin
2 x 5000 sc.
Hari
ke-4
rawatan,
pasien
masih
mengeluhkan kurang pendengaran, telinga berdenging
belum berkurang, demam tidak ada, pusing berputar
tidak ada, mual muntah tidak ada, nyeri ulu hati tidak
ada. Tanda vital dalam batas normal. Tes penala 512
Hz didapatkan Rinne positif pada telinga kanan dan
telinga kiri, Weber lateralisasi ke kiri, Schwabach
telinga kanan memendek dan kiri normal. Kesan tuli
sensorineural pada telinga kanan. Terapi diteruskan.
Hari
ke-10
rawatan,
pasien
masih
mengeluhkan
kurangnya
pendengaran,
telinga
berdenging berkurang, pusing tidak ada, demam tidak
ada, mual muntah tidak ada, nyeri ulu hati tidak ada,
merah pada wajah tidak ada. Tanda vital dalam batas
normal. Tes penala didapatkan Rinne positif pada
telinga kanan dan telinga kiri, Weber lateralisasi ke
kiri, Schwabach telinga kanan memendek dan kiri
normal. Kesan tuli sensorineural pada telinga kanan.
Pemeriksaan audiometri nada murni didapatkan
telinga kanan tuli sensorineural derajat sangat berat
dengan ambang dengar 97,5 dB (gambar 4).
Gambar 3. Audiogram saat pasien masuk, Tuli
sensorineural derajat sangat berat dengan ambang
dengar 103,75 dB pada telinga kanan
Hasil pemeriksaan laboratorium darah
lengkap didapatkan Hb 12,6 gr/dl, leukosit 9100/mm3,
trombosit 279.000/mm3, hematokrit 39%, PT 8,9’’,
APTT 31,2’’, INR 0,8, gula darah sewaktu 136 mg/dl,
kolesterol total 185 mg/dl, HDL 55 mg/dl, LDL 120
mg/dl, trigliserida 52 mg/dl, ureum 24 mg/dl, kreatinin
0,6 mg/dl, natrium 139 mmol/dl, kalium 3,6 mmol/dl,
klorida 105 mmol/dl, SGOT 11 u/L, SGPT 10 u/L.
Kesan : PT memendek. Pasien didiagnosis dengan tuli
mendadak telinga kanan dengan hiperkoagulasi.
Pasien dirawat dan diberi terapi sesuai protap
tuli mendadak di bagian THT-KL FK Unand RSUP. Dr.
M. Djamil Padang yaitu oksigen intranasal 4 liter
permenit selama 15 menit tiap 6 jam, metilprednison
tablet 16mg peroral tappering off dengan dosis hari IIII dosis 1 x 4 tablet, hari IV-VI dosis 1 x 3 tablet, hari
VII-IX dosis 1 x 2 tablet, hari X-XII dosis 1 x 1 tablet.
Drip pentoksifilin 300 mg dalam RL 500 cc selama 8
jam, injeksi mekobalamin 3 x 500 mcg IV, injeksi
vitamin C 3 x 100 mg IV, gingko biloba 2 x 80 mg
peroral, lanzoprazol 1 x 1 tablet peroral. Pasien
dianjurkan istirahat total. Pasien dikonsulkan ke dokter
Gambar 4. Audiogram hari ke-10 rawatan, Tuli
sensorineural derajat sangat berat dengan ambang
dengar 97,5 dB pada telinga kanan
Hasil audiometri mengambarkan adanya
perbaikan sebesar 6,25 dB. Hasil laboratorium ulang
PT 9,9’, APTT 29,3’, INR 0,93, kesan normal. Pasien
minta rawat jalan karena alasan keluarga. Terapi
metilprednison tapering off diteruskan, mekobalamin 3
x 500 mg peroral, pentoksifilin 3 x 400 mg peroral,
neurobion 1 x 1 tablet peroral, vitamin C 3 X 100 mg
peroral, gingko biloba 2 x 80mg peroral. Terapi
heparin dihentikan. Pasien disarankan tetap istirahat
total dirumah dan dianjurkan kontrol 5 hari lagi.
Pengobatan hari ke-15, pasien kontrol ke poli
THT-KL. Pasien masih merasakan kurangnya
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pendengaran pada telinga kanan, kemudian
dianjurkan untuk menjalani terapi penyuntikan
deksametason intratimpani sebanyak 4 kali 0,3 ml (1,5
mg) selang 3 hari.
Prosedur penyuntikan sebagai berikut:
Pasien tidur telentang dengan kepala diputar sekitar
30o menjauhi telinga yang sakit, telinga dibersihkan,
lalu dilakukan aseptik / antiseptik menggunakan
betadin,
kemudian
dilakukan
anestesi
lokal
menggunakan xylocain spray yang disemprotkan ke
kapas gulung lalu dimasukkan ke liang telinga sampai
ke membran timpani, dibiarkan selama 10 menit lalu
kapas diangkat. 0,3 ml deksametason 5 mg/ml
disuntikkan ke kavum timpani melalui kuadran
posterorinferior membran timpani menggunakan spuit
1 ml dengan jarum no. 23G yang ujungnya
dibengkokkan,
dengan
panduan
mikroskop.
Sebelumnya larutan deksametason dipanaskan sesuai
suhu tubuh (37oC). Setelah penyuntikan, posisi pasien
dipertahankan selama 1 jam. Prosedur penyuntikan
selesai, evaluasi respon pasien.
Pasien kontrol ulang setelah 1 minggu
tindakan dengan membawa hasil audiometri, yaitu tuli
sensorineural derajat berat dengan ambang dengar
77,5 dB (Gambar 5). Pasien merasakan pendengaran
lebih baik dibandingkan sebelum diterapi, telinga
berdengung berkurang, pusing berputar tidak ada,
nyeri pada telinga tidak ada, keluar cairan dari telinga
tidak ada. Tes penala didapatkan Rinne telinga kanan
dan kiri positif, Weber lateralisasi ke kiri, Schwabach
telinga kanan memendek dan kiri sama dengan
pemeriksa. Pasien disarankan kontrol 1 bulan lagi
untuk evaluasi perbaikan pendengaran, tetapi pasien
tidak kontrol dikarenakan masalah pekerjaan. Evaluasi
keluhan pasien melalui telepon mengatakan
pendengaran sudah lebih baik dari kontrol terakhir.
Gambar 5. Audiogram 1 minggu setelah tindakan, tuli
sensorineural derajat berat dengan ambang dengar
77,5 dB pada telinga kanan
PEMBAHASAN
Telah dilaporkan satu kasus pasien
perempuan usia 36 tahun dengan diagnosis tuli
mendadak telinga kanan. Tuli mendadak dapat terjadi
pada semua usia dengan usia rata-rata 50-60 tahun.
Insiden pada laki-laki sama dengan perempuan.
Pada kasus ini pasien mendapat terapi
kortikosteroid oral selama dua minggu kemudian
dilanjutkan dengan terapi penyuntikan deksametason
intratimpani sebanyak empat kali secara selang 3 hari,
dikarenakan tidak terdapat perbaikan atau gagal
terapi. Sebelum diterapi ambang dengar 103,75 dB
setelah terapi kortikonsteroid oral menjadi 97,5 dB.
Pasien ini menjalani salvage therapy, terapi
penyuntikan deksametason intratimpani sebanyak
empat kali, (selang 3 hari) dengan dosis 0,3 ml
deksametason 5 mg/ml. Hal ini sesuai dengan yang
dilakukan Wang pada penelitian di China. Besar dosis
yang digunakan tergantung dari volume telinga tengah
pasien sebesar 0,66 ml. Penelitian Zhou juga
melakukan penyuntikan intratimpani sebanyak 4 kali
secara selang hari tetapi dengan metilprednisolon.
Efek samping penyuntikan yang dirasakan pada
pasien adalah nyeri setelah penyuntikan, pasien
mendapat terapi asam mefenamat 500 mg bila
nyeri.18,19
Penelitian Lim menyimpulkan bahwa antara
terapi oral kortikosteroid, injeksi intra timpani dan
terapi kombinasi memberikan hasil yang relatif sama,
sehingga dapat digunakan sebagai terapi pertama
dalam pengobatan tuli mendadak.20 Penelitian Lee
menyimpulkan bahwa terapi kombinasi antara injeksi
intra timpani dengan kortikosteroid siistemik lebih baik
dari pada kortikosteroid sistemik saja.21 Sedangkan
menurut Dispenza dkk menyarankan salvage therapy
menggunakan injeksi deksametason intratimpani pada
pasien yang mengalami kegagalan terapi pertama
kortikosteroid sistemik.22
Pada pasien ini terdapat perbaikan
pendengaran dibandingkan awal terapi kortikosteroid 4
minggu setelah tindakan sebesar 26,25 dB. Pada awal
terapi 103,75 dB, 2 minggu setelah oral kortikosteroid
tindakan 97,5 dB dan 2 minggu setelah tindakan
terdapat perbaikan pendengaran menjadi 77,5 dB..
Menurut kriteria Siegel pasien ini termasuk perbaikan
ringan.17 Menurut kriteria Kallinen : Baik, apabila rerata
perbaikan 10-30 dB pada 5 frekuensi. Pada pasien ini
masing masing frekuensi terdapat perbaikan 40 dB
(250Hz), 35 dB (500Hz), 35 dB (1000Hz), 20 dB
(2000Hz), dan 25 dB (4000Hz).11 Sedangkan menurut
Ho seperti yang dikutip oleh Filipo; pulih minimal
dengan peningkatan ambang dengar sekitar 10-30
dB.16 Pada pasien ini perbaikan ambang dengar
26,25 dB.
Prognosis tergantung pada usia, onset
kehilangan pendengaran, ada tidaknya vertigo, jenis
audiogram pertama, respon 2 minggu pengobatan
pertama, derajat ketulian, adanya tinnitus dan faktor
predisposisi. Pasien ini berusia 36 tahun, dengan
onset kehilangan pendengaran 2 hari, Tidak ada
Jurnal Kesehatan Andalas. 2018; 7(Supplement 3)
101
http://jurnal.fk.unand.ac.id
vertigo, tetapi terdapat tinitus. Derajat ketulian pasien
ini menurut ISO adalah tuli sangat berat (>90dB), yaitu
103,75dB, dengan tipe pendengaran menurut Atay
adalah profound (>70dB pada masing-masing
frekuensi).1 Mulai pengobatan pasien ini termasuk
early treatment dikarenakan kurang dari 5 hari dari
onset pertama.
Setelah terapi kortikosteroid sistemik dari
ambang dengar 103,75 dB menjadi 97,5 dB, dengan
makna tidak ada perbaikan dikarenakan peningkatan 6
dB. Setelah dilanjutkan dengan penyuntikan
deksametason intra-timpani ambang dengar menjadi
77,5 dB, terdapat peningkatan 20 dB. Menurut Haynes
keberhasilan injeksi intratimpani pada salvage therapy
apabila terdapat peningkatan minimal 20 dB.6 Pada
pasien ini sesudah terapi terdapat peningkatan 20dB,
dapat dikatakan bahwa terapi pada pasien ini berhasil.
8.
9.
10.
11.
SIMPULAN
Pemberian kortikosteroid intratimpani sebagai
salvage therapy bermanfaat sebagai penatalaksanaan
pada pasien tuli mendadak yang setelah pengobatan
oral kortikosteroid gagal. Salvage therapy dikatakan
berhasil apabila terdapat peningkatan minimal 20 dB
setelah terapi
12.
13.
DAFTAR PUSTAKA
1.
2.
3.
4.
5.
6.
7.
Atay G, Kayahan B, Cınar BC, Sarac S,
Sennaroglu L. Prognostic Factors in Sudden
Sensorineural Hearing Loss. Balkan Med J. 2016;
33:87-93.
Novita S, Yuwono N. Diagnosis dan Tata Laksana
Tuli Mendadak. Cermin Dunia Kedokteran. 2013;
40(11):820-6.
Chau JK, Lin JRJ, Atashband S, Irvine RA,
Westerberg BD. Systematic review of the
evidence for the etiology of adult sudden
sensorineural hearing loss. The Laryngoscope.
2010; 120.
Oliver ER, Hashisalri GT. Sudden Sensory
Hearing Loss. In: Johnson JT, Rosen CA, editors.
Bailey's Head & Neck Surgery Otolaryngology.
Otology. 2. 5 ed. Philadelphia: Lippincott Williams
& WJ.lkins; 2014. p.2589-96.
Raymundo IT, Filho JB, Maia A, Pinheiro TG,
Oliveira CA. Intratympanic Methylprednisolone as
Rescue Therapy in Sudden Sensorineural Hearing
Loss. Braz J Otorhinolaryngol. 2010; 76(4):499509.
Haynes DS, O’Malley M, Cohen S, Watford K,
Labadie RF. Intratympanic Dexamethasone for
Sudden Sensorineural Hearing Loss After Failure
of Systemic Therapy. The Laryngoscope. 2006;
117:1-15.
Weber PC, Khariwala S. Anatomy and Physiology
of Hearing. In: Johnson JT, Rosen CA, editors.
Bailey's Head and Neck Surgery Otolaryngology.
Otology 2. 5 ed. Philadelphia: Lippincott Williams
& WJ.lKins; 2014. p.2253-73.
14.
15.
16.
17.
18.
19.
Fedriani J. Steroid Intratimpani untuk Penanganan
Tuli Mendadak. Cermin Dunia Kedokteran. 2015;
42(2):149-51.
Stachler RJ, Chandrasekhar SS, Archer SM,
Rosenfeld RM, Schwartz SR, Barrs DM, et al.
Clinical Practice Guideline: Sudden Hearing Loss.
Otolaryngology–Head and Neck Surgery. 2012;
146(1S):1-35.
Soetirto I, Hendarmin H, Bashiruddin J. Gangguan
Pendengaran (Tuli). In: Soepardi EA, Iskandar N,
Bashiruddin J, Restuti RD, editors. Buku Ajar Ilmu
Kesehatan Telinga Hidung Tenggorok Kepala dan
Leher. 6 ed. Jakarta: Fakultas Kedokteran
Universitas Indonesia; 2007. p.10-22.
Bashiruddin J, Soetirto I. Tuli Mendadak. In:
Soepardi EA, Iskandar N, Bashiruddin J, Restuti
RD, editors. Buku Ajar Ilmu Kesehatan Telinga
Hidung Tenggorok Kepala dan Leher. 6 ed.
Jakarta:
Fakultas
Kedokteran
Universitas
Indonesia; 2007. p.46-8.
Battaglia A, Burchette R, Cueva R. Combination
Therapy (Intratympanic Dexamethasone + HighDose Prednisone Taper) for the Treatment of
Idiopathic Sudden Sensorineural Hearing Loss.
Otol Neurotol. 2008; 29:453-60.
Lalwani AK, McGuire JF. Pharmacologic and
Molecular Therapies of the Cochlear and
Vestibular Labyrinth. In: Flint PW, Haughey BH,
Lund VJ, Niparko JK, Richardson MA, Robbins
KT, et al., editors. Cummings Otolaryngology
Head & Neck Surgery. Otology, Neuro-otology,
and Skull Base Surgery 3. 5 ed. Philadelphia:
Elsevier; 2010. p.2179-202.
Kasapoglu F, Tuzemen G, H›zalan I, Erisen L,
Basut O, Onart S, et al. Prognosis in Sudden
Hearing Loss: Is it the Disease or the Treatment
that Determines the Prognosis? Int Adv Otol.
2009; 5(2):187-94.
Conlin AE, Parnes LS. Treatment of Sudden
Sensorineural Hearing Loss. Arch Otolaryngol
Head Neck Surg. 2007; 133:582-6.
Filipo R, Covelli E, Basalamo G, Attanasio G.
Intratympanic prednisolone therapy for sudden
sensorineural hearing loss: A new protocol. Acta
Oto-Laryngologica. 2010; 130:1209-13.
Ferri E, Frisina A, Fasson AC, Armato E,
GiacomoSpinato, Amadori M. Intratympanic
Steroid Treatment for Idiopathic Sudden
SensorineuralHearing Loss after Failure of
Intravenous Therapy. ISRN Otolaryngology. 2011;
2012:1-6.
Kara E, Cetik F, Tarkan O, Surmelioglu O.
Modified intratympanic treatment for idiopatic
sudden sensorineural hearing loss. Eur Arch
Otorhinolaryngol. 2010; 267:701-7.
Zhou Y, Zheng H, Zhang Q, Campione PA. Early
transtympanic steroid injection in patients with
‘poor prognosis’ idiopathic sensorineural sudden
hearing loss. ORL. 2011; 73:31-7.
Jurnal Kesehatan Andalas. 2018; 7(Supplement 3)
102
http://jurnal.fk.unand.ac.id
20. Lim HJ, Kim YT, Choi SJ, Lee JB, Park HY, Park
K, et al. Efficacy of 3 Different Steroid Treatments
for Sudden Sensorineural Hearing Loss: A
Prospective, Randomized Trial. Otolaryngology–
Head and Neck Surgery. 2012; 148(1):121-7.
21. Lee JB, Choi SJ. Potential Benefits of
Combination Therapy as Primary Treatment for
Sudden
Sensorineural
Hearing
Loss.
Otolaryngology–Head and Neck Surgery. 2016;
154(2):328-34.
22. Dispenza F, Stefano AD, Costantino C, Marchese
D, Riggio F. Sudden Sensorineural Hearing Loss:
Results of intratympanic steroids as salvage
treatment. Am J Otolaryngol Head Neck Med
Surg. 2013; 34:296-300.
Jurnal Kesehatan Andalas. 2018; 7(Supplement 3)
103
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Absence of vaccinia virus detection in a remote region of the Northern Amazon forests, 2005-2015
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Archives of virology
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cc-by
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To cite this version: Galileu Barbosa Costa, Anne Lavergne, Edith Darcissac, Vincent Lacoste, Betania Paiva Drumond, et
al.. Absence of vaccinia virus detection in a remote region of the Northern Amazon forests, 2005-2015. Archives of Virology, 2017, 162 (8), pp.2369 - 2373. 10.1007/s00705-017-3353-x. pasteur-01585618 Galileu Barbosa Costa, Anne Lavergne, Edith Darcissac, Vincent Lacoste,
Betania Paiva Drumond, Jônatas S. Abrahão, Erna Geessien Kroon, Benoît
de Thoisy, Giliane de Souza Trindade Distributed under a Creative Commons Attribution 4.0 International License HAL Id: pasteur-01585618
https://riip.hal.science/pasteur-01585618v1
Submitted on 18 Sep 2017 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
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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 1 Title: Absence of Vaccinia virus detection in a remote region from Northern Amazon forests
1
2005-2015
2
Galileu Barbosa Costa1†, Anne Lavergne2, Edith Darcissac2, Vincent Lacoste2, Betânia Paiva
3
Drumond1, Jônatas Santos Abrahão1, Erna Geessien Kroon1, Benoit de Thoisy2, Giliane de Souza
4
Trindade1†
5
6
1 – Laboratório de Vírus, Departamento de Microbiologia, Instituto de Ciências Biológicas
7
Universidade Federal de Minas Gerais. Av Antônio Carlos, nº 6627, Pampulha, Belo Horizonte
8
Minas Gerais, Brazil. CEP: 31270-901. 9
2 – Laboratoire des Interactions Virus-Hôtes, Institut Pasteur de la Guyane, 23 Avenue Pasteur, BP
10
6010, 97306 Cayenne, French Guiana. 11
†Corresponding authors. E-mail address: galileuk1@gmail.com; gitrindade@yahoo.com.br
12
giliane@icb.ufmg.br; Tel: +553134092747; Fax: +553134436482. Full mailing address: Avenida
13
Presidente Antônio Carlos, 6627 - Pampulha, Belo Horizonte - MG, 31270-901
14
15
16
Abstract
17
Vaccinia virus (VACV) circulates in Brazil and other South America countries, being responsible
18
for a zoonotic disease that usually affects dairy cattle and humans, causing economic losses and
19
impacting animal and human health. Furthermore, VACV was already detected in wild areas from
20
Brazilian Amazon. To better understand the natural history of VACV, we investigated it
21
circulation in wildlife from French Guiana, a remote region in Northern Amazon forests. ELISA
22
and plaque reduction neutralization test were performed to detect anti Orthopoxvirus antibodies
23 1 – Laboratório de Vírus, Departamento de Microbiologia, Instituto de Ciências Biológicas,
7
Universidade Federal de Minas Gerais. Av Antônio Carlos, nº 6627, Pampulha, Belo Horizonte,
8
Minas Gerais, Brazil. CEP: 31270-901. 9 2 – Laboratoire des Interactions Virus-Hôtes, Institut Pasteur de la Guyane, 23 Avenue Pasteur, BP
10
6010, 97306 Cayenne, French Guiana. 11 2 – Laboratoire des Interactions Virus-Hôtes, Institut Pasteur de la Guyane, 23 Avenue Pasteur, BP
10
6010, 97306 Cayenne, French Guiana. HAL Id: pasteur-01585618
https://riip.hal.science/pasteur-01585618v1
Submitted on 18 Sep 2017 11 2 – Laboratoire des Interactions Virus-Hôtes, Institut Pasteur de la Guyane, 23 Avenue Pasteur, BP
10
6010, 97306 Cayenne, French Guiana. 11 †Corresponding authors. E-mail address: galileuk1@gmail.com; gitrindade@yahoo.com.br;
12
giliane@icb.ufmg.br; Tel: +553134092747; Fax: +553134436482. Full mailing address: Avenida
13
Presidente Antônio Carlos, 6627 - Pampulha, Belo Horizonte - MG, 31270-901
14 †Corresponding authors. E-mail address: galileuk1@gmail.com; gitrindade@yahoo.com.br;
12
giliane@icb.ufmg.br; Tel: +553134092747; Fax: +553134436482. Full mailing address: Avenida
13
Presidente Antônio Carlos, 6627 - Pampulha, Belo Horizonte - MG, 31270-901
14 Vaccinia virus (VACV) circulates in Brazil and other South America countries, being responsible
18
for a zoonotic disease that usually affects dairy cattle and humans, causing economic losses and
19
impacting animal and human health. Furthermore, VACV was already detected in wild areas from
20
Brazilian Amazon. To better understand the natural history of VACV, we investigated its
21
circulation in wildlife from French Guiana, a remote region in Northern Amazon forests. ELISA
22
and plaque reduction neutralization test were performed to detect anti-Orthopoxvirus antibodies. 23
Real-time and standard PCR targeting C11R, A56R and A26L were applied to detect VACV DNA
24
in serum, saliva and tissue samples. VACV was not evidenced in any samples tested. These
25 2 findings provide additional information on the VACV epidemiological puzzle. The virus could
26
nevertheless be circulating at low levels that were not detected in areas with no human or cattle
27
presence. 28
29
30
Vaccinia virus (VACV) is the prototype of the Orthopoxvirus (OPV) genus, Poxviridae
31
family, and was used as a live vaccine around the world to eradicate smallpox. VACV circulates in
32
zoonotic cycles involving bovine herd and dairy workers in rural areas from Brazil, causing a
33
disease named Bovine Vaccinia (BV) which accounts for several economical losses and increasing
34
the public health concerns [1]. 35 In the last seventeen years, VACV spread throughout all Brazilian territory [1], though its
36
occurrence varies among different regions. Furthermore, VACV has been detected in countries
37
bordering Brazil, such as Argentina [2], Uruguay [3], and Colombia [4]. Although since 1999,
38
several BV outbreaks affecting mainly dairy cattle and humans have been reported [1], natural
39
VACV circulation had already been described in a rodent of Oryzomys genus captured in the 60´s in
40
the Amazon region [5]. HAL Id: pasteur-01585618
https://riip.hal.science/pasteur-01585618v1
Submitted on 18 Sep 2017 41 During these last years in which reports of BV outbreaks have been observed, VACV
42
circulation has been described in the Amazon region, in mammal species such as bovines, primates,
43
coatis, and also humans [6–12]. Nevertheless, there is little information on a possible VACV
44
sylvatic cycle and on its natural reservoirs since the origin of VACV is still unknown and its natural
45
reservoir is still unidentified [13–16]. Given that VACV has a broad host range and that wild
46
animals might be implicated in its transmission chain, we decided to search for VACV circulation
47
in wildlife animals (rodents, marsupials, and bats) captured in pristine forest areas with the absence
48
or limited anthropogenic impacts of Northern Amazon forests. 49 3 We analyzed a total of 2,365 wild mammals from French Guiana, captured between 2005
50
and 2015 in tropical upland rain forest, Amazon region [17, 18]. Captured sites were located in
51
remote forests, slightly disturbed forests (facing selective logging, hunting pressure, and low
52
fragmentation level), secondary forests and edge between forest and open habitats (Figure 1). Wild
53
mammals included rodents (n = 420), marsupials (n = 606) and Chiroptera (n = 1,339) (Table 1). 54
All samples underwent molecular testing for VACV. To detect VACV DNA we used real-time PCR
55
targeting the C11R and A56R genes, and semi-nested PCR targeting C11R and A26L genes [19]. A
56
molecular approach was performed in serum and tissue samples from rodents and marsupials, and
57
in DNA extracted from saliva and blood clots of bats. The positive controls used on molecular assays
58
were DNA extracted from serum and liver of experimental infected Balb/c mice with Vaccinia virus Western
59
Reserve (VACV-WR) strain and Brazilian Vaccinia (Br-VACV) group 1 and 2 viruses [20]. For those for
60
which we were not able to have an experimental infected animal (marsupials and bats), we provided
61
an experimentally contamination of serum and liver samples as in the protocol previously described
62
by Dutra and co-authors [21]. Briefly, it was added different concentrations of VACV-WR and Br-
63
VACV strains (with a range from 104 pfu/µL to 1 pfu/µL, respectively). The experimentally
64
contaminated samples were tested by PCR, compared to the Balb/c controls, and then used as
65
positive controls for marsupials and bats. HAL Id: pasteur-01585618
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Submitted on 18 Sep 2017 66 50 In addition, serum samples from the rodents and marsupials were screened for anti-OPV IgG
67
by ELISA and neutralizing antibodies by ≥50% plaque reduction neutralization test (PRNT50) [19]. 68
Sera were grouped into pools of 2–5 serum samples from animals belonging to the same species
69
that were from the same collection area. No serum samples from bats were available for serological
70
tests. For serological assays, the positive controls were also those provided by experimental infected
71
mouse with VACV-WR and Br-VACV strains [20]. For the ELISA assay particularly, we used
72
purified recombinant A/G protein peroxidase conjugated as secondary antibody. We run rodent and
73 4 marsupial samples together in the same plate (for ELISA and PRNT), comparing the results with
74
known positive and negative rodents’ serum samples. 75 Most of the rodents captured belong to the species Proechimys guyannensis (46,4%),
76
followed by Proechimys cuvieri (11,2%) and Zygodontomys brevicauda (9,5%). The main
77
marsupials studied were Didelphis marsupialis (34,1%) and Marmosa murina (26,2%). For bat
78
species, most of them were Desmodus rotundus (32,3%), Carollia perspicillata (17,2%) and
79
Pteronotus spp. (14,0%). All animals tested by both serological and molecular techniques were
80
negative for VACV. 81 In this study, we assessed numerous forest mammal species and found no serological or
82
molecular evidence for VACV circulation among them. Increasing efforts have been done regarding
83
VACV ecology in South America, and some studies have highlighted the importance of wild
84
animals and peridomestic rodents as VACV carriers [7, 22, 23, Miranda JB, personal
85
communication/in peer review]. Our negative results contrast with the above-mentioned ones and
86
some hypotheses can be considered in explaining the lack of evidence for VACV circulation in wild
87
animals in French Guiana during the 2005-2015 period. One hypothesis is that wildlife was
88
investigated in a highly preserved Northern Amazon region, where natural geographic barriers of
89
unfavorable forest habitats exist, preventing VACV dissemination from Brazil to French Guiana. 90
This could also be emphasized by the “dilution effect” hypothesis, taking into account that in a
91
highly preserved environment, the maintenance of a highly diverse mammal community may
92
prevent infection of putative more sensitive species. HAL Id: pasteur-01585618
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Submitted on 18 Sep 2017 A great diversity of mammal species was
93
studied, but with only a small sampling of species from which VACV has been detected in Brazil,
94
such as Mus musculus [20], Oryzomys sp [5], and Caluromys philander [Miranda JB, personal
95
communication/in peer review]. Furthermore, the species Didelphis albiventris from which VACV
96
was recently detected in São Paulo State, Southeast region of Brazil [24], is not present in French
97 5 Guiana. Nevertheless, two closely related species Didelphis imperfecta (n = 2) and D. marsupialis
98
(n = 207) were tested. 99 Guiana. Nevertheless, two closely related species Didelphis imperfecta (n = 2) and D. marsupialis
98
(n = 207) were tested. 99 Another explanation for the absence of VACV detection in this study could be the extremely
100
low number of bovines in French Guiana. One hypothesis for the broad circulation of VACV in
101
Brazil is that bovines are acting as VACV amplifiers, excreting high loads of viable particles, being
102
a source of infection for themselves and other animals [25]. VACV can be detected in animals
103
without clinical disease since it has been detected in blood samples and feces in animals without
104
lesions but presenting neutralizing antibodies [25]. Due to the potential role of cattle as a viral
105
amplifier, the presence of bovines could facilitate VACV persistence and dissemination into the
106
environment. 107 Another explanation for the absence of VACV detection in this study could be the extremely
100
low number of bovines in French Guiana. One hypothesis for the broad circulation of VACV in
101
Brazil is that bovines are acting as VACV amplifiers, excreting high loads of viable particles, being
102
a source of infection for themselves and other animals [25]. VACV can be detected in animals
103
without clinical disease since it has been detected in blood samples and feces in animals without
104
lesions but presenting neutralizing antibodies [25]. Due to the potential role of cattle as a viral
105
amplifier, the presence of bovines could facilitate VACV persistence and dissemination into the
106
environment. 107 VACV has recently been identified at the borders of Brazil, in countries such as Argentina
108
[2], Uruguay [3], and Colombia [4]. HAL Id: pasteur-01585618
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Submitted on 18 Sep 2017 Even though the circumstances of VACV detection in South
109
American countries are still uncertain, the cattle trade and translocation has to be considered, as
110
highlighted in Amazon region (Mato Grosso and Rondônia states) [9]. Furthermore, some authors
111
have hypothesized that the seroprevalence of OPV-antibodies in human populations from the
112
Amazon basin (Acre state) could be linked to agriculture and livestock practices, reinforcing the
113
role of cattle on VACV maintenance [8]. 114 VACV has recently been identified at the borders of Brazil, in countries such as Argentina
108
[2], Uruguay [3], and Colombia [4]. Even though the circumstances of VACV detection in South
109
American countries are still uncertain, the cattle trade and translocation has to be considered, as
110
highlighted in Amazon region (Mato Grosso and Rondônia states) [9]. Furthermore, some authors
111
have hypothesized that the seroprevalence of OPV-antibodies in human populations from the
112
Amazon basin (Acre state) could be linked to agriculture and livestock practices, reinforcing the
113
role of cattle on VACV maintenance [8]. 114 Compared to other South American countries where VACV has been detected, a different
115
situation is observed in French Guiana where a lower deforestation rate is observed and where only
116
a few cattle or horses are present [26] (http://www.globalforestwatch.org/) (Figure 1). Nevertheless,
117
our findings do not definitively establish that VACV is not circulating, and also do not exclude the
118
possibility that small mammals can act as its reservoir in nature. However, data presented here
119
strongly suggest the absence of circulation of VACV, or at low levels that were not detected, in
120
remote Amazonian areas with no human impact over a long period of time. The monitoring and
121 6 surveillance of VACV circulation is important to ensure early warning of zoonotic outbreaks for
122
vulnerable human populations. 123
124
125
Legend for figure:
126
An overview of French Guiana with the location of the studied regions, where were implemented
127
several trapping sites. The background of the map highlights the human footprint index expected to
128
summarize the anthropic threats on biodiversity. These threats (accessibility, land use, human
129
density) are summed and allowed producing an index with lower pressures strenght in light and
130
higher pressures darker. For each studied region, the dominant habitat is given. HAL Id: pasteur-01585618
https://riip.hal.science/pasteur-01585618v1
Submitted on 18 Sep 2017 131
132
Acknowledgements
133
We thank colleagues from the Laboratoire des Interactions Virus-Hôtes, Institut Pasteur de la
134
Guyane for their excellent technical support. We also thank Pró-Reitoria de Pesquisa from
135
Universidade Federal de Minas Gerais (PRPq/UFMG), CAPES, FAPEMIG and CNPq. JS Abrahão,
136
EG Kroon, and GS Trindade are researchers from CNPq. 137
138
Funding
139
This study was funded by two European projects, CAROLIA and RESERVOIRS, through ERDF
140
funds and a European Commission "REGPOT-CT-2011-285837-STRonGer" grant within the FP7. 141
This work also benefited from an "Investissements d’Avenir' grant managed by Agence Nationale
142
de la Recherche, France (CEBA, ref. ANR-10-LABX-25-01). 143
144
Conflict of interest
145
Authors declare no conflict of interest. 146 surveillance of VACV circulation is important to ensure early warning of zoonotic outbreaks for
122
vulnerable human populations. 123 Acknowledgements
133 Acknowledgements
133
We thank colleagues from the Laboratoire des Interactions Virus-Hôtes, Institut Pasteur de la
134
Guyane for their excellent technical support. We also thank Pró-Reitoria de Pesquisa from
135
Universidade Federal de Minas Gerais (PRPq/UFMG), CAPES, FAPEMIG and CNPq. JS Abrahão,
136
EG Kroon, and GS Trindade are researchers from CNPq. 137
138
Funding
139
This study was funded by two European projects, CAROLIA and RESERVOIRS, through ERDF
140
funds and a European Commission "REGPOT-CT-2011-285837-STRonGer" grant within the FP7. 141
This work also benefited from an "Investissements d’Avenir' grant managed by Agence Nationale
142
de la Recherche, France (CEBA, ref. ANR-10-LABX-25-01). 143
144
Conflict of interest
145
Authors declare no conflict of interest. 146 We thank colleagues from the Laboratoire des Interactions Virus-Hôtes, Institut Pasteur de la
134
Guyane for their excellent technical support. We also thank Pró-Reitoria de Pesquisa from
135
Universidade Federal de Minas Gerais (PRPq/UFMG), CAPES, FAPEMIG and CNPq. JS Abrahão,
136
EG Kroon, and GS Trindade are researchers from CNPq. 137 7 147 147
Ethical approval
148
Captures of rodents, bats and marsupials do not require ethic committee in French Guiana. 149
150
References
151
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in cattle and humans in São Paulo, Brazil. Arch Virol 158:2433-41. 249
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Vincent AF, Fonseca CR, Megid J (2016) Dogs and Opossums Positive for Vaccinia virus during
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Outbreak Affecting Cattle and Humans, São Paulo State, Brazil. Emerg Infect Dis 22:271-3. 253
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255
ZI, (2013) Detection of Vaccinia virus in Blood and Faeces of Experimentally Infected Cows. 256
Transbound Emerg Dis 60:552-5. 257
258
26. Hansen MC, Potapov PV, Moore R, Hancher M, Turubanova SA, Tyukavina A, Thau D,
259
Stehman SV, Goetz SJ, Loveland TR, Kommareddy A, Egorov A, Chini L, Justice CO, Townshend
260
JR (2013) High-resolution global maps of 21st-century forest cover change. Science 342:850-3. 261 piece in the VACV ecological puzzle: could peridomestic rodents be the link between wildlife and
243
bovine vaccinia outbreaks in Brazil? PLoS One 4:e7428. 244
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https://research.tue.nl/files/197580534/polymers_14_00798_v2.pdf
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English
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NMR Profiling of Reaction and Transport in Thin Layers: A Review
|
Polymers
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follow below link for the End User Agreement: Download date: 24. Oct. 2024 NMR Profiling of Reaction and Transport in Thin Layers
Citation for published version (APA):
Nicasy, R., Huinink, H., Erich, B., & Olaf, A. (2022). NMR Profiling of Reaction and Transport in Thin Layers: A
Review. Polymers, 14(4), Article 798. https://doi.org/10.3390/polym14040798 Citation for published version (APA):
Nicasy, R., Huinink, H., Erich, B., & Olaf, A. (2022). NMR Profiling of Reaction and Transport in Thin Layers: A
Review. Polymers, 14(4), Article 798. https://doi.org/10.3390/polym14040798 polymers polymers polymers
Citation: Nicasy, R.; Huinink, H.;
Erich, B.; Olaf, A. NMR Profiling of
Reaction and Transport in Thin
Layers: A Review. Polymers 2022, 14,
798. https://doi.org/10.3390/
polym14040798 Article Ruben Nicasy 1
, Henk Huinink 1,*
, Bart Erich 1,2
and Adan Olaf 1,2 Ruben Nicasy 1
, Henk Huinink 1,*
, Bart Erich 1,2
and Adan Olaf 1,2 1
Applied Physics Department, Eindhoven University of Technology, P.O. Box 513,
5600 MB Eindhoven, The Netherlands; r.j.k.nicasy@tue.nl (R.N.); s.j.f.erich@tue.nl (B.E.);
o.c.g.adan@tue.nl (A.O.) 2
Organization of Applied Scientific Research, TNO The Netherlands, P.O. Box 49,
2600 AA Delft, The Netherlands 2
Organization of Applied Scientific Research, TNO The Netherlands, P.O. Box 49,
2600 AA D lft Th N th
l
d Abstract: Reaction and transport processes in thin layers of between 10 and 1000 µm are important
factors in determining their performance, stability and degradation. In this review, we discuss the
potential of high-gradient Nuclear Magnetic Resonance (NMR) as a tool to study both reactions and
transport in these layers spatially and temporally resolved. As the NMR resolution depends on
gradient strength, the high spatial resolution required in submillimeter layers can only be achieved
with specially designed high-gradient setups. Three different high-gradient setups exist: STRAFI
(STRay FIeld), GARField (Gradient-At-Right-angles-to-Field) and MOUSE (MObile Universal Surface
Explorer). The aim of this review is to provide a detailed overview of the three techniques and their
ability to visualize reactions and transport processes using physical observable properties such as
hydrogen density, diffusion, T1- and T2-relaxation. Finally, different examples from literature will be
presented to illustrate the wide variety of applications that can be studied and the corresponding
value of the techniques. Keywords: high-resolution nuclear magnetic resonance (NMR); thin layer; transport; reaction;
GARField; STRAFI; MOUSE
Citation: Nicasy, R.; Huinink, H.;
Erich, B.; Olaf, A. NMR Profiling of
Reaction and Transport in Thin
Layers: A Review. Polymers 2022, 14,
798. https://doi.org/10.3390/
polym14040798
Academic Editor: Andrea Mele
Received: 1 November 2021
Accepted: 14 February 2022
Published: 18 February 2022
Citation: Nicasy, R.; Huinink, H.;
Erich, B.; Olaf, A. NMR Profiling of
Reaction and Transport in Thin
Layers: A Review. Polymers 2022, 14,
798. https://doi.org/10.3390/
polym14040798
Academic Editor: Andrea Mele
Received: 1 November 2021
Accepted: 14 February 2022
Published: 18 February 2022 www.tue.nl/taverne Take down policy
If you believe that this document breaches copyright please contact us at:
openaccess@tue.nl
providing details and we will investigate your claim. Download date: 24. Oct. 2024 1. Introduction Thin layers (10–1000 µm) are very important in a broad range of applications [1] that
can be found in building materials [2], energy storage [3,4], photovoltaic devices [5,6],
art [7,8], flexible electronics [9], optics [10] and most importantly in coatings [11–13]. The
reason to incorporate thin layers can be manifold, for example, down-scaling [14], intro-
ducing novel technologies [15], increasing performance and stability. Another important
feature when using thin polymer films is the ability to incorporate a huge variety of func-
tional molecules which allow electrical [16–18], magnetic [19] or even color-like [20] like
features in the thin layer. Performance and stability of these thin layers will be determined
by their final structure and underlying physical processes. In literature, the study of these
processes is mostly categorized in different groups belonging to polymer or non-polymer
thin layers. Here, we also differentiate between polymer and non-polymer films. Within
the polymer films, we identify two main groups of processes, firstly, the ones during film
formation and, secondly, processes that occur when the film is formed. The process of film
formation is a multistage process which in literature can be defined in different ways. In
this review, we categorize two types of film formation, both starting with the evaporation
of the solvent followed by either curing or coalescence. After the film has formed, other
processes such as transport of liquid and chemical reaction take place. In non-polymer
films, such as cements or printing paper, liquid uptake and deformation are the most
important processes. A more fundamental understanding of these processes will lead to
more cost-effective, efficient and stable applications. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. 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/). https://www.mdpi.com/journal/polymers Polymers 2022, 14, 798. https://doi.org/10.3390/polym14040798 Polymers 2022, 14, 798 2 of 32 Film formation [21,22] is crucial in determining the final structure of the film and its
stability. If film formation happens incorrectly, the layer can suffer from non-uniformities,
skin formation [23,24], precipitation [25], chemical differences and other mechanical mal-
functions [26] that all reduce performance. In most cases, applying thin layers starts with a
liquid solution that covers the surface, containing both the film material (polymer, latex)
and a solvent [21]. 1. Introduction In the mid-1900s, volatile organic components were mostly used as
solvent. However, since 1950, concerns about their toxicity [27], flammability and envi-
ronmental effects [28,29] have led to a change towards using water as solvent [30]. When
the layer is applied, film formation happens in a two-stage process where first the solvent
has to evaporate, allowing close contact between the polymers, followed by a type of
mechanism to overcome their mutual repulsion and form a rigid layer. The rate of solvent
evaporation is critical for film formation and depends on multiple factors [31,32]. At first,
the rate of evaporation will depend on vapor pressure, temperature, surface area and air
flow. In a second stage, the release of solvent becomes limited by transport through the thin
layer [31,33]. If the solvent evaporates too quickly, the polymer will not have time to adhere
to the surface or form a continuous film. In contrast, a slow evaporation rate will induce
overwetting in the subsurface. When enough solvent has evaporated, a continuous layer is
formed by curing (cross-linking) or coalescence in the case of latex [34,35]. Both cross-links
and coalescence will be responsible for the strength and characteristics of the initial thin
layers. A wide variety of studies focus on cross-linking [36–38], and coalescence [22] to
gain a better understanding and improve the final structure. g
g
p
Processes after film formation such as liquid transport [39,40] and reactions within
these films are responsible for performance, degradation and instabilities. A wide variety of
degradation processes can be identified, including biodegradation [41], photodegradation
by light [42] and chemical degradation. A more fundamental understanding of the transport
of liquids and chemical reactions within the thin layer will contribute to better performance
and stability of the thin layer. Studies on the penetration and diffusion of liquids and the
corresponding effect on the film morphology are the focus of a large group of studies and
will be discussed in this review [43]. Other types of non-polymer thin layers can be found in the printing industry [44–46]
(printing paper with a thickness of 100 µm). In these printing papers, the penetration of ink
is of great importance as it influences the final print quality [47,48]. The wide variety of
coatings [49], hydrophobicity [50] and basis weight found in these papers will all influence
the printing process and the resulting print quality. 1. Introduction Another type of thin layer can be
found in the human skin [51,52] where layers such as the stratum corneum and viable
epidermis, both around 50 µm thick, can be found. In these skin layers, processes such
as the penetration of skin care products [53] or oils take place. As a final example, in
cement pastes [54,55] used in building applications, strength is based upon the internal
morphology and transport of ions. gy
Experimental techniques able to study film formation, reaction and transport processes
are necessary to gain the insights allowing improvement of performance and stability. Since many processes in thin layers such as diffusion, curing and chemical reactions will
happen with a so-called front, gaining spatial and time-dependent information is a crucial
requirement. Conventional techniques are not able to gain both spatial and time-resolved
information coherently. Techniques such as weight measurements [56], microscopy or
ASA (Automatic Scanning Absorptometer) measurements [57] can only measure global
properties such as mass or liquid uptake in the case of ASA, but will not gain spatial
information. High-spatial-resolution MRI has proven to be a versatile tool for studying
these processes. Nuclear Magnetic Resonance (NMR) is a well-established technique that
started in 1946 where for the first time NMR was used for the detection of magnetic
properties of atoms by Bloch [58,59] and Purcell [60]. The method was later extended
to two different areas, namely, nuclear Magnetic Resonance Imaging (MRI) and NMR
spectroscopy, which led to Nobel Prizes in Medicine [61] (Paul C. Lauterbur and Sir Peter Polymers 2022, 14, 798 3 of 32 3 of 32 Mansfield) and Chemistry [62] (Richard R. Ernst and Kurt Wuthrich). In this review, the
focus lies on the first one (MRI). MRI has made its way into material research, as well as in polymer research [22,63–66]. NMR can be used to measure density profiles, atoms’ mobility, and probe their environment
which enables to study reactions and transport processes in thin layers. Reactions in thin
films, such as cross-linking [67–69], glass transitions, curing and many more chemical
reactions, will all influence the mobility of the atoms, which is visible in the NMR signal. Additionally, transport processes can be studied by measuring time-dependent density
profiles. We will discuss different studies to demonstrate how chemical and physical
information of thin polymer films between 10 and 1000 µm can be extracted using high-
spatial-resolution NMR setups. 1. Introduction Characterizing polymers with NMR has been the focus of many studies. Most research
is conducted with NMR spectroscopy which is already well known and described in many
books [70–73]. Here, the focus lies upon the different high-resolution NMR setups that
are able to extract information with high spatial resolution. Three different high-gradient
NMR setups exist with resolution in the micrometer range that are able to extract physical
and chemical information about thin films: STRAFI (STRay FIeld), GARField (Gradient-At-
Right-angles to Field) and MOUSE (MObile Universal Surface Explorer). This paper will
begin by explaining the basic theory behind NMR and how hydrogen atoms can be used to
extract spacial information. p
In Section 3, the GARField [74], STRAFI [75] and MOUSE [76] setup will be discussed,
where an overview will be given about their similarities and differences. Lastly, in Section 4,
different thin layer studies will be discussed in order to provide a better understanding of
the methods’ possibilities. 2.1. NMR Spatial Encoding The frequency
#»f [MHz] depends on the magnetic field and is given by #»f =
#»ω
2π = γ
2π
# »
B0
(4) (4) where #»ω [rad s−1] is the Larmor frequency.# » where #»ω [rad s−1] is the Larmor frequency.# »
#» By applying a second magnetic field »
B1 perpendicular to the main magnetic field ( »B0),
the hydrogen atoms will precess along the new established magnetic field which allows
manipulation of the hydrogen atoms. The excitation is best when the radio-frequency (RF)
pulse exactly coincides with the Larmor frequency #»ω from Equation (4). In most cases,
this manipulation is used to bring the magnetic moments of the hydrogen atoms in the
xy-plane. In an NMR measurement, the total magnetisation # »
M along this xy-plane can be
measured, which is a sum of all the magnetic moments, # »
M = ∑
i
#»
µi
(5) (5) Measuring this signal is mostly conducted in the form of spin echoes, firstly introduced
by Hahn [80,81] in 1950. After a combination of pulses that will be discusses in Section 2.3,
the signal is recorded at some echo time te[s]. Figure 1. Left: Larmor frequency, middle: conventional NMR setup (Helmholtz coils) with condition
|B|/G ≫1 where the gradient field −→
G and applied magnetic field −→
B0 are shown and approximated
by straight lines because the radii of curvature in this case are negligible. Depicted in red is the
measurement area used in NMR experiments. Right: The same Helmholtz coils as the middle, but
with |B|/G ≪1. In this particular case, the radius of curvature cannot be neglected which can be
seen in the field lines from −→
B0. −→
G is not drawn because it lies in correspondence with the middle
figure, not in one direction, which would make the graph too complicated. Figure 1. Left: Larmor frequency, middle: conventional NMR setup (Helmholtz coils) with condition
|B|/G ≫1 where the gradient field −→
G and applied magnetic field −→
B0 are shown and approximated
by straight lines because the radii of curvature in this case are negligible. Depicted in red is the
measurement area used in NMR experiments. Right: The same Helmholtz coils as the middle, but
with |B|/G ≪1. In this particular case, the radius of curvature cannot be neglected which can be
seen in the field lines from −→
B0. 2.1. NMR Spatial Encoding The existence of a nuclear spin was demonstrated by Bloch and Purcell in 1946 when
they measured for the first time nuclear magnetic resonance (NMR) [58,77]. They received
the Nobel Prize in Physics for this work in 1952. The basics of NMR are often explained
using a classical vector model [65]. In this classical model, the nuclei can be described by a
small magnetic dipole with magnetic moment #»µ [Am2], also known as “spin”. The nuclear
spin #»µ is the consequence of a moving charge within the atoms nucleus. A relation exists
between the angular momentum #»b and the magnetic moment of the nucleus #»µ, #»µ = γ#»b
(1) (1) where the proportionality constant γ [rad s−1 T−1] is the gyromagnetic ratio. The most
studied nucleus (also abundant in polymers) is hydrogen (1H) where γ/2π = 42.58 MHz/T. where the proportionality constant γ [rad s−1 T−1] is the gyromagnetic ratio. The most
studied nucleus (also abundant in polymers) is hydrogen (1H) where γ/2π = 42.58 MHz/T. We want to emphasize that although hydrogen is the most abundant and well known atom,
there also exist studies on other elements such as fluorine [78] (40.08 MHz/T) or carbon-13
(10.71 MHz/T) [79]. # » We want to emphasize that although hydrogen is the most abundant and well known atom,
there also exist studies on other elements such as fluorine [78] (40.08 MHz/T) or carbon-13
(10.71 MHz/T) [79]. » (
/ ) [
]
When these nuclear spins enter an external applied magnetic field # »
B0 [T], they will
experience a torque #»τ [Nm] related to the magnetic field by #»τ = #»µ × # »
B0
(2) (2) As the torque equals the time derivative of the angular momentum #»b , Equations (1)
and (2) can be combined to:
d#» As the torque equals the time derivative of the angular momentum #»b , Equations (1)
and (2) can be combined to: d#»µ
dt = γ#»µ × # »
B0
(3) (3) Since all the nuclei will experience this same time dependence in their magnetic
moment #»µ, they will start to align and precess around the applied magnetic field # »
B0, which
by normal convention is pointing in the z-direction. This precession is called Larmor Polymers 2022, 14, 798 4 of 32 precession. A schematic picture of this precession is shown in Figure 1 left. 2.2. NMR Relaxation In the previous section, the formula for the signal intensity (Equation (7)) introduced
two characteristic time scales that described signal loss after excitation. This signal loss
is known as relaxation and will be described in this section. There are two typical char-
acteristic relaxation processes called T1- or spin–lattice relaxation and T2- or spin–spin
relaxation. In the first one, energy between the hydrogen spins and the surroundings is exchanged. This energy exchange will relax the spins back to their original z-direction. How fast Mz is
restored is characterized by this T1-time. The energy transfer will be most effective when
surrounding atoms vibrate at the Larmor frequency. The second relaxation process will
account for losses in the transversal component of the magnetization vector (Mxy). The rate
at which this relaxation occurs is defined by a T∗
2 -relaxation time that is strongly correlated
to the molecular motion and the local environment. For example, dipolar interactions
between neighboring atoms induces a small difference in magnetic field [82], which results
in dephasing and a faster signal decay. Both relaxation times will be influenced by different local factors that enable to extract
physical information about their surroundings and their mobility. To understand how
these times can be used to gain physical information, a deeper look at both relaxation times
is provided. p
The total T∗
2 - and T1-relaxation time can be written as the contribution of different
factors as [83] p
The total T∗
2 - and T1-relaxation time can be written as the contribution of different 1
T∗
2
= 1
T2
+ 1
T2i
=
1
T2dip
+
1
T2pores
+
1
T2di f
+ 1
T2i
(8)
1
T1
=
1
T1dip
+
1
T1pores
(9) (8) (9) where T∗
2 and T1 are the relaxation times found in Equation (7). Here, T∗
2 is a combination
of both T2 and T2i, where the second one is attributed to inhomogeneities in the magnetic
field introduced by the setup [84]. When using Hahn [80,81], CPMG [85,86] or other
pulse sequences (Section 2.3), one can compensate for field inhomogeneities and the T2i
contribution can be neglected. Therefore, T∗
2 can be replaced by T2. 2
The first relaxation mechanism comes from neighboring dipoles (T1dip, T2dip). Dipoles
will introduce small changes in the local magnetic field that lead to a faster relaxation. 2.1. NMR Spatial Encoding −→
G is not drawn because it lies in correspondence with the middle
figure, not in one direction, which would make the graph too complicated. One of the advantages of NMR is the ability to achieve spatial resolution along the
y-direction, perpendicular to the thin layer. Encoding spatial information can be achieved
by applying a magnetic field gradient #»G[T/m] ≡(∂Bz/∂x, ∂Bz/∂y, ∂Bz/∂z) perpendicular
to the # »
B0 field, see Figure 1 middle. Depending on the strength of the gradient (G) and
magnetic field (|B0|), there exist two different scenarios, |B|/G ≫1 and |B|/G ≪1. The rest of this section will cover the special case where |B|/G ≫1, found in conven-
tional NMR setups. In this particular case, the curvature of the magnetic field lines is so
small that the following assumptions can be made: the magnetic field is constant, pointing
in the z-direction, and the gradient along the y-direction can be assumed to be constant and
equal to ∂Bz/∂y (Figure 1 middle). Both assumptions will simplify the explanation. How-
ever, for most high-gradient NMR setups used for thin film profiling where |B|/G ≪1 [74],
these assumptions are not valid anymore. This will be discussed in Section 2.4. In the case where |B|/G ≫1, the gradient will cause a change in magnetization along
the y-direction, indicated by the black arrows in Figure 1 middle, which leads to a change
in Larmor frequency, (6) ω(y) = 2π f (y) = γ(B0 + Gy · y)
(6) ω(y) = 2π f (y) = γ(B0 + Gy · y) Polymers 2022, 14, 798 5 of 32 with Gy—the gradient of the z-component of the magnetic field along the y-direction
(∂Bz/∂y ≈∂B0/∂y) and f—the frequency amplitude. The total measured NMR signal
is a combination of all different nuclei that resonate with different Larmor frequencies. When applying a Fourier transform of the recorded echo, the separate contributions of all
different Larmor frequencies can be extracted and linked to a specific y-position through
Equation (6), providing the spatial density distribution ρ(y). After excitation, the total
signal intensity S(y, te) at position y will decay over a time due to environmental effects
and is given by S(y, te) ∝ρ(y)
1 −exp
−tr
T1
! exp
−te
T∗
2
! (7) (7) where T1 and T∗
2 are characteristic timescales that define the signal decay. 2.1. NMR Spatial Encoding More information
about these times and the underlying processes can be found in the next section. 2.2. NMR Relaxation In most cases, this relaxation mechanism can be linked to the molecular mobility. This
molecular mobility is quantified by a motional correlation time (τc) which is defined as the
average time necessary for a molecule to rotate one radian. The influence of dipole–dipole
interaction was described by Bloembergen, Purcell and Pound (BPP) in 1948 [87], where Polymers 2022, 14, 798 6 of 32 they established relationships between T1dip and T2dip and the motional correlation time
(τc), Figure 2 left:
2 1
T1dip
= 3
10
γ4¯h2
r6 [
τc
1 + ω2
0τ2c
+
τc
1 + 4ω2
0τ2c
]
(10)
1
T2dip
= 3
20
γ4¯h2
r6 [3τc +
5τc
1 + ω2
0τ2c
+
2τc
1 + 4ω2
0τ2c
]
(11) (10) 1
T2dip
= 3
20
γ4¯h2
r6 [3τc +
5τc
1 + ω2
0τ2c
+
2τc
1 + 4ω2
0τ2c
]
(11) (11) These relaxation times can be used to discriminate between different polymer motional
regimes inside a thin film, providing information about different structures, mobility,
chemical reactions and mechanical responses. In a rigid environment, such as solids or
polymers below their glass transition temperature, hydrogen atoms will experience the
same deviations in magnetic field for a longer time, leading to a larger phase difference and
faster decay. On the contrary, when molecules are mobile, for example in liquid water, the
differences in magnetization will be averaged out over time leading to a longer relaxation
time. This was used to observe for example different polymer substances [66], crosslinked
and non-crosslinked materials and skin layers within thin polymer films [88,89], see also
Section 4. This could ultimately be used to observe any difference between materials where
the molecular mobility of the studied atoms is different. The second term appearing in both relaxations is the relaxation caused by a porous
matrix (T1pores, T2pores) which was described in detail by Brownstein and Tarr [90]. When
atoms diffuse through a porous matrix they will encounter the pore surface, where re-
laxation due to dipoles or other effects occurs. How effectively this surface relaxation
occurs depends on the particular medium and is quantified by the surface relaxivity σ
[m/s] [91–95]. Depending on the diffusion constant (D [m2/s]), pore radius (r[m]) and
surface relaxation, different regimes can be excluded [96]. 2.2. NMR Relaxation There is a fast diffusion regime
with σr/D ≪1, where the surface relaxation will become dominant, and a slow diffusion
regime, with σr/D ≫1 where the surface relaxation can be neglected. Here, the factor r/D
is related to the number of encounters with the surface. Therefore, the surface relaxation
will become important when the surface relaxivity is high or many encounters are present. In the fast diffusion regime, Brownstein and Tarr found that 1/T1pores and 1/T2pores can be
approximated by 1
T2pores
= σ2
S
V
(12)
1
T1pores
= σ1
S
V
(13) (12) (13) where the factor S/V is the pore surface-to-volume ratio. These relaxation times can be
used to gain useful information about the porous matrix [83,91–94,97] and possible changes
over time when subjected to different environmental factors. A third parameter only found in T2-relaxation comes from diffusion of molecules. When atoms diffuse, they will encounter a different field strength due to the setups gradient. This leads to dephasing and an accelerated decay of Mxy. This diffusion-induced decay will
also influence the resulting T2-time. This effect will become larger with increased gradient
strength, diffusion constant (D[m/s2]) and diffusion time (te), which is described by the
following formula [98,99], 1
T2di f = αγ2G2t2
e D
(14) (14) Probing relaxation times can also be useful for studying different pools of atoms at
the same time, a trick commonly used in thin film studies [100–104]. When the relaxation
times of different hydrogen groups are far enough apart, their relative contributions to the
signal can be separated. Figure 2 right demonstrates this principle by showing a decay of a
latex film [105]. The decay clearly shows a two-component exponential decay. From the
BPP-theory we know that the latex can be attributed to short relaxation time while the long
T2-relaxation time can be linked to free water. 7 of 32 Polymers 2022, 14, 798 Figure 2. Left: Dependence of T1 and T2 upon τc (Reproduced from Chem. Soc. Rev., 2014,43,
1627–1659 with permission from the Royal Society of Chemistry.) [106]. Right: Signal decay of a
latex film measured with a GARField NMR. Both soft and hard-type latex are visible and show a
multi-exponential decay where the short and long T2– component are marked. The short relaxation
time is attributed to hydrogen atoms within the polymer, while the long relaxation time comes from
free water. 2.2. NMR Relaxation (Reprinted from Progress in Organic Coatings, Volume 123, Benjamin Voogt, Henk Huinink,
Bart Erich, Jurgen Scheerder, Paul Venema and Olaf Adan, Water mobility during drying of hard and
soft type latex: Systematic GARField 1H NMR relaxometry studies, Pages 111–119, Copyrights 2018,
with permission from Elsevier [107]). Figure 2. Left: Dependence of T1 and T2 upon τc (Reproduced from Chem. Soc. Rev., 2014,43, Figure 2. Left: Dependence of T1 and T2 upon τc (Reproduced from Chem. Soc. Rev., 2014,43,
1627–1659 with permission from the Royal Society of Chemistry.) [106]. Right: Signal decay of a
latex film measured with a GARField NMR. Both soft and hard-type latex are visible and show a
multi-exponential decay where the short and long T2– component are marked. The short relaxation
time is attributed to hydrogen atoms within the polymer, while the long relaxation time comes from
free water. (Reprinted from Progress in Organic Coatings, Volume 123, Benjamin Voogt, Henk Huinink,
Bart Erich, Jurgen Scheerder, Paul Venema and Olaf Adan, Water mobility during drying of hard and
soft type latex: Systematic GARField 1H NMR relaxometry studies, Pages 111–119, Copyrights 2018,
with permission from Elsevier [107]). By exploiting this method, the relative contribution and relaxation time of different
pools of hydrogen atoms can be followed over time using the same experiment. This is
by far the most powerful tool in studying thin films over time [108]. Using this technique,
M.R.Halse [109] showed that they could follow three groups of hydrogen atoms in a
decane/rubber system at the same time: dry rubber (T2 = 0.7 ms), swollen (T2 = 1.8 ms)
rubber and solvent (T2 = 200 ms). 2.3. Pulse Sequences In the beginning, the magnetization vector is in equilibrium, pointing in the same
direction as the main magnetic field #»B0. Bringing the magnetisation out of equilibrium
and creating an NMR signal is achieved by using a radio frequency (RF) wave. This RF
irradiation will have a magnetic component (#»B1) along the xy-plane exerting a torque on
the magnetization (Equation (2)). Applying this RF-field for a certain amount of time is
called an “RF-pulse”. The angle by which the magnetization will rotate is called the “flip
angle” (α[◦]) and depends on both the pulse time (tp[s]) and the magnitude of the RF-field
(B1[T]) given by (15) α = γB1tp
(15) Applying different RF-pulses is referred to as a pulse sequence and can be used to
measure all the above mentioned parameters such as signal intensity, relaxation times and
diffusion. As the focus of this review lies on the techniques, only the main sequences and
some basics will be discussed. More details and explanations on the different types of pulse
sequences can be found in more advanced studies [43,88,110] and reviews [83,111]. q
Measurements of the signal intensity are mostly performed using Hahn spin
echoes [80,81] with the following pulse sequences [90◦-τ-180◦-τ-echo] [111]. At t = 0,
a 90◦pulse rotates the spins into the xy-plane. After this pulse, the transverse magneti-
zation starts to dephase due to the environment, field inhomogeneity and the gradient
according to Equation (7). At t = te/2, a second 180◦is given that refocuses the spins. At
t = te, the spins are refocused and a so-called spin echo is created from which the transverse
magnetization can be measured. g
To measure the T2-relaxation, the Carr–Purcell–Meiboom–Gilll sequence (CPMG) [85,86]
is performed. The sequence start exactly the same as the Hahn spin echo, followed by a
train of 180◦pulses [90◦-τ-(180◦-τ-echo-τ)n]. Every 180◦pulse refocuses the spins, resulting Polymers 2022, 14, 798 8 of 32 in a series of spin echoes. The echo intensity drops due to T2-relaxation. The signal intensity
at the nth echo is given by S(nte) = S0exp(−nte/T2)
(16) (16) from which the T2-relaxation time can be calculated. In literature, adjustments to this
well-known sequence are reported. A specific pulse sequence that should be mentioned
here is the Ostroff–Waugh (OW) pulse sequence where the 180◦pulse is exchanged by
another 90◦pulse [112]. from which the T1-relaxation time can be calculated. from which the T1-relaxation time can be calculated. from which the T1-relaxation time can be calculated. 2.3. Pulse Sequences In high-gradient measurements, this sequence is chosen above the
more conventional CPMG sequence, for reasons that will be explained in Section 2.5. To measure the T1-relaxation time, conventional NMR setups use mainly two se-
quences, the saturation recovery and inversion recovery sequence. However, in the high-
resolution setups described in this review, the saturation recovery sequence is chosen
for reasons that will be explained in Section 2.5. The pulse sequence of the saturation
recovery sequence is given by [(α)m-τ2-90◦-τ-180◦-τ-echo] [113]. The measurement starts
by saturating the nuclear spins, setting the magnetization in the transverse plane to zero. This is achieved by m pulses with a certain angle α. After complete saturation, Hahn spin
echoes will be measured at different time intervals specified by τ2. The measured echo
intensity is then given by S(τ2) = S0(1 −exp(−τ2/T1))
(17) (17) m which the T1-relaxation time can be calculated. from which the T1-relaxation time can be calculated. 2.4. High Resolution Spatial Encoding To profile thin films with a high-enough resolution, a high gradient is required, see
Equation (6). Three NMR setups are able to generate high-enough gradients to profile
films between 10–1000 µm, namely, STRAFI (STRAy FIeld) [114], GARField (Gradient-At-
Right-angles-to-Field) [74] and MOUSE (MObile Universal Surface Explorer) [115]. In these
high-gradient setups, where |B|/G ≪1, Equation (6) cannot be used anymore because the
curvature in the magnetic field lines is significant, see Figure 1 right. g
g
g
g
To understand this, we need to consider Maxwell in free space with a static magnetic
field and no current,
» ∇× #»B = 0. (18) (18) According to this condition, the following relation should hold for the gradient in the
y-direction (Gy = ∂Bz/∂y), ∂Bz
∂y = ∂By
∂z
(19) (19) Therefore, by introducing a gradient in the y-direction (∂Bz/∂y), there must be a
gradient in another direction (∂By/∂z) resulting in a curvature into the mean magnetic
field, which is of the order of |B|/G [74]. In conventional NMR setups with a low gradient,
this curvature is around 102m and can be neglected as in the previous section. When the
gradient becomes much larger, the assumptions made in the previous section are not valid
anymore. Consequently, it follows from Equation (6) that #»f (#»r ) = γ
2π (#»B0 + #»G · #»r )
(20) (20) where we use a vector notation to account for the fact that the Larmor frequency is different
along the same horizontal plane. As signals are excited and resolved according to their
respective Larmor frequency, this inhomogeneous Larmor frequency will lead to some
problematic effects within high-resolution setups. The RF-pulses that normally excite a
rectangular region will now excite curved slices that depend on the shape of the magnetic
field lines. The curvature in STRAFI, for example, is around 0.1 m [74], much lower than
in conventional NMR setups (102 m). Figure 3 shows the sensitive region of an original
NMR-MOUSE where a clear curvature can be observed [116]. 9 of 32 Polymers 2022, 14, 798 Figure 3. Left: sensitive region of an original NMR-MOUSE. 2.5. Resolution and Field of View In the previous section, we discussed how a gradient is used to encode spatial informa-
tion in a measurement. The resulting resolution is mostly determined by the high gradient
and its ability to encode and read out spatial information. However, there are some other
limiting factors that determine the maximum achievable resolution of the experimental
setup. Another important parameter is field of view (FOV) which provides the maximum
area that can be measured with the NMR setup. This section will cover some of the most
important factors that determine the final resolution and FOV. Both parameters will be
discussed for two measurement strategies—a Fourier measurement and a slice selective
pulse measurement. In the first one, the spatial information is collected via a Fourier
transformation of the NMR signal, mostly found in GARField [105,117] and sometimes in
STRAFI [118]. In the slice selective pulse measurement, a slice selective pulse will collect
the NMR signal of a thin slice without the need for a Fourier transformation. Here, the
profile is built up slice-by-slice which requires a mechanical movement of the sample or
magnet, mostly found in STRAFI [119] and MOUSE [120]. In a Fourier transformation measurement, the maximum theoretical resolutions that
can be achieved are determined by the lowest frequency difference (∆f [Hz]) that can
be differentiated. This difference will depend on the window width or acquisition time
(∆ta [s]) of the measurement (∆f ≈1/∆ta) [121]. The dimension that corresponds to this
frequency difference is determined by the gradient [121] and is given by ∆z =
1
γGz∆ta
(21) (21) Thus, for a gradient of 40 T/m and a window width of 100 µs, the maximum achievable
resolution becomes 5.9 µm. It can therefore be seen that both a higher gradient or acquisition
time will increase the maximum theoretical resolution. However, ∆ta should always be
lower than the T∗
2 found in Equation (8). When ∆ta > T∗
2 , the signal is limited by a fast
T2-relaxation and not by the acquisition time. In this particular case, ∆ta in Equation (21)
should be replaced by T∗
2 . As the typical ∆ta of high-gradient setups are around 100 µs,
much lower then conventional NMR setups, these cases almost never appear. In the slice selective measurements, the resolution is determined by the frequency
bandwidth (∆f) of the RF-pulse. The frequency bandwidth from the RF-pulse is inversely
proportional to the pulse length (tp). 2.4. High Resolution Spatial Encoding Right: Slice thickness of the signal
amplitude for a conventional NMR-MOUSE (Reprinted from Magnetic Resonance Imaging, Volume
23, Issue 2, Bernhard Blümich, Federico Casanova, Juan Perlo, Sophia Anferova, Vladimir Anferov,
Kai Kremer, Nicolae Goga, Klaus Kupferschläger, Michael Adams, Advances of unilateral mobile
NMR in nondestructive materials testing, pages 197–201, Copyright 2005, with permission from
Elsevier. [116]). Figure 3. Left: sensitive region of an original NMR-MOUSE. Right: Slice thickness of the signal Figure 3. Left: sensitive region of an original NMR-MOUSE. Right: Slice thickness of the signal
amplitude for a conventional NMR-MOUSE (Reprinted from Magnetic Resonance Imaging, Volume
23, Issue 2, Bernhard Blümich, Federico Casanova, Juan Perlo, Sophia Anferova, Vladimir Anferov,
Kai Kremer, Nicolae Goga, Klaus Kupferschläger, Michael Adams, Advances of unilateral mobile
NMR in nondestructive materials testing, pages 197–201, Copyright 2005, with permission from
Elsevier. [116]). amplitude for a conventional NMR-MOUSE (Reprinted from Magnetic Resonance Imaging, Volume
23, Issue 2, Bernhard Blümich, Federico Casanova, Juan Perlo, Sophia Anferova, Vladimir Anferov,
Kai Kremer, Nicolae Goga, Klaus Kupferschläger, Michael Adams, Advances of unilateral mobile
NMR in nondestructive materials testing, pages 197–201, Copyright 2005, with permission from
Elsevier. [116]). 2.5. Resolution and Field of View Thus, for a 10 µs pulse time, the excited frequency
bandwidth (∆f) is around 0.1 MHz which for a gradient of 40 T/m would excite a slice of
58.7 µm = (∆r ≈1/tpγG). p
A problem arises in high-gradient fields because of the curvature induced on the
magnetic field (see Section 2.1) that limits the homogeneity of |#»B0| and therefore the
resolution. In the STRAFI and MOUSE, these inhomogeneities will be the limiting factor
for the resolutions. Determining these resolutions can be achieved experimentally by
measuring the profile of a thin slice, containing NMR active atoms, and analyzing the Polymers 2022, 14, 798 10 of 32 10 of 32 resulted profile [3]. An example of this limitation is shown in Figure 3, where on the left
the sensitive area for a normal NMR-MOUSE is shown. The corresponding depth profile
can be viewed on the right. In most cases, the experimental resolution is determined by
taking the half-width of this profile which in this case would be around 1 mm and limits the
maximum achievable resolution. As will be explained later, GARField introduces specially
designed poles in order to make |#»B0| homogeneous [74]. For this reason, the achievable
resolution in GARField is not limited by inhomogeneities and is mostly higher than in a
STRAFI and MOUSE setup. p
The high gradients will also induce a large spread in resonant frequencies and unlike
a conventional NMR imaging setup, the gradients cannot be switched off. The spread
in resonance frequencies can be of the order of 25 MHz cm−1 [121] which severely limits
the single-shot field of view (FOV) that can be achieved with one RF-pulse. In a Fourier
measurement, the theoretical FOV is determined by the slice selective pulse (∆r ≈1/tpγG)
which can be increased by lowering the pulse time. Now, we can explain why in a high-
gradient setup, an OW-sequence and saturation recovery sequence are chosen above the
more conventional CPMG sequence and inversion recovery sequence. Since the OW- and
saturation recovery sequences use only 90◦pulses, always the same volume will be excited. In a CPMG- and inversion-recovery sequence where 90◦and 180◦pulses are used, this
is not the case. Another problem in the case of the inversion recovery arises in the first
pulse, used to invert the magnetization from Mz to M−z, which will not be exactly 180◦
throughout the sample which can interfere with the T1-measurement. 3. Methods In this section, the three mentioned high-resolution NMR setups (STRAFI, GARField
and MOUSE) will be discussed in more detail. The goal is to provide a general guideline
that helps in deciding which setup is most suitable for studying a specific application
or material. 2.5. Resolution and Field of View However, in real
measurements, limitations arise because the sensitivity drops significantly when moving
away from the RF-coil, limiting the FOV for a GARField setup to around 500 µm depending
on the signal-to-noise ratio. In a slice selective measurement, the sample can be moved through the sensitive area
which in essence could result in a limitless FOV. However, the setup design will mostly be
the limiting factor leading to a FOV in the order of a few millimeters. The slight differences between the three setups will lead to slight changes into the
achievable resolution and FOV. A more detailed explanation can be found in Section 4
where the setups are discusses separately. However, in most cases, the achievable resolution
and FOV can be approximated using the above-mentioned formulas. 3.1. STRAFI (STRAy Field Imaging) Stray field imaging was introduced by A. A. Samoilenko et al. [75] in 1988. The STRAFI
technique uses the stray field (fringe field) of a superconducting magnet to produce its high
gradient. Figure 4 left is a schematic representation of a STRAFI setup. Indicated with black
dotted lines are the field lines from the superconducting magnet indicating the direction
of the main magnetic field #»B0. The measurement area lies in the stray field just below
the superconducting magnet where a large gradient can be found that lies along the same
direction as #»B0. Typical gradients that can be achieved are between 30 and 60 [Tm−1] [114]. Indicated with red lines in Figure 4 are the field lines from the RF-pulse. From here there
exist mainly two different ways to acquire a complete profile, leading to two types of
STRAFI, conventional STRAFI [122,123] and Fourier transform STRAFI [118]. Polymers 2022, 14, 798 11 of 32 Figure 4. Left: schematic representation of STRAFI. Middle: schematic representation of GARField. Right: schematic representation of MOUSE. In all figures, the measurement area is depicted with
an orange rectangle. The magnetic field lines are indicated with black for the B0-field while the
ones from the RF-pulse are indicated with red. In all setups, the direction of the gradient (G), main
magnetic field (B0) and RF-pulse field (B1) in the measurement area are indicated with black arrows. Figure 4. Left: schematic representation of STRAFI. Middle: schematic representation of GARField. Figure 4. Left: schematic representation of STRAFI. Middle: schematic representation of GARField. Right: schematic representation of MOUSE. In all figures, the measurement area is depicted with
an orange rectangle. The magnetic field lines are indicated with black for the B0-field while the
ones from the RF-pulse are indicated with red. In all setups, the direction of the gradient (G), main
magnetic field (B0) and RF-pulse field (B1) in the measurement area are indicated with black arrows. g
p
p
Right: schematic representation of MOUSE. In all figures, the measurement area is depicted with
an orange rectangle. The magnetic field lines are indicated with black for the B0-field while the
ones from the RF-pulse are indicated with red. In all setups, the direction of the gradient (G), main
magnetic field (B0) and RF-pulse field (B1) in the measurement area are indicated with black arrows. In the conventional STRAFI, the profile is acquired by recording the NMR signal
slice-by-slice [122,123]. 3.1. STRAFI (STRAy Field Imaging) One method of imaging slice-by-slice is by moving the sample,
which can be seen in Figure 4. As slices are imaged separately, no Fourier transform is
required, which leads to a resolution that is limited by the frequency bandwidth of the
excitation pulse, see Section 2.5. The fact that the resolution is pulse-time-dependent
involves some drawbacks and limitation on the resolutions. As the pulse lengths can
never be longer than the T2 of the material, the resolution for polymers with very short
T2 times is limited. Since the frequency bandwidth of the pulse is inversely proportional
to the pulse time, this method allows to have rather good resolutions at the expense of
long measurement times. Another time restraint comes from the mechanical movement
required in slice-by-slice measurements that has a huge influence on the measurement time. This becomes problematic when measuring fast dynamic processes. However, there are
some tricks to lower the time required to measure a single profile. For example, during the
repetition time, one can already start to measure other slices which means that the time
is limited by the T1 of the sample [114]. As slices are measured separately and changing,
some parameters will always influence measurement time. For example, increasing the
resolution or sample thickness will also increase measurement time as more slices need to
be measured. Therefore, for slow processes, good resolution can be achieved, while for fast
processes, the measurement time is too long. Thus, measuring with STRAFI is easier for
slow processes and lower resolutions (above 50 µm). p
In the special case, when the frequency band with of the excitation pulse is large enough
to capture the complete sample, a Fourier transform STRAFI can be implemented [118]. Here, the echo is collected from the whole sample and the profile is reconstructed by
a Fourier transform. Using this method, measurements are only limited by T1 and not
because of mechanical movement. This technique was mostly used to image thin film below
400 µm. The theoretical resolutions in these measurements are determined by Equation (21). However, the resolution is mostly limited by the shape of the sensitive area and should be
determined experimentally. A major drawback of STRAFI is that the sensitive area where
the #»B0-field is most homogeneous does not coincide with the optimum gradient position. 3.2. GARField (Gradient-At-Right-Angles-to-Field) 3.2. GARField (Gradient-At-Right-Angles-to-Field) To solve the high-curvature problem found in STRAFI that limits the maximum
resolution, the GARField NMR was introduced in 1999 by P. M. Glover et al. [74]. In this
setup, the researchers changed from a gradient along one component (y-direction) to a
gradient in the magnetic field amplitude |#»B0|. To achieve this, the setup uses electromagnets
with specially designed pole tips. The specific shape was calculated by P.M. Glover et al.,
using a specific solution of the Laplace equation ∆2φ = 0 where φ is the scalar potential
defined by B = grad φ. For a detailed calculation, we refer to the original paper [74]. The specially designed pole tips from the electromagnets are able to generate a horizontal
plane where |B0| is constant along the xy-plane and perpendicular to the gradient, Figure 4
middle. When making these pole tips, it was found that the ratio G/|B| is always constant
and independent of y and z. This allows to operate with different gradient strengths without
losing the in-plane uniformity of the magnetic field, allowing for an easy adaptation of the
resolution. Shown in Figure 4, middle, is a schematic representation of the setup where the
shape of the magnetic pole tips can be seen. Implementing these magnetic poles was only
possible when the orientation of the magnetic field and gradient were slightly different
compared to STRAFI. Where in STRAFI the B0 and G are aligned, they are perpendicular in
the GARField setup. As the RF-pulse should be perpendicular to the main magnetic field,
the RF-coil from the STRAFI and GARField will have different orientations. The highest
sensitivity is obtained when the sample is placed at the end of the RF-coil. This is only
possible in the GARField setup. In STRAFI, this arrangement is impossible and the sample
should be placed apart from the RF-coil, which lowers the sensitivity. p
p
y
Compared to conventional STRAFI and MOUSE, a depth profile is measured in one
single measurement without repositioning the sample or magnet. The time required to
measure a single profile depends on the different parameters used in the pulse sequence
but is typically shorter than in STRAFI. A drawback, however, is the limited FOV. Without
the possibility to reposition the sample, the FOV is mostly limited by the reduced sensitivity
when moving away from the coil, mostly around 500 µm. 3.2. GARField (Gradient-At-Right-Angles-to-Field) As some applications such as coatings are mostly used on metallic surfaces, a special
note should be made. Metallic surfaces interfere with the magnetic signals in the setup,
which introduces artifacts in the measurement. Artifacts can be the result of differences
in magnetic susceptibility between polymer and metal and from eddy currents generated
in the metal. To address this problem, H. Zhu et al. [125] investigated the effect of metal
substrates on the NMR signal. The researchers found through simulations and experimental
work that magnetic susceptibility can be neglected when measuring with a GARField NMR,
but that the eddy currents interfere with the pulse field. Therefore, it was calculated that
measuring on metallic surfaces required more pulse power to manage these eddy currents. 3.1. STRAFI (STRAy Field Imaging) Therefore, the actual gradient is mostly lower than the maximum gradient that is reported
for the particular electromagnet used in the STRAFI setups. After a trade-off between
measurement speed and resolution, the most encountered studies reported resolutions
between 50 [124]–450 [122] µm. Polymers 2022, 14, 798 12 of 32 12 of 32 3.4. Guideline Choosing the proper setup depends on multiple parameters, such as the sample
material, required resolutions and measurement time. When resolution is most important, GARFIeld is the best option. By solving the curva-
ture problems found in STRAFI, GARField will have the best resolution of all setups—the
most reported resolutions lie somewhere between 5 and 15 µm (Table 1). In addition to a
good resolution, the Fourier measurements used in GARField offers faster measurement
speed then slice-selective measurements (Table 1). Profiles are mostly measured at times
between 1 and 10 min. It should be mentioned that STRAFI also has a Fourier implementa-
tion, but with lower resolutions (most reported resolutions between 24–60 µm) and lower
sensitivity coming from the RF-coils orientation. Therefore, for a dynamical process or if a
high resolution is required, GARField would be the best option. A drawback of GARField
is the rather low FOV (<500 µm) with a sensitivity that lowers when moving away from
the RF-coil. When larger samples should be measured, STRAFI would be a better choice. Due to
the slice-selective excitation, samples can be moved through the sensitive area, making the
FOV, in essence, limitless. While at 400 µm the GARField already loses a lot of sensitivity,
the STRAFI does not encounter this signal loss. In these measurements, resolution always
comes with a trade-off towards measurement time. Higher resolutions require to measure
more slices when keeping the FOV constant. When measurements are required on-site or if a small piece of the sample is not
available, MOUSE is the only suitable option. For most applications such as walls, oil/water
wells [130–132], PE pipes [116] or paintings, samples small enough for the STRAFI or
GARField setup are not available, leaving MOUSE as the only option. An overview of the most frequently encountered parameters is given in Table 1. It
should be mentioned that in all cases, trade-offs between different parameters are made. Therefore, the best achievable resolution is almost never achieved. However, the table
should give an idea about different ranges that are commonly used for the different setups. Table 1. The table summarizes the most important characteristics of the different setups. p
p
|# »
B0|
|#»
G[T/m]| Measure Time Resolution
Portable Sample Size
STRAFI
2.3–7
30–60
>3 min
24–60 µm
no
limitless
GARField 0.7–1.4
17–44
1–10 min
5–15 µm
no
50–400 µm
MOUSE
0.025–0.7 11.5
60 min
50–1000 µm yes
100–4000 µm 3.3. The MOUSE Different from the STRAFI and GARField, the NMR-MOUSE (MObile Universal
Surface Explorer) is a portable device invented to investigate large objects in a nondestruc-
tive manner [7,8]. The NMR-MOUSE consists of a compact permanent magnet (red and
blue in Figure 4), which generates the B0-field. Inside the magnets, the magnetic field is
homogeneous but when moving to the fringe field, the field starts to become more inho-
mogeneous, creating a rather high gradient of about 22 T/m depending on the specifics
of the magnets [7]. The small size and low weight make it suitable for on-site testing in a
non-destructive manner. This makes the NMR-MOUSE suitable to carry out measurements
on large surfaces such as walls or paintings without the need for collecting samples and
destroying the object. This magnetic field is typically quadratic along the x- and z-direction and with a main
gradient along the y-direction. This variation in the y-direction can be used to extract depth
profiles of a specific material. The B1-field is obtained from a built-in RF-coil, see Figure 4
right. By calculating the exact magnetic field, is has been found that there is a sensitive
region just outside the device [126]. Just like a conventional STRAFI, slice-selective pulses Polymers 2022, 14, 798 13 of 32 13 of 32 are used to extract spatial information, therefore, recording a signal is fully equivalent to
STRAFI [114]. However, the sensitive region lies outside the magnets, which limits the final
depth that can be measured. Different devices are available to measure different depths
ranging from 3 to 25 mm [7]. Selecting the best device depends on the application as more
depth coincides with a lower sensitivity. A drawback of the simple magnetic design is the fact that the sensitive region is
oddly shaped, varying in thickness, see Figure 3. This results in low sensitivity and huge
restrictions on the maximum achievable resolution. Spatial resolutions better than half
a millimeter are hard to achieve [127,128]. Attempts have been made in order to reduce
the resolution. J. Perlo et al. [129] reported resolutions as low as 2.3 µm by making a new
magnetic geometry with four permanent magnets. Measuring with these high resolutions
in a slice-selective manner is, however, very time consuming. Another drawback of the
design is the limitation of the sensitive volume, which limited the FOV to 50 µm. 4.1. Structure and Structural Evolution Studying thin layers starts by identifying their internal structure and structural evolu-
tion. Therefore, the first NMR measurements performed on thin layers aimed to determine
the moisture content and different polymer states such as crystallinity or cross-linking. Determining the structure of thin layers can be achieved using the signal intensity, dif-
fusivity and T1/T2-relaxation times. All these parameters provide information about the
hydrogen content, their environment and micro structures, as described in Section 2.2. In
the first part of this section, two studies on cement pastes performed by P. J. McDonald
et al. [54,55] are used to explain how the relaxation times can be used to obtain structural
information, followed by a summary of some important studies using similar principles. In
these studies, GARField was only used to measure in one particular slice. In principle, this
information could have been obtained via conventional NMR instruments. Nevertheless,
we value this particular study as GARField has the potential to perform a similar study,
but with high spatial resolution in depth. Lastly, a study performed by B. Voogt et al. [107]
is used to explain how diffusivity can be used to determine structural information using
the above mentioned setups. In studies performed by P. J. McDonald et al. [54,55], the researchers characterized
hydrated cement paste using different T1- and T2-relaxation studies. T1- and T2-relaxation
times for different cement paste were determined using a saturation recovery and OW-
sequence with the GARField-setup. Using the GARField-setup, a slice selective measure-
ment was performed 10 mm below the surface of the sample with a slice thickness of
0.6 mm. When plotted in a T1-T2 correlation spectrum, the researches could relate the relax-
ation times to different groups of hydrogen atoms with varying pore radii (Equations (12)
and (13)). Such plots for a white cement sample cured under water are shown in Figure 5. They identified that the cement paste had pores with the following length scales: gel pores
(T2 = 400 µs) and multiple capillary pores (T2 > 400 µs) with different pore sizes. Observing
the different relaxation times, they found that after 6 days, 2 peaks are visible, both with
low T1 and T2 times corresponding to gel pores. At day 7, capillary pores with longer
relaxation times also start to become visible (Figure 5). Characterizing bulk properties like
this can also be performed using a normal NMR spectrometer [133]. 4. Applications The following sections will provide a deeper look into the most common types of
measurement performed by these three NMR setups on thin layers. Every section will start Polymers 2022, 14, 798 14 of 32 14 of 32 by discussing the general concepts using a representative study as an example, followed by
a summary of related studies on a wide variety of samples and materials. by discussing the general concepts using a representative study as an example, followed by
a summary of related studies on a wide variety of samples and materials. 4.1. Structure and Structural Evolution Red corre-
sponds to water in capillary-sized pores, green to gel-sized pores and blue for interlayer
pores and black is the total amount of moisture. The authors observed that after 1 hour,
the moisture uptake already reaches 90% of its total amount. A striking fact is that they
saw for the first time that moisture will start to redistribute between the different pores. In
this research, they only looked at a particular part in the layer, however, with GARField,
MOUSE or STRAFI, the same information at different positions is available at every position
in the layer. Studies on cement like-materials such as pastes and Portland cement have also been
performed on STRAFI [134–136] and MOUSE [135,137]. In other studies, this separation
based on relaxation times was used to separately study water and polymer contribution in
latex films [105,107], human skin [138] and another group of polymers such as Polyurethane
coatings [139], plasticizing of nylon-6 [140,141], semi-crystalline polyethylene [142] and
photo-polymerization of methacrylate [143]. Additionally, medical applications, such as
the binding behavior of collagen-binding liposomes, have been studied [144,145]. Even
layers with very low relaxation times, such as ice (T2 = 3.5 µs), have been imaged during
melting using a STRAFI setup [122]. g
g
p
In addition to T2- and T1-relaxation, another way to characterize the structure of thin
layers is via the diffusion coefficient D of the hydrogen atoms. In materials, water can be
present in a wide variety of states with different diffusion coefficients such as free water,
water confined in pores or water bound to the polymer matrix. When materials undergo a
structural transition, the water diffusivity will be altered, giving insight into the internal
structure of the layer. A study performed by B. Voogt et al. [107] demonstrates this principle. The researchers used the proton local mobility and diffusivity to characterize the structure
of hard and soft latex during drying. By measuring the T2-relaxation time with different
echo times, the diffusion coefficient was determined, see Equation (14). Figure 7 shows
the diffusion coefficient measured at different times steps during drying. From t = 30 min
onward, the diffusion constant was too low to be determined. As a result of drying, the
particles’ mobility will be restricted lowering the auto-diffusion D (Figure 7) and the T2
of the atoms. The T2- of latex remained rather constant around 0.1 ms, indicating that the
proton mobility is constant. 4.1. Structure and Structural Evolution In this particular case,
the researchers identified the correlation maps at a specific location of 10 mm below the
surface. The advantage of the GARField NMR could be to perform the correlation maps at
different positions and study the different pore fraction at different positions, however, this
was not done in this particular study. Figure 5. T1-T2-correlation maps of white cement with water–cement ratio of 0.4. Figures are
taken from the curing process under water after 2 days (left), 6 days (middle) and 7 days (right). A development in pore space can be observed by the separation of the single peak into smaller
peaks when approaching 6 and 7 days. (Reprinted from Magnetic Resonance Imaging, Volume 25,
Issue 4, Peter J. McDonald, Jonathan Mitchell, Michael Mulheron, Luc Monteilhet, Jean-Pierre Korb,
Two-dimensional correlation relaxation studies of cement, pages 470–473, Copyrights 2007, with
permission from Elsevier [54]). Figure 5. T1-T2-correlation maps of white cement with water–cement ratio of 0.4. Figures are
taken from the curing process under water after 2 days (left), 6 days (middle) and 7 days (right). A development in pore space can be observed by the separation of the single peak into smaller
peaks when approaching 6 and 7 days. (Reprinted from Magnetic Resonance Imaging, Volume 25,
Issue 4, Peter J. McDonald, Jonathan Mitchell, Michael Mulheron, Luc Monteilhet, Jean-Pierre Korb,
Two-dimensional correlation relaxation studies of cement, pages 470–473, Copyrights 2007, with
permission from Elsevier [54]). Polymers 2022, 14, 798 15 of 32 15 of 32 In a follow-up study [55], the dynamic porosity in cement paste during water up-
take and drying was studied, using T2-relaxation. Three main groups were found that
correspond to different pore sizes: hydrates inner layers of 1 nm (T2 = 120 µs), gel pores
of 3–5 nm (T2 = 360 µs) and capillary pores larger than 5 nm (T2 = 1080 µs). Figure 6,
left, shows typical signal decays where the solid line is the total decay and marked with
dashed lines are the contributions of the different pores. For completeness, the authors
added a dotted line representing the part coming from the crystalline solid. Using this
multi-exponential decay, the researchers could follow the different types of hydrogen atoms
during evaporation (Figure 6 middle) and moisture uptake (Figure 6 right). 4.1. Structure and Structural Evolution By analyzing the diffusion constants and relaxation times,
they found that water changes from a free state towards pore water and finally water that
is physically bound to the polymer matrix. The researchers also observed a difference in
packing between the soft and hard type of latices. The relative concentration of the polymer
increased in the soft type but remained constant in the hard type. This indicates that in the
soft type, the particles come closer together, forming a close packed structure, while for the
hard type, this is not possible due to the hardness. These measurements clearly show the
restricted movement of the hydrogen atoms. In a similar study performed by V. Baukh et al. [141], multilayered coatings were
investigated. The authors found that the diffusion coefficient increased with water content
and that binding of water to the polymer was stronger at low water concentrations. In
other studies, diffusion coefficients were measured in order to determine the amount of
bacteria [123] or structural changes in water-swollen cellophane [146]. As the diffusion coefficient depends on the structure of the material, it can also be
used to differentiate layers in heterogeneous structures. P. J. McDonald et al. [52] used this
method to determine the diffusion constants and profiles of the human skin in vivo. The Polymers 2022, 14, 798 16 of 32 measurements revealed a clear contrast between the stratum corneum and viable epidermis. This contrast was largely attributed to a difference in diffusion constant between both
layers. Comparable studies on human skin that characterized the different skin layers were
performed on GARField [51,147] and MOUSE [138,148]. In addition to water and polymers,
studies on small penetrants such as toluene and n-hexane in PE pellets have also been
performed [149]. measurements revealed a clear contrast between the stratum corneum and viable epidermis. This contrast was largely attributed to a difference in diffusion constant between both
layers. Comparable studies on human skin that characterized the different skin layers were
performed on GARField [51,147] and MOUSE [138,148]. In addition to water and polymers,
studies on small penetrants such as toluene and n-hexane in PE pellets have also been
performed [149]. Figure 6. Left: Signal intensity decay measured by a quadrature echo train. The solid line is the
total fit. The dashed lines represent the fractions with different T2 times (120 µs, 360 µs and 1080 µs). 4.1. Structure and Structural Evolution Middle and right: Pore water fractions measured 600 µm below the surface during drying (middle)
and wetting (right). Red corresponds to water in capillary pores, green to gel sized pores and blue to
interlayer spaces. Black represents the total hydrogen content. (Reprinted from Cement and Concrete
Research, Volume 133, Peter J. McDonald, Ors Istok, Magdalena Janota, Agata M. Gajewicz-Jaromin,
David A. Faux, Sorption, anomalous water transport and dynamic porosity in cement paste: A
spatially localised 1H NMR relaxation study and a proposed mechanism [55]). Figure 6. Left: Signal intensity decay measured by a quadrature echo train. The solid line is the Figure 6. Left: Signal intensity decay measured by a quadrature echo train. The solid line is the
total fit. The dashed lines represent the fractions with different T2 times (120 µs, 360 µs and 1080 µs). Middle and right: Pore water fractions measured 600 µm below the surface during drying (middle)
and wetting (right). Red corresponds to water in capillary pores, green to gel sized pores and blue to
interlayer spaces. Black represents the total hydrogen content. (Reprinted from Cement and Concrete
Research, Volume 133, Peter J. McDonald, Ors Istok, Magdalena Janota, Agata M. Gajewicz-Jaromin,
David A. Faux, Sorption, anomalous water transport and dynamic porosity in cement paste: A
spatially localised 1H NMR relaxation study and a proposed mechanism [55]). Figure 6. Left: Signal intensity decay measured by a quadrature echo train. The solid line is the
total fit. The dashed lines represent the fractions with different T2 times (120 µs, 360 µs and 1080 µs). Middle and right: Pore water fractions measured 600 µm below the surface during drying (middle)
and wetting (right). Red corresponds to water in capillary pores, green to gel sized pores and blue to
interlayer spaces. Black represents the total hydrogen content. (Reprinted from Cement and Concrete
Research, Volume 133, Peter J. McDonald, Ors Istok, Magdalena Janota, Agata M. Gajewicz-Jaromin,
David A. Faux, Sorption, anomalous water transport and dynamic porosity in cement paste: A
spatially localised 1H NMR relaxation study and a proposed mechanism [55]). Figure 7. Diffusion coefficient of free water within the hard- and soft-type latex. (Reprinted from
Progress in Organic Coatings, Volume 123, Benjamin Voogt, Henk Huinink, Bart Erich, Jurgen Scheerder,
Paul Venema and OlafAdan, Water mobility during drying of hard and soft type latex: Systematic
GARField 1H NMR relaxometry studies, Pages 111–119, Copyrights 2018, with permission from
Elsevier [107]). 4.1. Structure and Structural Evolution Figure 7. Diffusion coefficient of free water within the hard- and soft-type latex. (Reprinted from
Progress in Organic Coatings, Volume 123, Benjamin Voogt, Henk Huinink, Bart Erich, Jurgen Scheerder,
Paul Venema and OlafAdan, Water mobility during drying of hard and soft type latex: Systematic
GARField 1H NMR relaxometry studies, Pages 111–119, Copyrights 2018, with permission from
Elsevier [107]). 4.2. Film Formation—Drying Film formation is the process in which a polymer emulsion or colloidal dispersion will
overcome their stabilizing forces to form a continuous film. These stabilizing forces can be
overcome when the continuous liquid phase evaporates, forcing the emulsion droplets or
polymer particles together. The evaporation of excess liquid is referred to as the drying
phase of the film formation process. When enough liquid has evaporated, the polymers
or colloidal particles need to form a continuous film. After solvent evaporation, there are
two types of processes: curing and coalescence [150]. In the curing process, a stable film is
formed by cross-links between reactive polymers. On the other hand, in coalescence, the
polymer particles will first coalescence and deform to form a close packed structure. In Polymers 2022, 14, 798 17 of 32 17 of 32 order to form a homogeneous film, the deformation is followed by entanglement of the
polymer chains. Particle boundaries disappear, which will lower the film roughness [22]. All these combination of drying, curing and coalescence make film formation a complicated
process. Since most applied films and coatings undergo this film formation, this section
is dedicated to studies that are focused this topic. First, drying experiments and then the
subsequent processes are discussed. During drying, water or solvent evaporates from the polymer film, mostly reducing
the overall thickness. The drying process can be monitored with an NMR signal. A
representative example of a study on the drying processes of waterborne colloidal films was
performed by J.-P. Gorce et al. [151]. Measurements were performed on a GARField setup
using an OW-sequence with a 8.7 µm resolution. Profiles of the NMR signal measured at
different times are shown in Figure 8. The width of the signal corresponds to the thickness of
the polymer film. First of all, evaporation leads to film shrinkage due to the disappearance
of hydrogen atoms. When all water has evaporated, the profile reaches its final thickness,
corresponding to 150 µm. This decrease in profile thickness can be used to characterize
a drying front and a corresponding drying rate. Secondly, the maximum signal intensity
reduces over time from 0.7 to 0.4. To understand the reduced signal, we need to introduce
ρ = ρa + ρb in Equation (7), S ∝ρaexp(−te/T2a) + ρbexp(−te/T2b)
(22) (22) S ∝ρaexp(−te/T2a) + ρbexp(−te/T2b) where we have left out the T1-term. 4.2. Film Formation—Drying Additionally, the water fractions where determined
for every profile, where for the left figure the water fraction were 0.48 (62 min), 0.33 (602 min), 0.23
(821 min), 0.15 (956 min) and 0.09 (1061 mi.), while for the right figure, the water fractions were
0.44 (2 min), 0.25 (7 min), 0.15 (13 min) and 0.10 (31 min). (Reprinted by permission from Springer
Nature Customer Service Centre GmbH Springer Nature: The European Physical Journal E—Soft Matter,
Vertical water distribution during the drying of polymer films cast from aqueous emulsions, J.-P. Gorce et al., 2014 [151]). Figure 8. NMR profiles measured during film drying of an alkyd emulsion in a closed environmen Figure 8. NMR profiles measured during film drying of an alkyd emulsion in a closed environment
with Pe = 0.2 (left) and with Pe = 16 (right). Additionally, the water fractions where determined
for every profile, where for the left figure the water fraction were 0.48 (62 min), 0.33 (602 min), 0.23
(821 min), 0.15 (956 min) and 0.09 (1061 mi.), while for the right figure, the water fractions were
0.44 (2 min), 0.25 (7 min), 0.15 (13 min) and 0.10 (31 min). (Reprinted by permission from Springer
Nature Customer Service Centre GmbH Springer Nature: The European Physical Journal E—Soft Matter,
Vertical water distribution during the drying of polymer films cast from aqueous emulsions, J.-P. Gorce et al., 2014 [151]). g
p
g
y
g
y
with Pe = 0.2 (left) and with Pe = 16 (right). Additionally, the water fractions where determined
for every profile, where for the left figure the water fraction were 0.48 (62 min), 0.33 (602 min), 0.23
(821 min), 0.15 (956 min) and 0.09 (1061 mi.), while for the right figure, the water fractions were
0.44 (2 min), 0.25 (7 min), 0.15 (13 min) and 0.10 (31 min). (Reprinted by permission from Springer
Nature Customer Service Centre GmbH Springer Nature: The European Physical Journal E—Soft Matter,
Vertical water distribution during the drying of polymer films cast from aqueous emulsions, J.-P. Gorce et al., 2014 [151]). In a comparable study performed by P. Ekanayake et al. [152], density profiles of
drying colloidal films revealed that the particle concentration gradient inside this dense
layer of colloidal particles scales with Pe0.8. Furthermore, in this study, the GARField setup
was chosen as it has the best resolution, allowing to see this thin dense top layer. 4.2. Film Formation—Drying Here, the signal is split into a term coming from the
solvent (ρa, T2a) and one coming from the polymer or colloidal particles (ρb, T2b). When
solvent is released, the total active hydrogen atoms ρ is almost unchanged as hydrogen
atoms from the water are replaced by the ones from the polymer. Therefore, the signal
decrease cannot be attributed to a loss in active hydrogen atoms. However, the relative
contributions do change, where during drying ρa reaches zero, ρb reaches its maximum. As
the mobility of the polymer is much lower then that of free water, the T2 of the polymer is
lower then that of free water by at most one order of magnitude (see Section 2.2). Faster
relaxation leads to a lower signal, where the final signal amplitude is closely related to the
mobility of the polymer phase which in this particular case was very low. In the beginning,
the signal intensity (0.7) is dominated by the solvent (ρaexp(−te/T2a)); after drying, the
signal will be determined by the polymer (ρbexp(−te/T2b)) which due to a faster decay
will have a lower signal intensity (0.4). g
y
In the same study, the influence of the Peclet number Pe = vLe f /D was investigated. v[m/s] is the speed of the receding water front, Le f [m]—the thickness of the layer and
D[m2/s]—the diffusion coefficient. By controlling the thickness, the speed of the receding
water front and the diffusion coefficient, the Pe number could be adjusted. When Pe > 1,
advection dominates, but for Pe < 1, diffusion is more important. Figure 8 left shows
profiles for the case were Pe = 0.2, whereas right represents Pe = 16. A difference in profile
shape was observed that indicates that diffusion towards the surface can keep up with the
evaporation (flat profiles) when Pe = 0.2, but not when Pe = 16, resulting in a concentration
gradient of water molecules near the surface. The formation of this dense polymer layer to
the surface showed limited diffusion towards the surface, slowing down evaporation. 18 of 32 18 of 32 Polymers 2022, 14, 798 Figure 8. NMR profiles measured during film drying of an alkyd emulsion in a closed environment
with Pe = 0.2 (left) and with Pe = 16 (right). 4.2. Film Formation—Drying g
p
y
In some cases, drying can induce the formation of a skin layer that can trap water. These skin layers reduce solvent evaporation and significantly slow down the drying
process [23,24]. E. Ciampi et al. [23] investigated skin formation upon drying of poly(vinyl
alcohol) (PVOH). Profiles were measured with a GARField NMR for an initial PVOH
content of 10 WT% and 25 WT%. The profiles for the 10 WT% dried similarly to the
ones measured by [151]. In case of high-polymer-weight fractions (25 WT%), a skin layer
developed that trapped water inside the profile, leading to slower evaporation. Similar studies of the drying behavior of different polymers, such as alkyd lay-
ers [89,117,153,154], latex layers [107], gelatin layers [155] and even dental resins [119,156–158],
have been conducted using the GARField, STRAFI and MOUSE NMR setups. 4.3. Film Formation—Curing In case of reactive compounds, a curing reaction can start when enough water evapo-
rates and the polymer content is high enough. Measurements with a GARField on drying
alkyd coatings [153] illustrate the capability to measure the drying–curing film formation
process. In this study performed be S. J. F. Erich et al., NMR profiles on alkyd coatings
were measured every 10 min with an OW-sequence. Measured profiles can be seen in
Figure 9. The profiles reveal this two-stage process where the polymer layer shrinks and the
intensity drops due to solvent evaporation, as explained in previous section. This process
is indicated by the arrows in Figure 9 and it accounts for a rapid loss in signal intensity in
the beginning. After drying, a (reaction) curing front moves into the polymer film. The
signal loss due to curing of polymers can be attributed to the loss in mobility of the polymer. When polymers are cured, they become more rigid, the T2 drops and signal is lost when
the T2 approaches te. 19 of 32 19 of 32 Polymers 2022, 14, 798 Figure 9. Hydrogen profiles of a solvent-borne alkyd sample measured during drying. The top of
the coating corresponds to the left side, while the cover glass can be found on the right. The vertical
dashed line indicates the top of the film after drying. (Reprinted from S. J. F. Erich, J. Laven, L. Pel, H. P. Huinink, and K. Kopinga, “Dynamics of cross linking fronts in alkyd coatings”, Appl. Phys. Lett. 86,
134105 (2005) https://doi.org/10.1063/1.1886913, with permission of AIP Publising [153]). Figure 9. Hydrogen profiles of a solvent-borne alkyd sample measured during drying. The top of
the coating corresponds to the left side, while the cover glass can be found on the right. The vertical
dashed line indicates the top of the film after drying. (Reprinted from S. J. F. Erich, J. Laven, L. Pel, H. P. Huinink, and K. Kopinga, “Dynamics of cross linking fronts in alkyd coatings”, Appl. Phys. Lett. 86,
134105 (2005) https://doi.org/10.1063/1.1886913, with permission of AIP Publising [153]). In the study of S. J. F. Erich et al. [153], curing experiments are performed on water-
borne and solvent-borne alkyds. Both systems showed the same behavior: a clear evapora-
tion and curing stage could be distinguished. 4.3. Film Formation—Curing In a related paper [89], NMR and confocal
Raman Microscopy were compared, and the curing could be related to the disappearance
of double bonds and the formation of oxidative cross-links. These cross-links lowered the
mobility and reduced the NMR signal, giving rise to the observed curing front. Tracking
these fronts in different environmental conditions revealed that oxygen supply was the
limiting factor in the curing front dynamics. g
g
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Further, the effect of drying on porous media [117], catalysts [159–162] and pigment
volume concentrations [163] was studied. Curing also plays a role in dental resins. By exposing the resin to a light source, it
will begin to polymerize and shrink. This polymerization shrinkage (PS) is crucial for
the durability of the material, as it will determine stresses and strains in the film and the
uptake of fluids and bacteria. A polymer that is widely used as a dental bonding agent is
dimethacrylic acid. Curing studies on this polymer have been performed with a STRAFI
NMR [119,156–158]. In an experiment performed by T. Nunes et al. [119], glass vials
were filled with a liquid resin. The liquid resin was exposed to light. Different groups of
hydrogen atoms could be discriminated based on their T2- and T1-relaxation times: mobile
molecules (free monomers) with long relaxation times and rigid molecules (cured AB2). It was found that oxygen strongly influenced the kinetic behavior. Using STRAFI, the
influence of different cements and curing protocols could be determined and all were
shown to have an effect on the particular curing process, and they should be chosen with
care in medical applications. Other curing measurements can be found for the curing of wood glue layers [154] and
gelatin layers (biopolymer) [155]. 4.4. Film Formation—Coalescence In some applications, film formation involves coalescence (e.g., with latex particles). In case of latex dispersions, film formation occurs due to the interdiffusion of polymer
chains rather then curing. Well-known applications are water-based paints [164–167] or
pressure-sensitive adhesives [24,168]. The formation of dry, homogeneous films from colloidal dispersions such as latex in
water can be described by a three-step process [22]. First, water evaporates and the particles
concentrate and overcome their colloidal stability [169]. Next, the particles deform [170], Polymers 2022, 14, 798 20 of 32 20 of 32 trying to fill the void volume in the film. Lastly, interdiffusion of individual polymer
chains overcomes the particles’ boundaries, forming a continuous and rigid film [171]. The deformation and interdiffusion of these polymers depens on the mobility of the latex
particles, reflected by their glass transition temperature (tg) [107]. The sequence of events
can overlap in time. The processes can also influence each other. For instance, the water
fraction will influence the mobility of the polymer chains [107,172], leading to a decrease
in deformation and interdiffusion upon drying [85]. Studying these processes with NMR
can give crucial information, leading to a better understanding of unwanted effects such as
film cracking [173], trapped water [24] and irregular particle formation [174]. g
pp
g
p
To illustrate the use of high-resolution NMR for latex film formation, we use a study
by B. Voogt et al. [107] as an example. In this study, drying of both soft (tg < room tem-
perature) and hard (tg > room temperature) latices was studied inside a GARField NMR. Measurements were performed with an OW-sequence with te = 2τ = 40 µs. Figure 10
shows profiles measured during the drying of both soft- (left) and hard-type (right) latices
at an RH of 80%. As discussed in Section 4.2, a step-wise process is observed where first
evaporative drying takes place. This will induce film shrinkage due to the evaporation of
water that can be observed via a receding front. This will lead to higher concentration of
latex particles. In both films, thickness (d) and maximum signal intensity (Smax) decreased
over time. It was observed that the hard-type latex dried much faster. Hard-type latex
thickness decreased until 20 min, while for the soft latex, this only happened at 40 min. 4.4. Film Formation—Coalescence The
maximum intensity of the hard-type latex also lowered faster and ended lower at around
0.2, while for the soft latex a signal intensity of 0.6 was observed, both leveling off at 40 min. The difference in leveling off between Smax and d for hard latex suggests a continuation of
bulk water evaporation, while the latex particles are fixed in position and are not able to
keep concentrating. This is understandable as particles below their tg will form brittle and
porous networks [22] and are unable to form a continuous film. Figure 10. NMR signal intensity profiles during drying of soft- (left) and hard-type (right) latex. Indicated by Smax and d are the final maximum signal intensities and thicknesses of the coatings. (Reprinted from Progress in Organic Coatings, Volume 123, Benjamin Voogt, Henk Huinink, Bart Erich,
Jurgen Scheerder, Paul Venema and OlafAdan, Water mobility during drying of hard and soft type
latex: Systematic GARField 1H NMR relaxometry studies, Pages 111–119, Copyrights 2018, with
permission from Elsevier [107]). Figure 10. NMR signal intensity profiles during drying of soft- (left) and hard-type (right) latex. Indicated by Smax and d are the final maximum signal intensities and thicknesses of the coatings. (Reprinted from Progress in Organic Coatings, Volume 123, Benjamin Voogt, Henk Huinink, Bart Erich,
Jurgen Scheerder, Paul Venema and OlafAdan, Water mobility during drying of hard and soft type
latex: Systematic GARField 1H NMR relaxometry studies, Pages 111–119, Copyrights 2018, with
permission from Elsevier [107]). In this study by B. Voogt et al., the protons in free water and polymer where studied
simultaneously, using a multi-exponential decay analysis Section 2.2. A typical OW-decay
is shown in Figure 2 right. For both latices, a clear multi-exponential decay was observed
where the short relaxation time could be linked to the polymer and the long relaxation
time to the free water phase. Both short relaxation times are fixed at 0.1 ms, showing that
both latices have proton pools with mobilities that are not affected by drying. These are the
protons embedded within the latex. Using this multi-exponential decay, the different concentrations could be followed
over time. The researchers found that a loss of free water (ρlong) due to evaporation is Polymers 2022, 14, 798 21 of 32 independent of the lattice type. However, the latex fraction shows an increase for the
soft latex type (ρshort), indicating a further increase in concentration. 4.4. Film Formation—Coalescence This effect was not
observed in the hard latex type. The increase in proton density is the consequence of
coalescence of the particles. When film formation happens above the polymers tg, the
polymer mobility is high enough that the polymer chains can interdiffuse and form a
polymer film. This indicates that the tg of latex has a huge influence on the film formation
processes and the coalescence of the particles. This study illustrates that coalescence
of latex particles can be made visual inside the NMR setups. This makes it possible to
study the effect of multiple parameters on the coalescence kinetics and improve latex film
applications. Other studies have focused on the coalescence of particles at the liquid–air interface,
leading to a ”skin-layer” [24,175]. In certain compositions, coalescence appears near the
liquid–air interface during the drying stage. This is schematically represented in Figure 11
left. This skin-layer will block the evaporation, leading to trapped water within the film. R. Rodriguez et al. [175] studied the film formation for different compositions: acrylic
copolymer (SM0), a hybrid latex containing 25% PDMS (SM25), and a blend of the acrylic
(SM0) with 11 wt% PDMS emulsion. Drying measurements on two of these compositions
are shown in Figure 11, namely, SM0 (b) and SM25 (c). The SM0 polymer has a much lower
mobiltiy than the SM25. Therefore, the signal intensity for SM0 is almost zero after drying,
while for the SM25 there is still signal. By setting the echo time to 180 µs, the researchers
made sure that almost no signal was attributed to the SM0 polymer. The signal could be
attributed to free water. In both drying experiments, the signal intensity increases in the
depth of the film. This increase was also observed in other studies [24] and was attributed
to a gradient in free water. In the neat acrylic dilution (SM0), a deviation from this linear
behavior is observed where a layer with lower signal intensity appears at the top. This
indicates a step in the free water concentration and a denser packing of latex particles at
the surface (Figure 11a). This layer increased in thickness over time. This drastic step in
particle density was not observed in the SM25, see Figure 11c. Here, a more uniform profile
is observed that reached its final form at 55 min. Figure 11. 4.4. Film Formation—Coalescence (a): schematic representation of latex packing near the surface accompanied by a represen-
tation of the corresponding NMR signal. NMR profiles measured over time for (b) acrylic copolymer
latex (SM0) and (c) 25 wt% PDMS on the acrylic monomer(SM25) (Reprinted with permission from
Macromolecules 2012, 45, 4, 1937–1945. Copyright 2012 American Chemical Society [175]). Figure 11. (a): schematic representation of latex packing near the surface accompanied by a represen-
tation of the corresponding NMR signal. NMR profiles measured over time for (b) acrylic copolymer
latex (SM0) and (c) 25 wt% PDMS on the acrylic monomer(SM25) (Reprinted with permission from
Macromolecules 2012, 45, 4, 1937–1945. Copyright 2012 American Chemical Society [175]). Other studies used similar NMR measurements to study the effect of glass transition
temperature [176], surfactant [168], tackifying resins (TR) [24], salts [177], different amounts
of carboxylic acid functional groups [105] and the Peclet number [151] on the film formation
process. GARField NMR seems to be the best technique for these processes, as it has a
high-enough FOV to cope with the latex films and can give the best resolution. However,
measurements with a MOUSE were also performed on latex films [120]. This, however, led
to lower resolutions (30 µm) and was hampered by long measurement times. 4.5. Diffusion/Penetration Liquid uptake and diffusion in thin layers is important for coatings, the printing
industry or sustainability of materials. Water and solvents can weaken interactions between
neighboring polymers and ultimately lead to failure of the thin layer by softening or
cracking. Both the amount of liquid and the speed of penetration are crucial parameters in Polymers 2022, 14, 798 22 of 32 22 of 32 characterizing the stability of thin layer. In this section, it will be shown how NMR profiling
has been used to measure liquid penetration over time. A crucial parameter for solvent
penetration is the diffusion coefficient. This parameter is, however, difficult to measure
because it depends on morphology such as crystallinity [178] or temperature. p
p
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y
y
p
To illustrate the capability of high-resolution NMR depth profiling in studying pen-
etration and diffusion experiments, a study by N. J. W. Reuvers et al. [179] is used. The
water uptake in thin nylon-6 films was measured with a GARField NMR. p
y
The water uptake was measured using an OW-sequence. A water uptake experiment
in a 200 µm thick nylon-6 is shown in Figure 12 left. When going from right to left, the
glass plate, glue layer, nylon and water can be observed which can also be seen in the
schematic picture of the setup. While the glass plate cannot be imaged by the NMR, a
clear difference between the silicon glue, nylon film and water is observed. The observed
differences in signal intensities can be linked to varying T1 and T2-times. Shown with a bold
line is the signal intensity measured before the experiment. The researchers distinguished
three different processes. (1) A liquid fronts develops, traveling towards the bottom of
the layer (t < 6 h), (2) water distributes equally over the film (6 h < t < 10 h) and (3) a
slower processes occurs where a small signal increase is observed near the glass–polymer
interface. Using these profiles, the researchers determined the actual diffusion coefficient
within the layer. To be able to do this, the researchers needed to convert the NMR signal
intensity to moisture content (θ). They could link the NMR signal to a moisture profile using
gravimeter calibration [179]. The resulting relationship was nonlinear and was attributed
to plasticization and a change in relaxation of free water. 4.5. Diffusion/Penetration The NMR signal profiles in
Figure 12 can be viewed as a supper position of a liquid front and plasticization front. Using the relation between moisture content and NMR signal, the original NMR profiles
(Figure 12 left) are converted to moisture content profiles (Figure 12 right). The small signal
increase observed before (3) could be attributed to polymers that become more mobile and
contribute to the signal. Figure 12. Left: Liquid profiles measured during the uptake of water into 200 µm thick nylon
membranes. Right: same profiles as in the middle graph but after replacing the signal intensity
with the moisture content. (Reprinted with permission from Macromolecules 2012, 45, 4, 1937–1945. Copyright 2012 American Chemical Society. [179]). Figure 12. Left: Liquid profiles measured during the uptake of water into 200 µm thick nylon
membranes. Right: same profiles as in the middle graph but after replacing the signal intensity
with the moisture content. (Reprinted with permission from Macromolecules 2012, 45, 4, 1937–1945. Copyright 2012 American Chemical Society. [179]). The moisture profiles were used to extract the diffusion coefficient of water within
the nylon film. The calculated diffusion coefficients are shown in Figure 13. The diffusion
coefficient increases with increasing moisture content. To study the signal change due to plasticizing, the excess water is replaced by D2O. D2O has the same characteristics as water but will not contribute to the NMR signal. It was
observed that half the NMR signal in the profiles was linked to hydrogen atoms of water
and half of the signal to mobilized polymer. Additionally, the glass transition temperature
and T2-relaxation could be linked with moisture content (Figure 13 right). In a follow-up Polymers 2022, 14, 798 23 of 32 study, it was found that the plasticization lags behind the water migration in these nylon-6
thin films [180]. Figure 13. Left: the diffusion coefficient (D) in function of moisture content. Right: The glass
transition temperature (tg) and average relaxation time (T2) in function of moisture content. (Reprinted
with permission from Macromolecules 2008, 41, 22, 8537–8546. Copyright 2012 American Chemical
Society [175]). Figure 13. Left: the diffusion coefficient (D) in function of moisture content. Right: The glass
transition temperature (tg) and average relaxation time (T2) in function of moisture content. (Reprinted
with permission from Macromolecules 2008, 41, 22, 8537–8546. Copyright 2012 American Chemical
Society [175]). Similar penetration studies have been performed on dental resins. Using STRAFI,
G. 4.5. Diffusion/Penetration Hunter et al. [158] studied the uptake of water and water/ethanol mixtures into a
commercial dental resin. Measurements with different ethanol concentrations revealed
that the diffusion coefficient increased with ethanol content. Additionally, transport in
ceramic substrates [181], multilayer coatings [141,182], glassy pellets of the starch polymer
amylose [124], cement pastes [136] glue lines [154], vulcanized rubber [183] and nylon-
6 [140,141] have been studied. In addition to the penetration of water, the transport of
different ions such as Mn2+ and Cu2+ [184], different salt solutions [185] and the ingress of
vapours [186,187] have also been studied with the NMR setups. Finally, the influence of
stress [188] on the penetration behavior was studied by V. Baukh et al. 4.6. Thin Films on Site: Cultural Heritage In some applications, measurements of thin layers need to be performed outside the
lab because it is impossible to collect small samples or to recreate a similar structure. As the
STRAFI and GARField setups are located within the lab and require samples of a specific
shape and size, measurements on site are performed by NMR-MOUSE. The portability of
the NMR-MOUSE makes it possible to measure samples with infinite lateral dimensions
where limits are mostly bound because of time constraints. One big category of such
applications studied with the NMR-MOUSE is cultural heritage [189]: for example, in case
of paintings on wood. A significant issue with cultural heritage is structural damage during
conservation. Using the NMR-MOUSE, the internal structure of paint layers can be studied. These studies can help in characterizing different sources of damage and identifying the
best conservation environment. Damage of cultural heritage is mainly caused by moisture uptake which, for example,
in wooden painting or walls, can cause degradation over time. Measuring the moisture
distribution can provide information on the conditions in which the painting should be
stored and can help in preserving the cultural heritage [8]. In this section, a closer look
at some analysis methods will be given, demonstrating the use of the NMR-MOUSE on
cultural heritage. g
B. Blumich et al. [7] demonstrated the use of the NMR-MOUSE for studying the layered
structure of paintings. They started with a wood panel covered by a primer and one or
multiple layers of paint. They tried to mimic the structure found in old paintings. Figure 14
shows a picture of the structure where two positions are marked where either one (1) or
two (2) layers of paint are used. Depicted in Figure 14b are the depth profiles measured by
an NMR-MOUSE of the two sides shown in Figure 14a. Both paint layers had good signal
and the thickness of the layers is also reflected in the width of the NMR signals. Imaging B. Blumich et al. [7] demonstrated the use of the NMR-MOUSE for studying the layered
structure of paintings. They started with a wood panel covered by a primer and one or
multiple layers of paint. They tried to mimic the structure found in old paintings. Figure 14
shows a picture of the structure where two positions are marked where either one (1) or
two (2) layers of paint are used. 4.6. Thin Films on Site: Cultural Heritage Depicted in Figure 14b are the depth profiles measured by
an NMR-MOUSE of the two sides shown in Figure 14a. Both paint layers had good signal
and the thickness of the layers is also reflected in the width of the NMR signals. Imaging Polymers 2022, 14, 798 24 of 32 24 of 32 different layers was performed both with total amplitude measurement and characterizing
the signal decays (CPMG). In Figure 14e, the total NMR signal is measured along the
thickness of the painting revealing a layered structure where paint, primer and wood are
observed. Using this method, the thickness of the different layers could be monitored at
different points, revealing damage at certain positions. The CPMG could give information
about changes within the layers over time. g
y
In the same paper, the authors also demonstrated the ability to characterize dam-
aged old paper structures by measuring the hydrogen atoms in the cellulose fibres, time-
dependent water uptake and drying in stone. Even old master violins such as a Stradivari
were investigated in this way. The study revealed that the master violins’ wood density
increased with age, which determined the quality of the instrument. The possibilities of
this measurement technique was also used for the ancient Roman fresco and the bricks in
the walls of the cryptoporticus at Colle Oppio in Rome [190], the degradation of historical
paper [76] and the conservation treatments on paintings [191–193]. 4.7. Conclusions In GARField,
the problem with the inhomogenous field is solved by specially designed poles leading Polymers 2022, 14, 798 25 of 32 to the best space and time resolution found in all the setups. Moreover, Fourier STRAFI
exists, which can measure much faster then conventional STRAFI. However, due to the
arrangement of the magnetic field, a solenoid-shaped coil is needed that compared to the
surface coil in GARField will always have lower sensitivity. A drawback of this special
design is that GARField lacks the ability to measure samples larger then 400 µm. To
measure larger samples, STRAFI would be more appropriate. Finally, the NMR-MOUSE
has proven to be valuable in cultural heritage because it is the only mobile device able to
measure depth profile on-site in a non-invasive manner. p
p
A variety of applications such as drying, film formation, penetration and measure-
ments on cultural heritage have been reviewed illustrating the potential of these NMR-
profiling tools. By measuring hydrogen profiles over time, the setups were able to follow
drying, curing and penetration fronts. Using these profiles, the diffusion coefficient and
front kinetics could be determined for a wide variety of parameters. Detailed structural
information about thin layers could be gathered by measuring T2, T1 relaxation times and
signal intensity. The information could be used to follow film formation, film degradation,
penetration and diffusion experiments. p
p
The current measurement techniques lack the ability to measure below 1 min. Future
improvements towards higher time resolutions is, for example, useful in the printing
industry where the penetration occurs at timescales of 100 ms. Great potential was shown
by R. J. K. Nicasy et al. [194] that demonstrated the use of high-speed NMR to profile
liquid uptake in non-transparent porous media with time resolutions of 10 ms. This was
only shown for liquid penetration but could be extended towards the characterization of
chemical structures. In addition to measuring speed, improvement towards the amount of
chemical information obtained with the setup can be achieved by combining the above-
mentioned techniques with NMR spectroscopy. Currently, no such studies are available,
but they could be very valuable to determine chemical components and reactions in
much more detail. Lastly, by combining the NMR techniques with other measurement
setups, a better understanding of the information can be achieved. 4.7. Conclusions Three different high-resolution NMR setups able to study thin layers between 10 and
1000 µm were reviewed, namely, STRAFI, GARField and MOUSE. These NMR techniques
are all able to give spatial and time-resolved information about thin layers. Figure 14. (a): Easel painting model with a wooden background covered by a primer and one (1)
or two (2) paint layers. (b): measured depth profiles for the position marked in Figure 14a. A clear
difference in thickness is observed between both layers where one layer is thinner than the two layers. (d) Signal decay measurements at position 2 for different depths. (e) Complete depth profile through
the painting indicating the different layers. (Reprinted with permission from Acc. Chem. Res. 2010, 43,
6, 761–770. Copyright 2010 American Chemical Society [7]). Figure 14. (a): Easel painting model with a wooden background covered by a primer and one (1)
or two (2) paint layers. (b): measured depth profiles for the position marked in Figure 14a. A clear
difference in thickness is observed between both layers where one layer is thinner than the two layers. (d) Signal decay measurements at position 2 for different depths. (e) Complete depth profile through
the painting indicating the different layers. (Reprinted with permission from Acc. Chem. Res. 2010, 43,
6, 761–770. Copyright 2010 American Chemical Society [7]). The setups used two different acquisition methods: slice-selective pulse and Fourier-
based measurements. In the slice-selective pulse measurements performed by MOUSE
and in conventional STRAFI, the signal is recorded in a step-wise manner, whereby the
pulse excites the region of interest. In this case, the resolution is determined by the pulse
length given by ∆r ≈1/tpγG. High resolution can be achieved at the expense of long
measurement times. The advantage of the step-wise acquisition is that sample size is only
limited by the aperture used. Unfortunately, the resolution is limited by the large curvatures
found in the main magnetic field # »
B0 of both STRAFI and MOUSE and should be determined
experimentally. In GARField and in some cases in STRAFI, the acquisition is achieved by
a Fourier analysis. The sample is excited with one pulse (limiting the total measurement
area) where the resolution is determined by the acquisition time, given by ∆r ≈1/taγG. As the sample is measured using one pulse, measurements are much faster. 4.7. Conclusions This has already been
shown to be valuable with confocal Raman microscopy [89] and electrochemical impedance
spectroscopy [195], but could be extended towards other techniques. Author Contributions: Conceptualization, R.N., H.H., B.E. and A.O.; writing—original draft prepa-
ration, R.N., H.H., B.E. and A.O.; writing—review and editing, R.N., H.H., B.E. and A.O.; supervision,
H.H., B.E. and A.O.; project administration, H.H., B.E. and A.O.; funding acquisition, H.H., B.E. and
A.O. All authors have read and agreed to the published version of the manuscript. Funding: This publication is part of the project PQP (Print Quality and Particles) (with project number
17099) of the research programme Collaboration program High Tech Systemen en Materialen (HTSM)
2018 TTW] which is (partly) financed by the Dutch Research Council (NWO). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 2.
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BioMed Central BioMed Central Open Ac
Research article
Expressed sequence tags (ESTs) from immune tissues of turbot
(Scophthalmus maximus) challenged with pathogens
Belén G Pardo1, Carlos Fernández1, Adrián Millán1, Carmen Bouza1,
Araceli Vázquez-López1, Manuel Vera1, José A Alvarez-Dios2, Manuel Calaza2,
Antonio Gómez-Tato3, María Vázquez4, Santiago Cabaleiro4,
Beatriz Magariños5, Manuel L Lemos5, José M Leiro5 and Paulino Martínez*1 Address: 1Departamento de Genética. Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus de Lugo, 27002 Lugo, Spain,
2Departamento de Matemática Aplicada, Facultad de Matemáticas, Universidad de Santiago de Compostela, 15782 Santiago de Compostela,
Spain, 3Departamento de Geometría y Topología, Facultad de Matemáticas, Universidad de Santiago de Compostela, 15782 Santiago de
Compostela, Spain, 4Cluster de la Acuicultura de Galicia (CETGA), Punta de Couso s/n, 15965, Aguiño (Ribeira), A Coruña, Spain and
5Departamento de Microbiología y Parasitología. Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain Email: Belén G Pardo - mariabelen.gomez@usc.es; Carlos Fernández - carlos.fernandez.lopez@usc.es; Adrián Millán - adrian.millan@usc.es;
Carmen Bouza - mcarmen.bouza@usc.es; Araceli Vázquez-López - aravazq@usc.es; Manuel Vera - manuel.vera@usc.es; José A Alvarez-
Dios - joseantonio.alvarez.dios@usc.es ; Manuel Calaza - mcalaza@usc.es; Antonio Gómez-Tato - antonio.gomez.tato@usc.es;
María Vázquez - ruizocenda@cetga.org; Santiago Cabaleiro - cabaleiro@cetag.org; Beatriz Magariños - beatriz.magarinos@usc.es;
Manuel L Lemos - manuel.lemos@usc.es; José M Leiro - josemanuel.leiro@usc.es; Paulino Martínez* - paulino.martinez@usc.es
* Corresponding author * Corresponding author Received: 23 June 2008
Accepted: 25 September 2008 Received: 23 June 2008
Accepted: 25 September 2008 This article is available from: http://www.biomedcentral.com/1746-6148/4/37 This article is available from: http://www.biomedcentr © 2008 Pardo 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. © 2008 Pardo 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. BMC Veterinary Research Open Access Research article Open Ac
Research article
Expressed sequence tags (ESTs) from immune tissues of turbot
(Scophthalmus maximus) challenged with pathogens
Belén G Pardo1, Carlos Fernández1, Adrián Millán1, Carmen Bouza1,
Araceli Vázquez-López1, Manuel Vera1, José A Alvarez-Dios2, Manuel Calaza2,
Antonio Gómez-Tato3, María Vázquez4, Santiago Cabaleiro4,
Beatriz Magariños5, Manuel L Lemos5, José M Leiro5 and Paulino Martínez*1 Background
h
b
( for the development of EST resources in Japanese flounder
(8856 ESTs) and Atlantic halibut (17659 ESTs) [21-28],
EST resources in turbot are scarce (3171 ESTs). Less than
800 sequences have been deposited to date in the NCBI
nucleotide database, most of them from anonymous mic-
rosatellite searching [29]. g
The turbot (Scophthalmus maximus; Scophthalmidae; Pleu-
ronectiformes) is a commercially valuable flatfish that has
been intensively cultured for the last decade. Its produc-
tion has steadily increased to 7120 tonnes in 2006 (80%
from Spain; FEAP, 2006) and represents one of the most
promising aquaculture species in Europe. However, dis-
ease outbreaks in turbot have occurred frequently and
losses due to infections constitute a serious problem for its
culture [1,2]. The use of antibiotics, vaccines and fish
health management practices has partially solved the
problem, but the achievement of large-scale production in
the highly competitive world market requires enhancing
resistance of cultured fish to diseases. Information on the
immune response of turbot is still limited, and little is
known about host-pathogen interactions in fish species. The screening and identification of immune-relevant
genes is essential to analyze the genetic basis for infection,
immunity and resistance to pathogens of economic rele-
vance in aquaculture. Expressed sequence tag (EST) anal-
ysis is a powerful approach to provide a rapid and efficient
method to go from expressed sequences to genes. ESTs are
essential for studies of gene function [3,4], but are also
useful to identify polymorphic gene markers, such as mic-
rosatellites and single nucleotide polymorphisms (SNPs)
[5-8]. These markers are the basis for genetic and physical
mapping, and for comparative genome analysis [9-11]. From a practical perspective, maps can be applied for
assisted selection programmes (MAS) and eventually for
identification of genes related with quantitative traits
(QTL) [12,13]. In addition, ESTs constitute the basic
resources to develop microarrays for functional genomics
studies [14]. With the aim of increasing the genomic resources in tur-
bot and identifying relevant genes for immunity, three
cDNA libraries were constructed from mRNA isolated
from immunity-related tissues of turbot (liver, spleen and
head kidney) at different times after infection with Aerom-
onas salmonicida and the scuticociliate parasite Philasterides
dicentrarchi. These pathogens are responsible for impor-
tant disease outbreaks in turbot, as well as in other culture
fish species [30-33]. Our main goal was to obtain the most
accurate information possible to address functional
genomic studies on disease resistance. Background
h
b
( However, the use of
non-normalized cDNA libraries made it feasible to get a
preliminary picture of the turbot genetic response to path-
ogens through analyzing transcript distribution among
infected vs. control libraries. A total of 12584 ESTs were
sequenced and compared to GenBank database and a
large array of defence or immune-related genes was iden-
tified. Also, this large scale EST study increased the
number of putative markers for mapping. A total of 191
microsatellites, of which 104 exhibited sufficient flanking
sequences for primer design, and 2197 good quality SNPs
were identified for the first time in turbot. The cDNA
sequences generated will serve as a basis for microarray
construction. This first EST study in turbot will provide the
support for further research into the genetics, genomics
and even proteomics of this important aquaculture spe-
cies. EST sequence resources are rapidly growing in molecular
databases. However, the number of ESTs in fish is gener-
ally scarce, excluding some model species and Atlantic
salmon among cultured fish [15-19]. The scarcity of EST
resources in cultured fish limits the use of modern func-
tional genomic approaches for selective breeding pur-
poses [20]. Among flatfish, aquaculture production has
been successfully achieved in turbot, Japanese flounder
and Atlantic halibut. Compared to the very large efforts Abstract Background: The turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish
species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish,
very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides
dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the
turbot industry. Little is known about the molecular mechanisms for disease resistance and host-
pathogen interactions in this species. In this work, thousands of ESTs for functional genomic studies
and potential markers linked to ESTs for mapping (microsatellites and single nucleotide
polymorphisms (SNPs)) are provided. This information enabled us to obtain a preliminary view of
regulated genes in response to these pathogens and it constitutes the basis for subsequent and
more accurate microarray analysis. Results: A total of 12584 cDNAs partially sequenced from three different cDNA libraries of
turbot (Scophthalmus maximus) infected with Aeromonas salmonicida, Philasterides dicentrarchi and
from healthy fish were analyzed. Three immune-relevant tissues (liver, spleen and head kidney)
were sampled at several time points in the infection process for library construction. The
sequences were processed into 9256 high-quality sequences, which constituted the source for the
turbot EST database. Clustering and assembly of these sequences, revealed 3482 different putative
transcripts, 1073 contigs and 2409 singletons. BLAST searches with public databases detected
significant similarity (e-value ≤ 1e-5) in 1766 (50.7%) sequences and 816 of them (23.4%) could be
functionally annotated. Two hundred three of these genes (24.9%), encoding for defence/immune-
related proteins, were mostly identified for the first time in turbot. Some ESTs showed significant Page 1 of 12
(page number not for citation purposes) BMC Veterinary Research 2008, 4:37 http://www.biomedcentral.com/1746-6148/4/37 http://www.biomedcentral.com/1746-6148/4/37 differences in the number of transcripts when comparing the three libraries, suggesting regulation
in response to these pathogens. A total of 191 microsatellites, with 104 having sufficient flanking
sequences for primer design, and 1158 putative SNPs were identified from these EST resources in
turbot. Conclusion: A collection of 9256 high-quality ESTs was generated representing 3482 unique
turbot sequences. A large proportion of defence/immune-related genes were identified, many of
them regulated in response to specific pathogens. Putative microsatellites and SNPs were identified. These genome resources constitute the basis to develop a microarray for functional genomics
studies and marker validation for genetic linkage and QTL analysis in turbot. Page 2 of 12
(page number not for citation purposes) Gene annotation
d
f ESTs were identified by BLAST searches against nucleotide
database at GenBank. Due to the low representation of
fish genes, a protein-based homology strategy was also
used in the international database searches. Protein
sequences have been demonstrated to be more suitable to
detect homology over long periods of evolutionary time
[37]. Our EST database pprovided a graphical view of all
contigs, their PROSITE/PRINTS protein patterns and a
search interface by keywords, microsatellite, gene, and
UniGene/GO/KEGG information. Tools to search for
sequences and markers based on annotations, to perform
local BLAST searches, and to select sequences for a pro-
spective microarray were also included. These tools used
RepeatMasker http://www.repeatmasker.org/ for masking
low-complexity sequences and OligoArray 2.1 http://
berry.engin.umich.edu/oligoarray2_1/ to predict second-
ary structures and potential cross-hybridization. EST projects generate a large number of redundant
sequences due to the random selection of cDNAs from tis-
sue libraries, especially when libraries are non-normal-
ized and a high number of clones are sequenced. Clustering redundant sequences is a critical step to iden-
tify genes. The program CAP3 http://seq.cs.iastate.edu./
was used to cluster EST sequences using the default
parameters. As shown in Table 1, clustering yielded 3482
unique turbot sequences: 2409 singletons (69.2%) and
1073 contigs (30.8%) comprising 6847 ESTs (6.4
sequences/contig) and an average length of 527 bp. Figure
1A shows the histogram distribution of contig sizes. Although most contigs showed two (46.7%) or three-to-
five (30.3%) sequences, a small number of highly As shown in Table 1, 1716 (49.3%) unique sequences dis-
played no significant similarity to known sequences or
ESTs in the public databases, whereas the remaining 1766
(50.7%) showed significant matches with e-values ≤ 1e-5. Among these, BLAST database searches allowed assign-
ment of putative function to 816 sequences (23.4%). In
spite of their lower frequency among unique sequences,
contigs were annotated more frequently (59.9%) than
singletons (40.1%). As in other ESTs fish studies, the
lower percentage of annotated singletons suggests that
these are either novel fish-specific or rapidly evolving
genes [38]. Also, it is possible that bioinformatic errors
could have a greater impact on singletons, since they are
unique sequences whose information cannot be con-
trasted with other sequences in the database. The lower
annotation success regarding similar genomic projects
[28,39] was probably related to the read length (around
500 bp) and specially the direction (from the 3'end) of
sequencing. http://www.biomedcentral.com/1746-6148/4/37 http://www.biomedcentral.com/1746-6148/4/37 BMC Veterinary Research 2008, 4:37 [26-28]. However, this order comprises around 600 spe-
cies, many of them of great economic value both for fish-
eries and farming. In this study, we have addressed the
construction of an EST database for the identification of
genes related with immunity and defence in turbot. expressed genes were also detected. As shown in Figure
1B, the beta-globin contig (407 ESTs) and five others con-
tained more than 100 ESTs, which represents a high pro-
portion (1212/9256 = 13.1%) over the total redundancy
(74.0%). Most of these ESTs shared homology with genes
involved in transport, protein metabolism and response
to stress, all of which related to defence. At the other
extreme, 77.0% of the 1073 contigs were ≤ 5 times redun-
dant, indicating that most of these unique sequences rep-
resent rare mRNAs and that these libraries provide a rich
source of sequence information. The three cDNA libraries constructed held at least 2.5 ×
106 primary recombinant clones. The directional cloning
approach used for construction of cDNA libraries ensured
that cDNA inserts appeared mostly in the same orienta-
tion within the vector. Clones were sequenced from the 3'
end with the vector primer T7 to obtain large gene-specific
genomic regions for future oligo-microarray design. The
libraries used were non-normalized and, as usually
observed [16], substantial redundancy was obtained
(around 74%). This approach allowed an analysis of the
turbot response to specific pathogens by comparing the
amount of transcripts across all genes or groups of genes
classified in functional categories. A total of 12584 ESTs
were sequenced. After trimming and vector removal 9256
high quality ESTs were obtained (Table 1), showing an
average length of 409 bp. Their sequences are available in
the dbEST NCBI database under numbers FE943103-
FE952358. Results and Discussion
cDNA libraries and ESTs EST analysis is an efficient and fast method for gene dis-
covery [15,17]. In Pleuronectiformes, this approach has
been recently applied in Japanese flounder (Paralichthys
olivaceus; [21,22,24,25]), winter flounder (Pseudopleu-
ronectes americanus; [34]), flounder (Platichthys flesus;
[35,36]) and Atlantic halibut (Hippoglossus hippoglossus; Page 2 of 12
(page number not for citation purposes) Page 2 of 12
(page number not for citation purposes) http://www.biomedcentral.com/1746-6148/4/37 Sequence prevalence distribution of the identified contigs from turbot libraries
Figure 1
Sequence prevalence distribution of the identified contigs from turbot libraries. (A) Absolute frequency histogram
showing contig size (number of sequences) distribution. (B) Functional and BLAST hits confidence characteristics of the ten
largest contigs. Only biological function according to GO terms has been included. Gene annotation
d
f The 3' untranslated region (UTR) is approxi-
mately double the length of the 5'UTR according to Gen-
Bank fish entries. So, what is gained in specificity for
microarray oligo-design is lost for gene annotation. Table 1: Summary statistics of ESTs from turbot libraries Table 1: Summary statistics of ESTs from turbot libraries
Number
%
Good-quality ESTs
9256
Redundant sequences
6847
74.0
Unique sequences
3482
36.0
Contigs
1073
30.8
Singletons
2409
69.2
Unique sequences with no BLAST hits
1716
49.3
Unique sequences with BLAST hits
1766
50.7
BLASTN
1091
61.8
BLASTX
675
38.2
Unique sequences with functional annotation
816
23.4
Contigs
489
59.9
Singletons
327
40.1 Page 3 of 12
(page number not for citation purposes) Page 3 of 12
(page number not for citation purposes) BMC Veterinary Research 2008, 4:37 http://www.biomedcentral.com/1746-6148/4/37 A Sequence prevalence distribution of the identified contigs from turbot libraries
Figure 1
Sequence prevalence distribution of the identified contigs from turbot libraries. (A) Absolute frequency histogra
showing contig size (number of sequences) distribution. (B) Functional and BLAST hits confidence characteristics of the ten
argest contigs. Only biological function according to GO terms has been included. B
Gene
Contig size e-value
% Hom
Biological process
BLAST
Hepcidin precursor
70
3e-21
83
-
BLASTN
Lysozyme C precursor
76
8e-42
100
Response to stress
BLASTX
Trypsinogen 1 precursor
86
7e-21
67
Proteolysis
BLASTX
Serotransferrin
88
5e-11
85
Ion transport
BLASTN
Trypsinogen 1
114
4e-88
69
Proteolysis
BLASTX
cDNA clone D. labrax
148
8e-22
82
-
BLASTN
Ribosomal protein S27a
155
3e-32
68
Prot. metabol. process
BLASTX
Ribosomal protein S2
171
0.0
93
Biosyntethic process
BLASTN
Hemoglobin alpha-1 chain
219
6e-32
86
Transport
BLASTX
Beta type globin
407
3e-127
81
Transport
BLASTN Sequence prevalence distribution of the identified contigs from turbot libraries
Figure 1
Sequence prevalence distribution of the identified contigs from turbot libraries. (A) Absolute frequency histo
showing contig size (number of sequences) distribution. (B) Functional and BLAST hits confidence characteristics of the t
largest contigs Only biological function according to GO terms has been included
B
Gene
Contig size e-value
% Hom
Biological process
BLAST
Hepcidin precursor
70
3e-21
83
-
BLASTN
Lysozyme C precursor
76
8e-42
100
Response to stress
BLASTX
Trypsinogen 1 precursor
86
7e-21
67
Proteolysis
BLASTX
Serotransferrin
88
5e-11
85
Ion transport
BLASTN
Trypsinogen 1
114
4e-88
69
Proteolysis
BLASTX
cDNA clone D. labrax
148
8e-22
82
-
BLASTN
Ribosomal protein S27a
155
3e-32
68
Prot. http://www.biomedcentral.com/1746-6148/4/37 BMC Veterinary Research 2008, 4:37 All unique sequences were annotated based on similarity
using BLASTX or BLASTN [40] in the public databases
GenBank NR and Unigene. The multiple annotations pro-
vided greater assurance about gene description and fre-
quency of annotation than in a single database. The use of
consensus sequences allowed sequences without signifi-
cant similarity regions with a known protein (e.g., 5' or 3'
noncoding regions) to be annotated if they were members
of an annotated contig. All hits with e-value ≤ 1e-5 and
their associated alignments were stored in the database
and tracked with any associated functional annotation. We also ran AutoFACT [41] on all sequences in the data-
base. It is interesting to note that while AutoFACT was
able to come up with more function-specific information
than our online tool (provided external database hits were
found), it was not able to annotate as many sequences as
our online custom tool. defence/immunity were always retained given their inter-
est in this study. Biological process, molecular function
and cellular component categories are shown in Figures 2,
3 and 4, respectively. According to these criteria, biologi-
cal processes associated with proteins (synthesis, metabo-
lism and proteolysis) were the largest annotated
categories, though a significant group related to transport
and response to stress did appear. In accordance with this,
a large proportion of sequences were classified into struc-
tural constituent of ribosome and protein-related func-
tions (binding, peptidase activity) regarding molecular
function. A remarkably high proportion of annotated
sequences were categorized as oxidoreductase activity. Finally, most gene activity was located into the ribosome,
followed by the membrane, nucleus and the extracellular
and cytoplasm constituents of the cell. Gene annotation
d
f metabol. process
BLASTX
Ribosomal protein S2
171
0.0
93
Biosyntethic process
BLASTN
Hemoglobin alpha-1 chain
219
6e-32
86
Transport
BLASTX
Beta type globin
407
3e-127
81
Transport
BLASTN B http://www.biomedcentral.com/1746-6148/4/37 Immune genes Page 5 of 12
(page number not for citation purposes) BMC Veterinary Research 2008, 4:37 http://www.biomedcentral.com/1746-6148/4/37 Classification of turbot unique sequences in molecular function categories following Gene Ontology (GO)
Figure 3
Classification of turbot unique sequences in molecular function categories following Gene Ontology (GO). Molecular Function
0
20
40
60
80
100
Motor activity
Catalytic activity
Isomerase activity
Lyase activity
Oxidoreductase activity
Transferase activity
Kinase activity
Hydrolase activity
Peptidase activity
Enzyme regulator activity
Receptor activity
Transporter activity
Structural molecule activity
Structural constituent of ribosome
Transcription regulator activity
Chemokine activity
Binding
Protein binding
Cofactor binding
Heme binding
Carbohydrate binding
Nucleic acid binding
RNA binding
DNA binding
Translation factor activity, nucleic acid binding
Calcium ion binding
Ion binding
Lipid binding
Nucleotide binding
Oxigen binding ot unique sequences in molecular function categories following Gene Ontology (GO)
rbot unique sequences in molecular function categories following Gene Ontology (GO). Classification of turbot unique sequences in molecular function categories following Gene Ontology (GO)
Figure 3
Classification of turbot unique sequences in molecular function categories following Gene Ontology (GO). genes with defence or immunity using scientific literature
information available on vertebrates. According to this
analysis, 203 genes out of 816 annotated (24.9%)
appeared related to defence or immunity in our work
(Table 2). This observation is in agreement with the
important role of liver, spleen and head kidney in the
immune response of fish. though some of them have important roles in the
immune response like B-cell linker, chemotaxin, comple-
ment components, IgD, IgM, interferon stimulated gene
12 (b2), lipopolysaccharide (LPS)-binding protein, natu-
ral killer (NK)-lysine type 1, peptidoglycan recognition
protein, skin mucus lectin and tumour necrosis factor
(TNF) receptor associated factor2, among others. Previ-
ously, only a hepcidin [44] and a natural killer cell
enhancing factor [45] had been characterized in turbot. Like in mammals, the immune system of fish is composed
of non-specific and specific defence. The innate immune
response is an important and highly developed defence
mechanism against pathogens in fish [43]. Examples of
innate immunity include anatomic barriers, mechanical
removal of pathogens, bacterial antagonism, pattern-rec-
ognition receptors, antigen-nonspecific defence com-
pounds, complement pathways, phagocytosis, and
inflammation [43]. In Table 2, a list of defence/immune-
related genes that were found three or more times in our
libraries is shown. Complement related genes were pre-
dominant (7.9%) followed by apoptosis (4.9%) and
immunoglobulin (Ig)-related (3.9%) genes. Immune genes Annotated ESTs (816) were classified into functional cat-
egories according to GO terms [42] (77.0%), 367 among
contigs (75.1%) and 261 among singletons (79.8%). A
single sequence very often showed several GO terms in the
same ontology, so we try to group them using a single
more general category. Overly specific categories were also
collapsed into more general terms. Any terms related to Systematic classification of annotated sequences using
available bioinformatics tools (GO, KEGG) provides a
useful information to analyze the functional profile of
annotated genes. In our study, we were especially inter-
ested in identifying immune genes or in a broader sense
those genes regulated in response to specific pathogens of
turbot. So, we analyzed the relationship of our annotated Classification of turbot unique sequences in biological processes categories following Gene Ontology (GO)
Figure 2
Classification of turbot unique sequences in biological processes categories following Gene Ontology (GO). Biological Process
0
20
40
60
80
10
Biological process
Reproduction
Pathogenesis
Biological adhesion
Developmental process
Cell differentiation
Regulation of biological process
Cellular homeostasis
Cellular process
Cell adhesion
Metabolic process
Primary metabolic process
Cellular metabolic process
Biosyntethic process
Protein metabolic process
Lipid metabolic process
Nucleobase, nucleoside, nucleotide and nucleic acid metabolic process
Carbohydrate metabolic process
Immune system process
Transport
Ion trasnport
Electron trasnport
Protein transport
Lipid transport
Proteolysis
Response to stress
Response to stimulus
Signal transduction
Transcription
Cytoskeleton organization & biogenesis
Organelle organization & biogenesis
Chromosome organization & biogenesis
0 Biological Process
0
20 Classification of turbot unique sequences in biological processes categories following Gene Ontology (GO)
Figure 2
Classification of turbot unique sequences in biological processes categories following Gene Ontology (GO). Page 5 of 12
(page number not for citation purposes)
Classification of turbot unique sequences in biological processes categories following Gene Ontology (GO)
Figure 2
Classification of turbot unique sequences in biological processes categories following Gene Ontology (GO). sification of turbot unique sequences in biological processes categories following Gene Ontology (GO
re 2
sification of turbot unique sequences in biological processes categories following Gene O Classification of turbot unique sequences in biological processes categories following Gene Ontology (GO)
Figure 2
Classification of turbot unique sequences in biological processes categories following Gene Ontology (GO). Page 6 of 12
(page number not for citation purposes) Immune genes Glutathione
S-transferase, heat shock proteins and cytochrome P450,
elastases, major histocompatibility complex (MHC) and
coagulation factors involved in innate immunity were
also present, as well as others like interferon, perforin,
hepcidin, nephrosin or alpha-2-macroglobulin. Interest-
ingly, the majority of our immune-related cDNAs
(75.0%) were reported for the first time in turbot, even The availability of a large number of sequences from
immune-related cDNA libraries, both from non-infected
fish (controls) as well as from fish challenged with specific
pathogens, suggested a comparison of transcript profiles
to identify genes regulated in response to these pathogens. Four or more sequences per gene (contig) are the mini-
mum necessary to get statistical power to check the null
hypothesis of even distribution of sequences among the
three libraries. Ninety six genes presented four or more
sequences in our libraries. However, taking into account
the large number of tests performed, we decided to use a
more conservative set of genes (with 6 or more sequences)
to avoid type I errors. This new set comprised 72 genes,
whose distribution among libraries is shown in Addi-
tional file 1 together with their probability of departure
from the null hypothesis using a chi-square test. To use all
information available and to get higher statistical power,
we decided to perform an additional analysis by compar- Page 6 of 12
(page number not for citation purposes) Page 6 of 12
(page number not for citation purposes) BMC Veterinary Research 2008, 4:37 http://www.biomedcentral.com/1746-6148/4/37 Classification of turbot unique sequences in cellular component categories following Gene Ontology (GO)
Figure 4
Classification of turbot unique sequences in cellular component categories following Gene Ontology (GO). Cellular Component
0
20
40
60
80
Extracellular region
Membrane
Macromolecular complex
Intracellular
Cytoplasm
Cytosol
Ribosome
Cytoskeleton
Mitochondrion
Nucleus
Chromosome
100 40 Classification of turbot unique sequences in cellular component categories following Gene Ontology (GO)
Figure 4
Classification of turbot unique sequences in cellular component categories following Gene Ontology (GO). Classification of turbot unique sequences in cellular component categories following Gene Ontology (GO)
Figure 4
Classification of turbot unique sequences in cellular component categories following Gene Ontology (GO). nique sequences in cellular component categories following Gene Ontology (GO)
ot unique sequences in cellular component categories following Gene Ontology (GO). Table 2: Defence and immune-related annotated ESTs from
turbot libraries
Genes
No. Markers-containing ESTs EST studies can also provide resources for the identifica-
tion of polymorphic DNA markers such as microsatellites
and SNPs. In our study, screening of EST sequences for
short tandem repeats (2–6 bp) identified 191 microsatel-
lites using a conservative criterion (≥ 6 and ≥ 8 repeats for
tri/tetra/penta and dinucleotide motifs, respectively). Of
these, 120 had significant hits in BLAST with e-value cut
off ≤ 1e-5 and 71 were annotated. Most microsatellites
were dinucleotide (128) and trinucleotide (56), while
only 6 tetranucleotide and 1 pentanucleotide were found. Among these 191 ESTs sequences, 104 contained suffi-
cient flanking sequences length for primer design. Fifty of
these 104 microsatellites-containing ESTs were contigs,
therefore the in silico comparison of the sequences
included in these contigs allowed us to identify 11 puta-
tive polymorphic microsatellites. However, the strongest genetic signature was observed in
response to P. dicentrarchi infection. Specific genes were
exclusively detected in this library and sometimes at high
frequencies, like those homologous to Oryzias latipes and
Paralichthys olivaceous cDNA clones and to mitochondrial
ATP synthase alpha-subunit gene. Functional annotation
of these unknown genes now appears relevant to under-
stand the response of turbot to this pathogen. The same
was reflected when GO categories were analyzed (see
Additional file 2). A large number of differentially regu-
lated gene categories (P = 0) appeared associated to P. dicentrarchi library according to the different GO criteria. Cytoskeleton organization and biogenesis, as well as car-
bohydrate metabolic process appeared up-regulated cate-
gories among biological processes. Transporter and
hydrolase activities, as well as protein, ion and heme
binding also were overexpressed regarding molecular
function classification. Finally, cytoskeleton and cytosol
appeared as the highest active cellular components. Look-
ing at specific categories, a significant increase in the
expression of antioxidant genes, like glutathione peroxi-
dase and glutathione-S-transferase, was observed. These
genes play a pivotal role to prevent cellular damage due to
the increased reactive oxygen species (ROS) during infec-
tion [47]. Paramá et al. [48] have recently demonstrated
the increase in intracellular ROS by proteases of turbot
kidney in response to this pathogen. Tissue trauma or
invasion by pathogens induces changes in the quantities
of several macromolecules in animal body fluids, which
comprise one aspect of the acute phase response (APR). In
fish, APR proteins include pentraxins, serum amyloid P,
several components of the complement system, transfer-
rin and thrombin. http://www.biomedcentral.com/1746-6148/4/37 BMC Veterinary Research 2008, 4:37 general down-regulation of genes involved in proteolytic
activity in response to this bacterium (chymotrypsin B
precursor, trypsinogen1, chymotrypsinogen 2, trypsyno-
gen-like serin protease and elastase precursor) was
observed, in turn correlated to the lower presence of pepti-
dase and proteolysis GO terms in this library. The possi-
bility that A. salmonicida could be blocking these genes
cannot be ruled out, since it would facilitate the infection
process. Additionally, it was noteworthy that several iron
metabolism-related genes turned out to be up-regulated
after infection with A. salmonicida (haptoglobin fragment
1, globin-related proteins, hepcidin precursor). The high
relevance of iron for bacteria infection could explain this
observation. The up-regulation of hepcidin after bacteria
infection has been recently described in turbot and gilt-
head seabream [44,46]. Finally, some other immune rele-
vant genes like MHC II alpha and beta antigens,
complement C9, thrombin, chemotaxin, bactericidal per-
meability-increasing protein, apolipoprotein A-IV3, heat
shock protein 90 beta and chemotaxin showed significant
up-regulation in response to A. salmonicida. transferrin was observed in response to P. dicentrarchi in
our study. Transferrin up-regulation could be related to
the increase in inflammation and enhanced oxidative
stress typical of infections with this parasite [48]. Finally,
up-regulation of other important components of the
immune response such the profilin and lysozyme was
observed. Profilin-like protein has been reported as a toll-
like receptor (TLR) 11 ligand in some parasites [49]. TLRs
are evolutionary conserved transmembrane proteins that
recognize a unique pattern of molecules derived from
pathogens or damaged cells, triggering robust but defined
innate immune responses [49]. Immune genes unique sequences
%
Complement related
16
7.9
Apoptosis related
10
4.9
Immunoglobulin related
8
3.9
Glutathione S-transferase
7
3.5
Elastase
6
2.9
Cytochrome P450
6
2.9
Major histocompatibility complex
5
2.5
Coagulation factor
5
2.5
Interferon related
3
1.5
Perforin
3
1.5
Hepcidin precursor
3
1.5
Nephrosin
3
1.5
Alpha-2-macroglobulin
3
1.5
Other genes
119
58.6
Total
203
24.9
"No. unique sequences" refers to the total amount of the different
annotated contigs and singletons for each gene class listed in the
Table. "Total percentage" of defence/immune-related genes is
referred to the number of unique annotated sequences from turbot
libraries (816). Table 2: Defence and immune-related annotated ESTs from
turbot libraries ing the profiles of all genes grouped according to GO cat-
egories (see Additional file 2). Though this approach
could be mixing genes with opposite expression patterns,
it could provide significant trends associated to specific
functions for complementing the "individual-level gene"
analysis. Bonferroni correction for multiple tests was con-
sidered in this case, because of the higher statistical power
achieved with this approach. As expected, defence/immune-related genes appeared
overrepresented in the set of 72 genes (37.5%) with regard
to the total number of annotated genes in our libraries
(24.9%). Some genes apparently responded in a similar
way to both pathogens, being down- (myosin, nephrosin
and several peptidases) or up-regulated (actin and lys-
ozyme)
regarding
control. However,
most
genes
responded to only one pathogen. The different infection
profile of the pathogens used in this study, a bacterium
(A. salmonicida) and a parasite (P. dicentrarchi), is expected
to stimulate a specific set of genes in the host. So, protein
biosynthesis was up-regulated in response to Aeromonas
salmonicida, as suggested the increased amount or ribos-
ome-related and elongation factor proteins, and their cor-
respondent GO terms (see Additional file 2). Also, a Page 7 of 12
(page number not for citation purposes) Page 7 of 12
(page number not for citation purposes) http://www.biomedcentral.com/1746-6148/4/37 Page 8 of 12
(page number not for citation purposes) Conclusion To our knowledge, this is the first report on a large tran-
scriptional analysis in turbot providing new genomic
resources in this important European aquaculture species. This study describes a collection of 9256 ESTs represent-
ing 3482 unique sequences obtained from three direc-
tionally cloned cDNA libraries from S. maximus, all of
these being novel ESTs for this species. Therefore, this is a
valuable EST collection, which increases genomic
resources of turbot and enhances the genomic tools avail-
able for non-model fish species. The transcript profile
comparison among the three libraries allowed the identi-
fication of putative genes generally or specifically related
with infections in turbot. In addition, a high number of
putative microsatellite and SNP EST-markers are now
available for turbot map and highly useful for compara-
tive mapping. These ESTs will be the basis for the develop- Tissue source and challenge g
Two batches of 40 individuals (20–30 g each) obtained
from a mixture of heterogeneous genetic families were
collected at a specialized turbot fish farm. Fish from each
batch were challenged intraperitoneally with A. salmonic-
ida subsp. salmonicida and P. dicentrarchi, respectively
[53,54]. The dose was adjusted to obtain around 50% sur-
vival (LD50). The challenges were performed at the
CETGA facilities in quarantine tanks. Fish were sacrificed
prior to organ extraction using a lethal dose of MS222
anesthetic. In order to obtain mRNA representative of
both innate and adaptive immune systems across the
infection process, liver, spleen and head kidney tissues
were collected from 5 sacrificed fish at five sampling
points along the infective process for A. salmonicida (12 h,
1 day, 3 days, 7 days and 21 days post-inoculation) and at
four sampling points for P. dicentrarchi (1 day, 3 days, 7
days and 15 days post-inoculation). Analogous batches of
control fish were injected with saline serum and sampled
at the same days of challenged fish. For each sample time,
equal amounts of tissue from liver, kidney and spleen of
each fish were pooled and immediately frozen in liquid
nitrogen, constituting 15 pools for A. salmonicida (3 tis-
sues × 5 sampling points), 12 for P. dicentrarchi (3 tissues
× 4 sampling points) and the same pools for their respec-
tive controls. Each sample of pooled tissues was ground to
a fine powder in a mortar and pestled with liquid nitrogen
and stored at -80°C until being used for RNA extraction. The use of pools of individuals at each sampling point was
planned, both for identifying putative SNPs, as well as for
averaging individual effects in future microarray analysis
on turbot immune response. Real SNPs are those which passed quality filters 1 and 2 using the
pipeline QualitySNP and true SNPs are the highest quality SNPs
passing the three filters (see Methods). http://www.biomedcentral.com/1746-6148/4/37 BMC Veterinary Research 2008, 4:37 Table 3: Summary statistics of SNP identification from turbot
EST resources
Real SNPs
True SNPs andSNPs
Total sequences analysed
12584
Number of contigs
257
255
Total SNPs detected
2197
1158
SNP frequency
1.39/100 bp
0.74/100 bp
Total number of transitions
749
453
C/T
556
344
A/G
193
109
Total number of transversions
974
558
A/T
161
87
A/C
352
214
T/G
251
130
C/G
210
127
Total number of indels
366
125
Tri-allelic polymorphisms
99
21
Tetra-allelic polymorphisms
9
1
Real SNPs are those which passed quality filters 1 and 2 using the
pipeline QualitySNP and true SNPs are the highest quality SNPs
passing the three filters (see Methods). Table 3: Summary statistics of SNP identification from turbot
EST resources Table 3: Summary statistics of SNP identification from turbot
EST resources ment
of
a
turbot
microarray,
focused
on
the
characterization of the transcriptional response to patho-
gen exposure. Markers-containing ESTs Up-regulation of pentraxin and sero- Because of their abundance within the genome, SNPs are
the most common type of genetic markers [50] for study-
ing complex genetic traits and genome evolution [51]. In
turbot there have no been reports on SNP identification
so far. The use of non-normalized libraries and a large
number of individuals in library construction made possi-
ble the identification of 2197 good quality SNPs. After the
three filters used in the QualitySNP pipeline [52], we
finally detected 2197 real and 1158 true SNPs (Table 3),
representing a rate of 1.39 and 0.74 SNPs per 100 bp,
respectively. Real and true SNPs included 749 and 453
transitions, 974 and 558 transversions and 366 and 125
indels, respectively. With this pipeline, only clusters with
at least 4 EST sequences were selected to minimize the
detection of SNPs caused by sequencing errors. As shown
in Table 4 the majority of SNPs were detected in contigs
involving a larger number of sequences, which provides
an additional proof of our SNP quality. The identification
of microsatellite and SNPs markers within turbot ESTs
will contribute to extend the turbot genetic map [10] after
linkage analysis in reference families. Since these markers
are linked to genes, they will be useful as Type I markers
for population genomics scanning in this species and for
comparative mapping and fish evolutionary studies. Page 8 of 12
(page number not for citation purposes) Page 8 of 12
(page number not for citation purposes) http://www.biomedcentral.com/1746-6148/4/37 Authors' contributions This work represents a collaboration between different
institutions (Xunta de Galicia -local Government- and
Universities) and turbot industry (CETGA- Centro Tec-
nológico Gallego de Acuicultura-). PM and CB identified
the need for this investigation, designed and supervised
the project. BGP and AV constructed the cDNA libraries. BGP, CF, and MVE performed all sequencing work, and
revised and compiled this information for the subsequent
bioinformatics analysis. Database for editing, clustering
and functional annotation of genes was developed by JAA,
MC, and AG in close connection with PM, BGP, CB, CF
and AM. These last two authors performed all bioinfor-
matics analysis using this database. BM and MLL, for
Aeromonas salmonicida, and JL, for Philasterides dicentrarchi,
supplied virulent strains and performed all challenges at
CETGA facilities. Maintenance, sacrifice, organ and RNA
extraction from fish was done by MV and SC. The paper
was written by BGP and mostly revised by PM and JAA. All
authors read the manuscript and gave their approval. A bioinformatic tool was developed in order to process all
data. Basecalling from chromatogram traces was per-
formed by using PHRED [55,56]. Vector, poly-A tails and
low-quality regions were trimmed from EST sequences
using a custom Perl script, a local BLAST search engine
and the trimmest utility from the EMBOSS suite. High
quality ESTs (at least 100 bp and PHRED score ≥ 20 after
removal of vector sequence, adapter, and poly-A tail) of
both normal and infected libraries were combined and
assembled
to
form
clusters
using
CAP3
http://
seq.cs.iastate.edu./[57] with the overlapping identity per-
centage and minimum overlapping length parameters set
to > 85.0% and 50 bp, respectively, in order to obtain
highly reliable contig sequences. These contigs were man-
ually revised to detect possible errors all along the bioin-
formatics process. ESTs that did not form contigs
(singletons) and the contigs resultant of assemblage of
multiple sequences were referred to as unique sequences. Singletons and consensus sequences of each contig
(unique sequences) were compared against public data-
bases. Unique sequences were searched by both Blastn
and Blastx, and the corresponding outputs subsequently
parsed using BioPerl for fish-relevant hits and significant
UniGene information. GO, KEGG and COG terms were
extracted from AutoFACT output, which in turn feeds
from Tblastx and RPS-blast output. All results were stored
in a mySQL database to be consulted and searched using
a custom-designed, friendly AJAX web interface. The e-
value cut off was ≤ 1e-5. Authors' contributions All annotations thus obtained RNA isolation and cDNA library construction Glycerol stocks of overnight cul-
tures were prepared in 96 well plates and stored at -80°C. Bioinformatic mining of microsatellites and SNPs The set of 3482 unique sequences was searched for micro-
satellites using the program SPUTNIK http://espressosoft
ware.com/pages/sputnik.jsp. The
minimum
repeat
number used for this search was 8 for dinucleotide and 6
for tri-, tetra-and pentanucleotide microsatellites. Micros-
atellite-containing ESTs were identified as candidates for
marker development if they presented enough flanking
sequences on either side of the repeats for primer design. Detection of single nucleotide polymorphisms (SNPs)
was done using the pipeline QualitySNP with default set-
tings [52]. This program uses three filters for the identifi-
cation of reliable SNPs: filter 1 screens for all potential
SNPs; filter 2 uses a haplotype-based strategy to detect reli-
able SNPs; and filter 3 screens SNPs by calculating a con-
fidence score based on sequence redundancy and quality. SNPs that pass filters 1 and 2 are called real SNPs, and
those that pass all of the three filters are called true SNPs. Sequencing and bioinformatics Plasmid DNA was isolated from around 4000 clones from
each library using the DirectPrep® 96 Miniprep kit (QIA-
GEN) and the Plasmid Miniprep96 kit (MILLIPORE) with
a robotic platform (BIOMEK 3000), and checked by elec-
trophoresis in 1% agarose gels. One part of the purified
DNA was sequenced following the ABI Prism BigDye™
Terminator v3.1 Cycle Sequencing Kit protocol on an ABI
3100 DNA sequencer (Applied Biosystems) and the other
with the DTCS kit protocol on a CEQ2000 DNA
sequencer (BECKMAN COULTER). All clones were
sequenced from their 3' ends using a standard T7 primer
to obtain the highest specific sequences of genes for oligo-
microarray design. Those clones which suffered a system-
atic drop on sequencing signal after poly-A tails were
sequenced from 5' end. RNA isolation and cDNA library construction y
Total RNA was extracted from pooled tissues of control
and infected fish using TRIZOL Reagent (Life Technolo-
gies) according to manufacturer's recommendations. RNA
quality was assessed in a Bioanalyzer (Bonsai Technolo-
gies). RNA was quantified using gel and NanoDrop® ND-
1000 spectrophotometer (NanoDrop® Technologies Inc)
estimations. Poly-A mRNA was isolated using the Dyna-
beads® mRNA Purification Kit (INVITROGEN). Three
cDNA libraries (from A. salmonicida and P. dicentrarchi
infected fish and control) were directionally constructed
(5' EcoRI, 3' XhoI), with equal amounts of RNA from each
tissue at each sampling time, using the ZAP-cDNA Library
Construction Kit (STRATAGENE) following manufac-
turer's instructions except for size fractioning. This was
performed on cDNAs prior to ligation into vector and car-
ried out with the SizeSep 400 Spun Columns (GE Table 4: Real and true quality SNP distribution in contigs with 4
or more ESTs
Number of contigs
Real SNPs
True SNPs
with 4 sequences
22
58
58
with 5–10 sequences
94
295
235
with 11–20 sequences
75
437
266
with 21–30 sequences
28
322
215
with 31–50 sequences
21
289
110
with > 50 sequences
17
796
274
Total
257
2197
1158
Real SNPs are those which passed quality filters 1 and 2 using the
pipeline QualitySNP and true SNPs are the highest quality SNPs
passing the three filters (see Methods). Table 4: Real and true quality SNP distribution in contigs with 4
or more ESTs Page 9 of 12
(page number not for citation purposes) Page 9 of 12
(page number not for citation purposes) http://www.biomedcentral.com/1746-6148/4/37 BMC Veterinary Research 2008, 4:37 were complemented with AutoFACT output, whenever
available. HEALTHCARE). To allow characterization of the inserts in
a plasmid system the library was mass excised according
to the manufacturer's recommendations, converting it
from plaque forming units to a phagemid in E. coli SOLR
strain. Manually isolated colonies were randomly picked
and arrayed into 96-well microtiter plates containing liq-
uid selective medium. Glycerol stocks of overnight cul-
tures were prepared in 96 well plates and stored at -80°C. HEALTHCARE). To allow characterization of the inserts in
a plasmid system the library was mass excised according
to the manufacturer's recommendations, converting it
from plaque forming units to a phagemid in E. coli SOLR
strain. Manually isolated colonies were randomly picked
and arrayed into 96-well microtiter plates containing liq-
uid selective medium. Additional file 2 Comparison of ESTs grouped according to GO terms among the three
turbot cDNA libraries. contigs of annotated genes are shown grouped
according to GO term categories (Molecular function; Biological process;
Cellular component) and split into the three turbot libraries from which
sequences were obtained. The expression pattern of each gene at each
library is evaluated as the amount of sequences found. This pattern is com-
pared among libraries and examined for its deviation from an even distri-
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latipes) liver mRNA. Mol Mar Biol Biotechnol 1997, 6:345-350. p
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Kuipers N, Busby M, Beetz-Sargent M, Alberto R, Gibbs AR, Hunt P,
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QualitySNP: a pipeline for detecting single nucleotide poly-
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W2772001229.txt
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https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/ijc.31192
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|
Acne in late adolescence and risk of prostate cancer
|
International journal of cancer
| 2,017
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cc-by
| 4,536
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IJC
International Journal of Cancer
Acne in late adolescence and risk of prostate cancer
Henrik Ugge
Katja Fall2,5†
1
n1, Scott Montgomery2,3,4, Sabina Davidsson1† and
, Ruzan Udumyan2, Jessica Carlsson1, Ove Andre
€
€
Department of Urology, Faculty of Medicine and Health, Orebro
University, Orebro,
Sweden
€
€
Clinical Epidemiology and Biostatistics, School of Medical Sciences, Orebro University, Orebro,
Sweden
3
Clinical Epidemiology Unit, Karolinska University Hospital, Karolinska Institutet, Stockholm, Sweden
4
Department of Epidemiology and Public Health, University College London, London, United Kingdom
5
Department of Medical Epidemiology, Karolinska Institutet, Stockholm, Sweden
1
Cancer Epidemiology
2
Accumulating evidence suggest that Propionibacterium acnes may play a role in prostate carcinogenesis, but data are so far limited and inconclusive. The aim of this population-based cohort study was therefore to test whether presence of acne vulgaris during late adolescence is associated with an increased risk of prostate cancer later in life. We identified a large cohort of young
men born in Sweden between 1952 and 1956, who underwent mandatory assessment for military conscription around the age of
18 (n 5 243,187). Test information along with health data including medical diagnoses at time of conscription was available
through the Swedish Military Conscription Register and the National Patient Register. The cohort was followed through linkages
to the Swedish Cancer Register to identify the occurrence of prostate cancer until December 31, 2009. We used Cox regression to
calculate adjusted hazard ratios (HR) and 95% confidence intervals (95% CI) for the association between acne in adolescence and
prostate cancer risk. A total of 1,633 men were diagnosed with prostate cancer during a median follow-up of 36.7 years. A diagnosis of acne was associated with a statistically significant increased risk for prostate cancer (adjusted HR: 1.43 95%; CI: 1.06–
1.92), particularly for advanced stage disease (HR: 2.37 95%; CI 1.19–4.73). A diagnosis of acne classified as severe conferred a
sixfold increased risk of prostate cancer (HR: 5.70 95% CI 1.42–22.85). Data from this large prospective population-based cohort
add new evidence supporting a role of P. acnes infection in prostate cancer.
While prostate cancer is the most common cancer among men in
the western world, its etiology is still poorly understood. Chronic
inflammation is thought to play an important role in the pathogenesis of different types of cancer,1 and accumulating evidence
Key words: Propionibacterium acnes, prostate cancer, acne vulgaris,
inflammation, acne vulgaris
Abbreviations: BMI: body mass index; CI: confidence interval;
ESR: erythrocyte sedimentation rate; HR: hazard ratio; P. acnes:
Propionibacterium acnes; PIA: proliferative inflammatory atrophy;
PIN: prostate intraepithelial neoplasia; PH: proportional hazards
Additional Supporting Information may be found in the online
version of this article.
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.
S.D. and K.F. contributed equally and share the last authorship
Grant sponsor: UK Economic and Social Research Council (ESRC)
as grants to the International Centre for Life Course Studies; Grant
numbers: RES-596-28-0001, ES/JO19119/1
DOI: 10.1002/ijc.31192
History: Received 10 July 2017; Accepted 17 Nov 2017; Online 4
Dec 2017
Correspondence to: Henrik Ugge, MD, Department of Urology,
€
€
Orebro
University Hospital, 701 85 Orebro,
Sweden, Email: henrik.
ugge@regionorebrolan.se
suggests a role of inflammation also in prostate cancer.2 Both acute
and chronic inflammation is often observed in prostate tumor
specimens and inflammatory cells are frequently found near areas
of proliferative glandular prostate epithelium with the morphological appearance of atrophy.3 This type of atrophy (PIA) is thought
of as a precursor lesion that may progress to prostate cancer
directly or via prostate intraepithelial neoplasia (PIN).4
Propionibacterium acnes, most commonly associated with
acne vulgaris, have been identified as the most prevalent microorganisms in prostate specimens,5–8 and have been associated
with presence of inflammatory foci.9 We have recently observed
that P. acnes are more common in prostatic tissue from prostate
cancer patients than in samples obtained from men without the
disease.7 Further, a longitudinal cohort study has demonstrated
that men who had received antibiotics as treatment for severe
acne were at increased risk of prostate cancer later in life.10
While a few other epidemiological studies support a link
between acne and prostate cancer,11 the literature is inconsistent,12–14 possibly due to the use of study designs relying on selfreported exposure information or retrospective data collection.
This is to our knowledge the first prospective large-scale population-based study using clinically diagnosed acne in order to test
if the condition is associated with increased prostate cancer risk.
Methods
This study is based on a cohort of 284,198 men born
between January 1, 1952 and December 31, 1956 that were
C 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of
Int. J. Cancer: 142, 1580–1585 (2018) V
Union for International Cancer Control
1581
Ugge et al.
What’s new?
Some evidence has suggested that acne vulgaris may increase the risk of prostate cancer, but the data have been inconsistent. The results of this prospective cohort study support a link between acne in adolescence and prostate cancer later in life,
potentially implicating the bacteria Propionibacterium acnes and resulting inflammation in prostate carcinogenesis. These
results also suggest that the association may be stronger for the most severe type of acne and for advanced prostate cancer.
Ethical approval
The study was approved by the regional Ethical Review
Board, Uppsala, Sweden (decision reference 2014/324).
Exposure assessment
At the time of conscription examination, participants were
subject to a health questionnaire and an interview performed
by a medical doctor, who recorded medical conditions
according to the 8th edition of the International Classification of Diseases (ICD-8). Diagnoses were recorded on a practical clinical basis, with emphasis on health problems possibly
affecting the participant’s performance during military service. Acne was identified using ICD-8 codes 706.00, 706.01,
706.10, 706.11, 706.12, and 706.20 from the Swedish Military
Conscription Register (n 5 4,847 in the estimation sample)
and using ICD-10 codes L70 from the Swedish National Outpatient register15 (added n 5 189 to the estimation sample).
Severe acne was defined using ICD-8 codes 706.00, 706.01,
706.11, 706.20 and ICD-10 codes L700C, L70.0 C, L70.1, and
L70.2 (see Online Appendix).
Outcome assessment
Prostate cancer cases were identified using ICD-7 code 177
from the Swedish National Cancer Register, with a completeness exceeding 95% for common cancer types.16 Patients
with stages T3–4, N1 or M1 were classified as advanced prostate cancer and stages T1-T2 as localized prostate cancer.
Prostate cancer diagnoses registered before December 31,
2009 (end of follow-up) were included.
Other measures
Swedish Military Conscription Register. The Swedish Mili-
Figure 1. Inclusion and exclusion flow chart.
tary Conscription Register17 records extensive and standardized physical and psychological examinations by physicians
and psychologists, as described in detail previously.18 Measures included height (cm) and body mass index (BMI) (kg/
m2), a physical working capacity score (0–9), a cognitive
function score (1–9), a stress resilience score (1–9) and a
summary disease score (0–9). The physical working capacity
score was based on a standardized ergometer test, where the
participant was subject to a 5 min sub maximal test, followed
by a 5 min maximal test with gradually increasing load. A
semistructured interview with a psychologist, covering issues
such as emotional stability and social maturity, was used as
an assessment of the conscript’s potential ability to cope with
stress during military service and summarized in a stress
resilience score. Cognitive function was obtained from standardized tests covering logical, verbal, technical and spatial
abilities. The summary disease score was used to quantify the
presence and severity of disease relevant to the capacity to
C 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of
Int. J. Cancer: 142, 1580–1585 (2018) V
Union for International Cancer Control
Cancer Epidemiology
included in the Swedish Military Conscription Register. These
men underwent conscription examinations in the 1970s,
except for 80 who were conscripted in 1969 and 1,163 in the
1980s. Examinations were performed at ages 18 and 19 years,
with a small number at later ages. At the time, assessment
for military service was compulsory in Sweden. Fewer than
4% of the male population were excluded from the enlistment examination due to chronic illness or disability.
In the cohort, 2,564 were excluded due to data inconsistencies such as errors in the personal identification number,
female sex or uncertain vital status. Further, 182 men were
excluded due to improbable measures at the conscription
assessment. We also excluded individuals with ill-defined
summary disease score (a standardized score based on presence and severity of health problems, n 5 35), neoplasms at
the time of conscription examination (n 5 599), and those
who had a malignant cancer diagnosis preceding prostate
cancer diagnosis (n 5 47). Next, we excluded men with missing data for covariates used in the analysis (n 5 37,435), leaving a sample of 243,187 available for complete case analysis
(Figure 1).
1582
Acne in late adolescence and risk of prostate cancer
Table 1. Characteristics of the exposed and unexposed to acne among Swedish men born 1952–1956 (n 5 243,187)
No acne n 5 238,151
Characteristics
(%)
n
Acne n 5 5,036
(%)
N
Head of household’s occupation
<0.001
Manual worker
98,525
(41.4)
2,130
(42.3)
Agricultural worker
9,242
(3.9)
165
(3.3)
Farm owner/manager
23,696
(9.9)
429
(8.5)
Office worker
66,320
(27.8)
1,497
(29.7)
Business owner/managers
25,734
(10.8)
541
(10.7)
Other/unknown
14,634
(6.1)
274
(5.4)
52016
(21.8)
1,076
(21.4)
Underweight (<18.5)
27,680
(11.6)
562
(11.2)
Normal weight (18.5–25.0)
192,519
(80.8)
4,138
(82.2)
Overweight and obese (>25.0)
17,952
(7.5)
336
(6.7)
Household crowding (persons/room) 1960 census
>2 people/room
0.419
Cancer Epidemiology
BMI categories (kg/m2)
0.032
Height (cm)
Mean (SD)
<0.001
178.68 (6.43)
179.57 (6.50)
5.20 (1.97)
5.38 (1.98)
6.31 (1.81)
6.23 (1.82)
2.00 (1.00–89.00)
3.00 (1.00–48.00)
Cognitive function score
Mean (SD)
<0.001
Physical working capacity score
Mean (SD)
0.002
Erythrocyte sedimentation rate (mm/h)
Median (min–max)
1
p1
<0.001
p values from v2 test (categorical), t test (continuous) or median test (ESR).
undertake military duty. Erythrocyte sedimentation rate
(ESR) was used as a marker of systemic inflammation, and
was in the analyses adjusted for erythrocyte volume fraction
(EVF).
Socioeconomic and demographic characteristics. Demographic
data, including information on vital status and emigration,
were provided by the government organization Statistics Sweden, using the Total Population Register.19 Markers of material and social circumstances in childhood were taken from
the 1960 census. Household crowding during childhood was
calculated (people/habitable room) and dichotomized. Occupation of the head of household in 1960 was classified as
manual, agricultural, farm owners/managers, office workers,
business owners/managers and others. Region of residence
was taken from the 1970 census.
Statistical analysis
Descriptive statistics included frequencies, proportions, means
and standard deviations. We used time-dependent Coxregression to examine unadjusted and adjusted associations
between acne and prostate cancer onset later in life. The
functional form of the measures was explored using multivariable fractional polynomial modeling20 which indicated a
linear relationship with the log hazard of the outcome for
height, stress resilience score, cognitive function score, physical working capacity score and erythrocyte volume fraction.
The adjusted model included the following measures: birth
year, household crowding, head of household’s occupation,
region of residence, BMI, summary disease score, erythrocyte
sedimentation rate (modelled as categorical variables), height,
stress resilience score, physical working capacity score, cognitive function score and erythrocyte volume fraction (modeled
as linear measures). In sensitivity analysis, we used Coxregression to examine the associations with acne identified
from conscription records only. The assumption of proportional hazards (PH) for Cox regression was evaluated by
using a Grambsch–Therneau test.21
The statistical software used was Stata version 14/SE for
Windows (StataCorp, College Station, Texas). Tests were
two-sided and statistical significance was defined as p < 0.05
and 95% confidence intervals that do not include 1.00.
Results
During follow-up, 1,633 men were diagnosed with prostate
cancer of which 1,317 had localized disease, 203 advanced
prostate cancer, and 113 unknown stage. Median follow-up
time was 36.7 years, median age at study-exit was 55 years
C 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of
Int. J. Cancer: 142, 1580–1585 (2018) V
Union for International Cancer Control
1583
Ugge et al.
Table 2. Hazard ratio (HR) with 95% confidence interval (CI) for the association between acne, and characteristics in late adolescence and
prostate cancer among Swedish men assessed for military conscription and born 1952–1956 (n 5 243,187)
N events
Unadjusted HR [95% CI]
Adjusted HR [95% CI]1
No acne
1,586
1.00
1.00
Acne
Characteristic
Acne in and after adolescence
47
1.41 [1.06,1.89]
1.43 [1.06,1.92]
Nonsevere
45
1.37 [1.02,1.84]
1.38 [1.02,1.87]
Severe
2
5.88 [1.47,23.55]
5.70 [1.42,22.85]
1
439
1.00 [1,00, 1.00]
1.00 [1,00, 1.00]
2–6
1,044
0.90 [0.81, 1.01]
0.93 [0.82, 1.05]
7–10
92
0.79 [0.63, 0.99]
0.83 [0.66, 1.05]
11–14
35
0.95 [0.67, 1.33]
1.02 [0.71, 1.45]
15–89
23
0.73 [0.48, 1.11]
0.80 [0.52, 1.22]
-
1.04 [1.01, 1.07]
1.03 [1.00, 1.06]
Underweight (<18.5)
178
0.87 [0.74, 1.02]
0.96 [0.81, 1.13]
Normal weight (18.5–25.0)
1,355
1.00 [1.00, 1.00]
1.00 [1.00, 1.00]
Overweight and obese (>25.0)
100
0.82 [0.67, 1.01]
0.85 [0.69, 1.04]
<2
1,349
1.00 [1.00, 1.00]
1.00 [1.00, 1.00]
2
284
0.77 [0.67, 0.87]
0.86 [0.75, 0.98]
Physical working capacity score (per one unit increase)
2
BMI (kg/m )
Household crowding (persons per room)
1
Adjusting for birth year, head of household’s occupation in 1960, household crowding in 1960, height, BMI, physical capacity score, summary disease score, summary cognitive score, stress resilience score, erythrocyte sedimentation rate, erythrocyte volume fraction and region of residence in
1970. The models assume proportional hazards and use 243,187 observations.
and median age at prostate cancer diagnosis was 53 years. Of
the 243,187 men eligible for the study, 4,847 (2%) were registered with a diagnosis of acne at time of conscription assessment, likely representing more severe cases. Another 189
men were later diagnosed with acne in the National Patient
Register. A slightly higher proportion of men with acne were
sons of office workers and a somewhat lower proportion to
agricultural workers or farm owners. Men with acne tended
to have higher cognitive function and ESR, but lower physical
working capacity compared with men without acne. Men
with acne further tended to be taller (Table 1). There was no
difference in the prevalence of significant health problems or
stress resilience in men with and without acne or in stress
resilience score (data not shown).
We found that men with acne had a statistically significant increased risk of prostate cancer later in life compared
with men without acne (HR 5 1.43; 95% CI: 1.06–1.92)
(Table 2). We further noted a higher magnitude association
for men diagnosed with severe acne (adjusted HR: 5.70; 95%
CI: 1.42–22.85). We found a modest but statistically significant trend of higher risk with increasing levels of physical
capacity. There was no clear association of ESR levels or BMI
with prostate cancer risk. When prostate cancer cases were
categorized according to disease stage, we found a statistically
significant association between acne and advanced prostate
Table 3. Hazard ratio (HR) with 95% confidence interval (CI) for the
association between acne and prostate cancer among Swedish men
assessed for military conscription and born 1952–1956
(n 5 243187), by tumor stage
Exposure
N events
Unadjusted
HR [95% CI]
Adjusted
HR [95% CI]1
Advanced prostate cancer
Acne
No
194
1.00
1.00
Yes
9
2.21 [1.13,4.32]
2.37 [1.19,4.73]
Localized prostate cancer
Acne
No
1,280
1.00
1.00
Yes
37
1.38 [0.99,1.91]
1.39 [1.00,1.94]
1
Adjusting for birth year, head of household’s occupation in 1960,
household crowding in 1960, height, BMI, physical capacity score,
summary disease score, summary cognitive score, stress resilience
score, erythrocyte sedimentation rate, erythrocyte volume fraction and
region of residence in 1970.
cancer (adjusted HR: 2.37; 95% CI: 1.19–4.73). The association with localized disease was of lower magnitude and borderline statistically significant (adjusted HR: 1.39; 95% CI:
1.00–1.94) (Table 3). In a sensitivity analysis, where we
C 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of
Int. J. Cancer: 142, 1580–1585 (2018) V
Union for International Cancer Control
Cancer Epidemiology
Erythrocyte sedimentation rate (mm/h)
1584
excluded men who were diagnosed after conscription
(n 5 189), the overall association was unchanged (adjusted
HR: 1.40; 95% CI: 1.06–1.92).
Cancer Epidemiology
Discussion
In this population-based study using prospectively recorded
data, we observed an association between acne in late adolescence and development of prostate cancer later in life. The
data suggest that the association may be stronger for the
most severe type of acne and for advanced prostate cancer.
The literature is currently limited and somewhat conflicting regarding the association between acne and prostate cancer. The results from a prospective cohort study suggest that
acne during young adulthood is associated with an increased
risk of prostate cancer-specific death.11 Another cohort study
using prospectively collected data shows an increased prostate
cancer risk for men who reported treatment with tetracycline
for more than four years as a marker of severe acne.10 A few
case-control studies, where subjects have been asked regarding
their history of acne, have on the other hand not shown any
associations between acne and prostate cancer,12–14 and one
case–control study reported an inverse association between
acne-related facial scarring and later prostate cancer.22 In the
same cohort, high serum titers of antibodies directed against P.
acnes were observed to be inversely associated with prostate
cancer risk.23 Studies using retrospectively collected and selfreported data are sensitive to exposure misclassification, which
may explain the reported null findings.12–14,22 Reliance on selfreported information on acne has also been shown to be a less
reliable measure of the disease.24
The prevalence of acne in this cohort (2%) may seem
low compared with other studies, and compared with the
overall incidence of acne among adolescent men. Acne vulgaris is considered to affect a majority of, if not all, adolescents to some extent, and some 20% are said to be affected
by moderate to severe lesions.25,26 The latter figure corresponds roughly to the number counted as exposed in other
studies examining the relationship between self-reported
history of acne and prostate cancer.12–14 It is however likely
that men registered with acne by a physician in the setting
of conscription examination constitute a group with more
severe acne. The prevalence of severe acne based on physical
examinations in similar settings has earlier been estimated
to 2.9–6.9 percent.27,28 While the present study may underestimate the number of exposed, it should accurately have
identified clinically significant cases of acne. The other study
reporting a positive association between acne and prostate
cancer10 could be argued to have used a similarily strict definition of exposure since prescription of tetracycline would
have required physical examination. Identification of more
severe cases may thus help explain discrepancies with results
of studies based on self-reported, less severe acne. Indeed,
although based on very small numbers, our results suggest a
stronger link with what specifically has been classified as
Acne in late adolescence and risk of prostate cancer
more severe forms of acne. The focus of the study is further
on acne later during adolescence (age 18–19 years), and the
results may not be generalizable to persons with acne earlier
in life.
P. acnes can induce inflammatory reactions, through complement activation and induction of proinflammatory cytokines.29,30 Induction of cytokines such as IL-6 and IL-8,
partly through activation of toll-like receptors (TLRs), is
thought to be involved in the pathogenesis of acne,31 and has
been demonstrated to occur in both skin and prostate cells
infected by P. acne.7,32,33 Although the exact mechanism of
P. acnes-associated pathogenesis is not completely understood
in either skin or prostate, there is evidence pointing to the
importance of the host inflammatory response.32,34 P. acnes
is ubiquitous on the skin, but only a smaller proportion of
the population develop severe acne lesions as a consequence
of colonization, which further suggests that individual variation in immune response may influence the outcome of
infection.35 Immunologic phenotypes predisposing an intense
dermatologic reaction could also be prone to a similar
inflammatory response in the prostate. We found that the
mean erythrocyte sedimentation rate (ESR), a marker of systemic inflammation, was higher among men with than without acne, but not an independent determinant of prostate
cancer risk.
An alternative explanation of our result may involve
androgens. Hormonal activity plays a role in the pathology of
acne36,37 and the central role of androgen signalling in prostate cancer and prostate cancer treatment has been known
for a long time.38 The role of androgen signalling in prostate
carcinogenesis is however not as clearly established.39 We
were unfortunately not able to address this potentially important confounder in this study, which is a limitation.
The strengths of this study include its large study population, prospective design and long, essentially complete,
follow-up. Another advantage is that the exposure, the diagnosis of acne, was made as part of the physical examination
during the assessment for conscription and collected independently of a later diagnosis of prostate cancer. A weakness
is that the number of exposed was low and the power for
detailed analyses limited. The study population is furthermore relatively young which results in a low number of
prostate cancer cases, possibility leading to an underestimation of the true association. Specifically, our study could be
said to have examined the association between acne in late
adolescence and early prostate cancer diagnosis. Whether the
association remains for prostate cancer at later ages is
unknown.
In conclusion, the results of this prospective cohort-study
indirectly support the hypothesis that P. acnes may play a
role in prostate cancer. Whether the association is explained
by P. acnes, host factors associated with specific immune
responses, hormonal or other factors remains to be investigated further.
C 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of
Int. J. Cancer: 142, 1580–1585 (2018) V
Union for International Cancer Control
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Cancer Epidemiology
References
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https://openalex.org/W4311220768
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https://jurnal.uns.ac.id/jmme/article/download/64421/38258
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Analysis of Distance Learning Activities to Teach Collaboration Ability of Undergraduate Students of Mathematics Education Study Program
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Journal of Mathematics and Mathematics Education
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| ERROR: type should be string, got "https://jurnal.uns.ac.id/jmme https://jurnal.uns.ac.id/jmme https://jurnal.uns.ac.id/jmme p-ISSN 2089-8878; e-ISSN 2715-8276 \nVol. 12, No. 02, December 2022 * Correspondence purposes, email: dewiisabella5@gmail.com * Correspondence purposes, email: dewiisabella5@gmail.com Abstrak: Pembelajaran jarak jauh adalah pembelajaran yang dilakukan secara terpisah antara \npeserta didik dengan pendidik, pembelajaran yang dilakukan didesain menggunakan alat \nkomunikasi atau media manajemen belajar untuk mendukung pembelajaran. Pada abad ke – 21 \nini terdapat kemampuan-kemampuan yang harus dikuasai oleh seseorang yang disebut \nKompetensi Abad 21 yang salah satunya adalah kemampuan kolaborasi. Tujuan dari penelitian \nini adalah mendeskripsikan aktivitas pembelajaran jarak jauh untuk membelajarkan kemampuan \nkolaborasi mahasiswa dan mendeskripsikan pengaruh aktivitas pembelajaran pada pembelajaran \njarak jauh dalam mengembangkan kemampuan kolaborasi. Jenis penelitian ini adalah deskriptif \nkualitatif dengan metode kajian literatur yang dilengkapi data lapangan. Hasil penelitian \nmenunjukkan ada beberapa hal yang perlu diperhatikan dalamn merancang aktivitas pembelajaran \njarak jauh untuk membelajarkan kemampuan kolaborasi mahasiswa, yaitu 1) membagi \nmahasiswa menjadi beberapa kelompok, 2) digunakan media yang mendukung mahasiswa untuk \nmemberikan tanggapan, gagasan, komentar dan sebagainya baik berupa forum video conference \nmaupun LMS, 3) instruksi tentang aktivitas umpan balik, dan 4) budaya yang dibangun dalam \naktivitas pembelajaran. Diperoleh juga indikator kemampuan kolaborasi yaitu 1) kerjasama dan \nsaling ketergantungan untuk mencapai tujuan bersama seperti kerjasama dalam kelompok, \nberdiskusi, berkontribusi aktif, dan fleksibel, 2) bertanggung jawab atas dasar belajar dan perilaku \nmasing-masing seperti bertanggung jawab dalam pekerjaan dan bekerja secara produktif, dan 3) \nterjadinya pelaksanaan suatu aktivitas kerja yang kohesif seperti dapat beradaptasi, \nberkomunikasi dengan baik, dan menghargai sesama. Selain itu, terdapat kelebihan dan \nkekurangan dari rancangan aktivitas pembelajaran jarak jauh ini. Accepted: July 01, 202x \nApproved: July 24, 2022 \nPublished: Dece\nDOI: 10.20961/jmme.v12i2.64421 \n(*)This study was presented at ICMME 2022 on 24-26 July 2022 \n[ 61 ] ANALYSIS OF DISTANCE LEARNING ACTIVITIES TO \nTEACH COLLABORATION ABILITY OF \nUNDERGRADUATE STUDENTS OF MATHEMATICS \nEDUCATION STUDY PROGRAM(*) Dewi Isabella Palma1*, Elina Nadiasari1 \n1Sanata Dharma University, Yogjakarta, Indonesia Kata kunci : Pembelajaran jarak jauh, Kompetensi abad 21, Kolaborasi Abstract: Distance learning is learning that is carried out separately between students and \neducators, the learning carried out is designed using communication tools or learning management \nmedia to support learning. In the 21st century, there are abilities that must be mastered by someone \ncalled 21st Century Competencies, one of which is the ability to collaborate. The purpose of this \nstudy is to describe distance learning activities to teach student collaboration skills and describe \nthe influence of learning activities on distance learning in developing collaboration skills. This \ntype of research is descriptive qualitative with a literature review method equipped with field data. The results showed that there are several things that need to be considered in designing distance \nlearning activities to teach students' collaboration skills, namely 1) dividing students into groups, Published: December 01, 2022 Accepted: July 01, 202x \nApproved: July 24, 2022 \nDOI: 10.20961/jmme.v12i2.64421 \n(*)This study was presented at ICMME 2022 on 24-26 July 2022 \n[ 61 ] 62 62 p-ISSN 2089-8878; e-ISSN 2715-8276 p-ISSN 2089-8878; e-ISSN 2715-8276 62 2) using media that supports students to provide responses, ideas, comments and so on in the form \nof video conference forums and LMS, 3) instructions on feedback activities, and 4) the culture \nbuilt in learning activities. Indicators of collaboration ability are also obtained, namely 1) \ncooperation and interdependence to achieve common goals such as cooperation in groups, \ndiscussion, active contribution, and flexibility, 2) being responsible for the basis of learning and \nbehavior of each such as being responsible for work and working productively, and 3) the \nimplementation of a cohesive work activity such as being able to adapt, communicate well, and \nrespect for others. In addition, there are advantages and disadvantages of this design of distance \nlearning activities. Keywords: Distance Learning, 21st Century Competencies, Collaboration INTRODUCTION Distance learning is an institution-based formal education where study groups are separated and \ninteractive telecommunications systems are used to connect learners, resources and instructors \n(Simonson et al., 2019). According to Abidin (Abidin et al., 2020), distance learning is a learner who is \nplanned in another place or outside his place of teaching and when the learning process does not occur \nface-to-face directly between the teacher and learner, so that learning emphasizes independent learning \nand uses special techniques in designing materials and special methodologies for communication \nthrough media such as computers, telephones, television and so on. Then, according to Law No. 20 of \n2003 concerning the National Education System Article 1 (Yuangga & Sunarsi, 2020) states that distance \neducation is an education whose students are separated from educators and learning is carried out using \nvarious learning resources through telecommunications, information and other media. Based on some of \nthe opinions above, it can be concluded that distance learning is learning that is carried out separately \nbetween students and educators (not face-to-face in person), where learning is carried out independently \nby students and the learning carried out is designed using communication tools or learning management \nmedia such as Whatsapp, Zoom, Moodle, and so on to support learning both synchronously and \nasynchronously. Based on this, the concept of distance learning has begun to be developed before the \nCOVID-19 pandemic which began at the tertiary level. This is stated in Law No. 12 of 2012 with the \naim of providing Higher Education services to groups of people who cannot attend regular face-to-face \neducation and expanding access and facilitating Higher Education and learning services. On the other hand, along with the times, in the 21st century there are competencies that must be \nmastered by students from an early age. P21 (Partnership for 21st Century Learning) develops a learning \nframework in the 21st century that requires students to have skills, knowledge and abilities in the fields \nof technology, media and information, learning skills and innovation as well as life and career skills \n(Wijaya et al., 2016). Within the framework, there are four abilities commonly referred to as 21st century Vol. 12, No. 02, December 2022 Dewi Isabella Palma and Eliana Putri, Analysis of Learning… \n \n63 63 Dewi Isabella Palma and Eliana Putri, Analysis of Learning… 63 competencies, which students must master in learning, namely critical thinking, communication, \ncollaboration, and creativity. INTRODUCTION Triling and Fadel (Wijaya et al., 2016)describe that critical thinking and \novercoming problems are students able to use reason for various situations, use system thinking methods, \nmake decisions and overcome problems; communication and collaboration, namely students are able to \ncommunicate clearly and collaborate with other group members; creativity and innovation, namely \nstudents are able to think creatively, work creatively and create new innovations. Even though distance \nlearning is growing and exists at every level of education, educators still have to make sure students \nmaster these four abilities. The importance of mastering and developing these four abilities, so that \nstudents can become superior and qualified human beings. One of the important 21st century competencies is collaboration. P21 (Roekel, 2010) defines \ncollaboration as the ability to work effectively and respectfully with diverse teams, exercise flexibility \nand willingness to assist in making the necessary compromises to achieve common goals, and \nresponsibility for joint work and respect for the individual contributions made by each member of the \ngroup. Roekel (2010) explains that collaboration is important because a large group of diverse \nindividuals will produce better estimates and collaboration can create knowledge for many people. Based \non this, learning that supports collaboration will have a good impact on student learning, because through \ncollaboration, students will get a variety of knowledge from the results of collaboration. There are several \nstudies that examine the application of collaborative learning models to distance learning. In a study \nconducted by Farah (Fauziah, 2021) found that collaborative learning carried out in the experimental \ngroup was in the category of quite effective, namely 63.7%. Then research conducted by Thompson and \nKu (Thompson & Ku, 2006), found that there was ineffective communication, conflicts between group \nmembers, and negative attitudes that posed challenges in collaborating online, but more collaborative \ngroups produced better quality projects and had a more positive attitude towards online learning. Based \non this, it can be seen that collaboration is a challenge in distance learning. Based on the importance of the current ability of collaboration and development of distance \nlearning, this study will discuss distance learning activities to teach student collaboration skills and \ndescribe the influence of learning activities on distance learning in developing collaboration skills. JMME Halaman 61-71 RESEARCH METHOD This research is a type of qualitative descriptive research. Qualitative descriptive is a type of \nresearch that gives an idea of an object that is observed on the basis of qualitatively visible facts as they \nare. The main source of data in the study is words. In addition, the source of this research data is the \nresult of a literature review supported by interviews with lecturers and surveys with students of the JMME Halaman 61-71 64 \n \n \nMathematics Education Study Program, one of the private universities in Yogyakarta who are the \nsubjects of this study. p-ISSN 2089-8878; e-ISSN 2715-8276 64 p-ISSN 2089-8878; e-ISSN 2715-8276 Mathematics Education Study Program, one of the private universities in Yogyakarta who are the \nsubjects of this study. Mathematics Education Study Program, one of the private universities in Yogyakarta who are the \nsubjects of this study. The data collection methods in this study are literature review, direct interview methods and \nsurvey methods. Interviews were conducted to explore information related to distance learning activities \nto improve student collaboration skills. Then a survey was conducted to collect information from \nstudents regarding the response and effects of distance learning activities to improve student \ncollaboration skills. The instruments used in this study are interview guidelines and questionnaires where \nthe instruments have been declared valid by validators. RESULTS AND DISCUSSION According to (Simonson et al., 2015) there are four characteristics of distance learning. First, \ndistance learning is carried out by accredited institutions equivalent to institutions that offer traditional \nface-to-face learning and not independent learning or learning in a nonacademic learning environment. Secondly, learners and teachers are separated geographically and perhaps even time so accessibility and \nconvenience are essential in distance learning. Third, interactive telecommunications connects students \nwith each other and also connects students with teachers. Whatever the medium, interaction is also very \nimportant in distance learning as learning in general, the relationship between students and teachers no \nlonger depends on the physical but on the sophistication of communication technology. Fourth, like other \neducation, distance learning also creates a learning community consisting of students, teachers, and \nlearning resources such as books, modules, videos, and others. Garrison and Shale (Simonson et al., \n2015) stated the criteria in determining the characteristics of the distance learning process. First, distance \nlearning implicitly suggests that the educational communication created between teachers and students \ndoes not occur in physical proximity. Second, distance learning should involve two-way communication \nbetween teachers and students in order to support and facilitate the learning process. Third, distance \nlearning uses media to mediate the need for two-way communication. In teaching collaboration skills, it is necessary to design the right activities. according to \n(Nurhayati et al., 2019) learning is carried out in groups with the aim of training cooperation between \nstudents in solving a problem. In addition, collaboration skills according to (Zubaidah, 2018)include \ngiving and receiving feedback from colleagues or fellow group members to perform the same task, so in \ndesigning learning activities it is necessary to facilitate students to provide feedback to fellow friends, \nespecially group members. Media is also needed in teaching collaboration skills in distance learning, \nthis is in accordance with the characteristics of Distance Learning according to (Simonson et al., 2015) \nabove. Vol. 12, No. 02, December 2022 Dewi Isabella Palma and Eliana Putri, Analysis of Learning… \n \n65 Dewi Isabella Palma and Eliana Putri, Analysis of Learning… Dewi Isabella Palma and Eliana Putri, Analysis of Learning… 65 Indicators of collaboration ability according to Suryani (Sunardi et al., 2017) are 1) each member \nworks together to achieve common goals and interdependence, 2) individuals are responsible for each \nother's learning and behavior, 3) Classes or groups are encouraged towards the implementation of \ncohesive group work activities. RESULTS AND DISCUSSION In addition, Trilling and Fadel (Mariamah et al., n.d.), there are five \nindicators of collaboration ability, namely cooperation in groups, adjusting to group friends, being \nresponsible for group work, discussing in making decisions, and communicating well in groups. . Then \nGreenstein (Hidayat et al., 2021) also revealed that the indicators that indicate the ability of collaboration \nare to actively contribute, work productively, show flexibility and compromise, show responsibility and \nshow respect. Based on these three opinions, it can be concluded that the indicators of collaboration \nability are 1) Cooperation and interdependence to achieve common goals such as cooperation in groups, \ndiscussion, active contribution, and flexibility, 2) being responsible for each other's learning and \nbehaviors such as being responsible for work and working productively, and 3) implementing cohesive \nwork activities such as being able to adapt, communicate well, and value others. To support a literature review of distance learning activities in teaching collaboration skills, we \nanalyzed distance learning activities designed by lecturers and their impact on students' collaboration \nability based on indicators of collaboration ability. Based on the results of interviews with lecturers at \none of the private universities, it was found that learning activities designed online require collaboration \nskills from students to carry out some learning activities or assignments given. The learning media used \ndepends on the type of activity and task being designed. For example, each small group provided a forum \non ZOOM such as a breakout room to facilitate student discussions. In addition, you can use the Learning \nManagement System (LMS) to provide a place for students to share work, share ideas, comments, \ncriticisms and suggestions with each other as well as a place to collect assignments. The encouragement \nof cooperation between students in groups leads students to use media that help collaboration such as \ngoogle documents that can be accessed simultaneously. Then, the learning activities that are designed \nrequire some attention so that the learning activities can run well. First, the activities carried out are to \ndivide students into small groups and it is also offered that in each small group there is a group leader to \norganize activities and tasks in the group to solve problems or projects. Furthermore there is an emphasis \ngiven to students verbally that the contribution between students is equal or evenly distributed. Then the \nsecond is peer-to-peer feedback activity. RESULTS AND DISCUSSION This feedback activity facilitates students to be able to convey \nideas, correct, observe, make comments, and assess fellow students. Research conducted by Ching & \nHsu (Purwadi, 2020) revealed that peer feedback has a positive impact on student learning and can \nincrease active participation in distance learning because of the freedom to express ideas and ideas they \nhave. Purwadi (2020) found that accustoming students to dare to think critically can develop cognitive JMME Halaman 61-71 66 p-ISSN 2089-8878; e-ISSN 2715-8276 66 skills to be able to think at a higher level. From these two explicit instructions for collaborating, it slowly \ndisappears so that students do not rely on instructions from lecturers. The culture applied in distance \nlearning is to support the development of collaboration, that is, a culture of learning and a culture of \nmutual respect. skills to be able to think at a higher level. From these two explicit instructions for collaborating, it slowly \ndisappears so that students do not rely on instructions from lecturers. The culture applied in distance \nlearning is to support the development of collaboration, that is, a culture of learning and a culture of \nmutual respect. 1. A learning culture is a learning culture that invites feedback givers to realize that the results \nare still learning outcomes. Likewise, for recipients of input, they are invited to realize that \nthe comments, values, suggestions or criticisms given are the result of learning from judging \nor correcting things that are lacking. 2. A culture of mutual respect is a culture of respect that invites feedback givers to realize that \nthe work done requires a long process and requires thought, time and effort. Similarly, the \nrecipient of the feedback is invited to realize that the comments, values, suggestions and \ncriticisms given are the efforts of the feedback giver to correct, observe, comment and assess \nthe shortcomings contained in his work. In addition to the learning activities carried out, the interview also explained the assessment \ncarried out by the lecturer in assessing learning activities that require cooperation. First, the assessment \nof contributions between peers. This assessment is delivered at the beginning of the lesson with the aim \nthat students are motivated to contribute well in each group. This assessment contains a range of scores \n(1 to 5) to rate each friend in a group. RESULTS AND DISCUSSION In the interview it was also mentioned that there are collaborative activities that are easier to do \nonline than offline. For example, when students present their answers online, it will be easier because \nstudents can directly share their answers through the screen sharing feature. In terms of time, sharing \nanswers is more efficient online than offline. In this activity, it was also found that one of the learning \nactivities that can be done offline and online is the presentation of student answers. Then, there are \nlearning activities that cannot be done online, but can be done offline, which is to monitor the \ncontribution of each student in a small group. Not all students can contribute for reasons that are often \ngiven by students with signal constraints, lecturers cannot be sure of the truth of these reasons. What \nlecturers can do is observe the consistency of student reasons. On the other hand, offline monitoring can \nbe easily done as it is enough to go around visiting each group and checking the answers. if the same or \nsimilar errors are found in each group, the lecturer can easily give a classical explanation for the mistakes \nmade. Furthermore, to find out the effectiveness of the lecture activities designed, a questionnaire was \ndistributed to students who took courses taught by the lecturer. There were 34 students who were \nrespondents. From the common questions regarding online learning that students do, several things were \nfound. The most widely used media is WhatsApp which is 94.1% and after that uses Moodle with 64.7% \nstatement; some of the other media students mentioned were zoom, google classroom, and desmos. In \naccessing online lectures, as many as 94.1% of students stated that they used laptops, all students stated \nthat they also accessed online lectures using mobile phones. And in accessing online lectures, 11.4% of \nstudents stated that they experienced difficulties due to network constraints. In lectures, students are \ndivided into groups which usually consist of 2-5 students. In large groups sometimes consist of 6-8 \nstudents. Students also stated that after being divided into groups, the lecturer will visit each group. Some of the difficulties experienced by students, the first is difficulty in communicating, this is \ncharacterized by the presence of group friends who are passive or do not respond when group discussions \nare running either via Whatsapp or via zoom. RESULTS AND DISCUSSION For example, in one group there are three students A, B, and C, \nthen A will assess B and C, as well as students B and C. Through this kind of assessment, it will be seen \nhow much contribution each student has in each group. Although there are some cases where students \nare reported to lecturers for not making any contribution to the group. The effort that can be made by \nthe lecturer is to ask the student directly concerned about the reason for not making any contribution in \nthe group and this is a consideration for the lecturer to the members of the group concerned. Second, \nassessment in the form of frequency in classical discussions. At the beginning of learning, lecturers will \nprovide multiple-choice questions that are conceptual and tricky. This trap question is given so that \nstudents do not lean towards one of the correct answers. So that several groups of students are formed \nwho answer different questions. Then the activity carried out is that the lecturer asks one of the student's \nanswer representatives to give an idea, then other students who have different answers are welcome to \nrespond or comment on the answer. Activities like this assess the frequency of student participation in a \ngiven activity. Third, the assessment of the analysis of social networks. Social network analysis is a way \nof assessing activities by visualizing the interactions that occur, for example student A comments on \nstudent B, student B comments on student C, this interaction gets a link or network. If this is visualized \nto all students, a picture will be obtained in the form of webs that visualize the relationship. Through this Vol. 12, No. 02, December 2022 Dewi Isabella Palma and Eliana Putri, Analysis of Learning… 67 visualization, you will see a circle that represents a student, the larger the circle, the student is the most \nactive student. The results of this analysis are also presented and explained to students which ones give \nthe most responses, receive responses and so on. This will give an indication that it is the student who \ngets the most responses, so that the topic or discussion given attracts the attention of the student. As for \nstudents who responded a lot, it was indicated that the student was active in discussion activities. RESULTS AND DISCUSSION Second, students experience difficulties in the internet \nnetwork which is sometimes constrained. Third, students sometimes find it more difficult to understand \nthe material being studied so that they have difficulty in finding solutions in groups. Some students also \nfeel that when studying offline, students can be more free to chat and joke and can coordinate in groups JMME Halaman 61-71 p-ISSN 2089-8878; e-ISSN 2715-8276 68 better. However, most students also stated that the advantage of online lectures is that online lectures are \nmore effective in terms of time and place. Students feel that online lectures become less tense and dare \nto express ideas in class. Students also state that they can learn new technologies through online lectures. In addition, online lectures are considered effective because they require students to be active, activities \nin online lectures are also considered to still make it easier for students to discuss, students can study in \ngroups or through other groups. The specific questions are based on the three indicators of collaboration capabilities used. The \nresults obtained show that all three indicators of collaboration ability are met. The first indicators are \ncooperation and interdependence to achieve common goals such as cooperation in groups; Discuss; \nactively contribute; and flexibility is met, this is shown from the percentage of students' ability to work \ntogether and express opinions in groups is 100%, but 8.8% of students stated that they did not work in \ngroups and felt that they could only work with a few groups. member. In decision making, all students \nstated that decisions are taken jointly and not unilaterally with a 100% percentage of answers. The second \nindicator is responsibility on the basis of learning and behavior of each such as being responsible for \nworking and working productively. All students stated offering to divide assignments into groups with \na 100% statement percentage, but 5.2% of students said they did not work on assigned assignments in \ngroups. However, most students work on assignments that have been shared in groups. In presenting \nideas in groups, most students stated that they conveyed the idea of completing the task, which was \n97.1%. The third indicator is the implementation of cohesive work activities such as being able to adapt, \ncommunicate well, and respect others. The fulfillment of these indicators is shown by the results of the \nquestionnaire which states that all students state that they accept every opinion submitted. RESULTS AND DISCUSSION All students \nalso stated that they could accept and appreciate the work of each group member. Most students also felt \nadaptable to group members although 5.9% of students stated they were unable to adapt. Based on the above results, it was found that distance learning can develop student collaboration \nskills. Distance learning activities should be designed by requiring cooperation from students, namely \n1) dividing students into groups either through direct instruction delivered or formed from the given \nanswer choices, 2) using media that supports students to provide responses, ideas, comments and so on. both in the form of video conference forums and LMS, 3) instructions for feedback activities, and 4) \nculture built in learning activities such as learning culture and mutual respect culture. It was also \nconveyed by students that they were divided into groups with members between 2-5 people for small \ngroups or 6-8 people for large groups, and in lectures adequate technology was used to support learning \nactivities such as using Whatsapp, Moodle, Google classes, and Desmos. Vol. 12, No. 02, December 2022 69 Dewi Isabella Palma and Eliana Putri, Analysis of Learning… 69 The assessment of cooperation that can be carried out also depends on the activities or tasks given, \nnamely the assessment of contributions between colleagues, the assessment of the frequency of classical \ndiscussions, and the assessment of social network analysis. In assessing the contribution of peers, it can \nhelp lecturers to know that there are unbalanced student contributions. This is also conveyed by students, \nthat students experience some communication problems because there are group members who are \npassive or do not respond when discussing. The opinion of this student is in accordance with what is \nconveyed by the lecturer regarding peer assessment related to the contribution of his group of friends. Advantages and disadvantages of distance learning activities that require cooperation in learning \nactivities and assignments. The advantage of this activity is the efficiency of learning time. If learning \nactivities can run well, learning time will be more efficient than offline. the shortcomings experienced \nare aspects of supervision of the contributions of students in the group. According to the results of \nlecturer interviews and student questionnaires, it was found that signal problems are one of the most \nfrequently conveyed reasons and it is difficult to ascertain the truth. This causes the contribution of \nstudents to be uneven. RESULTS AND DISCUSSION It is also conveyed by students that they are experiencing internet network \ndifficulties. JMME Halaman 61-71 CONCLUSIONS AND SUGGESTIONS Based on the results and discussion above, it can be concluded that there are several things that \nneed to be considered in designing learning activities to teach student collaboration skills, namely 1) \ndividing students into groups either through instructions directly delivered or formed from the selection \nof answers given, 2) media that supports students to provide responses, ideas, comments and so on both \nin the form of video conference forums and so on. LMS, 3) instruction on feedback activities, and 4) \ncultures built into learning activities such as culture of learning and culture of mutual respect. These four \nthings are found to be able to help teach students collaborative abilities as measured by three indicators, \nnamely, 1) cooperation and interdependence to achieve common goals such as cooperation in groups, \ndiscussion, active contribution, and flexibility, 2) being responsible for the basis of learning and behavior \nof each such as being responsible for work and working productively, and 3) the implementation of a \ncohesive work activity as can adapt, communicate well, and value others. In addition, the assessment \nthat can be done depends on the activity or task given, some assessments that can be given are the \nassessment of contributions between peers, an assessment in the form of frequency in classical \ndiscussions, and an assessment of social network analysis. The effectiveness of learning activities to teach student collaboration skills can be seen from the \nadvantages and disadvantages shown. The advantage of this learning activity is that the time and place JMME Halaman 61-71 70 p-ISSN 2089-8878; e-ISSN 2715-8276 of learning becomes more efficient. The disadvantage of this activity is the lack of supervision aspects \nthat lecturers can carry out regarding the correctness of the reasons for network constraints. ACKNOWLEDGMENTS The researcher would like to thank all parties who helped carry out research activities. This \ngratitude is addressed to the research subject who volunteered to be the subject of this study, Dr. M. Andy Rudhito, S.Pd who has provided guidance in conducting this research and Sanata Dharma \nUniversity who has contributed to the researcher. Zubaidah, S. (2018). Mengenal 4C: Learning and Innovation Skills untuk Menghadapi Era Revolusi 4.0. \n2nd Science Education National Conference, 13(April), 1–18. JMME Halaman 61-71 REFERENCES Abidin, Z., Hudaya, A., & Anjani, D. (2020). Efektivitas Pembelajaran Jarak Jauh Pada Masa Pandemi \nCovid-19. Research \nand \nDevelopment \nJournal \nof \nEducation, \n1(1), \n131. https://doi.org/10.30998/rdje.v1i1.7659 Fauziah, F. (2021). Implementasi Pembelajaran Kolaboratif Menggunakan Breakout Room Zoom \nMeeting Pada Pembelajaran Jarak Jauh. Edutech : Jurnal Inovasi Pendidikan Berbantuan \nTeknologi, 1. https://doi.org/https://doi.org/10.51878/edutech.v1i3.683 Hidayat, A. K., Yulianti, D., & Herpratiwi. (2021). Penggunaan Google Sites Dalam Membangun \nKolaborasi Pada Materi Korosi Ditinjau Dari Kemandirian Belajar Siswa. Jurnal Edukasi \nMatematika Dan Sains), 9(2), 440–451. https://doi.org/10.25273/jems.v9i2.10997 Mariamah, S., Bachtiar, M. Y., Pgpaud, P. P. G., & Makassar, U. N. (n.d.). Penerapan Project Based \nLearning Untuk Meningkatkan Kemampuan Kolaborasi Anak Usia Dini. 125–130. Nurhayati, D. I., Yulianti, D., & Mindyarto, B. N. (2019). Bahan Ajar Berbasis Problem Based Learning \npada Materi Gerak Lurus untuk Meningkatkan Kemampuan Komunikasi dan Kolaborasi Siswa. Unnes Physics Education Journal, 8(2), 218. Purwadi, I. M. A. (2020). Pengaruh Pemberian Umpan Balik Dari Teman Sejawat Terhadap \nKemampuan Kemampuan Berpikir Kritis Dan Komunikasi Matematis Siswa Smp Laboratorium \nUndiksha \nSingaraja. Jurnal \nPendidikan \nMatematika \nUndiksha, \n11(1), \n63–72. https://ejournal.undiksha.ac.id/index.php/JJPM/article/view/24382 Roekel, D. Van. (2010). Preparing 21st Century Students for a Global Society: An Educator’s Guide to \nthe \n“Four \nCs.” \nNational \nEducation \nAssociation. https://www.nea.org/professional-\nexcellence/student-engagement/tools-tips/being-21st-century-educator Simonson, M., Smaldino, S., & Zvacek, S. (2015). Teaching and Learning at a Distance (Sixth Edition). Information Age Publishing, Inc. Simonson, M., Smaldino, S., & Zvacek, S. (2019). Teaching and Learning at a Distance: Foundations of \nDistance Education (7th ed.). Information Age Publishing. Vol. 12, No. 02, December 2022 Dewi Isabella Palma and Eliana Putri, Analysis of Learning… \n71 Dewi Isabella Palma and Eliana Putri, Analysis of Learning… 71 Sunardi, Kurniati, D., Sugiarti, T., Yudianto, E., & Nurmaharani, R. (2017). Pengembangan Indikator \n4c’s Yang Selaras Dengan Kurikulum 2013 Pada Mata Pelajaran Matematika Sma/Ma Kelas X \nSemester 1. Jurnal Ilmiah AdMathEdu, 7. Thompson, L., & Ku, H.-Y. (2006). A Case Study Of Online Collaborative Learning. Quarterly Review \nof Distance Education, 7(4). Wijaya, E. Y., Sudjimat, D. A., & Nyoto, A. (2016). Transformasi Pendidikan Abad 21 Sebagai Tuntutan \nPengembangan Sumber Daya Manusia di era Global. Jurnal Pendidikan, 1, 263–278. Yuangga, K. D., & Sunarsi, D. (2020). Pengembangan Media Dan Strategi Pembelajaran Untuk \nMengatasi Permasalahan Pembelajaran Jarak Jauh Di Pandemi Covid- 19. Jurnal Guru Kita, 4(3), \n51–58. Zubaidah, S. (2018). Mengenal 4C: Learning and Innovation Skills untuk Menghadapi Era Revolusi 4.0. 2nd Science Education National Conference, 13(April), 1–18. JMME Halaman 61-71"
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‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools in Canada
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Journal of Religious Education (2022) 70:327–340
https://doi.org/10.1007/s40839-022-00182-w Journal of Religious Education (2022) 70:327–340
https://doi.org/10.1007/s40839-022-00182-w Abstract Pope Francis met representatives of the Indigenous peoples of Canada in Rome in April
2022 and in Canada in July 2022. At these meetings he offered sincere apologies for the
ways in which the Catholic Church had colluded with the strategy of cultural assimila
tion of the Indigenous people in Canada. This was especially abhorrent in the residential
schools, operated by the Catholic Church (and other Christian churches), that aimed to
‘evangelise’ and ‘civilise’ the Indigenous children. He emphasised that this was a ‘disas
trous error’ and the ‘overall effects of the policies linked to the residential schools were
catastrophic’. He was very clear at both meetings that this abuse was ‘incompatible with
the Gospel of Jesus Christ’. These Catholic residential schools and the impact of these
schools on the Indigenous people are discussed in this article. Keywords Catholic residential schools · Canada · Pope Francis Keywords Catholic residential schools · Canada · Pope Francis ‘…and yet there’s still no peace’ Catholic Indigenous
Residential Schools in Canada Stephen J. McKinney1 Accepted: 15 September 2022 / Published online: 29 October 2022
© The Author(s) 2022 Stephen J. McKinney
Stephen.mckinney@glasgow.ac.uk 1
School of Education, University of Glasgow, Glasgow, Scotland Stephen J. McKinney
Stephen.mckinney@glasgow.ac.uk
1
School of Education, University of Glasgow, Glasgow, Scotland 2 Forgotten histories and missing voices There are a number of ways to frame or contextualise the history of the Catholic residen
tial schools in Canada and other parts of the world. They can be framed as a (misguided)
opportunity for evangelisation, for spreading the word of God, or as destructive products of
a rapacious colonial system and as instruments of power and social control (Aquash, 2013;
Johnson, 2018). These are, of course, highly relevant frames and will be referred to in this
article. There are other ways to frame this history using the lenses of forgotten histories and
missing voices. Forgotten histories and missing voices are very closely related, but there is
a distinction to be drawn between them. All remembering is selective, and this can induce selective forgetting (Stone et al., 2017). Remembering is often used to support the sense of the ‘self-concept’ for an individual per
son. The self-concept includes the following dimensions but is not synonymous with any of
them: self-esteem; self-worth; self-image; ideal self; identities or roles and personal traits
and qualities (Ackerman, 2018). A person acts and interacts with others based on their own
self-concept and, if others accept this self-concept in interactions, they help to affirm the
self-concept for that person. The remembering associated with the self-concept can be a
combination of positive and negative memories, but an emphasis on more positive memo
ries can preclude more negative memories. By extension and adaptation, this can be applied to the concept of an institution that can
be held by those in leadership positions or positions of power – and this enables a deeper
understanding of the internal conception and external projection of the institution. The insti
tution can act and interact based on the held concept and this can be affirmed in interactions. The held concept and projection of an institution, for example, can be premised on the view
that the institution is socially just, and the remembering and recall will be about the positive
times that demonstrate this social justice. This then can lead to a hesitancy or a failure to
recall times when social justice was not demonstrated – which would obviously contradict
the held concept of the institution. This leads to a forgetting or supressing of histories or
parts of histories. Forgotten histories refers to unacknowledged, overlooked, obscured or hidden histories
that have not been recalled or remembered. 1 Introduction The visit of Pope Francis to Canada in July 2022 has assumed significant historical impor
tance. During the visit, the Pope apologised to the Indigenous people for the role of the
Catholic Church and religious communities in the cultural and religious abuses they have
experienced. These abuses were experienced by many children who had been placed in the
Indigenous residential schools. This article discusses the context for these apologies and the
apologies themselves in three parts. First, the article will use forgotten histories and missing
voices as a framework. This framework will enable a deeper understanding of the institu
tional significance of the revelations about the treatment of the Indigenous children in the
residential schools. Second, the rationale for the residential schools and the treatment of the
Indigenous children will be scrutinised, with a focus on religious abuse. Third, the apologies 1 3 3 3 328 S. J. McKinney of Pope Francis will be examined, and some concluding remarks are offered about why it is
important to remember this history. of Pope Francis will be examined, and some concluding remarks are offered about why it is
important to remember this history. 1 3 2 Forgotten histories and missing voices They may have been forgotten because they
were about persons or groups of persons who were not deemed to be important or relevant
in the ways in which the narratives of the history of the institution have been configured
and been projected. For example, the stories of lower placed members of the institution or
women may have been forgotten because other narratives were more prominent, such as
hierarchical and male dominated narratives. Or they were forgotten because the history was
supressed as it exposed uncomfortable or shameful attitudes, actions or events that disrupted
the held concept and projection of the institution. There is a danger that the held conception
becomes a form of self-deception. These forgotten histories will include the missing voices
from the past which were not considered to be important or were deliberately excluded. It would be a mistake to consign missing voices solely to past periods of time, to periods
of forgotten history. Missing voices can refer to the present and voices that are missing 1 3 ‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools… 329 in the contemporary world. Within the Catholic Church, missing voices still refers to the
voices of women, including Catholic women religious who are scrutinised, at times, and
excluded by male hierarchies (Brigham, 2015). There are signs that Pope Francis is attempt
ing to address this, and good recent examples have included the appointment of four women
(three religious and a lay woman) as consultors to the secretariat of the Synod of Bishops
and, at the time of writing, his intention to appoint two women to the Dicastery that helps
him select bishops (Lamb, 2019, Vatican news, 2022a). Missing voices still refers to the
voices of children and groups of children who are not heard. Very recently it has referred
to the voices of children who identify as LGBTQ in Catholic schools (Huchting & Fisher,
2019). A focus on forgotten histories and missing voices can have a positive outcome in the
sense that some extraordinary histories can be uncovered, and missing voices heard and
highlighted. The life of Saint Katherine Drexel, foundress of the sisters of the Blessed Sac
rament in America is a good example of a Catholic educator who began to listen to the
missing voices. She divided opinion during her lifetime and her legacy continues to divide
opinion (Lipperini, 2013; Morton, 2014, 2022). 2 Forgotten histories and missing voices For some she brought the message of Christ
to the unchurched black masses, to others she ‘imposed the U.S. Catholic Church’s Jim
Crow policies on Black education in the South’. Morton (2022, 19–20) argues for a move
away from binary understandings and a more nuanced vision of the process of her life work: In the Afro-Catholic Diaspora, Mother Katherine is neither hero nor villain; she is a
beloved witness to this self-determined Black Catholic education…Mother Katherine
experienced a shift from being a missionary to unchurched black souls to becoming an
accomplice to the holistic survival of Black people – mind, body, and spirit. Within this analysis of the work of Saint Katherine one of the greatest parts of the devel
opment of her vocation was that she began to listen to the missing voices of the African
Americans. Within this analysis of the work of Saint Katherine one of the greatest parts of the devel
opment of her vocation was that she began to listen to the missing voices of the African
Americans. While it is helpful to examine the more positive outcomes of a nuanced approach to for
gotten histories, it is clear that forgotten histories and missing voices often refer to darker
histories that have been deliberately forgotten or suppressed. Forgotten histories and abuses
of power are being uncovered in the sex abuse scandals that have emerged in many Chris
tian churches. The sex abuse has taken many forms and the impact of the scandals, certainly
in the Catholic Church, has been exacerbated by the attempts to cover up or to deny, and
even to bury the scandals (Dale & Alpert, 2007; Formicola, 2016) In other words trying to
ensure they remain forgotten histories and that the missing voices of the victims of abuse
are suppressed. This is beginning to be researched from different sociological, legal and
theological perspectives (Moran, 2019; Formicola, 2020). 3 Indigenous children in Catholic schools – Australia and the USA Forgotten histories and missing voices are revealed in the contemporary histories of the
treatment of the children of Indigenous peoples by the Catholic Church in various locations
including Canada, USA and Australia. In all three countries, the aim was to assimilate, not
integrate, the Indigenous children into state languages, cultures and even accepted forms of 1 3 330 S. J. McKinney religion in the 19th and 20th centuries. This was partly a legacy of the Doctrine of Discovery
and a series of papal bulls that had been used from the 15th century to legally justify the
colonisation by the European powers (Miller, 2019). religion in the 19th and 20th centuries. This was partly a legacy of the Doctrine of Discovery
and a series of papal bulls that had been used from the 15th century to legally justify the
colonisation by the European powers (Miller, 2019). This proposed assimilation was of a limited nature, in that it did not provide equal oppor
tunities for the Indigenous children. It usually meant assimilation into the lowest socio-
economic status and conditions: the boys were prepared for manual labour or farming, and
the girls were prepared for some form of domestic service (Smith, 2009). It is useful to note
that the assimilation in the early days did not extend to citizenship as this was only granted
to all Indians in the United States in 1924 and in Canada in 1960 (Dawson, 2012). In Aus
tralia the 1967 Referendum granted rights and equality to the Aboriginal people and Torres
Strait Islanders – citizenship was granted in different states at different times prior to this
(Gordon, 2018). Zucker (2008) notes that the assimilation policy in Australia did not recognise the
Aboriginal culture. The Aborigines were considered to have no culture, no history and to
be without spirituality. The Indigenous people would be improved by adopting the superior
white way of life and the Christian religion. Indigenous children were separated from their
parents to be educated into Christianity and in many places by Catholic missionaries. While
different stories have emerged, and some suggest that the children were often treated better
in the homes run by the Catholic Church than government institutions, the children were still
separated from their families and for a specific purpose – evangelisation and civilisation. 3 Indigenous children in Catholic schools – Australia and the USA A similar story unfolds in the United States of America where a recent investigation has
confirmed that the United States was responsible for supporting 408 boarding schools across
37 states between 1819 and 1969 (United States Department of the Interior, 2022). The
Catholic Church was funded to run some of the Federal Indian boarding schools, as were
other Christian denominations. Smith (2009, 5–6) argues that this was conceived as part of a
deliberate choice of cultural genocide rather than physical genocide because, in the context
of the United States of America, cultural genocide was perceived to be more economically
efficient. Cultural genocide or ‘ethnocide’ does not involve the mass death of physical geno
cide but the attempt to ‘destroy culture, language and religion’ and steal land and outlaw
customs (MacDonald & Hudson, 2012, 442). The rejection of the spirituality and religious
practices of the children and the imposition of Catholic Christianity were components of
the cultural genocide that had tragic consequences. The recent investigation has prompted
the United States bishops to seek a greater understanding of this history, promising to share
their findings and create a greater culture of inclusion (Ruff, 2022). 4 Indigenous children in Catholic schools – Canada In Canada the strategies to ‘educate’ the Indigenous children (First Nations, Métis, and Inuit
children) began before the opening of the residential schools in the late nineteenth century. There was an unsuccessful attempt at a Catholic boarding school for Aboriginal boys in the
early 17th century (The Truth and Reconciliation Commission of Canada, 2015a). Sir Pere
grine Maitland, the lieutenant Governor of Upper Canada proposed a plan in the early 1820s
to ‘civilise’ the Indians that would focus on the young as it was expected that this would
be more successful than focussing on adults (Hutchings, 2016). Maitland’s chief advisor,
John Strachan, who would become Toronto’s first Anglican bishop, believed that Aboriginal 1 3 ‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools… 331 children should be separated from their families and raised by pious white people. A number
of small boarding schools, operated by different denominations, were set up in the early to
mid 19th century. children should be separated from their families and raised by pious white people. A number
of small boarding schools, operated by different denominations, were set up in the early to
mid 19th century. In the latter part of the 19th century, Nicholas Davin was commissioned by Sir John A. Macdonald Canada’s first prime minister, to write a report on Industrial Schools for Indi
ans and Half-Breeds (published in 1879) (Indian Residential School History and Dialogue
Centre Collections, 2022). Davin drew on the example of the Industrial schools in America
which were deemed to be a ‘principal feature of the policy of aggressive civilisation’ (Davin,
1979). The evidence from America was that the day school did not work ‘because the influ
ence of the wigwam was stronger than the influence of the school’. Christian churches were,
in the opinion of Davin, best placed to supplant Aboriginal spirituality with a better one
(Johnson, 2018). Macdonald justified the residential schools in 1883 as follows: When the school is on the reserve the child lives with its parents, who are savages;
he is surrounded by savages, and though he may learn to read and write his habits,
and training and mode of thought are Indian. He is simply a savage who can read
and write. 4 Indigenous children in Catholic schools – Canada It has been strongly pressed on myself, as the head of the Department, that
Indian children should be withdrawn as much as possible from the parental influence,
and the only way to do that would be to put them in central training industrial schools
where they will acquire the habits and modes of thought of white men. The first industrial residential school was opened by the Canadian government in Battlefield
in 1883 and the strategy to ‘Christianise’ and ‘civilise’ would be put into operation in many
more Government funded residential schools, the majority of which were operated by the
Christian churches until 1969 (White & Peters, 2009; Morcom, 2017). Over 150,000 chil
dren attended these schools between the late nineteenth century and the late 1990s when the
remaining federally supported schools were finally closed. These schools were founded on
the principles of the inherent superiority of whites over the uncivilised and savage Indians
and attempted to implement the policy of the ‘aggressive civilisation’ of the children, as
described by Davin in his 1879 report (Bombay et al., 2014; Hanson et al., 2020). Similar
to America, this was an attempt at cultural genocide. Part of the impetus for the cultural
genocide was to ensure the acquisition of the lands occupied by the Indigenous peoples and
the natural resources on these lands (Rose, 2018). The Indian Act of 1920 allowed the government to ‘compel any First Nations child to
attend residential school’, but it was never compulsory for the children to attend residential
schools and some children attended day schools (The Truth and Reconciliation Commission
of Canada, 2015a, 62). These government funded residential schools were run on behalf
of the government by the Catholic Church, the Anglican Church, the Presbyterian Church,
the Methodist Church and later the United Church and other denominations. The Catholic
Church was responsible for approximately 60% of the residential schools, the Anglican
Church 30% and the remaining 10% was between the other denominations (Feir, 2016). The
schools provided religious instruction and basic academic and industrial skills. On arrival the children were given a new name (Euro-Canadian) to replace their Aborigi
nal name and were given a number (The Truth and Reconciliation Commission of Canada,
2015a). It is reported that in many schools the numbers were used daily rather than names. Their hair was cut, and they swapped their traditional clothing for uniform. 4 Indigenous children in Catholic schools – Canada In many of the 1 3 3 332 S. J. McKinney schools, children were punished if they practiced their own customs/culture or used their
own language. Most of the children spoke an Aboriginal language and had scant or no
understanding of French or English, the languages used in the schools. This meant that the
arrival at the school was a bewildering and frightening experience for the children as they
could not communicate and could not understand what was being said to them. Over time,
some children lost fluency in their own language or abandoned their own tongue. There
were exceptions and in one of the Alberta schools, Fr Mullen translated the Bible and hymns
into Cree for the children. In some schools the children were free to converse in their own
languages in the playground. Brothers and sisters were separated in the schools. There was a high death rate due to
maltreatment, poor sanitation, under nourishment and inadequate health care (Mosby &
Galloway, 2017; Wilk et al., 2017). Clearly the basic needs were not met for many of the
children in terms of care, nourishment and health. Many of the survivors of the residential
schools would suffer from long term physical and mental health problems. 5 Truth and Reconciliation Commission of Canada The Truth and Reconciliation Commission of Canada was set up in 2008 to reveal the
‘complex history and on-going legacy of the church-run residential schools to the Canadian
people’ and ‘guide and inspire’ a process that would lead to reconciliation. The Commission
travelled throughout Canada for six years to hear the voices of the Aboriginal people who
had been educated in the residential schools and produced a lengthy report in 2015 with
ninety-four Calls to Action (The Truth and Reconciliation Commission of Canada, 2015a). A previous Commission had produced the Report of the Royal Commission on Aboriginal
Peoples in 1996 that urged for a reconciliation process in Canada. This 400-page report and
the 440 recommendations initially received some media attention but was largely ignored by
the government (Hurley & Wherrett, 1999). Nevertheless, this report did begin the process
of raising wider awareness of the treatment of the Aboriginal people (McGregor, 2018). The
Truth and Reconciliation Commission of Canada provided a second opportunity to examine
and understand how the Aboriginal people had been treated and seek reconciliation. The Truth and Reconciliation Commission of Canada (2015a, 1) used the term cultural
genocide and distinguished cultural genocide from physical and biological genocide: Physical genocide is the mass killing of the members of a targeted group, and biologi
cal genocide is the destruction of the group’s reproductive capacity. Cultural genocide
is the destruction of those structures and practices that allow the group to continue as
a group. States that engage in cultural genocide set out to destroy the political and
social institutions of the targeted group. Land is seized, and populations are forcibly
transferred, and their movement is restricted. Languages are banned. Spiritual lead
ers are persecuted, spiritual practices are forbidden, and objects of spiritual value are
confiscated and destroyed. And, most significantly to the issue at hand, families are
disrupted to prevent the transmission of cultural values and identity from one genera
tion to the next. 1 3 ‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools… 333 The Commission concluded that this separation of children from their families meant that
‘the Canadian Government essentially declared the Aboriginal people to be unfit parents’
(The Truth and Reconciliation Commission of Canada, 2015a 4). The Commission heard
over 6,000 testimonies from former students of the residential schools, family members,
community members and interested participants. 5 Truth and Reconciliation Commission of Canada Some of the testimonies were from former
staff of the residential schools. The missing voices began to be heard in the testimonies of the adults who had been
forced to attend the residential schools as children (Sedehi, 2019). The testimonies speak
about physical, sexual, mental, cultural and religious abuse. 6 Religious abuse This section draws on the testimonies of survivors of Catholic residential schools as
recounted to The Truth and Reconciliation Commission of Canada (The Truth and Recon
ciliation Commission of Canada, 2015b). Some former students appreciated the Christian
upbringing they received through religious education and the daily rituals of prayer. How
ever, there is also evidence of religious abuse that took a number of forms. First, the children
were not allowed to practice their own religion and spirituality, and some were threatened
with punishment if they participated in their own rituals. In some cases, this even applied to
participation in rituals that were practiced outside school term time (Johnson, 2018). Sarah
McLeod recalls returning to Kamloops school with a small totem pole, a birthday present. This was taken from her by one of the nuns and discarded; the nun described it as being ‘all
devil’. Second, the imposition of a strict regime of daily Christian prayer was common: morn
ing and evening prayers, before and after meals and before classes. Antonette White who
had attended the Catholic school at Kuper Island near Vancouver Island, British Columbia
commented: I think the worst thing, is the praying. It’s, it’s like you pray, pray, pray, and yet there’s
still no peace in that prayer of what they made you do. Noel Starblanket attended the Lebret (Qu’Appelle, St. Paul’s, Whitecalf) Industrial School. This was operated by the Missionary Oblates of Mary Immaculate and the Grey Nuns from
1884 to 1973 (University of Regina, 2022). He recalls a very similar strict regime, as does
Victoria Boucher-Grant who attended Fort William (St. Joseph’s) school, Ontario: I learned how to pray. I learned how to, this became a way of life, kneeling on my
knees, and praying to, to some, some God that made me feel guilty because I was, I
was not a very clean person. Third, fear of the judgment of God and the images of the devil and hell were used to coerce
children in a number of ways. Joseph Martin Larocque at the Qu’Appelle school remembers
being scared of images of the devil with a pitchfork in religious education classes. Father
Lacombe’s instructional ladder, a pictorial catechism, was used in a number of the Catholic
schools. This represented stairs leading to heaven and Jesus and the angels. At the bottom of 1 3 334 S. J. 6 Religious abuse McKinney the stairs was fire, and according to Fred Bass, Indian people. Bass stated that he was told at
the Roman Catholic school, Kamsack, Sastatchewan that if he and the other children did not
change their ways, they would end up in the fire. Strict prohibition of the children’s spiri
tuality and the denial of their right to practice their rituals and the imposition of Catholic
religious instruction and practice, then, were used as weapons to evangelise and catechise. 7 The Christian churches and Canada apologise The apologies by the Christian churches were for the collusion with the assimilationist poli
cies of colonialism and for the work of the churches in the residential schools. These apolo
gies began with the United Church of Canada in 1986 apologising for being an agent of
colonialism (Bush, 2015). At this point, there was no explicit apology for the collusion with
the Residential school system. Later apologies included contrition for involvement in the
residential schools. Two Anglican bishops apologised in 1991 and 1993 and the Presbyte
rian Church in 1994. In the Catholic Church the apologies were issued, most notably, by the
Missionary Oblates of Mary Immaculate and different dioceses. On 24 July, 1991 Fr Doug
Crosby, then President of the Oblates apologised on behalf of the members of the order for
the damage caused by the schools they operated (Robinson, 2019). He apologised for three
things. In the first instance, he apologised for the existence of the schools that separated chil
dren from their families. In the second instance, he apologised for the physical and sexual
abuse. In the third instance, he apologised for dismissing the Indigenous religious tradition
and the attempts at assimilation. On June 11, 2008 Stephen Harper, the Prime Minister of
Canada, apologised on behalf of Canada for the role of the residential schools in the policy
of assimilation. 1 3 9 Pope Francis and the apologies to the Indigenous people in Canada The Truth and Reconciliation Commission of Canada (2015a, 221) noted that that the Roman
Catholic Church did not have a single spokesperson and the apologies had been issued by
different diocese and the religious organisations. On April 29, 2009, Pope Benedict XVI
met with Mr Phil Fontaine, the Grand Chief of the Assembly of First Nations of Canada and
The Most Reverend James Weisbgerber, President of the Canadian Conference of Bishops
(Vatican, 2009). In the short communique that was released, the Pope expressed his ‘sorrow
at the anguish caused’ by the church involvement in the residential schools. These words of
regret were not an official apology and he did not use the word apology. The Commission
contrasted this with the pastoral letter of Benedict issued in Ireland in 2010 as a response
to the child abuse in the church in Ireland. In this letter, the Pope stated: ‘You have suffered
grievously and I am truly sorry’ (Pope Benedict, 2010, Sect. 6). This led the Commission
to develop Call to Action number 58 which called upon the Pope to issue an apology to
survivors and their families and communities for the ‘spiritual, cultural, emotional, physi
cal and sexual abuse’ of the children in the Catholic residential schools. They called for an
apology similar to the pastoral letter of 2010 and that the apology should be delivered by the
Pope one year after he received the report of the Commission (The Truth and Reconciliation
Commission of Canada, 2015a, 223, 330). On July 24 to 30, 2022, Pope Francis visited Canada on an apostolic journey. On July 25,
he met with the Indigenous people (First Nation, Métis and Inuit) of Canada and described
his visit as a penitential pilgrimage (Pope Francis, 2022a). The Pope had previously met
some representatives of the Indigenous people in Rome in March/April 2022. At that meet
ing in Rome, the Pope apologised for the ‘suffering, hardship, discrimination’ and abuse
experienced by the people and he drew attention to the role of the residential schools in the
attempt to rob the Indigenous people of their cultural identity. Pope Francis stated that all
of this had made him feel indignation and shame very strongly (Pope Francis, 2022b). He
was emphatic that when confronted with historical memory we must have a commitment to
learn from past mistakes. 8 The unmarked graves The report of the discovery of unmarked graves of 215 Indigenous children in 2021 at the
site of Kamloops Indian Residential School in South Central British Columbia, and later at
the sites of other former residential schools, once again highlighted the tragic consequences
of the injustices of the residential schools (Thorne & Moss, 2022). Kamloops was run by
the Catholic Church. The exact number of children who died may never be known because
of the practice of poor record keeping (Supernant, 2022). There are parallels in other parts of the world. In Scotland, the unmarked grave of possi
bly as many as 400 children is in the cemetery of St. Mary’s Parish Church Lanark (Gamble,
2018). The children had been residents at the Smyllum Park School run by the Daughters
of Charity as an orphanage and for poor children (1864–1981) and they were buried in the
‘Smyllum plot’ in the graveyard. While the children appeared to have died from natural
causes and economic cost may explain the lack of headstones, this is within the context of
the grim evidence of emotional, physical and sexual abuse towards children in the school
that has been uncovered in the Scottish Child Abuse Inquiry Case Study no. 1 (Scottish
Child Abuse Inquiry, 2018). The unmarked graves in Canada and Scotland are chilling indicators that the children
were not to be remembered and would be part of forgotten history. This recovered history 1 3 1 3 ‘…and yet there’s still no peace’ Catholic Indigenous Residential Schools… 335 of the unmarked graves and the abuse experienced by many of the Indigenous children in
the residential homes in Canada demonstrates how far removed the care for the children
was from the conception of social justice that is espoused by Catholic Social Teaching. The
inherent God-given human dignity of all individuals as revealed in sacred scripture which
underpins contemporary Catholic Social Teaching was not recognised (Genesis 1:26–27;
Pontifical Council for Justice and Peace, 2004). Christian ‘dignity’ was imposed with a
European cultural form of Catholic Christianity with terrible consequences. 9 Pope Francis and the apologies to the Indigenous people in Canada The Pope was very clear that the abuses the Indigenous people had
experienced were ‘contrary to the Gospel of Jesus Christ’. In his address at Maskwacis in Canada on July 25, 2022, the Pope apologised again for
the devastation of the Indigenous families and their culture caused by the assimilationist
policies and the residential schools. He drew attention to the role of Catholic Church and
the religious communities in the cultural destruction and the physical, verbal, psychological
and spiritual abuse of the children in their care. Pope Francis emphasised that the ‘overall
effects of the policies linked to the residential schools were catastrophic’ and that this was a
‘disastrous error’. Once again, he was very clear that this abuse was ‘incompatible with the 3 3 336 S. J. McKinney Gospel of Jesus Christ’. Pope Francis recognised the importance of memory and recovering
the forgotten histories (Pope Francis, 2022a). The Pope acknowledged that the public apol
ogy is only a first step in a process that must seek to ensure there is no repetition of the dam
aging culture and practices that have caused such harm for a vulnerable group of people. In the second address on July 25, 2022, at a meeting with Indigenous peoples and mem
bers of the parish community of Sacred Heart Edmonton, the Pope again recalled the role of
the residential schools in ‘robbing communities and individuals of their cultural and spiri
tual identity’ (Pope Francis, 2022c). This was part of the ‘violation of dignity, the experience
of evil, the betrayal of trust’ and shameful for believers. He does not offer a simple solution
but urges that the way forward is to be reconciled in the crucified Christ, and he used this
powerful image to stress the betrayal that took place in the residential schools: This is the way forward: to look together to Christ, to love betrayed and crucified for
our sake; to look to Christ, crucified in the many students of the residential schools. If
we truly want to be reconciled with one another and with ourselves, to be reconciled
with the past, with wrongs endured and memories wounded, with traumatic experi
ences that no human consolation can ever heal, our eyes must be lifted to the crucified
Jesus; peace must be attained at the altar of his cross. 9 Pope Francis and the apologies to the Indigenous people in Canada Pope Francis, despite limited physical mobility, undertook the penitential pilgrimage to
deliver frequent apologies to the Indigenous Peoples for the harm inflicted on them and
damage caused by the cultural suppression in the residential schools. The repeated apologies
emphasised the severity of the harm and damage. His final public statements were delivered
in a Homily at Vespers with Bishops, Priests, Deacons, Consecrated Persons, Seminarians,
and Pastoral Workers in the Cathedral of Notre Dame in Quebec on July 28, 2022 (Pope
Francis, 2022d). He discussed the cultural coercion of the Indigenous peoples and also
asked forgiveness of the victims of sexual abuse. 10 Concluding remarks This article has examined the history of the residential schools in Canada, the meetings of
Pope Francis with the Indigenous people of Canada and his apologies for the cultural and
religious injustices imposed on the Indigenous people and the grievous harm caused by the
residential schools. This has been discussed within the frameworks of forgotten histories
and the missing voices of the survivors of the Catholic residential schools. The Pope has
listened to the voices and urges that the history is investigated, and a healing process can
begin. The Pope did not refer to the term cultural genocide during the encounters with Indig
enous people in Rome and Canada, despite this being the term used by the Truth and Recon
ciliation Commission of Canada. However, he did use this term in his response to a reporter
on the flight back to Rome from Canada. Pope Francis said the following (Vatican News,
2022b): …I didn’t use the word because it didn’t come to my mind, but I described the geno
cide and asked for forgiveness, pardon for this activity that is genocidal. For example, 1 3 337 …and yet there’s still no peace’ Catholic Indigenous Residential Schools… I condemned this too: taking away children, changing culture, changing mentality,
changing traditions, changing a race, let’s put it that way, an entire culture. Yes, geno
cide is a technical word. I didn’t use it because it didn’t come to my mind, but I
described it… It’s true, yes, yes, it’s genocide. It is vitally important that the forgotten history of the Indigenous Residential schools is
being recovered and the missing voices are heard. Education is needed to ensure that people,
especially non-Indigenous people in Canada and beyond, are aware of this history (Neufeld
et al., 2022). This history impacts not only on the Canadian people but also on the associ
ated Christian churches and, for the purposes of this study, especially the Catholic Church. Education in this history is crucial for current and future generations – that they avoid the
deeply harmful mistakes of the past (Kuhl, 2017).i Significant numbers of the children did not receive an adequate education in the harsh
environment of many of the residential schools. 10 Concluding remarks The education in the residential schools, for
the most part, did not cohere with the vision of education described by Pope Francis in his
address to the parish community of Sacred Heart Edmonton (Pope Francis, 2022c): Education must always start from respect and the promotion of talents already present
in individuals. It is not, nor can it ever be, something pre-packaged and imposed. For
education is an adventure, in which we explore and discover together the mystery of
life. Further, the Catholic culture and religious education in the residential schools did not cohere
with the vision of Catholic education based on the love of Christ that is outlined in the series
of contemporary church documents on Catholic education. This series begins with Gravissi
mum Educationis (1965) through The Catholic School (1977) to the Identity of the Catholic
School for a Culture of Dialogue (2022) (Pope Paul VI, 1965; The Sacred Congregation for
Catholic Education, 1977; Congregation for Catholic Education 2022).f These documents offer a vision of Catholic education that is free of coercion; an educa
tion that is not imposed to support a strategy of cultural genocide. This education respects
the inherent God-given dignity of every person and aims to ensure that all people have
opportunities to develop their talents and gifts, enabling them to contribute to the com
mon good of society. Often, the children in the residential schools were not provided with
sufficient opportunities to develop their talents and gifts - opportunities which would have
enabled them to fulfil their potential and make a meaningful contribution to the common
good of society (Brady, 2010). Funding The author did not receive support from any organisation for the submitted work. Declarations Conflict of interest The author declares that he has no conflict of interest. The author has no relevant financial
or non-financial interests to disclose. 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 3 3 338 S. J. McKinney 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/. 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
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institutional affiliations. irshdc.ubc.ca/index.php/Detail/objects/9427 (2017). Residential schools and the effects on Indigenous health and
well-being in Canada—a scoping review. Public Health Reviews, 38(8), 1–23. https://doi.org/10.1186/
s40985-017-0055-6 Zucker, M. (2008). Open hearts: the Catholic church and the stolen generation in the Kimberley. Journal
of the Australian Catholic Historical Society, 29, 23–37. https://link.gale.com/apps/doc/A190243415/
AONE?u=glasuni&sid=googleScholar&xid=eca28645 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. 1 3 1
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